(19) TEPZZ 67Z447B_T (11) EP 2 670 447 B1 (12) EUROPEAN PATENT SPECIFICATION (4) Date of publication and mention of the grant of the patent: 01.07. Bulletin /27 (21) Application number: 1270236.9 (22) Date of filing: 03.02.12 (1) Int Cl.: A61L 27/14 (06.01) A61L 31/04 (06.01) A61K 8/73 (06.01) A61Q /00 (06.01) A61Q 19/08 (06.01) A61K 47/22 (06.01) A61K 47/42 (06.01) A61K 9/00 (06.01) A61L 27/4 (06.01) A61L 31/16 (06.01) A61L 27/ (06.01) (86) International application number: PCT/EP12/0187 (87) International publication number: WO 12/4419 (09.08.12 Gazette 12/32) (4) Hyaluronic acid composition comprising an ascorbic acid derivative Hyaluronsäurezusammensetzung, die ein Ascorbinsäurederivat enthält Composition à base d acide hyaluronique contenant un dérivé d acide ascorbique (84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR () Priority: 03.02.11 EP 113232 (43) Date of publication of application: 11.12.13 Bulletin 13/0 (73) Proprietor: Q-Med AB 72 28 Uppsala (SE) (72) Inventors: EDSMAN, Katarina S-77 6 Uppsala (SE) WIEBENSJÖ, Åsa S-77 9 Uppsala (SE) (74) Representative: Kånge, Lars Rikard Awapatent AB Box 86 4 Stockholm (SE) (6) References cited: EP-A1-1 884 231 WO-A1-97/012 WO-A2-09/00790 US-A1-06 122 147 EMANUEL ABRAMS AND JOHNSON SANDSOM: "Effect of ascorbic acid on rheumatoid synovial fluid", ANN. RHEUM. DIS., vol. 23, 1964, pages 29-299, XP002670273, EP 2 670 447 B1 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). Printed by Jouve, 7001 PARIS (FR)
Description Field of the invention [0001] The present invention relates to the field of injectable hyaluronic acid compositions and the use of such compositions in cosmetic applications. Background 2 [0002] One of the most widely used biocompatible polymers for medical use is hyaluronic acid. It is a naturally occurring polysaccharide belonging to the group of glycosaminoglycans (GAGs). Hyaluronic acid and the other GAGs are negatively charged heteropolysaccharide chains which have a capacity to absorb large amounts of water. Hyaluronic acid and products derived from hyaluronic acid are widely used in the biomedical and cosmetic fields, for instance during viscosurgery and as a dermal filler. [0003] Water-absorbing gels, or hydrogels, are widely used in the biomedical field. They are generally prepared by chemical crosslinking of polymers to infinite networks. While native hyaluronic acid and certain crosslinked hyaluronic acid products absorb water until they are completely dissolved, crosslinked 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. [0004] Since hyaluronic acid is present with identical chemical structure except for its molecular mass in most living organisms, it gives a minimum of reactions and allows for advanced medical uses. Crosslinking and/or other modifications of the hyaluronic acid molecule is necessary to improve its duration in vivo. Furthermore, such modifications affect the liquid retention capacity of the hyaluronic acid molecule. As a consequence thereof, hyaluronic acid has been the subject of many modification attempts. [000] Hyaluronic acid products for injection are often combined with a suitable anaesthetic, e.g. lidocaine, to reduce pain or discomfort experienced by the patient due to the injection procedure. Description of the invention 3 4 0 [0006] It is an object of the present invention to provide an improved injectable hyaluronic acid composition for medical and/or non-medical applications. [0007] Hyaluronic acid compositions for use in injection need to be sterilized before use. Sterilization is generally performed by heat treatment, such as autoclaving. The heat treatment generally leads to a reduction of the rigidity or viscosity of the hyaluronic acid composition. As mentioned above, hyaluronic acid products for injection are often combined with a suitable anaesthetic, e.g. lidocaine, to reduce pain or discomfort experienced by the patient due to the injection procedure. It has been observed that the addition of some commonly used anaesthetics, e.g. lidocaine, counteract the effect of the heat treatment on the rheology of a hyaluronic acid composition such that the resulting composition becomes more rigid or viscous than a gel without lidocaine. This change in rheology may be disadvantageous in some applications, for example in applications where shallow injection of the gel is required or desired, and/or where a very fine gauge needle is required or desired. Examples of such applications include skin revitalization and soft tissue augmentation, for example filling of wrinkles or contouring of the face or body, by hyaluronic acid gel injections. US06/1221474 discloses injectable compositions comprising hyaluronic acid, a local anaesthetic, like lidocaine and an ascorbic acid derivative as a radical interceptor. [0008] It has now been found that addition of a relatively small amount of an ascorbic acid derivative selected from the group consisting of ascorbyl phosphates, ascorbyl sulfates and ascorbyl glycosides to a hyaluronic acid composition comprising a local anesthetic selected from the group consisting of amide and ester type local anesthetics may effectively reduce the "viscosity increase" of the hyaluronic acid composition caused by the local anesthetic upon sterilization of the composition by autoclave. Thus, the addition of a relatively small amount of one of these ascorbic acid derivatives to a hyaluronic acid composition comprising a local anesthetic may facilitate the use of finer needles for injection without increasing the force required to expel the composition and without making changes to the hyaluronic acid component. Also, the reduction of the viscosity and/or elastic modulus G of the solution is advantageous in applications where the composition is injected close to the surface of the skin, for example in skin revitalization or soft tissue augmentation, for example filling of wrinkles or contouring of the face or body, by hyaluronic acid gel injection. [0009] The effect of the ascorbic acid derivative on the viscosity and/or elastic modulus G on the composition has been shown for both unmodified hyaluronic acids and modified, for example crosslinked, hyaluronic acids, which indicates that it is common to all compositions comprising hyaluronic acid. [00] Besides the advantageous effect on the viscosity and/or elastic modulus G on the composition, the addition of one of these ascorbic acid derivatives to the composition may also provide further benefits. Ascorbic acid (also known as vitamin C) and its derivatives can act as reducing agents and scavenge aggressive oxidizing agents and radicals. 2
