CN104105474A - Dermal filler compositions for fine line treatment - Google Patents

Abstract

The present invention provides highly injectable, long-lasting hyaluronic acid-based hydrogel dermal filler compositions which are particularly advantageous for correction of fine lines in the face.

Description

For the corium bulking agent compositions of microgroove treatment

Related application

The application requires the U.S. Provisional Patent Application No.61/534 of JIUYUE in 2011 submission on the 14th, 780 priority and interests, and be the U.S. Patent Application Serial Number 13/593 of submitting on August 23rd, 2012,313 part continuation application, the latter is the U.S. Patent Application Serial Number 13/486 of submitting on June 1st, 2012,754 part continuation application, it has required the U.S. Provisional Patent Application No.61/493 submitting on June 3rd, 2011,309 priority and interests, in these application cases, the whole disclosures of each are incorporated to herein by this specific reference in its entirety.

Background

The present invention relates generally to corium bulking agent compositions, and relate more specifically to the effective injectable corium of the microgroove bulking agent compositions on treatment skin.

Skin aging is a progressive phenomenon, occurs and affected by life style factor along with passage of time, for example, drink, smoking and sunshine.Available atrophy, laxly and fat characterize the aging of skin of face.Atrophy reduces greatly corresponding to skin histology thickness.Subcutaneous tissue is lax causes skin too much and sagging and cause occurring cheek and blepharoptosis.Obesity refers to increase excess weight because of face and cervical region bottom swelling.These change conventionally to be dried, follow the string coarse relevant with skin texture.

Hyaluronic acid (HA), also referred to as hyaluronan, is that the knot that is distributed widely in whole human body is formed, the non-sulfuric acid glycosaminoglycans of epithelium and nervous tissue.Hyaluronic acid is all very abundant in different skin layer, and wherein hyaluronic acid has several functions, for example, guarantee good hydration, helps the formation of extracellular matrix, plays packing material and participates in tissue repair mechanism.But the amount of other matrix polymer existing in hyaluronic acid, collagen protein, elastin laminin and skin reduced with the age.For example, be repeatedly exposed to (for example) and cause that from the ultraviolet light of the sun hypodermal cell reduces the generation of its hyaluronan and increased the degradation rate of hyaluronan.This material unaccounted-for (MUF) causes various skins, for example, corrugate, recess, water loss and contribute to other undesirable condition of aging appearance.

Injectable corium filler has successfully been used for the treatment of aging skin.Filler can replace the endogenous matrix polymer losing, or strengthens/promote the function of existing matrix polymer, to treat these skins.Become and become more and more popular based on hyaluronic corium filler, because hyaluronic acid is the material existing whole human body natural.The common well-tolerated of these filleies, impermanency, and be the quite low-risk treatment to various skin situation.

Tyndall effect (Tyndall effect) is the adverse events occurring in some patients of the corium filler based on hyaluronic acid (HA) having used.Tyndall effect is characterised in that, occurs blue variable colour at the skin part of having injected corium filler, and this expression is seen visible hyaluronic acid by translucent epidermis.Clinical report shows, filler application technique and skin properties can affect the performance of this adverse events.High rigidity and elastomeric filler are successfully upper as regions such as nasolabial fold, cheek and chins for revising face, without change color being had to any worry, because material is injected in middle part and corium district, deep.But, when using these packing material corrections shallow tables, tiny wrinkle, for example tear ditch, glabella stricture of vagina, canthus stricture of vagina, laugh line or forehead, or when wrong application the in too shallow table ground, usually observe the light blue variable color of skin in upper part of dermis region.This phenomenon, is considered to the result of Tyndall effect, causes the variable color of application site semipermanent, and sometimes only after the hyaluronidase of having used degraded packing material, disappears.Therefore, Tyndall effect is more common in the patient of the tiny wrinkle of the shallow table for the treatment of.As long as gel maintains in skin, Tyndall effect performance extends, and some months is conventionally a reason of main focus in patient.

Prepare especially gel filled " the tiny wrinkle " found with treatment tear ditch, forehead, canthus stricture of vagina, glabella stricture of vagina etc. of corium based on HA around.Commercially available HA " microgroove " gel comprise Juv é derm Refine (G '～67Pa; G "/G '～0.59, HA concentration 18mg/ml), Belotero Soft (G '～28Pa; G "/G '～1.1, HA concentration 20mg/ml), Emervel Touch (G '～56Pa; G "/G '～0.64, HA concentration 20mg/ml), Stylage S (G '～192Pa; G "/G '～0.20, HA concentration 16mg/ml), Teosyal First Lines (G ' 59Pa; G "/G '～0.53, HA concentration 20mg/ml), Restylane Touch (G '～489Pa; G "/G '～0.24, HA concentration 18mg/ml).Although (for example), by making linear HA chain and a small amount of cross-linking agent slightly be cross-linked and/or reduce the final HA concentration of these gels, these gels are mixed with and have low elastic modulus, but most of commercially available " microgroove " gel still demonstrates Tyndall effect in some patients, particularly in the time of shallow table injection, for example, be less than the degree of depth of about 1mm.

In the treatment of shallow table wrinkle, can adopt the gel based on collagen protein and seem can not cause Tyndall effect.Gel based on collagen protein is not subject to highly favor, because their persistent period in skin are relatively poor and need to test in advance in individuality. (calcium hydroxy apetite) is subcutaneous, injectable implant, and its key component is synthesizing hydroxylapatite calcium, but not hyaluronic acid.From different based on hyaluronic corium filler, calcium hydroxy apetite is opaque, has therefore avoided the complication of Tyndall effect.But if placement is too shallow, this filler can be regarded as being located immediately at the whiteness under skin.In addition, with compared with hyaluronic filler, need to inject and conventionally not recommend for ocular compared with minute hand head.

By needs provide can not show owing to the light blue variable color of Tyndall effect based on hyaluronic injectable corium filler.

General introduction

The invention describes preparation can use in the corium of top, does not produce any light blue variable color of skin, or compositions and the compound method of the corium filler based on HA of at least not remarkable or unconspicuous light blue variable color.Further, have been found that many of the present invention gel filled persistent period in vivo of current description is obviously longer than commercially available gel on Vehicles Collected from Market.Aspect more of the present invention, provide strengthening the useful optically transparent corium filler of skin appearance, it has increased volume and richness, and has reduced the appearance of even tiny wrinkle, there is no " Tyndall effect ".This compositions can be introduced the microgroove of skin, even in thin skin region and quite shallow table, can not cause the negative blue variable colour relevant to the optically transparent corium filler of many routines.

More specifically, in one aspect of the invention, provide long-acting treatment corium bulking agent compositions, it comprises biocompatible polymer conventionally, for example cross-linked-hyaluronic acid component and the additive merging with hyaluronic acid component.

In one embodiment, polymer is polysaccharide, for example hyaluronic acid.Hyaluronic acid comprises crosslinking component and can further comprise non-crosslinked component.Additive can comprise vitamin, for example vitamin C (for example, the vitamin C of stable form) or vitamin C derivatives (for example, L-AA 2-glucoside (AA2G), ascorbic acid 3-aminopropyl phosphate ester (vitagen (Vitagen)) or sodium ascorbyl phosphate (AA2P)).

In one aspect of the invention, additive is vitamin derivative, and it for example, by being applicable to course of reaction, etherificate, amidatioon or esterification and polymer covalent bond.

Of the present invention one extensive aspect, corium bulking agent compositions is provided, described compositions comprises the hyaluronic acid component crosslinked with crosslinking component and the additive except described crosslinking component.Hyaluronic acid component can be combined with additive chemistry.Further, when in the corium district that is administered to patient, with respect to except not having described additive, identical compositions substantially, described compositions table reveals Tyndall effect and reduces.Described compositions can further comprise other additive, for example anesthetis (for example lignocaine).In one embodiment, additive is vitamin C derivatives, for example AA2G.In another embodiment, additive is vitagen.

In one embodiment, hyaluronic acid component is combined with additive chemistry, and conjugation is between about 3mol% and about 40mol, for example, between about 3mol% and about 10mol%.

Described compositions optical clear substantially.G ' the value of described compositions between about 40Pa and about 100Pa, for example, is not more than about 100Pa conventionally, for example, be not less than about 40Pa.

The method of the microgroove on treatment patient skin is provided in another aspect of this invention.In one embodiment, described method comprises in patient's skin introduces the step that comprises hyaluronic acid component, is cross-linked the compositions of the mixture of described hyaluronic crosslinking component and the additive except described crosslinking component, described compositions optical clear substantially, and wherein with respect to except not having described additive, identical compositions substantially, described compositions table reveals Tyndall effect and reduces.

In another aspect of this invention, the method of improving face appearance is provided, and described method generally includes the following step: use the optically transparent corium bulking agent compositions that does not show or show unconspicuous Tyndall effect substantially to patient's corium district.Described compositions is made through the following steps: hyaluronic acid is provided, cross-linking agent is reacted with vitamin C derivatives, the cross-linking agent having reacted and vitamin C derivatives are added in described hyaluronic acid and comprise covalently bound ascorbic cross-linked-hyaluronic acid compositions to form; And homogenize also neutralizes described cross-linked-hyaluronic acid compositions to obtain injectable corium bulking agent compositions.In some embodiments, vitamin C derivatives is AA2G.In other embodiments, vitamin C derivatives is vitagen.

In still another aspect of the invention, provide and reduced the method that in patient's thin skin district, microgroove occurs, wherein said method is usually included in the degree of depth that is not more than about 1mm, use corium bulking agent compositions to described patient, one is optically transparent based on hyaluronic corium bulking agent compositions substantially, and it comprises vitamin C or vitamin C derivatives.In some embodiments, being not more than 0.8mm, be not more than 0.6mm or be not more than compositions described in the deeper injection of 0.4mm.

In still another aspect of the invention, provide a kind of corium bulking agent compositions, its substantially optical clear and conventionally comprise the hyaluronic acid component crosslinked with crosslinking component and with the covalently bound vitamin C derivatives of hyaluronic acid component.In an exemplary, the G ' of described compositions is worth between about 40Pa and about 100Pa.Further, the hyaluronic acid concentration of described compositions may be between about 18mg/g and about 30mg/g.At treatment skin, for example, even very thin skin, for example thickness is not more than in the microgroove or shallow table gauffer on the skin of about 1mm, and these compositionss may be particularly useful and effective.In some embodiments, compositions of the present invention continues at least 3 months introducing after skin, at least 6 months or reach 1 year.

May be easier to understand and be familiar with these and other aspect of the present invention and advantage with reference to following accompanying drawing and detailed description.

Accompanying drawing summary

Fig. 1 is the structural representation of L-AA 2-glucoside (AA2G).

Fig. 2 is the structural representation of ascorbic acid 3-aminopropyl phosphate ester (vitagen).

Fig. 3 is the structural representation of sodium ascorbyl phosphate (AA2P).

Fig. 4 is the structural representation of BDDE (BDDE).

Fig. 5 is the structural representation of tetramethylolmethane glycidyl ether (Star-PEG epoxide).

Fig. 6 is the structural representation of tetramethylolmethane (3-aminopropyl) ether (Star-PEG amine).

Fig. 7 shows according to the table of the conjugation of various corium bulking agent compositions of the present invention and G ' value.

Fig. 8 shows according to the table of conjugation, HA concentration and the G ' value of HA-AA2G of the present invention (BDDE) corium bulking agent compositions.

Fig. 9 is for 4 kinds of different alpha-glucosaccharase enzyme concentrations, at (Minute) aspect the time, and the pictorial diagram of percentage ratio that the AsA of the solution in PBS from AA2G observing discharges.

Figure 10 shows from according to the release profiles schematic diagram (sustained release) of the free AsA of combination corium filler of the present invention (AA2G transform mol%) compared with the response time.

Figure 11 A and 11B show the additional release data according to various corium filleies of the present invention.

Figure 12 shows after gels that the corium that the present invention is based on HA in shallow table injection is gel filled and some commercially available confession microgrooves are applied, the image of skin.

Figure 13 shows the vision Tyndall score that the present invention is based on gel filled and some the commercially available gel for microgroove application of the corium of HA.

Figure 14 shows the gels that corium is gel filled and some commercially available confession microgrooves are applied that the present invention is based on HA from the blue light % of skin outgoing.

Figure 15 shows to implant and the present invention is based on gel filled and some the commercially available gels for microgroove application of the corium of HA after 1 week, total % of residue gel.

Figure 16 shows in implantation and the present invention is based on gel filled and some the commercially available gels for microgrooves application of the corium of HA the 0th week, the 12nd week, the 24th week and the 40th week, remains total % of gel.

Describe in detail

In one aspect of the invention, provide corium bulking agent compositions, described compositions comprises biocompatible polymer conventionally, for example polysaccharide (for example cross-linked-hyaluronic acid) and with the covalently bound vitamin C derivatives of described polymer.Described compositions provides vitamin C and other treatment or the beauty treatment interests of sustained release for skin collagen hypertrophy.In the time introducing skin, for example Intradermal, described compositions is reacted with the endogenous enzyme in body, and passes in time, generates in vivo bioactive vitamin C through enzymatic lysis.Because vitamin C discharges from described compositions in a few weeks or months, thing followed interests can be used to health.

Described polymer can be selected from protein, peptide and polypeptide, polylysine, collagen protein, procollagen, elastin laminin and laminin，LN.

Described polymer can be selected from the synthetic polymer with hydroxyl, amine and carboxyl functional group: poly-(vinyl alcohol), Polyethylene Glycol, polyvinylamine, polyallylamine, deacetylate polyacrylamide, polyacrylic acid and polymethylacrylic acid.Described polymer can be selected from dendroid or branched polymer, comprises dendroid polyhydric alcohol and dendroid polyamine.Described polymer can be selected from the surface of solids with hydroxyl, amine and carboxyl functional group.

Described polymer can be polysaccharide, for example, be selected from the polysaccharide of following group, comprises starch and derivant thereof; Glucosan and derivant thereof; Cellulose and derivant thereof; Chitin, chitosan and alginate and derivant thereof.

