KR20210125119A - Dermal filler compositions for fine line treatment - Google Patents

Abstract

The present invention provides a highly injectable, long-lasting, hyaluronic acid-based hydrogel dermal filler composition that is particularly advantageous for correcting facial fine lines.

Description

Dermal filler composition for the treatment of fine lines {DERMAL FILLER COMPOSITIONS FOR FINE LINE TREATMENT}

Related applications

This application claims the priority and benefit of U.S. Provisional Patent Application No. 61/534,780, filed September 14, 2011, which is a continuation-in-part of U.S. Provisional Patent Application No. 13/486,754, filed June 1, 2012, 2011 It is a continuation-in-part of U.S. Provisional Patent Application No. 13/593,313, filed on August 23, 2012, claiming the priority and benefit of U.S. Provisional Patent Application No. 61/493,309, filed on June 3, 2012, and the full text of each of these basic applications The disclosure of which is incorporated herein by this specific reference in its entirety.

Technical Field of the Invention

The present invention relates generally to a dermal filler composition, and more specifically to an injectable dermal filler composition effective for the treatment of fine lines in the skin.

Skin aging is a progressive phenomenon, occurs over time, and can be influenced by lifestyle factors such as alcohol consumption, tobacco and sun exposure. Aging of the facial skin can be characterized by atrophy, sagging and thickening. Atrophy corresponds to a massive decrease in skin tissue thickness. The sagging of the subcutaneous tissue causes excessive skin and eyelid sagging, resulting in sagging of the cheeks and eyelids. Thickening refers to excessive weight gain due to swelling of the lower face and neck. These changes are typically associated with dryness, loss of elasticity and rough texture.

Hyaluronic acid (HA), also known as hyaluronan, is a non-sulfated glycosaminoglycan that is widely distributed throughout the human body in joints, epithelium, and nervous tissue. Hyaluronic acid, for example, ensures good hydration, aids in the organization of the extracellular matrix, acts as a filler material; It is common in different layers of skin if it has a variety of actions, such as participating in tissue damage mechanisms. However, with age, the amount of hyaluronic acid, collagen, elastin and other matrix polymers present in the skin decreases. For example, repeated exposure to ultraviolet light, such as sunlight, causes dermal cells to decrease the production of hyaluronan as well as increase the rate of its degradation. This loss of material results in a variety of skin conditions such as, for example, wrinkles, hollowness, water loss and other unwanted conditions that contribute to the appearance of aging.

Injectable dermal fillers have been successfully used to treat aging skin. To treat these skin conditions, fillers can replace the lost endogenous matrix polymer, or enhance/promote the function of an existing matrix polymer. Hyaluronic acid-based dermal fillers have become increasingly popular because hyaluronic acid is a substance found naturally throughout the body. These fillers are generally well tolerated, non-permanent, and fairly low risk treatments for a wide variety of skin conditions.

The Tyndall effect is a side effect that occurs in some patients treated with hyaluronic acid (HA)-based dermal fillers. The Tyndall effect is characterized by the appearance of a bluish discoloration at the skin site where the dermal filler has been injected, indicating visible hyaluronic acid visible through the translucent epidermis. Clinical reports suggest that filler administration technique and skin characteristics may influence the manifestation of this side effect. Fillers with high stiffness and elasticity are successfully used in precise areas on the face, such as nasolabial folds, cheeks, and chin, without any fear of facial discoloration, as this material is injected into the middle and deep dermal regions. However, when these filler materials are used to repair, for example, lacrimal sulcus, glabellar lines, crow's feet, smile lines or non-deep wrinkle in the forehead, or are accidentally applied not too deep in the upper region of the dermis, A bluish discoloration of the skin is often observed. This phenomenon, thought to be a result of the Tyndall effect, leaves a semi-permanent discoloration at the site of application, which sometimes disappears only after administration of hyaluronidase to break down the filler material. Consequently, Tyndall effect is more common in patients being treated for non-deep fine lines. Long-term signs of Tyndall effect, where the gel lasts on the skin for as long as 7 months, typically, is a major cause of concern for patients.

The HA-based dermal filler gel is specially formulated to treat the "fine lines" lines found around the tear sulcus, forehead, eye lines, glabellar lines, and the like. Commercially available HA "fine lines" gels include Juvederm Refine (G' -67 Pa; G"/G' -0.59, HA concentration 18 mg/ml), Belotero Soft (G') ~28 Pa; G"/G' ~1.1, HA concentration 20 mg/ml), Emervel Touch (G' ~56 Pa; G"/G' ~0.64, HA concentration 20 mg/ml), Style Age S (G' ~192 Pa; G"/G' ~0.20, HA concentration 16mg/ml), Teosyal First Lines (G' 59 Pa; G"/G' ˜0.53, HA concentration 20 mg/ml), Restylane Touch (G′ ˜489 Pa; G″/G′ ˜0.24, HA concentration 18 mg/ml). Although these gels are formulated to have a low modulus of elasticity, for example by lightly crosslinking the linear HA chains with a small amount of crosslinking agent and/or reducing the final HA concentration of these gels, most commercially available "fine line" gels are: It still exhibits Tyndall effect in some patients when injected not particularly deep, eg at a depth of less than about 1 mm.

The collagen-based gel can be used for the treatment of non-deep wrinkles and was not shown to cause the Tyndall effect. Collagen-based gels are highly undesirable because they have a relatively poor duration on the skin and require prior testing in subjects. Radiesse® (calcium hydroxyapatite) is a subcutaneously injectable implant, the principal component of which is synthetic calcium hydroxyapatite, not hyaluronic acid. Unlike hyaluronic acid-based dermal fillers, calcium hydroxyapatite is not transparent, thus avoiding the problem of Tyndall effect. However, if placed not too deep, this filler can be seen as a white matter just under the skin. Furthermore, compared to hyaluronic acid-based fillers, Radiesse® requires a larger needle for injection and is typically not recommended for use in the eye area.

It is desirable to provide an injectable hyaluronic acid-based dermal filler that does not show bluish discoloration due to the Tyndall effect even when injected deeply.

US Patent Application Publication No. 2011/0171286 (2011.07.14)

US Patent Application Publication No. 2011/0171311 (2011.07.14)

Park, D. J. et al. 2010, Orthopedic Proceedings, Vol. 92-B, No. SUPP I

The present invention describes compositions and formulation methods for preparing HA-based dermal fillers that can be administered to the upper dermis without producing any bluish discoloration of the skin or at least a significant or notable bluish discoloration. Additionally, many of the currently described filler gels of the present invention have been found to last significantly longer in vivo than currently commercially available gels. In some aspects of the present invention, optically clear dermal fillers useful for enhancing the appearance of the skin are provided, which add volume and fullness, and reduce the appearance of constant fine lines and wrinkles without "tyndling". The composition can be introduced into fine lines in the skin, even in thin skin areas, rather not deeply, without causing the negative blue discoloration associated with many conventional optically clear dermal fillers.

More specifically, in one aspect of the present invention, a long lasting therapeutic dermal filler composition is provided, which generally comprises a biocompatible polymer such as a crosslinked hyaluronic acid component and an additive in combination with a hyaluronic acid component.

In one embodiment, the polymer is a polysaccharide, such as hyaluronic acid. Hyaluronic acid includes a crosslinking component, and may further include a non-crosslinking component. The additive may be a vitamin, e.g., vitamin C, e.g., a stabilized form of vitamin C or a vitamin C derivative, e.g., L-ascorbic acid 2-glucoside (AA2G), ascorbyl 3-aminopropyl phosphate ( Vitagen) or sodium ascorbyl phosphate (AA2P).

In one aspect of the invention, the additive is a vitamin derivative covalently conjugated to a polymer by a suitable reaction procedure, for example etherification, amidation or esterification.

In a broad aspect of the present invention, a dermal filler composition is provided, wherein the composition comprises a crosslinking component and a crosslinked hyaluronic acid component, and an additive other than the crosslinking component. The hyaluronic acid component may be chemically conjugated to the additive. Additionally, the composition exhibits reduced Tyndall effect when administered to a dermal region of a patient, relative to a composition that is substantially the same except without the additive. The composition may further comprise other additives, for example an anesthetic such as lidocaine. In one embodiment, the additive is a vitamin C derivative, such as AA2G. In another embodiment, the additive is Vitagen.

In one embodiment, the hyaluronic acid component is chemically conjugated to an additive having a degree of conjugation of from about 3 mol% to about 40 mol%, for example from about 3 mol% to about 10 mol%.

The composition may be substantially optically clear. The composition generally has a G' value from about 40 Pa to about 100 Pa, such as less than or equal to about 100 Pa and, for example, greater than or equal to about 40 Pa.

In another aspect of the invention, a method of treating fine lines in the skin of a patient is provided. In one embodiment, the method comprises introducing into the skin of a patient a composition comprising a hyaluronic acid component, a crosslinking component that crosslinks the hyaluronic acid, and an additive other than the crosslinking component, wherein the composition is substantially optically clear, the composition comprising: For substantially the same composition except without the additive, it exhibits reduced Tyndall effect when administered to the dermal region of a patient.

In another aspect of the present invention, a method for improving the appearance of the face is provided, the method comprising administering a substantially optically clear dermal filler that generally exhibits no or insignificant Tyndall effect to a dermal region of a patient. include The composition comprises the steps of providing hyaluronic acid, reacting a crosslinking agent with a vitamin C derivative, adding the reacted crosslinking agent and vitamin C derivative to hyaluronic acid to form a crosslinked hyaluronic acid composition comprising covalently conjugated vitamin C; and homogenizing and neutralizing the crosslinked hyaluronic acid composition to obtain an injectable dermal filler composition. In some embodiments, the vitamin C derivative is AA2G. In another embodiment, the vitamin C derivative is vitagen.

In another aspect of the present invention, there is provided a method of reducing the appearance of fine lines in a thin skin region of a patient, the method generally optically clear comprising a dermal filler composition, i.e., vitamin C or a vitamin C derivative. and administering the hyaluronic acid-based dermal filler composition to the patient at a depth of about 1 mm or less. In some embodiments, the composition is injected at a depth of about 0.8 mm or less, about 0.6 mm or less, or about 0.4 mm or less.

In another aspect of the present invention, there is provided a dermal filler composition that is substantially optically clear and generally includes a hyaluronic acid component crosslinked with a crosslinking component and a vitamin C derivative covalently conjugated with a hyaluronic acid component. In an exemplary embodiment, the composition has a G' value from about 40 Pa to about 100 Pa. Additionally, the composition may have a hyaluronic acid concentration of from about 18 mg/g to about 30 mg/g. These compositions may be particularly useful and effective in treating fine lines or moderate creases in the skin, for example even very thin skin, for example skin having a thickness of about 1 mm or less. In some embodiments, a composition of the present invention persists for at least 3 months, at least 6 months or up to 1 year after being introduced into the skin.

These and other aspects of the invention may be more readily understood and appreciated by reference to the following drawings and detailed description.

1 shows the structure of L-ascorbic acid 2-glucoside (AA2G);
Figure 2 shows the structure of ascorbyl 3-aminopropyl phosphate (Vitagen);
Figure 3 shows the structure of sodium ascorbyl phosphate (AA2P);
Figure 4 shows the structure of 1,4-butanediol diglycidyl ether (BDDE);
Figure 5 shows the structure of pentaerythritol glycidal ether (star-PEG epoxide);
Figure 6 shows the structure of pentaerythritol (3-aminopropyl) ether (star-PEG amine);
7 is a table showing the degree of conjugation and G' value for various dermal filler compositions according to the present invention;
8 is a table showing the degree of conjugation, HA concentration and G' value for the HA-AA2G (BDDE) dermal filler composition according to the present invention;
9 shows a graphical representation of the observed percent release of AsA from a solution of AA2G in PBS versus time (minutes) for four different α-glucosidase concentrations;
Figure 10 depicts a representation of the release profile (sustained release) of free AsA from a conjugated dermal filler according to the present invention (AA2G conversion in mol% versus reaction time);
11A and 11B show additional release data for various dermal fillers according to the present invention;
12 shows an image of the skin after sub-injection of the HA-based dermal filler gel of the present invention and some commercially available gels for fine line application;
Figure 13 shows the visual Tyndall score of the HA-based dermal filler gels of the present invention and certain commercially available gels for fine line applications;
Figure 14 shows the % of blue light emitted from the skin of the HA-based dermal filler gels of the present invention and some commercially available gels for fine line applications;
Figure 15 shows the overall % of gel remaining one week after implantation of an HA-based dermal filler gel of the present invention and some commercially available gels for fine line applications;
16 shows the overall % of gel remaining at weeks 0, 12, 24 and 40 of an implanted HA-based dermal filler gel of the present invention and some commercially available gels for fine line applications. drawing.

In one aspect of the present invention, a dermal filler composition is provided, wherein the composition generally comprises a biocompatible polymer, such as a polysaccharide, such as cross-linked hyaluronic acid, and a vitamin C derivative covalently conjugated to the polymer. The composition provides sustained release of vitamin C for skin neocollagenesis as well as other therapeutic or cosmetic benefits. When introduced into the skin, for example into the dermis, the composition reacts with endogenous enzymes in the body, and over time, bioactive vitamin C is made in vivo through enzymatic cleavage. As vitamin C is released from the composition over a period of weeks or months, its attendant benefits are made available to the body.

The polymer may be selected from the group of polymers consisting of proteins, peptides and polypeptides, polylysine, collagen, pro-collagen, elastin and laminin.

Polymers are synthetic polymers having hydroxyl, amine, and carboxyl functionality: poly(vinyl alcohol), polyethylene glycol, polyvinylyl amine, polyallylamine, deacetylated polyacrylamide, polyacrylic acid, and polymethacrylic acid may be selected from the group of polymers consisting of The polymer may be selected from the group of polymers consisting of dendritic or branched polymers including dendritic polyols and dendritic polyamines. The polymer may be selected from the group of polymers consisting of a solid surface bearing hydroxyl, amine and carboxyl functional groups.

Polymers include, for example, starch and derivatives thereof; dextran and its derivatives, cellulose and its derivatives; polysaccharides selected from the group of chitin and chitosan and alginates and derivatives thereof.

In an exemplary embodiment of the invention, the polymer is a glycosaminoglycan. The hydrogel compositions disclosed herein may further comprise two or more different glycosaminoglycan polymers. The term "glycosaminoglycan" as used herein is synonymous with "GAG" and "mucopolysaccharide" and refers to a long unbranched polysaccharide composed of repeating disaccharide units. The repeating unit consists of a hexose (hexose) or hexuronic acid and pharmaceutically acceptable salts thereof, linked to a hexosamine (a hexose containing nitrogen). Members of the GAG family differ in the type of hexosamine, hexose or hexuronic acid unit they contain, e.g. glucuronic acid, iduronic acid, galactose, galactosamine, glucosamine), and also the geometry of the glycosidic bond. may be different from Any glycosaminoglycan polymer is useful in the hydrogel compositions disclosed herein, provided that the glycosaminoglycan polymer improves a skin condition. Non-limiting examples of glycosaminoglycans include chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronan. Non-limiting examples of acceptable salts of glycosaminoglycans include sodium salts, potassium salts, magnesium salts, calcium salts, and combinations thereof. Glycosaminoglycans and their resulting polymers useful in the hydrogel compositions and methods disclosed herein are disclosed, for example, in US Patent Publication No. 2003/0148995 to Piron and Tholin, entitled Polysaccharide Crosslinking, Hydrogel Preparation, Resulting Polysaccharides(s) and Hydrogel(s), uses Thereof); Lebreton (Title of the Invention: Cross-Linking of Low and High Molecular Weight Polysaccharides Preparation of Injectable Monophase Hydrogels); US Patent Publication No. 2008/0089918 to Lebreton entitled Viscoelastic Solutions Containing Sodium Hyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses; US Patent Publication No. 2010/0028438 to Lebreton et al. entitled Hyaluronic Acid-Based Gels Including Lidocaine; and US Patent Publication No. 2006/0194758 entitled Polysaccharides and Hydrogels thus Obtained; and International Patent Publication No. WO 2004/073759 to Di Napoli, Composition and Method for Intradermal Soft Tissue Augmentation, each of which is incorporated herein by reference in its entirety. GAGs useful in the hydrogel compositions and methods disclosed herein include, for example, hyaluronan-based dermal fillers JUVEDERM®, JUVEDERM® 30, JUVEDERM®. (Trademark) Ultra, JUVEDERM(R) Ultra Plus, JUVEDERM(R) Ultra XC, and JUVEDERM(R) Ultra Plus XC (Al, Irvine, CA) commercially available, such as Allergan Inc. Table 1 lists representative GAGs.