As ascorbic acid and its derivatives can improve the collagen formation, they may enhance skin morphology. They may also improve epidermal barrier formation, reduce transepidermal water loss, improve wound healing, and thus play an important role in prevention of skin aging and associated dry skin conditions. Ascorbic acid and its derivatives are known for their anti-inflammatory and photoprotective properties as well as their action on the improvement of UV-induced skin damage. It has also been shown that ascorbic acid and its derivatives can clinically improve dermatologic conditions that have inflammation as a component of the disease process, such as psoriasis and asteototic eczema. As ascorbic acid and its derivatives can suppress the formation of melanin, they may also have whitening effect of the skin, and they have been demonstrated to clinically improve melasma and senile freckles. They may also promote hair growth. Ascorbic acid and its derivatives have also been suggested to have anti-cancer properties. [0011] Addition of an ascorbic acid derivative to the hyaluronic acid composition generally has no effect, or little effect, on the stability of the composition. Notably, it has been observed that the addition of an ascorbic acid derivative does not increase the stability of the hyaluronic acid composition. Studies by the inventors have shown that the addition of one of these ascorbic acid derivatives may sometimes result in a slight decrease in stability of the hyaluronic acid composition. However, the inventors have found that the advantages associated with adding the ascorbic acid derivative outweigh the slight decrease in stability caused by the addition in some cases. In order to avoid unnecessary decrease in stability of the hyaluronic acid composition the concentration of the ascorbic acid derivative should be kept below the maximum concentrations as set out below. [0012] According to aspects illustrated herein, there is provided an injectable hyaluronic acid composition comprising: 2 3 4 0 a hyaluronic acid, a local anesthetic selected from the group consisting of amide and ester type local anesthetics or a combination thereof, and an ascorbic acid derivative selected from the group consisting of ascorbyl phosphates, ascorbyl sulfates and ascorbyl glycosides [0013] in an amount which prevents or reduces the effect on the viscosity and/or elastic modulus G of the composition caused by the local anesthetic upon sterilization by heat. [0014] The term "injectable" means that the hyaluronic acid composition is provided in a form which is suitable for parenteral injection, e.g. into soft tissue, such as skin, of a subject or patient. An injectable composition should be sterile and free from components that may cause adverse reactions when introduced into soft tissue, such as the skin, of a subject or patient. This implies that no, or only very mild, immune response occurs in the treated individual. That is, no or only very mild undesirable local or systemic effects occur in the treated individual. [00] The viscosity and/or elastic modulus G of the hyaluronic acid composition may be measured according to various methods, well known to the person skilled in the art. Viscosity may for example be measured as the "Zero shear viscosity, η 0 " by rotational viscometry using a Bohlin VOR rheometer (Measuring system C14 or PP, Gap 1.00 mm). Other methods of measuring viscosity may also be applicable. The elastic modulus G may for example be measured using a Bohlin VOR Reometer (Measure system PP, Gap 1.00 mm) by performing a strain sweep to find the linear viscoelastic region (LVR) and then measuring the viscoelastic properties within the LVR. Other methods of measuring elastic modulus G may also be applicable. [0016] The injectable hyaluronic acid composition is preferably aqueous and the hyaluronic acid, the local anesthetic and the ascorbic acid derivative are preferably swelled, dissolved or dispersed in the aqueous phase. [0017] The injectable hyaluronic acid composition comprises a hyaluronic acid. The hyaluronic acid may be a modified, e.g. branched or crosslinked, hyaluronic acid. According to certain embodiments the hyaluronic acid is a crosslinked hyaluronic acid. According to specific embodiments the hyaluronic acid is a hyaluronic acid gel. [0018] 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 crosslinking. 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 -CH 2 OH groups to -CHO and/or -COOH; periodate oxidation of vicinal hydroxyl groups, optionally followed by reduction, e.g. reduction of-cho to -CH 2 OH 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; crosslinking; substitutions with various compounds, e.g. using a crosslinking 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. [0019] The hyaluronic acid can be obtained from various sources of animal and non-animal origin. Sources of nonanimal origin include yeast and preferably bacteria. The molecular weight of a single hyaluronic acid molecule is typically in the range of 0.1- MDa, but other molecular weights are possible. 3
