WO2025088047A1

WO2025088047A1 - Dermal filler formulations

Info

Publication number: WO2025088047A1
Application number: PCT/EP2024/080094
Authority: WO
Inventors: Birgit FUHLENDORFF; Khadija Schwach-Abdellaoui
Original assignee: Hyamedix
Current assignee: Hyamedix
Priority date: 2023-10-24
Filing date: 2024-10-24
Publication date: 2025-05-01
Anticipated expiration: 2026-04-24

Abstract

The present invention relates to an injectable dermal filler formulation for providing an improved and homogeneous volumizing and filling effect. In particular, the formulation comprises a combination of hyaluronic acid and poly(orthoester) and may be used for cosmetic purposes.

Description

Dermal filler formulations

Technical field of the invention

Background of the invention

Soft tissue augmentation finds its use both as a proactive treatment for attaining or preserving a youthful appearance and as responsive treatment to repair damage caused by disease or injury. The most common treatments are performed in the face where dermal fillers may be injected to increase tissue volume and reduce aesthetics signs of facial aging.

Dermal fillers should provide an efficient and persistent filling effect, be easily injectable, and cause minimal instances of adverse effects. One challenge is to obtain formulations of sufficiently low viscosity to be administered via a needle and syringe while at the same time have satisfactory elasticity in situ to retain their injected volume and provide the desirable filling effect. This inherent paradox is a crucial design challenge for dermal fillers.

One option is to use a shear thinning polymer such as hyaluronic acid (HA). Hyaluronic acid is a natural occurring polymer with a linear, non-branched structure of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine linked via glycosidic bonds. HA is found in many living organisms, from bacteria to higher animals such as humans, and is marketed in applications within the cosmetic, biomedical, and pharmaceutical fields. It is biocompatible, non-immunogenic and can easily be broken down by natural enzymes within the body, making it a safe compound that over the last 20 years has been injected into millions of patients.

Hydrogels may be prepared from hyaluronic acid by chemically crosslinking the polymers and subjecting them to swelling in an aqueous medium. Crosslinked HA is widely used in dermal fillers for the correction of age-related volume loss, moderate to severe facial wrinkles and folds and lip augmentation. The success of HA-based dermal fillers is rooted in attractive properties such as low antigenicity and good filling capacity. Following injection beneath the skin, the HA-based dermal fillers incorporate in the dermal layer and attract and bind water molecules to provide the desired volume.

However, HA-based dermal fillers often require the use of additional lubricants, such as non-crosslinked HA, to facilitate injection of the formulation, i.e. to lower the required extrusion force. Unfortunately, the addition of excessive lubricants also impedes the desired rheological properties of the formulation leading to a diminished filling effect. Moreover, the natural occurrence of hyaluronidase may cause degradation of HA-based dermal fillers at a rate that is counterproductive for a dermal filler formulation intended to provide a long-lasting effect.

Hence, it would be advantageous to provide an improved dermal filler formulation that is safe and easy to inject and provide a long-lasting and homogenous volumizing and filling effect.

Summary of the invention

Herein are provided an injectable dermal filler formulation that are based on the mixture of a crosslinked hyaluronic acid (HA)-based hydrogel with a poly(orthoester) (POE). The formulation provides great filling effect, high stability a physiological pH, is easily injectable and particularly suitable for use in sensitive areas. The injectable dermal filler formulation may be prepared by a multi-step method that optimise the performance of the crosslinked HA.

Thus, an object of the present invention relates to the provision of a safe and easily injectable dermal filler formulation with improved rheological properties and great volume effect.

Thus, an aspect of the present invention relates to an injectable dermal filler formulation comprising:

- crosslinked hyaluronic acid (HA), and

- at least one poly(orthoester) (POE).

Another aspect of the present invention relates to a method for preparing an injectable dermal filler formulation as described herein, said method comprising the following steps:

(i) providing a first solution comprising hyaluronic acid (HA),

(ii) reacting said first solution with a crosslinking agent to form a hydrogel comprising crosslinked HA, and (iii) adding at least one poly(orthoester) (POE) to said hydrogel followed by mixing, thereby providing said injectable dermal filler formulation.

Yet another aspect of the present invention relates to an injectable dermal filler formulation as described herein obtainable by a method as described herein.

Still another aspect of the present invention relates to a kit comprising:

- an injectable dermal filler formulation as described herein, and

- optionally, instructions for use.

A further aspect of the present invention relates to use of an injectable dermal filler formulation or a kit as described herein for cosmetic applications.

Brief description of the figures

Figure 1 shows rheometer measurement of storage modulus (G') and loss modulus (G") of a set of dermal filler formulations over a frequency sweep from 0.01-10 Hz. Dermal filler formulations with a ratio between HA and crosslinking agent ("crosslinking ratio") of (A) 5: 1, (B) 10: 1, and (C) 20: 1 were tested. Samples without a "TEG-POE %" were controls without addition of TEG-POE to the formulation.

Figure 2 shows measurement of extrusion force of a set of dermal filler formulations. (A) The extrusion force is recorded as the value of the maximum load. (B) Maximum load of dermal filler formulations of different crosslinking ratio as a function of content of TEG-POE.

Figure 3 shows the storage modulus over time for hydrogel formulation containing different amounts of TEG-POE when exposed to hyaluronidase. Degradation of the hydrogel formulation is expressed as the ratio between the storage modulus of the degraded sample and a control sample wherein PBS buffer is added instead of hyaluronidase.

Figure 4 shows (A) cell viability for cells growing in absence of a hydrogel formulation (CTR), cells growing in presence of a hydrogel formulation without TEG-POE (HA:DVS 10: 1) and cells growing in presence of a hydrogel formulation contain 2.5% or 5% TEG-POE (TEG-POE 2.5%, TEG-POE 5%). (B) Optical microscopy shows the morphology of each of the four samples. The present invention will in the following be described in more detail.

Detailed description of the invention

Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

Dermal filler

In the present context, the term "dermal filler" relates to a material that has the capacity to add volume to areas of soft tissue deficiency. Dermal fillers are not limited by the location and type of injection, and may be targeted at several levels below the dermis, including, but limited to, submuscularly, subcutaneous, and supraperiostally. The soft tissue to which the dermal filler is injected include, but is not limited to, muscles, tendons, fibrous tissues, fat, blood vessels, nerves, and synovial tissues. In particular, the dermal filler may be injected into fat or tissues.

Injectable

In the present context, the term "injectable" when used in conjunction with the dermal filler formulation means that the formulation is suitable for injection into the skin or tissue of a subject. In particular, an injectable formulation can be dispensed from a syringe without undue application of feree.

It is to be understood that lubricants, such as non-crosslinked HA, may be further added to the dermal filler formulation to tailor the rheological properties of the formulation and to enable injectability e.g. depending on the site or depth of injection.

Hyaluronic acid

In the present context, the term "hyaluronic acid" (HA) refers to polysaccharides with different molecular weights constituted by residues of D-glucuronic acid and N-acetyl- D-glucosamine acid. Hyaluronic acid occurs naturally in cell surfaces, in the basic extracellular substances of the connective tissue of vertebrates, in the synovial fluid of the joints, in the endobulbar fluid of the eye, and in human umbilical cord tissue.

Hyaluronic acid is defined herein as a non-sulfated glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GIcNAc) and glucuronic acid (GIcUA) linked together by alternating beta-1,4 and beta-1,3 glycosidic bonds. Hyaluronic acid is also known as hyaluronan or abbreviated as HA. In the present context, these terms will cover also the conjugate base hyaluronate, and accordingly the terms are used interchangeably herein. Accordingly, hyaluronic acid may be provided as sodium hyaluronate.

Crosslinked hyaluronic acid

In the present context, the term "crosslinked hyaluronic acid" refers to hyaluronic acid crosslinked with a crosslinking agent. Crosslinked hyaluronic acid is therefore understood to comprise a plurality of HA polymeric strands connected by a crosslinking agent capable of reacting with one or more functional groups in the hyaluronic acid polymeric structure. The functional groups in the hyaluronic acid backbone are hydroxyl, carboxylate and acetamide.

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

The degree of crosslinking can be controlled by adjusting the ratio between hyaluronic acid and the crosslinking agent. A higher amount of crosslinking agent will cause a higher degree of crosslinking and therefore a tighter polymeric network.

Hyaluronic acid is preferably crosslinked using a single type of crosslinking agent, but crosslinked hyaluronic acid may also be obtained by reaction with two or more crosslinking agents.

Crosslinking agent

In the present context, the term "crosslinking agent" refers to any compound that is capable of chemically linking hyaluronic acid polymers together in a crosslinked polymeric network. The reaction between the hyaluronic acid and the crosslinking agent is preferably covalent. Thus, crosslinking agents preferably comprise functional groups capable of forming a covalent bond with hydroxyl, carboxylate and/or acetamide functional groups of the hyaluronic acid polymer.

Crosslinking agents are capable of linking two hyaluronic acid polymers together and therefore comprises at least two functional groups. Some variants of crosslinking agents comprise at least two identical functional groups.

Examples of crosslinking agents include, but are not limited to, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polylactic acid, polyethylene glycol, l-Ethyl-3- (3-dimethylaminopropyl)carbodiimide (EDC), and carboxymethylcellulose (CMC), and modifications thereof.

