Skin filler formulations

Abstract

This invention relates to an injectable dermal filler formulation for providing improved and uniform plumping and filling effects. Specifically, the formulation comprises a combination of hyaluronic acid and polyorthoesters and can be used for cosmetic purposes.

Description

Technical Field

[0001] This invention relates to an injectable dermal filler formulation for providing improved and uniform plumping and filling effects. Specifically, the formulation comprises a combination of hyaluronic acid and polyorthoesters and can be used for cosmetic purposes. Background Technology

[0002] Soft tissue fillers have been found to be used both as an active treatment to achieve or maintain a youthful appearance and as a responsive treatment to repair damage caused by disease or trauma. The most common treatments are performed on the face, where dermal fillers are injected to increase tissue volume and reduce aesthetic signs of aging.

[0003] Dermal fillers should provide effective and long-lasting filling effects, be easy to inject, and minimize adverse reactions. One challenge is obtaining formulations with sufficiently low viscosity for administration via needles and syringes, while simultaneously maintaining satisfactory elasticity in situ to retain their injection volume and deliver the desired filling effect. This inherent contradiction is a key challenge in dermal filler design.

[0004] One option is to use shear-thinning polymers, such as hyaluronic acid (HA). Hyaluronic acid is a naturally occurring polymer with a linear, unbranched structure composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked by glycosidic bonds. HA is present in many living organisms, from bacteria to higher animals such as humans, and has been commercially used in the cosmetic, biomedical, and pharmaceutical fields. Its biocompatibility, lack of immunogenicity, and ease of degradation by natural enzymes in the body make it a safe compound that has been injected into millions of patients over the past 20 years.

[0005] Hydrogels can be prepared from hyaluronic acid by chemically crosslinking a polymer and causing it to swell in an aqueous medium. Crosslinked HA is widely used in dermal fillers to correct age-related volume loss, moderate to severe facial wrinkles and folds, and for lip augmentation. The success of HA-based dermal fillers stems from their attractive properties, such as low antigenicity and good filling capacity. After injection into the subcutaneous layer, HA-based dermal fillers integrate into the dermis and provide the desired volume by attracting and binding water molecules.

[0006] However, HA-based dermal fillers typically require additional lubricants, such as non-crosslinked HA, to facilitate injection, i.e., reduce the squeezing force required for injection. Unfortunately, adding excessive lubricant can also impair the desired rheological properties of the formulation, leading to reduced filling effect. Furthermore, naturally occurring hyaluronidase can cause degradation of HA-based dermal fillers, and the degradation rate is detrimental to dermal filler formulations designed to provide long-lasting effects.

[0007] Therefore, it is advantageous to provide an improved dermal filler formulation that is safe and easy to inject, and provides a long-lasting, uniform plumping and filling effect. Summary of the Invention

[0008] This article presents an injectable dermal filler formulation based on a mixture of cross-linked hyaluronic acid (HA)-based hydrogel and polyorthoester (POE). This formulation provides excellent filling effect, exhibits high stability at physiological pH, is easy to inject, and is particularly suitable for use in sensitive areas. This injectable dermal filler formulation can be prepared via a multi-step process to optimize the performance of the cross-linked HA.

[0009] Therefore, the object of the present invention relates to providing a safe and readily injectable dermal filler formulation having improved rheological properties and excellent volume-filling effect.

[0010] Therefore, one aspect of the present invention relates to an injectable dermal filler formulation comprising: - Cross-linked hyaluronic acid (HA); and - At least one polyorthoester (POE).

[0011] Another aspect of the present invention relates to a method for preparing an injectable dermal filler formulation as described herein, the method comprising the following steps: (i) Provide a first solution comprising hyaluronic acid (HA); (ii) Reacting the first solution with a crosslinking agent to form a hydrogel containing crosslinked HA; and (iii) Add at least one polyorthoester (POE) to the hydrogel, and then mix; This provides the injectable dermal filler formulation.

[0012] Another aspect of the invention relates to injectable dermal filler formulations as described herein, which can be obtained by methods as described herein.

[0013] Another aspect of the invention relates to a kit comprising: - Injectable dermal filler formulations as described herein; and - Optional, use the instruction manual.

[0014] A further aspect of the invention relates to the use of injectable dermal filler formulations or kits as described herein for cosmetic applications. Attached Figure Description

[0015] Figure 1 Rheometer measurements of the storage modulus (G') and loss modulus (G'') of a group of dermal filler formulations in the frequency range of 0.01–10 Hz are presented. Dermal filler formulations with HA to crosslinking agent ratios (“crosslinking ratios”) of (A) 5:1, (B) 10:1, and (C) 20:1 were tested. Samples without “TEG-POE %” served as a control group; no TEG-POE was added to the formulations.

[0016] Figure 2 The following table shows the measurement results of the extrusion pressure of a group of dermal filler formulations. (A) The extrusion pressure is recorded as the maximum load value. (B) The maximum load of dermal filler formulations with different crosslinking ratios as a function of TEG-POE content.

[0017] Figure 3 The changes in storage modulus over time of hydrogel formulations containing different amounts of TEG-POE upon exposure to hyaluronidase are shown. Degradation of the hydrogel formulation is expressed as the ratio of the storage modulus of the degraded sample to that of the control sample (in which PBS buffer was added instead of hyaluronidase).

[0018] Figure 4 The following figures are shown: (A) Cell viability of cells grown in the absence of the hydrogel formulation (CTR), in the presence of the TEG-POE-free hydrogel formulation (HA:DVS 10:1), and in the presence of the hydrogel formulation containing 2.5% TEG-POE or 5% TEG-POE (TEG-POE 2.5%, TEG-POE 5%). (B) The morphology of each of the four samples is shown in an optical microscope.

[0019] The invention will be described in more detail below. Detailed Implementation

[0020] definition Before outlining the invention in more detail, a set of terms and general definitions are first defined: dermal fillers In the context of this article, the term "dermal filler" refers to materials capable of increasing the volume of areas of soft tissue defects. Dermal fillers are not limited by injection site or type and can act on multiple layers below the dermis, including but not limited to the subcutaneous, subcutaneous, and supraperitoneal layers. Injectable dermal fillers can be found in soft tissues including but not limited to muscles, tendons, fibrous tissue, fat, blood vessels, nerves, and synovial tissue. Specifically, dermal fillers can be injected into fat or other tissues.

[0021] Injectable In the context of this article, when the term "injectable" is used in conjunction with dermal filler formulations, it means that the formulation is suitable for injection into the skin or tissue of a subject. Specifically, an injectable formulation can be expelled from a syringe without applying excessive external force.

[0022] It should be understood that lubricants, such as non-crosslinked HA, can be further added to dermal filler formulations to modulate the rheological properties of the formulation and make it injectable, for example, depending on the injection site or depth.

[0023] Hyaluronic acid In the context of this article, the term "hyaluronic acid" (HA) refers to a polysaccharide of varying molecular weights composed of residues of D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid is naturally found on cell surfaces, as a basic extracellular substance in vertebrate connective tissue, in synovial fluid, vitreous fluid of the eye, and in human umbilical cord tissue.

[0024] In this paper, hyaluronic acid is defined as a non-sulfated glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GIcNAc) and glucuronic acid (GIcUA) linked together by alternating β-1,4 and β-1,3 glycosidic bonds. Hyaluronic acid is also known as hyaluronic acid, or simply HA. In the context of this paper, these terms will also cover its conjugate base, hyaluronic acid salt, and therefore these terms are used interchangeably herein. Hyaluronic acid can therefore be provided in the form of sodium hyaluronate.

[0025] Cross-linked hyaluronic acid In the context of this article, the term "crosslinked hyaluronic acid" refers to hyaluronic acid crosslinked with a crosslinking agent. Therefore, crosslinked hyaluronic acid should be understood as comprising multiple HA polymer chains linked by a crosslinking agent capable of reacting with one or more functional groups in the hyaluronic acid polymer structure. The functional groups in the hyaluronic acid backbone are hydroxyl, carboxylate, and acetamide groups.

[0026] As a non-limiting example, the crosslinking agent divinyl sulfone (DVS) can covalently crosslink hyaluronic acid polymers by reacting its vinyl groups with the primary hydroxyl groups of N-acetyl-D-glucosamine.

[0027] The degree of crosslinking can be controlled by adjusting the ratio between hyaluronic acid and the crosslinking agent. A higher content of crosslinking agent will result in a higher degree of crosslinking, thus producing a tighter polymer network.

[0028] Hyaluronic acid is preferably cross-linked using a single type of cross-linking agent, but cross-linked hyaluronic acid can also be obtained by reacting it with two or more cross-linking agents.

[0029] Crosslinking agent In the context of this document, the term "crosslinking agent" refers to any compound capable of chemically linking hyaluronic acid polymers together to form a crosslinked polymer network. The reaction between hyaluronic acid and the crosslinking agent is preferably a covalent reaction. Therefore, the crosslinking agent preferably contains functional groups capable of forming covalent bonds with the hydroxyl, carboxyl, and / or acetamide functional groups of the hyaluronic acid polymer.

[0030] Crosslinking agents are capable of linking two hyaluronic acid polymers together, and therefore include at least two functional groups. Some variants of crosslinking agents include at least two identical functional groups.

[0031] Examples of crosslinking agents include, but are not limited to, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polylactic acid, polyethylene glycol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and carboxymethyl cellulose (CMC) and their modifications.

[0032] hydrogel In the context of this article, the term "hydrogel" refers to a network of macromolecular polymers that swells in aqueous solutions, buffer solutions, or biofluids. The degree of hydration depends on the degree of cross-linking. Hydrogels exhibit flowability between that of liquids and solids at room temperature.

[0033] Hydrogels can be processed to alter their properties. Specifically, hydrogels can be pulverized and micronized to reduce the particle size of the macromolecular network and facilitate their mixing with other components of dermal filler formulations, such as polyorthoesters and non-crosslinked hyaluronic acid.

[0034] Polyorthoester In the context of this article, the term "polyorthoester," also abbreviated as "POE," refers to a family of polymers containing orthoester bonds. Polyorthoesters are formed through transesterification of an orthoester with a diol, or through polyaddition of diketene acetate with a diol. The compound has the general chemical formula: -[-ROC(R1, OR2)-O-R3–] n –

[0035] Type IV polyorthoesters are well-known and generally accepted, typically classified as Type I through Type IV. In the context of this document, Type IV polyorthoesters are preferred POEs. They are formed by reacting diketene acetal 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU) with a diol modified with a short-chain sequence of polyglycolic acid or polylactic acid. The type of diol can modulate the mechanophysical state of the resulting POE IV. Therefore, POEs can be provided in semi-solid or gel form, for example, in a form with a low glass transition temperature (Tg).

[0036] The preferred polyorthoester is triethylene glycol-polyorthoester (TEG-POE).

[0037] In the context of this article, polyorthoesters (such as TEG-POE) are not considered as lubricants.