2 3 4 0 [00] In certain embodiments the concentration of said hyaluronic acid is in the range of 1 to 0 mg/ml. In some embodiments the concentration of said hyaluronic acid is in the range of 2 to 0 mg/ml. In specific embodiments the concentration of said hyaluronic acid is in the range of to mg/ml or in the range of to mg/ml. In certain embodiments, the hyaluronic acid is crosslinked. Crosslinked hyaluronic acid comprises crosslinks between the hyaluronic acid chains, which creates a continuous network of hyaluronic acid molecules which is held together by the covalent crosslinks, physical entangling of the hyaluronic acid chains and various interactions, such as electrostatic interactions, hydrogen bonding and van der Waals forces. [0021] Crosslinking of the hyaluronic acid may be achieved by modification with a chemical crosslinking agent. The chemical crosslinking agent may for example selected from the group consisting of divinyl sulfone, multiepoxides and diepoxides. According to embodiments the chemical crosslinking 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 crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE). [0022] The crosslinked hyaluronic acid product is preferably biocompatible. This implies that no, or only very mild, immune response occurs in the treated individual. That is, no or only very mild undesirable local or systemic effects occur in the treated individual. [0023] The crosslinked 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 crosslinked system of hyaluronic acid molecules when subjected to a liquid, typically an aqueous liquid. [0024] 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 crosslinked 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. [002] As mentioned, crosslinking of hyaluronic acid to form the crosslinked hyaluronic acid gel may for example be achieved by modification with a chemical crosslinking agent, for example BDDE (1,4-butandiol diglycidylether). The hyaluronic acid concentration and the extent of crosslinking affects the mechanical properties, e.g. the elastic modulus G, and stability properties of the gel. Crosslinked hyaluronic acid gels are often characterized in terms of "degree of modification". The degree of modification of hyaluronic acid gels generally range between 0.1 and mole%. It has been found that the effect of the ascorbic acid derivative on the viscosity and/or elastic modulus G on the composition in accordance with the present invention is particularly pronounced in crosslinked hyaluronic acid gels with a low degree of modification. The most pronounced effect is obtained in hyaluronic acid gels with a degree of modification of 2 mole% or less, such as 1. mole% or less, such as 1.2 mole% or less, for example in the range of 0.1 to 2 mole%, such as in the range of 0.2 to 1. mole%, such as in the range of 0.3 to 1.2 mole%, as compared to more crosslinked hyaluronic acid gels. The degree of modification (mole%) describes the amount of crosslinking agent(s) that is bound to HA, i.e. molar amount of bound crosslinking 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 crosslinking agent. Reaction conditions for crosslinking 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 0.1-2% and verify the resulting product characteristics with respect to the degree of modification. A BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel may for example be prepared according to the method described in Examples 1 and 2 of published international patent application WO 97012. [0026] In a preferred embodiment the hyaluronic acid of the composition is present in the form of a crosslinked hyaluronic acid gel crosslinked by a chemical crosslinking agent, wherein the concentration of said hyaluronic acid is in the range of to mg/ml and the degree of modification with said chemical crosslinking agent is in the range of 0.1 to 2 mole%. [0027] Hyaluronic acid gels may also comprise a portion of hyaluronic acid which is not crosslinked, i.e not bound to the three-dimensional crosslinked hyaluronic acid network. However, it is preferred that at least 0 % by weight, preferably at least 60 % by weight, more preferably at least 70 % by weight, and most preferably at least 80 % by weight, of the hyaluronic acid in a gel composition form part of the crosslinked hyaluronic acid network. [0028] The injectable hyaluronic acid composition further comprises a local anesthetic selected from the group consisting of amide and ester type local anesthetics or a combination thereof. A local anesthetic is a drug that causes reversible local anesthesia and a loss of nociception. When it is used on specific nerve pathways (nerve block), effects such as analgesia (loss of pain sensation) and paralysis (loss of muscle power) can be achieved. The local anesthetic may be added to the hyaluronic acid composition to reduce pain or discomfort experienced by the patient due to the injection procedure. The groups of amide (also commonly referred to as aminoamide) type local anesthetics and ester (also commonly referred to as aminoester) type local anesthetics are well defined and recognized in the art. [0029] Amide and ester type local anesthetic molecules are built on a simple chemical plan, consisting of an aromatic part linked by an amide or ester bond to a basic side-chain. The only exception is benzocaine which has no basic group. All other anesthetics are weak bases, with pka values mainly in the range 8-9, so that they are mainly but not completely, 4