Hydrogel

In the present context, the term "hydrogel" refers to a macromolecular polymeric network swollen in an aqueous solution, buffer, or biological fluids. The degree of hydration is dependent on the degree of crosslinking. A hydrogel has a fluidity at room temperature between that of a liquid and of a solid.

The hydrogel may be processed to modify the properties of the hydrogel. In particular, the hydrogel may be comminuted and micronized to reduce particle size of the macromolecular network and facilitate mixing with additional components of the dermal filler formulation, such as poly(orthoesters) and non-crosslinked hyaluronic acid.

Poly(orthoester)

In the present context, the term "poly(orthoester)", also abbreviated "POE", refers to the polymer family containing an orthoester linkage. Poly(orthoesters) are formed by transesterification of orthoesters with diols or by polyaddition of a diketene acetate and diol. The compounds have the general chemical structure -[-R-O-C(Ri, ORz)-O- R3-]n~.

Four classes of poly(orthoesters) are well known and generally accepted as type I-IV. In the present context, poly(orthoesters) of type IV are the preferred POE. These are formed by reaction between the diketene acetal 3,9-diethylidene-2,4,8,10- tetraoxaspiro[5.5]undecane (DETOSU) and a diol modified by short sequences of polyglycolide or polylactide. The type of diol can guide the mechano-physical state of the resulting POE IV. Accordingly, the POE may be provided as a semi-solid or gel, e.g. with a low glass transition temperature Tg.

A preferred poly(orthoester) is tri(ethylene glycol)-poly(orthoester) (TEG-POE).

In the present context, poly(orthoesters), such as TEG-POE, are not considered to be lubricants.

Semi-solid

In the present context, the term "semi-solid" refers to the mechano-physical state of a material which is flowable under moderate stress. A semi-solid material have the ability to support its own weight and hold its shape, but conforms its shape and has the ability to flow if pressure is applied upon it, i.e. it has gel-like behaviour. A semisolid material, such as a poly(orthoester), should have a viscosity in the range of about 10 Pa*s to about 500 Pa*s.

Below the glass transition temperature, Tg, a semi-solid poly(orthoester) exists in a glassy state. In contrast, above the Tg, a poly(orthoester) exists in a liquid state. Notably, semi-solid poly(orthoesters) are not thermoplastic polymers, which are solid (hard) when below the Tg and soft and mouldable above the Tg.

The terms semi-solid" and "quasi-solid" may be used interchangeably.

Storage modulus

In the present context, the term "storage modulus" refers to the storage modulus G' (also known as elastic modulus) in Pascal (Pa) determined from frequency sweep with fixed strain and varying frequencies. A storage modulus that remains constant over increasing frequencies indicates resistance to deformation.

Herein storage modulus is reported as the value at a frequency of 1 Hz and may be measured on a rotational rheometer (RheoStress 6000, HAAKE Rheometer, Waltham, MA, USA) equipped with a Cone-Plate and Plate-Plate geometry with the following settings:

- Temperature: 25°C

- Gap size: 1000 pm

- Geometry/plate size: PP20

- Strain: 0.005

- Frequency sweep: 0.01-10 Hz

Viscosity

In the present context, the term "viscosity" (shear dynamic viscosity) is determined by shear and refers to how resistant a fluid (here gel) is to flow. Accordingly, viscosity influences the injectability of a fluid or material. The viscosity is reported as Pa*s.

The viscosity can, as for the storage modulus, be measured using a rotational rheometer (RheoStress 6000, HAAKE Rheometer, Waltham, MA, USA) equipped with a Cone-Plate and Plate-Plate geometry with following settings; plate-plate and gap 1000 pm, 25°C , steady state shear analysis performed measuring the viscosity as a function of shear rate y ' in a range 0.1-1000 s^-1. Mean particle size

In the present context, the term "mean particle size" refers to the D50 value (or median diameter) of a particle size distribution. The D value is a percentile value that can be read directly form a differential particle size distribution, and D50 thus represents the value at which 50% of the particle have a smaller diameter and 50% of the particles have a larger diameter. The D50 value as used herein refers to the number weighted distribution of particles.

Particle size distribution can be measured by laser diffraction. Measurements can be performed using a Malvern Mastersizer 2000 coupled with a Hydro2000 dispersion unit. It is possible to extract the D50 value from these measurements.

By way of example, the particle size distribution can be determined by diluting the hydrogel comprising crosslinked HA to a concentration of 0.1-2% (w/w) of HA using a PBS buffer containing NaCI in the range of 0.5-15% (w/w). Measurements are carried out under constant stirring and assuming spherical particles (refractive index of 1.343) and in the interval 0.02 - 2000 pm.

About

Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%).

Injectable dermal filler formulations

Herein are provided injectable dermal filler formulation that provide a safe, voluminous, and long-lasting filler effect. The dermal filler formulations can be provided as a bulk dermal filler formulation that may be further formulated to suit the final application, e.g. the site of injection, depth of injection or intended function. For instance the desired rheological properties and extrusion force of the final formulation may be adjusted or additives such as local anaesthetics can be included. The final formulations are easy to inject, stable and highly biocompatible, and therefore improve patient compliance. The injectable dermal filler formulations disclosed herein are based on the mixture of a hydrogel component comprising crosslinked HA and poly(orthoester) (POE). Hydrogels are three-dimensional networks of polymers that can swell in aqueous medium and retain a large amount of water while maintaining a well-defined structure. The structure of hydrogels is well suited for providing a voluminous filler effect. Poly(orthoesters) are polymers that may be provided as semi-solid materials and when mixed with the HA-based hydrogel amongst others provide the benefits of a homogeneous and longer lasting filling effect and improved biocompatibility. In particular, POEs do not release acidic products, which may be preferred when the dermal filler formulation is utilized in sensitive areas, such as nasolabial folds and nasal areas, lips and oral commissure.

It has surprisingly been found that the inclusion of POEs in the HA hydrogel result in increased resistance to enzymatic degradation and improved biocompatibility with dermal cells without compromising the rheological properties of the bulk dermal filler formulation. As such, the combination of a HA-based hydrogel with POEs is an advantageous starting point for formulation of a broad range of dermal filler products, i.e. spanning a broad range of applications.

- a hydrogel comprising crosslinked hyaluronic acid (HA), and

- at least one poly(orthoester) (POE)

Another aspect of the present invention relates to a bulk dermal filler formulation comprising:

- a hydrogel comprising crosslinked hyaluronic acid (HA), and

- at least one poly(orthoester) (POE)

An embodiment of the present invention relates to the bulk dermal filler formulation as described herein, said bulk dermal filler formulation being further formulated into an injectable dermal filler formulation.

Another embodiment of the present invention relates to an injectable dermal filler formulation comprising a bulk dermal filler formulation comprising:

- a hydrogel comprising crosslinked hyaluronic acid (HA), and

- at least one poly(orthoester) (POE) The POE is preferably provided as a semi-solid material for better integration with the HA-based hydrogel upon mixing and improved flow mechanics. The POE can be incorporated in the formulation by a simple mixing step, with the resulting the dermal filler formulations being homogeneous and easily injectable at room temperature.

Thus, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the at least one POE is semi-solid.

A particular beneficial class of semi-solid POEs are those of class IV (POE IV), which are synthesized from the diketene acetal DETOSU and a diol, preferably modified with short sequences of polyglycolide and/or polylactide. POE IVs offer improved stability over other types of POEs.

Thus, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the at least one POE is a POE of type IV (POE IV).

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

A further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein said diol is modified by short sequences of polyglycolide and/or polylactide.

The injectable dermal filler formulation is not limited to one specific POE compound but the advantages can be achieved by a broader class of POE compounds. In contrast to polyesters, which will undergo bulk degradation, most POEs degrade via erosion on the surface. The reason is that hydrophobicity prevents water from dissolving and entering the bulk polymeric material, which ultimately limits hydrolysis of the orthoester bonds to the accessible surface layers of the material. Accordingly, POEs display good stability at physiological pH and aqueous conditions.

Without being bound by theory, it is contemplated that the integration of POE with the HA-based hydrogel improves the longevity of the dermal filler formulation due to the complementary degradation mechanisms. In particular, the POE may not only degrade slower than HA upon injection but could also protect HA from degradation, e.g. by hyaluronidases. This generic mechanism will apply to the broad class of POEs that may be described by a generic structural formula.

Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the at least one POE is represented by formula (I) or formula (II) :

wherein :

R is a bond, — (CHzja— , or — (CHzjb—O— (CHzjn--; where a is an integer of 1 to 10, and b and c are independently integers of 1 to 5;

R* is a Ci-4 alkyl; n is an integer of at least 5; and

A is R¹, R², R³, or R⁴, wherein R¹ is:

p is an integer of 1 to 20;

R⁵ is hydrogen or Ci-4 alkyl; and

R⁶ is:

s is an integer of 0 to 30; t is an integer of 2 to 200; and R⁷ is hydrogen or Ci-4 alkyl; wherein R² is:

x is an integer of 0 to 30; y is an integer of 2 to 200;

R⁸ is hydrogen or Ci-4 alkyl;

R⁹ and R¹⁰ are independently C1-12 alkylene; R¹¹ is hydrogen or Ci-6 alkyl and R¹² is Ci-6 alkyl; or Rⁿ and R¹² together are C3- 10 alkylene; and wherein R⁴ is a the residue of a diol containing at least one functional group independently selected from amide, imide, urea, and urethane groups; in which at least 0.01 mol % of the A units are of the formula R¹.

An embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the at least one POE is represented by formula (I).

