KR20200116168A - 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 fine wrinkles on the face.

Description

Dermal filler composition for fine wrinkle treatment {DERMAL FILLER COMPOSITIONS FOR FINE LINE TREATMENT}

Related application

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

Technical field of the present invention

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

Skin aging is an ongoing 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 huge decrease in skin tissue thickness. Sagging of the subcutaneous tissue causes excessive skin and eyelid sagging, and causes 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 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 and acts as a filler substance; It is common within different layers of skin when 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 not only to reduce the production of hyaluronan, but also to increase the rate of its degradation. This loss of material leads to a variety of skin conditions, such as, for example, wrinkles, hollowness, moisture loss and other unwanted conditions that contribute to the appearance of aging.

Injectable dermal fillers have been used successfully to treat aged skin. To treat such skin conditions, the filler can replace the missing endogenous matrix polymer, or enhance/promote the function of the existing matrix polymer. Hyaluronic acid-based dermal fillers have become more and more popular because hyaluronic acid is a substance found naturally throughout the human body. These fillers are generally well tolerated, non-persistent, and are a fairly low risk treatment for a wide variety of skin conditions.

The Tyndall effect is a side effect that occurs in some patients receiving hyaluronic acid (HA)-based dermal fillers. The Tyndle effect is characterized by the appearance of a blue discoloration at the skin site injected with the dermal filler, indicating visible hyaluronic acid seen through the translucent epidermis. Clinical reports suggest that filler administration techniques and skin characteristics may influence the signs 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 areas. However, when these filler substances are used, for example, to fix tear furrows, glabellar wrinkles, laugh wrinkles or small wrinkles in the forehead, or when accidentally applied not too deep in the upper area of the dermis, A bluish discoloration of the skin is often observed. This phenomenon, thought to be a result of the Tindle effect, leaves a semi-permanent discoloration of the application site, and sometimes disappears only after administration of hyaluronidase to break down the filler substance. As a result, the Tindle effect is more common in patients treated for minor fine lines. Long-term signs of the Tindle effect, which the gel lasts in the skin as typically as long as 7 months, are a major cause of concern in patients.

The HA-based dermal filler gel is specially formulated to treat "fine wrinkles" wrinkles found around the tear sulcus, forehead, crow's feet, glabellar lines and the like. Commercially available HA "fine wrinkles" 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), Stylage S (G' ~192 Pa; G"/G' ~0.20, HA concentration 16 mg/ml), Teosyal First Lines (G' 59 Pa; G"/G' ~0.53, HA concentration 20mg/ml), Restylane Touch (G' ~489 Pa; G"/G' ~0.24, HA concentration 18mg/ml). 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 by reducing the final HA concentration of these gels, but most commercially available "fine wrinkle" gels, It still exhibits the Tindle effect in some patients when injected not particularly deep, for example at a depth of less than about 1 mm.

Collagen-based gels can be used in the treatment of shallow wrinkles and have not been shown to cause the tindel effect. Collagen-based gels are not highly preferred because they have a relatively poor duration in the skin and require prior testing in the subject. Radiesse® (calcium hydroxyapatite) is a subcutaneous injectable implant, its principal component is synthetic calcium hydroxyapatite, not hyaluronic acid. Unlike hyaluronic acid-based dermal fillers, calcium hydroxyapatite is not transparent, thus avoiding the problem of the tindle effect. However, if placed not too deep, this filler can appear as a white substance 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 ocular area.

It is desirable to provide a hyaluronic acid-based dermal filler for injection that does not exhibit a bluish discoloration due to the tindle effect even when injected not 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, Orthopaedic 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 significant or noticeable 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, an optically clear dermal filler useful for improving the appearance of the skin is provided, which adds volume and plumpness, and reduces the appearance of certain fine wrinkles without "tindling". The composition can be introduced into fine lines in the skin, even in thin areas of skin, rather not deeply, without causing the negative blue discoloration associated with many conventional optically clear dermal fillers.

More specifically, in one aspect of the invention, there is provided a long lasting therapeutic dermal filler composition comprising a generally biocompatible polymer such as a crosslinked hyaluronic acid component and an additive in combination with the 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. Additives include vitamins, e.g. vitamin C, e.g. stabilized forms of vitamin C or vitamin C derivatives, e.g. L-ascorbic acid 2-glucoside (AA2G), ascorbyl 3-aminopropyl phosphate ( Vitagen) or sodium ascorbyl phosphate (AA2P).

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

In a broad aspect of the present invention, a dermal filler composition is provided, the composition comprising a crosslinking component and a crosslinked hyaluronic acid component, and an additive other than the crosslinking component. The hyaluronic acid component can be chemically conjugated to the additive. Additionally, the composition exhibits a reduced Tindle effect when administered to the dermal area of a patient for substantially the same composition except for the absence of additives. The composition may further contain 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 about 3 mol% to about 40 mol, for example, about 3 mol% to about 10 mol%.

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

In another aspect of the present invention, a method of treating fine wrinkles 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 transparent, wherein the composition is For substantially the same composition except for the absence of additives, it exhibits a reduced Tindle effect when administered to the dermal area of a patient.

In another aspect of the present invention, a method of improving the appearance of the face is provided, the method comprising administering a substantially optically transparent dermal filler that does not or does not significantly exhibit a Tindle effect to a dermal area 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 a dermal filler composition for injection. 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, a method of reducing the appearance of fine lines in a thin skin area of a patient is provided, wherein the method is generally a dermal filler composition, i.e., a substantially optically transparent composition comprising 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 comprising a substantially optically transparent, generally crosslinked hyaluronic acid component and a vitamin C derivative covalently conjugated with the hyaluronic acid component. In an exemplary embodiment, the composition has a G'value of about 40 Pa to about 100 Pa. Additionally, the composition may have a hyaluronic acid concentration of about 18 mg/g to about 30 mg/g. These compositions can be particularly useful and effective in treating fine lines or non-deep creases in the skin, for example even in very thin skin, for example with a thickness of about 1 mm or less. In some embodiments, the compositions of the present invention last 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 easily understood and recognized with reference to the following drawings and detailed description.

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

In one aspect of the invention, a dermal filler composition is provided, wherein the composition generally comprises a biocompatible polymer, such as a polysaccharide such as crosslinked hyaluronic acid, and a vitamin C derivative covalently conjugated to the polymer. The composition provides a sustained release of vitamin C for skin autocollagenesis 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. Because vitamin C is released from the composition over a period of weeks or months, its concomitant 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 with hydroxyl, amine, and carboxyl functional groups: poly(vinyl alcohol), polyethylene glycol, polyvinylylamine, polyallylamine, deacetylated polyacrylamide, polyacrylic acid and polymethacrylic acid. It 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 with hydroxyl, amine and carboxyl functional groups.

Polymers include, for example, starch and derivatives thereof; Dextran and derivatives thereof, cellulose and derivatives thereof; It may be a polysaccharide 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 made up of repetitive disaccharide units. The repeating unit consists of hexose (hexasaccharide) or hexuronic acid and pharmaceutically acceptable salts thereof linked to hexosamine (hexasaccharide containing nitrogen). Members of the GAG family differ in the type of hexosamine, hexose or hexuronic acid units they contain, e.g. glucuronic acid, iduronic acid, galactose, galactosamine, glucosamine), and also the geometry of glycoside bonds. Can be different from Any glycosaminoglycan polymer is useful in the hydrogel compositions disclosed herein, provided that the glycosaminoglycan polymer improves skin conditions. Non-limiting examples of glycosaminoglycans include chondroitin sulfate, dermatan sulfate, keratan sulfate, and 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, for example, Piron and Tholin, US Patent Publication No. 2003/0148995 (name of the invention: Polysaccharide Crosslinking, Hydrogel Preparation, Resulting Polysaccharides(s) and Hydrogel(s), uses Thereof); Lebreton (name of invention: Cross-Linking of Low and High Molecular Weight Polysaccharides Preparation of Injectable Monophase Hydrogels); US Patent Publication No. 2008/0089918 to Lebreton (name of the invention: Viscoelastic Solutions Containing Sodium Hyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses); US Patent Publication No. 2010/0028438 to Lebreton et al. (name of the invention: Hyaluronic Acid-Based Gels Including Lidocaine); And US Patent Publication No. 2006/0194758 (name of the invention: Polysaccharides and Hydrogels thus Obtained); And Di Napoli's International Patent Publication WO 2004/073759 (name of the invention: 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 are, for example, hyaluronan-based dermal filler Juvidum (JUVEDERM) (registered trademark), JUVEDERM (registered trademark) 30, Juvidum (JUVEDERM) (Registered trademark) Ultra, JUVEDERM (registered trademark) Ultra Plus, JUVEDERM (registered trademark) Ultra XC, and JUVEDERM (registered trademark) Ultra Plus XC (AL, located in Irvine, Calif.) It is commercially available such as Allergan Inc. Table 1 lists representative GAGs.