In an exemplary of the present invention, described polymer is glycosaminoglycans.Hydrogel composition disclosed herein can further comprise two or more different glycosaminoglycans polymer.As used herein, term " glycosaminoglycans " and " GAG " and " mucopolysaccharide " synonym and refer to the long linear polysaccharide that repetition disaccharide unit forms.Repetitive is made up of the hexose (hexose) or the hexuronic acid that are connected with hexosamine (nitrogenous hexose) and pharmaceutically acceptable salt thereof.GAG family member is different in the type of its contained hexosamine, hexose or hexuronic acid unit, for example glucuronic acid, iduronic acid, galactose, galactosamine, glucamine and also possible different in the geometric shape of glycosidic inkage.Any glycosaminoglycans polymer is all useful in hydrogel composition disclosed herein, and condition is that glycosaminoglycans polymer improves skin.The limiting examples of glycosaminoglycans comprises chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronan.The limiting examples of the acceptable salt of glycosaminoglycans comprises sodium salt, potassium salt, magnesium salt, calcium salt and combination thereof.For example, at Piron and Tholin, Polysaccharide Crosslinking, Hydrogel Preparation, Resulting Polysaccharides (s) and Hydrogel (s), uses Thereof, U.S. Patent Publication 2003/0148995; Lebreton, Cross-Linking of Low and High Molecular Weight Polysaccharides Preparation of Injectable Monophase Hydrogels; Lebreton, Viscoelastic Solutions Containing Sodium Hyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses, U.S. Patent Publication 2008/0089918; Lebreton, Hyaluronic Acid-Based Gels Including Lidocaine, U.S. Patent Publication 2010/0028438; With Polysaccharides and Hydrogels thus Obtained, U.S. Patent Publication 2006/0194758; With Di Napoli, Composition and Method for Intradermal Soft Tissue Augmentation, glycosaminoglycans useful in hydrogel composition disclosed herein and method and resulting polymers thereof have been described in international patent publications WO2004/073759, its separately accordingly by reference entirety be incorporated to.GAG useful in hydrogel composition disclosed herein and method is available on market, for example the corium filler based on hyaluronan 30, ultra, ultra Plus, ultra XC and ultra Plus XC (Allergan Inc, Irvine, California).Table 1 has been listed representative GAG.

Aspect of the present invention partly provides the hydrogel composition that comprises chondroitin sulfate polymer.As used herein; term " chondroitin sulfate polymer " refers to unbranched, the sulfated polymers of variable-length, its comprise two alternately monosaccharide be the disaccharide of D-Glucose aldehydic acid (GlcA) and N-ACETYL-D-GALACTOSAMINE (GalNAc) and pharmaceutically acceptable salt thereof.Chondroitin sulfate polymer also can comprise that epimerism turns to the D-Glucose aldehydic acid residue of L-iduronic acid (IdoA), is called dermatan sulfate by gained disaccharide in this case.Chondroitin sulfate can have the chains that exceed 100 independent sugar, and it is separately with variable position and amount sulphation.Chondroitin sulfate polymer is the important structure component of cartilage and its many resistances to compression is provided.Any chondroitin sulfate polymer is all useful in compositions disclosed herein, and condition is that chondroitin sulfate polymer improves skin.The limiting examples of chondroitin sulfate pharmaceutically acceptable salt comprises sodium chondroitin sulfate, chondroitin sulfate potassium, chondroitin sulfate magnesium, calcium chondroitin sulfate and combination thereof.

The aspect of this description partly provides the hydrogel composition that comprises keratan sulfate polymer.As used herein, term " keratan sulfate polymer " refers to the polymer of the variable-length that comprises disaccharide unit, itself comprises β-D-galactose and N-ACETYL-D-GALACTOSAMINE (GalNAc) and pharmaceutically acceptable salt thereof.Disaccharide in keratan sulfate duplicate block may cover the end at chain through fucosylation and N-acetyl-neuraminate.Any keratan sulfate polymer is all useful in compositions disclosed herein, and condition is that keratan sulfate polymer improves skin.The limiting examples of keratan sulfate pharmaceutically acceptable salt comprises keratan sulfate sodium, keratan sulfate potassium, keratan sulfate magnesium, keratan sulfate calcium and combination thereof.

The aspect of this description partly provides the hydrogel composition that comprises hyaluronan polymer.As used herein, term " hyaluronan polymer " and " HA polymer ", " hyaluronic acid polymer " and " hyaluronate polymer " synonym, refer to the anion, the non-sulfuric acid glycosaminoglycans polymer that comprise disaccharide unit, itself comprise β-1 by replacing, 4 and β-1, the D-Glucose aldehydic acid that 3 glycosidic bonds link together and D-N-acetyl glucosamine amine monomers and pharmaceutically acceptable salt thereof.Can be from animal and non-animal origin purification hyaluronan polymer.The polymer sizes scope of hyaluronan can be from approximately 5, and 000Da arrives approximately 20,000,000Da.Any hyaluronan polymer is all useful in compositions disclosed herein, and condition is that hyaluronan improves skin.The limiting examples of hyaluronan pharmaceutically acceptable salt comprises hyaluronan sodium, hyaluronan potassium, hyaluronan magnesium, hyaluronan calcium and combination thereof.

The aspect of this description partly provides the hydrogel composition that comprises crosslinked glycosaminoglycans polymer.As used herein, term " is cross-linked " and refers to that independent polymer molecule or monomer chain are connected into as gel more stable structure by intermolecular linkage.Thereby crosslinked glycosaminoglycans polymer has at least one intermolecular linkage that at least one independent polymer molecule is connected to another.The crosslinked hydrogel that conventionally causes of glycosaminoglycans polymer forms.This type of hydrogel viscosity is high and need sizable power to extrude fine needle.Can use dialdehyde and disulphide cross-linking agent to be cross-linked glycosaminoglycans polymer disclosed herein, include but not limited to multi-functional PEG based cross-linker, divinyl sulfone, glycidyl ether and di-epoxide, dual-carbodiimide.The limiting examples of hyaluronan cross-linking agent comprises multi-functional PEG based cross-linker, as tetramethylolmethane four glycidyl ethers (PETGE), divinyl sulfone (DVS), 1, 4-butanediol diglycidyl ether (BDDE), 1, 2-two (2, 3-glycidoxy) ethylene (EGDGE), 1, 2, 7, 8-diepoxy octane (DEO), (phenylene is two-(ethyl)-carbodiimide and 1, 6 hexylidenes two (ethyl carbodiimide), adipic dihydrazide (ADH), two (sulfosuccinic base) suberates (BS), hexamethylene diamine (HMDA), 1-(2, 3-glycidoxy)-2, 3-7-oxa-bicyclo[4.1.0, lysine, lysine methyl ester or its combination.At Stroumpoulis and Tezel, Tunably Crosslinked Polysaccharide Compositions, on October 22nd, 2010 submit to U.S. Patent application 12/910,466 in other useful cross-linking agent is disclosed, its by reference entirety be incorporated to.For example, at Piron and Tholin, Polysaccharide Crosslinking, Hydrogel Preparation, Resulting Polysaccharides (s) and Hydrogel (s), uses Thereof, U.S. Patent Publication 2003/0148995; Lebreton, Cross-Linking of Low and High Molecular Weight Polysaccharides Preparation of Injectable Monophase Hydrogels; Lebreton, Viscoelastic Solutions Containing Sodium Hyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses, U.S. Patent Publication 2008/0089918; Lebreton, Hyaluronic Acid-Based Gels Including Lidocaine, U.S. Patent Publication 2010/0028438; With Polysaccharides and Hydrogels thus Obtained, U.S. Patent Publication 2006/0194758; With Di Napoli, Composition and Method for Intradermal Soft Tissue Augmentation, the limiting examples of the method for crosslinked glycosaminoglycans polymer has been described in international patent publications WO2004/073759, and in compositions disclosed herein and method useful glycosaminoglycans polymer, its separately accordingly by reference entirety be incorporated to.

The aspect of this description partly provides the hydrogel composition that comprises the crosslinked glycosaminoglycans polymer with certain degree of cross linking.As used herein, term " degree of cross linking " refers to glycosaminoglycans polymer monomer unit, the percentage ratio of the hyaluronan disaccharide monomeric unit of being for example combined with cross-linking agent.The degree of cross linking is expressed as the percentage by weight of cross-linking agent and glycosaminoglycans.In some favourable embodiment of the present invention, the degree of cross linking is between approximately 3% with approximately between 12%, for example, between approximately 5% with approximately between 10%.

In one embodiment, hydrogel composition comprises crosslinked glycosaminoglycans polymer, for example cross-linked-hyaluronic acid, the concentration (for example) of the described crosslinked glycosaminoglycans polymer existing in wherein said compositions is between about 18mg/g and about 30mg/g.In some embodiments, the hyaluronic acid total concentration of described compositions is about 24mg/g or about 25mg/g.

The aspect of this description partly provides the hydrogel composition of the hyaluronan polymer of the hyaluronan polymer that comprises low-molecular-weight hyaluronan polymer, high molecular or low-molecular-weight and high molecular.As used herein, term in the time mentioning " hyaluronan " " high molecular " refers to that mean molecule quantity is 1,000,000Da or higher hyaluronan polymer.The limiting examples of high molecular hyaluronan polymer comprises approximately 1,500,000Da, approximately 2,000,000Da, approximately 2,500,000Da, approximately 3,000,000Da, approximately 3,500,000Da, approximately 4,000,000Da, approximately 4,500,000Da and approximately 5,000, the hyaluronan polymer of 000Da.As used herein, term in the time mentioning " hyaluronan " " low-molecular-weight " refers to that mean molecule quantity is less than the hyaluronan polymer of 1,000,000Da.The limiting examples of low-molecular-weight hyaluronan polymer comprises approximately 200,000Da, approximately 300,000Da, approximately 400,000Da, approximately 500,000Da, approximately 600,000Da, approximately 700,000Da, approximately 800,000Da and approximately 900, the hyaluronan polymer of 000Da.

In one embodiment, compositions comprises low-molecular-weight crosslinked hyaluronan polymer.Aspect this embodiment, compositions for example comprises mean molecule quantity, for () approximately 100,000Da, approximately 200,000Da, approximately 300,000Da, approximately 400,000Da, approximately 500,000Da, approximately 600,000Da, approximately 700,000Da, approximately 800,000Da or approximately 900, the crosslinked hyaluronan polymer of 000Da.In the other side of this embodiment, compositions for example comprises mean molecule quantity, for () at the most 100,000Da, at the most 200,000Da, at the most 300,000Da, at the most 400,000Da, at the most 500,000Da, at the most 600,000Da, at the most 700,000Da, at the most 800,000Da, at the most 900,000Da or at the most 950, the crosslinked hyaluronan polymer of 000Da.In the other side of this embodiment, compositions for example comprises mean molecule quantity, for () approximately 100, 000Da is to approximately 500, 000Da, approximately 200, 000Da is to approximately 500, 000Da, approximately 300, 000Da is to approximately 500, 000Da, approximately 400, 000Da is to approximately 500, 000Da, approximately 500, 000Da is to approximately 950, 000Da, approximately 600, 000Da is to approximately 950, 000Da, approximately 700, 000Da is to approximately 950, 000Da, approximately 800, 000Da is to approximately 950, 000Da, approximately 300, 000Da is to approximately 600, 000Da, approximately 300, 000Da is to approximately 700, 000Da, approximately 300, 000Da is to approximately 800, 000Da or approximately 400, 000Da is to approximately 700, the crosslinked hyaluronan polymer of 000Da.

In another embodiment, the crosslinked hyaluronan polymer that compositions comprises high molecular.Aspect this embodiment, compositions for example comprises mean molecule quantity, for () approximately 1,000,000Da, approximately 1,500,000Da, approximately 2,000,000Da, approximately 2,500,000Da, approximately 3,000,000Da, approximately 3,500,000Da, approximately 4,000,000Da, approximately 4,500,000Da or approximately 5,000, the crosslinked hyaluronan polymer of 000Da.In the other side of this embodiment, compositions for example comprises mean molecule quantity, for () at least 1,000,000Da, at least 1,500,000Da, at least 2,000,000Da, at least 2,500,000Da, at least 3,000,000Da, at least 3,500,000Da, at least 4,000,000Da, at least 4,500,000Da or at least 5, the crosslinked hyaluronan polymer of 000,000Da.In the other side of this embodiment, compositions for example comprises mean molecule quantity, for () approximately 1,000, and 000Da is to approximately 5,000,000Da, approximately 1,500,000Da is to approximately 5,000,000Da, approximately 2,000,000Da is to approximately 5,000,000Da, approximately 2,500,000Da is to approximately 5,000,000Da, approximately 2,000,000Da is to approximately 3,000,000Da, approximately 2,500,000Da is to approximately 3,000, the crosslinked hyaluronan polymer of 000Da.

In another embodiment, compositions comprises crosslinked hyaluronan polymer, the high molecular hyaluronan polymer that wherein said crosslinked hyaluronan polymer comprises different proportion and the combination of low-molecular-weight hyaluronan polymer.Aspect this embodiment, compositions comprises crosslinked hyaluronan polymer, and wherein said crosslinked hyaluronan polymer comprises high molecular hyaluronan polymer that ratio is about 20:1, about 15:1, about 10:1, about 5:1, about 1:1, about 1:5, about 1:10, about 1:15 or about 1:20 and the combination of low-molecular-weight hyaluronan polymer.

The aspect of this description partly provides the hydrogel composition that comprises uncrosslinked glycosaminoglycans polymer.As used herein, term " uncrosslinked " refers to lack the independent glycosaminoglycans polymer molecule of connection or the intermolecular linkage of monomer chain.Thereby uncrosslinked glycosaminoglycans polymer is not connected with any other glycosaminoglycans polymer by intermolecular linkage.Aspect this embodiment, compositions comprises uncrosslinked chondroitin sulfate polymer, uncrosslinked dermatan sulfate polymer, uncrosslinked keratan sulfate polymer, uncrosslinked heparan polymer, uncrosslinked Heparan sulfate polymer or uncrosslinked hyaluronan polymer.Uncrosslinked glycosaminoglycans polymer water soluble and conventionally keep in itself mobility.Thereby, uncrosslinked glycosaminoglycans polymer often with mix to promote compositions by the extrusion of fine needle as the hydrogel composition based on glycosaminoglycans polymer of lubricant.