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

The specific properties of the POE are largely guided by the "A moiety". It has been found that POEs with a large fraction of R³ substituents are favourable. In particular, the use of diols, such as low molecular weight polyethylene glycols or aliphatic diols, increases the softness of the POE.

Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein at least 50 mol%, such as at least 60 mol%, such as at least 70 mol%, such as approx. 80 mol%, of the A units are of the formula R³.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein R³ is a low molecular weight polyethylene glycol or aliphatic glycol, preferably a low molecular weight polyethylene glycol.

A preferred variety of POE is tri(ethylene glycol)-poly(orthoester) (TEG-POE), which yields a slightly more hydrophilic POE. Without being bound by theory, it is contemplated that the increased hydrophilicity improves integration with the HA-based hydrogel to form a more homogenous dermal filler formulation.

Thus, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein R³ is tri(ethylene glycol).

Another preferred embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the at least one POE is a tri(ethylene glycol)-poly(orthoester) (TEG-POE). A further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the at least one POE is represented by formula (III):

A still further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, 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%.

The amount of POE and/or HA included in the dermal filler formulation may be adjusted depending on the particular application and/or site of injection of the formulation. Thus, for some applications it may be desired to adjust the amount of POE and/or HA to the tailor filling effect, injectability and/or longevity of the formulation. For some formulations it is preferred that the amount of POE is less than the amount of crosslinked HA.

An embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the total concentration of crosslinked HA and POE is the range of about 0.1% (w/w) to about 12% (w/w), such as about 0.2% (w/w) to about 11% (w/w), such as about 0.4% (w/w) to about 10% (w/w), such as 0.6% (w/w) to about 6% (w/w), such as 0.8% (w/w) to about 4% (w/w), such as 1% (w/w) to about 2% (w/w), based on the total weight of the injectable dermal filler formulation.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the ratio between POE and HA in the formulation is in the range of about 1:50 (w/w) to about 1:0.5 (w/w), such as about 1:20 (w/w) to about 1 : 1 (w/w), such as about 1: 10 (w/w) to about 1 : 1.25 (w/w), preferably about 1:5 (w/w) to about 1: 1.5. Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the concentration of the at least one POE is in the range of about 0.01% (w/w) to about 3% (w/w), such as about 0.05% (w/w) to about 2.5% (w/w), such as about 0.1% (w/w) to about 2% (w/w), such as 0.2% (w/w) to about 1.5% (w/w), such as 0.3% (w/w) to about 1% (w/w), such as 0.4% (w/w) to about 0.8% (w/w), based on the total weight of the injectable dermal filler formulation.

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

Yet another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the concentration of the at least one POE is in about 5% (w/w).

Still another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the concentration of the at least one POE is in about 10% (w/w).

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

Yet another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the concentration of crosslinked HA is in the range of about 0.4% (w/w) to about 4% (w/w), such as about 1% (w/w) to about 3% (w/w), such as about 1.5% (w/w) to about 2.5% (w/w), such as about 1.8% (w/w) to about 2.2% (w/w), based on the total weight of the injectable dermal filler formulation. The dermal filler formulation can be used as an initial (or bulk) formulation that can be modulated based on its intended application by addition of further components, such as non-crosslinked HA, or active ingredients, such as local anaesthetics or vitamins. In the bulk formulation there is no need for lubricants, such as non-crosslinked HA, since these can be added upon final formulation of the dermal filler.

Thus, an embodiment of the present invention relates to the bulk dermal filler formulation as described herein, wherein said bulk dermal filler formulation does not comprise any lubricants.

Another embodiment of the present invention relates to the bulk dermal filler formulation as described herein, wherein the lubricants are non-crosslinked HA and/or a polyol, preferably non-crosslinked HA.

A further embodiment of the present invention relates to the bulk dermal filler formulation as described herein, wherein the polyol is selected from the group consisting of glycerin, mannitol, sorbitol, propylene glycol, erythritol, xylitol, maltitol, and lactitol, preferably glycerin and propylene glycol.

It has been found that the addition of POE to the hydrogel comprising crosslinked HA does not negatively impact the rheological properties of the dermal filler formulation. Therefore, a diverse set of dermal filler formulations can be produced based on the bulk dermal filler formulation without the need to use excessive lubricants (i.e. more than conventional dermal filler formulations), such as non-crosslinked HA, to facilitate injection thereof. This is advantageous because lubricants typically cause declining rheological properties resulting in formulations with a lessened filling effect.

However, for further formulation, i.e. to prepare the final dermal filler product, it is advantageous to include a fraction of non-crosslinked HA in the formulation to adjust the rheological properties of the formulation. In particular, formulations comprising non-crosslinked HA may be easier to inject. It is to be understood that non-crosslinked HA is added to the bulk dermal filler formulation as part of the final formulation of a dermal filler product. Depending on the intended purpose of the final dermal filler product, e.g. site and depth of injection, the amount of the non-crosslinked HA may be adjusted. Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation further comprises noncrosslinked HA.

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

A further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the ratio between non-crosslinked and crosslinked HA in the formulation is in the range of about 1 :50 (w/w) to about 1 :2 (w/w), such as about 1 :20 (w/w) to about 1 :3 (w/w), such as about 1 : 15 (w/w) to about 1:4 (w/w), preferably about 1 : 10 (w/w) to about 1 :5.

A still further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the total concentration of crosslinked HA and/or non-crosslinked HA is in the range of about 1 mg/g to about 50 mg/g, such as about 2 mg/g to about 40 mg/g, such as about 3 mg/g to about 30 mg/g, such as about 5 mg/g to about 25 mg/g, such as about 10 mg/g to about 20 mg/g.

An even further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the total concentration of crosslinked HA and/or non-crosslinked HA is about 15 mg/g to about 25 mg/g, such as about 18 mg/g to about 22 mg/g, preferably about 20 mg/g.

The size of the HA polymers can be varied to influence the structure of the polymeric network of the HA-based hydrogel, and therefore the dermal filler formulation. The size (molecular weight) of the HA polymers can be determined by measuring the intrinsic viscosity using the Mark Houwink Kuhn Sakurada (MHKS) equation or size exclusion chromatography coupled with multi angle laser light scattering (SEC-MALLS).

An embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the molecular weight of the HA of said first component and/or second component is the range of about 5 kDa to about 5000 kDa, such as 50 kDa to about 5000 kDa, such as about 100 kDa to about 3000 kDa, such as about 250 kDa to about 2000 kDa, such as about 500 kDa to about 1500 kDa, such as about 750 kDa to about 1250 kDa.

Herein it has been found that certain sizes of HA polymers may be favoured to provide hydrogels with structures that yield good filler effect.

Therefore, a preferred embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the molecular weight of the HA of said first component and/or second component is the range of about 750 kDa to about 1250 kDa, such as 800 kDa to about 1200 kDa, such as 900 kDa to about 1100 kDa, preferably about 1000 kDa.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the molecular weight of crosslinked HA and non-crosslinked HA is the same.

A further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the molecular weight of crosslinked HA and non-crosslinked HA is different.

However, the hydrogel is not limited to crosslinked HA polymers of only a single size but may comprise crosslinked HA of two or more different molecular weights. Hydrogels comprising HA polymers of different sizes may be formed by crosslinking all HA polymers in one step or by crosslinking HA polymers of different sizes in separate steps. By the latter approach it is possible to create hydrogels with sections of different density.

Accordingly, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the hydrogel comprises crosslinked HA of two or more different molecular weights.

The HA utilized in the hydrogel may in principle be any type of HA including, but not limited to, HA salified with organic or inorganic bases, HA esters with alcohols, HA amides, O-sulphated derivates of HA, deacetylated derivatives of HA, and percarboxylated derivatives of HA. HA ester may be with alcohols of the aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic series. HA amides may be with amine of the aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic and heterocyclic series.

Preferably, the HA is provided as an inorganic salt. Thus, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the HA is provided as an inorganic salt selected from the group consisting of sodium hyaluronate, potassium hyaluronate, ammonium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, and cobalt hyaluronate.

To avoid precipitation of the injectable dermal filler formulation, it is preferred to keep impurities at a minimum. In particular, calcium ions can contribute to unwanted precipitation in the hydrogel, such as calcium phosphate precipitates. Thus, minimizing the risk of precipitation should be kept in mind when selecting the HA salt and/or aqueous buffer of the hydrogel.

Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the HA is provided as an inorganic salt which does not comprise calcium.

A preferred embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the HA is provided as sodium hyaluronate.

The polymeric nature of the HA-based hydrogel can potentially make the injectable dermal filler formulation structurally inhomogeneous. For some applications this texture works well, e.g. wherein large needle sizes may not be prohibitive for its utilization. However, for use with smaller needle sizes which may be needed for more accurate delivery of dermal fillers, it is required that the injectable dermal filler formulation is highly homogenous. This may be accomplished by micronizing the hydrogel comprising crosslinked HA prior to mixing with the POE and, optionally noncrosslinked HA.

Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the mean particle size of the first component is less than about 1500 .m, such as less than about 1250 .m, such as less than about 1000 pirn, such as less than about 750 pirn, such as less than about 500 pirn.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the mean particle size of the hydrogel comprising crosslinked HA is in the range of about 50 .m to about 1500 .m, such as about 100 pirn to about 1250 pirn, such as about 150 pirn to about 1000 pirn, such as about 200 pirn to about 750 pirn, such as about 300 pirn to about 500 pirn.