Aspects of the present invention provide, in part, a hydrogel composition comprising a chondroitin sulfate polymer. As used herein, the term “chondroitin sulfate polymer” refers to a disaccharide of two alternative monosaccharides, D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc), and pharmaceutically acceptable salts thereof. Refers to an unbranched, sulfated polymer comprising a. The chondroitin sulfate polymer may comprise a D-glucuronic acid residue epimerized with L-iduronic acid (IdoA), in which case the resulting disaccharide is referred to as dermatan sulfate. A chondroitin sulfate polymer may have a chain of 100 or more individual sugars, each of which may be sulfated at variable positions and amounts. Chondroitin sulfate polymer is an important structural component of cartilage and provides its great resistance to compression. Any chondroitin sulfate polymer is useful in the compositions disclosed herein, provided that the chondroitin sulfate polymer improves a skin condition. Non-limiting examples of pharmaceutically acceptable salts of chondroitin sulfate include sodium chondroitin sulfate, potassium chondroitin sulfate, magnesium chondroitin sulfate, calcium chondroitin sulfate, and combinations thereof.

Aspects of the present specification provide, in part, a hydrogel composition comprising a keratan sulfate polymer. The term “keratan sulfate polymer” as used herein refers to a polymer of variable length comprising disaccharide units, which itself contains β-D-galactose and N-acetyl-D-galactosamine (GalNAc) and and pharmaceutically acceptable salts thereof. Within the repeat region of keratan sulfate, disaccharides can be fucosylated, and N-acetylneuraminic acid caps the ends of the chain. Any keratan sulfate polymer is useful in the compositions disclosed herein, provided that the keratan sulfate polymer improves a skin condition. Non-limiting examples of pharmaceutically acceptable salts of keratan sulfate include sodium keratan sulfate, potassium keratan sulfate, magnesium keratan sulfate, calcium keratan sulfate, and combinations thereof.

Aspects of the present specification provide, in part, a hydrogel composition comprising a hyaluronan polymer. As used herein, the term "hyaluronic acid polymer" is synonymous with "HA polymer", "hyaluronic acid polymer", and "hyaluronate polymer" is an anionic unsulfated glycosaminoglycan comprising disaccharide units. refers to a polymer, which itself includes D-glucuronic acid and DN-acetylglucosamine monomers and pharmaceutically acceptable salts thereof linked together via alternative β-1,4 and β-1,3 glycosidic bonds do. Hyaluronan polymers can be purified from animal and non-animal sources. Polymers of hyaluronan may range in size from about 5,000 Da to about 20,000,000 Da. Any hyaluronan polymer is useful in the compositions disclosed herein, provided that the hyaluronan polymer improves a skin condition. Non-limiting examples of pharmaceutically acceptable salts of hyaluronan include hyaluronan sodium, hyaluronan potassium, hyaluronan magnesium, hyaluronan calcium, and combinations thereof.

Aspects of the present specification provide a hydrogel composition comprising a partially crosslinked glycosaminoglycan polymer. The term “crosslinked” as used herein refers to intermolecular bonds in which individual polymer molecules or monomer chains are bound to a more stable structure, such as a gel. As such, the crosslinked glycosaminoglycan polymer has intermolecular bonds to which at least one individual polymer molecule is bound to each other. Crosslinking of the glycosaminoglycan polymer typically results in the formation of a hydrogel. These hydrogels have high viscosity and require considerable force to be extruded through a fine needle. The glycosaminoglycan polymers disclosed herein include polyfunctional PEG-based crosslinkers, dialdehydes and It can be crosslinked using a disulfide crosslinking agent. Non-limiting examples of hyaluronan crosslinking agents include polyfunctional PEG-based crosslinking agents like pentaerythritol tetraglycidyl ether (PETGE), divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE) , 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), (phenylenebis-(ethyl)-carbodiimide and 1, 6 Hexamethylenebis(ethylcarbodiimide), adipic dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxy) Propyl)-2,3-epoxycyclohexane, lysine, lysine methyl ester, or combinations thereof Other useful cross-linking agents include Stroumpoulis and Tezel, U.S. Patent Application Serial No. 12/910,466, filed on October 22, 2010; Title: Tunably Crosslinked Polysaccharide Compositions), which is incorporated by reference in its entirety.Any glycosaminoglycan polymer is useful in the hydrogel composition disclosed herein, provided that glycosa Minoglycan polymer improves skin condition.Non-limiting examples of glycosaminoglycan include chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronan.Acceptable of glycosaminoglycan Non-limiting examples of salt include sodium salt, potassium salt, magnesium salt, calcium salt and combinations thereof.Glycosaminoglycans and their resulting polymers useful in the hydrogel compositions and methods disclosed herein include, for example, For example, US Patent Publication Nos. 2003/0148995 to Piron and Tholin (Title: Polysaccharide Crosslinking, Hydrogel Preparation, Resulting Polysaccharides(s) and Hydrogel(s), uses Thereof); Lebreton (Title: Cross-Linking of Low) and High Molecular Weight Poly saccharides Preparation of Injectable Monophase Hydrogels); US Patent Publication No. 2008/0089918 to Lebreton entitled Viscoelastic Solutions Containing Sodium Hyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses; US Patent Publication No. 2010/0028438 to Lebreton et al. entitled Hyaluronic Acid-Based Gels Including Lidocaine; and US Patent Publication No. 2006/0194758 entitled Polysaccharides and Hydrogels thus Obtained; and International Patent Publication No. WO 2004/073759 to Di Napoli, Composition and Method for Intradermal Soft Tissue Augmentation, each of which is incorporated herein by reference in its entirety.

Aspects of the present specification provide, in part, a hydrogel composition comprising a glycosaminoglycan polymer having a degree of crosslinking. As used herein, the term "degree of crosslinking" refers to the percentage of glycosaminoglycan polymer monomer units, such as, for example, disaccharide monomer units of hyaluronan bound to a crosslinking agent. The degree of crosslinking is expressed as a weight percentage ratio of crosslinker to glycosaminoglycan. In certain advantageous embodiments of the invention the degree of crosslinking is from about 3% to about 12%, for example from about 5% to about 10%.

In an embodiment, the hydrogel composition comprises a crosslinked glycosaminoglycan polymer, e.g., crosslinked hyaluronic acid, wherein the crosslinked glycosaminoglycan polymer is, e.g., from about 18 mg/g to It is present in the composition at a concentration of about 30 mg/g. In some embodiments, the composition has a total hyaluronic acid concentration of about 24 mg/g or about 25 mg/g.

Aspects of the present specification provide, in part, a hydrogel composition comprising a low molecular weight hyaluronan polymer, a high molecular weight hyaluronan polymer, or a hyaluronan polymer of both low and high molecular weight. As used herein, the term “high molecular weight” when referring to “hyaluronan” refers to a hyaluronan polymer having an average molecular weight of 1,000,000 Da or more. Non-limiting examples of high molecular weight hyaluronan polymers include hyaluronan polymers of about 1,500,000 Da, about 2,000,000 Da, about 2,500,000 Da, about 3,000,000 Da, about 3,500,000 Da, about 4,000,000 Da, about 4,500,000 Da to about 5,000,000 Da. include As used herein, the term “low molecular weight” when referring to “hyaluronan” refers to a hyaluronan polymer having an average molecular weight of less than 1,000,000 Da. Non-limiting examples of low molecular weight hyaluronan polymers include hyaluronan polymers of about 200,000 Da, about 300,000 Da, about 400,000 Da, about 500,000 Da, about 600,000 Da, about 700,000 Da, about 800,000 Da, to about 900,000 Da. includes

In an embodiment, the composition comprises a low molecular weight crosslinked hyaluronan polymer. In aspects of this embodiment, the composition is, for example, about 100,000 Da, about 200,000 Da, about 300,000 Da, about 400,000 Da, about 500,000 Da, about 600,000 Da, about 700,000 Da, about 800,000 Da, or about 900,000 Da cross-linked hyaluronan polymer having an average molecular weight of In another aspect of this embodiment, the composition comprises, for example, at most 100,000 Da, at most 200,000 Da, at most 300,000 Da, at most 400,000 Da, at most 500,000 Da, at most 600,000 Da, at most 700,000 Da, at most 800,000 Da, at most 900,000 Da or a crosslinked hyaluronan polymer having an average molecular weight of up to 950,000 Da. In another aspect of this embodiment, the composition is, for example, from about 100,000 Da to about 500,000 Da, from about 200,000 Da to about 500,000 Da, from about 300,000 Da to about 500,000 Da, from about 400,000 Da to about 500,000 Da, about 500,000 Da Da to about 950,000 Da, about 600,000 Da to about 950,000 Da, about 700,000 Da to about 950,000 Da, about 800,000 Da to about 950,000 Da, about 300,000 Da to about 600,000 Da, about 300,000 Da to about 700,000 Da, about 300,000 Da to and a crosslinked hyaluronan polymer having an average molecular weight of about 800,000 Da, or about 400,000 Da to about 700,000 Da.

In another embodiment, the composition comprises a high molecular weight crosslinked hyaluronan polymer. In aspects of this embodiment, the composition comprises, for example, about 1,000,000 Da, about 1,500,000 Da, about 2,000,000 Da, about 2,500,000 Da, about 3,000,000 Da, about 3,500,000 Da, about 4,000,000 Da, about 4,500,000 Da, or about 5,000,000 Da a crosslinked hyaluronan polymer having an average molecular weight. In another aspect of this embodiment, the composition comprises, e.g., at least 1,000,000 Da, at least 1,500,000 Da, at least 2,000,000 Da, at least 2,500,000 Da, at least 3,000,000 Da, at least 3,500,000 Da, at least 4,000,000 Da, at least 4,500,000 Da or at least 5,000,000 Da a crosslinked hyaluronan polymer having an average molecular weight of Da. In another aspect of this embodiment, the composition is, for example, from about 1,000,000 Da to about 5,000,000 Da, from about 1,500,000 Da to about 5,000,000 Da, from about 2,000,000 Da to about 5,000,000 Da, from about 2,500,000 Da to about 5,000,000 Da, about 2,000,000 Da and a crosslinked hyaluronan polymer having an average molecular weight of from about 3,000,000 Da, about 2,500,000 Da to about 3,000,000 Da.

In another embodiment, the composition comprises a crosslinked hyaluronan polymer, wherein the crosslinked hyaluronan polymer comprises a combination of a high molecular weight hyaluronan polymer and a low molecular weight hyaluronan polymer at various ratios. do. In another embodiment, the composition comprises a crosslinked hyaluronan polymer, wherein the crosslinked hyaluronan polymer is about 20:1, about 15:1, about 10:1, about 5:1, about 1 a combination of a high molecular weight hyaluronan polymer and a low molecular weight hyaluronan polymer in a ratio of :1, about 1:5, about 1:10, about 1:15, or about 1:20.

Aspects of the present specification provide a hydrogel composition comprising a partially uncrosslinked glycosaminoglycan polymer. The term “uncrosslinked” as used herein refers to the lack of intermolecular bonds linking individual glycosaminoglycan polymer molecules or monomer chains. As such, the uncrosslinked glycosaminoglycan polymer is not linked to any other glycosaminoglycan polymer by intermolecular bonds. In aspects of this embodiment, the composition comprises an uncrosslinked chondroitin sulfate polymer, an uncrosslinked dermatan sulfate polymer, an uncrosslinked keratan sulfate polymer, an uncrosslinked heparan polymer, an uncrosslinked heparan sulfate polymer, or an uncrosslinked hyaluronan polymer. Uncrosslinked glycosaminoglycan polymers are water soluble and generally remain fluid in nature. As such, uncrosslinked glycosaminoglycan polymers are often mixed with glycosaminoglycan polymer-based hydrogel compositions as lubricants to facilitate extrusion of the composition through a fine needle.

In an embodiment, the composition comprises an uncrosslinked glycosaminoglycan polymer, wherein the uncrosslinked glycosaminoglycan polymer is, for example, about 2 mg/g, about 3 mg/g, about 4 mg/g, about 5 mg/g, about 6 mg/g, about 7 mg/g, about 8 mg/g, about 9 mg/g, about 10 mg/g, about 11 mg/g, about 12 mg/g g, about 13 mg/g, about 13.5 mg/g, about 14 mg/g, about 15 mg/g, about 16 mg/g, about 17 mg/g, about 18 mg/g, about 19 mg/g, at a concentration of about 20 mg/g, about 40 mg/g, or about 60 mg/g. In another aspect of this embodiment, the composition provides uncrosslinked glycosaminoglycan, wherein the uncrosslinked glycosaminoglycan comprises, e.g., at least 1 mg/g, at least 2 mg/g , at least 3 mg/g, at least 4 mg/g, at least 5 mg/g, at least 10 mg/g, at least 15 mg/g, at least 20 mg/g, at least 25 mg/g, at least 35 mg/g or at least 40 It is present at a concentration of mg/g. In another aspect of this embodiment, the composition comprises uncrosslinked glycosaminoglycan, wherein the uncrosslinked glycosaminoglycan is, for example, at most 1 mg/g, at most 2 mg/g. g, at most 3 mg/g, at most 4 mg/g, at most 5 mg/g, at most 10 mg/g, at most 15 mg/g, at most 20 mg/g, or at most 25 mg/g. In another aspect of this embodiment, the composition comprises uncrosslinked glycosaminoglycan, wherein the uncrosslinked glycosaminoglycan is, for example, from about 1 mg/g to about 60 mg/g. g, about 10 mg/g to about 40 mg/g, about 7.5 mg/g to about 19.5 mg/g, about 8.5 mg/g to about 18.5 mg/g, about 9.5 mg/g to about 17.5 mg/g, from about 10.5 mg/g to about 16.5 mg/g, from about 11.5 mg/g to about 15.5 mg/g, or from about 12.5 mg/g to about 14.5 mg/g.

Aspects of the present specification provide a hydrogel composition that is essentially free of partially crosslinked glycosaminoglycan polymers. The term “essentially free” (or “consisting essentially of”) as used herein refers to a composition in which only trace amounts of crosslinked matrix polymer can be detected. In aspects of this embodiment, the composition comprises chondroitin sulfate essentially free of cross-linked chondroitin sulfate polymer, dermatan sulfate essentially free of cross-linked dermatan sulfate polymer, keratan sulfate essentially free of cross-linked keratan sulfate polymer, cross-linked heparan essentially free of crosslinked heparan polymers, heparan sulfate essentially free of crosslinked heparan sulfate polymers, or hyaluronan sulfate essentially free of crosslinked hyaluronan polymers.

Aspects of the present specification provide a hydrogel composition that is completely free of partially crosslinked glycosaminoglycan polymers. As used herein, the term "completely free" refers to a composition within the detection range of the device or process used, wherein the crosslinked glycosaminoglycan polymer cannot be detected or its presence can be ascertained. . In aspects of this embodiment, the composition comprises chondroitin sulfate completely free of cross-linked chondroitin sulfate polymer, dermatan sulfate completely free of cross-linked dermatan sulfate polymer, keratan sulfate completely free of cross-linked keratan sulfate polymer, cross-linked heparan heparan completely free of polymer, heparan sulfate completely free of crosslinked heparan sulfate polymer, or hyaluronan sulfate completely free of crosslinked hyaluronan polymer.

Aspects of the present specification provide a hydrogel composition comprising a ratio of a partially crosslinked glycosaminoglycan polymer to an uncrosslinked glycosaminoglycan polymer. This ratio of crosslinked and uncrosslinked glycosaminoglycan polymers is known as the gel:fluid ratio. Any gel:fluid ratio is useful in the preparation of the compositions disclosed herein, provided that such ratios result in the compositions disclosed herein ameliorating the skin conditions disclosed herein. Non-limiting examples of gel:fluid ratios in compositions of the present invention include 100:0, 98:2, 90:10, 75:25, 70:30, 60:40, 50:50, 40:60, 30:70, 25:75, 10:90; 2:98, and 0:100.

In aspects of this embodiment, the composition comprises a crosslinked glycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymer, wherein the gel:fluid ratio is, for example, about 0:100, about 1:99, about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about 9:91, or about 10:90 . In another aspect of this embodiment, the composition comprises a crosslinked glycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymer, wherein the gel:fluid ratio is, for example, at most 1:99; Up to 2:98, up to 3:97, up to 4:96, up to 5:95, up to 6:94, up to 7:93, up to 8:92, up to 9:91, or up to 10:90. In aspects of this embodiment, the composition comprises a crosslinked glycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymer, wherein the gel:fluid ratio is, for example, from about 0:100 to about 3:97, about 0:100 to about 5:95, or about 0:100 to about 10:90.

In another aspect of this embodiment, the composition comprises a crosslinked glycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymer, wherein the gel:fluid ratio is, for example, about 15:85, about About 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, about 98:2, or about 100:0. In another aspect of this embodiment, the composition comprises a crosslinked glycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymer, wherein the gel:fluid ratio is, for example, at most 15:85; Up to 20:80, up to 25:75, up to 30:70, up to 35:65, up to 40:60, up to 45:55, up to 50:50, up to 55:45, up to 60:40, up to 65:35, Up to 70:30, up to 75:25, up to 80:20, up to 85:15, up to 90:10, up to 95:5, up to 98:2, or up to 100:0. In another aspect of this embodiment, the composition comprises a crosslinked glycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymer, wherein the gel:fluid ratio is, for example, from about 10:90 to about 70:30, about 15:85 to about 70:30, about 10:90 to about 55:45, about 80:20 to about 95:5, about 90:10 to about 100:0, about 75:25 to about 100:0, or from about 60:40 to about 100:0.