In one embodiment, compositions comprises uncrosslinked glycosaminoglycans polymer, and the concentration that wherein said uncrosslinked glycosaminoglycans polymer exists is (for example) about 2mg/g, about 3mg/g, about 4mg/g, about 5mg/g, about 6mg/g, about 7mg/g, about 8mg/g, about 9mg/g, about 10mg/g, about 11mg/g, about 12mg/g, about 13mg/g, about 13.5mg/g, about 14mg/g, about 15mg/g, about 16mg/g, about 17mg/g, about 18mg/g, about 19mg/g, about 20mg/g, about 40mg/g or about 60mg/g.In the other side of this embodiment, compositions comprises uncrosslinked glycosaminoglycans, and the concentration that wherein said uncrosslinked glycosaminoglycans exists is (for example) at least 1mg/g, at least 2mg/g, at least 3mg/g, at least 4mg/g, at least 5mg/g, at least 10mg/g, at least 15mg/g, at least 20mg/g, at least 25mg/g, at least 35mg/g or 40mg/g at least.In the other side of this embodiment, compositions comprises uncrosslinked glycosaminoglycans, and the concentration that wherein said uncrosslinked glycosaminoglycans exists is (for example) 1mg/g, 2mg/g, 3mg/g, 4mg/g, 5mg/g, 10mg/g, 15mg/g, at the most 20mg/g or 25mg/g at the most at the most at the most at the most at the most at the most at the most at the most.In the other side of this embodiment, compositions comprises uncrosslinked glycosaminoglycans, and the concentration of wherein said uncrosslinked glycosaminoglycans existence is extremely extremely extremely extremely extremely extremely extremely about 15.5mg/g or extremely about 14.5mg/g of about 12.5mg/g of about 16.5mg/g, about 11.5mg/g of about 17.5mg/g, about 10.5mg/g of about 18.5mg/g, about 9.5mg/g of about 19.5mg/g, about 8.5mg/g of about 40mg/g, about 7.5mg/g of about 60mg/g, about 10mg/g of (for example) about 1mg/g.

The aspect of this description partly provides the hydrogel composition that does not basically contain crosslinked glycosaminoglycans polymer.As used herein, term " does not basically contain " compositions that (or " substantially by ... composition ") refers to wherein only can to detect the crosslinked matrix polymer of trace.Aspect of this embodiment, compositions comprise do not basically contain crosslinked chondroitin sulfate polymer chondroitin sulfate, do not basically contain crosslinked dermatan sulfate polymer dermatan sulfate, do not basically contain crosslinked keratan sulfate polymer keratan sulfate, do not basically contain crosslinked heparan polymer heparan, do not basically contain the Heparan sulfate of crosslinked Heparan sulfate polymer or do not basically contain the sulphuric acid hyaluronan of crosslinked hyaluronan polymer.

The aspect of this description partly provides completely not containing the hydrogel composition that is cross-linked glycosaminoglycans polymer.As used herein, term " does not contain completely " and refers to that compositions is in the instrument using or technique detection range, can not detect that being cross-linked glycosaminoglycans polymer maybe can not confirm its existence.Aspect of this embodiment, compositions comprises completely containing the chondroitin sulfate of crosslinked chondroitin sulfate polymer, completely containing the dermatan sulfate of crosslinked dermatan sulfate polymer, completely containing the keratan sulfate of crosslinked keratan sulfate polymer, completely containing the heparan of crosslinked heparan polymer, completely containing the Heparan sulfate of crosslinked Heparan sulfate polymer or completely containing the sulphuric acid hyaluronan of crosslinked hyaluronan polymer.

The aspect of this description partly provides the hydrogel composition that comprises a certain proportion of crosslinked glycosaminoglycans polymer and uncrosslinked glycosaminoglycans polymer.This ratio crosslinked and uncrosslinked glycosaminoglycans polymer also can be described as gel: fluid ratio.Any gel in preparation compositions disclosed herein: fluid ratio is all useful, and condition is that this ratio produces the improvement disclosed herein compositions of skin as disclosed herein.Gel in the present composition: the limiting examples of fluid ratio comprises 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.

Aspect this embodiment, compositions comprises crosslinked glycosaminoglycans polymer and uncrosslinked glycosaminoglycans polymer, wherein 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 the other side of this embodiment, compositions comprises crosslinked glycosaminoglycans polymer and uncrosslinked glycosaminoglycans polymer, wherein gel: fluid ratio is (for example) 1:99,2:98,3:97,4:96,5:95,6:94,7:93,8:92, at the most 9:91 or 10:90 at the most at the most at the most at the most at the most at the most at the most at the most at the most.In the other side of this embodiment, compositions comprises crosslinked glycosaminoglycans polymer and uncrosslinked glycosaminoglycans polymer, wherein gel: fluid ratio is that (for example) about 0:100 is to extremely about 5:95 or extremely about 10:90 of about 0:100 of about 3:97, about 0:100.

In the other side of this embodiment, compositions comprises crosslinked glycosaminoglycans polymer and uncrosslinked glycosaminoglycans polymer, wherein 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 the other side of this embodiment, compositions comprises crosslinked glycosaminoglycans polymer and uncrosslinked glycosaminoglycans polymer, wherein gel: fluid ratio is (for example) 15:85,20:80,25:75,30:70,35:65,40:60,45:55,50:50,55:45,60:40,65:35,70:30,75:25,80:20,85:15,90:10,95:5, at the most 98:2 or 100:0 at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most.In the other side of this embodiment, compositions comprises crosslinked glycosaminoglycans polymer and uncrosslinked glycosaminoglycans polymer, wherein gel: fluid ratio be that (for example) about 10:90 is 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 extremely about 100:0 of about 60:40.

Hydrogel composition disclosed herein provides another kind of reagent or the agent combination of beneficial effect can further be included in to individual applying said compositions time.This type of beneficial agent includes but not limited to antioxidant, pruritus, the fat agent that disappears, anti-scar agent, antiinflammatory, anesthetis, counter-stimulus, vasoconstrictor, vasodilation, hemorrhage agent (as hemorrhage or anti-fibrolysis protein agent), remover, tensioning agent, anti-pox agent, pigmentation agent, anti-pigmentation agent or wetting agent.

For the purposes of the present invention, unless otherwise indicated, otherwise " % " in formula is defined as weight (, w/w) percentage ratio.

Aspect of the present invention partly provides disclosed herein can optionally comprise narcotic hydrogel composition.Anesthetis is preferably local anesthetic, causes reversibility local anesthesia and loses the anesthetis of nociception, for example amino amides local anesthetic and amino ester local anesthetic.The narcotic amount that compositions disclosed herein comprises is effectively to measure alleviate the individual pain of experiencing after applying said compositions.Thereby the narcotic amount that disclosed in this manual compositions comprises is between arriving approximately between 5% by general composition weight meter approximately 0.1%.Narcotic limiting examples comprises lignocaine, ambucaine (ambucaine), amolanone (amolanone), amylocaine (amylocaine), benoxinate (benoxinate), benzocaine (benzocaine), betoxycaine (betoxycaine), xenysalate (biphenamine), marcaine (bupivacaine), butacaine (butacaine), butamben (butamben), butanilicaine (butanilicaine), butethamine (butethamine), 2-diethylaminoethyl p-butoxybenzoate. (butoxycaine), carticaine (carticaine), chloroprocaine (chloroprocaine), hexyl benzoic acid Ai Kangyin (cocaethylene), cocaine (cocaine), cyclomethycaine (cyclomethycaine), cinchocaine (dibucaine), quotane (dimethysoquin), dimethocaine (dimethocaine), diperodon (diperodon), Neoquess (dycyclonine), bud that anhydrates subtracts (ecgonidine), bud subtracts (ecgonine), ethyl chloride, etidocaine (etidocaine), betaeucaine (beta-eucaine), euprocin (euprocin), fenalcomine (fenalcomine), formocaine, hexylcaine (hexylcaine), hydroxytetracaine (hydroxy teracaine), cycloform, leucinocaine mesylate (leucinocaine mesylate), levoxadrol (Ievoxadrol), lignocaine, mepivacaine (mepivacaine), meprylcaine (meprylcaine), metabutoxycaine (metabutoxycaine), chloromethanes, Myrtecaine (myrtecaine), receive her caine (naepaine), octacaine (octacaine), orthocaine (orthocaine), Mucaine (oxethazaine), parethoxycaine (parethoxycaine), phenacaine (phenacaine), phenol, piperocaine (piperocaine), piridocaine (piridocaine), polidocanol (polidocanol), pramoxine (pramoxine), prilocaine (prilocaine), procaine (procaine), propanocaine (propanocaine), proparacaine (proparacaine), Bing Veins caine (propipocaine), propoxycaine (propoxycaine), d-pseudococaine (psuedococaine), pyrrocaine (pyrrocaine), ropivacaine (ropivacaine), saligenin, tetracaine (tetracaine), tolycaine (toIycaine), trimecaine (trimecaine), zolamine (zolamine), its combination and salt thereof.Amino ester local anesthetic comprises procaine, chloroprocaine, cocaine, cyclomethycaine, cimethocaine (larocaine (larocaine)), propoxycaine, procaine (novocain (novocaine)), proparacaine, tetracaine (amethocaine (amethocaine)).The limiting examples of amino amides local anesthetic comprises articaine (articaine), marcaine, cinchocaine (cinchocaine) (cinchocaine), etidocaine, chirocaine (levobupivacaine), lignocaine (lignocaine (lignocaine)), mepivacaine, piperocaine, prilocaine, ropivacaine and trimecaine.Compositions disclosed herein can comprise single anesthesia agent or multiple anesthetis.The limiting examples of combination local anesthetic is lignocaine/prilocaine (EMLA).

Therefore in one embodiment, compositions disclosed herein comprises anesthetis and salt thereof.Aspect this embodiment, compositions disclosed herein comprises amino amides local anesthetic and salt or amino ester local anesthetic and salt thereof.In the other side of this embodiment, compositions disclosed herein comprises procaine, chloroprocaine, cocaine, cyclomethycaine, cimethocaine, propoxycaine, procaine, proparacaine, tetracaine or its salt or its any combination.In the other side of this embodiment, compositions disclosed herein comprises articaine, marcaine, cinchocaine, etidocaine, chirocaine, lignocaine, mepivacaine, piperocaine, prilocaine, ropivacaine, trimecaine or its salt or its any combination.Aspect this embodiment other, compositions disclosed herein comprises the combination of lignocaine/prilocaine.

In the other side of this embodiment, compositions disclosed herein comprises the weighing scale by total composition, measures for example, anesthetis into () approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1.0%, approximately 2.0%, approximately 3.0%, approximately 4.0%, approximately 5.0%, approximately 6.0%, approximately 7.0%, approximately 8.0%, approximately 9.0% or approximately 10%.Aspect other, compositions disclosed herein comprises the weighing scale by total composition, measures for example, anesthetis into () 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%, at least 0.8%, at least 0.9%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0% or at least 10%.Also having other side, compositions disclosed herein comprises the weighing scale by total composition, for example measures, as () 0.1%, at the most 0.2%, at the most 0.3%, at the most 0.4%, at the most 0.5%, at the most 0.6%, at the most 0.7%, at the most 0.8%, at the most 0.9%, at the most 1.0%, at the most 2.0%, at the most 3.0%, at the most 4.0%, at the most 5.0%, at the most 6.0%, at the most 7.0%, at the most 8.0%, at the most 9.0% or at the most 10% anesthetis at the most.In other side, compositions disclosed herein comprises the weighing scale by total composition, measures for example, anesthetis into () approximately 0.1% to approximately 0.5%, approximately 0.1% to approximately 1.0%, approximately 0.1% to approximately 2.0%, approximately 0.1% to approximately 3.0%, approximately 0.1% to approximately 4.0%, approximately 0.1% to approximately 5.0%, approximately 0.2% to approximately 0.9%, approximately 0.2% to approximately 1.0%, approximately 0.2% to approximately 2.0%, approximately 0.5% to approximately 1.0% or approximately 0.5% to approximately 2.0%.

In another embodiment, compositions disclosed herein does not comprise anesthetis.

In one aspect of the invention, provide injectable corium filler, it comprises polymer, for example glycosaminoglycans polymer, for example hyaluronic acid polymer, the hyaluronic acid that for example at least a portion is crosslinked, and additive or with the beneficial agent of described combination of polymers.

Comprise vitamin with the beneficial agent of described combination of polymers, for example vitamin C.The ascorbic limiting examples that is applicable to form comprises fat-soluble ester (ascorbyl palmitate or ascorbyl stearate), magnesium L-ascorbyl-2-phosphate (MAP), sodium ascorbyl phosphate (SAP) and the ascorbic acid 2-glucoside (AA2G of sodium salt, potassium salt and calcium salt, ascorbic acid and the long-chain fatty acid of ascorbic acid and ascorbic acid ^tM), sodium ascorbyl phosphate (AA2P), ascorbic acid sulphuric acid disodium and ascorbic acid 3-aminopropyl phosphate ester (vitagen).

In a particularly advantageous embodiment, beneficial agent and described polymer covalent bond.For example, beneficial agent can be vitamin C or vitamin C derivatives, itself and described polymer covalent bond and press the weighing scale of total composition, the amount existing in compositions between approximately 0.04% to approximately between 5.0%, the weighing scale of for example pressing total composition between approximately 0.1% to approximately between 4.0%, the weighing scale of for example pressing total composition between approximately 0.2% to approximately between 2.0%.In one embodiment, the ascorbic amount that compositions disclosed herein comprises by the weighing scale of total composition between approximately 0.3% to approximately between 1.2%.

Preferably, with the covalently bound vitamin C of described compositions comprise following at least one: ascorbic acid, L-AA, L-AA 2-sulfuric ester (AA-2S) and L-AA 2-phosphate ester (AA-2P), ascorbic acid 2-O-glucoside (AA-2G), 6-O-acyl group-2-O-α-D-glucopyranosyl-L-AA (6-acyl group-AA-2G), (ascorbic acid 3-aminopropyl phosphate ester, ascorbyl palmitate), its derivant and combination.Compositions disclosed herein can comprise single vitamin C reagent or multivitamin C reagent of planting.

In another embodiment of the present invention, corium filler is provided, wherein hyaluronic acid and BDDE are crosslinked.In this embodiment, conjugation can be between about 3mol% and about 10mol%, and about 15mol% is between about 40mol%.

In some embodiments, corium filler has lasting bioavailability.For example, provide in the time introducing in human skin, effectively discharged ascorbic acid or other vitamin at least about 1 month or reach approximately 20 months or the corium filler of longer time to the mankind.