2 ionized at physiological ph. As a result of their similarity they may be expected to have similar chemical and physical effects on the hyaluronic acid composition. [00] According to certain embodiments the local anesthetic is selected from the group consisting of amide and ester type local anesthetics, for example bupivacaine, butanilicaine, carticaine, cinchocaine (dibucaine), clibucaine, ethyl parapiperidinoacetylaminobenzoate, etidocaine, lignocaine (lidocaine), mepivacaine, oxethazaine, prilocaine, ropivacaine, tolycaine, trimecaine, vadocaine, articaine, levobupivacaine, amylocaine, cocaine, propanocaine, clormecaine, cyclomethycaine, proxymetacaine, amethocaine (tetracaine), benzocaine, butacaine, butoxycaine, butyl aminobenzoate, chloroprocaine, dimethocaine (larocaine), oxybuprocaine, piperocaine, parethoxycaine, procaine (novocaine), propoxycaine, tricaine or a combination thereof. [0031] According to certain embodiments the local anesthetic is selected from the group consisting amide type local anesthetics, for example bupivacaine, butanilicaine, carticaine, cinchocaine (dibucaine), clibucaine, ethyl parapiperidinoacetylaminobenzoate, etidocaine, lignocaine (lidocaine), mepivacaine, oxethazaine, prilocaine, ropivacaine, tolycaine, trimecaine, vadocaine, articaine, levobupivacaine or a combination thereof. According to some embodiments the local anesthetic is selected from the group consisting of bupivacaine, lidocaine, and ropivacaine, or a combination thereof. According to specific embodiments the local anesthetic is lidocaine. Lidocaine is a well-known substance, which has been used extensively as a local anesthetic in injectable formulations, such as hyaluronic acid compositions. [0032] The concentration of the amide or ester local anesthetic may be selected by the skilled person within the therapeutically relevant concentration ranges of each specific local anesthetic or a combination thereof. [0033] In certain embodiments the concentration of said local anesthetic is in the range of 0.1 to mg/ml. In some embodiments the concentration of said local anesthetic is in the range of 0. to mg/ml. [0034] When lidocaine is used as the local anesthetic, the lidocaine may preferably be present in a concentration in the range of 1 to mg/ml, more preferably in the range of 2 to 4 mg/ml, such as in a concentration of about 3 mg/ml. [003] The injectable hyaluronic acid composition further comprises an ascorbic acid derivative selected from the group consisting of ascorbyl phosphates, ascorbyl sulfates and ascorbyl glycosides. 3 [0036] The ascorbic acid derivative may be water soluble. The solubility of the ascorbic acid derivative in water under atmospheric conditions may preferably be sufficient to allow dissolution of a desired concentration of the ascorbic acid derivative in the composition. The solubility of the water soluble ascorbic acid derivative in water under atmospheric conditions may preferably be sufficient to allow a concentration of 0.001 mg/ml or more, and more preferably 0.01 mg/ml or more, in the hyaluronic acid composition. [0037] The ascorbic acid derivative may be capable of forming ascorbic acid or ascorbate in vivo, for example through enzymatic degradation mediated by phosphatases, glucosidases, etc. Thus, according to an embodiment, the ascorbic acid derivative is capable of forming ascorbic acid or ascorbate when placed in in vivo conditions. [0038] The ascorbic acid derivative is a compound having the general formula: 4 0 wherein R1, R2, R3, R4 are, independent of each other, H or an organic substituent and wherein at least one of R1, R2, R3 and R4 is where X is H, alkyl, alkenyl, alkynyl, aryl, an amine, an alcohol, a glycoside, or
where n can be 0 to 00. Counter ions can be, but are not limited to, Na +, K +, Ca 2+, Al 3+, Li +, Zn 2+ or Mg 2+. or wherein at least one of R1, R2, R3 and R4 is 2 Counter ions can be, but are not limited to, Na +, K +, Ca 2+, Al 3+, Li +, Zn 2+ or Mg 2+. 3 4 or wherein at least one of R1, R2, R3 and R4 is 0 carbohydrate residue, for example: 6
2 3 4 0 [0039] In certain embodiments the ascorbic acid derivative is selected from the group consisting of ascorbyl phosphates and ascorbyl glycosides, or a combination thereof. [00] In some embodiments the ascorbyl phosphate is selected from the group consisting of sodium ascorbyl phosphate (SAP) and magnesium ascorbyl phosphate (MAP), or a combination thereof. Ascorbyl phosphates convert to vitamin C in vivo by enzymatic hydrolysis by phosphatases. [0041] In some embodiments, the ascorbic acid derivative is an aminoalkyl ascorbyl phosphate. In certain embodiments, the ascorbic acid derivative is aminopropyl ascorbyl phosphate. [0042] In some embodiments, the ascorbic acid derivative is ascorbyl glucoside. Ascorbyl glucoside converts to vitamin C in vivo by enzymatic hydrolysis by glucosidases. [0043] The ascorbic acid derivatives described herein may be in unprotonated or fully or partially protonated form, or in the form a pharmaceutically acceptable salt. Specifically, the terms ascorbyl phosphate, ascorbyl sulfate, aminoalkyl ascorbyl phosphate, aminopropyl ascorbyl phosphate, ascorbyl glycoside and ascorbyl glucoside, as used herein, are intended to encompass the compounds in unprotonated or fully or partially protonated form, or in the form a pharmaceutically acceptable salt. Examples of suitable counter ions include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. [0044] The concentration of the ascorbic acid derivative may be selected by the skilled person depending on the specific ascorbic acid derivative used. [004] In certain embodiments the concentration of said ascorbic acid derivative is in the range of 0.001 to mg/ml. In certain embodiments the concentration of said ascorbic acid derivative is in the range of 0.001 to mg/ml. In some embodiments the concentration of said ascorbic acid derivative is in the range of 0.01 to mg/ml. A concentration of said ascorbic acid derivative of above 0.01 mg/ml is preferred since it provides a more marked reduction in viscosity and/or elastic modulus G of the hyaluronic acid composition. A concentration of said ascorbic acid derivative of below mg/ml is preferred since higher concentrations may result in unnecessary decrease of stability of the hyaluronic acid composition without additional benefits. [0046] The required concentration of the ascorbic acid derivative may vary within the above specified ranges depending on the particular