Field of the invention

The present invention is in the dermatological domain. The present invention relates to an injectable composition comprising a filler, preferably hyaluronic acid, and bupivacaine and/or a pharmaceutically acceptable salt, ester or prodrug thereof, and the use of said composition in the prevention or treatment of body and skin defects, in particular for prophylactic treatment of the immediate pain caused by injection of the composition. The present invention also relates to a manufacturing method which provides advantageous heat stability to the composition.

Background of the invention

Fillers such as hyaluronic acid gels are known as resorbable or slowly resorbable filling products, i.e. their effect is reversible since it will be degraded and absorbed by the body. It gives the possibility of filling structural body depressions, such as fine wrinkles, on the periphery of the mouth for example, but also deeper wrinkles like nasolabial folds.

However, administration by injection of fillers, such as of hyaluronic acid, may cause pain, red spots or blisters at the infiltrated areas and possibly bruises, or even bleeds at the needle-puncture site. The pain can persist for a few hours and the bruises for a week.

The drawbacks mentioned above therefore pose problems, in particular within the scope of aesthetical interventions. It is observed that such drawbacks represent a physiological, aesthetical and moral inconvenience for the subject who has received the injections. In particular, the management of pain is of importance to confer comfort and satisfaction to the patient who is generally requested to proceed to new injections some months later. Additionally, the management of secondary immediate reactions due to dermal or intradermal injection of fillers with vascular damages or vascular breaking wall inducing ecchymosis, bruising, leakage of blood components having immediate action on inflammation setting up, redness and oedema, are of particular interest. Such drawbacks also generate apprehension or even unsatisfaction.

Therefore, there is a need in the dermal industry for alleviating pain when fillers are injected.

Numerous dermal fillers already onto the market are made from hyaluronic acid with the addition of lidocaine to limit pain. However, lidocaine may e.g. present some impact on rheological properties of the gel specifically after the requested heat sterilization mandatory for injected products.

There is a need for improved dermal filler compositions with an anaesthetic compound to avoid these drawbacks.

Summary of the invention

It is an object of the present invention to find a remedy to the aforementioned drawbacks with further beneficial effects.

It is in particular an object of the present invention to provide a dermal filler composition with an anaesthetic compound having longterm stability.

It is a further object of the present invention to provide a process for

manufacturing an injectable filler composition with improved stability, in particular heat stability.

For these and other objects that will be evident from the following description, the present invention provides according to a first aspect an injectable filler composition comprising a cross-linked hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein the composition has a pH of below 7.1. The composition further comprises phosphate buffer at concentration of between 0.5 and 5 mM.

As a configuration of the first aspect, there is provided an injectable filler composition comprising a cross-linked hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein the composition has a pH 7.1 or lower and comprises phosphate buffer at concentration of below 1.5 mM.

The inventors have found that bupivacaine has a surprisingly strong protective effect on a hyaluronic acid gel and decreases the degradation rate during storage, as demonstrated in the Experimental Examples below. The measured effect of bupivacaine in a hyaluronic acid is larger than the effect observed when prior art anaesthetics lidocaine and mepivacaine are used.

Further, the inventors have found that the hyaluronic acid and bupivacaine composition preferably is formulated to a pH of 7.1 or lower, such as to a pH of about 6.8-7.0. This has been found to give a good formulation with low risk of precipitation of bupivacaine combined with high stability of the gel.

Further, the inventors have found that formulating the hyaluronic acid and the bupivacaine in a phosphate buffer having a concentration of between 0.5 and 5 mM is advantageous to use in a manufacturing process.

It is known that hyaluronic acid gels may be sensible to heat treatment and that incorporation of anaesthetics affect the properties of the gel.

However, the inventors have also found that bupivacaine has a stabilizing effect on a hydrogel formulation during sterilization.

In one preferred embodiment, the composition comprises between 0.5 % and 5 % (w/v), preferably between 1 % and 3 % (w/v), cross-linked hyaluronic acid, and between 0.025 % and 0.3%, preferably 0.05 % and 0.2 % (w/v) of bupivacaine.

According to another aspect, the present invention provides a process for manufacturing an injectable filler composition comprising a cross-linked hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof, comprising the following steps:

(a) preparing an aqueous mixture of cross-linked hyaluronic acid and a

therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof;

(b) adjusting the pH of the aqueous mixture to 7.1 or lower; and

(c) autoclaving the mixture to obtain an injectable filler composition.

It has surprisingly been realized that the use of bupivacaine in this procedure has an advantageous effect on product stability.

The process may be for manufacturing an injectable filler composition according to the first aspect above.

In one preferred embodiment, step (b) involves adjusting the pH of the aqueous mixture to 6.7 - 7.1. In one preferred embodiment of the manufacturing process, the injectable filler composition comprises phosphate buffer at concentration of below 10mM, preferably between 0.5 mM and 5 mM. Thus, the aqueous mixture of step a) may comprise phosphate buffer at concentration of between 0.5 mM and 5 mM. As an alternative, step b) may comprise formulating the hyaluronic acid and bupivacaine in a phosphate buffer at concentration of between 0.5 mM and 5 mM. According to a related aspect, the present invention provides an injectable filler composition comprising a cross-linked hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof, obtainable by the process according to the invention.

In an embodiment, this composition is for use in a prophylactic method of reducing the immediate pain caused by injection in a subject receiving an injection of said injectable filler composition.

According to a further aspect, the present invention provides a kit comprising i) at least one syringe and ii) a container containing an injectable filler composition according to the invention.