The dermal filler formulation comprises a solvent into which the mix of the hydrogel and POE are dispersed. The solvent may be any aqueous solvent which is compatible with the HA-based hydrogel and the POE, and which is biocompatible for administration to a subject. This includes isotonic aqueous buffers, including, but not limited to, a physiological saline solution and PBS. The buffer is preferably adjusted to a pH value suitable for administration to a subject. Moreover, the pH value is selected to avoid any degradation of hyaluronic acid and/or POE, such as in the range of pH 6.5-8.5.

Accordingly, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the solvent of the formulation is aqueous.

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

A further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein pH of the formulation is in the range of about pH 6.5 to about pH 8.5, such as about pH 7 to about pH 8, preferably about pH 7.5.

The rheological properties of the dermal filler formulation may be impacted by changing the crosslinking degree, i.e. the ratio of HA and crosslinking agent in the HA-based hydrogel. By increasing the relative amount of crosslinking agent, the hydrogel will contain a tighter polymeric network and consequently be more dense which can yield a good filling effect. However, a too dense hydrogel becomes difficult to inject and may not readily mix with other components of the formulation.

Thus, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the HA is crosslinked with a crosslinking agent.

Another embodiment of the present invention relates to the 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). A further embodiment of the present invention relates to the injectable dermal filler formulation 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), such as about 18: 1 % (w/w) to about 12: 1 % (w/w), ), such as about 16: 1 % (w/w) to about 14: 1 % (w/w).

A still further embodiment of the present invention relates to the method as 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).

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

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).

The crosslinking may also be expressed in terms of the degree of modification of the crosslinked HA. The degree of modification can be determined by NMR, such as performed at 500 MHz, at a pulse of 20 degree with several repetitions at ambient temperature. From the obtained spectrum, the degree of modification can be determined by calculating the ratio of the N-acetyl signals of HA to the characteristic signal of the crosslinking agent, e.g. by way of example, the methylene signals of BDDE.

Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the degree of modification of the crosslinked HA, expressed as the ratio of the sum of mono and double-linked crosslinking agents to the sum of HA disaccharide units, is in the range of about 0.5% to 25%.

Crosslinking of hyaluronic acid changes the form of the raw HA material and facilitates the transformation into non-dissolvable hydrogels that may absorb large amounts of water without losing a defined structure. The swollen structure provides a great voluminous filler effect. The principle of the crosslinking reaction of HA is that the polymer chains are covalently linked by reaction with a crosslinking agent, which can be selected from a number of different chemical compounds, including, but not limited to, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), l-ethyl-3-(3- dimethylaminopropyl) carbodimide (EDC), polyethylene glycol (PEG), polyethylene glycol diglycidyl ether, diepoxyoctane and p-Phenylene Biscarbodiimide.

Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the crosslinking agent is selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), formaldehyde, glutaraldehyde, polyanhydrides, polyaldehydes, polyhydric alcohols, carbodiimides, carboxylic acid chlorides, sulfonic acid chlorides, celluloses, dextrans, epichlorohydrin, ethylene glycol, diglycidyl ethers, polyglycerol polyglycidyl ethers, and bis- or polyepoxides, and derivatives thereof.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the crosslinking agent is selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), poly(lactic acid) (PLA), poly(ethylene glycol) (PEG), poly(ethylene glycol) bis(amine), polyethylene glycol diacrylate (PEGDA), poly(ethylene glycol)-dimethacrylate (PEGDM), poly(ethylene glycol) -diacrylamide (PEGDAA) and polyethylene glycol)- dimethacrylamide (PEGDMA), carboxymethylcellulose (CMC), dextran acrylate, dextran methacrylate, dextran glycidyl methacrylate, glycerol dimethacrylate, glycerol 1,3-diglycerolate diacrylate, sorbitol acrylate, and derivatives thereof.

A preferred embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the crosslinking agent is selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), poly(lactic acid) (PLA), and poly(ethylene glycol) (PEG), and derivatives thereof.

A preferred crosslinking agent is divinyl sulfone (DVS). For this crosslinking agent, the crosslinking reaction takes place between the primary hydroxyl group of N-acetyl-D- glucosamine, which reacts by a nucleophilic addition at the vinylic carbon atom at the DVS molecule. DVS contains two vinyl groups and is very reactive towards nucleophilic addition, which results in a ratio of two mole HA-disaccharide (N-acetyl-D- glucosamine) to one mole of DVS. Since DVS crosslinking does not involve the biologically reactive functional groups (carboxylate and acetamide) on the HA molecule, the gels largely preserve HA's natural polyanionic character, physiochemical and biological properties.

DVS is very reactive under aqueous alkaline conditions and is thus instantaneous fully reacted. The resulting bond between HA and DVS is a sulfonyl bis-ethyl ether linkage, which is known to be very stable towards hydrolysis (degradation) and stable towards basic treatment.

Accordingly, a preferred embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the crosslinking agent is divinyl sulfone (DVS).

The viscosity of a final dermal filler formulation plays a crucial role in how it can be administered to a subject. If the formulation is too viscous it limits significantly how the formulation may be administered to a subject, e.g., because it can no longer be injected with a syringe through a needle or would entail undesirable and unacceptable discomfort for the recipient of the formulation. If the content of crosslinked HA becomes too high, the formulation might become unsuitable e.g. for injection.

A fraction of non-crosslinked HA may be added to the bulk dermal filler formulation to promote the shear thinning properties of the final dermal filler product and improve injectability as force is applied to the syringe.

The storage modulus is one parameter that can be used to describe the viscoelastic properties of the dermal filler formulation. High crosslinking degree of the HA-based hydrogel will result in high storage modulus. While high storage modulus typically leads to an improved filler effect, too high storage modulus can cause the dermal filler to behave as a crystalline-like and inhomogeneous material which is not suitable for injection. Importantly, it has been found that the combination of POEs with a HA-based hydrogel does not negatively affect the injectability of the bulk dermal filler formulation.

Accordingly, the dermal filler formulation described herein is balanced in its composition to give viscoelastic properties suitable for injection and providing the desired voluminous effect of any final dermal filler products derived therefrom. As such, the combination of POEs with a HA-based hydrogel results in a storage modulus comparable to or better than formulations without POEs, and thus are beneficial for ensuring a good filling effect in the final dermal filler formulation.

As described herein, the bulk dermal filler formulation may be further formulated according to the final application of the formulation. Thus, viscosity and/or storage modulus may be adjusted for suitability for a specific site of injection or depth of injection. Preferably, viscosity and/or storage modulus is adjusted by addition of noncrosslinked HA.

Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation has a storage modulus in the range of about 5 Pa to about 4000 Pa, such as about 100 Pa to about 4000 Pa, such as about 150 Pa to about 3000 Pa, such as about 200 Pa to about 2500 Pa, such as about 200 Pa to about 2000 Pa, such as about 200 Pa to about 900 Pa, such as about 200 Pa to about 600 Pa.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation has a viscosity in the range of about 0.001 Pa*s to about 1000 Pa*s, such as about 25 Pa*s to about 1000 Pa*s, such as about 30 Pa*s to about 800 Pa*s, such as about 40 Pa*s to about 600 Pa*s, such as about 50 Pa*s to about 500 Pa*s.

Following final formulation to reach suitable rheological properties, the final dermal filler products can be injected in a recipient subject. The size of the needle of the syringe may be selected to suit the site and depth of injection. The rheological properties of the final dermal filler formulation should be adapted and the choice of needle size selected as to ensure no unnecessary patient discomfort. As demonstrated herein, the inclusion of POEs does not negatively impact injectability of the dermal filler formulation.

Thus, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation is injectable from a 1 mL syringe through a needle with a gauge (Birmingham gauge) in the range of about 18 G to about 30 G, such as about 22 G to about 28 G, such as about 24 G to about 27 G.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation is injectable from a 1 mL syringe through a needle with a gauge (Birmingham gauge) in the range of about 18 G to about 32 G, such as about 25 G to about 30 G, such as about 27 G to about 30 G. Yet another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation is injectable from a 1 mL syringe through a needle with a gauge (Birmingham gauge) of about 27 G.

A further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the hydrogel formulation is injectable from a 1 mL syringe through a needle with a gauge (Birmingham gauge) of about 27 G using an extrusion force of less than about 30N, preferably less than about 25N, more preferably less than about 20N, most preferably less than about 15N.

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

An even further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation has an extrusion force in the range of about ION to about 30N, such as about 15N to about 25N, such as about 18N to about 22N.

The bulk dermal filler formulation provides an advantageous balance between long- lasting volume effect, injectability and biocompatibility that can be passed on to final dermal filler formulations based thereupon. Accordingly, the final dermal filler formulations may have the rheological properties adjusted for its intended purpose and be injected in its "native" form meaning without holding any further ingredients. However, the dermal filler formulation may also comprise or be loaded with additives providing further benefits such as minimizing pain associated with injection or further improving the filler effect.

Thus, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation further comprises one or more additives.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the one or more additives are selected from the group consisting of resorbable biocompatible microparticles, local anaesthetics, polyols, vitamins, amino acids, metals, antioxidants, preservatives, and mineral salts. In particular, microparticles of calcium hydroxyapatite or polycaprolactone has previously been shown to promote new tissue formation similar to its surrounding environment. This process of neocollagenesis is advantageous and such microparticles are compatible with the dermal filler formulation described herein.