ascorbic acid derivative used. As an example, a suitable concentration of sodium ascorbyl phosphate (SAP) or magnesium ascorbyl phosphate (MAP) may be in the range of 0.01 to 1 mg/ml, while a suitable concentration of ascorbyl glucoside may be in the range of 0.1 to mg/ml. [0047] Thus, according to an embodiment, the ascorbic acid derivative is sodium ascorbyl phosphate (SAP) or magnesium ascorbyl phosphate (MAP) in a concentration in the range of 0.01 to 1 mg/ml and preferably in the range of 0.01 to 0. mg/ml. [0048] According to another embodiment, the ascorbic acid derivative is ascorbyl glucoside in a concentration in the range of 0.01 to 1 mg/ml, preferably in the range of 0.01 to 0.8 mg/ml, and more preferably in the range of 0.0 to 0.4 mg/ml. [0049] As mentioned, it has been observed the addition of an ascorbic acid derivative does not increase the stability of the hyaluronic acid composition. In other words, the injectable hyaluronic acid composition according to the present invention does not exhibit increased stability compared to the same composition without an ascorbic acid derivative. [000] The term stability, as used herein, is used to denote the ability of the hyaluronic acid composition to resist degradation during storage and handling prior to use. It is known that the addition of constituents to a hyaluronic acid or hyaluronic acid gel may affect the stability of said hyaluronic acid or hyaluronic acid gel. Stability of hyaluronic acid or hyaluronic acid gel composition can be determined by a range of different methods. Methods for determining stability include, but are not limited to, assessing homogeneity, color, clarity, ph, gel content and rheological properties of the composition. Stability of a hyaluronic acid composition is often determined by observing or measuring one or more of said parameters over time. Stability may for example be determined by measuring the viscosity and/or elastic modulus 7
2 3 4 0 G of the hyaluronic acid composition over time. Viscosity may for example be measured as the "Zero shear viscosity, η 0 " by rotational viscometry using a Bohlin VOR rheometer (Measuring system C14 or PP, Gap 1.00 mm). Other methods of measuring viscosity may also be applicable. The elastic modulus G may for example be measured using a Bohlin VOR Reometer (Measure system PP, Gap 1.00 mm) by performing a strain sweep to find the linear viscoelastic region (LVR) and then measuring the viscoelastic properties within the LVR. Other methods of measuring elastic modulus G may also be applicable. [001] There is thus provided an injectable hyaluronic acid composition, comprising: an aqueous hyaluronic acid gel comprising 2 to 0 mg/ml of a hyaluronic acid; 0. to mg/ml of lidocaine; and 0.01 to mg/ml of an ascorbic acid derivative selected from the group consisting of ascorbyl phosphates and ascorbyl glycosides, or a combination thereof. [002] In a more specific embodiment, there is provided an injectable hyaluronic acid composition, comprising: an aqueous hyaluronic acid gel comprising 2 to 0 mg/ml of a hyaluronic acid; 0. to mg/ml of lidocaine; and 0.01 to mg/ml of an ascorbyl phosphate, for example sodium or magnesium ascorbyl phosphate. [003] In another more specific embodiment, there is provided an injectable hyaluronic acid composition, comprising: an aqueous hyaluronic acid gel comprising 2 to 0 mg/ml of a hyaluronic acid; 0. to mg/ml of lidocaine; and 0.01 to mg/ml of an ascorbyl glycoside, for example ascorbyl glucoside. [004] In some embodiments, the composition has been subjected to sterilization. In certain embodiments is the composition sterilized, i.e. the composition has been subjected to heat and/or steam treatment in order to sterilize the composition. In some embodiments 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 F 0 -value 4. The F 0 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 posessing a Z-value of. [00] When hyaluronic acid compositions are subjected to sterilization by treatment with heat or steam, the viscosity and/or elastic modulus G are generally reduced. When an amide or ester type local anesthetic is added to the hyaluronic acid composition, this reduction in viscosity and/or elastic modulus G is decreased, resulting in a firmer or more viscous final product. The addition of the ascorbic acid derivative counteracts this effect of the local anesthetic, thereby producing a final product, having a viscosity and/or elastic modulus G more closely resembling those of the hyaluronic acid composition without the local anesthetic, without making changes to the hyaluronic acid component. [006] The crosslinked hyaluronic acid product according to the invention, 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 crosslinked hyaluronic acid composition and autoclaved. [007] The injectable hyaluronic acid compositions described herein may be employed in medical as well as nonmedical, e.g. purely cosmetic, procedures by injection of the composition into soft tissues of a patient or subject. The compositions have been found useful in, e.g., soft tissue augmentation, for example filling of wrinkles, by hyaluronic acid gel injection. The compositions have been found especially useful in a cosmetic treatment, referred to herein as skin revitalization, whereby small quantities of the hyaluronic acid 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 other aspects illustrated herein, there is provided an injectable hyaluronic acid composition as described above for use as a medicament. The composition is useful, for example in the treatment of various dermatological conditions. Particularly, there is provided an injectable hyaluronic acid composition as described above for use in a dermatological treatment selected from the group consisting of wound healing, treatment of dry skin conditions or sun-damaged skin, treatment of hyper pigmentation disorders, treatment and prevention