[0038] semi-solid In the context of this paper, the term "semi-solid" refers to the mechanophysical state of a material that exhibits fluidity under moderate pressure. Semi-solid materials are able to support their own weight and maintain their shape, but deform and become fluid when pressure is applied, exhibiting gel-like behavior. Semi-solid materials, such as polyorthoesters, should have a viscosity of approximately 10 Pa. s to approximately 500 Pa Within the range of s.

[0039] Below the glass transition temperature (Tg), semi-solid polyorthoesters exist in a glassy state. Conversely, above Tg, they exist in a liquid state. It is noteworthy that semi-solid polyorthoesters are not thermoplastic polymers; thermoplastic polymers are solid (rigid) below Tg, but become soft and moldable above Tg.

[0040] The terms "semi-solid" and "quasi-solid" are used interchangeably.

[0041] Energy storage modulus In the context of this paper, the term "storage modulus" refers to the storage modulus G' (also known as the elastic modulus) measured by frequency scanning at a fixed strain and varying frequency, and its unit is Pascal (Pa). The storage modulus remains constant as the frequency increases, indicating resistance to deformation.

[0042] In this paper, the storage modulus is expressed as a value at a frequency of 1 Hz and can be measured on a rotational rheometer (RheoStress 6000, HAAKE rheometer, Waltham, MA, USA) equipped with cone-plate and plate-plate geometries, with the following settings: - Temperature: 25℃ - Gap size: 1000 µm - Geometry / Plate Size: PP20 - Strain: 0.005, - Frequency scan: 0.01-10 Hz.

[0043] Viscosity In the context of this article, the term "viscosity" (shear dynamic viscosity), determined by shear testing, refers to the magnitude of resistance to flow of a fluid (here, a gel). Therefore, viscosity affects the injectability of a fluid or material. Viscosity is expressed in Pa. s represents.

[0044] Similar to storage modulus, viscosity can be determined using a rotational rheometer equipped with cone-plate and plate-plate geometries (RheoStress 6000, HAAKE rheometer, Waltham, MA, USA), with the following settings: plate-plate mode, 1000 µm gap, 25°C, for steady-state shear analysis in the range of 0.1–1000 s⁻¹. -1 The functional relationship between viscosity η and shear rate γ was determined within a certain range.

[0045] Average particle size In the context of this paper, the term "average particle size" refers to the D50 value (or median diameter) of the particle size distribution. The D value is a percentile value that can be directly read from the differential particle size distribution; therefore, D50 indicates that 50% of the particles have a diameter smaller than this value, and 50% of the particles have a diameter larger than this value. The D50 value used in this paper refers to the number-weighted distribution of the particles.

[0046] Particle size distribution can be measured using laser diffraction. This measurement can be performed using a Malvern Mastersizer 2000 equipped with a Hydro2000 dispersion unit. The D50 value can be extracted from these measurements.

[0047] As an example, particle size distribution can be determined by diluting a hydrogel containing cross-linked HA with PBS buffer containing 0.5-15% (w / w) NaCl to an HA concentration of 0.1-2% (w / w). Measurements were performed under continuous stirring, assuming spherical particles (refractive index 1.343), and the measurement range was 0.02-2000 µm.

[0048] about In this document, when the term “about” is used in contexts involving quantities, such as absolute quantities (e.g., numerical values, purity, weight, size, etc.), or relative quantities (e.g., percentages, equivalents, or ratios), time ranges, and parameters (e.g., temperature, pressure, etc.), it should be understood that such variables are approximate values ​​and therefore may vary from a specific actual value by ±10%, for example ±5%, preferably ±2% (e.g., ±1%). This is true even if such values ​​are primarily expressed as percentages (e.g., “about 10%” could be any value between 9% and 11% of the index 10).

[0049] Injectable dermal filler formulations This article provides an injectable dermal filler formulation that provides a safe, full, and long-lasting filling effect. This dermal filler formulation can be provided as a bulk formulation and can be further formulated to suit the final application, such as the injection site, injection depth, or intended function. For example, the rheological properties and extrusion pressure of the final formulation can be adjusted, or additives, such as local anesthetics, can be included. The final formulation is easy to inject, stable, and highly biocompatible, thus improving patient compliance.

[0050] The injectable dermal filler formulations disclosed herein are based on a mixture of a hydrogel component comprising cross-linked HA and a polyorthoester (POE). The hydrogel is a three-dimensional network of polymers that can swell in an aqueous medium and retain a significant amount of water while maintaining a defined structure. The structure of the hydrogel is well-suited for providing a full, plump filling effect. The polyorthoester can be a polymer provided in a semi-solid form, which, when mixed with the HA-based hydrogel, provides the benefits of a uniform and more durable filling effect, as well as improved biocompatibility. In particular, POE does not release acidic products, which may be preferred when the dermal filler formulation is used on sensitive areas such as the nasolabial folds and nose region, lips, and corners of the mouth.

[0051] Unexpectedly, the inclusion of POE in HA hydrogels enhanced their resistance to enzymatic degradation and improved their biocompatibility with dermal cells without impairing the rheological properties of the underlying dermal filler formulation. Therefore, the combination of HA-based hydrogels and POE provides a favorable starting point for formulating a wide range of dermal filler products, covering a broad spectrum of applications.

[0052] Therefore, one aspect of the present invention relates to an injectable dermal filler formulation comprising: - A hydrogel, said hydrogel comprising cross-linked hyaluronic acid (HA); and - At least one polyorthoester (POE).

[0053] Another aspect of the present invention relates to a basic dermal filler formulation comprising: - A hydrogel, said hydrogel comprising cross-linked hyaluronic acid (HA); and - At least one polyorthoester (POE).

[0054] Embodiments of the present invention relate to a basic dermal filler formulation as described herein, wherein the basic dermal filler formulation is further formulated into an injectable dermal filler formulation.

[0055] Another embodiment of the present invention relates to an injectable dermal filler formulation comprising a base dermal filler formulation, said base dermal filler formulation comprising: - A hydrogel, said hydrogel comprising cross-linked hyaluronic acid (HA); and - At least one polyorthoester (POE).

[0056] POE is preferably provided in the form of a semi-solid material to better integrate with HA-based hydrogels during mixing and to improve hydrodynamics. POE can be incorporated into formulations via a simple mixing step, resulting in a dermal filler formulation that is homogeneous and easy to inject at room temperature.

[0057] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein at least one POE is a semi-solid.

[0058] A particularly advantageous class of semi-solid POEs is type IV POE (POE IV), which is synthesized from diketene acetal (DETOSU) and a diol, preferably modified with short-chain sequences of polyglycolic acid and / or polylactic acid. POE IV offers improved stability compared to other types of POEs.

[0059] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein at least one POE is a type IV POE (POE IV).

[0060] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein POE is formed by reacting diketene acetal 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU) with a diol.

[0061] Further embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the diol is modified with short-chain sequences of polyglycolic acid and / or polylactic acid.

[0062] Injectable dermal filler formulations are not limited to a specific POE compound, but can achieve the aforementioned advantages through a wider range of POE compounds. Unlike polyesters, which undergo bulk degradation, most POEs degrade via surface erosion. This is because their hydrophobicity prevents water from dissolving and penetrating the bulk polymer material, ultimately confining the hydrolysis of orthoester bonds to the accessible surface of the material. Therefore, POEs exhibit good stability at physiological pH and in aqueous conditions.

[0063] Without being bound by theory, it can be argued that the combination of POE with HA-based hydrogels improves the durability of dermal filler formulations due to complementary degradation mechanisms. Specifically, POE not only degrades more slowly than HA after injection but also protects HA from degradation, such as by hyaluronidase. This general mechanism will apply to a wide range of POE types, which can be represented by a general structural formula.

[0064] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the at least one POE is as shown in formula (I) or formula (II): (I) (II) in: R represents a chemical bond, —(CH2) a —or—(CH2) b —O—(CH2) n —; where a is an integer from 1 to 10, and b and c are independent integers from 1 to 5; R* is C 1-4 alkyl; n is an integer that is at least 5; A is R 1 R 2 R 3 、or R 4 ; Among them, R 1 for: , p is an integer from 1 to 20; R 5 It is hydrogen or C 1-4 alkyl; R 6 for:

[0065] s is an integer from 0 to 30; t is an integer from 2 to 200; R 7 It is hydrogen or C 1-4 alkyl; Where R 2 for:

[0066] Where R 3 for:

[0067] x is an integer from 0 to 30; y is an integer from 2 to 200; R 8 It is hydrogen or C 1-4 alkyl; R 9 and R 10 Independently for C 1-12 Alkylene; R 11 It is hydrogen or C 1-6 Alkyl, R 12 C 1-6 Alkyl; or R 11 and R 12 Together for C 3-10 Alkylene; Among them, R 4 The residue is a diol residue containing at least one functional group, the functional group being independently selected from amide, imide, urea and carbamate groups; Among them, at least 0.01 mol% of unit A is of formula R. 1 .

[0068] Embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein at least one POE is shown in formula (I).

[0069] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein R* is ethyl.

[0070] The specific properties of POE are largely regulated by the "A portion." It has been found that it contains a large proportion of R... 3Substituent POEs are advantageous. In particular, the use of glycols, such as low molecular weight polyethylene glycol or aliphatic glycols, can increase the flexibility of POEs.

[0071] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein at least 50 mol%, for example at least 60 mol%, for example at least 70 mol%, for example about 80 mol%, of unit A is of formula R. 3 .

[0072] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein R 3 It is low molecular weight polyethylene glycol or aliphatic glycol, preferably low molecular weight polyethylene glycol.

[0073] A preferred POE variant is triethylene glycol-polyorthoester (TEG-POE), which forms a slightly more hydrophilic POE. Without being bound by theory, it can be assumed that the increased hydrophilicity improves its binding with HA-based hydrogels, resulting in a more uniform dermal filler formulation.

[0074] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein R 3 It is triethylene glycol.

[0075] Another preferred embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein at least one POE is triethylene glycol-polyorthoester (TEG-POE).

[0076] Further embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein at least one POE is shown in formula (III).

[0077] (III) in, .

[0078] Further embodiments of the invention relate to injectable dermal filler formulations as described herein, wherein X ranges from about 70% to about 90% and Y ranges from about 10% to about 30%; preferably X is about 80% and Y is about 20%.

[0079] The content of POE and / or HA in dermal filler formulations can be adjusted depending on the specific application and / or injection site. Therefore, for some applications, it may be necessary to adjust the content of POE and / or HA to regulate the filling effect, injectability, and / or durability of the formulation. For some formulations, it is preferable that the POE content is lower than the cross-linked HA content.

[0080] Embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein, based on the total weight of the injectable dermal filler formulation, the total concentration of crosslinked HA and POE is in the range of about 0.1% (w / w) to about 12% (w / w), for example about 0.2% (w / w) to about 11% (w / w), for example about 0.4% (w / w) to about 10% (w / w), for example 0.6% (w / w) to about 6% (w / w), for example 0.8% (w / w) to about 4% (w / w), for example 1% (w / w) to about 2% (w / w).