Other aspects and preferred embodiments of the present invention will be evident from the following detailed disclosure of the invention and the appended claims.

Detailed description of the invention

The applicant has developed an injectable composition comprising a filler and bupivacaine and/or a pharmaceutically acceptable salt, esters or prodrugs thereof. The injectable composition is useful to improve the body appearance, notably the appearance of the surface of the skin by reducing the depressions, such as wrinkles, or further by increasing the volume of certain portions of the body such as the lips. The injectable composition is also useful to alleviate physiological, aesthetical and moral inconveniences, notably to limit pain due to injection. In particular, the injectable composition is useful for prophylactic reduction of the immediate pain caused by injection in a subject receiving an injection of said injectable filler composition. Additionally, the applicant has developed a manufacturing process for a dermal filler composition with a bupivacaine compound with improved stability properties during and after sterilization.

The present invention is based on the injection of bupivacaine together with a filler, with improved appearance results. The inventors have found that bupivacaine has a surprisingly strong protective effect on a hyaluronic acid gel and decreases the degradation rate during storage. Additionally, a composition according to the invention reduces pain due to injection.

According to another embodiment, a composition according to the invention confers improved anesthetic properties to the dermal filler and maintain its requested rheological properties.

In a first aspect, the present invention concerns an injectable composition comprising a filler, preferably hyaluronic acid, and bupivacaine and/or a pharmaceutically acceptable salt, ester or prodrug thereof.

In a second aspect, the present invention concerns a kit comprising at least one syringe containing an injectable composition according to the invention. In a particular embodiment the syringe(s) may be prefilled with the composition to inject.

In a third aspect, the invention concerns the use of a composition or a kit according to the present invention, in preventing or treating skin defects, specially folds, wrinkles, skin depressions and scars, advantageously useful in diminishing, decreasing or avoiding skin reactions due to injection, specially pain.

Bupivacaine may thus be provided for use in diminishing, decreasing or avoiding skin reactions due to injection of a filler in a subject, preferably pain, by injection to a subject simultaneously with the filler.

The invention may thus also provide a method for diminishing, decreasing or avoiding skin reactions due to injection of an anaesthetic simultaneously with the injection of the filler.

The invention also provides a prophylactic method of reducing the immediate pain caused by injection in a subject receiving an injection of said injectable filler composition. In a preferred embodiment, the filler and bupivacaine are injected in a single composition, as defined herein.

However, the present disclosure also provides a kit comprising a container containing an injectable composition of a filler, and a container containing an injectable composition of bupivacaine. The containers could then be mixed and autoclaved by an operator and left for storage before injection.

The filler

A filler is generally defined as a biomaterial able to fill dermal tissues. The composition to be injected, comprising said filler in an aqueous medium and displaying filling properties, can also be defined as a“dermal filler”. Within the scope of the invention, a“filler” can include a mix of different individual fillers, such as individual glycosaminoglycans (GAGs).

In this context, compounds that can be used as dermal filler are resorbable polymers or molecules such as hyaluronic acid, collagen, alginate, dextran, elastine, polyurethane gels, chitosan, gelatin, carrageenans, or more permanent product as polyacrylamid gels, polymethylmethacrylate (PMMA) particles, microspheres or microparticles made of lactic acid polymers, glycolic acid polymers, or lactic acid- glycolic acid co-polymers, silicones, acrylic acid polymers, and derivatives thereof, this list not being exhaustive.

The filler may be a glycosaminoglycan (GAG), which is a negatively charged heteropolysaccharide chain which have a capacity to absorb large amounts of water. The GAG may for example be sulfated or non-sulfated glycosaminoglycans such as hyaluronan, chondroitin sulphate, heparan sulphate, heparosan, heparin, dermatan sulphate and keratan sulphate. In some embodiments the GAG is hyaluronic acid, chondroitin or chondroitin sulfate

The most preferred compounds are resorbable molecules such as hyaluronic acid, collagen, alginate, dextran, elastin or polyurethane gels.

Within the injectable composition, the concentration of said filler is

advantageously comprised between 0.5 mg/ml and 50 mg/ml, in particular between 10 mg/ml and 30mg/ml. For example, the claimed composition may comprise a concentration of filler of 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml, 26 mg/ml, 27 mg/ml, 28 mg/ml, 29 mg/ml, or 30 mg/ml.

An alternative way of measuring the quantity of the filler is using the “mass/volume percentage”. A solution with 1 g of solute dissolved in a final volume of 100 ml. of solution would be labeled as "1%" or "1 % m/v"

(mass/volume).

In embodiments, the filler represents 0.5 to 5 % (w/v) of the composition, preferably 1 to 3 % (w/v) of the composition.

For example, the filler may represent 1 %, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1 %, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0% expressed in w/v of the composition.

In a preferred embodiment of the invention, the filler is hyaluronic acid. Hyaluronic acid (HA) is a naturally occurring polysaccharide composed of a disaccharide motif comprising D-glucuronic acid and N-acetyl-D-glucosamine linked by alternating b(1 ,4)- and b(1 ,3)-glycosidic bonds.

Hyaluronic acid or hyaluronate is a non-sulfated glycosaminoglycan (GAG) widely distributed throughout connective, epithelial, and neural tissues. It is one of the chief components of the extracellular matrix. It contributes significantly to cell proliferation and migration. It plays an important role in skin hydration and skin elasticity. The level of hyaluronic acid decreases with ageing both in quantity and quality, inducing skin drying and wrinkles.