Therefore, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation comprises resorbable biocompatible microparticles.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the resorbable biocompatible microparticles comprise polycaprolactone or a calcium phosphate-based material, such as calcium hydroxyapatite.

A further embodiment of the present invention relate to the injectable dermal filler formulation as described herein, wherein the calcium phosphate-based material is selected from the group consisting of calcium hydroxyapatite, calcium fluoroapatite, calcium chloroapatite, calcium carbonate apatite, tetracalcium phosphate, calcium pyrophosphate, tricalcium phosphate, and octacalcium phosphate, preferably calcium hydroxyapatite.

The dermal filler formulation may also comprise other additives, such as polyols or local anaesthetics. Polyols, such as glycerin, may work as lubricant that promote injectability and local anaesthetics alleviate any potential pain or discomfort associated with injection of the dermal filler formulation.

Accordingly, an embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein formulation comprises one or more polyols selected from group consisting of glycerin, mannitol, sorbitol, propylene glycol, erythritol, xylitol, maltitol, and lactitol, preferably glycerin.

Another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the concentration of the one or more polyols is in the range of about 1% (vol/vol) to about 20% (vol/vol), such as 2% (vol/vol) to about 15% (vol/vol), such as 5% (vol/vol) to about 10% (vol/vol), based on the volume of the injectable dermal filler formulation. Yet another embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation comprises a local anaesthetics selected from the group consisting of ambucaine, amylocaine, benzocaine, bupivacaine, butacaine, butanilicaine, chloroprocaine, cocaine, cyclomethycaine, dibucaine, dimethocaine, diperodon, etidocaine, formocaine, hexylcaine, lidocaine, mepivacaine, meprylcaine, meta butoxycaine, orthocaine, oxethazaine, paraethoxycaine, phenacaine, piperocaine, piridocaine, procaine, proparacaine, propoxycaine, pyrrocaine, ropivacaine, tetracaine, tolycaine, and trimecaine, and salts thereof, preferably lidocaine.

A further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the formulation comprises lidocaine.

A still further embodiment of the present invention relates to the injectable dermal filler formulation as described herein, wherein the concentration of the local anaesthetics is in the range of about 0.1% (w/w) to about 5.0% (w/w), such as about 0.2% (w/w) to about 3.0% (w/w), such as about 0.3% (w/w) to about 2.0% (w/w), preferably about 0.5% (w/w) to about 1.0% (w/w), based on the total weight of the injectable dermal filler formulation.

It is to be understood that inclusion of larger aggregate additives, such as microparticles may change the rheological behaviour of the dermal filler formulation. Thus, for dermal filler formulations comprising microparticles it is expected that storage modules, viscosity and extrusion force will increase. For such formulations it may be preferable to inject with a slightly larger needle size.

The injectable dermal filler formulation described herein is prepared by mixing the hydrogel comprising crosslinked HA with the POE. The hydrogel is made and processed through a series of steps, including crosslinking of HA with a crosslinking agent, curing and comminuting the obtained hydrogel, neutralization and micronization of the fragmented hydrogel. The multi-step processing is advantageous in that it provides a hydrogel component with viscoelastic properties that are well suited for use in dermal filler formulation.

Thus, an aspect of the present invention relates to a method for preparing an injectable dermal filler formulation as described herein, said method comprising the following steps:

(i) providing a first solution comprising hyaluronic acid (HA), (ii) reacting said first solution with a crosslinking agent to form a hydrogel comprising crosslinked HA, and

(iii) adding at least one poly(orthoester) (POE) to said hydrogel followed by mixing, thereby providing said injectable dermal filler formulation.

This injectable dermal filler formulation comprising a hydrogel comprising crosslinked HA and a POE can also be considered a bulk dermal filler formulation, the further formulation of which can lead to final dermal filler formulations ready for use.

Thus, another aspect of the present invention relates to a method for preparing an injectable dermal filler formulation as described herein, said method comprising one or more steps of adding non-crosslinked HA and/or one or more additives to a bulk dermal filler formulation, wherein said bulk dermal filler composition formulation:

- a hydrogel comprising crosslinked hyaluronic acid (HA), and

- at least one poly(orthoester) (POE)

Crosslinking of HA by means of a crosslinking agent can be via covalent bonds with hydroxyl, carboxylate and/or acetamide functional groups of the hyaluronic acid polymer. By example, crosslinking may be achieved through the primary hydroxyl group of N-acetyl-D-glucosamine of HA under alkaline conditions. The base can be NaOH as in the case of using DVS or BDDE crosslinking agents. The HA is dissolved in the alkaline solution by mixing with a turbine mixer. Extended mixing for up to 90 min at 600 rpm is preferred to ensure that all HA are dissolved. However, mixing could be extended for as long as 180 min.

Thus, an embodiment of the present invention relates to the method as described herein, wherein said first solution is alkaline.

Another embodiment of the present invention relates to the method as described herein, wherein the pH of said first solution is in the range of about pH 9 to about pH 12, such as about pH 10 to about pH 12.

A further embodiment of the present invention relates to the method as described herein, wherein said first solution has a pH of at least 9. A still further embodiment of the present invention relates to the method as described herein, wherein said first solution comprises NaOH in a concentration between about 0.001 M to about 2.0 M, such as about 0.01 M to about 1.0 M, such as about 0.1 M to about 0.5 M, preferably about 0.2 M.

By another example, the crosslinking may be achieved through the carboxyl group on the D-glucuronic acid. The acid could be hydrochloric acid, as in the case of crosslinking using carbodiimides cross-linkers.

For formation of the HA-based hydrogel, the HA is mixed with a crosslinking agent and stirred intensely to ensure homogeneous distribution of the reactants. Mixing is preferably followed by an incubation period to cure the HA-based hydrogel before it is fragmented as part of the processing.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the ratio of HA to crosslinking agent in the range of about 25: 1 % (w/w) to about 2: 1 % (w/w), such as about 20: 1 % (w/w) to about 10: 1 % (w/w).

A further embodiment of the present invention relates to the method as described herein, wherein the ratio of HA to crosslinking agent is about 5: 1 % (w/w).

Yet 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).

A still further embodiment of the present invention relates to the method as described herein, further comprising a step of curing said hydrogel by heating.

Yet another embodiment of the present invention relates to the method as described herein, wherein curing comprises heating at about 35°C to about 45°C, such as at 40°C, for at least 1 hour, preferably 2 hours.

The HA hydrogel is divided into smaller pieces to yield a fragmented hydrogel. Typically, the hydrogel is broken down into chunks of 25x25x25 mm. It is to be understood that the chunks can be smaller or larger, such as down to about 10 mm or up to about 50 mm. This may be achieved by cutting the hydrogel on a plate of glass, plastic or metal. Alternatively, the hydrogel can be fragmented by mechanically forcing it through a cutting mesh (also known as an extrusion screen). The extrusion screen may have a mesh size of 25x25 mm. Thus, an embodiment of the present invention relates to the method as described herein, further comprising a step of comminuting said hydrogel.

Another embodiment of the present invention relates to the method as described herein, wherein said comminution is performed by cutting.

Comminution of the hydrogel is followed by rinsing of the fragmented hydrogel in excess volumes of an aqueous solvent. The solvent may be purified water or any type of suitable aqueous buffer with a pH in a range that serves to neutralize the fragmented hydrogel following the initial hydrogel formation in alkaline solution and wash out remaining unreacted crosslinking agent. During neutralization, the HA hydrogel absorb the aqueous medium and swell to a saturated form. The time required to saturate the fragmented HA hydrogel varies depending on the exact content and crosslinking degree of the hydrogel, but the neutralization and swelling is preferably performed for 19-25 hours.

Thus, an embodiment of the present invention relates to the method as described herein, further comprising a step of neutralizing said hydrogel in buffer.

Another embodiment of the present invention relates to the method as described herein, wherein said comminution is immediately followed by rinsing of the fragmented hydrogel.

Yet another embodiment of the present invention relates to the method as described herein, wherein said rinsing is achieved by immersion of the fragmented hydrogel in ultrapure water and/or rinsing buffer, and optionally agitating the immersed fragmented hydrogel.

A further embodiment of the present invention relates to the method as described herein, wherein the buffer agent is a phosphate buffer or a saline buffer.

A still further embodiment of the present invention relates to the method as described herein, wherein the buffer agent has a pH in the range of about 5.5 to about 9.

An even further embodiment of the present invention relates to the method as described herein, wherein the buffer agent has a pH in the range of about 5 to about 7.5, such as in the range of about 6 to about 7, preferable about 6.9. The neutralized (and swollen) hydrogel, which is still fragmented, is separated from excess and non-absorbed buffer by use of a sieve. The neutralized hydrogel is placed in the sieve and the non-absorbed liquid fraction is drained to provide a purified swollen hydrogel out of solution.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the separation of said neutralized hydrogel from said buffer is achieved by placement of the neutralized hydrogel in a sieve followed by drainage of the liquid fraction.

The purified hydrogel is micronized to secure a homogeneous material that can administered without any difficulties, such as blocking of needles. Thus, the dermal filler formulation described herein is readily injectable through various needle sizes, types or catheters without the need for applying strong force. Micronization can be achieved by extrusion or mixing.

Thus, an embodiment of the present invention relates to the method as described herein, further comprising a step of micronizing said hydrogel.

Another embodiment of the present invention relates to the method as described herein, wherein the micronization of said purified hydrogel is achieved by extrusion using an extrusion screen.