[0081] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the ratio of POE to HA in the formulation is in the range of about 1:50 (w / w) to about 1:0.5 (w / w), for example about 1:20 (w / w) to about 1:1 (w / w), for example about 1:10 (w / w) to about 1:1.25 (w / w), preferably about 1:5 (w / w) to about 1:1.5.

[0082] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein, based on the total weight of the injectable dermal filler formulation, the concentration of at least one POE is in the range of about 0.01% (w / w) to about 3% (w / w), for example about 0.05% (w / w) to about 2.5% (w / w), for example about 0.1% (w / w) to about 2% (w / w), for example 0.2% (w / w) to about 1.5% (w / w), for example 0.3% (w / w) to about 1% (w / w), for example 0.4% (w / w) to about 0.8% (w / w).

[0083] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the concentration of at least one POE is in the range of about 0.1% (w / w) to about 20% (w / w), for example about 0.5% (w / w) to about 18% (w / w), for example about 1% (w / w) to about 16% (w / w), for example about 2% (w / w) to about 15% (w / w), for example about 3% (w / w) to about 14% (w / w), for example about 4% (w / w) to about 12% (w / w), for example about 5% (w / w) to about 10% (w / w).

[0084] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the concentration of at least one POE is about 5% (w / w).

[0085] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the concentration of at least one POE is about 10% (w / w).

[0086] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein, based on the total weight of the injectable dermal filler formulation, the concentration of crosslinked HA is in the range of about 0.1% (w / w) to about 4% (w / w), for example about 0.2% (w / w) to about 3.5% (w / w), for example about 0.5% (w / w) to about 3% (w / w), for example about 0.7% (w / w) to about 2.8% (w / w), for example about 1% (w / w) to about 2.6% (w / w), for example about 1.2% (w / w) to about 2.4% (w / w), for example about 1.4% (w / w) to about 2.4% (w / w), for example about 1.6% (w / w) to about 2.2% (w / w).

[0087] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein, based on the total weight of the injectable dermal filler formulation, the concentration of crosslinked HA is in the range of about 0.4% (w / w) to about 4% (w / w), for example about 1% (w / w) to about 3% (w / w), for example about 1.5% (w / w) to about 2.5% (w / w), for example about 1.8% (w / w) to about 2.2% (w / w).

[0088] Dermal filler formulations can be used as initial (or base) formulations and can be modified according to their intended application by adding other components (such as non-crosslinked HA) or active ingredients (such as local anesthetics or vitamins). In base formulations, lubricants, such as non-crosslinked HA, are not required because these substances can be added in the final formulation stage of the dermal filler.

[0089] Therefore, embodiments of the present invention relate to basic dermal filler formulations as described herein, wherein the basic dermal filler formulations do not contain any lubricants.

[0090] Another embodiment of the invention relates to a basic dermal filler formulation as described herein, wherein the lubricant is a non-crosslinked HA and / or a polyol, preferably a non-crosslinked HA.

[0091] Further embodiments of the present invention relate to basic dermal filler formulations as described herein, wherein the polyol is selected from glycerol, mannitol, sorbitol, propylene glycol, erythritol, xylitol, maltitol, and lactitol, preferably glycerol and propylene glycol.

[0092] It has been found that adding POE to hydrogels containing cross-linked HA does not negatively affect the rheological properties of dermal filler formulations. Therefore, a variety of different dermal filler formulations can be prepared based on a base dermal filler formulation without using excessive (i.e., more than the amount used in conventional dermal filler formulations) lubricants (e.g., non-cross-linked HA) to facilitate injection. This is advantageous because lubricants typically lead to decreased rheological properties, which in turn reduces the filling effect of the formulation.

[0093] However, for further formulation, i.e., for preparing the final dermal filler product, it is advantageous to include a portion of non-crosslinked HA in the formulation to adjust its rheological properties. In particular, formulations containing non-crosslinked HA may be easier to inject. It should be understood that non-crosslinked HA is added to the base dermal filler formulation as part of the final formulation of the dermal filler product. The content of non-crosslinked HA can be adjusted according to the intended purpose of the final dermal filler product, such as the injection site and depth.

[0094] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the formulation further comprises non-crosslinked HA.

[0095] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein, based on the total weight of the injectable dermal filler formulation, the concentration of non-crosslinked HA is in the range of about 0.01% (w / w) to about 5% (w / w), for example about 0.02% (w / w) to about 3% (w / w), for example about 0.05% (w / w) to about 2% (w / w), for example about 0.1% (w / w) to about 1% (w / w), for example about 0.15% (w / w) to about 0.5% (w / w), for example about 0.2% (w / w) to about 0.4% (w / w).

[0096] Further embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the ratio of non-crosslinked HA to crosslinked HA in the formulation is in the range of about 1:50 (w / w) to about 1:2 (w / w), for example about 1:20 (w / w) to about 1:3 (w / w), for example about 1:15 (w / w) to about 1:4 (w / w), preferably about 1:10 (w / w) to about 1:5.

[0097] Further embodiments of the invention relate to injectable dermal filler formulations as described herein, wherein the total concentration of cross-linked HA and / or non-cross-linked HA is in the range of about 1 mg / g to about 50 mg / g, for example about 2 mg / g to about 40 mg / g, for example about 3 mg / g to about 30 mg / g, for example about 5 mg / g to about 25 mg / g, for example about 10 mg / g to about 20 mg / g.

[0098] Further embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the total concentration of cross-linked HA and / or non-cross-linked HA is from about 15 mg / g to about 25 mg / g, for example from about 18 mg / g to about 22 mg / g, preferably about 20 mg / g.

[0099] The size of HA polymers can be altered to influence the structure of the polymer network in HA-based hydrogels, and thus affect dermal filler formulations. The size (molecular weight) of HA polymers can be determined by measuring intrinsic viscosity using the Mark-Houwink-Kuhn-Sakurada (MHKS) equation, or by size exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS).

[0100] Embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the molecular weight of the HA in the first and / or second components is in the range of about 5 kDa to about 5000 kDa, for example, 50 kDa to about 5000 kDa, for example, about 100 kDa to about 3000 kDa, for example, about 250 kDa to about 2000 kDa, for example, about 500 kDa to about 1500 kDa, for example, about 750 kDa to about 1250 kDa.

[0101] This study found that certain sizes of HA polymers may be preferred to provide hydrogels with specific structures that produce good filling effects.

[0102] Therefore, preferred embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the molecular weight of the HA in the first and / or second components is in the range of about 750 kDa to about 1250 kDa, for example 800 kDa to about 1200 kDa, for example 900 kDa to about 1100 kDa, preferably about 1000 kDa.

[0103] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein cross-linked HA and non-cross-linked HA have the same molecular weight.

[0104] Further embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein cross-linked HA and non-cross-linked HA have different molecular weights.

[0105] However, hydrogels are not limited to having only a single size of cross-linked HA polymer, but can contain two or more cross-linked HA polymers of different molecular weights. Hydrogels containing HA polymers of different sizes can be formed by cross-linking all HA polymers in one step, or by cross-linking HA polymers of different sizes in separate steps. The latter method can produce hydrogels with regions of different densities.

[0106] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the hydrogel comprises two or more cross-linked HAs of different molecular weights.

[0107] In principle, the HA used in hydrogels can be any type of HA, including but not limited to HA that forms salts with organic or inorganic bases, esters formed from HA and alcohols, HA amides, O-sulfated derivatives of HA, deacetylated derivatives of HA, and percarboxylated derivatives of HA. HA esters can be esters of alcohols having aliphatic, aryl, cycloaliphatic, aromatic, cyclic, and heterocyclic series. HA amides can be amides of amines having aliphatic, aryl, cycloaliphatic, aromatic, cyclic, and heterocyclic series.

[0108] Preferably, HA is provided in the form of an inorganic salt. Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein HA is provided in the form of an inorganic salt selected from sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, and cobalt hyaluronate.

[0109] To avoid precipitation in injectable dermal filler formulations, it is preferable to keep impurities at a minimum. In particular, calcium ions can cause undesirable precipitation in hydrogels, such as calcium phosphate precipitation. Therefore, when selecting HA salts and / or aqueous buffers for hydrogels, minimizing the risk of precipitation should be kept in mind.

[0110] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein HA is provided in the form of an inorganic salt that does not contain calcium.

[0111] Preferred embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein HA is provided in the form of sodium hyaluronate.

[0112] The polymeric properties of HA-based hydrogels can potentially lead to structurally inhomogeneous injectable dermal filler formulations. For some applications, this texture performs well, for example, where large-sized needles may not hinder application. However, for applications requiring the use of smaller needles for more precise delivery of the dermal filler, a highly homogeneous injectable dermal filler formulation is desirable. This can be achieved by micronizing the hydrogel containing cross-linked HA prior to mixing it with POE and optionally uncrosslinked HA.

[0113] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the average particle size of the first component is less than about 1500 μm, for example less than about 1250 μm, for example less than about 1000 μm, for example less than about 750 μm, for example less than about 500 μm.

[0114] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the average particle size of the hydrogel containing crosslinked HA is in the range of about 50 μm to about 1500 μm, for example about 100 μm to about 1250 μm, for example about 150 μm to about 1000 μm, for example about 200 μm to about 750 μm, for example about 300 μm to about 500 μm.

[0115] The dermal filler formulation comprises a mixture of solvent, hydrogel, and POE dispersed in the solvent. The solvent can be any aqueous solvent compatible with HA-based hydrogels and POE, and biocompatible when administered to a subject. This includes isotonic buffer solutions, including but not limited to saline solutions and PBS. Preferably, the buffer solution is adjusted to a pH suitable for administration to the subject. Furthermore, the pH should be selected to avoid any degradation of hyaluronic acid and / or POE, for example, within the pH range of 6.5–8.5.

[0116] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the solvent of the formulation is an aqueous solvent.

[0117] Another embodiment of the invention relates to injectable dermal filler formulations as described herein, wherein the formulation comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0118] Further embodiments of the invention relate to injectable dermal filler formulations as described herein, wherein the pH of the formulation is in the range of about pH 6.5 to about pH 8.5, for example, about pH 7 to about pH 8, preferably about pH 7.5.

[0119] The rheological properties of dermal filler formulations can be influenced by altering the degree of crosslinking, i.e., the ratio of HA to the crosslinking agent in the HA-based hydrogel. Increasing the relative content of the crosslinking agent results in a denser polymer network in the hydrogel, leading to a more compact structure and thus a better filling effect. However, an overly dense hydrogel can become difficult to inject and may not mix well with other components of the formulation.

[0120] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the HA is cross-linked with a cross-linking agent.

[0121] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the ratio between HA and crosslinking agent is in the range of about 25:1% (w / w) to about 5:1% (w / w).