Aspect of the present invention partly provides the hydrogel composition of demonstration complex modulus disclosed herein, elastic modelling quantity, viscous modulus and tan δ.In the time applying power (stress, distortion), compositions tool viscoelasticity disclosed herein, because compositions has elastic component (solid, shaped for example crosslinked glycosaminoglycans polymer) and sticky ingredient (liquid for example uncrosslinked glycosaminoglycans polymer or carrier phase).The rheological attributes of describing this character is complex modulus (G*), the total resistance of its definitions section compound to distortion.Complex modulus is the plural number with real part and imaginary part: G*=G'+iG''.The absolute value of G* is Abs (G*)=Sqrt (G' ²+ G " ²).Complex modulus can be defined as to the summation of elastic modelling quantity (G ') and viscous modulus (G ").Falcone etc., Temporary Polysaccharide Dermal Fillers:A Model for Persistence Based on Physical Properties, Dermatol Surg.35 (8): 1238-1243 (2009); Tezel, the same, 2008; Kablik, the same, 2009; Beasley, the same, 2009; Its separately accordingly by reference entirety be incorporated to.

Elastic modelling quantity or modulus of elasticity refer to the ability of hydrogel material resistance to deformation, or on the contrary, the tendency of object to impermanency distortion in the time that it is applied to power.Elastic modelling quantity has characterized the fastness of compositions, and because it has described the energy storage from compositions motion, so the storage modulus of being also referred to as.Elastic modelling quantity has been described interaction between elasticity and intensity (G '=stress/strain), and therefore the quantitative measurement to composition hardness or pliability is provided.The elastic modelling quantity of object is defined as to the slope of its load-deformation curve in strain region: λ=stress/strain, wherein λ be Pascal's theorem (Pascal ' elastic modelling quantity in s); Stress is that transmutative force is divided by the area that applies power; And strain is the variation that causes of stress and the ratio of object original state.Although depend on the speed of the power of applying, compositions is harder, the elastic modelling quantity that has is higher and material is out of shape the power of Hua Geng great at distance to a declared goal, for example injection.Illustrate and how to measure stress, comprise direction, allow definition to be permitted eurypalynous elastic modelling quantity.3 main elastic modelling quantity are stretch modulus, modulus of shearing and bulk modulus.

Viscous modulus is also referred to as loss modulus, because it has described the energy of the loss with viscous dissipation.Tan δ is the ratio of viscous modulus and elastic modelling quantity, tan δ=G "/G '.Falcone, the same, 2009.For disclosed in this manual tan δ-value, tan δ is obtained by the dynamic modulus under 1Hz frequency.Lower tan δ is corresponding to harder, firmer or more resilient compositions.

In another embodiment, hydrogel composition disclosed herein demonstrates elastic modelling quantity.Aspect this embodiment, hydrogel composition demonstrates (for example) about 25Pa, about 50Pa, about 75Pa, about 100Pa, about 125Pa, about 150Pa, about 175Pa, about 200Pa, about 250Pa, about 300Pa, about 350Pa, about 400Pa, about 450Pa, about 500Pa, about 550Pa, about 600Pa, about 650Pa, about 700Pa, about 750Pa, about 800Pa, about 850Pa, about 900Pa, about 950Pa, approximately 1, 000Pa, approximately 1, 200Pa, approximately 1, 300Pa, approximately 1, 400Pa, approximately 1, 500Pa, approximately 1, 600Pa, about 1700Pa, about 1800Pa, about 1900Pa, approximately 2, 000Pa, approximately 2, 100Pa, approximately 2, 200Pa, approximately 2, 300Pa, approximately 2, 400Pa or approximately 2, the elastic modelling quantity of 500Pa.In the other side of this embodiment, hydrogel composition demonstrates (for example) at least 25Pa, at least 50Pa, at least 75Pa, at least 100Pa, at least 125Pa, at least 150Pa, at least 175Pa, at least 200Pa, at least 250Pa, at least 300Pa, at least 350Pa, at least 400Pa, at least 450Pa, at least 500Pa, at least 550Pa, at least 600Pa, at least 650Pa, at least 700Pa, at least 750Pa, at least 800Pa, at least 850Pa, at least 900Pa, at least 950Pa, at least 1, 000Pa, at least 1, 200Pa, at least 1, 300Pa, at least 1, 400Pa, at least 1, 500Pa, at least 1, 600Pa, at least 1700Pa, at least 1800Pa, at least 1900Pa, at least 2, 000Pa, at least 2, 100Pa, at least 2, 200Pa, at least 2, 300Pa, at least 2, 400Pa or at least 2, the elastic modelling quantity of 500Pa.Aspect this embodiment other, hydrogel composition demonstrates (for example) 25Pa at the most, 50Pa at the most, 75Pa at the most, 100Pa at the most, 125Pa at the most, 150Pa at the most, 175Pa at the most, 200Pa at the most, 250Pa at the most, 300Pa at the most, 350Pa at the most, 400Pa at the most, 450Pa at the most, 500Pa at the most, 550Pa at the most, 600Pa at the most, 650Pa at the most, 700Pa at the most, 750Pa at the most, 800Pa at the most, 850Pa at the most, 900Pa at the most, 950Pa at the most, at the most 1, 000Pa, at the most 1, 200Pa, at the most 1, 300Pa, at the most 1, 400Pa, at the most 1, 500Pa or at the most 1, the elastic modelling quantity of 600Pa.The other aspect of this embodiment, hydrogel composition demonstrates (for example) about 25Pa to about 150Pa, about 25Pa is to about 300Pa, about 25Pa is to about 500Pa, about 25Pa is to about 800Pa, about 125Pa is to about 300Pa, about 125Pa is to about 500Pa, about 125Pa is to about 800Pa, about 500Pa is to approximately 1,600Pa, about 600Pa is to approximately 1,600Pa, about 700Pa is to approximately 1,600Pa, about 800Pa is to approximately 1,600Pa, about 900Pa is to approximately 1,600Pa, approximately 1,000Pa is to approximately 1,600Pa, approximately 1,100Pa is to approximately 1,600Pa, approximately 1,200Pa is to approximately 1,600Pa, about 500Pa is to approximately 2,500Pa, approximately 1,000Pa is to approximately 2,500Pa, approximately 1,500Pa is to approximately 2,500Pa, approximately 2,000Pa is to approximately 2,500Pa, approximately 1,300Pa is to approximately 1,600Pa, about Isosorbide-5-Nitrae 00Pa is to approximately 1,700Pa, approximately 1,500Pa is to approximately 1,800Pa, approximately 1,600Pa is to approximately 1,900Pa, approximately 1,700Pa is to approximately 2,000Pa, approximately 1,800Pa is to approximately 2,100Pa, approximately 1,900Pa is to approximately 2,200Pa, approximately 2,000Pa is to approximately 2,300Pa, approximately 2,100Pa is to approximately 2,400Pa or approximately 2, and 200Pa is to approximately 2, the elastic modelling quantity of 500Pa.

In another embodiment, hydrogel composition disclosed herein demonstrates viscous modulus.Aspect this embodiment, hydrogel composition demonstrates the viscous modulus of (for example) about 10Pa, about 20Pa, about 30Pa, about 40Pa, about 50Pa, about 60Pa, about 70Pa, about 80Pa, about 90Pa, about 100Pa, about 150Pa, about 200Pa, about 250Pa, about 300Pa, about 350Pa, about 400Pa, about 450Pa, about 500Pa, about 550Pa, about 600Pa, about 650Pa or about 700Pa.In the other side of this embodiment, hydrogel composition demonstrates (for example) 10Pa, 20Pa, 30Pa, 40Pa, 50Pa, 60Pa, 70Pa, 80Pa, 90Pa, 100Pa, 150Pa, 200Pa, 250Pa, 300Pa, 350Pa, 400Pa, 450Pa, 500Pa, 550Pa, 600Pa, at the most 650Pa or viscous modulus of 700Pa at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most at the most.Aspect this embodiment other, hydrogel composition demonstrates (for example) about 10Pa to about 30Pa, about 10Pa is to about 50Pa, about 10Pa is to about 100Pa, about 10Pa is to about 150Pa, about 70Pa is to about 100Pa, about 50Pa is to about 350Pa, about 150Pa is to about 450Pa, about 250Pa is to about 550Pa, about 350Pa is to about 700Pa, about 50Pa is to about 150Pa, about 100Pa is to about 200Pa, about 150Pa is to about 250Pa, about 200Pa is to about 300Pa, about 250Pa is to about 350Pa, about 300Pa is to about 400Pa, about 350Pa is to about 450Pa, about 400Pa is to about 500Pa, about 450Pa is to about 550Pa, about 500Pa is to about 600Pa, about 550Pa is to about 650Pa or the extremely viscous modulus of about 700Pa of about 600Pa.

In another embodiment, hydrogel composition disclosed herein demonstrates tan δ.Aspect this embodiment, hydrogel composition demonstrates the tan δ of (for example) approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1.0, approximately 1.1, approximately 1.2, approximately 1.3, approximately 1.4, approximately 1.5, approximately 1.6, approximately 1.7, approximately 1.8, approximately 1.9, approximately 2.0, approximately 2.1, approximately 2.2, approximately 2.3, approximately 2.4 or approximately 2.5.In the other side of this embodiment, hydrogel composition demonstrates (for example) tan δ of 0.1, at the most 0.2, at the most 0.3, at the most 0.4, at the most 0.5, at the most 0.6, at the most 0.7, at the most 0.8, at the most 0.9, at the most 1.0, at the most 1.1, at the most 1.2, at the most 1.3, at the most 1.4, at the most 1.5, at the most 1.6, at the most 1.7, at the most 1.8, at the most 1.9, at the most 2.0, at the most 2.1, at the most 2.2, at the most 2.3, at the most 2.4 or at the most 2.5 at the most.Aspect this embodiment other, hydrogel composition demonstrates the tan δ of (for example) approximately 0.1 to approximately 0.3, approximately 0.3 to approximately 0.5, approximately 0.5 to approximately 0.8, approximately 1.1 to approximately 1.4, approximately 1.4 to approximately 1.7, approximately 0.3 to approximately 0.6, approximately 0.1 to approximately 0.5, approximately 0.5 to approximately 0.9, approximately 0.1 to approximately 0.6, approximately 0.1 to approximately 1.0, approximately 0.5 to approximately 1.5, approximately 1.0 to approximately 2.0 or approximately 1.5 to approximately 2.5.

The aspect of this description partly provides the hydrogel composition with the transparency and/or translucence disclosed herein.Optical transparence is to allow visible ray by the physical property of material, and translucence (also referred to as translucent or translucence) only allows light diffusion to pass through.Relative character is opacity.Transparent material is limpid, and trnaslucent materials can not clearly be understood thoroughly.Hydrogel disclosed herein is preferably optical clear or at least translucent.

In one embodiment, hydrogel composition optical clear disclosed herein.Aspect this embodiment, the light of hydrogel composition diffusion conduction (for example) approximately 75%, approximately 80% light, approximately 85% light, approximately 90% light, approximately 95% light or approximately 100% light.In the other side of this embodiment, the light of hydrogel composition diffusion conduction (for example) at least 75%, at least 80% light, at least 85% light, at least 90% light or at least 95% light.Aspect this embodiment other, the light of hydrogel composition diffusion conduction (for example) approximately 75% to approximately 100%, approximately 80% to approximately 100% light, approximately 85% to approximately 100% light, approximately 90% to approximately 100% light or approximately 95% to approximately 100% light.In one embodiment, the visible ray of hydrogel composition optical clear disclosed herein and conduction 100%.

Can further process hydrogel composition disclosed herein and optionally mix to form injectable or topical substance with carrier for example water of phase or saline solution by hydrogel being worn into granule, as solution, oil, washing liquid, gel, ointment, emulsifiable paste, slurry, ointment or paste.Thereby disclosed hydrogel composition can be single-phase or heterogeneous compositions.Hydrogel can be milled into the granularity of diameter 10 μ m to approximately 1000 μ m, for example extremely extremely extremely extremely extremely extremely extremely approximately 850 μ m or extremely approximately 1,000 μ m of approximately 900 μ m of approximately 700 μ m, approximately 750 μ m of approximately 550 μ m, approximately 600 μ m of approximately 300 μ m, approximately 450 μ m of approximately 150 μ m, approximately 200 μ m of approximately 75 μ m, approximately 100 μ m of approximately 30 μ m, approximately 50 μ m of approximately 15 μ m.

Aspect of the present invention partly provides injectable compositions disclosed herein.As used herein, term " injectable " refers to that material has the injection device using with fine needle to the necessary character of compositions described in individual's skin Zoned application.As used herein, term " fine needle " refers to 27G or less pin.Can be by determining that as discussed above the size of hydrogel particle realizes the syringeability of compositions disclosed herein.

Aspect this embodiment, hydrogel composition disclosed herein can be injected by fine needle.In the other side of this embodiment, hydrogel composition disclosed herein can for example, by the pin injection of () about 27G, about 30G or about 32G.Aspect this embodiment other, hydrogel composition disclosed herein can for example, by () 22G or less, 27G or less, 30G or less or 32G or less pin injection.Aspect this embodiment other, hydrogel composition disclosed herein can for example, by () about 22G pin injection to about 27G or about 27G to about 32G to about 35G, about 22G to about 34G, about 22G to about 33G, about 22G to about 32G, about 22G.

Aspect this embodiment, hydrogel composition disclosed herein can be by the extruding force of about 60N, about 55N, about 50N, about 45N, about 40N, about 35N, about 30N, about 25N, about 20N or about 15N, with the speed injection of 100mm/min.In the other side of this embodiment, hydrogel composition disclosed herein can pass through 27G pin, with 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, about 15N or less, about 10N or less or about 5N or less extruding force injection.Aspect this embodiment other, hydrogel composition disclosed herein can pass through 30G pin, with 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, about 15N or less, about 10N or less or about 5N or less extruding force injection.Aspect this embodiment other, hydrogel composition disclosed herein can pass through 32G pin, with 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, about 15N or less, about 10N or less or about 5N or less extruding force injection.