Hyaluronic acid and the other GAGs are negatively charged

heteropolysaccharide chains which have a capacity to absorb large amounts of water and form highly viscous solutions in water. Therefore, it is widely used as a pharmaceutical product. Moreover, since hyaluronic acid is present with identical chemical structure except for its molecular mass in most living organisms, this compound is considered to be very safe and no immunogenicity reaction has been observed. So far, few minor adverse events have been noticed.

The filler is hyaluronic acid or a pharmaceutically acceptable salt or derivative thereof, particularly the sodium or potassium salt. Hyaluronic acid can be used under different forms: salts thereof, derivatives thereof such as esters or amides, in a linear form or cross-linked. In particular, the molecular weight, typically comprised between 500 kDa and 5 000 kDa, and the degree of cross-linking depends on the application, especially on the depth of the wrinkles to be filled.

In a particular embodiment, the filler is modified hyaluronic acid, e.g. branched or cross-linked hyaluronic acid. Cross-linking and/or other modifications of the hyaluronic acid molecule is advantageous to improve its duration in vivo.

Furthermore, such modifications can modify the liquid retention capacity of the hyaluronic acid molecule. According to certain embodiments the hyaluronic acid is a cross-linked hyaluronic acid. According to specific embodiments the hyaluronic acid is a hyaluronic acid gel.

Unless otherwise provided, the term "hyaluronic acid" encompasses all variants and combinations of variants of hyaluronic acid, hyaluronate or hyaluronan, of various chain lengths and charge states, as well as with various chemical modifications, including cross-linking. That is, the term also encompasses the various hyaluronate salts of hyaluronic acid with various counter ions, such as sodium hyaluronate. Various modifications of the hyaluronic acid are also encompassed by the term, such as oxidation, e.g. oxidation of -CH2OH groups to - CHO and/or -COOH; periodate oxidation of vicinal hydroxyl groups, optionally followed by reduction, e.g. reduction of -CHO to -CH2OH or coupling with amines to form imines followed by reduction to secondary amines; sulphation; deamidation, optionally followed by deamination or amide formation with new acids; esterification; cross-linking; substitutions with various compounds, e.g. using a cross-linking agent or a carbodiimide assisted coupling; including coupling of different molecules, such as proteins, peptides and active drug components, to hyaluronic acid; and deacetylation. Other examples of modifications are isourea, hydrazide, bromocyan, monoepoxide and monosulfone couplings.

The hyaluronic acid can be obtained from various sources of animal and non- animal origin. Sources of non-animal origin include yeast and preferably bacteria. The molecular weight of a single hyaluronic acid molecule is typically in the range of 0.1-10 MDa, but other molecular weights are possible.

In one embodiment, the hyaluronic acid is cross-linked. Cross-linked hyaluronic acid comprises cross-links between the hyaluronic acid chains, which creates a continuous network of hyaluronic acid molecules which is held together by the covalent cross-links, physical entangling of the hyaluronic acid chains and various interactions, such as electrostatic interactions, hydrogen bonding and van der Waals forces.

Cross-linking of the hyaluronic acid may be achieved by modification with a chemical cross-linking agent. The chemical cross-linking agent may for example be selected from the group consisting of divinyl sulfone, multiepoxides and diepoxides. According to embodiments the chemical cross-linking agent is selected from the group consisting of 1 ,4-butanediol diglycidyl ether (BDDE),

1 ,2-ethanediol diglycidyl ether (EDDE) and diepoxyoctane. According to a preferred embodiment, the chemical cross-linking agent is 1 ,4-butanediol diglycidyl ether (BDDE).

Consequently, in embodiments the cross-linked hyaluronic acid is cross- linked by ether bonds.

Crosslinking may also be performed by amide coupling of

glycosaminoglycan molecules, such as amide coupling of hyaluronic acid molecules.

Consequently, in embodiments the cross-linked hyaluronic acid is cross- linked by amide bonds.

The amide coupling may be performed using a di- or multinucleophile functional crosslinker. Amide coupling using a di- or multiamine functional crosslinker together with a coupling agent is an attractive route for preparing crosslinked glycosaminoglycan molecules useful in the present invention.

Crosslinking can be achieved using a non-carbohydrate based di- or multinucleofile crosslinker, for example hexamethylenediamine (HMDA), or a carbohydrate based di- or multinucleofile crosslinker, for example

diaminotrehalose (DATH) together with a glycosaminoglycan.

Thus, the crosslinker comprising at least two nucleophilic functional groups may for example be a non-carbohydrate based di- or multinucleophilic crosslinker or a carbohydrate based di- or multinucleophilic crosslinker.

A preferred group of di- or multinucleophile functional crosslinker includes homo- or heterobifunctional primary amines, hydrazines, hydrazides, carbazates, semi-carbazides, thiosemicarbazides, thiocarbazates and aminoxy. The di- or multinucleophile functional di-, tri-, tetra-, and oligo-saccharides may be derived from nucleophile functional polysaccharides, such as chitobiose derived from chitin. The di- or multinucleophile functional di-, tri-, tetra-, and oligo

saccharides may also be di-, tri-, tetra-, and oligo-saccharides which have been modified by introduction of two or more nucleophile functional groups.

In embodiments, a glycosaminoglycan (GAG) of the second outer phase comprises crosslinks in which the glycosaminoglycan itself acts as a di- or multinucleofile crosslinker. As an example, a deacetylated glycosaminoglycan may itself act as the di- or multinucleofile crosslinker.