[0122] Further embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the ratio between HA and crosslinking agent is in the range of about 20:1% (w / w) to about 10:1% (w / w), for example about 18:1% (w / w) to about 12:1% (w / w), for example about 16:1% (w / w) to about 14:1% (w / w).

[0123] Further embodiments of the invention relate to the method described herein, wherein the ratio of HA to crosslinking agent is in the range of about 10:1% (w / w) to about 5:1% (w / w).

[0124] Another embodiment of the invention relates to the method described herein, wherein the ratio of HA to crosslinking agent is about 5:1% (w / w).

[0125] Another embodiment of the invention relates to the method described herein, wherein the ratio of HA to crosslinking agent is about 10:1% (w / w).

[0126] Crosslinking can also be expressed as the degree of modification of the crosslinked HA. The degree of modification can be determined by nuclear magnetic resonance (NMR), for example, by repeated measurements at 500 MHz with 20-degree pulses at ambient temperature. Based on the obtained spectrum, the degree of modification can be determined by calculating the ratio of the N-acetyl signal of HA to the characteristic signal of the crosslinking agent (e.g., the methylene signal of BDDE, as an example).

[0127] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the degree of modification of crosslinked HA is in the range of about 0.5% to 25%, expressed as the ratio of the total amount of crosslinking agent linked by single and double bonds to the total amount of HA disaccharide units.

[0128] Crosslinking of hyaluronic acid alters the morphology of the original HA material, promoting its transformation into an insoluble hydrogel that can absorb large amounts of water without losing its defined structure. This swollen structure provides excellent plumping and filling effects. The principle of the HA crosslinking reaction is that polymer chains are covalently linked by reacting with a crosslinking agent, which can be selected from many different compounds, including but not limited to divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), polyethylene glycol (PEG), polyethylene glycol diglycidyl ether, diepoxyoctane, and p-Phenylene Biscarbodiimide.

[0129] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the crosslinking agent is selected from divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), formaldehyde, glutaraldehyde, polyanhydride, polyaldehyde, polyol, carbodiimide, carboxylic acid chloride, sulfonic acid chloride, cellulose, dextran, epichlorohydrin, ethylene glycol, diglycidyl ether, polyglycerol polyglycidyl ether, and diepoxides or polyepoxides and their derivatives.

[0130] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the crosslinking agent is selected from divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polylactic acid (PLA), polyethylene glycol (PEG), polyethylene glycol diamine, polyethylene glycol diacrylate (PEGDA), polyethylene glycol dimethacrylate (PEGDM), polyethylene glycol diacrylamide (PEGDAA), polyethylene glycol dimethacrylamide (PEGDMA), carboxymethyl cellulose (CMC), acrylated dextran, methacrylated dextran, dextran glycidyl methacrylate, glyceryl dimethacrylate, glycerol 1,3-diglycerolate diacrylate, sorbitan acrylate, and derivatives thereof.

[0131] Preferred embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the crosslinking agent is selected from divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polylactic acid (PLA), and polyethylene glycol (PEG) and their derivatives.

[0132] The preferred crosslinking agent is divinyl sulfone (DVS). With this crosslinking agent, the crosslinking reaction occurs between the primary hydroxyl group of N-acetyl-D-glucosamine and the vinyl carbon atom in the DVS molecule, where the primary hydroxyl group reacts with the vinyl carbon atom via nucleophilic addition. DVS contains two vinyl groups and is highly reactive for nucleophilic addition, resulting in a 2:1 molar ratio of HA disaccharide (N-acetyl-D-glucosamine) to DVS. Because DVS crosslinking does not involve the bioreactive functional groups (carboxylate and acetamide groups) on the HA molecule, the gel largely retains the natural polyanionic properties, physicochemical and biological characteristics of HA.

[0133] DVS exhibits high reactivity under aqueous alkaline conditions, thus reacting completely instantaneously. The bond formed between HA and DVS is a sulfonyl diethyl ether bond, which is known to be highly stable against hydrolysis (degradation) and remains stable under alkaline conditions.

[0134] Therefore, preferred embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the crosslinking agent is divinyl sulfone (DVS).

[0135] The final viscosity of the dermal filler formulation plays a crucial role in how it is administered to subjects. If the formulation is too viscous, it will significantly limit how it is administered to subjects, for example, because it may be impossible to inject with a syringe through a needle, or it may cause undesirable and unacceptable discomfort to the recipient. If the content of cross-linked HA is too high, the formulation may become unsuitable, for example, for injection.

[0136] A portion of non-crosslinked HA can be added to the base dermal filler formulation to promote the shear-thinning properties of the final dermal filler product and improve its injectability when force is applied to the syringe.

[0137] Storage modulus is a parameter used to describe the viscoelastic properties of dermal filler formulations. The high degree of cross-linking in HA-based hydrogels leads to a high storage modulus. While a high storage modulus generally contributes to improved filling efficacy, excessively high storage moduli can cause the dermal filler to exhibit a crystalline and inhomogeneous material, making it unsuitable for injection. Importantly, it has been found that the combination of POE with HA-based hydrogels does not negatively impact the injectability of the underlying dermal filler formulation.

[0138] Therefore, the dermal filler formulations described herein are compositionally balanced to impart injectable viscoelastic properties, providing the desired full-filling effect for any final dermal filler product derived therefrom. Thus, the combination of POE with HA-based hydrogels produces a storage modulus comparable to or better than that of POE-free formulations, thereby contributing to ensuring good filling effects in the final dermal filler formulation.

[0139] As described herein, basic dermal filler formulations can be further formulated according to the final application of the formulation. Therefore, viscosity and / or storage modulus can be adjusted to suit specific injection sites or injection depths. Preferably, viscosity and / or storage modulus are adjusted by adding non-crosslinked HA.

[0140] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the storage modulus of the formulation is in the range of about 5 Pa to about 4000 Pa, for example about 100 Pa to about 4000 Pa, for example about 150 Pa to about 3000 Pa, for example about 200 Pa to about 2500 Pa, for example about 200 Pa to about 2000 Pa, for example about 200 Pa to about 900 Pa, for example about 200 Pa to about 600 Pa.

[0141] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the viscosity of the formulation is about 0.001 Pa. s to approximately 1000 Pa Within the range of s, for example, about 25 Pa s to approximately 1000 Pa s, for example, about 30 Pa s to approximately 800 Pa s, for example, about 40 Pa s to approximately 600 Pa s, for example, about 50 Pa s to approximately 500 Pa s.

[0142] After final formulation to achieve suitable rheological properties, the final dermal filler product can be injected into recipient subjects. The syringe needle size can be selected to suit the injection site and depth. The rheological properties of the final dermal filler formulation should be adjusted, and an appropriate needle size should be selected to ensure no unnecessary discomfort to the patient. As demonstrated in this paper, the inclusion of POE does not negatively affect the injectability of the dermal filler formulation.

[0143] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the formulation can be injected via a 1 mL syringe with a needle of a specification (Birmingham gauge) in the range of about 18 G to about 30 G, for example about 22 G to about 28 G, for example about 24 G to about 27 G.

[0144] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the formulation can be injected via a 1 mL syringe with a needle of a specification (Birmingham gauge) ranging from about 18 G to about 32 G, for example from about 25 G to about 30 G, for example from about 27 G to about 30 G.

[0145] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the formulation can be injected via a 1 mL syringe through a needle of approximately 27 G (Birmingham gauge).

[0146] Further embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the hydrogel formulation can be injected using a 1 mL syringe through a needle of about 27 G (Birmingham gauge) with a pressure of less than about 30 N, preferably less than about 25 N, more preferably less than about 20 N, and most preferably less than about 15 N.

[0147] A further embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the extrusion force of the formulation is less than about 30 N, preferably less than about 25 N, more preferably less than about 20 N, and most preferably less than about 15 N.

[0148] Further embodiments of the invention relate to injectable dermal filler formulations as described herein, wherein the extrusion force of the formulation is in the range of about 10 N to about 30 N, for example about 15 N to about 25 N, for example about 18 N to about 22 N.

[0149] Base dermal filler formulations offer a favorable balance between durable volume-filling effects, injectability, and biocompatibility, a balance that can be transferred to the final dermal filler formulation based on it. Therefore, the rheological properties of the final dermal filler formulation can be tailored to its intended purpose, injected in its "natural" form, meaning without any additional components. However, dermal filler formulations may also contain or be loaded with additives to provide further benefits, such as minimizing injection-related pain or further improving the filling effect.

[0150] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the formulation further comprises one or more additives.

[0151] Another embodiment of the invention relates to injectable dermal filler formulations as described herein, wherein one or more additives are selected from absorbable biocompatible microparticles, local anesthetics, polyols, vitamins, amino acids, metals, antioxidants, preservatives, and mineral salts.

[0152] In particular, hydroxyapatite calcium or polycaprolactone microparticles have previously been shown to promote the formation of new tissues similar to their surrounding environment. This novel collagen-generating process is advantageous, and such microparticles are compatible with the dermal filler formulations described herein.

[0153] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the formulation comprises absorbable biocompatible microparticles.

[0154] Another embodiment of the invention relates to injectable dermal filler formulations as described herein, wherein absorbable biocompatible microparticles comprise polycaprolactone or calcium phosphate-based materials, such as calcium hydroxyapatite.

[0155] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein the calcium phosphate-based material is selected from calcium hydroxyapatite, calcium fluorapatite, calcium chlorapatite, calcium carbonate, calcium tetracalcium phosphate, calcium pyrophosphate, calcium tricalcium phosphate, and calcium octacalcium phosphate, preferably calcium hydroxyapatite.

[0156] Dermal filler formulations may also contain other additives, such as polyols or local anesthetics. Polyols, such as glycerin, can act as lubricants to promote injectability; local anesthetics can reduce any potential pain or discomfort associated with injection of dermal filler formulations.

[0157] Therefore, embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the formulation comprises one or more polyols selected from glycerol, mannitol, sorbitol, propylene glycol, erythritol, xylitol, maltitol, and lactitol, preferably glycerol.

[0158] Another embodiment of the invention relates to an injectable dermal filler formulation as described herein, wherein, based on the volume of the injectable dermal filler formulation, the concentration of one or more polyols is in the range of about 1% (vol / vol) to about 20% (vol / vol), for example, 2% (vol / vol) to about 15% (vol / vol), for example, 5% (vol / vol) to about 10% (vol / vol).

[0159] Another embodiment of the present invention relates to an injectable dermal filler formulation as described herein, wherein the formulation comprises a local anesthetic selected from ambucaine, amicaine, benzocaine, bupivacaine, butacaine, butancaine, chloroprocaine, cocaine, cyclomicaine, dibucaine, dimethocaine, depiperazine, eticaine, formocaine, hecaine, lidocaine, mepivacaine, mepcocaine, metabutoxycaine, oxoxaine, paraethoxycaine, phenacaine, piperacaine, piridocaine, procaine, propylmicaine, propyloxycaine, pyrrolicaine, ropivacaine, tetracaine, tolicaine, trimethaine, and their salts, preferably lidocaine.