The hydrogel compositions disclosed herein may further comprise other agents or combinations of agents that provide a beneficial effect when the composition is administered to a subject. Such beneficial agents include antioxidants, anti-itch agents, anti-cellulite agents, anti-scarring agents, anti-inflammatory agents, anesthetics, anti-irritants, vasoconstrictors, vasodilators, anti-bleeding agents-like hemostatic or anti-fibrinolytic agents, exfoliants, swelling agents, anti-acne agents, pigmentation agents. , anti-pigmentation agents or moisturizers.

For the purposes of this specification, unless otherwise stated, "%" in a formulation is defined as a weight/weight (ie, w/w) percentage.

Aspects of the present specification provide, in part, a hydrogel composition disclosed herein that may optionally include an anesthetic. The anesthetic agent is preferably a local anesthetic, ie a reversible local anesthetic and an anesthetic that causes loss of nociception, eg, an aminoamide local anesthetic and an aminoester local anesthetic. The amount of anesthetic agent included in the compositions disclosed herein is an amount effective to relieve pain experienced by a subject upon administration of the composition. As such, the amount of anesthetic agent included in the compositions disclosed herein is from about 0.1% to about 5% by weight of the total composition. Non-limiting examples of anesthetics include lidocaine, ambucaine, amolanone, amylocaine, benoxynate, benzocaine, bethoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butetamine, Butoxycaine, Carticaine, Chloroprocaine, Cocoethylene, Cocaine, Cyclomethicaine, Dibucaine, Dimethisoquine, Dimethocaine, Diferodone, Dicyclonine, Euprocin, Phenalcomine, Formocaine, Hexyl Caine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, reboxadrol, lidocaine, mepivacaine, meprilcaine, metabutoxycaine, methyl chloride, myrthecaine, naepine , octacaine, orthocaine, oxetazane, parethoxycaine, phenacaine, phenol, piperocaine, pyridocaine, polydocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propiocaine, propoxycaine, pseudococaine, pyrocaine, lopivacaine, salicyl alcohol, tetracaine, tolicaine, trimecaine, zolamine, combinations thereof and salts thereof. Non-limiting examples of aminoester local anesthetics include procaine, chloroprocaine, cocaine, cyclomethicaine, cimethocaine (larocaine), propoxycaine, procaine (novocaine), proparacaine, tetracaine (ame tocaine). Non-limiting examples of aminoamide local anesthetics include articaine, bupivacaine, cinchocaine (dibucaine), etidocaine, levobupivacaine, lidocaine (lignocaine), mepivacaine, piperocaine, prill rocaine, lopivacaine and trimecaine. The compositions disclosed herein may include a single anesthetic agent or multiple anesthetic agents. A non-limiting example of a local anesthetic is lidocaine/prilocaine (EMLA).

Thus, in an embodiment, a composition disclosed herein comprises an anesthetic and a salt thereof. In aspects of this embodiment, a composition disclosed herein comprises an aminoamide local anesthetic and a salt thereof or an aminoester local anesthetic and a salt thereof. In another aspect of this embodiment, a composition disclosed herein comprises procaine, chloroprocaine, cocaine, cyclomethicaine, cytocaine, propoxycaine, procaine, proparacaine, tetracaine, or a salt thereof, or any combination thereof. In another aspect of this embodiment, a composition disclosed herein comprises articaine, bupivacaine, synchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine, ropivaca phosphorus, trimecaine, or a salt thereof, or any combination thereof. In another aspect of this embodiment, a composition disclosed herein comprises a lidocaine/prilocaine combination.

In other aspects of this embodiment, the composition disclosed herein comprises, e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6% by weight of the total composition. %, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1.0 wt%, about 2.0 wt%, about 3.0 wt%, about 4.0 wt%, about 5.0 wt%, about 6.0 wt%, about 7.0 wt% %, about 8.0% by weight, about 9.0% by weight, or about 10% by weight of the anesthetic agent. In another aspect, a composition disclosed herein comprises, for example, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7% by weight of the total composition. wt%, at least 0.8 wt%, at least 0.9 wt%, at least 1.0 wt%, at least 2.0 wt%, at least 3.0 wt%, at least 4.0 wt%, at least 5.0 wt%, at least 6.0 wt%, at least 7.0 wt%, at least 8.0 wt% %, at least 9.0% by weight or at least 10% by weight of an anesthetic. In another embodiment, a composition disclosed herein comprises, e.g., at most 0.1%, at most 0.2%, at most 0.3%, at most 0.4%, at most 0.5%, at most 0.6%, at most 0.7% by weight of the total composition. wt%, up to 0.8 wt% Up to 0.9 wt%, up to 1.0 wt%, up to 2.0 wt%, up to 3.0 wt%, up to 4.0 wt%, up to 5.0 wt%, up to 6.0 wt%, up to 7.0 wt%, up to 8.0 wt% %, up to 9.0% by weight, or up to 10% by weight of the anesthetic agent. In a further aspect, a composition disclosed herein comprises, e.g., about 0.1% to about 0.5%, about 0.1% to about 1.0%, about 0.1% to about 2.0%, about 0.1% by weight of the total composition. wt% to about 3.0 wt%, about 0.1 wt% to about 4.0 wt%, about 0.1 wt% to about 5.0 wt%, about 0.2 wt% to about 0.9 wt%, about 0.2 wt% to about 1.0 wt%, about 0.2 wt% anesthetic agent in an amount of from about 2.0% to about 2.0% by weight, from about 0.5% to about 1.0% by weight, or from about 0.5% to about 2.0% by weight.

In another embodiment, the compositions disclosed herein do not include an anesthetic.

In one aspect of the present invention, a polymer, e.g., a glycosaminoglycan polymer, e.g., a hyaluronic acid polymer, e.g., hyaluronic acid at least partially crosslinked, and an additive or beneficial agent in combination with the polymer An injectable dermal filler is provided.

Advantageous agents in combination with the polymer may include vitamins such as vitamin C. Non-limiting examples of suitable forms of vitamin C include ascorbic acid and sodium, potassium and calcium salts of ascorbic acid, fat soluble esters of ascorbic acid and long chain fatty acids (ascorbyl palmitate or ascorbyl stearate), magnesium ascorbyl phosphate ( MAP), sodium ascorbyl phosphate (SAP), and ascorbic acid 2-glucoside (AA2G™), sodium ascorbyl phosphate (AA2P), disodium ascorbyl sulfate, and ascorbyl 3-aminopropyl phosphate (Vitagen) ) is included.

In a particularly advantageous embodiment, the advantageous agent is covalently conjugated to the polymer. For example, the beneficial agent may be vitamin C, or a vitamin C derivative, which is covalently conjugated to a polymer and is from about 0.04% to about 5.0% by weight of the total composition, for example about 0.1% by weight of the total composition. % to about 4.0% by weight, for example from about 0.2% to about 2.0% by weight of the total composition. In one embodiment, the amount of vitamin C included in the compositions disclosed herein is from about 0.3% to about 1.2% by weight of the total composition.

Preferably, vitamin C covalently conjugated to the polymer is ascorbic acid, L-ascorbic acid, L-ascorbic acid 2-sulfate (AA-2S) and L-ascorbic acid 2-phosphate (AA- 2P), ascorbic acid 2-O-glucoside (AA-2G), 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acid (6-Acyl-AA-2G), (ascorbyl 3-aminopropyl phosphate, ascorbyl palmitate), derivatives and at least one of derivatives thereof. The compositions disclosed herein may include a single vitamin C agonist or multiple vitamin C agonists.

In another embodiment of the present invention, a dermal filler is provided wherein hyaluronic acid is crosslinked with BDDE. In this embodiment, the degree of conjugation may be from about 3 mol% to about 10 mol%, from about 15 mol% to about 40 mol%.

In some embodiments, the dermal filler has sustained bioavailability. For example, dermal fillers are provided that, when introduced into human skin, are effective to release ascorbic acid or other vitamins to humans for at least about 1 month to up to about 20 months or more.

Aspects of the present invention provide, in part, a hydrogel composition disclosed herein that exhibits a complex modulus, an elastic modulus, a viscous modulus, and/or a tan δ. Compositions as disclosed herein are viscoelastic, i.e., when a force is applied (stress, strain) the composition has an elastic component (solid-like, e.g., crosslinked glycosaminoglycan polymer) and a viscous component (liquid-like, for example, uncrosslinked glycosaminoglycan polymers or carrier phases). The rheological property that describes this property is the complex modulus (G*), which defines the overall resistance of the composition to deformation. The complex modulus is a complex number with real and imaginary parts: G*=G'+iG". The absolute value of G* is Abs(G*) = Sqrt(G' ² +G" ² ). The complex modulus can be defined as the sum of the elastic modulus (G') and the viscous modulus (G"). Falcone, et al., Temporary Polysaccharide Dermal Fillers: A Model for Persistence Based on Physical Properties , Dermatol Surg. 35 ( 8): 1238-1243 (2009); Tezel, supra, 2008; Kablik, supra, 2009; Beasley, supra, 2009; each of which is incorporated herein by reference in its entirety.

Modulus of elasticity or modulus of elasticity refers to the ability of a hydrogel material to resist the tendency of an object to deform or conversely non-permanently deform when a force is applied to the object. The modulus of elasticity characterizes the stiffness of a composition and is also known as the storage modulus because it accounts for the storage of energy from movement of the composition. The modulus of elasticity describes the interaction between elasticity and strength (G' = stress/strain) and, as such, provides a quantitative measure of the hardness or softening of a composition. The modulus of elasticity of an object is defined as the slope of its stress-strain curve in the elastic deformation region: λ = stress/strain, where λ is the modulus of elasticity in Pascals; Stress is the force that causes the deformation divided by the area to which the force is applied; Strain is the ratio of changes caused by stress to the original state of an object. Despite dependence on the rate at which the force is applied, a stiffer composition will have a higher modulus of elasticity and will take a greater force to deform a material over a given distance, such as by injection. Specifying how stress is measured, including direction, allows multiple types of modulus of elasticity to be defined. The three main modulus of elasticity are tensile modulus, shear modulus, and bulk modulus.

The viscous modulus is also known as the dissipation factor because it accounts for the energy lost as viscous dissipation. Tan δ is the ratio of the viscous modulus to the elastic modulus, tan δ = G"/G'. Falcone, supra, 2009. For the tan δ values disclosed herein, tan δ is the dynamic modulus at a frequency of 1 Hz. A lower tan δ corresponds to a stiffer, harder or more elastic composition.

In other embodiments, the compositions disclosed herein exhibit no elastic modulus. In aspects of this embodiment, the hydrogel composition is, for example, about 25 Pa, about 50 Pa, about 75 Pa, about 100 Pa, about 125 Pa, about 150 Pa, about 175 Pa, about 200 Pa, about 250 Pa, about 300Pa, about 350Pa, about 400Pa, about 450Pa, about 500Pa, about 550Pa, about 600Pa, about 650Pa, about 700Pa, about 750Pa, about 800Pa, about 850Pa, About 900Pa, about 950Pa, about 1,000Pa, about 1,200Pa, about 1,300Pa, about 1,400Pa, about 1,500Pa, about 1,600Pa, about 1700Pa, about 1800Pa, about 1900Pa, about 2,000Pa, about 2,100 It exhibits a modulus of elasticity of about Pa, about 2,200 Pa, about 2,300 Pa, about 2,400 Pa, or about 2,500 Pa. In other aspects of this embodiment, the hydrogel composition is, for example, at least 25 Pa, at least 50 Pa, at least 75 Pa, at least 100 Pa, at least 125 Pa, at least 150 Pa, at least 175 Pa, at least 200 Pa, at least 250 Pa Pa, at least 300 Pa, at least 350 Pa, at least 400 Pa, at least 450 Pa, at least 500 Pa, at least 550 Pa, at least 600 Pa, at least 650 Pa, at least 700 Pa, at least 750 Pa, at least 800 Pa, at least 850 Pa, At least 900Pa, at least 950Pa, at least 1,000Pa, at least 1,200Pa, at least 1,300Pa, at least 1,400Pa, at least 1,500Pa, at least 1,600Pa, at least 1700Pa, at least 1800Pa, at least 1900Pa, at least 2,000Pa, at least 2,100 an elastic modulus of Pa, at least 2,200 Pa, at least 2,300 Pa, at least 2,400 Pa, or at least 2,500 Pa. In another aspect of this embodiment, the hydrogel composition comprises, for example, at most 25 Pa, at most 50 Pa, at most 75 Pa, at most 100 Pa, at most 125 Pa, at most 150 Pa, at most 175 Pa, at most 200 Pa, Max 250Pa, Max 300Pa, Max 350Pa, Max 400Pa, Max 450Pa, Max 500Pa, Max 550Pa, Max 600Pa, Max 650Pa, Max 700Pa, Max 750Pa, Max 800Pa, Max 850Pa It exhibits an elastic modulus of Pa, up to 900 Pa, at most 950 Pa, at most 1,000 Pa, at most 1,200 Pa, at most 1,300 Pa, at most 1,400 Pa, at most 1,500 Pa, or at most 1,600 Pa. In another aspect of this embodiment, the hydrogel composition comprises, for example, about 25 Pa to about 150 Pa, about 25 Pa to about 300 Pa, about 25 Pa to about 500 Pa, about 25 Pa to about 800 Pa, about 125 Pa to about 300 Pa, about 125 Pa to about 500 Pa, about 125 Pa to about 800 Pa, about 500 Pa to about 1,600 Pa, about 600 Pa to about 1,600 Pa, about 700 Pa to about 1,600 Pa, about 800 Pa to about 1600 Pa, about 900 Pa to about 1,600 Pa, about 1,000 Pa to about 1,600 Pa, about 1,100 Pa to about 1,600 Pa, about 1,200 Pa to about 1,600 Pa, about 500 Pa to about 2,500 Pa, about 1,000 Pa to about 2,500 Pa, about 1,500 Pa to about 2,500 Pa, about 2,000 Pa to about 2,500 Pa, about 1,300 Pa to about 1,600 Pa, about 1,400 Pa to about 1,700 Pa, about 1,500 Pa to about 1,800 Pa, about 1,600 Pa to about 1900 Pa, about 1,700 Pa to about 2,000 Pa, about 1,800 Pa to about 2,100 Pa, about 1,900 Pa to about 2,200 Pa, about 2,000 Pa to about 2,300 Pa, about 2,100 Pa to about 2,400 Pa, or about 2,200 Pa to about 2,500 Pa represents the modulus of elasticity.

In other embodiments, the compositions disclosed herein exhibit no viscous modulus. In aspects of this embodiment, the hydrogel composition is, for example, about 10 Pa, about 20 Pa, about 30 Pa, about 40 Pa, about 50 Pa, about 60 Pa, about 70 Pa, about 80 Pa, about 90 Pa Pa, about 100Pa, about 150Pa, about 200Pa, about 250Pa, about 300Pa, about 350Pa, about 400Pa, about 450Pa, about 500Pa, about 550Pa, about 600Pa, about 650Pa, or about 700 Pa. In other aspects of this embodiment, the hydrogel composition is, for example, at most 10 Pa, at most 20 Pa, at most 30 Pa, at most 40 Pa, at most 50 Pa, at most 60 Pa, at most 70 Pa, at most 80 Pa, at most 90Pa, Maximum 100Pa, Maximum 150Pa, Maximum 200Pa, Maximum 250Pa, Maximum 300Pa, Maximum 350Pa, Maximum 400Pa, Maximum 450Pa, Maximum 500Pa, Maximum 550Pa, Maximum 600Pa, Maximum 650Pa or a viscous modulus of up to 700 Pa. In another aspect of this embodiment, the hydrogel composition is, for example, from about 10 Pa to about 30 Pa, from about 10 Pa to about 50 Pa, from about 10 Pa to about 100 Pa, from about 10 Pa to about 150 Pa, about 70 Pa to about 100 Pa, about 50 Pa to about 350 Pa, about 150 Pa to about 450 Pa, about 250 Pa to about 550 Pa, about 350 Pa to about 700 Pa, about 50 Pa to about 150 Pa, about 100 Pa to about 200 Pa, about 150 Pa to about 250 Pa, about 200 Pa to about 300 Pa, about 250 Pa to about 350 Pa, about 300 Pa to about 400 Pa, about 350 Pa to about 450 Pa, about 400 Pa to about 500 Pa, about 450 Pa to about 550 Pa, about 500 Pa to about 600 Pa, about 550 Pa to about 650 Pa, or about 600 Pa to about 700 Pa.

In another embodiment, a hydrogel composition disclosed herein exhibits a tan δ. In aspects of this embodiment, the hydrogel composition is, for example, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In other aspects of this embodiment, the hydrogel composition comprises, for example, at most 0.1, at most 0.2, at most 0.3, at most 0.4, at most 0.5, at most 0.6, at most 0.7, at most 0.8, at most 0.9, at most 1.0, at most 1.1, tan δ at most 1.2, at most 1.3, at most 1.4, at most 1.5, at most 1.6, at most 1.7, at most 1.8, at most 1.9, at most 2.0, at most 2.1, at most 2.2, at most 2.3, at most 2.4, or at most 2.5. In another aspect of this embodiment, the hydrogel composition is, for example, from about 0.1 to about 0.3, from about 0.3 to about 0.5, from about 0.5 to about 0.8, from about 1.1 to about 1.4, from about 1.4 to about 1.7, about 0.3 to about 0.6, from about 0.1 to about 0.5, from about 0.5 to about 0.9, from about 0.1 to about 0.6, from about 0.1 to about 1.0, from about 0.5 to about 1.5, from about 1.0 to about 2.0, or from about 1.5 to about 2.5; indicates.