The cross-linked hyaluronic acid product is preferably biocompatible. This implies that no, or only very mild, immune response occurs in the treated subject. That is, no or only very mild undesirable local or systemic effects occur in the treated subject.

The cross-linked hyaluronic acid product according to the invention may be a gel, or a hydrogel. That is, it can be regarded as a water-insoluble, but substantially dilute cross-linked system of hyaluronic acid molecules when subjected to a liquid, typically an aqueous liquid. While native hyaluronic acid and certain cross-linked hyaluronic acid products absorb water until they are completely dissolved, cross- linked hyaluronic acid gels typically absorb a certain amount of water until they are saturated, i.e. they have a finite liquid retention capacity, or swelling degree.

The gel contains mostly liquid by weight and can e.g. contain 90-99.9% water, but it behaves like a solid due to a three-dimensional cross-linked hyaluronic acid network within the liquid. Due to its significant liquid content, the gel is structurally flexible and similar to natural tissue, which makes it very useful as a scaffold in tissue engineering and for tissue augmentation.

As mentioned, cross-linking of hyaluronic acid to form the cross-linked hyaluronic acid gel may for example be achieved by modification with a chemical cross-linking agent, for example BDDE (1 ,4-butandiol diglycidylether). The hyaluronic acid concentration and the extent of cross-linking affects the mechanical properties, e.g. the elastic modulus G’, and stability properties of the gel. Cross-linked hyaluronic acid gels are often characterized in terms of“degree of modification”. The degree of modification (mole%) describes the amount of cross-linking agent(s) that is bound to HA, i.e. molar amount of bound cross-linking agent(s) relative to the total molar amount of repeating HA disaccharide units. The degree of modification reflects to what degree the HA has been chemically modified by the cross-linking agent. Reaction conditions for cross-linking and suitable analytical techniques for determining the degree of modification are all well known to the person skilled in the art, who easily can adjust these and other relevant factors and thereby provide suitable conditions to obtain a degree of modification in the range of 0.1- 10% and verify the resulting product characteristics with respect to the degree of modification. The degree of modification of hyaluronic acid gels generally range between 0.1 and 15 mole%. A BDDE (1 ,4-butandiol diglycidylether) cross-linked hyaluronic acid gel may for example be prepared according to the method described in Examples 1 and 2 of published international patent application WO 9704012.

In a preferred embodiment the hyaluronic acid of the composition is present in the form of a cross-linked hyaluronic acid gel cross-linked by a chemical cross-linking agent, wherein the concentration of said hyaluronic acid is in the range of 10 to 30 mg/ml and the degree of modification with said chemical cross- linking agent is in the range of 0.1 to 10 mole%.

Hyaluronic acid gels may also comprise a portion of hyaluronic acid which is not cross-linked, i.e. not bound to the three-dimensional cross-linked hyaluronic acid network. However, it is preferred that at least 50 % by weight, preferably at least 60 % by weight, more preferably at least 70 % by weight, of the hyaluronic acid in a gel composition form part of the cross-linked hyaluronic acid network.

In embodiments 40-80%, such as 40 - 70%, such as 40 - 60%, of the hyaluronic acid in a gel composition form part of the cross-linked hyaluronic acid network.

The anaesthetic compound

According to the present invention, the injectable dermal filler composition comprises an anesthetic chosen from bupivacaine and/or a pharmaceutically acceptable salt, ester or prodrug thereof. As an example, the injectable dermal filler composition may comprise bupivacaine and/or a pharmaceutically acceptable salt or ester thereof.

Bupivacaine is a widely used local anesthetic agent. The chemical name (IUPAC Name) is 1-butyl-N-(2,6-dimethylphenyl)piperidine-2-carboxamide having a molecular formula of C H N O. Injectable solutions of bupivacaine may be made from a salt (e.g. hydrochloride) or a hydrate (e.g. hydrochloride monohydrate).

Preferably, to be suitable for injection, bupivacaine is a salt, as hydrochloride, or a hydrate, hydrochloride monohydrate.

In certain embodiments the concentration of the bupivacaine is between 0.025 % and 0.3% (w/v), such as between 0.05 % and 0.2 % (w/v) bupivacaine. As an example, the concentration of the bupivacaine may be about 0.1 % (w/v). Such concentrations of bupivacaine may be beneficial due to the potency of bupivacaine after injection.

Additional components

According to an alternative embodiment, the claimed composition can also contain one or more additional components that man skilled in the art will select in order to keep the advantages of the composition according to the invention.

In alternative embodiments, the final compositions according to the invention have been subjected to sterilization, i.e. the final compositions according to the invention have been subjected to heat and/or steam and/or irradiation treatment in order to sterilize the composition. The sterilization of the composition is

advantageously performed to conserve the functional activity of the final

composition.

In some embodiments the final composition, or each component of the composition, has been subjected to sterilization by autoclaving or similar sterilization by heat or steam. Sterilization, e.g. autoclaving, may be performed at a Fo-value > 8. The Fo value of a saturated steam sterilisation process is the lethality expressed in terms of the equivalent time in minutes at a temperature of 121 °C delivered by the process to the product in its final container with reference to micro-organisms possessing a Z-value of 10.

According to the invention, bupivacaine used as the anesthetic in the dermal filler composition of the invention provides beneficial stability to the product during and after sterilization. Injectable compositions

Injectable compositions comprising a filler and bupivacaine and/or a pharmaceutically acceptable salt and/or a pharmaceutically acceptable ester are provided. In a preferred embodiment, the invention relates to the combination of a filler and bupivacaine in a single injectable composition. Unless otherwise specified, the term“composition” as used herein relates to any injectable composition comprising a filler and/or bupivacaine.