[0160] Further embodiments of the present invention relate to injectable dermal filler formulations as described herein, wherein the formulation comprises lidocaine.

[0161] Further embodiments of the invention relate to injectable dermal filler formulations as described herein, wherein, based on the total weight of the injectable dermal filler formulation, the concentration of the local anesthetic is in the range of about 0.1% (w / w) to about 5.0% (w / w), for example about 0.2% (w / w) to about 3.0% (w / w), for example about 0.3% (w / w) to about 2.0% (w / w), preferably about 0.5% (w / w) to about 1.0% (w / w).

[0162] It should be understood that additives containing larger aggregates, such as microparticles, can alter the rheological behavior of dermal filler formulations. Therefore, for dermal filler formulations containing microparticles, an increase in storage modulus, viscosity, and extrusion pressure can be expected. For such formulations, it may be preferable to use a slightly larger needle for injection.

[0163] The injectable dermal filler formulations described herein are prepared by mixing a hydrogel containing cross-linked HA with POE. The hydrogel is prepared and processed through a series of steps, including cross-linking the HA with a cross-linking agent, curing and pulverizing the resulting hydrogel, and neutralizing and micronizing the fragmented hydrogel. This multi-step processing is advantageous as it provides a hydrogel component with viscoelastic properties, making it ideal for use in dermal filler formulations.

[0164] Therefore, one aspect of the present invention relates to a method for preparing an injectable dermal filler formulation as described herein, the method comprising the following steps: (i) Provide a first solution containing hyaluronic acid (HA); (ii) Reacting the first solution with a crosslinking agent to form a hydrogel containing crosslinked HA; and (iii) Add at least one polyorthoester (POE) to the hydrogel, and then mix; This provides the injectable dermal filler formulation.

[0165] This injectable dermal filler formulation, which contains a hydrogel (which contains cross-linked HA) and POE, can also be considered a basic dermal filler formulation, which can be further formulated to produce a final dermal filler formulation available for use.

[0166] Therefore, another aspect of the present invention relates to a method for preparing an injectable dermal filler formulation as described herein, the method comprising one or more steps of adding non-crosslinked HA and / or one or more additives to a base dermal filler formulation; The basic dermal filler composition formulation comprises: - A hydrogel comprising cross-linked hyaluronic acid (HA); and - At least one polyorthoester (POE).

[0167] Crosslinking HA with the aid of a crosslinking agent can be achieved by forming covalent bonds with the hydroxyl, carboxyl, and / or acetamide functional groups of the hyaluronic acid polymer. For example, crosslinking can be achieved under alkaline conditions via the primary hydroxyl group of N-acetyl-D-glucosamine in HA. When using DVS or BDDE crosslinking agents, the base can be NaOH. HA is dissolved in the alkaline solution by mixing with a turbo mixer. It is preferable to extend the mixing at 600 rpm for up to 90 min to ensure that all HA is dissolved. However, mixing can be extended to 180 min.

[0168] Therefore, embodiments of the present invention relate to the method as described herein, wherein the first solution is alkaline.

[0169] Another embodiment of the invention relates to the method as described herein, wherein the pH of the first solution is in the range of about pH 9 to about pH 12, for example, about pH 10 to about pH 12.

[0170] Further embodiments of the present invention relate to the method as described herein, wherein the pH of the first solution is at least 9.

[0171] Further embodiments of the invention relate to the method described herein, wherein the first solution comprises NaOH in a concentration of about 0.001 M to about 2.0 M, for example about 0.01 M to about 1.0 M, for example about 0.1 M to about 0.5 M, preferably about 0.2 M.

[0172] As another example, crosslinking can be achieved via the carboxyl group on D-glucuronic acid. When using carbodiimide crosslinking agents, the acid can be hydrochloric acid.

[0173] To form an HA-based hydrogel, HA is mixed with a crosslinking agent and vigorously stirred to ensure uniform distribution of the reactants. Preferably, after mixing, the mixture is incubated for a period of time to solidify the HA-based hydrogel, which is then pulverized (as part of the processing).

[0174] Therefore, embodiments of the present invention relate to the method as described herein, wherein the ratio of HA to crosslinking agent is in the range of about 25:1% (w / w) to about 2:1% (w / w), for example about 20:1% (w / w) to about 10:1% (w / w).

[0175] Further embodiments of the present invention relate to the method as described herein, wherein the ratio of HA to crosslinking agent is about 5:1% (w / w).

[0176] Another embodiment of the present invention relates to the method as described herein, wherein the ratio of HA to crosslinking agent is about 10:1% (w / w).

[0177] A further embodiment of the invention relates to the method as described herein, which further includes a step of curing the hydrogel by heating.

[0178] Another embodiment of the invention relates to the method described herein, wherein curing includes heating at about 35°C to about 45°C, for example at 40°C, for at least 1 hour, preferably 2 hours.

[0179] The HA hydrogel is broken down into smaller fragments to produce a fragmented hydrogel. Typically, the hydrogel is pulverized into blocks of 25 × 25 × 25 mm. It should be understood that these blocks can be smaller or larger, for example, as small as about 10 mm or as large as about 50 mm. This step can be achieved by cutting the hydrogel on a glass, plastic, or metal plate. Alternatively, the hydrogel can be mechanically forced through a cutting screen (also known as an extrusion screen) to pulverize it. The mesh size of the extrusion screen can be 25 × 25 mm.

[0180] Therefore, embodiments of the present invention relate to the method as described herein, which further includes the step of pulverizing the hydrogel.

[0181] Another embodiment of the invention relates to the method as described herein, wherein the pulverization is performed by cutting.

[0182] After the hydrogel is fragmented, it is washed with an excess volume of aqueous solvent. The solvent can be purified water or any suitable aqueous buffer solution with a pH within a certain range. This is used to neutralize the fragmented hydrogel after initial hydrogel formation in an alkaline solution and to wash away any residual unreacted cross-linking agents. During neutralization, the HA hydrogel absorbs the aqueous medium and swells to saturation. The time required for the fragmented HA hydrogel to reach saturation varies depending on the specific composition and degree of cross-linking of the hydrogel, but neutralization and swelling are preferably carried out for 19-25 hours.

[0183] Therefore, embodiments of the present invention relate to the method as described herein, which further includes a step of neutralizing the hydrogel in a buffer solution.

[0184] Another embodiment of the invention relates to the method as described herein, wherein the fragmented hydrogel is washed immediately after the pulverization.

[0185] Another embodiment of the invention relates to the method as described herein, wherein the washing is accomplished by immersing the fragmented hydrogel in ultrapure water and / or a washing buffer, and optionally by agitating the immersed fragmented hydrogel.

[0186] Further embodiments of the present invention relate to the method as described herein, wherein the buffer is a phosphate buffer or a saline buffer.

[0187] Further embodiments of the invention relate to the method described herein, wherein the pH of the buffer is in the range of about 5.5 to about 9.

[0188] Further embodiments of the invention relate to the method described herein, wherein the pH of the buffer is in the range of about 5 to about 7.5, for example in the range of about 6 to about 7, preferably about 6.9.

[0189] Using a sieve, the neutralized (swollen) hydrogel, still in fragmented form, is separated from excess, unabsorbed buffer solution. The neutralized hydrogel is placed in the sieve to allow the unabsorbed liquid portion to drain, thus separating the purified swollen hydrogel from the solution.

[0190] Therefore, embodiments of the present invention relate to the method as described herein, wherein the separation of the neutralized hydrogel from the buffer solution is achieved by placing the neutralized hydrogel in a sieve and subsequently draining the liquid portion.

[0191] The purified hydrogel is micronized to obtain a homogeneous material that can be applied without difficulty, such as without clogging the needle. Therefore, the dermal filler formulations described herein can be easily injected through needles or catheters of various sizes and types without requiring significant force. Micronization can be achieved by extrusion or mixing.

[0192] Therefore, embodiments of the present invention relate to the method as described herein, which further includes the step of micronizing the hydrogel.

[0193] Another embodiment of the invention relates to the method described herein, wherein the micronization of the purified hydrogel is achieved by extrusion using an extrusion screen.

[0194] Another embodiment of the present invention relates to the method as described herein, wherein the mesh size of the extruded screen is in the range of 200-450 μm.

[0195] Further embodiments of the present invention relate to the method described herein, wherein micronization is achieved by mixing using a high-shear mixer.

[0196] If the dermal filler formulation is prepared using individual steps, the method includes the following steps in chronological order: (i) Crosslinking HA to provide a hydrogel; (ii) Curing the hydrogel; (iii) Pulverize the hydrogel; (iv) Neutralize the hydrogel; (v) Purify the hydrogel; (vi) Micronize the hydrogel; (vii) Add POE; After the preparation of the base dermal filler formulation, additional formulations of non-crosslinked HA and / or other components, such as local anesthetics, may be performed. These additional steps can be performed immediately after the base dermal filler formulation is prepared, or after the formulation has been stored.

[0197] Therefore, embodiments of the present invention relate to the method as described herein, which further includes the step of adding non-crosslinked HA to the hydrogel.

[0198] One aspect of the present invention relates to injectable dermal filler formulations as described herein, which are obtainable by methods as described herein.

[0199] Dermal filler formulations can be conveniently supplied in kit form, which also includes information on how to further formulate and / or apply the formulation. The formulation can be packaged for easy dispensing by adding, for example, non-crosslinked HA and / or other additives, or it can be pre-filled in one or more syringes for easy use.

[0200] Therefore, one aspect of the present invention relates to a kit comprising: (i) injectable dermal filler formulations as described herein; and (ii) Optional, use the instruction manual.

[0201] Another aspect of the invention relates to a kit comprising: (i) basic dermal filler formulations as described herein; and (ii) Optional, use the instruction manual.

[0202] Another embodiment of the invention relates to a kit as described herein, wherein the injectable dermal filler formulation is provided in a syringe.

[0203] The dermal filler formulations described herein are intended for use by subjects who wish to improve the appearance of their skin, such as to combat signs of aging. Therefore, dermal filler formulations can be used in a variety of cosmetic or soft tissue filling applications. These formulations are preferably injected by a qualified clinician.

[0204] Therefore, one aspect of the present invention relates to the use of injectable dermal filler formulations or kits as described herein for cosmetic applications.

[0205] Embodiments of the present invention relate to uses as described herein, wherein the cosmetic application is selected from one or more of the following: skin wrinkles and lines, frown lines, nasolabial folds, chin folds, marionette lines, jawline, corners of the mouth, perioral wrinkles, philtrum, crow's feet, skin depressions, scars, temples, subcutaneous support of the eyebrows, cheekbone and cheek fat pads, tear troughs, nose, lips, cheeks, cheekbones, chin, perioral region, infraorbital region, and cosmetic treatments for facial asymmetry.

[0206] Dermal filler formulations can be injected via several different routes of administration. The formulation can be injected as a single application, or small doses of dermal filler can be injected into adjacent sites to allow it to blend continuously along wrinkles or folds (serial puncture technique).