of hair loss, and treatment of conditions that have inflammation as a component of the disease process, such as psoriasis and asteototic eczema. In other words, there is provided an injectable hyaluronic acid composition as described above for use in the manufacture of a medicament for use in a dermatological treatment selected from the group consisting of wound healing, treatment of dry skin conditions or sundamaged skin, treatment of hyper pigmentation disorders, treatment and prevention of hair loss, and treatment of conditions that have inflammation as a component of the disease process, such as psoriasis and asteototic eczema. [008] According to another embodiment there is provided an injectable hyaluronic acid composition as described above for use in the treatment of a joint disorder by intraarticular injection. [009] According to other aspects illustrated herein, there is provided the use of an injectable hyaluronic acid composition as described above for cosmetic, non-medical, treatment of a subject by 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, preventing and/or treating hair loss, 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, skin revitalization, skin whitening and treatment of hyper pigmentation disorders such as senile freckles, melasma and ephelides. [0060] According to certain embodiments, there is provided the cosmetic, non-medical use of an injectable hyaluronic acid composition as described above for improving the appearance of skin, preventing and/or treating hair loss, filling 8
wrinkles or contouring the face or body of a subject. The use preferably comprises injecting the composition into the cutis and/or subcutis of a human subject. The use of the injectable hyaluronic acid composition for improving the appearance of skin, preventing and/or treating hair loss, filling wrinkles or contouring the face or body of a subject, is nonmedical, i.e. purely cosmetic. [0061] According to certain embodiments, there is provided the cosmetic, non-medical use of an injectable hyaluronic acid composition as described above for improving the appearance of skin. According to a preferred embodiment, there is provided the use of an injectable hyaluronic acid composition as described above for skin revitalization. [0062] According to certain embodiments, there is provided the cosmetic, non-medical use of an injectable hyaluronic acid composition as described above for preventing and/or treating hair loss. [0063] According to certain embodiments, there is provided the cosmetic, non-medical of an injectable hyaluronic acid composition as described above for filling wrinkles or contouring the face or body of a subject. [0064] According to other aspects illustrated herein, there is provided a cosmetic, non-medical of improving the appearance of skin, preventing and/or treating hair loss, filling wrinkles or contouring the face or body of a subject, comprising: a) providing an injectable hyaluronic acid composition as described above, and b) injecting said injectable hyaluronic acid composition into the skin of a subject. 2 [006] In certain embodiments the injectable hyaluronic acid composition is injected into the cutis and/or subcutis. [0066] According to certain embodiments, the cosmetic, non-medical method comprises improving the appearance of skin. According to a preferred embodiment, the method comprises skin revitalization. [0067] According to certain embodiments, the cosmetic, non-medical method comprises preventing and/or treating hair loss. [0068] According to certain embodiments, the cosmetic, non-medical method comprises filling wrinkles or contouring the face or body of a subject. [0069] According to other aspects illustrated herein, there is provided a method of manufacturing a hyaluronic acid composition comprising: a) mixing a hyaluronic acid, a local anesthetic selected from the group consisting of amide and ester type local anesthetics or a combination thereof, and an ascorbic acid derivative in an amount which prevents or reduces the effect on the viscosity and/or elastic modulus G of the composition caused by the local anesthetic upon sterilization by heat, and b) subjecting the mixture to sterilization by heat. 3 4 0 [0070] In the method of manufacturing the composition, said ascorbic acid derivative is operative for preventing or reducing the effect of the local anesthetic on the viscosity and/or elastic modulus G of the composition due to the sterilization by heat. [0071] The components of the composition, i.e. hyaluronic acid, local anesthetic and ascorbic acid derivative, may be further defined as described above for the injectable hyaluronic acid composition. [0072] The manufactured hyaluronic acid composition does not exhibit increased stability compared to the same composition without an ascorbic acid derivative. [0073] In some embodiments, the sterilization of step b) comprises subjecting the mixture to a heat treatment. In certain embodiments, the sterilization of step b) comprises autoclaving the mixture at a F 0 -value 4. The sterilization may be further characterized as described above for the composition. [0074] According to other aspects illustrated herein, there is provided the use of an ascorbic acid derivative in an injectable hyaluronic acid composition, further comprising a hyaluronic acid and a local anesthetic selected from the group consisting of amide and ester type local anesthetics or a combination thereof, for preventing or reducing the effect of the local anesthetic on the viscosity and/or elastic modulus G of the composition due to the sterilization by heat. [007] The components of the composition may be further defined as described above for the injectable hyaluronic acid composition. The sterilization may be further characterized as described above. [0076] The injectable hyaluronic acid composition formed by use of an ascorbic acid derivative does not exhibit increased stability compared to the same composition without an ascorbic acid derivative. Brief description of the drawings [0077] Figure 1 is a graph showing the effect of MAP (Magnesium Ascorbyl Phosphate) on a hyaluronic acid gel with lidocaine. 9