The compositions are administered to a subject by injection, preferably by dermal injection, in particular by intradermal injection. Intradermal injections are delivered into the dermis (more precisely in the superficial, middle or deep dermis), or the skin layer underneath the epidermis (which is the upper skin layer). Thus, the definition of intradermal in the context of the present invention excludes the transdermal or subcutaneous injections. Therefore, in the context of the instant invention the filler and bupivacaine are delivered to the target area of the skin in a pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier is any pharmaceutically acceptable formulation that can be applied to the skin for dermal, in particular for intradermal delivery of a pharmaceutical or medicament. The combination of a pharmaceutically acceptable carrier and a compound of the invention is designated an injectable formulation of the invention.

Typically, the composition consists of a solution or a gel, preferably an aqueous solution or gel, more preferably an aqueous gel.

The claimed composition is composed of or contains effective amounts of bupivacaine and fillers. As used herein, an“effective amount” means the minimum amount of the compound that is effective to obtain the desired effect in the context of the invention.

The compositions used in the invention can comprise any other

pharmaceutically acceptable components such as carriers, excipients, preservatives, etc.

The present invention provides according to a first aspect an injectable filler composition comprising a cross-linked hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein the composition has a pH of 7.1 or lower. As an example, the composition may have a pH of between 6.7 - 7.1 , such as about 7.0.

In embodiments, the osmolarity of the composition is between 270-350 mOsm, preferably between 290-310 mOsm.

In embodiments, the osmolarity of the composition is between 200-400 mOsm, preferably between 270-350 mOsm.

The bupivacaine and hyaluronic acid composition has been found to be stable within such osmolarity ranges.

The composition further comprises phosphate buffer at concentration of between 0.5 mM and 5 mM. The concentration relates to the concentration of phosphate ions in the composition.

The buffer may be phosphate-buffered saline that is commonly used in biological research and helps to maintain a substantially constant pH of a solution.

As an example, the composition comprises phosphate buffer at concentration of below 1.5 mM.

Further, the composition may comprise phosphate buffer at concentration of above 0.5 mM, such as between 0.5 mM and 1.5 mM, such as about 1.0 mM.

Furthermore, the composition may comprise phosphate buffer at a concentration of between 0.7 mM and 4 mM, such as between 0.8 mM and 3 mM, such as between 1 mM and 2 mM.

In embodiments the composition comprises between 0.5 % and 5 % (w/v) cross- linked hyaluronic acid and between 0.025 % and 0.3% (w/v) bupivacaine.

As an example, the composition comprises between 1 % and 3 % (w/v) cross- linked hyaluronic acid and between 0.025 % and 0.3% (w/v) bupivacaine.

As further example, the composition comprises between 0.5 % and 5 % (w/v) cross-linked hyaluronic acid and between 0.05 % and 0.2 % (w/v) bupivacaine.

Moreover, the composition may comprise about 2.0 % (w/v) cross-linked hyaluronic acid and about 0.1 % (w/v) bupivacaine.

Further, there is provided a prophylactic method of reducing the immediate pain caused by injection in a subject receiving an injection of an injectable filler composition comprising a cross-linked hyaluronic acid, comprising administering a therapeutically effective amount of bupivacaine in said injectable filler composition, wherein said injectable filler composition is a composition according to the first aspect above, comprising a cross-linked hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof.

Process for manufacturing the injectable filler composition

As another aspect of the invention, there is provided process for

manufacturing an injectable filler composition comprising a cross-linked

hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof, comprising the following steps:

(a) preparing an aqueous mixture of cross-linked hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof;

(b) adjusting the pH of the aqueous mixture to 7.1 or lower; and

(c) autoclaving the mixture to obtain an injectable filler composition.

Sterilization, e.g. autoclaving, may be performed at a Fo-value > 4, such as at a

Fo-value > 8. The Fo value of a saturated steam sterilisation process is the lethality expressed in terms of the equivalent time in minutes at a temperature of 121 °C delivered by the process to the product in its final container with reference to micro- organisms possessing a Z-value of 10.

In embodiments, step (b) involves adjusting the pH of the aqueous mixture to 6.7 - 7.1 , such as to a pH of about 7.0.

In one preferred embodiment, the injectable filler composition comprises phosphate buffer at concentration of between 0.5 mM and 5 mM. Thus, the aqueous mixture of step a) may comprise phosphate buffer at concentration of between 0.5 mM and 5 mM. As an alternative, step b) may comprise formulating the hyaluronic acid and bupivacaine in a phosphate buffer at concentration of between 0.5 mM and 5 mM.

The injectable filler composition obtained in step (c) may be an injectable filler as discussed in relation to the first aspect above.

As a further aspect of the invention, there is provided an injectable filler composition comprising a cross-linked hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof, obtainable by the process discussed above.

Further, there is provided a prophylactic method of reducing the immediate pain caused by injection in a subject receiving an injection of an injectable filler composition comprising a cross-linked hyaluronic acid, comprising administering a therapeutically effective amount of bupivacaine in said injectable filler composition, wherein said injectable filler composition is a composition obtainable by the process discussed above, comprising a cross-linked hyaluronic acid and a therapeutically effective amount of bupivacaine, or a pharmaceutically acceptable salt, ester or prodrug thereof.