[0207] Therefore, embodiments of the present invention relate to uses as described herein, wherein the formulation is administered subcutaneously, intradermally, or intramuscularly.

[0208] Another embodiment of the invention relates to the use as described herein, wherein the formulation is injected into the dermal region of a subject.

[0209] Another embodiment of the invention relates to the use as described herein, wherein the formulation is applied to increase the volume and fullness of the dermal region.

[0210] Further embodiments of the invention relate to uses as described herein, wherein the subject is a human.

[0211] Another aspect of the invention relates to a method of replacing or filling biological tissue, comprising administering an effective amount of an injectable dermal filler formulation as described herein to a subject in need.

[0212] It should be understood that the term "effective amount" refers to the amount of a dermal filler formulation sufficient to provide a favorable or desired cosmetic effect.

[0213] The listing or discussion of clearly prior art documents in this specification shall not be construed as an admission that such documents are part of the prior art or are common general knowledge.

[0214] Preferred, alternative, and implementation options for any given aspect, feature, or parameter of the invention are to be considered, unless the context otherwise requires, disclosed in conjunction with any and all preferred, alternative, and implementation options for all other aspects, features, and parameters of the invention. The description of injectable dermal filler formulations and all their features are particularly applicable, as they can readily form part of a base dermal filler formulation or injectable dermal filler formulation obtained as described herein, or as part of its use. Embodiments and features of the invention are also summarized in the following items.

[0215] project X1. An injectable dermal filler formulation comprising: - A hydrogel comprising cross-linked hyaluronic acid (HA); and - At least one polyorthoester (POE).

[0216] X2. The injectable dermal filler formulation according to item 1, wherein the at least one POE is a semi-solid.

[0217] X3. An injectable dermal filler formulation according to any one of items 1 or 2, wherein the at least one POE is a type IV POE (POE IV).

[0218] X4. An injectable dermal filler formulation according to any one of the preceding items, wherein the POE is formed by the reaction of diketene acetal 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU) with a diol.

[0219] X5. The injectable dermal filler formulation according to item X4, wherein the diol is modified with short-chain sequences of polyglycolic acid and / or polylactic acid.

[0220] X6. The injectable dermal filler formulation according to item X5, wherein the ratio of polylactic acid to polyglycolic acid is in the range of 80:20 to 20:80, for example 75:25 to 25:75, for example 70:30 to 30:70, for example 65:35 to 35:65, for example 60:40 to 40:60, for example 55:45 to 45:55, for example 50:50.

[0221] X7. An injectable dermal filler formulation according to any one of items X4-X6, wherein the diol is selected from oligoethylene glycol and / or organic diols.

[0222] X8. The injectable dermal filler formulation according to item X7, wherein the oligoethylene glycol is selected from triethylene glycol (TEG), oligoethylene glycol diglycolide, and oligoethylene glycol dilactide.

[0223] X9. The injectable dermal filler formulation according to item X7, wherein the organic diol has a hydrocarbon chain of 2 to 30 carbon atoms.

[0224] X10. An injectable dermal filler formulation according to any one of items X7-X9, wherein the organic diol is linear, branched, or cyclic.

[0225] X11. An injectable dermal filler formulation according to any one of items X7-X10, wherein the organic diol is saturated or unsaturated.

[0226] X12. An injectable dermal filler formulation according to any one of items X7-X11, wherein the organic diol is selected from 1,6-hexanediol, 1,10-decanediol, cis / trans-1,4-cyclohexanediol, p-menthane-3,8-diol, 1,4-butanediol, 1,5-pentanediol, 1,7-heptanediol, 1,8-octanediol, 1,10-decanediol, and 1,12-dodecanediol and any cyclic equivalent thereof.

[0227] X13. An injectable dermal filler formulation according to any one of items X2-X12, wherein the semi-solid POE is semi-solid at both room temperature and above room temperature.

[0228] X14. An injectable dermal filler formulation according to any one of the preceding items, wherein the glass transition temperature Tg of the dermal filler formulation is in the range of about -20°C to about 10°C.

[0229] X15. An injectable dermal filler formulation according to any one of the foregoing items, wherein the at least one POE is as shown in formula (I) or formula (II): (I) (II) in: R represents a chemical bond, —(CH2) a —or—(CH2) b —O—(CH2) n —; where a is an integer from 1 to 10, and b and c are independent integers from 1 to 5; R* is C 1-4 alkyl; n is an integer that is at least 5; A is R 1 R 2 R 3 、or R 4 ; Among them, R 1 for: , p is an integer from 1 to 20; R 5 It is hydrogen or C 1-4 Alkyl groups; and R 6 for:

[0230] s is an integer from 0 to 30; t is an integer from 2 to 200; R 7 It is hydrogen or C 1-4 alkyl; Where R 2 for:

[0231] Where R 3 for:

[0232] x is an integer from 0 to 30; y is an integer from 2 to 200; R 8 It is hydrogen or C 1-4 alkyl; R 9 and R 10 Independently for C 1-12 Alkylene; R 11 It is hydrogen or C 1-6 Alkyl, R 12 C 1-6 Alkyl; or R 11 and R 12 Together for C 3-10 Alkylene; Among them, R 4 The residue is a diol residue containing at least one functional group, the functional group being independently selected from amide, imide, urea and carbamate groups; Among them, at least 0.01 mol% of unit A is of formula R. 1 .

[0233] X16. The injectable dermal filler formulation according to item X15, wherein the at least one POE is as shown in formula (I).

[0234] X17. An injectable dermal filler formulation according to any one of items X15 or X16, wherein R* is ethyl.

[0235] X18. An injectable dermal filler formulation according to any one of items X15-X17, wherein unit A is of formula R, comprising at least 50 mol%, for example at least 60 mol%, for example at least 70 mol%, for example about 80 mol%. 3 .

[0236] X19. An injectable dermal filler formulation according to any one of items X15-X18, wherein R 3 It is low molecular weight polyethylene glycol or aliphatic glycol, preferably low molecular weight polyethylene glycol.

[0237] X20. An injectable dermal filler formulation according to any one of the preceding items, wherein the molecular weight of the POE is in the range of 1.5 kDa to 20 kDa, for example, 2.5 kDa to 10 kDa.

[0238] X21. An injectable dermal filler formulation according to any one of items X15-X20, wherein R 3 It is triethylene glycol.

[0239] X22. An injectable dermal filler formulation according to any one of the preceding items, wherein the at least one POE is triethylene glycol-polyorthoester (TEG-POE).

[0240] X23. An injectable dermal filler formulation according to any one of the foregoing items, wherein the at least one POE is as shown in formula (III): (III) in, .

[0241] X24. The injectable dermal filler formulation according to item X23, wherein X is in the range of about 70% to about 90% and Y is in the range of about 10% to about 30%; preferably X is about 80% and Y is about 20%.

[0242] X25. An injectable dermal filler formulation according to any one of items X23 or X24, wherein X is substantially 100%.

[0243] X26. An injectable dermal filler formulation according to any one of the preceding items, wherein the concentration of the at least one POE is in the range of about 0.1% (w / w) to about 20% (w / w), for example about 0.5% (w / w) to about 18% (w / w), for example about 1% (w / w) to about 16% (w / w), for example about 2% (w / w) to about 15% (w / w), for example about 3% (w / w) to about 14% (w / w), for example about 4% (w / w) to about 12% (w / w), for example about 5% (w / w) to about 10% (w / w).

[0244] X27. An injectable dermal filler formulation according to any one of the preceding items, wherein, based on the total weight of the injectable dermal filler formulation, the concentration of the at least one POE is in the range of about 0.01% (w / w) to about 3% (w / w), for example about 0.05% (w / w) to about 2.5% (w / w), for example about 0.1% (w / w) to about 2% (w / w), for example 0.2% (w / w) to about 1.5% (w / w), for example 0.3% (w / w) to about 1% (w / w), for example 0.4% (w / w) to about 0.8% (w / w).

[0245] X28. An injectable dermal filler formulation according to any one of the preceding items, wherein, based on the total weight of the injectable dermal filler formulation, the concentration of crosslinked HA is in the range of about 0.1% (w / w) to about 4% (w / w), for example about 0.2% (w / w) to about 3.5% (w / w), for example about 0.5% (w / w) to about 3% (w / w), for example about 0.7% (w / w) to about 2.8% (w / w), for example about 1% (w / w) to about 2.6% (w / w), for example about 1.2% (w / w) to about 2.4% (w / w), for example about 1.4% (w / w) to about 2.4% (w / w), for example about 1.6% (w / w) to about 2.2% (w / w).

[0246] X29. An injectable dermal filler formulation according to any one of the preceding items, wherein the formulation further comprises non-crosslinked HA.

[0247] X30. The injectable dermal filler formulation according to item X29, wherein, based on the total weight of the injectable dermal filler formulation, the concentration of non-crosslinked HA is in the range of about 0.01% (w / w) to about 5% (w / w), for example about 0.02% (w / w) to about 3% (w / w), for example about 0.05% (w / w) to about 2% (w / w), for example about 0.1% (w / w) to about 1% (w / w), for example about 0.15% (w / w) to about 0.5% (w / w), for example about 0.2% (w / w) to about 0.4% (w / w).

[0248] X31. An injectable dermal filler formulation according to any one of the preceding items, wherein the average particle size of the hydrogel comprising crosslinked HA is in the range of about 50 μm to about 1500 μm, for example about 100 μm to about 1250 μm, for example about 150 μm to about 1000 μm, for example about 200 μm to about 750 μm, for example about 300 μm to about 500 μm.

[0249] X32. An injectable dermal filler formulation according to any one of the preceding items, wherein the HA is cross-linked with a cross-linking agent.

[0250] X33. An injectable dermal filler formulation according to any one of the preceding items, wherein the crosslinking agent is selected from divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), formaldehyde, glutaraldehyde, polyanhydride, polyaldehyde, polyol, carbodiimide, carboxylic acid chloride, sulfonic acid chloride, cellulose, dextran, epichlorohydrin, ethylene glycol, diglycidyl ether, polyglycerol polyglycidyl ether, and diepoxides or polyepoxides and their derivatives.

[0251] X34. An injectable dermal filler formulation according to any one of the preceding items, wherein the crosslinking agent is divinyl sulfone (DVS) or 1,4-butanediol diglycidyl ether (BDDE).

[0252] X35. An injectable dermal filler formulation according to any one of the preceding items, wherein the crosslinking agent is divinyl sulfone (DVS).

[0253] X36. An injectable dermal filler formulation according to any one of the preceding items, wherein the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0254] X37. An injectable dermal filler formulation according to any one of items X32-X36, wherein the ratio between HA and crosslinking agent is in the range of about 25:1% (w / w) to about 5:1% (w / w).

[0255] X38. An injectable dermal filler formulation according to any one of the preceding items, wherein the storage modulus of the formulation is in the range of about 5 Pa to about 4000 Pa, for example about 20 Pa to about 3000 Pa, for example about 25 Pa to about 2000 Pa.