Yet another embodiment of the present invention relates to the method as described herein, wherein said extrusion screen has a mesh size in the range of 200-450 pm.

A further embodiment of the present invention relates to the method as described herein, wherein the micronization is achieved by mixing with a high shear mixer.

If the dermal filler formulation is prepared utilizing each step, the method comprises the following steps in chronological order:

(i) crosslinking HA to provide a hydrogel

(ii) curing the hydrogel

(iii) comminuting the hydrogel

(iv) neutralizing the hydrogel

(v) purifying the hydrogel

(vi) micronizing the hydrogel (vii) adding POE

Following preparation of the bulk dermal filler formulation, additional formulation with non-crosslinked HA and/or other ingredients, such as a local anaesthetics, can proceed. Such additional steps can be carried immediately after preparation of the bulk dermal filler formulation or after storage of said formulation.

Accordingly, an embodiment of the present invention relates to the method as described herein, further comprising a step of adding non-crosslinked HA to said hydrogel.

An aspect of the present invention relates to an injectable dermal filler formulation as described herein obtainable by a method as described herein.

The dermal filler formulation may conveniently be provided in a kit comprising also relevant information on how to further formulate and/or administer the formulation. The formulation may be packaged for easy modification via addition of e.g. non- crosslinked HA and/or further additives or may be preloaded in one or more syringes for easy use.

Accordingly, an aspect of the present invention relates to a kit comprising:

(i) an injectable dermal filler formulation as described herein, and

(ii) optionally, instructions for use.

Another aspect of the present invention relates to a kit comprising:

(i) a bulk dermal filler formulation as described herein, and

(ii) optionally, instructions for use.

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

The dermal filler formulation described herein is intended for use with a subject that wish to alter the appearance of the skin, e.g. to combat aesthetic signs of aging. Accordingly, the dermal filler formulation may find its utility in a large variety of cosmetic applications or soft tissue augmentation applications. The formulation is preferably injected by a professional clinician. Thus, an aspect of the present invention relates to use of an injectable dermal filler formulation or a kit as described herein for cosmetic applications.

An embodiment of the present invention relates to the use as described herein, wherein the cosmetic applications are one or more selected from the group consisting of cosmetic treatment of wrinkles and lines of the skin, glabellar lines, nasolabial folds, chin folds, marionette lines, jawlines, buccal commissures, perioral wrinkles, philtrum, crow's feet, cutaneous depressions, scars, temples, subdermal support of the brows, malar and buccal fat pads, tear troughs, nose, lips, cheeks, cheekbones, chin, perioral region, infraorbital region, and facial asymmetries.

The dermal filler formulation may be injected through several different routes of administration. The formulation may be injected as a single administration or in small aliquots of dermal filler close together in order for them to integrate continuously along a rhytid or fold (serial puncture technique).

Thus, an embodiment of the present invention relates to the use as described herein, wherein the formulation is injected subcutaneously, intradermally or intramuscularly.

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

Yet another embodiment of the present invention relates to the use as described herein, wherein the formulation is administered to add volume and fullness to said dermal region.

A further embodiment of the present invention relates to the use as described herein, wherein said subject is a human.

Another aspect of the present invention relates to a method for replacement or filling of a biological tissue comprising administering to a subject in need thereof an effective amount of the injectable dermal filler formulation as described herein.

It is to be understood that the term "effective amount" refers to a sufficient amount of the dermal filler formulation to provide a favourable or desired cosmetic result. The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the injectable dermal filler formulation and all its features, which may readily be part of the bulk dermal filler formulation or injectable dermal filler formulation obtained by the method or its use as described herein. Embodiments and features of the present invention are also outlined in the following items.

Items

XI. An injectable dermal filler formulation comprising:

- a hydrogel comprising crosslinked hyaluronic acid (HA), and

- at least one poly(orthoester) (POE).

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

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

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

X5. The injectable dermal filler formulation according to item X4, wherein said diol is modified by short sequences of polyglycolide and/or polylactide.

X6. The injectable dermal filler formulation according to item X5, wherein the ratio of polylactide to polyglycolide is in the range of 80:20 to 20:80, such as 75:25 to 25:75, such as 70:30 to 30:70, such as 65:35 to 35:65, such as 60:40 to 40:60, such as 55:45 to 45:55, such as 50:50.

X7. The injectable dermal filler formulation according to any one of items X4-X6, wherein the diol is selected from an oligoethylene glycol and/or an organic diol. X8. The injectable dermal filler formulation according to item X7, wherein the oligoethylene glycol is selected from the group consisting of triethylene glycol (TEG), oligoethylene glycol diglycolide, and oligoethylene glycol dilactide.

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

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

XI 1. The injectable dermal filler formulation according to any one of items X7-X10, wherein the organic diol is saturated or unsaturated.

X12. The 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-cyclohexane dimethanol, para-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.

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

X14. The 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.

X15. The injectable dermal filler formulation according to any one of the preceding items, wherein the at least one POE is represented by formula (I) or formula (II):

wherein :

R is a bond, — (CH2)a— , or — (CH2)b— O— (CH2)n— ; where a is an integer of 1 to 10, and b and c are independently integers of 1 to 5;

R* is a Ci-4 alkyl; n is an integer of at least 5; and

A is R¹, R², R³, or R⁴, wherein R¹ is:

p is an integer of 1 to 20;

R⁵ is hydrogen or Ci-4 alkyl; and

R⁶ is:

x is an integer of 0 to 30; y is an integer of 2 to 200;

R⁸ is hydrogen or Ci-4 alkyl;

R⁹ and R¹⁰ are independently C1-12 alkylene;

R¹¹ is hydrogen or C1-6 alkyl and R¹² is C1-6 alkyl; or Rⁿ and R¹² together are C3- 10 alkylene; and wherein R⁴ is a the residue of a diol containing at least one functional group independently selected from amide, imide, urea, and urethane groups; in which at least 0.01 mol % of the A units are of the formula R¹.

X16. The injectable dermal filler formulation according to item X15, wherein the at least one POE is represented by formula (I).

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

X18. The injectable dermal filler formulation according to any one of items X15-X17, wherein at least 50 mol%, such as at least 60 mol%, such as at least 70 mol%, such as approx. 80 mol%, of the A units are of the formula R³. X19. The injectable dermal filler formulation according to any one of items X15-X18, wherein R³ is a low molecular weight polyethylene glycols or aliphatic diols, preferably a low molecular weight polyethylene glycol.

X20. The injectable dermal filler formulation according to any one of the preceding items, wherein the molecular weight of the POE is the range of 1.5 kDa to 20 kDa, such as 2.5 kDa to 10 kDa.

X21. The injectable dermal filler formulation according to any one of items X15-X20, wherein R³ is tri(ethylene glycol).

X22. The injectable dermal filler formulation according to any one of the preceding items, wherein the at least one POE is a tri(ethylene glycol)-poly(orthoester) (TEG- POE).

X23. The injectable dermal filler formulation according to any one of the preceding items, wherein the at least one POE is represented by formula (III):

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%.

X25. The injectable dermal filler formulation according to any one of items X23 or X24, wherein X is essentially 100%.

X26. The 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), such as about 0.5% (w/w) to about 18% (w/w), such as about 1% (w/w) to about 16% (w/w), such as about 2% (w/w) to about 15% (w/w), such as about 3% (w/w) to about 14% (w/w), such as about 4% (w/w) to about 12% (w/w), such as about 5% (w/w) to about 10% (w/w).

X27. The 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 0.01% (w/w) to about 3% (w/w), such as about 0.05% (w/w) to about 2.5% (w/w), such as about 0.1% (w/w) to about 2% (w/w), such as 0.2% (w/w) to about 1.5% (w/w), such as 0.3% (w/w) to about 1% (w/w), such as 0.4% (w/w) to about 0.8% (w/w), based on the total weight of the injectable dermal filler formulation.

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

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

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

X31. The injectable dermal filler formulation according to any one of the preceding items, wherein the mean particle size of the hydrogel comprising crosslinked HA is in the range of about 50 pirn to about 1500 pirn, such as about 100 pirn to about 1250 pirn, such as about 150 pirn to about 1000 pirn, such as about 200 pirn to about 750 pirn, such as about 300 pirn to about 500 pirn.

X32. The injectable dermal filler formulation according to any one of the preceding items, wherein the HA is crosslinked with a crosslinking agent. X33. The injectable dermal filler formulation according to any one of the preceding items, wherein the crosslinking agent is selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), formaldehyde, glutaraldehyde, polyanhydrides, polyaldehydes, polyhydric alcohols, carbodiimides, carboxylic acid chlorides, sulfonic acid chlorides, celluloses, dextrans, epichlorohydrin, ethylene glycol, diglycidyl ethers, polyglycerol polyglycidyl ethers, and bis- or polyepoxides, and derivatives thereof.

X34. The 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).

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

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

X37. The 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).

X38. The injectable dermal filler formulation according to any one of the preceding items, wherein the formulation has a storage modulus in the range of about 5 Pa to about 4000 Pa, such as about 20 Pa to about 3000 Pa, such as about 25 Pa to about 2000 Pa.

X39. The injectable dermal filler formulation according to any one of the preceding items, wherein the formulation has a storage modulus in the range of about 100 Pa to about 4000 Pa, such as about 150 Pa to about 3000 Pa, such as about 200 Pa to about 2500 Pa, such as about 200 Pa to about 2000 Pa, such as about 200 Pa to about 900 Pa, such as about 200 Pa to about 600 Pa.