Kits

Another aspect of the invention is an article of manufacture that comprises a formulation of the invention in a suitable container with labelling and instructions for use. The container is advantageously a single dose syringe.

Preferably, instructions are packaged with the formulations of the invention, for example, a pamphlet or package label. The labelling instructions explain how to administer formulations of the invention, in an amount and for a period of time sufficient to treat the patient. Preferably, the label includes the dosage and administration instructions, the formulation's composition, the clinical pharmacology, drug resistance, pharmacokinetics, absorption, bioavailability, and contraindications.

The injectable composition according to the invention can then be integrated into a kit comprising one or more syringes containing said composition.

In another example of the present disclosure, the filler is contained in a first syringe, and bupivacaine and/or one or more component is/are contained in a separate syringe. The filler and bupivacaine can be contained in separate syringes for sequential administration or mixing.

In a particular embodiment, the filler, preferably the cross-linked hyaluronic acid, or an aqueous composition thereof, may be provided in the form of a pre-filled syringe, i.e. a syringe that is pre-filled with a filler composition, preferably the cross- linked hyaluronic acid, and autoclaved.

The filler composition and the bupivacaine composition can be used

simultaneously in the context of the present invention. When used simultaneously, the filler and bupivacaine are preferably presented as a mixture contained in a single syringe. Alternatively, they can be in the form of two separate compositions which are mixed extemporaneously, before injection.

In embodiments, the kit comprises i) at least one syringe and ii) a container containing an injectable filler composition according to the first aspect discussed above.

Aesthetic and other applications

The injectable compositions described herein are intended for use in preventing or treating body and skin defects, specially folds, wrinkles, skin depressions and scars. The treatment is considered cosmetic, i.e. non-medical.

The claimed composition is meant to be administered to a subject or a patient, especially by facial injection (such as to forehead, eyes, nasolabial fold). As used herein, the term“subject” or“patient” are used equivalently and means any animal, preferably a mammal, more preferably, a human to whom will be or has been administered compounds or formulations of the invention. The term “mammals” used herein encompasses any mammal.

The use preferably comprises injecting the composition(s) into the cutis or subcutis of a human subject, cutis being defined as the combination of the epidermal and the dermal outer layers of the skin. The use of the injectable composition(s) for improving the appearance of skin, filling wrinkles or contouring the face or body of a subject, may be essentially or totally non- medical, e.g. purely cosmetic.

When used separately, the composition of bupivacaine is injected at substantially the same site as the composition of filler, or in its vicinity.

The injectable compositions comprising the filler are useful in, e.g., soft tissue augmentation, for example filling of wrinkles, by a filler injection, preferably a hyaluronic acid gel injection. The compositions may be especially useful in a cosmetic treatment, referred to herein as skin revitalization, whereby small quantities of the filler composition are injected into the dermis at a number of injection sites distributed over an area of the skin to be treated, resulting in improved skin tone and skin elasticity. Skin revitalization is a simple procedure and health risks associated with the procedure are very low.

According to some aspects illustrated herein, there is provided the use of a composition as described above for cosmetic, non-medical, treatment of a subject by administration, preferably by dermal or intradermal injection, of the composition into the skin of the subject. A purpose of the cosmetic, non-medical, treatment may be for improving the appearance of the skin, filling wrinkles or contouring the face or body of a subject. The cosmetic, non-medical, use does not involve treatment of any form of disease or medical condition. Examples of improving the appearance of the skin include, but are not limited to, treatment of sun-damaged or aged skin and skin revitalization.

According to certain embodiments, there is provided the use of a composition as described above for improving the appearance of skin, filling wrinkles or contouring the face or body of a subject. According to a preferred embodiment, there is provided the use of a filler composition as described herein, for skin revitalization.

The cosmetic compositions are administered by dermal or intradermal injection into the skin of a subject, preferably into the cutis. Preferably the compositions are in the form of a gel.

Administration of gel structures may be performed in any suitable way, such as via injection from traditional hand-held syringes or any injection device for delivering liquid/viscous compositions. Any syringe may be equipped with standard cannula and needles of appropriate sizes or surgical insertion. The administration is performed where the soft tissue augmentation is desired, such as the chin, cheeks or elsewhere in the face or body.

For instance, the diameter of the injection needle ranges preferably from 17 to 34 gauge.

In an embodiment, the volume of the filler composition to be injected varies between 0.1 and 10 ml, typically between 0.5 and 4 ml. As an example, the volume of the filler may be about 1 ml. Preferably, said volume is presented as a single dose syringe. Said injection can be repeated, for example after 4 to 18 months.

There is provided a method for preventing or treating body and skin defects, specially folds, wrinkles, skin depressions and scars, by injecting in a subject in need thereof, comprising: 1 ) providing a filler composition comprising bupivacaine

2) administering said composition into the skin of a subject

According to certain embodiments, the method comprises improving the appearance of skin. According to a preferred embodiment, the method comprises skin revitalization.

According to certain embodiments, the method comprises filling wrinkles or contouring the face or body of a subject.

The invention further provides a method for diminishing, decreasing or avoiding skin reactions due to injection of a filler, preferably pain, simultaneously or subsequently to the injection of the filler.

Other potential benefits of combining the filler and bupivacaine for simultaneous use are as follows:

- By reducing the skin reactions, in particular inflammation, bupivacaine may allow the filler to persist longer, possibly due to its slower degradation: the more severe the tissue reaction, in particular the more inflammatory the filler is, the higher is the level of undesirable species (e.g. inflammatory species), thus degrading the filler faster.