An aspect of the present invention provides a hydrogel composition, in part, comprising a chondroitin sulfate polymer. The term "chondroitin sulfate polymer" as used herein refers to the disaccharides of two alternative monosaccharides and pharmaceutically acceptable salts thereof: D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc). Refers to an unbranched, sulfated polymer comprising. The chondroitin sulfate polymer may comprise a D-glucuronic acid residue epimerized with L-iduronic acid (IdoA), and the disaccharide obtained in this case is referred to as dermatan sulfate. Chondroitin sulfate polymers can have a chain of 100 or more individual sugars, each of which can be sulfated in varying 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 skin conditions. 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 a hydrogel composition comprising, in part, a keratan sulfate polymer. The term "keratan sulfate polymer" as used herein refers to a polymer of variable length comprising disaccharide units, itself β-D-galactose and N-acetyl-D-galactosamine (GalNAc) and And pharmaceutically acceptable salts thereof. Disaccharides can be fucosylated within the repeating region of keratan sulfuric acid, and N-acetylneuraminic acid caps the end of the chain. Any keratan sulfate polymer is useful in the compositions disclosed herein, provided that the keratan sulfate polymer improves skin conditions. 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 a hydrogel composition partially comprising a hyaluronan polymer. The term "hyaluronic acid polymer" as used herein is synonymous with "HA polymer", "hyaluronic acid polymer", and "hyaluronate polymer" is an anionic non-sulfurated 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 glycoside 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 skin conditions. Non-limiting examples of pharmaceutically acceptable salts of hyaluronan include sodium hyaluronan, potassium hyaluronan, magnesium hyaluronan, calcium hyaluronan, 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 an intermolecular bond in which individual polymer molecules or monomer chains are bonded to a more stable structure such as a gel. Thus, the crosslinked glycosaminoglycan polymer has an intermolecular bond in which at least one individual polymer molecule is bonded to each other. Crosslinking of the glycosaminoglycan polymer typically results in the formation of a hydrogel. These hydrogels have a high viscosity and require considerable force to be extruded through a fine needle. The glycosaminoglycan polymers disclosed herein include, but are not limited to, polyfunctional PEG-based crosslinking agents, divinyl sulfone, diglycidyl ether and bis-epoxides, biscarbodiimides, and dialdehydes and It can be crosslinked using a disulfide crosslinking agent. Non-limiting examples of hyaluronan crosslinking agents include multifunctional PEG-based crosslinkers-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), adip dihydrazide(ADH), bis(sulfosuccinimidyl)suberate(BS), hexamethylenediamine(HMDA), 1-(2,3-epoxy Propyl)-2,3-epoxycyclohexane, lysine, lysine methylester, or combinations thereof Other useful crosslinking agents are US Patent Application No. 12/910,466 filed October 22, 2010 by Stroumpoulis and Tezel ( Title of the Invention: Tunably Crosslinked Polysaccharide Compositions), which is incorporated by reference in its entirety. Any glycosaminoglycan polymer is useful in the hydrogel compositions disclosed herein, provided that glycosa Minoglycan polymers improve skin conditions Non-limiting examples of glycosaminoglycans include chondroitin sulfuric acid, dermatan sulfuric acid, keratan sulfuric acid, hyaluronan Acceptable glycosaminoglycans Non-limiting examples of salts include sodium salts, potassium salts, magnesium salts, calcium salts, and combinations thereof Glycosaminoglycans useful in the hydrogel compositions and methods disclosed herein and the resulting polymers thereof include, for example, For example, US Patent Publication No. 2003/0148995 of Piron and Tholin (name of invention: Polysaccharide Crosslinking, Hydrogel Preparation, Resulting Polysaccharides(s) and Hydrogel(s), uses Thereof); Lebreton (name of invention: Cross-Linking of Low) and High Molecular Weight Poly saccharides Preparation of Injectable Monophase Hydrogels); US Patent Publication No. 2008/0089918 to Lebreton (name of the invention: Viscoelastic Solutions Containing Sodium Hyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses); US Patent Publication No. 2010/0028438 to Lebreton et al. (name of the invention: Hyaluronic Acid-Based Gels Including Lidocaine); And US Patent Publication No. 2006/0194758 (name of the invention: Polysaccharides and Hydrogels thus Obtained); And Di Napoli's International Patent Publication WO 2004/073759 (name of the invention: Composition and Method for Intradermal Soft Tissue Augmentation), each of which is incorporated herein by reference in its entirety.

An aspect of the present specification provides a hydrogel composition comprising a glycosaminoglycan polymer having a degree of crosslinking in part. The term "degree of crosslinking" as used herein refers to the percentage of glycosaminoglycan polymer monomer units, such as, for example, the disaccharide monomer units of hyaluronan bound to a crosslinking agent. The degree of crosslinking is expressed as the ratio by weight of crosslinking agent to glycosaminoglycans. 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., a crosslinked hyaluronic acid, wherein the crosslinked glycosaminoglycan polymer, 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 both a low molecular weight and high molecular weight hyaluronan polymer. The term “high molecular weight” as used herein refers to a hyaluronan polymer having an average molecular weight of 1,000,000 Da or more when referring to “hyaluronan”. 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. The term “low molecular weight” as used herein refers to a hyaluronan polymer having an average molecular weight of less than 1,000,000 Da when referring to “hyaluronan”. Non-limiting examples of low molecular weight hyaluronan polymers are 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, and about 900,000 Da Includes.

In an embodiment, the composition comprises a low molecular weight crosslinked hyaluronan polymer. In an aspect 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 And a crosslinked hyaluronan polymer having an average molecular weight of. In another aspect of this embodiment, the composition is, 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 crosslinked hyaluronan polymers with an average molecular weight of up to 950,000 Da. In another aspect of this embodiment, the composition is, for example, about 100,000 Da to about 500,000 Da, about 200,000 Da to about 500,000 Da, about 300,000 Da to about 500,000 Da, about 400,000 Da to about 500,000 Da, about 500,000 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 Crosslinked hyaluronan polymers 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 is, 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. Crosslinked hyaluronan polymers having an average molecular weight. In another aspect of this embodiment, the composition is, for example, 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 Crosslinked hyaluronan polymers having an average molecular weight of Da. In another aspect of this embodiment, the composition is, for example, about 1,000,000 Da to about 5,000,000 Da, about 1,500,000 Da to about 5,000,000 Da, about 2,000,000 Da to about 5,000,000 Da, about 2,500,000 Da to about 5,000,000 Da, about 2,000,000 Da And crosslinked hyaluronan polymers having an average molecular weight of from about 3,000,000 Da to about 2,500,000 Da to about 3,000,000 Da.

In yet 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 yet 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 And a combination of a high molecular weight hyaluronan polymer and a low molecular weight hyaluronan polymer at 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 that bind 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 an aspect of this embodiment, the composition is 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, the uncrosslinked glycosaminoglycan polymer is often mixed with a glycosaminoglycan polymer-based hydrogel composition as a lubricant to facilitate the extrusion process 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, 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, It is present 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 an uncrosslinked glycosaminoglycan, wherein the uncrosslinked glycosaminoglycan is, for example, 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 an uncrosslinked glycosaminoglycan, wherein the uncrosslinked glycosaminoglycan is, for example, at most 1 mg/g, at most 2 mg/g. g, up to 3 mg/g, up to 4 mg/g, up to 5 mg/g, up to 10 mg/g, up to 15 mg/g, up to 20 mg/g, or up to 25 mg/g. In another aspect of this embodiment, the composition comprises an 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, It is present at a concentration of about 10.5 mg/g to about 16.5 mg/g, about 11.5 mg/g to about 15.5 mg/g, or about 12.5 mg/g to about 14.5 mg/g.

Aspects of the present specification provide a hydrogel composition 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 traces of the crosslinked matrix polymer can be detected. In an aspect of this embodiment, the composition comprises chondroitin sulfuric acid essentially free of crosslinked chondroitin sulfate polymer, dermatan sulfuric acid essentially free of crosslinked dermatan sulfate polymer, keratan sulfuric acid essentially free of crosslinked keratan sulfate polymer Heparan sulfuric acid essentially free of the resulting heparan polymer, heparan sulfuric acid essentially free of the crosslinked heparan sulfate polymer, or hyaluronan sulfuric acid essentially free of the crosslinked hyaluronan polymer.

Aspects of the present specification provide a hydrogel composition completely free of partially crosslinked glycosaminoglycan polymers. The term "completely free" as used herein refers to a composition within the detection range of the instrument or process used, and the crosslinked glycosaminoglycan polymer cannot be detected or its presence cannot be ascertained. . In an aspect of this embodiment, the composition comprises chondroitin sulfuric acid completely free of crosslinked chondroitin sulfate polymer, dermatan sulfate completely free of crosslinked dermatan sulfate polymer, keratan sulfate completely free of crosslinked keratan sulfate polymer, crosslinked 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 and 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 ratio results in a composition disclosed herein that ameliorates the skin conditions disclosed herein. Non-limiting examples of gel:fluid ratios in the compositions of the present invention are 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 an 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 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, Maximum 2:98, maximum 3:97, maximum 4:96, maximum 5:95, maximum 6:94, maximum 7:93, maximum 8:92, maximum 9:91, maximum 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 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, Max 20:80, Max 25:75, Max 30:70, Max 35:65, Max 40:60, Max 45:55, Max 50:50, Max 55:45, Max 60:40, Max 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 about 60:40 to about 100:0.

The hydrogel compositions disclosed herein may further include other agents or combinations of agents that provide beneficial effects when the composition is administered to a subject. Such advantageous agents are antioxidants, anti-itch agents, anti-cellulite agents, anti-scar agents, anti-inflammatory agents, anesthetics, anti-irritants, vasoconstrictors, vasodilators, anti-bleeding agents-like hemostatic agents or anti-fibrin dissolving agents, exfoliating agents, swelling agents, anti-acne agents, pigmentation agents. , Anti-pigmentation agents or moisturizing agents.

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

Aspects of the present specification provide a hydrogel composition disclosed herein that may optionally include an anesthetic agent in part. The anesthetic agent is preferably a local anesthetic, i.e., an anesthetic that causes a loss of reversible local anesthesia and nociception, for example aminoamide local anesthetics and aminoester local anesthetics. The amount of anesthetic agent included in the composition disclosed herein is an amount effective to relieve pain experienced by an individual upon administration of the composition. As such, the amount of anesthetic agent contained in the composition disclosed herein is from about 0.1% to about 5% by weight of the total composition. Non-limiting examples of anesthetics include lidocaine, ambucaine, amolaone, amilocaine, benoxynate, benzocaine, bethoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butetamine, Butoxycaine, Carticaine, Chloroprocaine, Cocoethylene, Cocaine, Cyclomethicine, Dibucaine, Dimethisoquine, Dimethocaine, Diperodone, Dicyclonaine, Euprosine, Phenalcomine, Formokine, Hexyl Caine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, reboxadrol, lidocaine, mepivacaine, meprilcaine, metabutoxycaine, methyl chloride, mirtecaine, naepine , Octacaine, orthocaine, oxetazine, parethoxycaine, phenakaine, phenol, piperocaine, pyridocaine, polydocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, Propiocaine, propoxycaine, pseudococaine, pyrocaine, ropivacaine, salicyl alcohol, tetracaine, tolicaine, trimecaine, zolamin, combinations thereof, and salts thereof. Non-limiting examples of aminoester local anesthetics include procaine, chloroprocaine, cocaine, cyclomethicine, cytokine (larocaine), propoxycaine, procaine (novocaine), proparacaine, tetracaine (ame. Tocaine). Non-limiting examples of aminoamide local anesthetics include articaine, bupivacaine, synchocaine (dibucaine), etidocaine, levobupivacaine, lidocaine (lignocaine), mepivacaine, piperocaine, prill. Locaine, ropivacaine 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 agent and salts thereof. In an aspect of this embodiment, the compositions disclosed herein comprise an aminoamide local anesthetic and salts thereof or an aminoester local anesthetic and salts thereof. In another aspect of this embodiment, the composition disclosed herein is procaine, chloroprocaine, cocaine, cyclomethicine, cytokine, propoxycaine, procaine, proparacaine, tetracaine, or salts thereof, or And any combination of these. In another aspect of this embodiment, the compositions disclosed herein are Articaine, Bupivacaine, Synchocaine, Etidocaine, Levobupivacaine, Lidocaine, Mepivacaine, Piperocaine, Prilocaine, Ropivaca Phosphorus, trimecaine, or salts thereof or any combination thereof. In another aspect of this embodiment, the composition disclosed herein comprises a lidocaine/prilocaine combination.