Aspects of the present specification provide, in part, a hydrogel composition disclosed herein having transparency and/or translucency. Optical transparency is the physical property that allows visible light to pass through a material, whereas translucency (also called translucency or translucency) only allows light to scatter and pass through. The opposite characteristic is opaque. Transparent materials are clear, while white translucent materials cannot be seen clearly. The hydrogels disclosed herein are preferably optically transparent or at least translucent.

In an embodiment, a hydrogel composition disclosed herein is optically translucent. In aspects of this embodiment, the hydrogel composition comprises, for example, about 75% light, about 80% light, about 85% light, about 90% light, about 95% light, or about 100% light diffusely transmits the light of In other aspects of this embodiment, the hydrogel composition is such that, for example, at least 75% of the light, at least 80% of the light, at least 85% of the light, at least 90% of the light, or at least 95% of the light is dispersed. transmit In another aspect of this embodiment, the hydrogel composition comprises, for example, about 75% to about 100% light, about 80% to about 100% light, about 85% to about 100% light, about 90% light % to about 100% of the light, or from about 95% to about 100% of the light is transmitted diffusely. In an embodiment, a hydrogel composition disclosed herein is optically transparent and transmits 100% of visible light.

The hydrogel compositions disclosed herein may further be treated by grinding the hydrogel into particles, optionally mixed with a carrier phase, such as, for example, water or saline, to prepare injectable or topical substance-like solutions, oils, lotions, Forms gels, ointments, creams, slurries, salves or pastes. As such, the disclosed hydrogel compositions may be monophasic or multiphasic compositions. The hydrogel has a diameter of about 10 μm to about 1000 μm, such as about 15 μm to about 30 μm, about 50 μm to about 75 μm, about 100 μm to about 150 μm, about 200 μm to about 300 μm, about 450 μm to It may be milled to a particle size of about 550 μm, about 600 μm to about 700 μm, about 750 μm to about 850 μm, or about 900 μm to about 1,000 μm.

Aspects of the present specification provide, in part, that a composition disclosed herein is injectable. The term “injectable” as used herein refers to a substance having the properties necessary to administer a composition to a skin region of a subject using an injection device having a fine needle. The term “fine needle” as used herein refers to a needle of 27 gauge or less. The injectability of the compositions disclosed herein can be accomplished by sizing the hydrogel particles as discussed above.

In aspects of this embodiment, the hydrogel compositions disclosed herein may be injected through a fine needle. In other aspects of this embodiment, a hydrogel composition disclosed herein is injectable through, for example, a needle of about 27 gauge, about 30 gauge, or about 32 gauge. In another aspect of this embodiment, a hydrogel composition disclosed herein is injectable through, for example, a 22 gauge or less, 27 gauge or less, 30 gauge or less, or 32 gauge or less needle. In another aspect of this embodiment, a hydrogel composition disclosed herein can be, for example, from about 22 gauge to about 35 gauge, from 22 gauge to about 34 gauge, from 22 gauge to about 33 gauge, from 22 gauge to about 32 gauge; Injectable through a needle of about 22 gauge to about 27 gauge, or about 27 gauge to about 32 gauge.

In aspects of this embodiment, a hydrogel composition disclosed herein comprises at a rate of about 60N, about 55N, about 50N, about 45N, about 40N, about 35N, about 30N, about 25N, about 20N, at a rate of 100 mm/min, Or it can be injected with an extrusion force of about 15N. In other aspects of this embodiment, a hydrogel composition disclosed herein is about 60N or less, about 55N or less, about 50N or less, about 45N or less, about 40N or less, about 35N or less, about 30N or less, about 25N or less, about 20N or less or less, about 15 N or less, about 10 N or less, or about 5 N or less, and can be injected through a 27 gauge needle. In another aspect of this embodiment, a hydrogel composition disclosed herein is about 60N or less, about 55N or less, about 50N or less, about 45N or less, about 40N or less, about 35N or less, about 30N or less, about 25N or less, about The injection may be through a 30 gauge needle having an extrusion force of 20 N or less, about 15 N or less, about 10 N or less, or about 5 N or less. In another aspect of this embodiment, a hydrogel composition disclosed herein is about 60N or less, about 55N or less, about 50N or less, about 45N or less, about 40N or less, about 35N or less, about 30N or less, about 25N or less, about It can be injected through a 32 gauge needle having an extrusion force of 20 N or less, about 15 N or less, about 10 N or less, or about 5 N or less.

Aspects of the present specification provide, in part, a hydrogel composition disclosed herein that exhibits adhesiveness. Adhesion, also referred to as cohesive cohesive force, cohesive force or compressive force, is a physical property of a material and is caused by the intermolecular attraction between like molecules within a material that acts to unite the molecules. Adhesion is expressed in terms of gram-force (gmf). The cohesive force is, inter alia, the molecular weight ratio of the initial free glycosaminoglycan polymer, the glycosaminoglycan polymer, the degree of crosslinking of the glycosaminoglycan polymer, the amount of free glycosaminoglycan polymer remaining after crosslinking, and the pH of the hydrogel composition. The composition should be sufficiently cohesive to remain localized to the site of administration. Additionally, in certain applications, sufficient cohesion is important for the composition to retain its shape and thus its functionality in the event of a mechanical load cycle. As such, in one embodiment, the compositions disclosed herein exhibit cohesive strength equivalent to water. In another embodiment, the hydrogel compositions disclosed herein exhibit sufficient adhesion to remain localized to the site of administration. In another embodiment, a hydrogel composition disclosed herein exhibits sufficient adhesion to retain its shape. In a further embodiment, a hydrogel composition disclosed herein exhibits sufficient adhesion to retain its shape and functionality.

Aspects of the present specification provide, in part, a hydrogel composition disclosed herein that exhibits a physiologically acceptable osmolarity. The term “osmolarity” as used herein refers to the concentration of an osmotically active solute in solution. The term "physiologically acceptable osmolarity" as used herein refers to a characteristic or consequent osmolarity of the normal functioning of a living organism. As such, administration of a hydrogel composition as disclosed herein exhibits an osmolarity that, when administered to a mammal, has a permanent deleterious or not substantially long-term effect. Osmolarity is expressed in terms of osmols of osmotically active solute per liter of solvent (Osmol/L or Osm/L). Osmolarity is independent of molarity because it measures moles of osmotically active solute particles rather than moles of solute. The difference arises because some compounds may dissociate in solution while others may not. The osmolarity of a solution can be calculated from the expression: Osmol/L = ∑ ϕ _i η _i C _i where ϕ is the osmotic coefficient that calculates the degree of non-ideality of the solution; η is the number of particles (eg ions) into which the molecule dissociates; C is the molar concentration of the solute; i is an index indicating the identity of a particular solute. The osmolarity of the hydrogel composition disclosed herein can be measured using a conventional method of treating the solution.

In an embodiment, a hydrogel composition disclosed herein exhibits a physiologically acceptable osmolarity. The term “osmolality” as used herein refers to the concentration of osmotically active solutes per kilo of solvent in the body. The term “physiologically acceptable osmolality” as used herein refers to an osmolality according to a characteristic or normal function of a living organism. As such, administration of a hydrogel composition disclosed herein exhibits an osmolality that, when administered to a mammal, has a permanent deleterious or not substantially long-term effect. Osmolality is expressed in terms of osmols of osmotically active solute per kilogram of solvent (osmol/kg or Osm/kg) and is equal to the sum of the molality of all solutes present in that solution. The osmolality of a solution can be measured using an osmometer. The most commonly used instrument in modern laboratories is the freezing point depression osmometer. This device measures the change in freezing point that occurs with increasing osmolality (freezing point depressing osmometer) or the change in water vapor pressure in a solution with increasing osmolality (vapor pressure drop osmometer).

In aspects of this embodiment, the hydrogel composition is, for example, about 100 mOsm/L, about 150 mOsm/L, about 200 mOsm/L, about 250 mOsm/L, about 300 mOsm/L, about 350 mOsm/L L, about 400 mOsm/L, about 450 mOsm/L, or about 500 mOsm/L. In other aspects of this embodiment, the hydrogel composition comprises, for example, at least 100 mOsm/L, at least 150 mOsm/L, at least 200 mOsm/L, at least 250 mOsm/L, at least 300 mOsm/L, at least 350 mOsm /L, at least 400 mOsm/L, at least 450 mOsm/L or at least 500 mOsm/L. In another aspect of this embodiment, the hydrogel composition is, for example, at most 100 mOsm/L, at most 150 mOsm/L, at most 200 mOsm/L, at most 250 mOsm/L, at most 300 mOsm/L, at most 350 mOsm It shows an osmolarity of /ℓ, a maximum of 400 mOsm/ℓ, a maximum of 450 mOsm/ℓ, or a maximum of 500 mOsm/ℓ. In another aspect of this embodiment, the hydrogel composition is, for example, from about 100 mOsm/L to about 500 mOsm/L, from about 200 mOsm/L to about 500 mOsm/L, from about 200 mOsm/L to about 400 mOsm /L, from about 300 mOsm/L to about 400 mOsm/L, from about 270 mOsm/L to about 390 mOsm/L, from about 225 mOsm/L to about 350 mOsm/L, from about 250 mOsm/L to about 325 mOsm/L , from about 275 mOsm/L to about 300 mOsm/L, or from about 285 mOsm/L to about 290 mOsm/L.

Aspects of the present specification provide, in part, a hydrogel composition disclosed herein that exhibits substantial stability. When referring to a hydrogel composition disclosed herein, the term “stability” or “stable” as used herein means that it cannot be degraded, degraded, or destroyed to any substantial or significant extent during storage prior to administration to a subject. It refers to a composition that is not easy. As used herein, the terms “substantially thermal stability,” “substantially thermal stability,” “autoclave stability,” or “steam sterilization stability,” refer to the present specification that is substantially stable when subjected to a heat treatment as disclosed herein. refers to the hydrogel composition disclosed in

The stability of a hydrogel composition disclosed herein can be determined by subjecting the hydrogel composition to a heat treatment, such as, for example, steam sterilization at normal pressure or under reduced pressure (eg, autoclaving). Preferably, the heat treatment is performed at a temperature of at least about 100° C. for from about 1 minute to about 10 minutes. Substantial stability of the hydrogel compositions disclosed herein is determined by 1) determining the change in the extrusion force (ΔF) of the hydrogel compositions disclosed herein after sterilization, wherein the change in the extrusion force of less than 2 N (hydrogel with specified additives) the extrusion force of the gel composition) - (indicating a substantially stable hydrogel composition as measured by the extrusion force of the hydrogel composition without added additives); and/or 2) determining a change in the rheological properties of the hydrogel composition disclosed herein after sterilization, wherein a change in tan δ 1 Hz of less than 0.1 (tan δ 1 Hz of the gel formulation by the additive) - (additive) indicating a substantially stable hydrogel composition as measured by the tan δ 1 Hz) of the gel formulation without As such, the substantially stable hydrogel compositions disclosed herein retain one or more of the following characteristics after sterilization: homogeneity, extrusion force, cohesive force, hyaluronan concentration, agent(s) concentration, osmolarity, pH , or other rheological characteristics desired by the hydrogel prior to heat treatment.

In an embodiment, a hydrogel composition comprising a glycosaminoglycan polymer and at least one agent disclosed herein is treated using a heat treatment that maintains the desired hydrogel properties disclosed herein. In aspects of this embodiment, a hydrogel composition comprising a glycosaminoglycan polymer and at least one agent disclosed herein is, for example, about 100° C., about 105° C., about 110° C., about 115° C. , a heat treatment of about 120° C., about 125° C., or about 130° C. In other aspects of this embodiment, a hydrogel composition comprising a glycosaminoglycan polymer and at least one agent disclosed herein is, for example, at least 100° C., at least 105° C., at least 110° C., at least 115° C. C., at least 120° C., at least 125° C. or at least 130° C. using a heat treatment. In another aspect of this embodiment, a hydrogel composition comprising a glycosaminoglycan polymer and at least one agent disclosed herein is, for example, from about 100°C to about 120°C, from about 100°C to about 125°C. °C, from about 100 °C to about 130 °C, from about 100 °C to about 135 °C, from about 110 °C to about 120 °C, from about 110 °C to about 125 °C, from about 110 °C to about 130 °C, from about 110 °C to about 135 °C, about 120 °C to about 125 °C, about 120 °C to about 130 °C, about 120 °C to about 135 °C, about 125 °C to about 130 °C, or about 125 °C to about 135 °C.

The stability of a hydrogel composition disclosed herein can be determined by subjecting the hydrogel composition to a heat treatment, such as, for example, steam sterilization at normal pressure or under reduced pressure (eg, autoclaving). Long-term stability of a hydrogel composition disclosed herein can be determined by subjecting the hydrogel composition to heat treatment, such as storage, for example, in an environment of about 45° C. for about 60 days. The long-term stability of the hydrogel compositions disclosed herein can be determined by 1) evaluating the clarity and color of the hydrogel composition after heat treatment at 45° C. (clear, colorless hydrogel compositions indicate substantially stable hydrogel compositions); 2) by determining the change in the extrusion force (ΔF) of the hydrogel composition disclosed herein after the 45 ° C heat treatment (the change in the extrusion force less than 2 N (extrusion force of the hydrogel composition with the specified additive prior to the 45 ° C heat treatment) - indicative of a substantially stable hydrogel composition as measured by (extrusion force of hydrogel composition with specified additives after 45° C. heat treatment); and/or 3) determining a change in the rheological properties of the hydrogel compositions disclosed herein after sterilization, wherein a change in tan δ 1 Hz of less than 0.1 (tan δ of the gel formulation with the specified additive prior to 45° C. heat treatment) 1 Hz) - (indicating a substantially stable hydrogel composition as measured by the tan δ 1 Hz of the gel formulation with specified additives after 45°C heat treatment). As such, the long-term stability of the hydrogel compositions disclosed herein is assessed by retention of one or more of the following characteristics after 45°C heat treatment: clarity (transparency and translucency), homogeneity and cohesion.

In aspects of this embodiment, the hydrogel composition is, for example, about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 27 months , is substantially stable at room temperature for about 30 months, about 33 months, or about 36 months. In other aspects of this embodiment, the hydrogel composition is, for example, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months, at least It is substantially stable at room temperature for 27 months, at least 30 months, at least 33 months or at least 36 months. In other aspects of this embodiment, the hydrogel composition is, e.g., from about 3 months to about 12 months, from about 3 months to about 18 months, from about 3 months to about 24 months, from about 3 months to about 30 months, about 3 months to about 36 months, about 6 months to about 12 months, about 6 months to about 18 months, about 6 months to about 24 months, about 6 months to about 30 months, about 6 months to about 36 months, about 9 months to about 12 months, about 9 months to about 18 months, about 9 months to about 24 months, about 9 months to about 30 months, about 9 months to about 36 months, about 12 months to about 18 months, about 12 months to about substantially stable at room temperature for 24 months, from about 12 months to about 30 months, from about 12 months to about 36 months, from about 18 months to about 24 months, from about 18 months to about 30 months, or from about 18 months to about 36 months .

The composition may optionally include other pharmaceutically acceptable ingredients including, but not limited to, buffers, preservatives, tonicity adjusting agents, salts, antioxidants, osmolality adjusting agents, emulsifying agents, wetting agents, and the like, but are not limited thereto. doesn't happen

Pharmaceutically acceptable buffers are buffers that can be used to prepare the hydrogel compositions disclosed herein, provided that the resulting formulation is pharmaceutically acceptable. Non-limiting examples of pharmaceutically acceptable buffers include acetate buffer, borate buffer, citrate buffer, neutral buffered saline, phosphate buffer and phosphate buffered saline. Any concentration of pharmaceutically acceptable buffer may be useful in formulating the pharmaceutical compositions disclosed herein, provided that a therapeutically effective amount of the active ingredient is recovered using such effective concentration of buffer. Non-limiting examples of physiologically acceptable buffer concentrations occur in the range of about 0.1 mM to about 900 mM. The pH of the pharmaceutically acceptable buffer may be adjusted, provided that the resulting formulation is pharmaceutically acceptable. It is understood that acids or bases may be used to adjust the pH of the pharmaceutical composition if desired. Any buffered pH level may be useful in formulating a pharmaceutical composition, provided that a therapeutically effective amount of the matrix polymer active ingredient is recovered using such effective pH level. Non-limiting examples of physiologically acceptable pH occur in the range of about pH 5.0 to about pH 8.5. For example, the pH of a hydrogel composition disclosed herein can be from about 5.0 to about 8.0, or from about 6.5 to about 7.5, from about 7.0 to about 7.4, or from about 7.1 to about 7.3.