Aspect of the present invention partly provides the hydrogel composition that demonstrates cohesiveness disclosed herein.Cohesiveness, also referred to as cohesion adhesion gravitation, bonding force or compression stress, is the physical property that plays the material that the intermolecular attraction of the effect of molecule synthesis one is caused in material between similar molecule.With fors (gmf) expression cohesiveness.Except other factors, caking property be subject to initially to dissociate glycosaminoglycans polymer molecular weight ratio, glycosaminoglycans polymer the degree of cross linking, crosslinked after the residual amount of free glycosaminoglycans polymer and the pH of hydrogel composition affect.Compositions should have enough caking property, uses part to remain in.In addition, in some applications, enough caking property keeps its shape to compositions, and therefore in the time there is mechanical load cycle, keeps functional very important.Thereby in one embodiment, hydrogel composition disclosed herein demonstrates cohesiveness, with water peer-level.In yet another embodiment, gel combination disclosed herein demonstrates enough cohesiveness and uses part to remain in.In yet another embodiment, gel combination disclosed herein demonstrates enough cohesiveness to keep its shape.In yet another embodiment, gel combination disclosed herein demonstrates enough cohesiveness to keep its shape and functional.

Aspect of the present invention partly provides the hydrogel composition that demonstrates physiologically acceptable M osmotic concentration (osmolarity) disclosed herein.As used herein, term " M osmotic concentration " refers to the concentration of osmotically active solute in solution.As used herein, term " physiologically acceptable M osmotic concentration " refers to conform to the normal function of live organism or its distinctive M osmotic concentration.Thereby, in the time being administered to mammal, using hydrogel composition disclosed herein and show the M osmotic concentration that there is no substantially long-term or permanent adverse effect.M osmotic concentration represents with the osmol(e) (Osmol/L or Osm/L) of osmotically active solute in every liter of solvent.M osmotic concentration is different from molar concentration, because it has measured the molal quantity of osmotically active solute granule instead of the molal quantity of solute.Because some compound can be in solution dissociation, and other can not, so there is difference.Can be calculated by following expression formula the M osmotic concentration of solution: , wherein for infiltration coefficient, it has illustrated the Non Ideal Degree of solution; η is the quantity of the granule (for example ion) of molecular dissociation one-tenth; And the molar concentration that C is solute; And i is the index that represents specific solute homogeneity.Can use the conventional method of measurement solution to measure the M osmotic concentration of hydrogel composition disclosed herein.

In one embodiment, hydrogel composition disclosed herein demonstrates physiologically acceptable M osmotic concentration.As used herein, term " osmolality " refers to the concentration of osmotically active solute in the interior every kilogram of solvent of body.As used herein, term " physiologically acceptable osmolality " refers to conform to or its distinctive osmolality with the normal function of live organism.Thereby, in the time being administered to mammal, using hydrogel composition disclosed herein and show the osmolality that there is no substantially long-term or permanent adverse effect.Osmolality represents and equals the weight-molal summation of all solutes that exist in this solution with the osmol(e) (osmol/kg or Osm/kg) of osmotically active solute in every kilogram of solvent.Can use osmometer to measure the osmolality of solution.In modern laboratory, the most frequently used instrument is that freezing point reduces osmometer.This apparatus measures increase the variation (freezing point reduction osmometer) that freezing point occurs in solution or increase with osmolality the variation (the low osmometer of steam drop) that in solution, vapour pressure occurs with osmolality.

Aspect this embodiment, hydrogel composition demonstrates the M osmotic concentration of (for example) about 100mOsm/L, about 150mOsm/L, about 200mOsm/L, about 250mOsm/L, about 300mOsm/L, about 350mOsm/L, about 400mOsm/L, about 450mOsm/L or about 500mOsm/L.In the other side of this embodiment, hydrogel composition demonstrates (for example) at least 100mOsm/L, at least 150mOsm/L, at least 200mOsm/L, at least 250mOsm/L, at least 300mOsm/L, at least 350mOsm/L, at least 400mOsm/L, at least 450mOsm/L or M osmotic concentration of 500mOsm/L at least.Aspect this embodiment other, hydrogel composition demonstrates (for example) 100mOsm/L, 150mOsm/L, 200mOsm/L, 250mOsm/L, 300mOsm/L, 350mOsm/L, 400mOsm/L, at the most 450mOsm/L or M osmotic concentration of 500mOsm/L at the most at the most at the most at the most at the most at the most at the most at the most.Aspect this embodiment other, hydrogel composition demonstrate (for example) about 100mOsm/L to about 500mOsm/L, about 200mOsm/L to about 500mOsm/L, about 200mOsm/L to about 400mOsm/L, about 300mOsm/L to about 400mOsm/L, about 270mOsm/L to about 390mOsm/L, about 225mOsm/L to about 350mOsm/L, about 250mOsm/L to about 325mOsm/L, about 275mOsm/L to about 300mOsm/L or about 285mOsm/L to the M osmotic concentration of about 290mOsm/L.

Aspect of the present invention partly provides the hydrogel composition of stability substantially that demonstrates disclosed herein.As used herein, term " stability " or " stablizing ", in the time mentioning hydrogel composition disclosed herein, refer to that compositions is before being administered to individuality, are not easy to degraded, decompose or are crushed to substantially any or significant degree in the time storing.As used herein, term " substantially heat stability ", " thermally-stabilised substantially ", " autoclave is stable " or " steam sterilization is stable " refer to that hydrogel composition disclosed herein is stable substantially in the time being subject to as disclosed herein heat treatment.

Can be by making hydrogel composition heat-treated, for example, for example, at normal pressure or add depress (, autoclaving) steam sterilization, measure the stability of hydrogel composition disclosed herein.Preferably at the temperature at least about 100 DEG C, heat-treat approximately 1 minute to approximately 10 minutes.Can assess by following manner the cardinal principle stability of hydrogel composition disclosed herein: 1) after mensuration sterilizing, the extruding force of hydrogel composition disclosed herein changes (Δ F), wherein deduct (the not extruding force of additivated hydrogel composition) by (having the extruding force of the hydrogel composition of specifying additive) and measure, the variation of extruding force is less than 2N and shows that hydrogel composition is stable substantially; And/or 2) after mensuration sterilizing, the rheological characteristic of hydrogel composition disclosed herein changes, wherein deduct (the tan δ 1Hz that there is no the gel preparation of additive) by (having the tan δ 1Hz of the gel preparation of additive) and measure, the variation of tan δ 1Hz is less than 0.1 and shows that hydrogel composition is stable substantially.Thereby stable hydrogel composition substantially disclosed herein has kept following one or more characteristics after sterilizing: other required rheological behavior of hydrogel before uniformity, extruding force, caking property, hyaluronan concentration, reagent concentration, M osmotic concentration, pH or heat treatment.

In one embodiment, the hydrogel composition that uses the heat treatment process that maintains required hydrogel character disclosed herein to comprise glycosaminoglycans polymer and at least one reagent disclosed herein.Aspect this embodiment, the hydrogel composition that uses the heat treatment process of (for example) approximately 100 DEG C, approximately 105 DEG C, approximately 110 DEG C, approximately 115 DEG C, approximately 120 DEG C, approximately 125 DEG C or approximately 130 DEG C to comprise glycosaminoglycans polymer and at least one reagent disclosed herein.In the other side of this embodiment, the hydrogel composition that uses the heat treatment process of (for example) at least 100 DEG C, at least 105 DEG C, at least 110 DEG C, at least 115 DEG C, at least 120 DEG C, at least 125 DEG C or at least 130 DEG C to comprise glycosaminoglycans polymer and at least one reagent disclosed herein.Aspect this embodiment other, the hydrogel composition that uses the heat treatment process of (for example) approximately 100 DEG C to approximately 120 DEG C, approximately 100 DEG C to approximately 125 DEG C, approximately 100 DEG C to approximately 130 DEG C, approximately 100 DEG C to approximately 135 DEG C, approximately 110 DEG C to approximately 120 DEG C, approximately 110 DEG C to approximately 125 DEG C, approximately 110 DEG C to approximately 130 DEG C, approximately 110 DEG C to approximately 135 DEG C, approximately 120 DEG C to approximately 125 DEG C, approximately 120 DEG C to approximately 130 DEG C, approximately 120 DEG C to approximately 135 DEG C, approximately 125 DEG C to approximately 130 DEG C or approximately 125 DEG C to approximately 135 DEG C to comprise glycosaminoglycans polymer and at least one reagent disclosed herein.

Can be by making hydrogel composition heat-treated, be for example stored in approximately 45 DEG C of environment approximately 60 days, measure the long-time stability of hydrogel composition disclosed herein.Can assess by following manner the long-time stability of hydrogel composition disclosed herein: 1) clarity and the color of hydrogel composition after 45 DEG C of heat treatments of estimation, hydrogel composition is limpid and not painted shows that hydrogel composition is stable substantially; 2) extruding force that is determined at hydrogel composition disclosed herein after 45 DEG C of heat treatments changes (Δ F), wherein by (before 45 DEG C of heat treatments, have the extruding force of the hydrogel composition of specifying additive) deduct (after 45 DEG C of heat treatments, have the extruding force of the hydrogel composition of specifying additive) to measure, the variation of extruding force is less than 2N and shows that hydrogel composition is stable substantially; And/or 3) after mensuration sterilizing, the rheological characteristic of hydrogel composition disclosed herein changes, wherein by (before 45 DEG C of heat treatments, have the tan δ 1Hz of the gel preparation of specifying additive) deduct (after 45 DEG C of heat treatments, have the tan δ 1Hz of the gel preparation of specifying additive) to measure, the variation of tan δ 1Hz is less than 0.1 and shows that hydrogel composition is stable substantially.Thereby, be used in the long-time stability that 45 DEG C of heat treatments have kept following one or more feature evaluations hydrogel composition disclosed herein afterwards: clarity (transparent and translucent), uniformity and caking property.

Aspect this embodiment, hydrogel composition is at room temperature stablized (for example) approximately 3 months, approximately 6 months, approximately 9 months, approximately 12 months, approximately 15 months, approximately 18 months, approximately 21 months, approximately 24 months, approximately 27 months, approximately 30 months, approximately 33 months or approximately 36 months substantially.In the other side of this embodiment, hydrogel composition is at room temperature stablized (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 27 months, at least 30 months, at least 33 months or at least 36 months substantially.In the other side of this embodiment, hydrogel composition is at room temperature stablized (for example) approximately 3 months to approximately 12 months substantially, approximately 3 months to approximately 18 months, approximately 3 months to approximately 24 months, approximately 3 months to approximately 30 months, approximately 3 months to approximately 36 months, approximately 6 months to approximately 12 months, approximately 6 months to approximately 18 months, approximately 6 months to approximately 24 months, approximately 6 months to approximately 30 months, approximately 6 months to approximately 36 months, approximately 9 months to approximately 12 months, approximately 9 months to approximately 18 months, approximately 9 months to approximately 24 months, approximately 9 months to approximately 30 months, approximately 9 months to approximately 36 months, approximately 12 months to approximately 18 months, approximately 12 months to approximately 24 months, approximately 12 months to approximately 30 months, approximately 12 months to approximately 36 months, approximately 18 months to approximately 24 months, approximately 18 months to approximately 30 months or approximately 18 months to approximately 36 months.

This compositions can optionally include but not limited to other pharmaceutically acceptable component, includes but not limited to buffer agent, antiseptic, tension regulator, salt, antioxidant, osmolality regulator, emulsifying agent, wetting agent etc.

Pharmaceutically acceptable buffer agent is the buffer agent that can be used for preparing hydrogel composition disclosed herein, and condition is that gained preparation pharmaceutically can be accepted.The limiting examples of pharmaceutically acceptable buffer agent comprises acetate buffer, borate buffer, citrate buffer agent, neutral buffered saline, phosphate buffer and phosphate buffered saline (PBS).The pharmaceutically acceptable buffer agent of any concentration can be used for preparing pharmaceutical composition disclosed herein, condition is to use the buffer agent of valid density to resume treatment the active component of effective dose.The limiting examples of physiologically acceptable buffer concentration is present in about 0.1mM to about 900mM.The pH of the pharmaceutically acceptable buffer agent of scalable, condition is that gained preparation is pharmaceutically can accept.Should understand the pH that can use as required acid or alkali regulating drug compositions.Can be by the pH level of any buffering for compounding pharmaceutical compositions, condition is to use this effective pH level to resume treatment the matrix polymer of effective dose.The limiting examples of physiologically acceptable pH is present in about pH5.0 to the scope of about pH8.5.For example, the pH of hydrogel composition disclosed herein can be approximately 5.0 to approximately 8.0 or approximately 6.5 to approximately 7.5, approximately 7.0 to approximately 7.4 or approximately 7.1 to approximately 7.3.

Pharmaceutically acceptable antiseptic includes but not limited to sodium pyrosulfite, sodium thiosulfate, acetylcysteine, anethole htpb and butylated hydroxytoluene.Pharmaceutically acceptable antiseptic includes but not limited to benzalkonium chloride, methaform, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, stabilisation oxygen chlorine compositions, for example (Allergan, Inc.Irvine, CA) and chelating agen, for example DTPA or DTPA-bisamide, DTPA calcium and CaNaDTPA-bisamide.

Pharmaceutically acceptable tension regulator for hydrogel composition disclosed herein includes but not limited to for example sodium chloride of salt and potassium chloride; And glycerol.Can provide described compositions as salt and can form together with much acid, including but not limited to hydrochloric acid, sulphuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid etc.Salt tends to or other proton solvent more soluble in water than corresponding free alkali form.Should be understood that and in pharmaceutical composition disclosed herein, can comprise these and other material known in pharmaceutical field.Other limiting examples of pharmaceutically acceptable component can be at for example Ansel, the same, (1999); Gennaro, the same, (2000); Hardman, the same, (2001); And Rowe, the same, in (2003), find, thus each mode entirety by reference is wherein incorporated to.

Aspect of the present invention partly provides the method for the treatment of individual soft tissue situation by using hydrogel composition disclosed herein.As used herein, term " treatment " refers to alleviate or eliminates beauty treatment or the clinical symptoms of the soft tissue situation that is characterised in that soft tissue defect, damaged, disease and/or disease in individuality; Or postpone or prevention is characterised in that beauty treatment or the clinical symptoms outbreak of the situation of soft tissue defect, damaged, disease and/or disease in individuality.For example, term " treatment " can refer to alleviate the symptom of the situation that is characterised in that soft tissue defects, disease and/or disease, and 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%.Can be by observing one or more beauty treatments, clinical symptoms and/or the physiological index determining disclosed herein hydrogel composition relevant to described situation be characterised in that the effect in the situation of soft tissue defects, disease and/or disease in treatment.Also can reduce and show that soft tissue defects, disease and/or disease improve by needs to synchronous therapy.One of skill in the art will appreciate that the suitable symptom relevant to specific soft tissue defects, disease and/or disease or index and whether be the candidate who treats by compound disclosed herein or compositions by understanding how to confirm individuality.