- When the composition is comprising bupivacaine, the efficiency of said anaesthetic may be improved: vasoconstrictive effect provided by the filler at lower concentrations may limit anaesthetic diffusion in a large area, thus making anaesthetic efficient in the strict injection site.

In addition to the above, the examples below are provided to illustrate particular embodiments and not to limit the scope of the invention.

Brief description of the drawings

Fig. 1 illustrates Gel content (GeIC) in percent measured on cross-linked HA-gels without caine, with Lidocaine 3 mg/ml_, Bupivacaine 1 mg/ml_ and Mepivacaine 3 mg/ml_. Light grey column represents non autoclaved product, grey columns are products directly after autoclave process and dark grey are products after storage at 60 °C for 6 days (accelerated stability corresponding to 2 years at 25°C).

Fig. 2 illustrates Swelling factor (mL/g) measured on cross-linked HA-gels without caine, with Lidocaine 3 mg/mL, Bupivacaine 1 mg/mL and Mepivacaine 3 mg/mL Light grey column represents non autoclaved product, grey columns are products directly after autoclave process and dark grey are products after storage at 60 °C for 6 days (accelerated stability corresponding to approximately 2 years at 25°C).

Fig. 3 illustrates Tan d measured on cross-linked HA-gels without caine, with Lidocaine 3 mg/mL, Bupivacaine 1 mg/mL and Mepivacaine 3 mg/mL. Light grey column represents non autoclaved product, grey columns are products directly after autoclave process and dark grey are products after storage at 60 °C for 6 days (accelerated stability corresponding to approximately 2 years at 25°C).

Fig. 4 illustrates Gel content (GeIC) in percent measured on cross-linked HA- gels without caine, with Lidocaine 3 mg/mL, Bupivacaine 1 mg/mL and Mepivacaine 3 mg/mL. Light grey column represents non autoclaved product, grey columns are products directly after autoclave process and dark grey are products after storage at 60 °C for 6 days (accelerated stability corresponding to approximately 2 years at 25°C).

Fig. 5 illustrates Swelling factor (mL/g) measured on cross-linked HA-gels without caine, with Lidocaine 3 mg/mL, Bupivacaine 1 mg/mL and Mepivacaine 3 mg/mL. Light grey column represents non autoclaved product, grey columns are products directly after autoclave process. The swelling factor of the gels stored at 60 °C for 6 days could not be measured reliably with this method due to the high degradation of the gels.

Fig. 6 illustrates Tan d measured on cross-linked HA-gels without caine, with Lidocaine 3 mg/mL, Bupivacaine 1 mg/mL and Mepivacaine 3 mg/mL. Light grey column represents non autoclaved product, grey columns are products directly after autoclave process and dark grey are products after storage at 60 °C for 6 days (accelerated stability corresponding to approximately 2 years at 25°C).

Examples

Example 1 - Preparation of filler compositions with different anaesthetic agents

Cross-linked hyaluronic acid (ΉA) gels were manufactured using hyaluronic acid, sodium hydroxide (NaOH) 1 % and butanediol diglycidyl ether (BDDE). The crosslinking reaction was performed at 50° C for 2 hours. The crosslinked hyaluronic acid was neutralized during swelling in a phosphate buffer and thereafter purified by dialysis. The whole batch was sieved and homogenized before the batch was splitted into four sub-batches. One batch was produced without caine, one with Lidocaine 3 mg/ml_ one with Bupivacaine 1 mg/ml_ and one with Mepivacaine 3 mg/ml_. The caines were added during mixing. Thereafter the gels were filled in syringes and sterilized by autoclaving. The final composition of all sub-batches were 20 mg HA/mL, 1 mM phosphate-buffer and a pH of 7.

Example 2 - Properties of prepared filler compositions

Gel content

The degradation behaviour of the prepared filler compositions of Example 1 were analysed by measuring the gel content (GeIC) as a function of time.

The GeIC describes in % the proportion of the total HA that is bound in gel form. Gel content is defined as the amount of HA in a sample that does not pass through a 0.22 pm filter. GeIC is calculated from the amount of HA that is collected in the filtrate and is given in percent of the total amount of HA in the gel sample. More degradation of a gel will lead to lower gel content.

During sterilization a certain amount of the gel is degraded resulting in a decrease of the gel content. During storage at elevated temperature further degradation of the gel occur. In this example the gels from Example 1 were analyzed before sterilization (only Lidocaine), directly after sterilization and after 6 days of storage at 60 °C. The gel without caine was only analyzed directly after sterilization. The results are presented in Figure 1.

Swelling factor

The swelling factor describes the maximum amount of water (more specific 0.9% sodium chloride) a gel can absorb. The swelling factor is measured by accurately weighing gel in a measuring cylinder, add 0.9% sodium chloride and after complete swelling of the gel read the maximum volume. More degradation of a gel will lead to a higher swelling factor. During sterilization a certain amount degradation of the gel occurs, resulting in an increase of the swelling factor. During storage further degradation of the gel occur. To correct for any variations in the final HA

concentration of the gels, a normalized swelling factor was calculated by multiplying the measured swelling factor by nominal HA-concentration (20 mg/mL) and divided by measured HA concentration. In this example the gels from Example 1 were analyzed before sterilization (only Lidocaine), directly after sterilization and after 6 days of storage at 60 °C. The gel without caine was only analyzed directly after sterilization. The results are presented in Figure 2.