Pharmaceutically acceptable preservatives include sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene. Pharmaceutically acceptable preservatives include benzalkonium chloride, chlorobutanol, thimerosal, phenylmercury acetate, phenylmercury nitrate, stabilized oxychloro compositions such as PURITE® (California). Allergan, Inc. of Irvine, Inc.) and chelating agents such as DTPA or DTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide.

Pharmaceutically acceptable tonicity adjusting agents useful in the hydrogel compositions disclosed herein include, for example, sodium chloride and potassium chloride; and salts such as glycerin. The composition may be provided as a salt and may be formed with a number of acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or other protic solvents than their corresponding free base forms. It is understood that these and other substances known in the pharmaceutical arts may be included in the pharmaceutical compositions disclosed herein. Other non-limiting examples of pharmacologically acceptable ingredients include, for example, Ansel, supra, (1999); Gennaro, supra, (2000); Hardman, supra, (2001); and Rowe, supra, (2003), each of which is incorporated herein by reference in its entirety.

Aspects of the present specification provide, in part, a method of treating a soft tissue condition in an individual by administering a hydrogel composition disclosed herein. The term “treating” as used herein refers to reducing or eliminating in a subject the cosmetic or clinical symptoms of a soft tissue condition characterized by a soft tissue imperfection, defect, disease and/or disorder; or to delaying or preventing in an individual the onset of cosmetic or clinical symptoms of a condition characterized by a soft tissue imperfection, defect, disease and/or disorder. For example, the term "treating" means, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% by reducing the symptoms of a condition characterized by a soft tissue defect, disease and/or disorder. The effectiveness of a hydrogel composition disclosed herein in treating a condition characterized by a soft tissue imperfection, defect, disease and/or disorder can be determined by observing one or more cosmetic, clinical symptoms and/or physiological indicators associated with the condition. have. Improvements in soft tissue imperfections, defects, diseases and/or disorders may also be indicated by a reduced need for concomitant treatment. One of ordinary skill in the art would know the appropriate symptoms or indicators associated with a specific soft tissue defect, disease and/or disorder, and would know how to determine whether an individual is a candidate for treatment with a compound or composition disclosed herein.

The hydrogel composition according to the present invention is administered to a subject. The subject is typically a human of any age, sex, or race. Any individual that is typically a candidate for conventional procedures for treating soft tissue disease is a candidate for the methods disclosed herein. Even if a subject experiencing signs of aging skin is an adult, a subject experiencing premature aging or other skin conditions suitable for treatment (eg, scarring) can be treated with a hydrogel composition disclosed herein. Additionally, the presently disclosed hydrogel compositions and methods may be applied to individuals seeking small/medium enlargement, shape change or contour alteration of a body part or region, which is not technically possible by existing soft tissue implantation techniques or It may not be aesthetically acceptable. Pre-procedural evaluations typically include routine history and physiological examinations in addition to informed consent disclosing all relevant risks and benefits of the procedure.

The hydrogel compositions and methods disclosed herein are useful for treating soft tissue conditions. Soft tissue conditions include, but are not limited to, soft tissue imperfections, defects, diseases and/or disorders. Non-limiting examples of soft tissue conditions include breast incompleteness, defect, disease and/or disorder, eg, breast incompleteness, defect, disease and/or disorder, eg, breast enlargement, breast reconstruction, breast fixation, micromammia, thoracic hypoplasia. Defects due to transplant complications such as sciatica, Poland's syndrome, capsule contractions and/or ruptures; Facial imperfections, defects, diseases and/or disorders such as facial enlargement, facial reconstruction, mesotherapy, Parry-Romberg syndrome, lupus erythematosus, dermal divot, scarring, sunken check , thin lips, nasal imperfections or conjoints, posterior orbital imperfections or defects, facial folds such as glabellar lines, wrinkles and/or folds, nasolabial folds, upper lip wrinkles, and/or nasolabial folds and/or other contour malformations or facial features of incompleteness; neck imperfections, defects, diseases and/or disorders; skin imperfections, defects, diseases and/or disorders; Other soft tissue imperfections, defects, diseases and/or disorders, e.g., upper arm, lower arm, hand, shoulder, back, torso, including abdomen, buttock, upper leg, lower leg, including calf, sole enlargement or reconstruction of the feet, eyes, genitals or other body parts, areas or areas, or diseases or disorders affecting those body parts, areas or areas; urinary incontinence, fecal incontinence, and other forms of incontinence; and gastroesophageal reflux disease (GERD). As used herein, the term “mesotherapy” refers to the non-surgical cosmetic treatment of the skin involving intradermal, intradermal and/or subcutaneous injection of an agent administered as a number of small droplets into the epidermis, dermal-epidermal junction and/or into the dermis. It refers to a therapeutic technique.

The amount of the hydrogel composition used by any of the methods disclosed herein typically depends on the desired alteration and/or amelioration, the desired reduction and/or elimination of soft tissue disease symptoms, the clinical and It will be determined based on the cosmetic effect and/or the body part or area being treated. The effectiveness of administration of the composition may be indicated by one or more of the following clinical and/or cosmetic measures: altered and/or improved soft tissue shape, altered and/or improved soft tissue size, altered and/or improved soft tissue contour , altered and/or improved tissue function, tissue ingrowth and/or new collagen deposition, sustained engraftment of the composition, improved patient satisfaction and/or quality of life, and reduced use of implantable foreign materials.

The effectiveness of the compositions and methods in treating facial soft tissue may be indicated by one or more of the following clinical and/or cosmetic measures: Facial features, such as increased size, shape and/or contour of the lips, cheeks, or eye area. increased size, shape and/or contour of altered size, shape and/or contour of a facial feature, such as altered size, shape and/or contour of the shape of the lips, cheeks, or eye region; reduction or elimination of wrinkles, folds, or wrinkles in the skin; resistance to wrinkles, folds, or wrinkles in the skin; rehydration of the skin; increased elasticity to the skin; reduction or elimination of skin roughness; increased and/or improved skin tone; reduction or elimination of stretch marks or marks; increased and/or improved skin tone, brilliance, brightness and/or radiance; increased and/or improved skin color, reduction or elimination of skin paleness; persistence of the composition; reduced side effects; improved patient satisfaction and/or quality of life.

As another example, for the course of urinary incontinence, the effectiveness of the compositions and methods for sphincter support may be indicated by one or more of the following clinical measures: reduced cycles of incontinence, sustained engraftment, improved patient satisfaction, and/or Quality of life and reduced use of implantable ambulatory fillers.

In aspects of this embodiment, the amount of the hydrogel composition administered is, for example, about 0.01 g, about 0.05 g, about 0.1 g, about 0.5 g, about 1 g, about 5 g, about 10 g, about 20 g, about 30 g, about 40 g, about 50 g, about 60 g, about 70 g, about 80 g, about 90 g, about 100 g, about 150 g or about 200 g. In other aspects of this embodiment, the amount of the hydrogel composition administered is, for example, from about 0.01 g to about 0.1 g, from about 0.1 g to about 1 g, from about 1 g to about 10 g, from about 10 g to about 100 g, or from about 50 g to It is about 200 g. In another aspect of this embodiment, the amount of hydrogel composition administered is, for example, about 0.01 ml, about 0.05 ml, about 0.1 ml, about 0.5 ml, about 1 ml, about 5 ml, about 10 ml, about 20 ml, about 30 ml, about 40 ml, about 50 ml, about 60 ml, about 70 g, about 80 ml, about 90 ml, about 100 ml, about 150 ml, or about 200 ml. In other aspects of this embodiment, the amount of the hydrogel composition administered is, for example, from about 0.01 ml to about 0.1 ml, from about 0.1 ml to about 1 ml, from about 1 ml to about 10 ml, from about 10 ml to about 100 ml , or from about 50 ml to about 200 ml.

The duration of treatment will typically be determined based on the cosmetic and/or clinical effect desired by the individual and/or physician and the body part or area being treated. In aspects of this embodiment, administration of a hydrogel composition disclosed herein is, for example, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, The soft tissue condition may be treated for about 13 months, about 14 months, about 15 months, about 18 months, or about 24 months. In other aspects of this embodiment, administration of a hydrogel composition disclosed herein is, e.g., at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, and treat the soft tissue condition for at least 13 months, at least 14 months, at least 15 months, at least 18 months, or at least 24 months. In other aspects of this embodiment, administration of a hydrogel composition disclosed herein is administered, e.g., from about 6 months to about 12 months, from about 6 months to about 15 months, from about 6 months to about 18 months, about 6 months. to about 21 months, about 6 months to about 24 months, about 9 months to about 12 months, about 9 months to about 15 months, about 9 months to about 18 months, about 9 months to about 21 months, about 6 months to about 24 months, about 12 months to about 15 months, about 12 months to about 18 months, about 12 months to about 21 months, about 12 months to about 24 months, about 15 months to about 18 months, about 15 months to about 21 months , from about 15 months to about 24 months, from about 18 months to about 21 months, from about 18 months to about 24 months, or from about 21 months to about 24 months.

Aspects herein provide, in part, administering a hydrogel composition disclosed herein. The term “administering” as used herein refers to any delivery mechanism that provides a composition disclosed herein to a subject that potentially results in a clinically, therapeutically or experimentally beneficial outcome. The actual delivery mechanism used to administer the composition to a subject depends on the type of skin condition, the location of the skin condition, the cause of the skin condition, the severity of the skin condition, the degree of relief desired, the particular composition used, and the rate of excretion of the particular composition used. , the pharmacokinetics of the particular composition employed, the properties of other compounds included in the particular composition employed, the particular route of administration, particular characteristics, the subject's history and risk factors, such as age, weight, general health, etc., or any of these It can be determined by one of ordinary skill in the art by considering factors including, but not limited to, combinations. In aspects of this embodiment, a composition disclosed herein is administered to a skin region of an individual by injection.

The route of administration of the hydrogel composition to an individual patient will typically be determined based on the cosmetic and/or clinical effect desired by the individual and/or physician and the body part or region being treated. The compositions disclosed herein can be administered by any means known to those of skill in the art, including, but not limited to, needleed syringes, pistols (eg, hand-compressed pistols), catheters, either locally or by direct surgical implantation. can be administered by A hydrogel composition disclosed herein can be administered into a skin region, such as, for example, a dermal region or a subcutaneous region. For example, a hydrogel composition disclosed herein can be injected using a needle having a diameter ranging from about 0.26 mm to about 0.4 mm and a length ranging from about 4 mm to about 14 mm. Alternatively, the needle may be between 21G and 32G and have a length of between about 4 mm and about 70 mm. Preferably, the needle is a disposable needle. The needle may be combined with a syringe, catheter and/or pistol.

Additionally, the compositions disclosed herein may be administered once or in multiple doses. Ultimately, the time used will comply with quality control standards. For example, a hydrogel composition disclosed herein may be administered in one or more several working hours, with working hours separated by several days or weeks. For example, a subject may be administered a hydrogel composition disclosed herein every 1, 2, 3, 4, 5, 6, or 7 days or every 1, 2, 3, or 4 weeks. Administration of a hydrogel composition disclosed herein to a subject may be administered monthly or on a bi-monthly basis or every 3, 6, 9 or 12 months.

Aspects herein provide, in part, a dermal region. The term “dermal region” as used herein refers to the region of the skin comprising the epidermis-dermal junction and the dermis, including the non-deep dermis (papillary region) and the deep dermis (reticular region). The skin is composed of three main layers: the epidermis, which provides waterproofing and acts as a barrier to infection; the dermis, which acts as a site for the exosphere of the skin; and harpy (subcutaneous fat layer). The epidermis contains no blood vessels and is nourished by diffusion from the dermis. The main types of cells that make up the epidermis are keratinocytes, melanocytes, Langerhans cells and Merkel cells.

The dermis is the layer of skin beneath the epidermis that is made up of connective tissue and relieves the body's impact from stress and strain. The dermis is tightly bound to the epidermis by the underlying membrane. It also harbors a number of mechanoreceptors/nerve terminals that provide sensations of touch and heat. It contains pores, sweat glands, sebaceous glands, apocrine glands, lymphatic vessels and blood vessels. The blood vessels in the dermis provide nutrients and waste removal from the basal layer of the epidermis as well as from its own cells. The dermis is structurally divided into two regions: a supercritical region adjacent to the epidermis, called the papillary region, and a deeper, thicker region known as the reticular region.

The papillary region consists of loose areolar tissue and connective tissue. It is named for the finger-like projections called papillae that extend towards the epidermis. The papilla has a "ragged" surface that interlocks with the epidermis, providing the dermis to strengthen the bond between the two layers of skin. The reticular region lies deeper than the papillary region, and is usually much thicker. It is composed of dense, irregular connective tissue, and gets its name from the dense concentration of collagenous, elastic and reticular fibers woven throughout it. These protein fibers provide the dermis with its properties of strength, extensibility and elasticity. Also located within the reticular region are hair roots, sebaceous glands, sweat glands, receptors, nails and blood vessels. Tattoo ink is retained in the dermis. Stretch marks from pregnancy are also located in the dermis.

The harpy lies beneath the dermis. Its purpose is to attach the dermal regions of the underlying bones and muscles, as well as supply them with blood vessels and nerves. It consists of loose connective tissue and elastin. The main cell types are fibroblasts, macrophages and adipocytes (the hypodermis contains 50% of body fat). Fat acts as a pad and insulator for the body.

In aspects of this embodiment, a hydrogel composition disclosed herein is administered to a skin region of an individual by injection into a dermal region or subcutaneous tissue region. In aspects of this embodiment, a hydrogel composition disclosed herein is administered to a dermal region of a living body, for example, by injection into the epidermal-dermal junction region, papillary region, reticular region, or any combination thereof.

Advantageously, some of the compositions are particularly useful and effective in reducing fine lines, for example in thin skin areas of a patient. For example, a method for treating fine lines is provided comprising administering to a patient a dermal filler composition as described elsewhere herein at a depth of about 1 mm or less.

Another aspect of the present disclosure includes administering, in part, a hydrogel composition disclosed herein to a subject suffering from a skin condition, wherein the administration of the composition ameliorates the skin condition, thereby treating the skin condition. In an aspect of this embodiment, the skin condition comprises administering to an individual suffering from skin dehydration a hydrogel composition disclosed herein, wherein administration of the composition rehydrates the skin, thereby treating skin dehydration. In another aspect of this embodiment, a method of treating a lack of skin elasticity comprises administering to a subject suffering from a lack of skin elasticity a hydrogel composition disclosed herein, wherein administration of the composition increases skin elasticity, thereby Treats the lack of skin elasticity by In another aspect of this embodiment, a method of treating rough skin comprises administering to an individual suffering from skin roughness a hydrogel composition disclosed herein, wherein administration of the composition reduces skin roughness, thereby Treat rough skin. In another aspect of this embodiment, a method of treating a lack of skin roughness comprises administering a hydrogel composition disclosed herein to an individual suffering from a lack of skin tone, wherein administration of the composition produces skin tone and , thereby treating the lack of skin tension.

In a further aspect of this embodiment, a method of treating skin stretch marks or marks comprises administering a hydrogel composition disclosed herein to an individual suffering from skin stretch marks or marks, wherein administration of the composition treats skin stretch marks or marks. reduce or eliminate, thereby treating skin stretch marks or marks. In another aspect of this embodiment, a method of treating pallor of the skin comprises administering to an individual suffering from pallor of the skin a hydrogel composition disclosed herein, wherein administration of the composition increases skin tone or radiance, This treats the paleness of the skin. In another aspect of this embodiment, a method of treating skin wrinkles comprises administering to an individual suffering from skin wrinkles a hydrogel composition disclosed herein, wherein administration of the composition reduces or eliminates skin wrinkles; , thereby treating skin wrinkles. In another aspect of this embodiment, a method of treating skin wrinkles comprises administering to an individual a hydrogel composition disclosed herein, wherein administration of the composition creates skin resistance to skin wrinkles, thereby causing skin wrinkles. treat

In some embodiments, the dermal filler has sustained bioavailability. For example, when introduced into the skin of a human (eg, intradermally or subcutaneously into a human to repair soft tissue defects of the facial space), ascorbic acid (or other A dermal filler that releases vitamins) is provided.

For example, to predict sustained vitamin C efficacy with filler duration, an assessment of the degree of conjugation is made. This evaluation was based on the formulation of AA2G conjugation to HA via etherification. Although the formulation is stable under physiological conditions, it initiates the release of ascorbic acid (AsA) by α-glucosidase attached to the cell membrane. Due to the fact that α-glucosidase is attached to the cell membrane, the AsA is released. Further release of AsA from HA-AA2G will entail HA degradation to make AA2G available to fibroblasts. Therefore, the release of AsA depends on the degree of AA2G conjugation and the duration of HA. Gels with a degree of conjugation of approximately 5 mol% were able to release active vitamin C in a period of at least 1 month, for example 3 to 5 months; Gels with a degree of 10 mol% conjugation were able to release active vitamin C in a period of up to 6 to 8 months; A gel with a degree of 15 mol% conjugation can release active vitamin C in a period of up to 10 months; 30 mol% can be released up to a year and a half.