Use according to hydrogel composition of the present invention to individuality.Individual normally any age, sex or race's the mankind.Conventionally be that any individuality of the candidate of conventional program treatment soft tissue situation is also the candidate of method disclosed herein.Be adult although experience the experimenter of skin aging sign, experience the premature aging of applicable treatment or the experimenter of other skin (for example, cicatrix) and also can treat with hydrogel composition disclosed herein.In addition, current disclosed hydrogel composition and method can be applicable to seek body part or region little/moderate is expanded, change of shape or profile change individuality, this,, by existing soft tissue implanted prosthetics, can not or can not accept technically on aesthetic.Except inform that assentment discloses all relevant risks and interests of described program comprehensively, preoperative assessment generally includes conventional history and physical examination.

Hydrogel composition disclosed herein and method are useful to treatment soft tissue situation.Soft tissue situation includes but not limited to soft tissue defect, damaged, disease and/or disease.The limiting examples of soft tissue situation comprises breast defect, damaged, disease and/or disease, and for example breast increase, breast reconstruction, breast are fixed, breast is too small, incomplete, the Polish Cotard of breast development (Poland ' s syndrome), damagedly shrink and/or break as pouch due to what complication of implant caused; Face defect, damaged, disease or disease, for example face increases, face reproduces, U.S. rope therapy, parry-Romberg syndrome (Parry-Romberg syndrome), lupus erythematosus profundus, corium pit, cicatrix, have sunken cheeks, defect or damaged, facial fold, microgroove and/or wrinkle after thin lip, nose defect or damaged, socket of the eye, as glabella stricture of vagina, muffle stricture of vagina, mouthful all stricture of vaginas and/or corners of the mouth stricture of vagina, and/or other profile deformity or defect of face; Cervical region defect, damaged, disease or/or disease; Skin defect, damaged, disease or/or disease; Other soft tissue defect, damaged, disease or/or disease, the for example increase of upper arm, underarm, hands, shoulder, back, trunk (comprising abdominal part), buttocks, thigh, shank (comprising calf), foot (comprising vola fat pad), eyes, genitals or other body part, region or area or reproduce, or affect disease or the disease of these body parts, region or area; The incontinence of urinary incontinence, fecal incontinence, other form; And gastroesophageal reflux disease (GERD).As used herein, term " U.S. rope therapy " refers to the non-operation beauty therapeutic technology of skin, involves to epidermis, corium-epidermis intersection and/or corium in epidermis, intradermal and/or subcutaneous injection be the reagent that multiple droplets are used.

Conventionally by based on required change and/or improvement, required soft tissue situation symptom alleviate and/or the amount of the hydrogel composition using together with any method disclosed herein is determined in the body part of elimination, individuality and/or doctor required clinical and/or cosmetic result and treatment or region.The effect that compositions is used can represent by following one or more clinical and/or beauty treatment modules: soft tissue alteration of form and/or improvement, the change of soft tissue size and/or improvement, the change of soft tissue profile and/or improvement, function of organization's change and/or improvement, tissue ingrowth support and/or new collagen deposition, compositions SE, patient satisfaction and/or quality of life improve and the use of implantable foreign body reduces.

The effect of described compositions and method treatment facial soft tissue can represent by following one or more clinical and/or beauty treatment modules: size, shape and/or the profile of facial characteristics improve, as the size of lip, buccal or ocular, shape and/or profile improve; Size, shape and/or the profile of facial characteristics change, as the size of lip, buccal or ocular, shape and/or profile change; Wrinkle, fold or microgroove on skin reduce or eliminate; Wrinkle on skin, fold or microgroove are had to resistance; Skin moisturizing; Skin elasticity increases; Pachylosis alleviates or eliminates; Tight skin soundness increases and/or improves; Drag line or striae gravidarum reduce or eliminate; The colour of skin, gloss, brightness and/or honorable enhancing and/or improvement; Skin color enhancing and/or improvement, palor alleviate or eliminate; Compositions SE; Side effect reduces; Patient satisfaction and/or quality of life improve.

As another example, for urinary incontinence operation, the compositions of supporting for sphincter and the effect of method can be with following one or more clinical measures canonical representations: incontinence frequency reduces, SE, patient satisfaction and/or quality of life improves and the use minimizing of implantable external filler.

Aspect this embodiment, the amount of the hydrogel composition of using is (for example) about 0.01g, about 0.05g, about 0.1g, about 0.5g, about 1g, about 5g, about 10g, about 20g, about 30g, about 40g, about 50g, about 60g, about 70g, about 80g, about 90g, about 100g, about 150g or about 200g.In the other side of this embodiment, the amount of the hydrogel composition of using is that (for example) about 0.01g is to about 0.1g, about 0.1g to about 1g, about 1g to extremely about 100g or extremely about 200g of about 50g of about 10g, about 10g.Aspect this embodiment other, the amount of the hydrogel composition of using is (for example) about 0.01mL, about 0.05mL, about 0.1mL, about 0.5mL, about 1mL, about 5mL, about 10mL, about 20mL, about 30mL, about 40mL, about 50mL, about 60mL, about 70g, about 80mL, about 90mL, about 100mL, about 150mL or about 200mL.In the other side of this embodiment, the amount of the hydrogel composition of using is that (for example) about 0.01mL is to about 0.1mL, about 0.1mL to about 1mL, about 1mL to extremely about 100mL or extremely about 200mL of about 50mL of about 10mL, about 10mL.

Conventionally by the persistent period of the body part based on individuality and/or the required beauty treatment of doctor and/or clinical effectiveness and treatment or area test treatment.Aspect this embodiment, use hydrogel composition disclosed herein and can treat soft tissue situation, for example approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 12 months, approximately 13 months, approximately 14 months, approximately 15 months, approximately 18 months or approximately 24 months.In the other side of this embodiment, use hydrogel composition disclosed herein and can treat soft tissue situation, 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, at least 13 months, at least 14 months, at least 15 months, at least 18 months or at least 24 months.In the other side of this embodiment, use hydrogel composition disclosed herein and can treat soft tissue situation, for example approximately 6 months to approximately 12 months, approximately 6 months to approximately 15 months, approximately 6 months to approximately 18 months, approximately 6 months to approximately 21 months, approximately 6 months to approximately 24 months, approximately 9 months to approximately 12 months, approximately 9 months to approximately 15 months, approximately 9 months to approximately 18 months, approximately 9 months to approximately 21 months, approximately 6 months to approximately 24 months, approximately 12 months to approximately 15 months, approximately 12 months to approximately 18 months, approximately 12 months to approximately 21 months, approximately 12 months to approximately 24 months, approximately 15 months to approximately 18 months, approximately 15 months to approximately 21 months, approximately 15 months to approximately 24 months, approximately 18 months to approximately 21 months, approximately 18 months to approximately 24 months or approximately 21 months to approximately 24 months.

Aspect of the present invention partly provides uses hydrogel composition disclosed herein.As used herein, term administering " point to individual any delivery mechanism that compositions disclosed herein is provided, it has caused clinically potentially, useful result in treatment or experimentally.Can consider to include but not limited to by those of ordinary skill in the art following because of usually definite for actual delivery mechanism from compositions to individuality that use: the type of skin, the position of skin, the reason of skin, the seriousness of skin, required alleviation degree, the persistent period of required alleviation, the particular composition using, the excretion rate of the particular composition using, the drug effect of the particular composition using, the character of other compound that the particular composition using comprises, particular route of administration, particular characteristics, individual medical history and risk factor (for example age, body weight, health status etc.) or its any combination.Aspect of this embodiment, use compositions disclosed herein by injection to individual skin area.

Conventionally body part or region based on individuality and/or the required beauty treatment of doctor and/or clinical effectiveness and treatment are determined to the approach of individual patient application of water gel combination.Compositions disclosed herein can be used by any mode known to persons of ordinary skill in the art, includes but not limited to be with needle injection, pistol (for example, hydropneumatic compression pistol), conduit, part or implants by Direct Surgery.Can be to skin area, for example hydrogel composition disclosed herein is used in corium district or rim surface zone.For example, can utilize diameter 0.26mm to about 0.4mm and length range from about 4mm the pin to about 14mm inject hydrogel composition disclosed herein.Alternatively, to can be 21-32G and length be that about 4mm is to about 70mm to pin.Preferably, pin is disposable use pin.Described pin can combine with syringe, conduit and/or pistol.

In addition, compositions disclosed herein can once or several times be used.Finally, the timing of use will be according to nursing quality standard.For example, hydrogel composition disclosed herein can be once or point several periods use, time spacer segment several days or a few week.For example, individuality can be used hydrogel composition disclosed herein for every 1,2,3,4,5,6 or 7 day or every 1,2,3 or 4 week.To individuality use hydrogel composition disclosed herein can one month or two months once or every 3,6,9 or use once for 12 months.

Aspect of the present invention partly provides corium district.As used herein, term " corium district " refers to the skin that comprises epidermis-corium intersection and comprises the region of the corium of shallow table corium (mamillary region) and deep dermis (webbed region).Skin is made up of three main layers: epidermis, and it provides water proofing property the barrier as infection; Corium, it is as the position of cutaneous appendage; And subcutaneous tissue (subcutaneous layer of fat).Epidermis does not contain blood vessel, and is nourished by the diffusion from corium.The main cell type of composition epidermis is keratinocyte, melanocyte, Langerhans cell (Langerhans cell) and Merkel cell (Merkels cell).

Corium is the skin layer being made up of connective tissue under epidermis and is giving of body stress and strain.Corium is tightly connected by basement membrane with epidermis.It also comprises many mechanoceptor/teleneurons that sense of touch and hotness are provided.It contains hair follicle, sweat gland, sebaceous gland, apocrine gland, lymphatic vessel and blood vessel.Blood vessel in corium provides nutrition and has removed refuse from it self cell and from the basal layer of epidermis.Corium is structurally divided into Liang Ge district: contiguous epidermis, be called Qian Biao district, mamillary region and be called the compare Hou district, deep of webbed region.

Mamillary region is made up of the areolar connective tissue that relaxes.It is called as nipple because of its finger-like outthrust, and it extends to epidermis.Nipple provides " rugged " that intermesh with epidermis surface for corium, has strengthened the connection between two-layer skin.Webbed region is positioned at depths, mamillary region, and conventionally thick a lot.It is made up of fine and close irregular connective tissue, and is gained the name by collagen, elasticity and the reticular fiber of its dense concentrations of braiding.These azelons give corium intensity, ductility and elastic property.Also be positioned within webbed region is root of hair, sebaceous gland, sweat gland, sensor, fingernail and blood vessel.The ink of tatooing is retained in corium.The striae gravidarum being produced by pregnancy is also arranged in corium.

Subcutaneous tissue is positioned at below corium.Its effect is to connect dermis of skin district and bone below and muscle and for it provides blood vessel and nerve.It is made up of loose connective tissue and elastin laminin.Main cell type is fibroblast, macrophage and lipocyte (subcutaneous tissue contains 50% body fat).Fat is as bedding and padding (padding) and the insulation material of health.

Aspect of this embodiment, by the corium district injection to subcutaneous area, use hydrogel composition disclosed herein to individual skin area.Aspect this embodiment, by for example, to () epidermis-corium junctional area, mamillary region, webbed region or its any combination injection, use hydrogel composition disclosed herein to individual corium district.

Advantageously, some compositions of the present invention is for example, to the appearance of microgroove in minimizing () patient's thin skin district particularly useful and effective.For example, provide the method for microgroove treatment, be included in the degree of depth that is not more than about 1mm, used the step of other local corium bulking agent compositions of describing herein to patient.

The other side of this description partly discloses the method for the treatment of skin, comprises step from hydrogel composition disclosed herein to the individuality of suffering from skin that use, and wherein applying said compositions has improved skin, thereby has treated skin.Aspect of this embodiment, the method of (skin is) treatment skin dehydration comprises step from hydrogel composition disclosed herein to the individuality of suffering from skin dehydration that use, wherein applying said compositions is skin moisturizing, thereby has treated skin dehydration.In this embodiment on the other hand, treatment skin inelastic method comprises to the step of suffering from the inelastic individuality of skin and use hydrogel composition disclosed herein, wherein applying said compositions has increased skin elasticity, lacks flexibility thereby treated skin.Aspect this embodiment another, the method for the treatment of pachylosis comprises step from hydrogel composition disclosed herein to the individuality of suffering from pachylosis that use, and wherein applying said compositions has reduced pachylosis, thereby has treated pachylosis.Aspect this embodiment another, the method that treatment skin lacks tautness comprises step from hydrogel composition disclosed herein to the individuality of suffering from skin shortage tautness that use, wherein applying said compositions makes skin tighter, lacks tautness thereby treated skin.

Aspect this embodiment another, the method for the treatment of skin stretch stricture of vagina or striae gravidarum comprises step from hydrogel composition disclosed herein to the individuality of suffering from skin stretch stricture of vagina or striae gravidarum that use, wherein applying said compositions has reduced or eliminated skin stretch stricture of vagina or striae gravidarum, thereby has treated skin stretch stricture of vagina or striae gravidarum.In this embodiment on the other hand, the method for the treatment of palor comprises step from hydrogel composition disclosed herein to the individuality of suffering from palor that use, and wherein applying said compositions has strengthened the colour of skin or brilliance, thereby has treated palor.In this embodiment on the other hand, the method for the treatment of wrinkle of skin comprises step from hydrogel composition disclosed herein to the individuality of suffering from wrinkle of skin that use, and wherein applying said compositions has reduced or eliminated wrinkle of skin, thereby has treated wrinkle of skin.Aspect this embodiment another, the method for the treatment of wrinkle of skin comprises step from hydrogel composition disclosed herein to individuality that use, and wherein applying said compositions makes the anti-wrinkle of skin of skin, thereby has treated wrinkle of skin.