In another aspect of this embodiment, the composition disclosed herein comprises, for example, 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%, about 0.8%, about 0.9%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0% %, about 8.0%, about 9.0%, or about 10% by weight of an anesthetic agent. In another aspect, the 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% or at least 10% by weight of an anesthetic agent. In another embodiment, the composition disclosed herein comprises a total composition of, 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 Wt%, max 0.8 wt% max 0.9 wt%, max 1.0 wt%, max 2.0 wt%, max 3.0 wt%, max 4.0 wt%, max 5.0 wt%, max 6.0 wt%, max 7.0 wt%, max 8.0 wt %, up to 9.0% by weight, or up to 10% by weight of an anesthetic agent. In a further aspect, the composition disclosed herein comprises, for example, 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 An anesthetic agent in an amount of from about 0.5% to about 1.0%, or from about 0.5% to about 2.0% by weight.

In other embodiments, the compositions disclosed herein do not include an anesthetic agent.

In one aspect of the 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 advantageous agent in combination with the polymer. Dermal fillers for injection are 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 (trade name)), sodium ascorbyl phosphate (AA2P), disodium ascorbyl sulfate, and ascorbyl 3-aminopropyl phosphate (vitagen ).

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 the 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 a composition 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 agent or multiple vitamin C agents.

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

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

Aspects of the present invention provide a hydrogel composition disclosed herein that partially exhibits a complex modulus, an elastic modulus, a viscous modulus, and/or tan δ. The composition as disclosed herein is viscoelastic, i.e. the composition is an elastic component (solid-like, e.g. crosslinked glycosaminoglycan polymer) and a viscous component (liquid-like, strain) when a force is applied For example uncrosslinked glycosaminoglycan polymer or carrier phase). The rheological property that explains this property is the complex modulus (G*), which defines the overall resistance of the composition to deformation. The complex modulus of elasticity 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 modulus of elasticity (G') and the modulus of viscosity (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.

The modulus 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 is characterized by the stiffness of the composition, and is also known as the storage modulus, as 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 the composition. The modulus of elasticity of an object is defined as the slope of its stress-strain curve in the elastically deformed region: λ = stress/strain, where λ is the modulus of elasticity (Pascal); Stress is the force that causes the deformation divided by the area to which the force is applied; Deformation is the ratio of the change caused by stress to the original state of an object. Despite the dependence of the speed at which the force is applied, a stiffer composition will have a higher modulus of elasticity and will take a greater force to deform the material over a given distance, for example by injection. Specifying how the stress, including direction, is measured allows many types of modulus to be defined. The three main modulus are tensile modulus, shear modulus and volume modulus.

The viscous modulus is also known as the loss rate, as it accounts for the energy lost as viscous dissipation. Tan δ is the ratio of the viscous modulus and 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 do not exhibit an 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 300 Pa, about 350 Pa, about 400 Pa, about 450 Pa, about 500 Pa, about 550 Pa, about 600 Pa, about 650 Pa, about 700 Pa, about 750 Pa, about 800 Pa, about 850 Pa, About 900 Pa, about 950 Pa, about 1,000 Pa, about 1,200 Pa, about 1,300 Pa, about 1,400 Pa, about 1,500 Pa, about 1,600 Pa, about 1700 Pa, about 1800 Pa, about 1900 Pa, about 2,000 Pa, about 2,100 It exhibits an elastic modulus of Pa, about 2,200 Pa, about 2,300 Pa, about 2,400 Pa, or about 2,500 Pa. In another aspect 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, 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 900 Pa, at least 950 Pa, at least 1,000 Pa, at least 1,200 Pa, at least 1,300 Pa, at least 1,400 Pa, at least 1,500 Pa, at least 1,600 Pa, at least 1700 Pa, at least 1800 Pa, at least 1900 Pa, at least 2,000 Pa, at least 2,100 It exhibits 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 is, 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 850 It represents an elastic modulus of Pa, maximum 900 Pa, maximum 950 Pa, maximum 1,000 Pa, maximum 1,200 Pa, maximum 1,300 Pa, maximum 1,400 Pa, maximum 1,500 Pa, or maximum 1,600 Pa. In another aspect of this embodiment, the hydrogel composition is, for example, from about 25 Pa to about 150 Pa, from about 25 Pa to about 300 Pa, from about 25 Pa to about 500 Pa, from 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 1,600 Pa, from about 900 Pa to about 1,600 Pa, from about 1,000 Pa to about 1,600 Pa, from about 1,100 Pa to about 1,600 Pa, from about 1,200 Pa to about 1,600 Pa, from about 500 Pa to about 2,500 Pa, from about 1,000 Pa to about 1,000 Pa 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 1,900 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 do not exhibit a viscosity 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, about 100 Pa, about 150 Pa, about 200 Pa, about 250 Pa, about 300 Pa, about 350 Pa, about 400 Pa, about 450 Pa, about 500 Pa, about 550 Pa, about 600 Pa, about 650 Pa, Or it shows a viscosity of 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, Max 100Pa, Max 150Pa, Max 200Pa, Max 250Pa, Max 300Pa, Max 350Pa, Max 400Pa, Max 450Pa, Max 500Pa, Max 550Pa, Max 600Pa, Max 650Pa Or, it shows a viscosity modulus of up to 700 Pa. In another aspect of this embodiment, the hydrogel composition is, for example, about 10 Pa to about 30 Pa, about 10 Pa to about 50 Pa, about 10 Pa to about 100 Pa, 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 It exhibits a viscosity modulus of 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 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 another aspect of this embodiment, the hydrogel composition is, 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, A tan δ of 1.2 max, 1.3 max, 1.4 max, 1.5 max, 1.6 max, 1.7 max, 1.8 max, 1.9 max, 2.0 max, 2.1 max, 2.2 max, 2.3 max, 2.4 max or 2.5 max. In another aspect of this embodiment, the hydrogel composition is, for example, about 0.1 to about 0.3, about 0.3 to about 0.5, about 0.5 to about 0.8, about 1.1 to about 1.4, about 1.4 to about 1.7, about 0.3 To about 0.6, about 0.1 to about 0.5, about 0.5 to about 0.9, about 0.1 to about 0.6, about 0.1 to about 1.0, about 0.5 to about 1.5, about 1.0 to about 2.0, or about 1.5 to about 2.5 Show.

Aspects of the present specification provide a hydrogel composition disclosed herein having partially transparency and/or translucency. Optical transparency is a physical property that allows visible light to pass through a material, whereas translucency (also called translucency or translucency) only allows light to diffuse 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, the hydrogel composition disclosed herein is optically translucent. In aspects of this embodiment, the hydrogel composition is, for example, about 75% light, about 80% light, about 85% light, about 90% light, about 95% light, or about 100% Transmits the light of In another aspect of this embodiment, the hydrogel composition is configured to disperse, for example, at least 75% light, at least 80% light, at least 85% light, at least 90% light, or at least 95% light. Deliver. 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 % To about 100% of light, or about 95% to about 100% of light is transmitted in a diffuse manner In an embodiment, the hydrogel composition disclosed herein is optically transparent and transmits 100% visible light.

The hydrogel composition disclosed herein can be further treated by grinding the hydrogel into particles, and optionally, mixed with a carrier phase such as water or saline, for injection or topical substance-like solutions, oils, lotions, Forms gels, ointments, creams, slurries, plasters or pastes. As such, the disclosed hydrogel composition may be a single phase or a multiphase composition. 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 the compositions disclosed herein are for injection. The term “injectable” as used herein refers to a substance having the properties that require administration of the composition to an area of the subject's skin using an injection device with a fine needle. The term “fine needle” as used herein refers to a needle of 27 gauge or less. The injection ability of the compositions disclosed herein can be accomplished by sizing the hydrogel particles as discussed above.

In an aspect of this embodiment, the hydrogel composition disclosed herein can be injected through a fine needle. In another aspect of this embodiment, the hydrogel composition disclosed herein is injectable through a needle of, for example, about 27 gauge, about 30 gauge, or about 32 gauge. In another aspect of this embodiment, the hydrogel composition disclosed herein is injectable through a needle of, for example, 22 gauge or less, 27 gauge or less, 30 gauge or less, or 32 gauge or less. In another aspect of this embodiment, the hydrogel composition disclosed herein may 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, It is injectable through a needle of about 22 gauge to about 27 gauge, or about 27 gauge to about 32 gauge.

In an aspect of this embodiment, the hydrogel composition disclosed herein is 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 15 N. In another aspect of this embodiment, the 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. It can be injected through a 27 gauge needle with an extrusion force of about 15N or less, about 10N or less, or about 5N or less. In another aspect of this embodiment, the 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 30 gauge needle with an extrusion force of 20N or less, about 15N or less, about 10N or less, or about 5N or less. In another aspect of this embodiment, the 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 with an extrusion force of 20N or less, about 15N or less, about 10N or less, or about 5N or less.

Aspects of the present specification provide a hydrogel composition disclosed herein that partially exhibits adhesiveness. Adhesion, also referred to as cohesive, cohesive 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). Cohesive strength, in particular, the molecular weight ratio of the initial free glycosaminoglycan polymer, the degree of crosslinking of the glycosaminoglycan polymer, the glycosaminoglycan polymer, the amount of the remaining free glycosaminoglycan polymer after crosslinking, And the pH of the hydrogel composition. The composition should be sufficiently cohesive, such as remaining localized at the site of administration. Additionally, in certain applications, sufficient cohesive force is important for retaining its shape and hence functionality for the composition in the event of a mechanical loading cycle. As such, in one embodiment, the compositions disclosed herein exhibit a cohesive force equivalent to water. In another embodiment, a hydrogel composition disclosed herein exhibits sufficient adhesion that remains localized at the site of administration. In another embodiment, the hydrogel composition disclosed herein exhibits sufficient adhesion to retain its shape. In a further embodiment, the 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 osmoticly active solute in a solution. The term "physiologically acceptable osmolarity" as referred to herein refers to a characteristic of the normal function of a living organism or the osmolarity accordingly. As such, administration of a hydrogel composition as disclosed herein exhibits an osmolar concentration that has a permanent deleterious or substantially non-long-term effect when administered to a mammal. Osmolarity is expressed in terms of the osmolality of the osmoly active solute per liter of solvent (Osmol/L or Osm/L). Osmolarity is independent of molarity because it measures the moles of solute particles that are osmolytically active rather than the moles of solute. The difference is because some compounds can dissociate in solution, while others may not. The osmolality of a solution can be calculated from the following expression: Osmol/L = ∑ φ _i η _i C _i where φ is the osmotic coefficient that calculates the degree of abnormality of the solution; η is the number of particles (eg, ions) from which the molecule dissociates; C is the molar concentration of the solute; i is an index indicating the identity of a specific solute. The osmolarity of the hydrogel composition disclosed herein can be measured using a conventional method of treating a solution.