In an embodiment of the invention, a vitamin C derivative comprising hyaluronic acid cross-linked with star-PEG epoxide and conjugated to hyaluronic acid to an extent of from about 3 mol% to about 40 mol% (e.g., AA2G (ascorbic acid) 2-glucoside), Vitagen (3-aminopropyl-L-ascorbyl phosphate) and SAP (one of sodium ascorbyl phosphate) are provided.

The method for preparing this dermal filler comprises a suitable ratio of reaction temperature and reaction time to achieve a composition containing AA2G with 4-arm epoxide (AA2G-4 arm epoxide), unreacted 4-arm epoxide and free AA2G. reacting pentaerythritol glycidal ether (Star-PEG epoxide) with ascorbic acid 2-glucoside (AA2G) in A four arm epoxide capped AA2G (AA2G-4 arm epoxide) is conjugated to hyaluronic acid via an epoxyl group. The unreacted 4 arm epoxide acts as a crosslinking agent for crosslinking hyaluronic acid and further as a conjugation agent for conjugating AA2G.

In another embodiment of the invention, a vitamin C derivative comprising hyaluronic acid cross-linked with BDDE and conjugated to hyaluronic acid at a conjugation level of 3 mol% to about 10 mol% (e.g., AA2G (ascorbic acid 2-glucoside) ), Vitagen (3-aminopropyl-L-ascorbyl phosphate) and SAP (sodium ascorbyl phosphate).

The preparation of this dermal filler comprises BDDE ascorbic acid 2-glucoside at a reaction temperature and reaction time ratio suitable to achieve a composition containing BDDE (AA2G-BDDE), unreacted BDDE and AA2G capped by free AA2G. (AA2G). BDDE capped AA2G (AA2G-BDDE) is conjugated to hyaluronic acid via an epoxyl group. Unreacted BDDE acts as a crosslinking agent to crosslink hyaluronic acid and as a conjugation agent to further conjugate AA2G.

9 is a table showing the effect of α-glucosidase concentration on AsA release from AA2G-PBS solution. Conversion of AA2G to AsA is dependent on the concentration of α-glycosidase. When the α-glycosidase concentration is 6.3 units per gram of gel, AA2G is almost completely converted to AsA after 15 minutes. When the α-glycosidase concentration is 4.7 units per gram of gel, AA2G takes 30 minutes to completely convert to AsA. Further reduced α-glycosidase concentration resulted in slow conversion of AA2G to AsA.

Figure 10 depicts a representation of the release profile (sustained release) of free AsA from a conjugated dermal filler according to the present invention (AA2G conversion in mol% versus reaction time); AA2G is fully converted to AsA in the AA2G/HA mix at 40 min. The AA2H/HA conjugate showed a time dependence of AA2G conversion to AsA.

11A and 11B show additional release data for various dermal fillers according to the present invention. More specifically, the conversion of AA2G to AsA in the HA-AA2G gel is dependent on the α-glycosidase concentration. High α-glycosidase concentration caused rapid conversion of AA2G to AsA. For a given α-glycosidase concentration, different formulations showed different profiles of AA2G versus AsA.

In one aspect of the present invention, the appearance of fine lines, for example, wrinkle lines that are not relatively deep in the skin, such as, but not limited to, fine lines around the eyes, lacrimal sulcus area, forehead, around the eyes, glabellar lines, etc. And, a dermal filler that is particularly effective for removal is provided.

The appearance of a blue discoloration at the skin site where dermal fillers are injected (Tyndall effect) is an important side effect experienced by some dermal filler patients. The Tyndall effect is more common in patients being treated for non-deep fine lines. An embodiment of the present invention has been developed that provides a long lasting translucent filler that can be injected deep to treat fine lines and wrinkles even in areas of relatively thin skin without causing any blue discoloration from the Tyndall effect. Fine lines or non-deep wrinkles are those wrinkles or folds on the skin (forehead, lateral canthus, erythema marginal/upper lip wrinkles) typically found in the areas of the face where the skin is usually the thinnest, i.e. the skin has a dermal thickness of less than 1 mm. understood to be a line. On the forehead, the average dermal thickness is about 0.95 mm for normal skin and about 0.81 mm for wrinkled skin. The dermis around the lateral canthus is even thinner (eg, about 0.61 mm for normal skin, about 0.41 mm for wrinkled skin). The average outer diameter of a 30 or 32 gauge needle (needles typically used for fine line gel applications) is from about 0.30 to about 0.24 mm.

The present invention provides a dermal filler composition as described elsewhere herein that does not cause a Tyndall effect. For example, the composition of the present invention comprises a crosslinking component, a crosslinked hyaluronic acid component, and an additive other than the crosslinking component; wherein the composition exhibits a reduced Tyndall effect when administered into a dermal region of a patient for a composition that is substantially the same except without the additive. The composition may be substantially optically clear.

In one embodiment, the additive is a vitamin C derivative, for example AA2G, which may be chemically conjugated to hyaluronic acid as described elsewhere herein.

In some embodiments, the crosslinking component is BDDE and the degree of conjugation is from about 3 mol% to about 10 mol%, or up to or greater than 15 mol%. In some embodiments, the composition further comprises an anesthetic, eg, lidocaine, in an amount suitable to provide comfort upon injection in the patient.

A method of treating fine lines in the skin of a patient is also provided. This method is usually introducing a composition as described herein into the skin of the patient. For example, the composition comprises a mixture of a hyaluronic acid component, a crosslinking component that crosslinks the hyaluronic acid, and an additive other than the crosslinking component, wherein the composition is substantially optically transparent; The dermal filler composition exhibits a reduced Tyndall effect for a composition that is substantially the same except without the additive.

In some embodiments of the present invention, the composition comprises a hyaluronic acid component crosslinked with a di or muldaamine crosslinker using EDC chemistry. For example, the crosslinking agent may be HMDA.

In certain embodiments of the present invention, the crosslinking agent is HMDA, wherein the composition has a G' of up to about 70 Pa, a G"/G' of about 0.65 to about 0.75, an extrusion force of about 24 N or less, and about 24 mg/g to about 25 mg/g. It has a final HA concentration of mg/g.

In certain embodiments of the present invention, the additive is an HA-AA2G conjugate or an HA-vitagen conjugate, wherein the degree of conjugation is from about 3 mol% to about 10 mol%, or up to about 15 mol%, or about 40 mol% is up to These compositions contain at least about 30 Pa, more preferably at least about 40 Pa, a G' of about 100 Pa, a G"/G' of about 0.30 to about 0.50, an extrusion force of about 27 N or less and about 24 mg/g to It may have a final HA concentration of about 25 mg/g.

For the purposes of this disclosure, "degree of conjugation" as used herein is defined as the molar percentage of a conjugate agent, e.g., AA2G, to repeat units (e.g., HA dimers) of hyaluronic acid. Thus, a degree of 10 mol% conjugation means that every 100HA repeat unit contains 10 conjugated AA2G. The degree of conjugation can be calculated using the method exemplified in Example 2 below or other methods known to those skilled in the art.

Example

Example 1: Conjugation of crosslinked AA2G to HA gel using BDDE as crosslinker

400.6 mg of low molecular weight hyaluronic acid (LMW HA) was hydrated in 1802 mg of 1 wt % NaOH in a syringe for -30 min. 800.7 mg of AA2G was placed in a vial followed by 713.7 mg of BDDE and 1416.8 mg of 10% NaOH. The solution (pH >12) was reacted in a 50° C. water bath for ˜20 min, then hydrated HA was added. After addition, the mixture was mixed -20 times by passing it back and forth between two syringes. The mixed paste was placed in a vial and a 50° C. water bath for ˜2.5 hours. 223.5 mg of 12M HCl was added to 9.05 g PBS, pH7.4. After ~2.5 h, an HA-AA2G gel was formed. The gel was cut into pieces and HCl-PBS solution was added to it. The gel was neutralized and allowed to swell overnight on an orbital shaker. The gel was sized through a -60 μm screen and mixed -20 times by passing it back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH7.4 buffer. Dialysis was performed for ~185 hours with frequent changes in PBS buffer. After dialysis, the gel was placed in a syringe and stored in a refrigerator at 4°C.

Example 2: Determination of AA2G Conjugation

The weight of the gel as described in Example 1 was recorded immediately before and after dialysis. The gel after dialysis was estimated to be ˜1 g/ml. > Dialysis was stopped at the point where no notable AA2G was produced in 1 L of PBS per 8 hours. AA2G was measured at 260 nm using a UV/Vis spectrophotometer (Nanodrop 2000C, ThermoScientific). A calibration curve of AA2G was calculated using different concentrations of AA2G in 2% HA (A@260 nm = 1.4838 [AA2G(mM)])

Weight of HA after dialysis: starting weight of HA x (actual weight before dialysis/theoretical weight)

mmol of AA2G after dialysis: Put the absorbance at 260 nm after dialysis in the formula (A@260nm = 1.4838 [AA2G(mM)]).

Conjugation in AA2G: (mmol of AA2G/mmol of HA)x100%

As described in Example 1, the degree of AA2G conjugation in the gel is 14.7 mol%.

Example 3: Determination of gel rheological properties

The properties of the gel obtained in Example 1 were measured using a vibrating parallel plate rheometer (Anton Paar, Physica MCR 301). The diameter of the plate used was 25 mm. The gap between the plates was set at 1 mm. For each measurement, a frequency sweep was first performed at a constant strain, followed by a strain sweep at a fixed frequency. G' (storage modulus) was obtained from the strain sweep curve at 1% strain. The value was 1450 Pa for the gel.

Example 4: AA2G conjugation to crosslinked HA gels using BDDE as crosslinker with variable degree of conjugation and gel rheology properties

The procedure was similar to that described in Example 1. The degree of conjugation is modified by controlling the crosslinker to HA and AA2G mole ratios. Gel properties were measured as described in Example 3. The details are as follows:

400.8 mg of LMW HA was hydrated in 1752.1 mg of 1% NaOH in a syringe for -30 min. 800.3 mg of AA2G was placed in a vial followed by 354.1 mg of BDDE and 1402.0 mg of 10% NaOH. The solution (pH >12) was reacted in a 50° C. water bath for ˜20 min, then hydrated HA was added. After addition, the mixture was mixed -20 times by passing it back and forth between two syringes. The mixed paste was placed in a vial and a 50° C. water bath for ˜2.5 hours. 140.9 mg of 12M HCl was added to 9.0053 g PBS, pH7.4. After ~2.5 h, an HA-AA2G gel was formed. The gel was cut into pieces and HCl-PBS solution was added to it. The gel was neutralized and allowed to swell overnight on an orbital shaker. The gel was sized through a -60 μm screen and mixed -20 times by passing it back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH7.4 buffer. Dialysis was performed for ~164.5 hours with frequent changes in PBS buffer. After dialysis, the gel was placed in a syringe and stored in a refrigerator at 4°C. The degree of conjugation is 13%. The gel storage modulus (G') is 803 Pa.

Example 5: AA2G conjugation to cross-linked HA gel using BDDE as cross-linking agent, the degree of conjugation is 5.3%, and G' is ˜300 Pa.

400.3 mg of LMW HA was hydrated in 3002.0 mg of 1% NaOH in a syringe for -30 min. 800.5 mg of AA2G was placed in a vial followed by 264.3 mg of BDDE and 1100.0 mg of 10% NaOH. The solution (pH >12) was reacted in a 50° C. water bath for ˜20 min, then hydrated HA was added. After addition, the mixture was mixed -20 times by passing it back and forth between two syringes. The mixed paste was placed in a vial and a 50° C. water bath for ˜2.5 hours. 104.2 mg of 12M HCl was added to 8.5128 g PBS, pH7.4. After ˜2.5 hours, a HA-AA2G gel was formed and HCl-PBS solution was added to it. The gel was neutralized and allowed to swell over the weekend (˜55 hours) on an orbital shaker. The gel was sized through a -60 μm screen and mixed -20 times by passing it back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH7.4 buffer. Dialysis was performed for ~114 hours with frequent changes in PBS buffer. After dialysis, the gel was placed in a syringe and stored in a refrigerator at 4°C. The degree of conjugation and the gel rheological properties are determined in the procedure as described in Examples 2 and 3. The degree of conjugation is 5.3%. The gel storage rate (G') is ~300 Pa.

Example 6: AA2G conjugation to crosslinked HA gel using star-PEG epoxide as crosslinker, the degree of conjugation is 29.4%, and G' is -235 Pa.

200.4 mg of LMW HA was hydrated in 2000 mg of 1% NaOH in a syringe for -30 min. 400 mg of AA2G was placed in a vial followed by 312.7 mg of star-PEG epoxide and 1026.5 mg of 10% NaOH. The solution was reacted in a 50° C. water bath for ˜20 min, then added to the hydrated HA. After addition, the mixture was mixed -20 times by passing it back and forth between two syringes. The mixed paste was placed in a vial and a 50° C. water bath for ˜2.5 hours. 187.4 mg of 12M HCl was added to 3.034 g PBS, pH7.4. After ˜2.5 hours, a HA-AA2G gel was formed and HCl-PBS solution was added to it. The gel was neutralized and allowed to swell over the weekend (˜68 hours) on an orbital shaker. The gel was sized through a -60 μm screen and mixed -20 times by passing it back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH7.4 buffer. Dialysis was performed for ~95 hours with frequent changes in PBS buffer. After dialysis, the gel was placed in a syringe and stored in a refrigerator at 4°C. The degree of conjugation and the gel rheological properties are determined in the procedure as described in Examples 2 and 3. The degree of conjugation is 29.4%. The gel storage modulus (G') is -235 Pa.

Example 7: AA2G conjugation to crosslinked HA gel using star-PEG epoxide as crosslinker, the degree of conjugation is 27.8%, and G' is ˜363 Pa.

200.3 mg of LMW HA was hydrated in 2000 mg of 1% NaOH in a syringe for -30 min. 400.2 mg of AA2G was placed in a vial followed by 313.4 mg of star-PEG epoxide and 1022.6 mg of 10% NaOH. This solution was added to the hydrated HA. After addition, the mixture was mixed -20 times by passing it back and forth between two syringes. The mixed paste was placed in a vial and a 50° C. water bath for ˜2.5 hours. 196.5 mg of 12M HCl was added to 3.016 g PBS, pH7.4. After ˜2.5 hours, a HA-AA2G gel was formed and HCl-PBS solution was added to it. The gel was neutralized and allowed to swell overnight (~24 h) on an orbital shaker. The gel was sized through a -60 μm screen and mixed -20 times by passing it back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH7.4 buffer. Dialysis was performed for ~98.5 hours with frequent change of PBS buffer. After dialysis, the gel was placed in a syringe and stored in a refrigerator at 4°C. The degree of conjugation and the gel rheological properties are determined in the procedure as described in Examples 2 and 3. The degree of conjugation is 27.8%. The gel storage modulus (G') is ˜363 Pa.

Example 8: AA2G conjugation to cross-linked HMW HA gel using BDDE as cross-linking agent, the degree of conjugation is 10 mol%, and G' is -240 Pa.

400.3 mg of LMW HA was hydrated in 2501.3 mg of 4% NaOH in a syringe for -30 min. 1200 mg of AA2G was placed in a vial followed by 304.7 mg of BDDE and 1178.6 mg of 16% NaOH. The solution (pH >12) was reacted in a 50° C. water bath for ˜20 min, transferred to a 20 cc syringe, and hydrated HA was added. After addition, the mixture was mixed -20 times by passing it back and forth between two syringes. The mixed paste was placed in a 20 cc vial and in a 50° C. water bath for ˜2.5 hours. After ~2.5 hours, a HA-AA2G gel was formed. Then, 226.6 mg of 12M HCl was added to 8492.2 mg of 10X PBS, pH7.4 to obtain a HCl-PBS solution, neutralized by addition of HCl-PBS solution, and the gel swelled. The gel was neutralized and swelled on an orbital shaker over 48 hours. The gel was sized through a -60 μm screen and mixed -20 times by passing it back and forth between two syringes. The gel was placed in a 20,000 MWCO CE dialysis bag and dialyzed in PBS, pH7.4 buffer. Dialysis was performed for ~114 hours with frequent changes in PBS buffer. After dialysis, the gel was placed in a syringe and stored in a refrigerator at 4°C. The degree of conjugation and the gel rheological properties are determined in the procedure as described in Examples 2 and 3. The degree of conjugation is 10 mol%. The gel storage modulus (G') is -240 Pa.

Example 9: Vitagen conjugation to crosslinked HMW HA gel using BDDE as crosslinking agent, the degree of conjugation is 15 mol%, and G' is ˜365 Pa.

398.2 mg of LMW HA was hydrated in 1753.24 mg of 1 wt% NaOH in a syringe for -40 min. BDDE (311.7 mg) was added to swell the HA and for another 80 min. The expanded HA/BDDE mixture was pre-reacted at 50° C. for 20 minutes.