In some embodiments, corium filler has lasting bioavailability.For example, provide in the time introducing in human skin, (for example, damaged to revise soft tissue hole on face through Intradermal or the subcutaneous introducing mankind), discharge ascorbic acid (or other vitamin) at least about 1 month or reach approximately 20 months or the corium filler of longer time to the mankind.

For example, for the prediction vitamin C continues the effect consistent with the filler persistent period, conjugation is assessed.The preparation that this assessment is combined with HA through etherificate based on AA2G.Described preparation is stable under physiological condition, but starts to start to discharge ascorbic acid (AsA) by the alpha-glucosidase being attached on cell membrane.Because alpha-glucosidase is attached on cell membrane, so filler/cell interface that is released in of AsA occurs.Further, AsA will degrade to make AA2G can use fibroblast with HA from the release of HA-AA2G.Therefore AA2G conjugation and the persistent period of HA depended in the release of AsA.Conjugation is that the gel of about 5mol% can at least reach 1 month, for example, within approximately 3～5 month periods, discharge active vitamin C; The gel of 10mol% conjugation can discharge active vitamin C at least reaching in 6～8 months periods; The gel of 15mol% conjugation can discharge active vitamin C at least reaching in 10～individual month period; 30mol%, reaches a year and a half.

* the parameter based on gel: volume, 0.1cc; Concentration, 24mg/ml.(0.1×24×3％×1000)/(338*0.1)＝2.13(mM)

* hypothesis:

AsA discharges with constant rate of speed.

The valid density of AsA is 0.05mM and the 2 days 2.13*2/ (0.05*30)=2.8 (individual month) of > that remain valid

In one embodiment of the invention, a kind of corium filler is provided, it comprises the hyaluronic acid epoxy crosslinked with Star-PEG and for example has the vitamin C derivatives of being combined with hyaluronic acid with the conjugation between about 3mol% and about 40mol%, one of AA2G (ascorbic acid 2-glucoside), vitagen (3-aminopropyl-L-AA phosphate ester) and SAP (sodium ascorbyl phosphate).

The method of preparing this corium filler comprises makes tetramethylolmethane glycidyl ether (Star-PEG epoxide) react by a certain percentage with ascorbic acid 2-glucoside (AA2G), and reaction temperature and response time are applicable to obtaining contains the compositions that is added the AA2G (AA2G-4 armlet oxide) of cap, unreacted 4 armlet oxides and free AA2G by 4-armlet oxide.The AA2G (AA2G-4 armlet oxide) that 4-armlet oxide adds cap is combined with hyaluronic acid through epoxy radicals.Unreacted 4 armlet oxides as cross-linking agent with cross-linked-hyaluronic acid and as bonding agent with further combined with AA2G.

In another embodiment of the present invention, a kind of corium filler is provided, it comprises the hyaluronic acid crosslinked with BDDE and for example has the vitamin C derivatives of being combined with hyaluronic acid with the conjugation between about 3mol% and about 10mol%, one of AA2G (ascorbic acid 2-glucoside), vitagen (3-aminopropyl-L-AA phosphate ester) and SAP (sodium ascorbyl phosphate).

The method of preparing this corium filler comprises makes BDDE react by a certain percentage with ascorbic acid 2-glucoside (AA2G), and reaction temperature and response time are applicable to obtaining contains the compositions that is added the AA2G (AA2G-BDDE) of cap, unreacted BDDE and free AA2G by BDDE.The AA2G (AA2G-BDDE) that BDDE adds cap is combined with hyaluronic acid through epoxy radicals.Unreacted BDDE as cross-linking agent with cross-linked-hyaluronic acid and as bonding agent with further combined with AA2G.

Fig. 9 shows that alpha-glucosaccharase enzyme concentration is on discharging the table of the impact of AsA from AA2G-PBS solution.AA2G depends on the concentration of alpha-glucosidase to the conversion of AsA.In the time that alpha-glucosidase concentration is 6.3 unit/g gels, AA2G is almost converted into AsA completely in 15min.In the time that alpha-glucosidase concentration is 4.7 unit/g gels, need 30min that AA2G is converted into AsA completely.Further reducing alpha-glucosidase concentration causes AA2G slow to the conversion of AsA.

Figure 10 shows from according to the release profiles schematic diagram (sustained release) of the free AsA of combination corium filler of the present invention (AA2G transform mol%) compared with the response time.In AA2G/HA mixture, AA2G is converted into AsA completely in 40min.AA2H/HA conjugate demonstrates AA2G and is converted into the time dependence of AsA.

Figure 11 A and 11B show the additional release data according to various corium filleies of the present invention.More specifically, in HA-AA2G gel, AA2G depends on alpha-glucosidase concentration to the conversion of AsA.High alpha-glucosidase concentration causes AA2G to AsA rapid conversion.For specifying alpha-glucosidase concentration, different preparations demonstrate the heterogeneity that AA2G transforms to AsA.

In one aspect of the invention, provide corium filler, its appearance to treatment and elimination microgroove is effective especially, and for example gauffer of relatively shallow table on skin, such as but not limited near microgroove eyes, Lei Gou district, forehead, canthus stricture of vagina, glabella stricture of vagina etc.

Occur that at the skin part of having injected corium filler blue variable colour (Tyndall effect) is the great adverse events of some corium filler patient experiences.Tyndall effect is more common in the patient of the tiny wrinkle of the shallow table for the treatment of.Researched and developed embodiment of the present invention, it provides and can inject to treat microgroove and wrinkle at shallow table, even in relatively thin skin area, injects, without any long-acting, the translucent filler of the blue variable colour being produced by Tyndall effect.Microgroove or shallow table wrinkle are generally understood to conventionally the thinnest at skin, and the dermis thickness of skin is less than wrinkle or the gauffer on the skin of finding in the facial zone (forehead, outer canthus, vermilion border/mouth contour) of 1mm.At forehead, Average True skin thickness for normal skin for about 0.95mm is about 0.81mmm for wrinkling skin.Outer canthus corium around even thinner (for example for normal skin about 0.61mm and for wrinkling skin about 0.41mm).30 or the mean outside diameter of 32G pin (being generally used for the pin of microgroove gel application) be approximately 0.30 and about 0.24mm.

The invention provides for example at other local corium bulking agent compositions that can not cause Tyndall effect of describing of this paper.For example, compositions of the present invention comprises the hyaluronic acid component crosslinked with cross-linking agent, the additive except crosslinking component; When in the corium district that is administered to patient, with respect to except not having described additive, identical compositions substantially, described compositions table reveals Tyndall effect and reduces.Described compositions optical clear substantially.

In one embodiment, additive is vitamin C derivatives, the chemically combined AA2G of hyaluronic acid that for example can describe with other place of this paper.

In some embodiments, crosslinking component is that BDDE and conjugation are between about 3mol% and about 10mol%, or up to 15mol% or higher.In some embodiments, described compositions further comprises the anesthetis that is suitable for after injection providing for patient comfortable amount, for example lignocaine.

The method of the microgroove on treatment patient skin is also provided.Described method generally includes to the step of introducing for example compositions as herein described in patient skin.The mixture of the crosslinking component that for example described compositions comprises hyaluronic acid component, cross-linked-hyaluronic acid and the additive except crosslinking component, described compositions optical clear substantially; And wherein with respect to except not having described additive, identical compositions substantially, corium bulking agent compositions shows Tyndall effect and reduces.

In some embodiments of the present invention, described compositions comprises the hyaluronic acid component that uses EDC chemistry and diamidogen or polyamines cross-linking agent crosslinked.For example, described cross-linking agent can be HMDA.

In the certain embodiments of the present invention that are HMDA at cross-linking agent, the G ' of described compositions is up to about 70Pa, G "/G ' is between approximately 0.65 with approximately between 0.75, the about 24N of extruding force or less, and final HA concentration is between about 24mg/g and about 25mg/g.

Be in certain embodiments of the present invention of HA-AA2G conjugate or HA-vitagen conjugate at additive, conjugation is between about 3mol% and about 10mol%, or up to about 15mol%, or up to about 40mol%.The G ' of these compositionss is from least about 30Pa, more preferably at least about 40Pa to about 100Pa, G "/G ' is between approximately 0.30 with approximately between 0.50, the about 27N of extruding force or less, and final HA concentration is between about 24mg/g and about 25mg/g.

For the purposes of the present invention, as used herein " conjugation " is defined as the molar percentage of coalition, for example AA2G and hyaluronic acid repetitive (for example, HA dimer).Therefore, 10mol% conjugation means the AA2G of every 100 HA repetitives containing 10 combinations.Can use the method for explanation in embodiment 2 below, or other method calculations incorporated degree well known by persons skilled in the art.

Embodiment

embodiment 1: use BDDE to be combined with crosslinked HA gel as cross-linking agent AA2G

Make hydration～30min in the 1wt%NaOH of low-molecular-weight hyaluronic acid (LMW HA) 1802mg in syringe of 400.6mg.The AA2G of 800.7mg is put into bottle, then put into the BDDE of 713.7mg and the 10%NaOH of 1416.8mg.Before adding in hydration HA, make above solution (pH>12) reaction～20min in 50 DEG C of water-baths.After interpolation, by transmitting back and forth mixture is mixed～20 times between 2 syringes.The paste of mixing is put into bottle and 50 DEG C of water-bath～2.5h.The 12M HCl of 223.5mg is added in 9.05g PBS (pH7.4).After～2.5h, form HA-AA2G gel.Gel is cut into slices, and add wherein HCl-PBS solution.Make gel neutralization and expand whole night on orbital shaker.Pass through～60 μ m sieves sieve gels and pass through and transmit and mix～20 times back and forth between 2 syringes.Gel is put in 15,000MWCO RC bag filter and in PBS (pH7.4) buffer and dialysed.Carry out～185h of dialysis, frequently changes PBS buffer.After dialysis, gel is put into syringe and be stored in 4 DEG C of refrigerators.

the mensuration of embodiment 2:AA2G combination

Before dialysis and after dialysis, correctly record the weight of gel as described in Example 1.After supposing dialysis, gel is～1g/mL.Every >8h in 1L PBS, stops dialysis while not there is apparent AA2G.Use UV/Vis spectrophotometer (Nanodrop2000C, ThermoScientific) to measure AA2G under 260nm.Use the variable concentrations (A@260nm=1.4838[AA2G (mM)]) of AA2G in 2%HA to calculate the calibration curve of AA2G.

The starting weight of the weight of HA: HA after dialysis × (actual weight/theoretical weight before dialysis)

The mmol of AA2G after dialysis: in equation (A@260nm=1.4838[AA2G (mM)]), absorption is located under 260nm after dialysis.

The combination@of AA2G: (HA of the mmol/mmol of AA2G) × 100%

AA2G conjugation in gel is 14.7mol% as described in example 1 above.

embodiment 3: the mensuration of gel rheology

Use vibration parallel-plate rheometer (Anton Paar, Physica MCR301) to measure the character of the gel obtaining in embodiment 1.The diameter of plate used is 25mm.Between plate, gap is made as 1mm.For each measurement, under fixed frequency, before strain sweep, first carry out the frequency scanning under constant strain.Under 1% strain, obtain G ' (storage modulus) by strain sweep curve.Be 1450Pa for gel value.

embodiment 4: use BDDE as cross-linking agent, with adjustable conjugation and gel rheology AA2G be combined with crosslinked HA gel

Program is similar to description in embodiment 1.By adjusting cross-linking agent and HA and AA2G mol ratio change conjugation.Measure as described in example 3 above gelling properties.Details as Follows:

Make hydration～30min in the 1%NaOH of LMW HA 1752.1mg in syringe of 400.8mg.The AA2G of 800.3mg is put into bottle, then put into the BDDE of 354.1mg and the 10%NaOH of 1402.0mg.Before adding in hydration HA, make above solution (pH>12) reaction～20min in 50 DEG C of water-baths.After interpolation, by transmitting back and forth mixture is mixed～20 times between 2 syringes.The paste of mixing is put into bottle and 50 DEG C of water-bath～2.5h.The 12M HCl of 140.9mg is added in 9.0053g PBS (pH7.4).After～2.5h, form HA-AA2G gel.Gel is cut into slices, and add wherein HCl-PBS solution.Make gel neutralization and expand whole night on orbital shaker.Pass through～60 μ m sieves sieve gels and pass through and transmit and mix～20 times back and forth between 2 syringes.Gel is put in 15,000MWCO RC bag filter and in PBS (pH7.4) buffer and dialysed.Carry out～164.5h of dialysis, frequently changes PBS buffer.After dialysis, gel is put into syringe and be stored in 4 DEG C of refrigerators.Conjugation is 13%.Gel storage modulus (G ') be 803Pa.

embodiment 5: use BDDE as cross-linking agent, AA2G is combined with crosslinked HA gel, conjugation is that 5.3%, G ' is～300Pa.

Make hydration～30min in the 1%NaOH of LMW HA 3002.0mg in syringe of 400.3mg.The AA2G of 800.5mg is put into bottle, then put into the BDDE of 264.3mg and the 10%NaOH of 1100.0mg.Before adding in hydration HA, make above solution (pH>12) reaction～20min in 50 DEG C of water-baths.After interpolation, by transmitting back and forth mixture is mixed～20 times between 2 syringes.The paste of mixing is put into bottle and 50 DEG C of water-bath～2.5h.The 12M HCl of 104.2mg is added in 8.5128g PBS (pH7.4).After～2.5h, form HA-AA2G gel, and add wherein HCl-PBS solution.Make gel neutralization and on orbital shaker, expand whole weekend (～55h).Pass through～60 μ m sieves sieve gels and pass through and transmit and mix～20 times back and forth between 2 syringes.Gel is put in 15,000MWCO RC bag filter and in PBS (pH7.4) buffer and dialysed.Carry out～114h of dialysis, frequently changes PBS buffer.After dialysis, gel is put into syringe and be stored in 4 DEG C of refrigerators.Press the program of describing in embodiment 2 and 3 and measure conjugation and gel rheology.Conjugation is 5.3%.Gel storage modulus is～300Pa.

embodiment 6: use star-PEG epoxide as cross-linking agent, AA2G and crosslinked HA gel combination, conjugation is that 29.4%, G ' is～235Pa.