[0256] X39. An injectable dermal filler formulation according to any one of the preceding items, wherein the storage modulus of the formulation is in the range of about 100 Pa to about 4000 Pa, for example about 150 Pa to about 3000 Pa, for example about 200 Pa to about 2500 Pa, for example about 200 Pa to about 2000 Pa, for example about 200 Pa to about 900 Pa, for example about 200 Pa to about 600 Pa.

[0257] X40. An injectable dermal filler formulation according to any one of the foregoing items, wherein the viscosity of the formulation is about 0.001 Pa. s to approximately 1000 Pa Within the range of s, for example, approximately 0.01 Pa s to approximately 1000 Pa s, for example, about 0.1 Pa s to approximately 1000 Pa s, for example, about 1 Pa s to approximately 1000 Pa s, for example, about 10 Pa s to approximately 1000 Pa s, for example, about 25 Pa s to approximately 1000 Pa s, for example, about 30 Pa s to approximately 800 Pa s, for example, about 40 Pa s to approximately 600 Pa s, for example, about 50 Pa s to approximately 500 Pa s.

[0258] X41. An injectable dermal filler formulation according to any one of the preceding items, wherein the extrusion force of the formulation is in the range of about 10 N to about 30 N, for example about 15 N to about 25 N, for example about 18 N to about 22 N.

[0259] X42. An injectable dermal filler formulation according to any one of the preceding items, wherein the formulation further comprises one or more additives.

[0260] X43. The injectable dermal filler formulation according to item X42, wherein the one or more additives are selected from absorbable biocompatible microparticles, local anesthetics, polyols, vitamins, amino acids, metals, antioxidants, preservatives, and mineral salts.

[0261] X44. The injectable dermal filler formulation according to item X43, wherein the absorbable biocompatible microparticles comprise polycaprolactone or a calcium phosphate-based material, such as calcium hydroxyapatite.

[0262] X45. An injectable dermal filler formulation according to any one of the preceding items, wherein the formulation comprises one or more polyols selected from glycerol, mannitol, sorbitol, propylene glycol, erythritol, xylitol, maltitol and lactitol, preferably glycerol.

[0263] X46. An injectable dermal filler formulation according to any one of the preceding items, wherein the formulation comprises a local anesthetic selected from ambucaine, amicaine, benzocaine, bupivacaine, butacaine, butancaine, chloroprocaine, cocaine, cyclomicaine, dibucaine, dimethicaine, diphenoxycaine, eticaine, formocaine, hecaine, lidocaine, mepivacaine, mepivacaine, mepivacaine, oxocaine, oxoxicaine, paraethoxycaine, phenacaine, piperacaine, pidocaine, procaine, propylmicaine, propyloxycaine, pyrrolicaine, ropivacaine, tetracaine, tolicaine, trimethaine, and their salts, preferably lidocaine.

[0264] Y1. A method for preparing an injectable dermal filler formulation according to any one of the preceding items, the method comprising the steps of: (i) Provide a first solution containing hyaluronic acid (HA); (ii) Reacting the first solution with a crosslinking agent to form a hydrogel containing crosslinked HA; and (iii) Add at least one polyorthoester (POE) to the hydrogel, and then mix; This provides the injectable dermal filler formulation.

[0265] Y2. The method according to Project Y1, wherein the ratio of HA to crosslinking agent is in the range of about 25:1% (w / w) to about 2:1% (w / w), for example about 20:1% (w / w) to about 10:1% (w / w).

[0266] Y3. The method according to any one of items Y1 or Y2, further comprising the step of curing the hydrogel by heating.

[0267] Y4. The method according to any one of items Y1-Y3, further comprising the step of pulverizing the hydrogel.

[0268] Y5. The method according to any one of items Y1-Y4, further comprising the step of neutralizing the hydrogel in a buffer solution.

[0269] Y6. The method according to any one of items Y1-Y5, further comprising the step of micronizing the hydrogel.

[0270] Y7. The method according to any one of items Y1-Y6, further comprising the step of adding non-crosslinked HA to the hydrogel.

[0271] Z1. An injectable dermal filler formulation according to any one of items X1-X46, which is obtainable by any one of items Y1-Y7.

[0272] W1. A kit comprising: - An injectable dermal filler formulation according to any one of items X1-X46 or Z1; and - Optional, use the instruction manual.

[0273] U1. Use of an injectable dermal filler formulation or a kit according to any one of items X1-X46 or Z1 for cosmetic applications.

[0274] U2. According to the use described in item U1, wherein the cosmetic application is selected from one or more of the following cosmetic treatments: skin wrinkles and lines, frown lines, nasolabial folds, chin wrinkles, marionette lines, jawline, corners of the mouth, perioral wrinkles, crow's feet, skin depressions, scars, temples, subcutaneous support of the eyebrows, cheekbone and cheek fat pads, tear troughs, nose, lips, cheeks, chin, perioral area, infraorbital area, and facial asymmetry.

[0275] U3. The use according to any one of items U1 or U2, wherein the formulation is administered subcutaneously, intradermally or intramuscularly.

[0276] The invention will now be described in further detail in the following non-limiting embodiments.

[0277] Example Example 1: Preparation of injectable dermal filler formulation Dermal filler formulations were prepared using a custom-designed process to evaluate their performance, including injectability and filling effect.

[0278] method: Sodium hyaluronate (1 MDa) was dissolved in 0.2 M NaOH solution under mechanical stirring in an Ika mixer equipped with a turbine mixer to prepare a 5-8% (w / w) HA solution. The solution was stirred until no HA clumps were present. Divinyl sulfone (DVS) was added to the solution to achieve the desired HA:DVS ratio (5:1, 10:1, or 20:1), and then mixed until it was completely dispersed in the solution.

[0279] The HA:DVS mixture was then transferred to a plastic tray to form a uniform layer approximately 25-40 mm thick, and cured at 40°C for 2 hours while the tray was covered. After curing, the crosslinked hydrogel was left at ambient temperature for 10 minutes, then cut into fragments (approximately 25 × 40 mm) using a cutting wheel. The fragmented hydrogel was transferred to a beaker containing 12 mM PBS (137 mM NaCl, 2.7 mM KCl, 10.2 mM Na₂HPO₄, 2.0 mM KH₂PO₄), pH 6.9, and swollen for up to 25 hours. Swelling and neutralization were performed to neutralize the pH of the gel and wash away residual DVS and any DVS adducts that might be present. The total swelling time for the fragmented hydrogel was set to 19-25 hours, depending on the degree of crosslinking. All gels with a crosslinking degree greater than 10:1 should be neutralized for at least 24 hours, while fragmented hydrogels with a crosslinking degree less than 10:1 should be neutralized for 19 hours.

[0280] Separate the neutralized hydrogel (pH 6.9–7.5) from the buffer solution using a sieve. Place the hydrogel in the sieve for 10 minutes to allow all buffer solution to seep out. Weigh the remaining hydrogel and, if necessary, perform a mass correction with buffer solution to ensure the overall purified hydrogel reaches the specified HA concentration.

[0281] The purified hydrogel was then micronized by high-shear mixing or extrusion. Micronization by high-shear mixing was performed using a Silverson mixer with an attached Ultramixer head. Micronization by extrusion was performed by passing the hydrogel through an extrusion sieve with a mesh size of 200-450 μm. Support sieves with a mesh size of 1.5 mm can be placed on each side of the extrusion sieve for support. The hydrogel was processed for 20 strokes (10 strokes in each direction) at a pressure of approximately 6 bar. The micronized hydrogel particles were then autoclaved at 121°C for 15 min to provide a hydrogel containing cross-linked HA for mixing with other components of dermal filler formulations.

[0282] Triethylene glycol-polyorthoester (TEG-POE) was added to a hydrogel containing cross-linked HA and with or without non-cross-linked HA, and the components were mixed at room temperature in a solvent-free manner. Mixing continued until a homogeneous, flowable, and non-sticky semi-solid mixture was obtained. Formulations containing 2.5 wt%, 5 wt%, 7.5 wt%, 10 wt%, 12.5 wt%, or 20 wt% TEG-POE were prepared. These formulations correspond to variants of the basic dermal filler formulations described herein.

[0283] For some (not all) samples of dermal filler formulations, a non-crosslinked (linear, 1 MDa) HA (2% (w / w)) component was added to 12 mM PBS (137 mM NaCl, 2.7 mM KCl, 10.2 mM Na2HPO4, 2.0 mM KH2PO4) at pH 6.9, and the hydrogel composition was autoclaved at 121°C for 15 min.

[0284] Figure 1-4 The dermal filler formulations shown in Tables 1-2 do not contain non-crosslinked HA.

[0285] Average particle size of hydrogel The average particle size of the micronized hydrogel particles was measured using a Malvern Mastersizer 2000 in conjunction with a Hydro2000 dispersion unit in a buffer solution containing 0.9% NaCl. Measurements were performed under the assumption that the particles were spherical, and the measurement range was 0.02–2000 µm.

[0286] Example 2: Rheology and injectability of dermal filler formulations The rheological properties and injectability of the dermal filler formulation of Example 1 were tested to identify formulations with properties particularly suitable as dermal fillers.

[0287] Rheological measurement The storage modulus of the dermal filler formulation of Example 1 was measured using a rotational rheometer (RheoStress 6000, HAAKE rheometer, Waltham, MA, USA) equipped with cone-plate and plate-plate geometries. Samples were tested under the following conditions: frequency scan range of 0.01–10 Hz, strain of 0.005, 21 test points, gap of 1000 µm, operating temperature of 25 °C, and a plate-plate geometry of 20 mm. The viscoelastic properties were investigated by recording the functional relationships between the storage modulus G' and loss modulus G'' as a function of oscillation frequency under controlled deformation γ = 0.01 (under which all materials exhibited a linear viscoelastic response in the 0.1–10 Hz range).

[0288] Steady-state shear analysis was performed, with measurements taken in the range of 0.1–1000 s. -1 The functional relationship between viscosity η and shear rate γ within a certain range.

[0289] Injectable The injectability of dermal filler formulations was assessed by measuring the extrusion force of the formulation, i.e., the force required to propel the formulation through a syringe with a predetermined needle size. Testing was conducted using an INSTRON 5566 universal testing machine equipped with a test setup consisting of a 1 mL syringe fitted with a 27 G needle. Testing was performed at 25°C and a test speed of 57 mm / min, measuring the load required to inject the material through the needle. Friction between the piston and the syringe was assessed using a preliminary test on an empty syringe as a control. The extrusion force was recorded as the maximum load value. Figure 2 A). Perform three repeated measurements for each syringe.

[0290] result Measurement results show that the storage modulus (G') and loss modulus (G'') of all tested dermal filler formulations are essentially independent of frequency, with the storage modulus being higher than the loss modulus. Figure 1 A- Figure 1 C), meaning the formulation is a gel material.