801.9 mg of Vitagen was separated and dissolved in 1459.7 mg of 10 wt% NaOH and mixed with HA pre-reacted with BDDE. The reaction was continued at 50° C. for another 2.5 hours. After ˜2.5 hours, a HA-vitagen gel was formed. Then, 195 mg of 12M HCl was added to 9004.0 mg of 10X PBS, pH7.4 to obtain a HCl-PBS solution, neutralized by addition of HCl-PBS solution, and the gel swelled. The gel was neutralized and swelled on an orbital shaker over 48 hours. The gel was sized through a -60 μm screen and mixed -20 times by passing it back and forth between two syringes. The gel was placed in a 20,000 MWCO CE dialysis bag and dialyzed in PBS, pH7.4 buffer. Dialysis was carried out for ~120 hours with frequent change of PBS buffer. After dialysis, the gel was placed in a syringe and stored in a refrigerator at 4°C. Gel rheological properties are measured in the procedure as described in Example 3. The degree of conjugation was determined to be about 15 mol% using a method similar to AA2G crystals as described in Example 2. The gel storage modulus (G') is ˜365 Pa.

Example 10: Vitagen Conjugation to Linear HA Via Amidation Chemistry

200.3 mg of HMW HA was hydrated in 10 ml of water in a 60 cc syringe. 500 mg of Vitagen was dissolved in 0.5 ml of water and the solution was neutralized to pH 4.8. 197.7 mg of EDC and 149 mg of NHS were individually dissolved in 6 ml of water. Add the above solution (dissolver and EDC/NHS solution) to another 60cc syringe containing 23.5 ml of water. Mix the two syringes ~20 times by passing them back and forth between the two syringes. The mixture was stored in one syringe and immersed in a 37° C. bath for 4 hours. The solution was finally dialyzed against PBS pH7.4 buffer until no apparent vitagen was observed. The degree of conjugation was determined by a method similar to that described in Example 3. The degree of conjugation is about 10 mol%.

Example 11: AA2PG Conjugation Crosslinked to HA Gel

200.4 mg of LMW HA was hydrated in 1000 mg of MES 5.2 buffer in a syringe for -30 minutes. 292 mg of AA2P was placed in a vial followed by addition of 300 mg of star-PEG amine. The solution was reacted overnight at room temperature. The gel was hydrated with PBS buffer and dialyzed against PBS buffer to remove unreacted AA2P. Finally, the gel was characterized as described in Examples 2 and 3 to determine the degree of conjugation and the gel rheological properties. The degree of conjugation is about 20 mol%. The gel storage modulus (G') is about 500 Pa.

Example 12

Formulation of HA/BDDE Dermal Fillers with AA2G to Reduce Appearance of Fine Lines

For any of the gels described in the examples above after dialysis, an appropriate amount of free HA gel was added to the gel to improve or modify the gel cohesion and/or injectability. For example, free HA fibers were swelled in a phosphate buffer solution to obtain a uniform viscoelastic gel (“free” HA gel). Add 1 of this crosslinked gel to the HA/BDDE crosslinked gel obtained in Example 1 before the dialysis step (eg, to obtain a composition having from about 1% to about 5% w/w free HA). The resulting gel is then filled into ready-to-fill sterile syringes and autoclaved at a temperature and pressure sufficient for sterilization for at least about 1 minute. After autoclaving, the final HA/AA2G product is packaged and distributed to physicians for use as a dermal filler for non-deep injection to improve the appearance of fine lines in the orbit or other facial areas.

Example 13

Formulation of HA-AA2G Dermal Filler Containing Lidocaine

Following the procedure of Example 12, but after the dialysis step and before addition of the free HA gel, lidocaine chlorhydrate (lidocaine HCl) is added to the mixture. In powder form (lidocaine HCl) can first be solubilized in WFI and filtered through a 0.2 μm filter. A dilute NaOH solution is added to the cohesive HA/AA2G gel to reach a slightly basic pH (eg, a pH of about 7.5 to about 8). A solution of lidocaine HCl is then added to the slightly basic gel to reach a final desired concentration, eg, about 0.3% (w/w). The resulting pH of the HA/AA2G/lidocaine mixture is then about 7, and the HA concentration is about 24 mg/g. Mechanical mixing is carried out to obtain adequate uniformity in a standard reactor equipped with an appropriate compounding machine.

Example 14

Conjugation of additives containing carboxyl functional groups to HA hydrogels

Additives such as retinoic acid (AKA, tretinoin), appearance and alpha-lipoic acid contain a carboxyl functional group (-COOH). These additives are conjugated to HA hydrolysis via esterification using EDC chemistry. An example for conjugation according to the present invention is described as follows:

200 mg of HMW HA was hydrated in 10 ml of pH 4.8 MES buffer in a 60 cc syringe. In another syringe, 200 mg of retinoic acid is dissolved in 5 ml of a water-acetone mixture (water/acetone volume ratio 1:3). The two syringes are mixed through the syringe connector for about 20 times. Then 197.7 mg of EDC and 149 mg of NHS were individually dissolved in 6 ml of water. Connect the syringe containing EDC and NHS to the syringe containing HA and retinoic acid to mix the reactants for at least 20 times by passing the two syringes back and forth. The mixture was stored in one syringe and immersed in a 37° C. bath for 4 hours. The gel is dialyzed against isopropanol to remove unconjugated retinoic acid, then dialyzed against PBS buffer under azeotropic conditions. The gel is packaged into sterile syringes and stored at 4°C.

Example 15

Conjugation of additives containing hydroxyl functional groups to HA hydrogels.

Additives such as retinol (AKA, tretinoin), catalase, dimethylaminoethanol and g-tocopherol contain hydroxyl functionality (-OH). These additives are conjugated to HA hydrolysis via esterification using EDC chemistry. A typical example for conjugation is described as follows:

200 mg of HMW HA is hydrated in 10 ml of pH 4.8 2-(N-morpholino) ethanesulfonic acid (MES) buffer in a 60 cc syringe. In another syringe, 200 mg of retinoic acid is dissolved in 5 ml of a water-acetone mixture (water/acetone volume ratio 1:3). The two syringes are mixed through the syringe connector for about 20 times. Then 197.7 mg of EDC and 149 mg of NHS were individually dissolved in 6 ml of water. Connect the syringe containing EDC and NHS to the syringe containing HA and retinol to mix the reactants for at least 20 times by passing the two syringes back and forth. The mixture was stored in one syringe and immersed in a 37° C. bath for 4 hours. The gel is dialyzed against isopropanol to remove unconjugated retinol, then dialyzed against PBS buffer under azeotropic conditions. The gel is packaged into sterile syringes and stored at 4°C.

Example 16

Conjugation of additives containing hydroxyl functional groups to HA hydrogels after modification.

This is a two-step process.

Step 1: Cross-linked HA gel, e.g., a commercial HA-based dermal filler, e.g., JUVEDERM(R), Allergan or Restylane(R), Irvine, CA, Medicis Aesthetics Treated with EDC/NHS (Medicis Aesthetics, Inc.) to activate the carboxyl group of HA.

Step 2: The activated HA hydrogel is treated with an additive containing hydroxyl groups. Additives containing hydroxyl groups are retinol, catalase, dimethylaminoethanol and g-tocopherol hydroxyl functionality (-OH).

Typical examples for conjugation of additives to crosslinked HA gels are:

2 gm of Juvederm gel is mixed with 200 gm of EDC and 150 mg of NHS at room temperature. Then 200 mg of retinol in 3 ml of acetone-water mixture is added. The mixture was reacted at 37° C. for 4 hours. The gel is dialyzed against isopropanol to remove unconjugated retinol, then dialyzed against PBS buffer under azeotropic conditions. The gel is packaged into sterile syringes and stored at 4°C.

Example 17

Conjugation of growth factors, peptides or elastin to HA hydrogels

Additives such as epidermal growth factor (EGF), transforming growth factor (TGF) and peptides contain functional amine groups and are conjugated to HA to form advantageous dermal fillers. These additives are conjugated to HA via amidation chemistry. A typical example for conjugation is described as follows:

200.3 mg of HMW HA is hydrated in 10 ml of MES pH 5.4 buffered water. Add 20 mg of EGF to 100 mg of MES solution. To this mixture, 197.7 mg of EDC and 149 mg are added. The resulting reaction mixture was reacted at 37° C. for 4 hours. After the reaction is complete, the gel is dialyzed against isopropanol and then against PBS buffer under azeotropic conditions. The gel is packaged into sterile syringes and stored at 4°C.

The present invention provides a method for improving the viability of transplanted adipose tissue. The method generally comprises the step of introducing a composition into the patient's skin adjacent to the transplanted adipose tissue, wherein the composition is a composition as described elsewhere herein. For example, the composition may include hyaluronic acid and a vitamin C derivative covalently conjugated to hyaluronic acid, wherein the degree of conjugation is from about 3 mol% to about 40 mol%. In another aspect of the invention, a method for treating skin comprises introducing into the skin a composition comprising adipose tissue, hyaluronic acid and vitamin C conjugated to hyaluronic acid.

Example 18

Conjugation of growth factors, peptides or elastin to HA hydrogels

To evaluate the mitogenic effect of vitamin C and its derivatives on human adipose tissue derived stem cells (hASC), vitamin C (ascorbic acid) or its derivatives (Vitagen) in free form or AA2G) or without hASCs were cultured on tissue culture plastics for 4 days in complete MesenPro medium. Fields as described by the manufacturer (ATCC, Manassas, VA) were assessed for proliferation by MTT assay. After 4 days, concentrations of ascorbic acid, 0.25, 0.5 and 1 mM, were found to enhance the proliferation of the controls lacking ascorbic acid by 60%, 80% and 96%, respectively (yellow tetrazolium by dehydrogenase). Measure the amount of MTT converted to purple formazan (purple formazan is solubilized by detergent). The same concentration of AA2G was used to obtain proliferation of 70%, 60% and 50% of the control, respectively. Similar results were obtained with Vitagen, which showed increases of 70%, 60% and 30% respectively over the control group. In summary, vitamin C and its derivatives, AA2G and vitagen in the presence of growth factor containing medium enhance hASC proliferation in cell culture.

Cross-linked HA gel with conjugated vitamin C

According to certain embodiments of the present invention exhibiting Tyndall effect and other advantages, cross-linked HA-based gels with conjugated vitamin C using 1,4-butanediol diglycidyl ether (BDDE) as cross-linking agent The preparation is described in Examples 19 and 20 below. In Example 19, the vitamin C derivative is ascorbic acid 2-glucoside (AA2G), and in Example 20, the vitamin C derivative is ascorbyl 3-aminopropyl phosphate (Vitagen). These gels have optimal rheological properties, good injectability and high HA concentration (25 mg/g). Without wishing to be bound by any particular theory, it has been discovered by the present inventors that crosslinking BDDE and HA in the presence of AA2G or vitagen significantly alters the properties of the gel, compared to commercial HA gels crosslinked with BDDE. It has high crosslinking density, high HA concentration, low viscosity and low pressure force. As AA2G or vitagen is present during crosslinking, the gel formed has these ascorbic acid derivatives bound either alone or via BDDE as a suspending group and as a crosslinking agent bridging the HA chains. The microscopic structure of the gel changes significantly, which results in a gel with very low extrusion force even through needles as fine as 30 gauge. In addition, the gel has from about 3 mol% to about 10 mol%, or up to about 15 mol%, of vitamin C conjugated to HA. When the gels are injected, they release active vitamin C by endogenous enzymes such as α-glucosidase from fibroblasts or phosphotase. Active vitamin C can trigger skin collagenization and act as a radical scavenger to inhibit gel degradation.

Example 19

Formulation of HA/AA2G gels with reduced Tyndall effect

The mixture of 400.1 mg LMW HA and 402.3 mg AA2G in the syringe was hydrated for 60 minutes after addition of 1764.0 mg 5 wt% NaOH solution. In a separate vial, 800.8 mg of AA2G was added followed by 1401.1 mg of a 9.1 wt% NaOH solution and 252.6 mg of BDDE. The resulting solution (pH >12) was reacted in a 50° C. water bath for ˜20 min and then transferred to hydrated HA. After addition, the mixture was mixed -20 times by passing it back and forth between two syringes. The paste was then transferred to a vial and then placed in a 50° C. water bath for ˜2.5 hours. After cross-linking, a solution containing 197.0 mg of 12 M HCl and 9.18 g 10X PBS, pH 7.4 was added to neutralize the base and the gel swelled on an orbital shaker for 72 hours. The size was changed by forcing the gel through a ˜60 μm pore size mesh. The gel, which was resized by passing two syringes back and forth, was mixed ~20 times, then transferred to a cellulose ester dialysis bag, MWCO ~ 20 kDa, dialyzed against PBS, pH 7.4 buffer for 5 days, buffer changed twice a day did. After dialysis, the gel was dispensed into a 1 ml COC syringe, centrifuged at 5000 RPM for 5 minutes to remove air bubbles, and sterilized with water steam. The gel had a final HA concentration of 25 mg/g, AA2Gmol% calculated as described in Example 2 of about 10mol%, and a G' of about 80Pa. Another gel was made in a similar manner and had a G' value of about 60 Pa to about 80 Pa.

Example 19A

Formulation of HA/AA2G with lidocaine gel with reduced Tyndall effect

To the gel of Example 19, an amount of lidocaine was added to produce HA/AA2G by lidocaine gel with 0.3% ww lidocaine. Lidocaine solutions were prepared by dissolving lidocaine HCl in PBS buffer pH-7.4. An aliquot of lidocaine solution was added to the gel in Example 19 after dialysis but prior to sterilization. The gel was then thoroughly mixed to obtain a homogenized mixture with 0.3% lidocaine concentration.

Example 20

Formulation of HA/Vitagen Gels with Reduced Tyndall Effect

401.0 mg of LMW HA was hydrated in 2355.0 mg of 1 wt% NaOH solution in a syringe for ˜45 minutes. 303.8 mg BDDE was added to the hydrated HA and mixed 10 times by syringe-to-syringe mixing. The mixture was pre-reacted in a 50° C. water bath for 15 minutes. 800.1 mg of Vitagen was individually dissolved in 950.6 mg of 15 wt % NaOH followed by 510.1 Milli-Q water. The Vitagen solution was mixed back and forth 30 times with the pre-heated hydrated HA/BDDE mixture using a syringe-to-syringe mixture. The mixture was placed back in a 50° C. water bath and the reaction allowed to proceed for another 2 hours before a solution containing 148.1 mg of 12M HCl and 8523.1 mg of 10× PBS, pH7.4 was added to the crosslinker. HCl-PBS solution was added to neutralize and the gel swelled. The gel was neutralized and swelled on an orbital shaker over 48 hours. The gel was sized through a -60 μm screen and mixed -20 times by passing it back and forth between two syringes. The gel was placed in a 20,000 MWCO CE dialysis bag and dialyzed in PBS, pH7.4 buffer. Dialysis was performed for ~197 hours with frequent changes in PBS buffer. After dialysis, the gel was transferred into a 1 ml COC syringe, centrifuged at 5000 RPM for 5 minutes, and sterilized with water steam. The final HA concentration of the gel was 24 mg/g.

HA gel crosslinked via 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide

Hydrochloride (EDC) Chemistry

The preparation of cross-linked HA-based gels, in accordance with certain embodiments of the present invention exhibiting the Tyndall effect and other advantages, is described in Examples 21 and 22 below. In Example 21, the gel made via EDC chemistry using a crosslinking agent was hexamethylene diamine (HMDA), and in Example 20, 3-[3-(3-aminopropoxy)-2,2-bis( 3-Amino-propoxymethyl)-propoxy]-propylamine (4 arm amine-4 AA). The crosslinking agent is carried out under mild conditions, for example at room temperature and for example at pH 5.4. Reaction conditions can be adjusted to produce a highly reticulated gel, with optimal rheological properties, good injectability and high HA concentration (24 mg/g). Crosslinks HA at very low hydration or reaction concentrations and the binders 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) or sulfonyl It has been discovered by the inventors that it may be advantageous to have a moderate amount of HMDA or 4 AA along with -NHS(sulfo-NHS). The gels will have crosslinking points away from each other, so highly crosslinked materials have a high damping force. In contrast, crosslinking of BDDE with HA at these low hydration or reaction concentrations may be impractical due to the relative inefficiency of the crosslinking agent.

Example 21

Formulation of HA/HMDA gels with reduced Tyndall effect

20.0 g of 100 mM MES buffer pH 5.2 was added to a syringe containing 1000.0 mg of LMW HA. HMDA solution was prepared by dissolving 260.9 mg HMDA.HCl in 200.5 mg 100 mM MES buffer pH 5.2 and adding 2 μl of 1M NaOH to pH 5.2. EDC solutions were prepared by dissolving 254.2 mg of EDC in 1188.4 mg 100 mM MES buffer pH 5.2, and in a separate vial, 44.3 mg of NHS was dissolved in 1341.8 mg of 100 mM MES buffer pH 5.2. Upon full hydration of the HA, for ˜1 hour, 790 μl of HMDA solution was added to the hydrated HA. The mixture was homogenized by syringe-to-syringe mixing 10 times. Then 490 μl EDC and 490 μl NHS solution were added to the homogenized paste and again mixed 10 times by syringe-to-syringe mixing. The mixture was then transferred to a vial and cross-linked at room temperature for 5 hours before addition of 17.9 ml of IX PBS buffer pH 7.4. The gel was allowed to swell for 3 days on a roller and then a force was applied through a 60 μm pore size mesh. Resized gels were placed in a cellulose ester membrane tubing MWCO 20 KDa and dialyzed against IX PBS for 4 days with changing buffer twice a day. Gels were prepared in 1 ml COC syringes, centrifuged at 5000 RPM for 5 minutes, and sterilized with water steam. The final HA concentration of the gel was 25 mg/g.