X40. The injectable dermal filler formulation according to any one of the preceding items, wherein the formulation has a viscosity in the range of about 0.001 Pa*s to about 1000 Pa*s, such as about 0.01 Pa*s to about 1000 Pa*s, such as about 0.1 Pa*s to about 1000 Pa*s, such as about 1 Pa*s to about 1000 Pa*s, such as about 10 Pa*s to about 1000 Pa*s, such as about 25 Pa*s to about 1000 Pa*s, such as about 30 Pa*s to about 800 Pa*s, such as about 40 Pa*s to about 600 Pa*s, such as about 50 Pa*s to about 500 Pa*s.

X41. The injectable dermal filler formulation according to any one of the preceding items, wherein the formulation has an extrusion force in the range of about 10N to about 30N, such as about 15N to about 25N, such as about 18N to about 22N.

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

X43. The injectable dermal filler formulation according to item X42, wherein the one or more additives are selected from the group consisting of resorbable biocompatible microparticles, local anaesthetics, polyols, vitamins, amino acids, metals, antioxidants, preservatives, and mineral salts.

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

X45. The injectable dermal filler formulation according to any one of the preceding items, wherein formulation comprises one or more polyols selected from group consisting of glycerin, mannitol, sorbitol, propylene glycol, erythritol, xylitol, maltitol, and lactitol, preferably glycerin.

X46. The injectable dermal filler formulation according to any one of the preceding items, wherein the formulation comprises a local anaesthetics selected from the group consisting of ambucaine, amylocaine, benzocaine, bupivacaine, butacaine, butanilicaine, chloroprocaine, cocaine, cyclomethycaine, dibucaine, dimethocaine, diperodon, etidocaine, formocaine, hexylcaine, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, orthocaine, oxethazaine, paraethoxycaine, phenacaine, piperocaine, piridocaine, procaine, proparacaine, propoxycaine, pyrrocaine, ropivacaine, tetracaine, tolycaine, and trimecaine, and salts thereof, preferably lidocaine.

Yl. A method for preparing an injectable dermal filler formulation according to any one of the preceding items, said method comprising the following steps:

(i) providing a first solution comprising hyaluronic acid (HA),

Y2. The method according to item Yl, wherein the ratio of HA to crosslinking agent in the range of about 25: 1 % (w/w) to about 2: 1 % (w/w), such as about 20: 1 % (w/w) to about 10: 1 % (w/w).

Y3. The method according to any one of items Y1 or Y2, further comprising a step of curing said hydrogel by heating.

Y4. The method according to any one of items Y1-Y3, further comprising a step of comminuting said hydrogel.

Y5. The method according to any one of items Y1-Y4, further comprising a step of neutralizing said hydrogel in buffer.

Y6. The method according to any one of items Y1-Y5, further comprising a step of micronizing said hydrogel.

Y7. The method according to any one of items Y1-Y6, further comprising a step of adding non-crosslinked HA to said hydrogel.

Zl. An injectable dermal filler formulation according to any one of items X1-X46 obtainable by a method according to any one of items Y1-Y7.

Wl. A kit comprising:

- an injectable dermal filler formulation according to any one of items X1-X46 or

Zl, and

- optionally, instructions for use.

Ul. Use of an injectable dermal filler formulation according to any one of items X1-X46 or Zl or a kit according to any one of item W1 for cosmetic applications.

U2. The use according to item Ul, wherein the cosmetic applications are one or more selected from the group consisting of cosmetic treatment of wrinkles and lines of the skin, glabellar lines, nasolabial folds, chin folds, marionette lines, jawlines, buccal commissures, perioral wrinkles, crow's feet, cutaneous depressions, scars, temples, subdermal support of the brows, malar and buccal fat pads, tear troughs, nose, lips, cheeks, chin, perioral region, infraorbital region, and facial asymmetries.

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

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1: Preparation of injectable dermal filler formulation

Dermal filler formulations were prepared by a custom designed process to evaluate their performance, hereunder injectability and filler effect.

Method:

Sodium Hyaluronate (1 MDa) were dissolved in a 0.2M NaOH solution under mechanical stirring by an Ika-mixer with a turbine mixer to prepare a 5-8% (w/w) HA solution. The solution was stirred until no HA lumps were present in the solution. Divinyl sulfone (DVS) was added to the solution in an amount to reach the desired HA to DVS ratio (5: 1, 10: 1 or 20: 1) followed by mixing until completely dispersed in the solution.

The HA: DVS mix was then transferred to a plastic tray in an even layer with a thickness of approximately 25-40 mm, and cured at 40°C for 2 hours while covering the tray with a lid. Subsequent to curing, the crosslinked hydrogel was placed at ambient temperature for 10 min and thereafter cut in pieces (approx. 25x40 mm) with a cutting wheel. The fragmented hydrogel was transferred to a beaker containing 12 mM PBS (137 mM NaCI, 2.7 mM KCI, 10.2 mM Na₂HPO₄, 2.0 mM KH₂PO₄) pH 6.9 and swollen for up to 25 hours. The swelling and neutralization are done to neutralize the pH of the gel and wash out remaining DVS and possible DVS adducts. The total swelling time for the fragmented hydrogel is set to be 19-25 hours, depending on the crosslinking degree. All gels of a higher crosslinking degree than 10: 1 should be neutralized for at least 24 hours, whereas fragmented hydrogels of a lower crosslinking degree than 10: 1 should be neutralized for 19 hours.

The neutralized hydrogel (pH 6.9-7.5) was separated from the buffer in a mesh. The hydrogel was left in the mesh for 10 min to let all buffer seep out. The remaining hydrogel was weighed and, if necessary, mass corrected by buffer if needed to reach a specific HA concentration in the bulk purified hydrogel.

The bulk purified hydrogel was then micronized by either high shear mixing or extrusion. Micronization by high shear mixing was performed by using a Silverson mixer attached with a Ultramixer workhead. Micronization by extrusion was performed by passing the hydrogel through an extrusion screen with a mesh size of 200-450 pm. A support screen with a mesh size of 1.5 mm may be placed on each side of the extrusion screen for support. The hydrogel can be treated with 20 strokes (10 in each direction) at a pressure of approx. 6 bars. The micronized hydrogel particles were autoclaved at 121 °C for 15 min to provide the hydrogel comprising crosslinked HA for mixing with additional components of the dermal filler formulation.

Tri(ethylene glycol)-poly(orthoester) (TEG-POE)was added to the hydrogels comprising crosslinked HA and with or without non-crosslinked HA and the components were mixed at room temperature in absence of solvents. Mixing was continued until a homogenous, flowable and non-tacky semi-solid blend was achieved. Formulations comprising 2.5 wt%, 5 wt%, 7.5 wt%, 10 wt%, 12.5 wt% or 20 wt% TEG-POE were prepared. These formulations corresponded to variants of the bulk dermal filler formulation described herein.

For some (not all) samples of the dermal filler formulation a component of noncrosslinked (linear, IMDa) HA (2% (w/w)) in 12 mM PBS (137 mM NaCI, 2.7 mM KCI, 10.2 mM Na₂HPO₄, 2.0 mM KH2PO4) pH 6.9 was added, and the hydrogel composition was autoclaved at 121 °C for 15 min.

No non-crosslinked HA was added to the dermal filler formulations presented in figures 1-4 and Tables 1-2.

Mean particle size of hydrogel

The mean particle size of the micronized hydrogel particles was measured in a buffer with 0.9% NaCI using a Malvern Mastersizer 2000 coupled with a Hydro2000 dispersion unit. Measurements were performed assuming spherical particles and in the interval 0.02 - 2000 pm.

Example 2: Rheology and injectability of dermal filler formulations The rheological properties and injectability of the dermal filler formulations of Example 1 was tested to identify formulations with characteristics particularly suitable as dermal fillers.

Rheology measurements

The storage modulus of the dermal filler formulations of Example 1 was measured on a rotational rheometer (RheoStress 6000, HAAKE Rheometer, Waltham, MA, USA) equipped with a Cone-Plate and Plate-Plate geometry.. The samples were tested at the following conditions: Frequency sweep from 0.01-10 Hz, strain 0.005, 21 points, Gap 1000 pm and running temperature of 25°C, Geometry is plate-plate 20mm. The viscoelastic properties were investigated by registering storage modulus G' and loss modulus G" as a function of the oscillation frequency at controlled deformation y=0.01, at which all the materials exhibited linear viscoelastic response, in the range of 0.1-10 Hz.

Steady state shear analysis was performed measuring the viscosity as a function of shear rate y in the range of 0.1-1000 s^-1.

Injectability

Injectability of the dermal filler formulations were assessed by measurement of the extrusion force of the formulations, i.e. the force required to push the formulation through a syringe with a predetermined needle dimension. The tests were performed by means of Universal Testing Machine INSTRON 5566 equipped with an experimental setup constituted by a syringe (ImL) with a 27G needle mounted. The tests were performed at 25°C at a test speed of 57 mm/min, measuring the load necessary to inject the material throughout the needle. The friction between piston and syringe was evaluated by means of a preliminary test on empty syringe as control. The extrusion force was recorded as the value of the maximum load (Figure 2A). Measurements were replicated in triplicates for each syringe.