In an embodiment, a hydrogel composition disclosed herein exhibits a physiologically acceptable osmolarity. The term "osmolar concentration" as used herein refers to the concentration of osmoly active solute per kilo of solvent in the body. The term "physiologically acceptable osmolality" as referred to herein refers to the osmolyte concentration according to the characteristic or normal function of a living organism. As such, administration of the hydrogel composition disclosed herein exhibits an osmotic concentration that has a permanent deleterious or substantially non-long term effect when administered to a mammal. The osmolality is expressed in terms of the osmolality of the osmoly active solute per kilogram of solvent (osmol/kg or Osm/kg), and is equal to the sum of the molar concentrations of all solutes in the solution. The osmolality of the solution can be measured using an osmometer. The most commonly used instrument in modern laboratories is the freezing point drop osmometer. This instrument measures the change in the freezing point that occurs as the osmotic concentration increases (the freezing point drop osmometer) or the change in the water vapor pressure in the solution as the osmotic concentration increases (the water 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. It represents an osmolar concentration of ℓ, about 400 mOsm/ℓ, about 450 mOsm/ℓ, or about 500 mOsm/ℓ. In another aspect of this embodiment, the hydrogel composition is, 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, up to 100 mOsm/L, up to 150 mOsm/L, up to 200 mOsm/L, up to 250 mOsm/L, up to 300 mOsm/L, up to 350 mOsm /ℓ, maximum 400 mOsm/ℓ, maximum 450 mOsm/ℓ, or maximum 500 mOsm/ℓ osmolar concentration. 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 , About 275 mOsm/L to about 300 mOsm/L, or 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 “stable” or “stable” as indicated herein means that any substantial or significant degree is degraded, degraded or destroyed during storage prior to administration to a subject. It refers to a composition that is not easy. The terms "substantial thermal stability", "substantially thermal stability", "autoclave stability" or "steam sterilization stability" as referred to herein refer to the present specification that is substantially stable when a heat treatment as disclosed herein is applied. It refers to the hydrogel composition disclosed in.

The stability of the hydrogel composition disclosed herein can be determined by subjecting the hydrogel composition to a heat treatment such as steam sterilization, for example 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 about 1 minute to about 10 minutes. The substantial stability of the hydrogel composition disclosed herein is 1) by determining the change in the extrusion force (ΔF) of the hydrogel composition disclosed herein after sterilization (where the change in the extrusion force less than 2N is (hydrogel with specified additives The extrusion force of the gel composition)-(indicating a substantially stable hydrogel composition as measured by (extrusion force of the hydrogel composition without added additives)); And/or 2) by determining the change in the rheological properties of the hydrogel composition disclosed herein after sterilization (wherein the change at tan δ 1 Hz less than 0.1 is (tan δ 1 Hz of the gel formulation by the additive)- (Indicating a substantially stable hydrogel composition as measured by tan δ 1 Hz) of the gel formulation in the absence of) can be evaluated. As such, the substantially stable hydrogel composition disclosed herein possesses one or more of the following characteristics after sterilization: homogeneity, extrusion force, cohesive force, hyaluronan concentration, agent(s) concentration, osmolarity, pH. , Or other rheological features 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. , About 120°C, about 125°C, or about 130°C. In another aspect 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. It is treated using a heat treatment of at least 120C, at least 125C or at least 130C. In another aspect of this embodiment, a hydrogel composition comprising a glycosaminoglycan polymer and at least one agent disclosed herein is, for example, about 100° C. to about 120° C., about 100° C. to about 125° C. ℃, about 100 ℃ to about 130 ℃, about 100 ℃ to about 135 ℃, about 110 ℃ to about 120 ℃, about 110 ℃ to about 125 ℃, about 110 ℃ to about 130 ℃, about 110 ℃ to about 135 ℃, It is treated using a heat treatment of 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 the hydrogel composition disclosed herein can be determined by subjecting the hydrogel composition to a heat treatment such as steam sterilization, for example at normal pressure or under reduced pressure (eg, autoclaving). The long-term stability of the hydrogel composition disclosed herein can be determined by subjecting the hydrogel composition to a heat treatment such as storage in an environment of about 45° C. for about 60 days. The long-term stability of the hydrogel composition disclosed herein is 1) by evaluating the clarity and color of the hydrogel composition after heat treatment at 45° C. (clear, colorless hydrogel composition indicates a substantially stable hydrogel composition); 2) By determining the change in the extrusion force (ΔF) of the hydrogel composition disclosed in the present specification after heat treatment at 45°C (change in the extrusion force less than 2N is (extrusion force of the hydrogel composition having the specified additives before heat treatment at 45°C) -(Represents a substantially stable hydrogel composition as measured by the extrusion force of the hydrogel composition with the specified additive after heat treatment at 45° C.); And/or 3) by determining the change in the rheological properties of the hydrogel composition disclosed herein after sterilization (wherein, the change at tan δ 1 Hz less than 0.1 is (the tan δ of the gel formulation with the specified additives prior to heat treatment at 45° C.) 1 Hz)-(indicates a substantially stable hydrogel composition as measured by the tan δ 1 Hz of the gel formulation with the specified additives after heat treatment at 45° C.)). 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 heat treatment at 45° C.: clarity (transparency and translucency), homogeneity and cohesiveness.

In an aspect 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 another aspect 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 another aspect of this embodiment, the hydrogel composition is, for example, about 3 months to about 12 months, about 3 months to about 18 months, about 3 months to about 24 months, 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 Is substantially stable at room temperature for 24 months, about 12 months to about 30 months, about 12 months to about 36 months, about 18 months to about 24 months, about 18 months to about 30 months, or about 18 months to about 36 months .

The composition may optionally contain other pharmaceutically acceptable ingredients including, but not limited to, buffers, preservatives, tonicity modifiers, salts, antioxidants, osmolality modifiers, emulsifiers, wetting agents, etc. It doesn't work.

Pharmaceutically acceptable buffers are buffers that can be used to prepare the hydrogel compositions disclosed herein, provided that the obtained preparation 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 the 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 can be adjusted, provided that the resulting formulation is pharmaceutically acceptable. It is understood that an acid or base can be used to adjust the pH of the pharmaceutical composition if necessary. Any buffered pH level may be useful in formulating a pharmaceutical composition, provided that a therapeutically effective amount of the matrix polymeric active ingredient is restored using this 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 the hydrogel composition disclosed herein may be about 5.0 to about 8.0, or about 6.5 to about 7.5, about 7.0 to about 7.4, or about 7.1 to about 7.3.

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

Pharmaceutically acceptable tonicity modifiers useful in the hydrogel compositions disclosed herein include, for example, sodium chloride and potassium chloride; And salts such as glycerin, but are not limited thereto. 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 the corresponding free base form. It is understood that these and other substances known in the pharmaceutical world may be included in the pharmaceutical compositions disclosed herein. Other non-limiting examples of pharmacologically acceptable ingredients are, 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 a method of treating a soft tissue condition in a subject, in part, by administering a hydrogel composition disclosed herein. The term “treating” as referred to herein refers to reducing or eliminating in a subject the cosmetic or clinical symptoms of a soft tissue condition characterized by soft tissue imperfections, defects, diseases and/or disorders; Or to delay or prevent the onset of cosmetic or clinical symptoms of a condition characterized by soft tissue imperfections, defects, diseases and/or disorders in a subject. 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 may mean 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 soft tissue imperfections, defects, diseases and/or disorders 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 concurrent treatment. One of skill in the art will know the appropriate symptoms or indicators associated with a specific soft tissue defect, disease and/or disorder, and will 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 individual is typically a human of any age, sex or race. Any individual that is typically a candidate for conventional procedures for treating soft tissue diseases 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 (eg, scars) suitable for treatment can be treated with the hydrogel composition disclosed herein. In addition, the currently disclosed hydrogel compositions and methods can be applied to individuals seeking small/medium magnification, shape change or contour change of a body part or area, which is not technically possible by existing soft tissue implantation techniques, or It may not be aesthetically acceptable. Pre-procedure evaluation typically includes routine history and physiological examination in addition to informed consent to disclose 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 imperfections, defects, diseases and/or disorders, such as breast imperfections, defects, diseases and/or disorders, such as breast augmentation, breast reconstruction, breast fixation, porosity, pleural hypoplasia. Defects due to transplant complications such as dystrophy, Poland syndrome, capsule contraction and/or rupture; Facial imperfections, defects, diseases and/or disorders, such as facial enlargement, facial reconstruction, mesotherapy, Parry-Romberg syndrome, lupus erythematosus, dermal divot, scars, sunken checks , Thin lips, nasal imperfections or consolidation, posterior orbital imperfections or defects, facial folds such as glabellar lines, wrinkles and/or wrinkles, nasolabial lines, upper lip wrinkles, and/or tail wrinkles and/or other contour anomalies or facial Incomplete; 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 arms, lower arms, hands, shoulders, back, torso including abdomen, buttocks, upper legs, lower legs including calves, including soles of feet 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). The term “mesotherapy” as referred to herein refers to the non-surgical beauty of the skin involving intradermal, intradermal and/or subcutaneous injection of an agent administered as a plurality of small droplets into the epidermis, dermal-epidermal junction and/or into the dermis. Refers to a therapeutic technique.