Example 22

Formulation of HA/4 AA gels with reduced Tyndall effect

32.55 of 100 mM MES buffer pH 5.2 was added to a syringe containing 1000.4 mg of LMW HA. A 4 AA solution was prepared by dissolving 256.3 mg 4 AA in 1039.8 mg 100 mM MES buffer pH 5.2 and adding 380 μl of 6M HCl to pH 5.2. An EDC solution was prepared by dissolving 251.2 mg of EDC in 1013.8 mg 100 mM MES buffer pH 5.2, and in a separate vial, 74.7 mg of NHS was dissolved in 200.0 mg 100 mM MES buffer pH 5.2. Upon full hydration of the HA, for ˜1 hour, 260 μl of a 4 AA solution was added to the hydrated HA. The mixture was homogenized by syringe-to-syringe mixing 10 times. Then 277 μl EDC and 273 μl NHS solution were added to the homogenized paste and again mixed 10 times by syringe-to-syringe mixing. The mixture was then transferred to a vial and cross-linked at room temperature for 5 hours before addition of 6.4 ml of 10X PBS buffer pH 7.4. The gel was allowed to swell for 3 days on a roller and then a force was applied through a 60 μm pore size mesh. Resized gels were placed in a cellulose ester membrane tubing MWCO 20 KDa and dialyzed against IX PBS for 4 days with changing buffer twice a day. Gels were prepared in 1 ml COC syringes, centrifuged at 5000 RPM for 5 minutes, and sterilized with water steam. The gel had a final HA concentration of 23 mg/g.

Example 23

Determination of the rheological properties of the gels of Examples 19-22.

The rheological properties of the gel were measured using an oscillating parallel plate rheometer, Anton Paar Physica MCR 301. A plate diameter of 25 mm was used with a gap height of 1 mm. Each measurement was performed at a constant temperature of 25°C. A frequency sweep of 1 Hz to 10 Hz at a constant strain of 2% and a strain sweep of 1% to 300% at a constant frequency of 5 Hz with a logarithmic increase in the frequency followed by a logarithmic increase in frequency. Storage modulus (G') and viscous modulus (G") were obtained from strain sweeps at 1% strain.

Example 24

Extrusion force measurement of the gels of Examples 19 to 22

The force required to extrude the gel through a 30 gauge needle was measured using an Instron 5564 and Bluehill 2 software. The gel was extruded from a 1 ml COC syringe through a 30G½ TSK needle. The plunger was pushed against 11.35 mm at a speed of 100 mm/min and the extrusion force was recorded.

Example 25

Bioavailability testing of the gels of Examples 19-22

A 50 μl bolus injection of the gel was implanted intradermally on the dorsal side of Sprague Dawley rats. The implants were removed at 1 week and analyzed histologically by hematoxylin and eosin (H&E) staining, and CD68 staining, a marker for mononuclear inflammatory cells. Three 20X images of CD68 were scored from 0 to 4 based on the degree of staining. These values were then averaged to provide a sample score. Four samples were analyzed from each gel.

Example 26

Cytotoxicity test of Examples 19-22, ISO 10993-5.

In vitro cytotoxicity testing of gels was performed by NAMSA according to Agarose Overlay Method of ISO 10993-5: biological Evaluation of Medical Devices - Part 5: Tests for In Vitro Cytotoxicity. Triplicate wells were dosed with 0.1 ml of test article placed on a filtered disc as well as 0.9% NaCl solution, 1 cm long high density polyethylene as negative control, and 1 x 1 cm portion of latex as positive control. Each was placed on an agarose surface directly overlaid on a monolayer of L929 mouse fibroblasts. After incubation for 24 hours at 37° C. in 5% CO ₂ , the cultures were tested macroscopically and microscopically for any aberrant cell morphology and cell lysis. Test articles were scored from 0 to 4 based on the nearest dissolution zone of the sample. The test material from Examples 1, 3 and 4, scored as 0 as the test article, did not show any evidence of causing any cell lysis or toxicity.

Quantitative analysis of Tyndall effect

To further support visual observations and to perform comparative performance analysis of HA fillers, it was deemed necessary to perform a quantitative analysis of Tyndall effect. No quantitative technique exists in the literature for Tyndall effect specific to dermal fillers. However, based on the existing scientific understanding of light scattering and light interaction with the skin, two distinct approaches based on (a) colorimetry and (b) spectroscopy were used to quantify the Tyndall effect in skin. Based on these techniques, three distinct quantitative parameters (outlined below) were established to measure the Tyndall effect in vivo.

와 관련됨)에 대해 최대 스코어 5가 주어진다. 시험 샘플을 스코어링하기 위해 맹검 전 스케일에 대해 3명의 독립적 관찰자를 훈련시켰다.a) Tyndall Effect Visual Score : The scale ranges from 1 to 5 in increments of 0.5. A score of 1 is given to injection sites with normal skin tone and no blue discoloration. Thick, pronounced blue discoloration (typically Restylane or Juvederm Ultra Plus)

A maximum score of 5 is given for Three independent observers were trained on a pre-blind scale to score test samples.

b) Blue component of skin color - "b" : A colorimeter (CM2600D, Konica Minolta, NJ) was used to quantify the blue component of light sent from skin sites injected with various fillers. This was achieved by using the "b" component on the Lab color scale.

c) "% Blue Light Emitted from Skin": A handheld spectrophotometer (CM2600D, Konica Minolta, NJ) was used to quantify the % blue light emitted from the skin over the entire visible range. This was achieved by integrating the region under 400 nm to 490 nm and normalizing by the total area under the spectrum (400 nm to 700 nm).

Example 27

Tyndall evaluation of gels

The gel was injected intradermally through a 27G½ TSK needle using a linear threading technique into the thighs of 2 month old hairless rats. The gel was implanted subtly to mimic the clinical fine line procedure. A test on Tyndall was performed 48 hours after gel implantation. Before performing the Tyndall test, the animals were euthanized to improve the contrast of the Tyndall effect due to the lack of hemoglobin.

Images of the gels from Examples 19-21 2 days after implantation are shown in FIG. 12 . Images for commercial Juvederm Refine and Restylane Touch are shown for comparison. The bluish line (Tyndall effect) is clearly visible in the images of the commercial gels Juvederm Refine and Restylan Touch. The gels from Examples 19, 19A (not shown) and 21 did not show Tyndall effect.

Injection sites were scored using a visual score of 1 to 5 in increments of 0.5. Injection sites with score 1 showed no skin discoloration, whereas injection sites with score 5 showed severe skin blue discoloration. Spectroscopic analysis was performed on the injection site with the aid of a colorimeter (CM2600D, Konica Minolta, NJ). The blue component of skin color "b" and the percentage of blue light emitted from the skin (400 nm to 700 nm) were independently measured. 13 and 14 show the visual Tyndall score and the % of blue light emitted. The gels from Examples 19 and 21 showed no Tyndall effect and had lower visual Tyndall scores and % of blue light emission values. Tyndall score and % of emitted blue light values were higher for Juvederm Refine and Restilan Touch. Belotero Soft did not show any tyndall, and the values were similar to those of Examples 19 and 21. See Figures 13 and 14.

Example 28

Evaluation of In Vivo Persistence of Gels by History

A 50 μl bolus injection of a gel of the present invention and a commercial gel was implanted intradermally on the dorsal side of Sprague Dawley rats. The implants were removed at 1 week and analyzed histologically by hematoxylin and eosin (H&E) staining. Sections were taken precisely at the injection site. Two sections were cut from each tissue sample, and H&E stained sections were stitched using a stitching scope. Samples were then grouped and scored as follows: none (0%), low (25%), medium (50%) and high (100%) depending on the amount of material remaining. See FIG. 15 .

Example 28A

Evaluation of In Vivo Persistence of Gels by MRI

After intradermal injection in female Sprague Dawley rats, magnetic resonance imaging (MRI) was used to evaluate the change in volume and surface area over time of gels of the present invention and commercial gels over a period of 40 weeks. The gel was injected in a target volume of 150 μl per implant. The implants were placed at two contralateral sites slightly caudal to the shoulder, two contralateral sites slightly cephalic from the knee, and two diametrically opposite sites midway between the head and tail. MRI scans were performed on a 7 Tesla 70/30 Bruker Biospec MRI scanner. Images were collected on the day of transplantation (week 0) and at 12, 24, and 40 weeks after transplantation. A plot of absolute volume of gel versus time is shown in FIG. 16 below. High release gels have a high absolute volume at 40 weeks of implantation.

Example 29

Compositions of the present invention as used in the treatment of periorbital wrinkles

A 40-year-old skinny woman has microscopic wrinkles in the periorbital area and requests dermal filler treatment. Using a 30 gauge needle, the doctor introduces 0.6 ml of an HA-based gel according to the invention (as described in Example 19) not deep into her under eye wrinkles and in the lacrimal sulcus area using a linear threading technique. . Although the gel is introduced not deeply, no blue discoloration was observed and the patient is satisfied with the result.

As can be seen, the compositions of the present invention, e.g., the compositions of Examples 19 and 21, had reduced or insignificant Tyndall effect, and certain HA-based commercial gels such as Juvederm Refine/Surgiderm 18 and Velotero Soft has a substantially longer duration in the body. For example, for the "fine line" formulation Velotero Soft, inventive Example 19 not only had the Tyndall effect at least beneficial to this commercial gel, but also advantageously had a substantially longer duration in vivo.

Example 30

Injectable composition of the present invention for improving the appearance of fine lines

Additives such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and derivatives thereof, alone and in combination, are conjugated to a cross-linked hyaluronic acid gel in a manner to produce a variety of substantially clear, injectable HA-based gels. . The HA component is at least 90% by weight, for example fully low molecular weight HA as defined elsewhere herein, or about 100% low molecular weight HA. These additives are conjugated to the HA hydrogel using any suitable means. having an HA concentration of at least about 20 mg/g, e.g., about 23 mg/g, about 24 mg/g, about 25 mg/g, about 30 mg/g, suitable for injection through a fine gauge needle; The conjugated gel is sized and processed to produce an injectable pH neutral, cohesive composition. The gel has a G' value of at least about 50 Pa, about 60 Pa, about 70 Pa, up to about 80 Pa, and less than or equal to about 100 Pa. The gel is packaged and sterilized using autoclave, UV light or other suitable means.

Each gel can be applied to the patient's wrinkle, e.g., in the periorbital region, nasolabial fold region, lacrimal sulcus region, neck region, or any other facial region where dermal peeling is beneficial, e.g. at a depth of less than or equal to about 1.0 mm. Useful for injection into the skin. No discoloration was observed due to Tyndall effect, despite not deep incorporation of the gel, and the patient is satisfied with the result.

Finally, while aspects herein have been described with reference to various embodiments, it should be understood that those skilled in the art will readily appreciate that the specific disclosed embodiments are merely illustrative of the subject principles disclosed herein. Accordingly, the subject matter disclosed herein is not limited in any way to specific methods, protocols and/or reagents, and the like. As such, numerous and varied modifications or changes can be made by those skilled in the art, or alternative constructions of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of this disclosure. Changes in the detailed description may be made without departing from the spirit of the invention as defined in the appended patent claims. Finally, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which is only defined by the claims. Additionally, all subject matter contained in the above description or shown in the accompanying drawings is intended to be construed as illustrative only and not restrictive. Accordingly, the present invention is not expressly limited to what has been shown and described.

Certain embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, modifications to these described embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventor intends the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto, as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein or otherwise clearly contradicted by context.

The groups of alternative components or embodiments of the invention disclosed herein should not be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from a group for reasons of convenience and/or patentability. When any such inclusions or deletions occur, the specification is deemed to contain groups that are modified and thus fulfills the written description of all Markush groups used in the appended claims.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weights, reaction conditions, etc. used in the specification and claims are to be understood as being modified in all instances by the term "about" unless otherwise indicated. The term “about” as used herein means that the item, variable or term so quantified includes a range of plus or minus 10% above and below the value of the stated item, variable or term. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weights, reaction conditions, etc. used in the specification and claims are to be understood as being modified in all instances by the term "about" unless otherwise indicated. The term “about” as used herein means that the item, variable or term so quantified includes a range of plus or minus 10% above and below the value of the stated item, variable or term. Accordingly, unless otherwise contradicted, the numerical parameters set forth in this specification and the appended claims are approximations which may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical variable should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. While the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as concisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements.

As used in the context of describing the invention, the singular and similar references (especially in the context of the following claims) refer to both the singular and the plural, unless otherwise indicated herein or otherwise clearly contradicted by context. should be construed as all-inclusive. Recitation of ranges in this specification are merely intended to serve as a shorthand way of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (eg, "such as") provided herein is intended merely to better illuminate the invention and not to limit the scope of the invention otherwise claimed. do not raise No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in the claims using language made up of or consisting essentially of. As used in the claims, whether as amended or supplemented, the expanding term "consisting of" excludes any element, step, or ingredient not specified in the claim. The unfolding term "consisting essentially of" limits the scope of the claims to the material or steps embodied and which do not materially affect the basic and novel feature(s). Embodiments of the invention so claimed are essentially or explicitly described and made possible herein.

All patents, patent publications, and other publications mentioned and identified herein are incorporated herein by reference in their entirety, for example, for the purpose of describing and disclosing the compositions and methods described in such publications for use in connection with the present invention. , and is clearly included. These publications are provided solely for their disclosure prior to the filing date of the present invention. Nothing in this regard should be construed as an admission that the inventors are entitled to antedate such disclosure by virtue of the present invention or for any other reason. All statements as to the date or representation of the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

Claims (16)

A dermal filler composition comprising hyaluronic acid and a vitamin C derivative covalently conjugated to hyaluronic acid, the composition comprising:
wherein the vitamin C derivative is L-ascorbic acid 2-glucoside (AA2G), ascorbyl 3-aminopropyl phosphate, sodium ascorbyl phosphate (SAP), L-ascorbic acid 2-sulfate (AA2S), L-ascorb acid 2-phosphate (AA2P), 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbicine (6-Acyl-AA2G) and ascorbyl palmitate;
wherein the hyaluronic acid is crosslinked, and
wherein the dermal filler composition shows a reduced Tyndall effect compared to the same composition except that there is no vitamin C derivative. The dermal filler composition according to claim 1, wherein the vitamin C derivative is conjugated to hyaluronic acid at a degree of conjugation of 3 mol% to 15 mol%. The dermal filler composition according to claim 1, wherein the vitamin C derivative is converted into vitamin C in vivo by enzymatic cleavage. The dermal filler composition according to claim 1, wherein the vitamin C derivative is ascorbic acid 2-glucoside (AA2G). The dermal filler composition according to claim 1, wherein the vitamin C derivative is ascorbyl 3-aminopropyl phosphate. The dermal filler composition according to claim 1, wherein the vitamin C derivative is sodium ascorbyl phosphate (SAP). According to claim 1, wherein the hyaluronic acid is 1,4-butanediol diglycidyl ether (BDDE), pentaerythritol glycidyl ether (star-PEG epoxide), pentaerythritol (3-aminopropyl) ether (Star-PEG amine) or a dermal filler composition that is cross-linked with lysine. The dermal filler composition according to claim 7, wherein the hyaluronic acid is cross-linked with 1,4-butanediol diglycidyl ether (BDDE). The dermal filler composition according to claim 7, wherein the hyaluronic acid is cross-linked with lysine. The dermal filler composition according to claim 1, further comprising an anesthetic. The dermal filler composition according to claim 10, wherein the anesthetic agent is lidocaine. A dermal filler composition comprising hyaluronic acid and a vitamin C derivative covalently conjugated to hyaluronic acid, useful in a method for treating a soft tissue condition in a subject in need thereof, comprising:
The method comprises administering the dermal filler composition, wherein the vitamin C derivative is L-ascorbic acid 2-glucoside (AA2G), ascorbyl 3-aminopropyl phosphate, sodium ascorbyl phosphate (SAP), L -ascorbic acid 2-sulfate (AA2S), L-ascorbic acid 2-phosphate (AA2P), 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acid (6-Acyl -AA2G) and ascorbyl palmitate, wherein the hyaluronic acid is crosslinked, and wherein the dermal filler composition shows a reduced Tyndall effect compared to the same composition except that there is no vitamin C derivative, and wherein the soft tissue A dermal filler composition for treating a soft tissue condition wherein the condition is a wrinkle or scar. The dermal filler composition according to claim 12, wherein the dermal filler composition releases the vitamin C derivative for at least 1 month and up to 20 months. The dermal filler composition according to claim 12, wherein the vitamin C derivative is covalently conjugated to hyaluronic acid via BDDE or lysine. The dermal filler composition according to claim 12, wherein the vitamin C derivative is ascorbic acid 2-glucoside (AA2G). The dermal filler composition according to claim 12, wherein the vitamin C derivative is sodium ascorbyl phosphate (SAP).

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