Results

The measurements showed that the storage modulus (G') and loss modulus (G") of all tested dermal filler formulations were substantially independent of frequency, with the storage modulus being higher than the loss modulus (Figure 1A-C), i.e. the formulations behaves as gel materials.

Investigating closer the storage modulus at a fixed frequency of 1 Hz (Table 1) as a function of TEG-POE content, it was surprisingly found that TEG-POE leads to an increased storage modulus compared to formulations without any TEG-POE. In particular, formulations with 10% TEG-POE had a high storage modulus.

Table 1. Storage modulus (G') at 1 Hz for dermal filler formulations containing no or different amounts of TEG-POE.

The viscosity n was measured as a function of shear rate y in the range of 0.1-1000 s- ¹ (Table 2). These flow curves is an indication of how the material flow, with lower viscosity making it easier for the material to distribute following injection. Surprisingly, the addition of TEG-POE in the 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 more significant for hydrogel formulations with a crosslinking ratio of 5: 1 and 10: 1.

Table 2. Viscosity n for dermal filler formulations containing no or different amounts of

TEG-POE.

Injectability experiments with formulations of varying amounts of TEG-POE showed that the injectability of the hydrogel formulations were substantially independent of the inclusion of TEG-POE in the hydrogel formulations (Figure 2B). This is surprising since TEG-POE on its own is not injectable and would have been expected to influence injectability.

Conclusion

This Example demonstrates that it was possible to prepare hydrogel formulations comprising a poly(orthoester) with excellent rheological properties which are required for use as a dermal filler. Moreover, the inclusion of a poly(orthoester) did not affect the injectability of the formulation.

In all, the tested formulations, being bulk dermal filler formulations, are considered to be excellent starting points for further formulation of injectable dermal fillers useful in a broad range of applications. This is especially true since the inclusion of a POE did not impair (if anything it improved) the rheological properties of the formulation, and it is shown in Examples 3 and 4 that new advantageous properties are obtained by the inclusion of a POE, such as TEG-POE.

Example 3: Stability of dermal filler formulation

The stability of the dermal filler formulations were assessed in a hyaluronidase assay to determine the longevity of the formulations. Resistance to enzymatic degradation was evaluated by mixing the formulations with a hyaluronidase solution, incubating them at 37°C and measuring the viscoelastic spectra at different times.

Method:

Hyaluronidase powder was reconstituted with PBS 2X to obtain a concentration of 50 U/mL. Hyaluronidase solution (200 pL) was added to 2 g of each formulation (no TEG- POE, or 2.5%, 5%, 10% or 20% TEG-POE) to obtain a final concentration of about 5 U/g, and then stored at 37 °C. For each sample was prepared a control by mixing 200 pL of PBS 2X instead of hyaluronidase solution. The enzymatic degradation was expressed as the ratio between the storage modulus of the degraded sample and the control sample at 1 Hz measured after 3 hours, after 6 hours and after 24 hours.

Results

The experiments revealed that all formulations containing TEG-POE were more resistant to hyaluronidase degradation than the control formulation without TEG-POE (Figure 3). In particular, the samples with 2.5%, 5% or 10% TEG-POE performed well, with the sample comprising 5% TEG-POE demonstrating the most resistance to hyaluronidase degradation.

Conclusion

This example demonstrates that inclusion of a poly(orthoester) in the hydrogel formulation protects the hyaluronic acid content from enzymatic degradation. This is important for the longevity of the hydrogel formulation (dermal filler formulation) upon injection into a subject.

Example 4: Biocompatibility of dermal filler formulation

The biocompatibility of the dermal filler formulations was assessed by seeding of cells on the hydrogel formulations and observing viability.

Method:

Cell culture

In order to evaluate the biological response of the hydrogel formulations, human dermal fibroblast (HDF) cells (Sigma-Aldrich, Burlington, MA, USA) were grown in a T- 75 cell culture flask (VWR, Radnor, PA, USA) at 37 °C and 5% CO2. Cell culture medium Dulbecco's Modified Eagle's medium (DMEM) (Microgem, Naples, Italy) supplemented with 20% of fetal bovine serum, 1% of non-essential amino acids (NEAA), and antibiotics (penicillin G sodium 100 U/mL, streptomycin 100 pg/mL) were used and changed every 3-4 days.

Cell viability assay

In order to study the cell viability, a density of 1 x 10⁴ cells/mL of HDF cells were seeded on a 96-well plate (World Precision Instruments, Inc., Sarasota, FL, USA). The hydrogel formulations were previously autoclaved at 121 °C and subsequently incubated with the DMEM for 24 h at 37 °C in incubator. The cells were then incubated for 24 and 48 h with 200 pL of the eluate of the selected formulations.

Then, the Alamar blue assay (AB) was performed by adding AB reagent, at 10% v/v with respect to the medium to the samples and incubated at 37 °C for 4 h. The absorbance of the samples was measured using a spectrophotometer plate reader (Multilabel Counter, 1420 Victor, Perkin Elmer, Waltham, MA, USA) at 570 nm and 600 nm. The AB reagent dye indicates an oxidation-reduction by changing colour in response to the chemical reduction in the growth medium, resulting from cell viability.

Data are expressed as the percentage difference between treated and control to evaluate the percentage of reduction (Reduction %), which is calculated with the following formula:

where Oi is the molar extinction coefficient (E) of oxidized AB at 570 nm; O2 is the E of oxidized AB at 600 nm; Ai is the absorbance of test wells at 570 nm; A2 is the absorbance of test wells at 600 nm; Pi is the absorbance of the control well at 570 nm; and P2 is the absorbance of the control well at 600 nm.

The percentage of reduction for each sample was normalized to the percentage of reduction for the control to obtain the cell viability percentage.

Moreover, HDF cells viability has been further monitored by observing their morphology after 48 hours using an optical microscope (magnification x20).

Results

The experiments show that growing human dermal fibroblast (HDF) cells in a hydrogel formulation without TEG-POE result in a similar cell viability (Figure 4A). Surprisingly, the inclusion of TEG-POE in the hydrogels improved cell viability, more so for the hydrogel formulation containing 5% TEG-POE (Figure 4A). This observation was supported also by microscopy images (Figure 4B).

Conclusion This example demonstrates that the inclusion of a poly(orthoester) in the hydrogel formulation is not detrimental to cell viability in any manner, but instead improves the viability of the dermal cells. This is very encouraging since the hydrogel formulations will inevitably either carry cells or come in contact with cells. In total, the examples show that inclusion of a poly(orthoester), such as TEG-POE, in the hydrogel formulation reinforces the polymeric network and improves elasticity and biocompatibility while increasing resistance against enzymatic degradation. Thus, the formulations presented herein has several advantages over formulations without a POE, and they can advantageously be used as a starting point for preparation of injectable dermal filler products, e.g. by further adjustment of properties according to the final use, such as by addition of non-crosslinked HA and/or additives, such as local anaesthetics.

Claims

1. An injectable dermal filler formulation comprising :

- a hydrogel comprising crosslinked hyaluronic acid (HA), and

- at least one poly(orthoester) (POE).

2. The injectable dermal filler formulation according to claim 1, wherein the at least one POE is 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 POE of type IV (POE IV).

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

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

6. The injectable dermal filler formulation according to any one of the preceding claims, wherein the at least one POE is represented by formula (I) or formula (II) :

wherein :

R is a bond, — (CHzja— , or ~-(CH2)b--O---(CH2)n--; where a is an integer of 1 to 10, and b and c are independently integers of 1 to 5;

R* is a Ci-4 alkyl; n is an integer of at least 5; and

A is R¹, R², R³, or R⁴, wherein R¹ is:

p is an integer of 1 to 20;

R⁵ is hydrogen or Ci-4 alkyl; and

R⁶ is:

wherein R³ is or

x is an integer of 0 to 30; y is an integer of 2 to 200;

R⁸ is hydrogen or Ci-4 alkyl;

R⁹ and R¹⁰ are independently C1-12 alkylene;

7. The injectable dermal filler formulation according to claim 6, wherein R³ is a low molecular weight polyethylene glycols or aliphatic diols, preferably a 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 a tri(ethylene glycol)-poly(orthoester) (TEG-POE).

9. The 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), such as about 0.5% (w/w) to about 18% (w/w), such as about 1% (w/w) to about 16% (w/w), such as about 2% (w/w) to about 15% (w/w), such as about 3% (w/w) to about 14% (w/w), such as about 4% (w/w) to about 12% (w/w), such as about 5% (w/w) to about 10% (w/w).

10. The injectable dermal filler formulation according to any one of the preceding claims, wherein the HA is crosslinked with a crosslinking agent selected from the group consisting of divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), formaldehyde, glutaraldehyde, polyanhydrides, polyaldehydes, polyhydric alcohols, carbodiimides, carboxylic acid chlorides, sulfonic acid chlorides, celluloses, dextrans, epichlorohydrin, ethylene glycol, diglycidyl ethers, polyglycerol polyglycidyl ethers, and bis- or polyepoxides, and derivatives thereof.

11. The injectable dermal filler formulation according to claim 10, wherein the ratio between HA and 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, said method comprising the following steps:

(i) providing a first solution comprising hyaluronic acid (HA),

(ii) reacting said first solution with a crosslinking agent to form a hydrogel comprising crosslinked HA, and

13. An injectable dermal filler formulation according to any one of claims 1-11 obtainable by a method according to claim 12.

14. A kit comprising:

- an injectable dermal filler formulation according to any one of claims 1-11 or 13, and

- optionally, instructions for use.

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