Rheology

Rheological properties of the gels were evaluated using tan d, a rheological parameter that will increase with increasing degradation of the gel. Tan d is measured as the tangent of the phase angle - the ratio of viscous modulus (G") to elastic modulus (G').

In this example the gels from Example 1 were analyzed before sterilization (only Lidocaine), directly after sterilization and after 6 days of storage at 60 °C. The gel without caine was only analyzed directly after sterilization. These results are presented in Figure 3.

Conclusions

The above Examples show that the investigated caines; Lidocaine,

Bupivacaine and Mepivacaine all have a stabilizing/shielding effect on the hydrogel formulation during sterilization.

During storage of the gels at elevated temperature (60 °C) Bupivacaine has the strongest protective effect on the gel and prevent it from degradation. This can be seen in that gel containing Bupivacaine has the smallest change of all measured gel parameters compared to Lidocaine and Mepivacaine during storage.

Example 3 - Preparation of filler compositions with different anaesthetic agents

Cross-linked hyaluronic acid (ΉA) gels were manufactured using hyaluronic acid, sodium hydroxide (NaOH) 1% and butanediol diglycidyl ether (BDDE). The crosslinking reaction was performed at 50° C for 2 hours, using a different, lower amount of BDDE than in Example 1. The crosslinked hyaluronic acid was neutralized during swelling and thereafter purified by dialysis in a phosphate buffer (1 mM). The whole batch was sieved and homogenized before the batch was splitted into four sub-batches. One batch was produced without caine, one with Lidocaine 3 mg/mL, one with Bupivacaine 1 mg/mL and one with Mepivacaine 3 mg/mL. The caines were added during mixing. Thereafter the gels were filled in syringes and sterilized by autoclaving. The final composition of all sub-batches were 20 mg HA/mL, 1 mM phosphate-buffer and a pH of 7. Example 4 - Properties of prepared filler compositions

Gel content

The degradation behaviour of the prepared filler compositions of Example 3 were analysed by measuring the gel content (GeIC) as a function of time.

The GeIC describes in % the proportion of the total HA that is bound in gel form. Gel content is defined as the amount of HA in a sample that does not pass through a 0.22 pm filter. GeIC is calculated from the amount of HA that is collected in the filtrate and is given in percent of the total amount of HA in the gel sample. Less crosslinking or more degradation of a gel will lead to lower gel content.

During sterilization a certain amount of the gel is degraded resulting in a decrease of the gel content. During storage at elevated temperature further degradation of the gel occur. In this example the gels from Example 3 were analyzed before sterilization, directly after sterilization and after 6 days of storage at 60 °C.

The results are presented in Figure 4.

Swelling factor

The swelling factor describes the maximum amount of water (more specific 0.9% sodium chloride) a gel can absorb. The swelling factor is measured by accurately weighing gel in a measuring cylinder, add 0.9% sodium chloride and after complete swelling of the gel read the maximum volume. Less crosslinking or more degradation of a gel will lead to a higher swelling factor. During sterilization a certain amount degradation of the gel occurs, resulting in an increase of the swelling factor. During storage further degradation of the gel occur. To correct for any variations in the final HA concentration of the gels, a normalized swelling factor was calculated by multiplying the measured swelling factor by nominal HA-concentration (20 mg/ml) and divided by measured HA concentration. In this example the gels from Example 3 were analyzed before sterilization and directly after sterilization. The results are presented in Figure 5.

Rheology

The rheological properties of the gels were evaluated using tan d, a rheological parameter that will increase with decreasing crosslinking or increasing degradation of the gel. Tan d is measured as the tangent of the phase angle - the ratio of viscous modulus (G") to elastic modulus (G').

In this example the gels from Example 3 were analyzed before sterilization, directly after sterilization and after 6 days of storage at 60 °C. These results are presented in Figure 6.

Conclusions

The above Examples show that bupivacaine has the strongest protective effect on the gel and prevent it from degradation. This can be seen in that gel containing Bupivacaine has the smallest change of all measured gel parameters, both during sterilization and during storage of the gels at elevated temperature (60 °C), compared to Lidocaine and Mepivacaine.

Example 5 - Preparation of filler compositions with different phosphate buffer concentrations

Cross-linked hyaluronic acid (HA) gel was manufactured using hyaluronic acid, sodium hydroxide (NaOH) 1 % and butanediol diglycidyl ether (BDDE). The crosslinking reaction was performed at 50° C for 2 hours. The crosslinked hyaluronic acid was neutralized during swelling and thereafter purified by dialysis in a phosphate buffer (1 mM). The whole batch was sieved and homogenized before the batch was splitted into two sub-batches. Phosphate buffer (1 M) was added to one sub-batch to a final concentration of 7 mM. Thereafter Bupivacaine was added during mixing to both sub-batches to the concentration 1 mg/ml_. After

homogenization the gels were filled in syringes and sterilized by autoclaving. The final composition of both sub-batches were 20 mg HA/mL, 1 mg/ml_ Bupivacaine and a pH of 7.

The Gel content in percent, the Swelling factor (mL/g) and Tan d were measured on both sub-batches after autoclaving. The results are shown in Table 1 below. Table 1

This experiment shows that the gel formulated in a phosphate buffer of 1 mM had a higher gel content as well as a lower normalized swelling factor and a lower Tan d compared to the gel formulated in a 7 mM phosphate buffer. Thus, it may be more advantageous to use the gel formulated in 1 mM phosphate buffer in a

manufacturing process.