The amount of hydrogel composition used by any of the methods disclosed herein is typically the desired alteration and/or amelioration, reduction and/or elimination of desired soft tissue disease symptoms, clinical and/or clinical and/or desired by the individual and/or physician /Or will be determined based on the cosmetic effect and 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 measurements: 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 can be indicated by one or more of the following clinical and/or cosmetic measurements: facial features such as increased size, shape and/or contour of the lips, cheeks, or eye area. Increased size, shape and/or contour of the; Altered size, shape and/or contour of facial features such as altered size, shape and/or contour of the shape of the lips, cheek or eye area; 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 tension; Reduction or removal of stretch marks or marks; Increased and/or improved skin tone, shine, brightness and/or radiance; Increased and/or improved skin color, reduction or elimination of skin paleness; Duration 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 compositions and methods for sphincter support may be indicated by one or more of the following clinical measures: reduced incontinence cycle, 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 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 40g, about 50g, about 60g, about 70g, about 80g, about 90g, about 100g, about 150g or about 200g. In another aspect of this embodiment, the amount of the hydrogel composition administered is, for example, about 0.01 g to about 0.1 g, about 0.1 g to about 1 g, about 1 g to about 10 g, about 10 g to about 100 g, or about 50 g to It is about 200g. 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 another aspect of this embodiment, the amount of the hydrogel composition administered is, for example, about 0.01 mL to about 0.1 mL, about 0.1 mL to about 1 mL, about 1 mL to about 10 mL, about 10 mL to about 100 mL. , Or from about 50 ml to about 200 ml.

The duration of treatment will typically be determined by the individual and/or physician based on the desired cosmetic and/or clinical effect and the body part or area being treated. In aspects of this embodiment, administration of the 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, Soft tissue conditions can be treated for about 13 months, about 14 months, about 15 months, about 18 months, or about 24 months. In another aspect of this embodiment, the administration of the hydrogel composition disclosed herein is, for example, 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, The soft tissue condition can be treated for at least 13 months, at least 14 months, at least 15 months, at least 18 months, or at least 24 months. In another aspect of this embodiment, administration of the hydrogel composition disclosed herein is, for example, about 6 months to about 12 months, about 6 months to about 15 months, 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 of the present specification provide, in part, a step of administering a hydrogel composition disclosed herein. The term “administering” as indicated herein refers to any delivery mechanism that provides a composition disclosed herein to an individual that potentially results in clinically, therapeutically or experimentally beneficial results. The actual delivery mechanism used to administer the composition to an individual is 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, the rate of excretion of the particular composition used. , The pharmacokinetics of the particular composition used, the properties of other compounds included in the particular composition used, the particular route of administration, particular characteristics, the history and risk factors of the individual, such as age, weight, general health, etc. It can be determined by one of ordinary skill in the art by considering factors including, but not limited to, combinations. In an aspect of this embodiment, the composition disclosed herein is administered to an area of the subject's skin by injection.

The route of administration of the hydrogel composition to an individual patient will typically be determined by the individual and/or physician based on the desired cosmetic and/or clinical effect and the part or area of the body being treated. The compositions disclosed herein include, but are not limited to, needled syringes, pistols (e.g., hand-compressed pistols), catheters, either locally or by direct surgical implantation, any means known to those skilled in the art. Can be administered by The hydrogel composition disclosed herein can be administered into a skin area such as, for example, a dermal area or a subcutaneous area. For example, a hydrogel composition disclosed herein can be injected using a needle having a diameter of about 0.26 mm to about 0.4 mm and a length in the range of about 4 mm to about 14 mm. Alternatively, the needle can 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 can be combined with a syringe, catheter and/or pistol.

Additionally, the compositions disclosed herein may be administered once or multiple times. Ultimately, the time spent will follow quality control standards. For example, the hydrogel compositions disclosed herein may be administered at one or more times several working times, with working times a few days or weeks away. 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 the hydrogel compositions disclosed herein to a subject may be administered on a monthly or two-month basis or every 3, 6, 9 or 12 months.

Aspects of the present specification provide, in part, a dermal region. The term “dermal region” as referred to herein refers to a region of the skin including the epidermal-dermal junction and the dermis including the non-deep dermis (papillary region) and the deep dermis (reticular region). The skin is made up of three main layers: the epidermis, which provides water resistance and acts as a barrier against infection; The dermis acting as a place for the external part of the skin; And the lower skin (subcutaneous fat layer). The epidermis does not contain blood vessels and is supplied with nutrients 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 a layer of skin under the epidermis that is made of connective tissue and relieves the body's impact from stress and deformation. The dermis is tightly bound to the epidermis by the lowermost membrane. It also hides a number of mechanical receptors/nerve endings that provide a sense 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 the removal of nutrients and waste products from their own cells as well as from the basal layer of the epidermis. The dermis is structurally divided into two areas: a supercritical area adjacent to the epidermis called the papillary area and a deeper, thicker area known as the reticular area.

The papillary area consists of loose areolar tissue connective tissue. It is termed a finger-like protrusion called a nipple extending toward the epidermis. The nipples have a "bumpy" surface that interlocks with the epidermis, providing the dermis that strengthens the bond between the two layers of the skin. The reticular area lies deeper than the nipple area and is usually much thicker. It consists of dense irregular connective tissue, and gets its name from the dense concentration of collagenous, elastic and reticulated fibers that are woven throughout it. These protein fibers give the dermis its strength, extensibility and elasticity properties. In addition, hair roots, sebaceous glands, sweat glands, receptors, nails, and blood vessels are located in the reticulated region. Tattoo ink is retained in the dermis. Also, stretch marks from pregnancy are located in the dermis.

The lower skin is placed under the dermis. Its purpose is to attach the dermal regions of the basal bone and muscles as well as supply blood vessels and nerves to it. 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 an aspect of this embodiment, the hydrogel composition disclosed herein is administered to a skin area of a subject by injection into a dermal area or subcutaneous tissue area. In an aspect of this embodiment, the hydrogel composition disclosed herein is administered to a dermal region of a living body by injection into, for example, an epidermal-dermal junction region, a papillary region, a reticulant 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 patients. For example, a method for treating fine wrinkles 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 specification includes the step of administering a hydrogel composition disclosed herein to a subject partially suffering from a skin disease, wherein the administration of the composition improves the skin disease, thereby treating the skin disease. In an aspect of this embodiment, the skin condition comprises administering a hydrogel composition disclosed herein to an individual suffering from skin dehydration, 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 an individual suffering from a lack of skin elasticity a hydrogel composition disclosed herein, wherein administration of the composition increases skin elasticity, thereby By treating the lack of elasticity of the skin. In another aspect of this embodiment, a method of treating skin roughness comprises administering a hydrogel composition disclosed herein to an individual suffering from skin roughness, wherein administration of the composition reduces skin roughness, thereby Treats skin roughness. In another aspect of this embodiment, a method of treating the lack of skin roughness comprises administering a hydrogel composition disclosed herein to an individual suffering from the lack of skin tension, wherein the administration of the composition creates skin tension and , Thereby treating the lack of skin tension.

In a further aspect of this embodiment, a method of treating a skin stretch mark or mark comprises administering a hydrogel composition disclosed herein to an individual suffering from the skin stretch mark or mark, wherein the administration of the composition Reduce or eliminate, thereby treating skin stretch marks or marks. In another aspect of this embodiment, a method of treating skin paleness comprises administering a hydrogel composition disclosed herein to an individual suffering from skin paleness, wherein administration of the composition increases skin tone or light, This cures the paleness of the skin. In another aspect of this embodiment, a method of treating skin wrinkles comprises administering a hydrogel composition disclosed herein to an individual suffering from skin wrinkles, wherein administration of the composition reduces or eliminates skin wrinkles and , Thereby treating wrinkles on the skin. In another aspect of this embodiment, a method of treating skin wrinkles comprises administering to a subject a hydrogel composition disclosed herein, wherein the administration of the composition creates skin resistance to skin wrinkles, thereby Cure.

In some embodiments, the dermal filler has sustained bioavailability. For example, when introduced into the skin of a human (e.g., intradermally or subcutaneously to a human to repair soft tissue defects in 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 persistence, an assessment of the degree of conjugation is made. This evaluation was based on the formulation of AA2G conjugation to HA via etherification. The formulation is stable under physiological conditions, but begins to release ascorbic acid (AsA) by α-glucosidase attached to the cell membrane, due to the fact that α-glucosidase adheres to the cell membrane at the filler/cell interface. The release of AsA occurs. Further release of AsA from HA-AA2G will entail HA degradation to make AA2G available to fibroblasts. Thus, 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 capable of releasing active vitamin C in a period of at least 1 month, eg 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; Gels with a degree of 15 mol% conjugation can release active vitamin C in a period of up to 10 months; 30㏖% can be released up to one and a half years.

In an embodiment of the invention, a vitamin C derivative (e.g., AA2G (ascorbic acid 2-glucoside), vitagen (3-aminopropyl-L-ascorbyl phosphate) and SAP (one of sodium ascorbyl phosphate)).

The preparation method of this dermal filler is a ratio of reaction temperature and reaction time suitable to achieve a composition containing AA2G by 4-arm epoxide (AA2G-4 arm epoxide), unreacted 4-arm epoxide and free AA2G. In a step of reacting pentaerythritol glycidal ether (Star-PEG epoxide) with ascorbic acid 2-glucoside (AA2G). 4 Arm Epoxide Capped AA2G (AA2G-4 arm epoxide) is conjugated to hyaluronic acid through an epoxy group. The unreacted 4 arm epoxide acts as a crosslinking agent to crosslink hyaluronic acid and as a conjugation agent to further conjugate AA2G.

In another embodiment of the present invention, a vitamin C derivative (e.g., AA2G (ascorbic acid 2-glucoside ), Vitagen (3-aminopropyl-L-ascorbyl phosphate) and SAP (ascorbyl sodium phosphate).

The preparation method of this dermal filler comprises BDDE ascorbic acid 2-glucoside at a ratio of reaction temperature and reaction time suitable to achieve a composition containing AA2G capped by BDDE (AA2G-BDDE), unreacted BDDE and free AA2G. It includes reacting with (AA2G). BDDE capping AA2G (AA2G-BDDE) is conjugated to hyaluronic acid through an epoxy 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 depends on the concentration of α-glycosidase. When the α-glycosidase concentration is 6.3 units per gram of gel, AA2G is converted to AsA almost completely 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 a slow conversion of AA2G to AsA.

10 shows a representation of the release profile (sustained release) of free AsA from conjugated dermal fillers according to the present invention (mol% versus AA2G conversion in reaction time); AA2G is completely converted to AsA in the AA2G/HA blend in 40 minutes. The AA2H/HA conjugate showed the 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. The high α-glycosidase concentration caused rapid conversion of AA2G to AsA. For a given α-glycosidase concentration, different formulations showed different profiles of AA2G against AsA.