Figure 2 is a graph showing the effect of MAP on a hyaluronic acid gel with lidocaine. Figure 3 is a graph showing the effect of MAP on a non-crosslinked hyaluronic acid with lidocaine. Figure 4 is a graph showing the effect of MAP on a non-crosslinked hyaluronic acid with lidocaine. Figure is a graph showing the effect of MAP on a hyaluronic acid gel with lidocaine autoclaved at various F 0 values. Figure 6 is a graph showing the effect of MAP on a hyaluronic acid gel with bupivacaine. Figure 7 is a graph showing the effect of MAP on a hyaluronic acid gel with tetracaine. Figure 8 is a graph showing the effect of SAP (Sodium Ascorbyl Phosphate) on a hyaluronic acid gel with lidocaine. Figure 9 is a graph showing the effect of Methylsilanol ascorbate on a hyaluronic acid gel with lidocaine. Figure is a graph showing the effect of Ascorbyl glucoside on a non-crosslinked hyaluronic acid with bupivacaine. Figure 11 is a graph showing the effect of different concentrations of SAP on a hyaluronic acid gel with lidocaine. Figure 12 is a graph showing the effect of L-Ascorbic acid acetonide on a hyaluronic acid gel with tetracaine. Figure 13 is a graph showing the effect of SAP on a hyaluronic acid gel with lidocaine. 2 Figure 14 is a graph showing the effect of Aminopropyl Ascorbyl Phosphate on a non-crosslinked hyaluronic acid with lidocaine. Figure is a graph showing the effect of Ascorbyl glucoside on a hyaluronic acid gel with lidocaine. Figure 16 is a graph showing the effect of Ascorbyl glucoside on a hyaluronic acid gel with lidocaine. Figure 17 is a graph showing the effect of Ascorbyl glucoside on a hyaluronic acid gel with lidocaine. 3 Figure 18 is a graph showing the effect of MAP on a hyaluronic acid gels with lidocaine. Figure 19 is a graph showing the effect of SAP on a hyaluronic acid gel with lidocaine in a stability study. Figure is a graph showing the effect of Ascorbyl glucoside on a hyaluronic acid gel with lidocaine in a stability study. Figure 21 is a graph showing the effect of MAP or Ascorbyl glucoside on a hyaluronic acid gel with lidocaine in a stability study. Figure 22 is a graph showing the effect of Ascorbyl glucoside on a hyaluronic acid gel with lidocaine in a stability study. 4 Examples 0 [0078] Without desiring to be limited thereto, the present invention will in the following be illustrated by way of examples. Since hyaluronic acid polymer and hyaluronic acid gel may always be subject to some batch to batch variations, each example has been performed with a single batch of hyaluronic acid polymer or hyaluronic acid gel in order to obtain comparable results. Slight variations in, e.g., rheological properties or viscosity between similar compositions in different examples may be due to such batch to batch variations. Example 1. Hyaluronic acid gel with lidocaine and MAP [0079] In this experiment, the rheological properties after autoclaving of hyaluronic acid gels without additives were compared to hyaluronic acid gels with added lidocaine and hyaluronic acid gels with added lidocaine and MAP respectively. [0080] Formulations having various concentrations lidocaine and MAP as outlined in Table 1 were prepared as de-
scribed below. Table 1. Formulation # HA Gel [mg/ml] Lidocaine [mg/ml] MAP [mg/ml] G at 1.0 Hz [Pa] 1a 0 0 239 1b 3 0 437 1c 3 0.07 394 1d 3 0.7 211 1e 1 0 4 1f 1 0.07 388 1g 1 0.7 6 2 [0081] In all formulations a BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel with a degree of modification of 1 mole% and a hyaluronic acid content of mg/ml was used. The degree of modification (mole%) describes the amount of crosslinking agent(s) that is bound to HA, i.e. molar amount of bound crosslinking 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 crosslinking agent. [0082] The BDDE (1,4-butandiol diglycidylether) crosslinked hyaluronic acid gel may for example be prepared according to the method described in Examples 1 and 2 of published international patent application WO 97012. [0083] A stock-solution of lidocaine hydrochloride monohydrate (CAS number 68-0-0, Sigma Aldrich, St Louis, USA) was prepared by dissolving lidocaine hydrochloride monohydrate in WFI (water for injection) and a stock-solution of Magnesium Ascorbyl Phosphate (MAP, CAS number 11-31-2, Nikko Chemicals co, Japan), was prepared by dissolving MAP in phosphate buffered saline (8 mm, 0.9% NaCl). Formulation 1 a: [0084] The hyaluronic acid gel was diluted to the same degree as 1 b-1 g by adding phosphate buffered saline (8 mm, 0.9% NaCl). 3 Formulation 1 b: [008] Stock-solution of lidocaine was added to the hyaluronic acid gel to a final concentration of 3 mg/ml gel. Formulation 1 c: 4 0 [0086] Stock-solution of lidocaine and stock-solution of MAP were added to the hyaluronic acid gel to the final concentrations of 3 mg lidocaine/ml and 0.07 mg MAP/ml gel. [0087] Formulations 1 d-1 g were prepared in the same manner by varying the amounts of lidocaine stock-solution and MAP stock-solution. To all formulations phosphate buffered saline (8 mm, 0.9% NaCl) was added to adjust the dilution to the same degree. [0088] The ph values of the formulations were adjusted to 7. 6 0.2 and the formulations were filled in 1 ml glass syringes (BD Hypak SCF) and autoclaved in a Getinge GEV 66 ERC-1 autoclave (F 0 ). [0089] The rheological properties of the formulations were analysed using a Bohlin VOR Reometer (Measure system PP, Gap 1.00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the viscoelastic properties were measured within the LVR. [0090] The results are presented in Figure 1. MAP counteracts the effect on the elastic modulus G of the composition caused by the local anesthetic upon sterilization by heat. The higher the concentration of MAP the larger is the decrease on the elastic G modulus. A higher concentration of lidocaine does not affect the increase on the elastic modulus G. Example 2. Hyaluronic acid gel with a higher degree of modification with lidocaine and MAP [0091] Formulations as outlined in Table 2 were prepared essentially according to the method described in Example 1, with the exception that a hyaluronic acid gel with a higher degree of modification (approximately 7%) was used. The hyaluronic acid gel may for example be prepared according to the method described in the examples of US patent 11