[0291] Further investigation into the functional relationship between storage modulus and TEG-POE content at a fixed frequency of 1 Hz (Table 1) unexpectedly revealed that TEG-POE led to an increase in storage modulus compared to formulations without any TEG-POE. In particular, the formulation with 10% TEG-POE exhibited a high storage modulus.

[0292]

[0293] Table 1. Storage modulus (G') of dermal filler formulations with or without different amounts of TEG-POE at 1 Hz.

[0294] In 0.1-1000 s -1 The viscosity η as a function of shear rate γ was determined within a certain range (Table 2). These flow curves indicate how the material flows, and the lower viscosity makes the material easier to diffuse and distribute after injection. Unexpectedly, the addition of TEG-POE to hydrogel formulations resulted in a decrease in viscosity for all formulations. This observation was independent of the ratio between HA and crosslinking agent (“crosslinking ratio”), but was more significant in hydrogel formulations with crosslinking ratios of 5:1 and 10:1.

[0295]

[0296] Table 2. Viscosity η of dermal filler formulations with or without different amounts of TEG-POE.

[0297] Injectability tests on formulations containing different amounts of TEG-POE showed that the injectability of hydrogel formulations was essentially independent of whether or not the hydrogel formulation contained TEG-POE. Figure 2 B). This was unexpected because TEG-POE itself is not injectable, so it was expected to affect injectability.

[0298] in conclusion This embodiment demonstrates that hydrogel formulations containing polyorthoesters and possessing the excellent rheological properties required for use as dermal fillers can be prepared. Furthermore, the inclusion of polyorthoesters does not affect the injectability of the formulation.

[0299] In summary, the tested formulations, as basic dermal filler formulations, are considered an excellent starting point for further formulation of injectable dermal fillers suitable for a wide range of applications. This is especially true since the inclusion of POE does not impair (if any, but rather improves) the rheological properties of the formulation, and Examples 3 and 4 demonstrate that new advantageous properties are obtained by including POE, such as TEG-POE.

[0300] Example 3: Stability of dermal filler formulations The stability of dermal filler formulations was evaluated using a hyaluronidase assay to determine their durability. Resistance to enzyme degradation was assessed by mixing the formulation with a hyaluronidase solution, incubating at 37°C, and then measuring viscoelastic spectra at different time points.

[0301] method: Hyaluronidase powder was reconstituted with 2X PBS to obtain a concentration of 50 U / mL. Hyaluronidase solution (200 μL) was added to 2 g of each formulation (without TEG-POE or containing 2.5%, 5%, 10%, or 20% TEG-POE) to obtain a final concentration of approximately 5 U / g, and then stored at 37°C. A control group was prepared for each sample by mixing with 200 µL of 2X PBS instead of the hyaluronidase solution. Enzyme degradation is expressed as the ratio of the storage modulus of the degraded sample to the control sample at 1 Hz, measured at 3, 6, and 24 hours. result Experiments showed that all formulations containing TEG-POE exhibited stronger resistance to hyaluronidase degradation than the control formulation without TEG-POE. Figure 3 In particular, samples containing 2.5%, 5%, or 10% TEG-POE performed well, with the sample containing 5% TEG-POE showing the strongest resistance to hyaluronidase degradation.

[0302] in conclusion This example demonstrates that including polyorthoesters in hydrogel formulations can protect hyaluronic acid components from enzymatic degradation. This is crucial for the durability of hydrogel formulations (dermal filler formulations) after injection into subjects.

[0303] Example 4: Biocompatibility of Dermal Filler Formulations The biocompatibility of dermal filler formulations was assessed by seeding cells onto hydrogel formulations and observing their viability.

[0304] method: Cell culture To evaluate the biological response of the hydrogel formulation, human skin fibroblasts (HDF) (Sigma-Aldrich, Burlington, MA, USA) were cultured in T-75 cell culture flasks (VWR, Radnor, PA, USA) at 37°C and 5% CO2. The cells were grown in Duchenne modified Eagle medium (DMEM, Microgem, Naples, Italy) supplemented with 20% fetal bovine serum, 1% non-essential amino acids (NEAA), and antibiotics (penicillin G sodium 100 U / mL, streptomycin 100 L amino acids), with the medium changed every 3–4 days.

[0305] Cell viability assay To study cell viability, a density of 1×10⁻⁶ cells was used. 4 HDF cells were seeded at a concentration of 10 cells / mL in 96-well plates (WorldPrecision Instruments, Inc., Sarasota, FL, USA). The hydrogel formulation was pre-sterilized at 121°C and then incubated with DMEM at 37°C for 24 h. The cells were then incubated with 200 cells of the selected formulation's elution buffer for 24 h and 48 h.

[0306] Almarin (AB) reagent was then added to the samples at a concentration of 10% v / v relative to the culture medium, and the samples were incubated at 37°C for 4 h. The absorbance of the samples was measured at 570 nm and 600 nm using a spectrophotometer (Multilabel Counter, 1420Victor, Perkin Elmer, Waltham, MA, USA). The AB reagent dye indicates redox conditions by changing color in response to chemical reduction in the growth medium, which is induced by cell viability.

[0307] Data are expressed as percentage differences between the treatment and control groups. To assess the percentage of restoration (restoration%), the following formula is used for calculation:

[0308] Wherein, O1 is the molar extinction coefficient (E) of oxidized AB at 570 nm; O2 is the E of oxidized AB at 600 nm; A1 is the absorbance of the test well at 570 nm; A2 is the absorbance of the test well at 600 nm; P1 is the absorbance of the control well at 570 nm; and P2 is the absorbance of the control well at 600 nm.

[0309] The percentage of reduction for each sample was normalized relative to the percentage of reduction for the control group to obtain the percentage of cell viability.

[0310] In addition, the viability of HDF cells was further monitored by observing the morphology of HDF cells using an optical microscope (magnification ×20) after 48 hours.

[0311] result Experiments showed that human skin fibroblasts (HDF) grown in a hydrogel formulation without TEG-POE exhibited similar cell viability. Figure 4 A). Surprisingly, the presence of TEG-POE in the hydrogel improved cell viability, with the hydrogel formulation containing 5% TEG-POE showing a more significant improvement. Figure 4 A). This observation is also supported by microscope images ( Figure 4 B).

[0312] in conclusion This example demonstrates that the inclusion of polyorthoesters in hydrogel formulations does not impair cell viability in any way; on the contrary, it improves skin cell viability. This is very encouraging because hydrogel formulations will inevitably carry or come into contact with cells.

[0313] In summary, these examples demonstrate that the inclusion of polyorthoesters, such as TEG-POE, in hydrogel formulations enhances the polymer network and improves elasticity and biocompatibility, while also increasing resistance to enzymatic degradation. Therefore, the formulations described herein offer several advantages over POE-free formulations and can serve as a favorable starting point for the preparation of injectable dermal filler products, whose properties can be further tailored to the end use, for example, by adding non-crosslinked HA and / or additives, such as local anesthetics.

Claims

1. An injectable dermal filler formulation comprising: - A hydrogel comprising cross-linked hyaluronic acid (HA); and - At least one polyorthoester (POE).

2. The injectable dermal filler formulation according to claim 1, wherein, The at least one POE is a semi-solid.

3. The injectable dermal filler formulation according to any one of claims 1 or 2, wherein, The at least one POE is a type IV POE (POE IV).

4. The injectable dermal filler formulation according to any one of the preceding claims, wherein, The POE is formed by the reaction of diketene acetal 3,9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU) with a diol.

5. The injectable dermal filler formulation according to any one of claims 2-4, wherein, The semi-solid POE is semi-solid at both room temperature and above room temperature.

6. An injectable dermal filler formulation according to any one of the preceding claims, wherein, The at least one POE is as shown in formula (I) or formula (II): (I) (II) in: R represents a chemical bond, —(CH2) a —or—(CH2) b —O—(CH2) n —; where a is an integer from 1 to 10, and b and c are independent integers from 1 to 5; R* is C 1-4 alkyl; n is an integer that is at least 5; A is R 1 R 2 R 3 、or R 4 ; Among them, R 1 for: ， p is an integer from 1 to 20; R 5 It is hydrogen or C 1-4 alkyl; R 6 for: s is an integer from 0 to 30; t is an integer from 2 to 200; R 7 It is hydrogen or C 1-4 alkyl; Where R 2 for: Where R 3 for: x is an integer from 0 to 30; y is an integer from 2 to 200; R 8 It is hydrogen or C 1-4 alkyl; R 9 and R 10 Independently for C 1-12 Alkylene; R 11 It is hydrogen or C 1-6 Alkyl, R 12 C 1-6 Alkyl; or R 11 and R 12 Together for C 3-10 Alkylene; Among them, R 4 The residue is a diol residue containing at least one functional group, the functional group being independently selected from amide, imide, urea and carbamate groups; Among them, at least 0.01 mol% of unit A is of formula R. 1 .

7. The injectable dermal filler formulation according to claim 6, wherein, R 3 It is low molecular weight polyethylene glycol or aliphatic glycol, preferably low molecular weight polyethylene glycol.

8. The injectable dermal filler formulation according to any one of the preceding claims, wherein, The at least one POE is triethylene glycol-polyorthoester (TEG-POE).

9. An injectable dermal filler formulation according to any one of the preceding claims, wherein, The concentration of the at least one POE is in the range of about 0.1% (w / w) to about 20% (w / w), for example about 0.5% (w / w) to about 18% (w / w), for example about 1% (w / w) to about 16% (w / w), for example about 2% (w / w) to about 15% (w / w), for example about 3% (w / w) to about 14% (w / w), for example about 4% (w / w) to about 12% (w / w), for example about 5% (w / w) to about 10% (w / w).

10. An injectable dermal filler formulation according to any one of the preceding claims, wherein, The HA is cross-linked with a cross-linking agent selected from divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), formaldehyde, glutaraldehyde, polyanhydride, polyaldehyde, polyol, carbodiimide, carboxylic acid chloride, sulfonic acid chloride, cellulose, dextran, epichlorohydrin, ethylene glycol, diglycidyl ether, polyglycerol polyglycidyl ether, diepoxide or polyepoxide and its derivatives.

11. The injectable dermal filler formulation of claim 10, wherein, The ratio between the HA and the crosslinking agent is in the range of about 25:1% (w / w) to about 5:1% (w / w).

12. A method for preparing an injectable dermal filler formulation according to any one of the preceding claims, the method comprising the following steps: (i) Provide a first solution containing hyaluronic acid (HA); (ii) React the first solution with a crosslinking agent to form a hydrogel containing crosslinked HA; as well as (iii) Add at least one polyorthoester (POE) to the hydrogel, and then mix; This provides the injectable dermal filler formulation.

13. An injectable dermal filler formulation according to any one of claims 1-11, which can be obtained by the method according to claim 12.

14. A kit comprising: - An injectable dermal filler formulation according to any one of claims 1-11 or 13; as well as - Optional, use the instruction manual.

15. Use of the injectable dermal filler formulation according to any one of claims 1-11 or 13, or the kit according to claim 14, for cosmetic applications.

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