In one aspect of the present invention, the appearance of fine wrinkles, for example, wrinkles that are not relatively deep in the skin, for example, but is not limited to the following, fine wrinkles around the eyes, lacrimal furrow area, forehead, eye circumference, glabellar wrinkles, etc. And a dermal filler that is particularly effective in removing is provided.

(Tindle effect), the appearance of a blue discoloration at the skin site where the dermal filler was injected, is an important side effect experienced by some dermal filler patients. The Tindle effect is more common in patients being treated for fine lines that are not deep. Embodiments of the present invention have been developed that provide long lasting translucent fillers that can be injected not deeply to treat fine lines and wrinkles even in areas of relatively thin skin without causing any blue discoloration from the Tindle effect. Fine lines or wrinkles are usually the thinnest, that is, those wrinkles or wrinkles in the skin (forehead, lateral canthal, red lip border/upper lip wrinkles) typically found in areas of the face where the skin has a dermal thickness less than 1 mm It is understood to be good. 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 canal is even thinner (eg, about 0.61 mm for normal skin and about 0.41 mm for wrinkled skin). The average outer diameter of a 30 or 32 gauge needle (needles typically used for fine wrinkle 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 the Tindle 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; The dermal filler composition, wherein the composition exhibits a reduced tindle effect when administered into the dermal area of a patient for substantially the same composition except for the absence of the additive. The composition may be substantially optically transparent.

In one embodiment, the additive is AA2G, which can be chemically conjugated to a vitamin C derivative, for example 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 such as lidocaine in an amount suitable to provide comfort upon injection to the patient.

Also provided is a method of treating fine lines in the skin of a patient. That method is usually Introducing a composition as described herein into the patient's skin. For example, the composition comprises a mixture of a hyaluronic acid component, a crosslinking component for crosslinking hyaluronic acid, and an additive other than the crosslinking component, wherein the composition is substantially optically transparent; The dermal filler composition exhibits a reduced tindle effect for substantially the same composition except for the absence of additives.

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 can be HMDA.

In certain embodiments of the 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 an extrusion force of about 24 mg/g to about 25 It has a final HA concentration of mg/g.

In certain embodiments of the invention, the additive is a HA-AA2G conjugate or HA-vitagen conjugate, but the degree of conjugation is from about 3 mol% to about 10 mol%, or up to about 15 mol%, or about 40 mol%. Until. These compositions comprise at least about 30 Pa, more preferably at least about 40 Pa, to about 100 Pa of G', about 0.30 to about 0.50 G"/G', about 27 N or less extrusion force, 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 the conjugate agent, eg AA2G, to repeat units of hyaluronic acid (eg, HA dimer). 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 illustrated in Example 2 below or other methods known to those skilled in the art.

Example

Example 1: AA2G conjugation crosslinked to HA gel using BDDE as crosslinking agent

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 minutes. After 800.7 mg of AA2G was put into the vial, 713.7 mg of BDDE and 1416.8 mg of 10% NaOH were added. The solution (pH >12) was reacted in a 50° C. water bath for -20 minutes, and 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, pH 7.4. After ˜2.5 hours, 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 swollen overnight on an orbital shaker. The gel was resized through a -60 μm screen and mixed -20 times by passing back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH 7.4 buffer. Dialysis was performed for ~185 hours with frequent changes in PBS buffer. After dialysis, the gel was put into 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. After dialysis, the gel was estimated to be -1 g/ml. > Dialysis was stopped at the point where no noticeable AA2G was produced in 1 liter of PBS per 8 hours. AA2G was measured at 260 nm using a UV/Vis spectrophotometer (Nanodrop 2000C, ThermoScientific). The calibration curve of AA2G was calculated using different concentrations of AA2G in 2% HA (A@260nm = 1.4838 [AA2G(mM)])

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

The mmol of AA2G after dialysis: Put the absorbance at 260nm 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 and then a strain sweep was performed 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 gel using BDDE as a crosslinking agent with variable degree of conjugation and gel rheological properties

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

400.8 mg of LMW HA was hydrated in 1752.1 mg of 1% NaOH in a syringe for -30 minutes. 800.3 mg of AA2G was put into the 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 minutes, and 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, pH 7.4. After ˜2.5 hours, 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 swollen overnight on an orbital shaker. The gel was resized through a -60 μm screen and mixed -20 times by passing back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH 7.4 buffer. Dialysis was performed for ~164.5 hours with frequent changes in PBS buffer. After dialysis, the gel was put into 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 a crosslinked HA gel using BDDE as a crosslinking 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 minutes. After 800.5 mg of AA2G was put into the vial, 264.3 mg of BDDE and 1100.0 mg of 10% NaOH were added. The solution (pH >12) was reacted in a 50° C. water bath for -20 minutes, and 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 8.5128g PBS, pH 7.4. After ˜2.5 hours, a HA-AA2G gel was formed, and a HCl-PBS solution was added to it. The gel was neutralized and swollen over the weekend (-55 hours) on an orbital shaker. The gel was resized through a -60 μm screen and mixed -20 times by passing back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH 7.4 buffer. Dialysis was performed for ~114 hours with frequent changes in PBS buffer. After dialysis, the gel was put into a syringe and stored in a refrigerator at 4°C. The degree of conjugation and the gel rheological properties are measured 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 a crosslinked HA gel using star-PEG epoxide as a crosslinking agent, the degree of conjugation is 29.4%, and G'is -235Pa.

200.4 mg of LMW HA was hydrated in 2000 mg of 1% NaOH in a syringe for -30 minutes. After 400 mg of AA2G was put into the vial, 312.7 mg of star-PEG epoxide and 1026.5 mg of 10% NaOH were added. The solution was reacted in a 50° C. water bath for ˜20 minutes, and 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, pH 7.4. After ˜2.5 hours, a HA-AA2G gel was formed, and a HCl-PBS solution was added to it. The gel was neutralized and swollen over the weekend (-68 hours) on an orbital shaker. The gel was resized through a -60 μm screen and mixed -20 times by passing back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH 7.4 buffer. Dialysis was performed for ~95 hours with frequent changes in PBS buffer. After dialysis, the gel was put into a syringe and stored in a refrigerator at 4°C. The degree of conjugation and the gel rheological properties are measured 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 a crosslinked HA gel using star-PEG epoxide as a crosslinking agent, 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 minutes. After 400.2 mg of AA2G was put into the vial, 313.4 mg of star-PEG epoxide and 1022.6 mg of 10% NaOH were added. 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, pH 7.4. After ˜2.5 hours, a HA-AA2G gel was formed, and a HCl-PBS solution was added to it. The gel was neutralized and swollen overnight (-24 hours) on an orbital shaker. The gel was resized through a -60 μm screen and mixed -20 times by passing back and forth between two syringes. The gel was placed in a 15,000 MWCO RC dialysis bag and dialyzed in PBS, pH 7.4 buffer. Dialysis was performed for -98.5 hours with frequent changes in PBS buffer. After dialysis, the gel was put into a syringe and stored in a refrigerator at 4°C. The degree of conjugation and the gel rheological properties are measured 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 a crosslinked HMW HA gel using BDDE as a crosslinking 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 minutes. 1200 mg AA2G was placed in the 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 minutes, transferred to a 20cc 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 10X PBS, pH 7.4 to obtain a HCl-PBS solution, which was neutralized by adding HCl-PBS solution, and the gel was swollen. The gel was neutralized and swollen over 48 hours on an orbital shaker. The gel was resized through a -60 μm screen and mixed -20 times by passing back and forth between two syringes. The gel was placed in a 20,000 MWCO CE dialysis bag and dialyzed in PBS, pH 7.4 buffer. Dialysis was performed for ~114 hours with frequent changes in PBS buffer. After dialysis, the gel was put into a syringe and stored in a refrigerator at 4°C. The degree of conjugation and the gel rheological properties are measured 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 a crosslinked HMW HA gel using BDDE as a 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 minutes. The HA was swollen by the addition of BDDE (311.7 mg), and the HA was swollen for another 80 minutes. 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 the HA pre-reacted with BDDE. The mixture was continued to react at 50° C. for another 2.5 hours. After -2.5 hours, an HA-vitagen gel was formed. Then, 195 mg of 12M HCl was added to 9004.0 mg 10X PBS, pH 7.4 to obtain a HCl-PBS solution, which was neutralized by adding HCl-PBS solution, and the gel was swollen. The gel was neutralized and swollen over 48 hours on an orbital shaker. The gel was resized through a -60 μm screen and mixed -20 times by passing back and forth between two syringes. The gel was placed in a 20,000 MWCO CE dialysis bag and dialyzed in PBS, pH 7.4 buffer. Dialysis was performed for ~120 hours with frequent changes in PBS buffer. After dialysis, the gel was put into 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 determination 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. This solution (Yunggae and EDC/NHS solution) is added to another 60cc syringe containing 23.5 ml of water. Mix the two syringes -20 times by passing it 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. After 292 mg of AA2P was placed in the vial, 300 mg of star-PEG amine was added. The solution was reacted at room temperature overnight. 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 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 filler with AA2G to reduce the appearance of fine lines

For any of the gels described in the above examples after dialysis, a suitable amount of free HA gel was added to the gel to improve or modify the degree of gel cohesion and/or injectability. For example, free HA fibers were swollen in a phosphate buffer solution to obtain a homogeneous viscoelastic gel ("free" HA gel). Prior to the dialysis step, this crosslinked gel is added 1 to the HA/BDDE crosslinked gel obtained in Example 1 (eg, to obtain a composition having from about 1% to about 5% w/w free HA). The resulting gel is then filled into a ready-to-fill sterile syringe 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 a physician 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 the addition of the free HA gel, lidocaine chlorine hydrate (lidocaine HCl) is added to the mixture. In powder form (lidocaine HCl) can be first solubilized in WFI and filtered through a 0.2 μm filter. Dilute NaOH solution is added to the cohesive HA/AA2G gel so that a slightly basic pH is reached (eg, a pH of about 7.5 to about 8). The lidocaine HCl solution is then added to the slightly basic gel to reach the final desired concentration, for example about 0.3% (w/w). Then the obtained pH of the HA/AA2G/lidocaine mixture is 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 blending 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 carboxyl functional groups (-COOH). These additives are conjugated to HA hydrolysis through esterification using EDC chemistry. Examples for conjugation according to the invention are 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. The reaction is mixed for at least 20 times by connecting the syringe containing EDC and NHS to the syringe containing HA and retinoic acid and passing 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 and then dialyzed against PBS buffer under azeotropic conditions. The gel is packaged into a sterile syringe and stored at 4°C.