6,921,819 B2. Table 2. Formulation # HA Gel [mg/ml] Lidocaine [mg/ml] MAP [mg/ml] G at 1.0 Hz [Pa] 2a 0 0 393 2b 3 0 417 2c 3 0.3 388 [0092] The ph values of the formulations were adjusted to 7. 6 0.2 and the formulations were filled in 1 ml glass syringes (BD Hypak SCF) and autoclaved in a Getinge GEV 66 ERC-1 autoclave (F 0 29). [0093] The rheological properties of the formulations were analysed using a Bohlin VOR Reometer (Measure system PP, Gap 1.00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the viscoelastic properties were measured within the LVR. [0094] The results are presented in Figure 2. MAP counteracts the effect on the elastic modulus G of the composition caused by the local anesthetic upon sterilization by heat. 2 Example 3. Non-crosslinked hyaluronic acid with lidocaine and MAP [009] Formulations as outlined in Table 3 were prepared essentially according to the method described in Example 1, with the exception that a non-crosslinked hyaluronic acid with an average molecular weight of 1 x 6 Da was used. Table 3. Formulation # HA [mg/ml] Lidocaine [mg/ml] MAP [mg/ml] Zero shear viscosity η 0 [Pas] 3a 0 0 3.83 3b 3 0 4.26 3c 3 0.07 2.4 3d 3 0.3 1.98 3 [0096] The ph values of the formulations were adjusted to 7. 6 0.2 and the formulations were filled in 1 ml glass syringes (BD Hypak SCF) and autoclaved in a Getinge GEV 66 ERC-1 autoclave (F 0 22). [0097] The viscosity of the formulations was studied using rotational viscometry using a Bohlin VOR rheometer (Measure system PP, Gap 1.00 mm). The results are presented in Figure 3. MAP counteracts the effect on the viscosity of the composition caused by the local anesthetic upon sterilization by heat. Example 4. Non-crosslinked hyaluronic acid with lidocaine and MAP at lower concentrations [0098] Formulations as outlined in Table 4 were prepared essentially according to the method described in Example 3, with the exception that lower concentrations of MAP were used. 4 0 Table 4. Formulation # HA [mg/ml] Lidocaine [mg/ml] MAP [mg/ml] Zero shear viscosity η 0 [Pas] 4a 0 0.13 4b 3 0 6.16 4c 3 0.03.27 4d 3 0.01.87 4e 3 0.00.91 [0099] The ph values of the formulations were adjusted to 7. 6 0.2 and the formulations were filled in 1 ml glass syringes (BD Hypak SCF) and autoclaved in a Getinge GEV 66 ERC-1 autoclave (F 0 22). The viscosity of the formulations was studied using rotational viscometry using a Bohling VOR rheometer (Measure 12
system PP, Gap 1.00 mm). [00] The results are presented in Figure 4. MAP counteracts the effect on the viscosity of the composition caused by the local anesthetic upon sterilization by heat. Example. Hyaluronic acid gel with lidocaine and MAP autoclaved at different F 0 -values [01] Formulations as outlined in Table were prepared essentially according to the method described in Example 1, with the exception that a different concentration of MAP was used. Table. Formulation # HA [mg/ml] Lidocaine [mg/ml] MAP [mg/ml] Average F 0 G at 1.0 Hz [Pa] a 0 0 22 194 b 3 0 22 269 c 3 0.3 22 2 d 0 0 6 317 e 3 0 6 363 f 3 0.3 6 332 2 [02] The ph values of the formulations were adjusted to 7. 6 0.2 and the formulations were filled in 1 ml glass syringes (BD Hypak SCF) and autoclaved in a Getinge GEV 66 ERC-1 autoclave at the different F 0 -values described in Table. [03] The rheological properties of the formulations were analysed using a Bohlin VOR Reometer (Measure system PP, Gap 1.00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the viscoelastic properties were measured within the LVR. [04] The results are presented in Figure. The effect on the elastic modulus G of the composition caused by the local anesthetic upon sterilization by heat is slightly larger for the higher F 0 -value. MAP counteracts the effect on the elastic modulus G of the composition caused by the local anesthetic upon sterilization by heat. Example 6. Hyaluronic acid gel with bupivacaine and MAP 3 [0] Formulations as outlined in Table 6 were prepared essentially according to the method described in Example 1, with the exceptions that lidocaine was replaced by bupivacaine (CAS-number 2180-92-9, Cambrex, Karlskoga, Sweden) and that a hyaluronic acid gel with a modification degree of < 1 %, with a hyaluronic acid content of 12 mg/ml was used. 4 0 Table 6. Formulation # HA Gel [mg/ml] Bupivacaine [mg/ml] MAP [mg/ml] G at 1.0 Hz [Pa] 6a 12 0 0 62 6b 12 1 0 90 6c 12 1 0.3 61 [06] The ph values of the formulations were adjusted to 7. 6 0.2 and the formulations were filled in 1 ml glass syringes (BD Hypak SCF) and autoclaved in a Getinge GEV 66 ERC-1 autoclave (F 0 22). [07] The rheological properties of the formulations were analysed using a Bohlin VOR Reometer (Measure system PP, Gap 1.00 mm). Initially a strain sweep was made to find the linear viscoelastic region (LVR) and then the viscoelastic properties were measured within the LVR. [08] The results are presented in Figure 6. Bupivacaine has similar effect on the elastic modulus G of the composition as lidocaine. MAP counteracts the effect on the elastic modulus G of the composition caused by the local anesthetic upon sterilization by heat. Example 7. Hyaluronic acid gel with tetracaine and MAP [09] Formulations as outlined in Table 7 were prepared essentially according to the method described in Example 13