Example 15

Conjugation of an additive containing a hydroxyl functional group to an HA hydrogel.

Additives such as retinol (AKA, tretinoin), catalase, dimethylaminoethanol and g-tocopherol contain hydroxyl functional groups (-OH). These additives are conjugated to HA hydrolysis through 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 retinoleic 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. The reaction is mixed for at least 20 times by connecting the syringe containing EDC and NHS to the syringe containing HA and retinol and 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 and then dialyzed against PBS buffer under azeotropic conditions. The gel is packaged into a sterile syringe and stored at 4°C.

Example 16

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

This is a two-step process.

Step 1: Cross-linked HA gel, such as a commercial HA-based dermal filler, such as JUVEDERM®, Allergan or Restylane®, Irvine, CA, Medisys Aesthetic 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 a hydroxyl group. Additives containing hydroxyl groups are retinol, catalase, dimethylaminoethanol and g-tocopherol hydroxyl functional groups (-OH).

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

2 gm of Juvidum 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 are added. The mixture was reacted at 37° C. for 4 hours. The gel is dialyzed against isopropanol to remove unconjugated retinol and then dialyzed against PBS buffer under azeotropic conditions. The gel is packaged into a sterile syringe and stored at 4°C.

Example 17

Conjugation of growth factor, peptide or elastin to HA hydrogel

Additives such as epidermal growth factor (EGF), transforming growth factor (TGF) and peptides contain functional amine groups and are conjugated to HA to form beneficial dermal fillers. These additives are conjugated to HA through 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 buffer. In 100 mg of MES solution, 20 mg of EGF is added. To the mixture, 197.7 mg of EDC and 149 mg are added. The obtained 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 a sterile syringe and stored at 4°C.

The present invention provides a method of improving the viability of transplanted adipose tissue. The method generally includes introducing the composition into the skin of a patient adjacent to the implanted adipose tissue, the composition being a composition as described elsewhere herein. For example, the composition may include hyaluronic acid and a vitamin C derivative covalently conjugated to hyaluronic acid, but 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 to the skin a composition comprising adipose tissue, hyaluronic acid and vitamin C conjugated to hyaluronic acid.

Example 18

Conjugation of growth factor, peptide or elastin to HA hydrogel

To evaluate the mitogen 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) supplemented with or without them were cultured on tissue culture plastics for 4 days in complete MesenPro medium. The 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 control group lacking ascorbic acid by 60%, 80% and 96%, respectively (yellow tetrazolium by dehydrogenase. Measures the amount of MTT converted to purple formazan (purple formazan is solubilized by a detergent). Using the same concentration of AA2G, proliferation of 70%, 60% and 50% of the control group was obtained, respectively. Similar results were obtained with Vitagen, which showed 70%, 60% and 30% increases over the control, respectively. In summary, vitamin C and its derivatives, AA2G and Vitagen, in the presence of medium-containing growth factors, enhance hASC proliferation in cell culture.

Crosslinked HA gel with conjugated vitamin C

According to a specific embodiment of the present invention showing the Tyndle effect and other benefits, of a crosslinked HA-based gel with conjugated vitamin C using 1,4-butanediol diglycidyl ether (BDDE) as a crosslinking 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 being bound by the manipulation of any specific theory, it has been found by the inventors that crosslinking BDDE and HA in the presence of AA2G or Vitagen greatly changes the properties of the gel, and the gel compared to commercial HA gels crosslinked with BDDE. It has high crosslinking density, high HA concentration, low viscosity and low pressure output. Since AA2G or Vitagen is present during crosslinking, the gel formed has these ascorbic acid derivatives bound either alone or via BDDE as a suspension group and as a crosslinking agent bridging the HA chain. The microscopic structure of the gel changes greatly, which results in a gel with very low extrusion force even through a needle 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 from fibroblasts or phosphorases by endogenous enzymes such as α-glucosidase. Active vitamin C can trigger skin collagenization and act as a radical scavenger to inhibit gel breakdown.

Example 19

Formulation of HA/AA2G gel with reduced tindle effect

A mixture of 400.1 mg LMW HA and 402.3 mg AA2G in a syringe was hydrated for 60 minutes after the addition of 1764.0 mg of 5 wt% NaOH solution. In a separate vial 800.8 mg of AA2G was added followed by 1401.1 mg of 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 minutes, and then transferred to hydrated HA. After addition, the mixture was mixed -20 times by passing it back and forth between two syringes. Then, the paste was transferred to the vial and placed in a 50° C. water bath for -2.5 hours. After crosslinking, 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 was swollen for 72 hours on an orbital shaker. The size was changed by applying force to the gel through a ˜60 μm pore size mesh. After mixing the gel whose size was changed by passing two syringes back and forth ~20 times, it was transferred to a cellulose ester dialysis bag, MWCO ~ 20kDa, and dialyzed against PBS, pH 7.4 buffer for 5 days, and the buffer was changed twice a day. I did. After dialysis, the gel was supplied 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, an AA2Gmol% calculated as described in Example 2 of about 10 mol%, and a G'of about 80 Pa. Another gel was made in a similar manner, which had a G'value of about 60 Pa to about 80 Pa.

Example 19A

Formulation of HA/AA2G by lidocaine gel with reduced Tyndle effect

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

Example 20

Formulation of HA/Vitagen Gel with Reduced Tindle Effect

401.0 mg of LMW HA was hydrated in a solution of 2355.0 mg of 1% NaOH by weight in a syringe for ˜45 minutes. 303.8 mg BDDE was added to 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, then in 510.1 Milli-Q water. The Vitagen solution was mixed back and forth 30 times with the preheated hydrated HA/BDDE mixture using a syringe to syringe mixture. The mixture was put back into a 50° C. water bath, and after the reaction was allowed to proceed for another 2 hours, a solution containing 148.1 mg of 12M HCl and 8523.1 mg of 10X PBS, pH 7.4 was added to the crosslinking agent. HCl-PBS solution was added to neutralize and the gel was swollen. The gel was neutralized and swollen over 48 hours on an orbital shaker. The gel was resized through a -60 μm screen and mixed -20 times by passing back and forth between two syringes. The gel was placed in a 20,000 MWCO CE dialysis bag and dialyzed in PBS, pH 7.4 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

According to a specific embodiment of the present invention showing the Tindle effect and other advantages, the preparation of a crosslinked HA-based gel is described in Examples 21 and 22 below. In Example 21, the gel made through EDC chemistry using a crosslinking agent is 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, eg at room temperature and eg at pH 5.4. Reaction conditions can be adjusted to produce highly reticulated gels and have optimal rheological properties, good injection capacity 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 with -NHS (sulfo-NHS). Gels will have crosslinking points that are separated from each other, so highly crosslinked materials have high damping power. In contrast, crosslinking of BDDE and HA at such low hydration or reaction concentrations may be impractical due to the relative inefficiency of the crosslinking agent.

Example 21

Formulation of HA/HMDA gel with reduced tindle 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 2010.5 mg of 100 mM MES buffer pH 5.2, and adding 2 μl of 1M NaOH to a pH of 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 total hydration of HA, for ˜1 hour, 790 μl of HMDA solution was added to the hydrated HA. The mixture was homogenized by 10 syringe to syringe mixing. Then 490 μl EDC and 490 μl NHS solution were added to the homogenized paste and mixed again 10 times by syringe to syringe mixing. The mixture was then transferred to a vial, cross-linked at room temperature for 5 hours, and 17.9 ml of 1X PBS buffer pH 7.4 was added. After the gel was expanded on a roller for 3 days, force was applied through a 60 μm pore size mesh. The sized gel was placed in a cellulose ester membrane tubing MWCO 20 KDa, and dialyzed against 1X PBS for 4 days while changing the buffer twice a day. The gel was prepared in 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 25 mg/g.

Example 22

Formulation of HA/4 AA gel with reduced tindle 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 of 100 mM MES buffer pH 5.2, and adding 380 μl of 6M HCl to a pH of 5.2. EDC solutions were 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 NHS was dissolved in 2020.0 mg 100 mM MES buffer pH 5.2. Upon total hydration of HA, for ˜1 hour, 260 μl of 4 AA solution was added to the hydrated HA. The mixture was homogenized by 10 syringe to syringe mixing. 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, crosslinked at room temperature for 5 hours, then 6.4 ml of 10X PBS buffer pH 7.4 was added. After the gel was expanded on a roller for 3 days, force was applied through a 60 μm pore size mesh. The sized gel was placed in a cellulose ester membrane tubing MWCO 20 KDa, and dialyzed against 1X PBS for 4 days while changing the buffer twice a day. The gel was prepared in a 1 ml COC syringe, 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. It consisted of 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 due to a logarithmic increase in the strain after a logarithmic increase of the frequency. The storage modulus (G') and viscous modulus (G") were obtained from the strain sweep at 1% strain.

Example 24

Measurement of the extrusion force of the gels of Examples 19 to 22

The force required to extrude the gel through a 30 gauge needle was measured using 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 at a rate of 100 mm/min for 11.35 mm and the extrusion force was recorded.

Example 25

Bioavailability test of gels of Examples 19-22

A 50 μl bolus injection of the gel was implanted intradermally on the back of Sprague Dolly rats. Implants were removed at 1 week, and analyzed systematically 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.

The in vitro cytotoxicity test of the gel was performed by NAMSA according to the literature [Agarose Overlay Method of ISO 10993-5: biological Evaluation of Medical Devices-Part 5: Tests for In Vitro Cytotoxicity]. In addition to 0.1 ml of test article placed on a filtered disc in triplicate wells, 0.9% NaCl solution, 1 cm long high density polyethylene as a negative control, and a 1×1 cm 2 portion of latex as a positive control were administered. Each was placed on an agarose surface laid directly on a monolayer of L929 mouse fibroblasts. After incubation at 37° C. in 5% CO ₂ for 24 hours, the cultures were tested macroscopically and microscopically for any abnormal cell morphology and cell lysis. Test articles were scored from 0 to 4 based on the proximal dissolution zone of the sample. Test materials from Examples 1, 3 and 4 scored as 0 as test articles showed no evidence of causing any cell lysis or toxicity.

Quantitative analysis of tindle effect

In order to further support visual observation and perform comparative performance analysis of the HA filler, it was considered necessary to perform a quantitative analysis of the Tindle effect. No quantitative technique exists in the literature for the specific Tyndle effect on dermal fillers. However, based on the existing scientific understanding of light scattering and light interactions with skin, we quantified the Tyndle effect in skin using two distinct approaches based on (a) colorimetric and (b) spectroscopy. Based on these techniques, three distinct quantitative variables (abbreviated below) were established to measure the effect of Tyndle in vivo.

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

Associated with) a maximum score of 5 is given. Three independent observers were trained on the pre-blind scale to score the test samples.

b) Blue component of skin color-"b" : The blue component of light sent from the skin area injected with various fillers was quantified using a colorimeter (CM2600D, Konica Minolta, NJ). This was achieved by using the "b" component of the Lab color scale.

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

Example 27

Gel tindle evaluation

The gel was injected intradermally through a 27G½ TSK needle using a linear threading technique into the femur of a 2-month-old hairless rat. The gel was implanted not deep to mimic the clinical fine wrinkle procedure. 48 hours after gel implantation, a test for tindle was performed. Before performing the Tindle test, animals were euthanized to improve the contrast of the Tindle effect due to the lack of hemoglobin.

Images of the gels from Examples 19 to 21 2 days after implantation are shown in FIG. 12. Images for commercial Juvidum Refine and Restylane Touch are shown for comparison. A bluish line (Tindle effect) can be clearly seen in the images of commercial gels Juvidum Refine and Restilan Touch. Gels from Examples 19, 19A (not shown) and 21 did not show the Tindle effect

The injection site was scored using a visual score of 1-5 in increments of 0.5. The injection site with score 1 did not show skin discoloration, while the injection site 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 percent of blue light emitted from the skin (400 nm to 700 nm) were independently measured. 13 and 14 show the visual Tindle score and the% of blue light emitted. The gels from Examples 19 and 21 did not show a Tindle effect and had a lower visual Tindle score and% of blue light emission values. The Tindle score and% of emitted blue light values were higher for Juvidum Refine and Restilan Touch. Belotero Soft did not show any tines and the values were similar to those of Examples 19 and 21. See FIGS. 13 and 14.

Example 28

In vivo persistence evaluation of gels by history

50 μl bolus injections of the gels of the present invention and commercial gels were implanted intradermally on the back of Sprague Dolly rats. Implants were removed at 1 week and analyzed systematically by hematoxylin and eosin (H&E) staining. Sections were accurately taken at the injection site. Two sections were cut from each tissue sample, and H&E stained sections were stitched using a stitching scope. The 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 Figure 15.

Example 28A

In vivo persistence evaluation of gels by MRI

After intradermal injection in female Sprague Dawley rats, magnetic resonance imaging (MRI) was used to evaluate the volume and surface area changes over time of the gels of the present invention and commercial gels over a period of 40 weeks. The gel was injected at a target volume of 150 μl per implant. The implants were placed in two diametrically opposite sites slightly to the tail with respect to the shoulder, two diametrically opposite sites slightly cranial to the knee, and at the midpoint 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 week 12, week 24, and week 40 after transplantation. A plot of the absolute volume of gel versus time is shown in Figure 16 below. The high persistence gel has a high absolute volume at 40 weeks of implantation.

Example 29

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

A 40-year-old skinny woman has fine wrinkles in the area around the orbit and requires dermal filler treatment. Using a 30 gauge needle, the doctor introduces 0.6 ml of the HA-based gel according to the invention (as described in Example 19) into the fine lines under her eyes and in the lacrimal sulcus area not deeply using a linear threading technique. . The gel was introduced not deeply, but no blue discoloration was observed, and the patient was satisfied with the result.

As shown, the compositions of the present invention, e.g. the compositions of Examples 19 and 21, had a reduced or insignificant Tindle effect, and certain HA based commercial gels, such as Juvidum Refine/Surgiderm It has a substantially longer duration in the body for 18 and Velotero Soft. For example, for the “fine wrinkles” formulation Velotero Soft, Example 19 of the present invention not only had at least an advantageous Tindle effect on this commercial gel, but advantageously had a substantially longer duration in vivo.

Example 30

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

Additives such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and derivatives thereof, alone and in combination, are conjugated to crosslinked hyaluronic acid gels in a manner to produce various substantially transparent, injectable HA based gels. . The HA component is at least 90% by weight, for example completely low molecular weight HA, or about 100% low molecular weight HA as defined elsewhere herein. These additives are conjugated to the HA hydrogel using any suitable means. Having HA concentrations up to at least about 20 mg/g, e.g., about 23 mg/g, about 24 mg/g, about 25 mg/g, about 30 mg/g, and suitable for injection through a fine gauge needle, The conjugated gel is sized and processed to produce a pH neutral, cohesive composition for injection. 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 about 100 Pa. Gels are packaged and sterilized using autoclave, UV light or other suitable means.

Each gel is injected into the patient's wrinkles, e.g., at a depth of about 1.0 mm or less in the periorbital area, nasolabial fold area, lacrimal furrow area, neck area or any other facial area where dermal peeling is advantageous. Useful for injection into the skin. Despite the shallow introduction of the gel, no discoloration due to the Tindle effect was observed, and the patient was satisfied with the results.

Finally, while aspects of the present specification 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 merely illustrate the subject principles disclosed herein. Accordingly, the subject matter disclosed herein is not limited in any way to a particular method, protocol and/or reagent, etc. As such, one of ordinary skill in the art can make numerous and various modifications or variations, or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Changes in the detailed description can be made without departing from the spirit of the present invention as defined in the appended claims. Finally, the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the invention as defined only by the claims. Additionally, all objects contained in the above description or shown in the accompanying drawings are intended to be interpreted as illustrative only and not limiting. Accordingly, the present invention is not expressly limited as shown and described.

Certain embodiments of the invention are described herein, including the best way known to the inventors for carrying out the invention. Of course, variations on these described embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to use such variations as appropriate, and the inventors intend for the invention to be practiced in addition to those specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto, if permitted by applicable law. Furthermore, 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.

A group of alternative elements or embodiments of the invention disclosed herein should not be construed as a limitation. Each group member may be referred to and claimed individually or by any combination with other members of the group or other elements found herein. One or more members of the group may be included in or deleted from the group for convenience and/or patentability reasons. When any such inclusion or deletion occurs, the present specification is deemed to contain the group to be modified, and thus meets the described description of all Markush groups used in the appended claims.

Unless otherwise indicated, all numbers expressing amounts of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term “about”. The term “about” as indicated 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 amounts of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as being modified in all instances by the term “about”. The term “about” as indicated 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. Thus, unless contradicted otherwise, the numerical variables set forth in this specification and the appended claims are approximations that may vary depending on 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 principles of equality to the scope of the claims, each numerical variable should be interpreted at least in light of the number of significant figures reported, and by applying ordinary rounding techniques. The numerical ranges and variables presenting the broad scope of the present invention are approximate values, but the numerical values presented in specific examples are reported as concisely as possible. However, any numerical value essentially contains certain errors resulting from the standard deviation found in its respective test measurement.

Singular terms and similar references (especially in the context of the following claims) used in the context of describing the present invention, unless otherwise indicated in the specification or clearly contradicted by the context, may be used as the singular and the plural. It should be interpreted as covering everything. Recitation of ranges in this specification is only intended to serve as a shortened way of referring individually to each distinct value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification 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 embodiments or illustrative language (eg, “such as”) provided herein is only intended to better describe the invention, and to limit the scope of the invention otherwise claimed. I do not raise it. No language in this specification is to be construed as representing any unclaimed element essential to the practice of the invention.

The specific embodiments disclosed herein may be further limited in the claims using language that is made or made essentially of. When used in the claims, whether an amendment is submitted or added, the developing term “consisting of” excludes any element, step or ingredient not specified in the claims. The developing term “consisting essentially of” limits the scope of the claims to those that do not materially affect the material or steps specified and the basic and novel feature(s). Embodiments of the present invention so claimed are essentially or explicitly described and made possible herein.

All patents, patent publications and other publications referenced and identified herein describe the compositions and methods described in such publications, e.g., used in connection with the present invention, and are incorporated herein in their entirety for purposes of disclosure. And clearly included. These publications are provided for their disclosure only prior to the filing date of the present invention. Nothing in this regard is to be construed as an admission that the inventors qualify as an precedent for this disclosure because of the invention or for any other reason. All references to dates or representations of the contents of these documents are based on information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

Claims (20)

A dermal filler composition comprising hyaluronic acid and a vitamin C derivative covalently conjugated to hyaluronic acid. The dermal filler composition of claim 1, wherein the vitamin C derivative is conjugated to hyaluronic acid with a degree of conjugation of about 3 mol% to about 15 mol%. The dermal filler composition of claim 1, wherein the vitamin C derivative is converted to vitamin C in vivo by enzymatic cleavage. The dermal filler composition of claim 1, wherein the vitamin C derivative is selected from ascorbic acid 2-glucoside (AA2G), ascorbyl 3-aminopropyl phosphate, and sodium ascorbyl phosphate (AA2P). The dermal filler composition of claim 4, wherein the vitamin C derivative is ascorbic acid 2-glucoside (AA2G). The dermal filler composition of claim 4, wherein the vitamin C derivative is ascorbyl 3-aminopropyl phosphate. The dermal filler composition of claim 4, wherein the vitamin C derivative is sodium ascorbyl phosphate (AA2P). The dermal filler composition of claim 1, wherein the hyaluronic acid is crosslinked. The method of claim 8, 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 crosslinked with lysine. The dermal filler composition of claim 9, wherein the hyaluronic acid is crosslinked with 1,4-butanediol diglycidyl ether (BDDE). The dermal filler composition of claim 9, wherein the hyaluronic acid is crosslinked with lysine. The dermal filler composition of claim 1, further comprising an anesthetic. The dermal filler composition of claim 12, wherein the anesthetic agent is lidocaine. A method for treating a soft tissue condition in a subject in need thereof, comprising administering a dermal filler composition comprising hyaluronic acid and a vitamin C derivative covalently conjugated to hyaluronic acid. The method of claim 14, wherein the soft tissue condition is wrinkles or scars. The method of claim 14, wherein the dermal filler composition releases the vitamin C derivative in a subject in need thereof for at least about 1 month and up to 20 months. 15. The method of claim 14, wherein the vitamin C derivative is covalently conjugated to hyaluronic acid via BDDE or lysine. The method of claim 14, wherein the vitamin C derivative is selected from ascorbic acid 2-glucoside (AA2G), ascorbyl 3-aminopropyl phosphate, and sodium ascorbyl phosphate (AA2P). 15. The method of claim 14, wherein the vitamin C derivative is AA2G. 15. The method of claim 14, wherein the vitamin C derivative is AA2P.

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