Make hydration～30min in the 1%NaOH of LMW HA 2000mg in syringe of 200.4mg.The AA2G of 400mg is put into bottle, then put into the star-PEG epoxide of 312.7mg and the 10%NaOH of 1026.5mg.Before adding in hydration HA, make above solution reaction～20min in 50 DEG C of water-baths.After interpolation, by transmitting back and forth mixture is mixed～20 times between 2 syringes.The paste of mixing is put into bottle and 50 DEG C of water-bath～2.5h.The 12M HCl of 187.4mg is added in 3.034g PBS (pH7.4).After～2.5h, form HA-AA2G gel, and add wherein HCl-PBS solution.Make gel neutralization and on orbital shaker, expand whole weekend (～68h).Pass through～60 μ m sieves sieve gels and pass through and transmit and mix～20 times back and forth between 2 syringes.Gel is put in 15,000MWCO RC bag filter and in PBS (pH7.4) buffer and dialysed.Carry out～95h of dialysis, frequently changes PBS buffer.After dialysis, gel is put into syringe and be stored in 4 DEG C of refrigerators.Press the program of describing in embodiment 2 and 3 and measure conjugation and gel rheology.Conjugation is 29.4%.Gel storage modulus is～235Pa.

embodiment 7: use star-PEG epoxide as cross-linking agent, AA2G and crosslinked HA gel combination, conjugation is that 27.8%, G ' is～363Pa.

Make hydration～30min in the 1%NaOH of LMW HA 2000mg in syringe of 200.3mg.The AA2G of 400.2mg is put into bottle, then put into the star-PEG epoxide of 313.4mg and the 10%NaOH of 1022.6mg.Above solution is added in hydration HA.After interpolation, by transmitting back and forth mixture is mixed～20 times between 2 syringes.The paste of mixing is put into bottle and 50 DEG C of water-bath～2.5h.The 12M HCl of 196.5mg is added in 3.016g PBS (pH7.4).After～2.5h, form HA-AA2G gel, and add wherein HCl-PBS solution.Make gel neutralization and on orbital shaker, expand (～24h) whole night.Pass through～60 μ m sieves sieve gels and pass through and transmit and mix～20 times back and forth between 2 syringes.Gel is put in 15,000MWCO RC bag filter and in PBS (pH7.4) buffer and dialysed.Carry out～98.5h of dialysis, frequently changes PBS buffer.After dialysis, gel is put into syringe and be stored in 4 DEG C of refrigerators.Press the program of describing in embodiment 2 and 3 and measure conjugation and gel rheology.Conjugation is 27.8%.Gel storage modulus is～363Pa.

embodiment 8: use BDDE as cross-linking agent, AA2G and crosslinked HMW HA gel in conjunction with, conjugation is about 10mol%, G ' is about 240Pa.

Make hydration～30min in the 4wt%NaOH of HMW HA 2501.3mg in syringe of 400.3mg.The AA2G of 1200mg is put into bottle, then put into the BDDE of 304.7mg and the 16wt%NaOH of 1178.6mg.Before adding hydration HA to, make above solution (pH>12) reaction～20min in 50 DEG C of water-baths.After interpolation, by transmitting back and forth mixture is mixed～20 times between 2 syringes.The paste of mixing is put into 20cc bottle and 50 DEG C of water-bath～2.5h.After～2.5h, form HA-AA2G gel.The 12M HCl of 226.6mg is added to 8492.2mg10 × PBS (pH7.4) middle acquisition HCl-PBS solution and adds HCl-PBS solution so that gel neutralization and expansion.Make gel neutralization and expand more than 48h on orbital shaker.Pass through～60 μ m sieves sieve gels and pass through and transmit and mix～20 times back and forth between 2 syringes.Gel is put in 20,000MWCO CE bag filter and in PBS (pH7.4) buffer and dialysed.Carry out～114h of dialysis, frequently changes PBS buffer.After dialysis, gel is put into syringe and be stored in 4 DEG C of refrigerators.Press the program of describing in embodiment 2 and 3 and measure conjugation and gel rheology.Conjugation is 10mol%.Gel storage modulus is about 240Pa.

embodiment 9: use BDDE as cross-linking agent, vitagen coagulates with crosslinked LMW HA cementing closing, conjugation is 15mol%, G ' is about 365Pa.

Make hydration～40min in the 1wt%NaOH of LMW HA 1753.24mg in syringe of 398.2mg.In the HA expanding, add BDDE (311.7mg) and continue to allow the HA 80min that expands again.Make the HA/BDDE mixture expanding at 50 DEG C, react in advance 20min.

The vitagen of 801.9mg is dissolved in separately in the 10wt%NaOH of 1459.7mg and and the HA that reacts in advance with BDDE mix.Make mixture continue to react again 2.5h at 50 DEG C.After～2.5h, form HA-vitagen.The 12M HCl of 195mg is added to 10 × PBS (pH7.4) middle acquisition HCl-PBS solution of 9004.0mg and adds HCl-PBS solution so that gel neutralization and expansion.Make gel neutralization and expand more than 48h on orbital shaker.Pass through～60 μ m sieves sieve gels and pass through and transmit and mix～20 times back and forth between 2 syringes.Gel is put in 20,000MWCO CE bag filter and in PBS (pH7.4) buffer and dialysed.Carry out～120h of dialysis, frequently changes PBS buffer.After dialysis, gel is put into syringe and be stored in 4 DEG C of refrigerators.Press the program of describing in embodiment 3 and measure gel rheology.Using and measuring similar method mensuration conjugation to the AA2G describing in embodiment 2 is about 15mol%.Gel storage modulus is about 365Pa.

embodiment 10: vitagen is combined with linear HA through amide chemistry

Make hydration in the water of HMW HA 10ml in 60cc syringe of 200.3mg.The vitagen of 500mg is dissolved in 0.5ml water and by solution and neutralizes pH4.8.The NHS of the EDC of 197.7mg and 149mg is dissolved in separately in 6ml water.Above solution (solution and EDC/NHS solution) is added in another 60cc syringe that 23.5ml water is housed.By transmitting and mix 20 times back and forth between 2 syringes.Mixture is stored in a syringe and immerses 4h in 37 DEG C of baths.Finally use PBS (pH7.4) buffer dialysis solution, until do not observe apparent vitagen.Measure conjugation by the similar approach of describing as embodiment 3.Conjugation is about 10mol%.

the combination of embodiment 11:AA2P and crosslinked HA gel

Make hydration～30min in the MES5.2 buffer of LMW HA 1000mg in syringe of 200.4mg.The AA2P of 292mg is put into bottle, then add the star-PEG amine of 300mg.Above solution is at room temperature reacted whole night.Make gel hydration and dialyse to remove unreacted AA2P with PBS buffer with PBS buffer.As described in embodiment 2 and 3, characterize last gel to measure conjugation and gel rheology.Conjugation is about 20mol%.Storage modulus (G ') be about 500Pa.

embodiment 12

the joining of the HA/BDDE corium filler product with AA2G occurring for reducing microgroove system

To any gel of describing in above embodiment, after dialysis, in gel, add appropriate free HA gel to improve change gel cohesiveness and/or syringeability.For example, free HA fiber is expanded in phosphate buffer, to obtain even viscoelastic gel (" dissociating " HA gel).Before dialysis step, this uncrosslinked gel is added to (for example,, to obtain the compositions with approximately 1% to about 5%w/w free HA) in the HA/BDDE cross-linked gel obtaining in embodiment 1.Then by gel-filled gained to be easy to fill asepsis injector in and under sufficient temp and pressure high pressure steam sterilization at least about 1min.After high pressure steam sterilization, packaging HA/AA2G final products are also distributed to doctor with as the shallow table injection of corium filler, improve the appearance of microgroove in socket of the eye week or other facial zone.

embodiment 13

comprise the preparation of the HA-AA2G corium filler of lignocaine

According to the program of embodiment 12, but after dialysis step and before adding free HA gel, in mixture, add lidocaine hydrochloride (lignocaine HCl).First (the lignocaine HCl) of powder type is dissolved in WFI and by 0.2 μ m filter and filters.To adding NaOH weak solution in viscosity HA/AA2G gel for example, to reach alkalescence pH (, between approximately 7.5 and the about pH between 8).Then lignocaine HCl solution is added in alkalescence gel to reach final desired concn to for example concentration of approximately 0.3% (w/w).Then the gained pH of HA/AA2G/ lignocaine mixture for approximately 7 HA concentration be about 24mg/g.In the standard reaction device that is equipped with agitation as appropriate machine, carry out mechanical mixture to obtain suitable uniformity.

embodiment 14

containing the additive of carboxyl functional group and the combination of HA hydrogel

Additive, for example tretinoin (AKA, retinoic acid), adapalene (adapalence) and alpha-lipoic acid contain carboxyl functional group (COOH).Use EDC chemistry through esterification, these additives are combined with HA hydrogel.The example of described combination according to an embodiment of the invention has below been described:

Make hydration in the pH4.8MES buffer of HMW HA 10ml in 60cc syringe of 200mg.In another syringe, the tretinoin of 200mg is dissolved in to the boiling mixture (water/acetone volume ratio 1:3) of 5ml.Mix above two syringes approximately 20 times by syringe adapter.Then the NHS of the EDC of 197.7mg and 149mg is dissolved in respectively in the 6ml water in independent syringe.The syringe that EDC and NHS are housed is connected with the syringe that HA and tretinoin are housed, to make reactant mixing at least 20 times by transmitting back and forth between 2 syringes.Mixture is stored in a syringe and immerses 4h in 37 DEG C of baths., then under aseptic condition, dialyse with PBS buffer to remove unconjugated tretinoin with isopropyl alcohol dialysis gel.Gel pack is stored in asepsis injector and at 4 DEG C.

embodiment 15

the combination of the additive of hydroxy functional groups and HA hydrogel.

Additive, for example retinol (AKA, retinoic acid), catalase, dimethylaminoethanol and g-tocopherol contain hydroxy functional group (OH).Use EDC chemistry through esterification, these additives are combined with HA hydrogel.The representative instance of combination has below been described:

Make the middle hydration of 2-(N-morpholine) ethyl sulfonic acid (MES) buffer (pH4.8) of HMW HA 10ml in 60cc syringe of 200mg.In another syringe, the retinol of 200mg is dissolved in to the boiling mixture (water/acetone volume ratio 1:3) of 5ml.Mix above two syringes approximately 20 times by syringe adapter.Then the NHS of the EDC of 197.7mg and 149mg is dissolved in respectively in the 6ml water in independent syringe.The syringe that EDC and NHS are housed is connected with the syringe that HA and retinol are housed, to make reactant mixing at least 20 times by transmitting back and forth between 2 syringes.Mixture is stored in a syringe and immerses 4h in 37 DEG C of baths., then under aseptic condition, dialyse with PBS buffer to remove unconjugated retinol with isopropyl alcohol dialysis gel.Gel pack is stored in asepsis injector and at 4 DEG C.

embodiment 16

the additive of hydroxy functional groups and HA hydrogel are by the combination of post-modification.

This is two step processes.

Step 1: process and be cross-linked HA gel with EDC/NHS, for example the commercial corium filler based on HA (for example, allergan, Irvine CA or medicis Aesthetics, Inc.) to activate the carboxyl of HA.

Step 2: with the additive treating activation HA hydrogel of hydroxyl.The additive of hydroxyl is retinol, catalase, dimethylaminoethanol and g-tocopherol hydroxy functional group (OH).

Additive is as follows with the representative instance that crosslinked HA gel is combined:

At room temperature make the Juvederm gel of 2gm mix with the EDC of 200gm and the NHS of 150mg.Then add the retinol of 200mg in 3ml acetone-water mixture.Make above mixture react 4h at 37 DEG C., then under aseptic condition, dialyse with PBS buffer to remove unconjugated retinol with isopropyl alcohol dialysis gel.Gel pack is stored in asepsis injector and at 4 DEG C.

embodiment 17

the combination of somatomedin, peptide or elastin laminin and HA hydrogel

The additive that contains amine functional group, for example epidermal growth factor (EGF), transforming growth factor (TGF) and peptide, can be combined to form with HA useful corium filler.These additives are combined with HA by amidatioon chemistry.The representative instance of combination has below been described:

Make HMW HA hydration in the MES of 10ml pH5.4 buffer of 200.3mg.Add the EGF of 20mg in 100mg MES solution.To above mixture, add EDC and the 149mg of 197.7mg.Make gained reactant mixture react 4h at 37 DEG C.After having reacted, with the isopropyl alcohol gel of further dialysing, then under aseptic condition with the dialysis of PBS buffer.Gel pack is stored in asepsis injector and at 4 DEG C.

The present invention further provides the method that strengthens transplant fat regular organization activity power.Described method may generally include step from compositions to the skin of the contiguous transplant fat tissue of patient that introduce, and described compositions is compositions that other place is described herein.For example, described compositions can comprise hyaluronic acid and with the covalently bound vitamin C derivatives of hyaluronic acid, wherein conjugation is between about 3mol% and about 40mol%.In other side of the present invention, the method for the treatment of skin comprises to the step of introducing the ascorbic compositions that comprises fatty tissue, hyaluronic acid and be combined with hyaluronic acid in skin.

embodiment 18

the combination of somatomedin, peptide or elastin laminin and HA hydrogel

For the mitogenesis effect of assessment vitamin C and the stem cell (hASC) of derivant to Human Adipose Tissue-derived thereof, hASC is in tissue culturing plastic, in the complete MesenPro culture medium (Invitrogen that supplements or do not supplement vitamin C (ascorbic acid) or derivatives thereof (vitagen or AA2G) that is free form, Carlsbad, CA) middle cultivation 4 days.Estimate propagation by MTT algoscopy as described in manufacturer (ATCC, Manassas, VA).After 4 days, find that the concentration of ascorbic acid 0.25,0.5 and 1mM makes propagation (by be converted into purple first by dehydrogenase the measurement amount of yellow tetrazolium MTT) (purple first being washed agent dissolves) improve 60%, 80% and 96% than the contrast that lacks ascorbic acid respectively.Use the AA2G of same concentrations to obtain respectively the propagation raising that exceedes contrast 70%, 60% and 50%.Obtain analog result with vitagen, show than contrast and increase by 70%, 60% and 30% respectively.In a word, vitamin C and derivant thereof, AA2G and vitagen have improved the hASC propagation in cell culture under the culture medium containing somatomedin exists.

have in conjunction with ascorbic crosslinked HA gel

In embodiment 19 and 20, describe below and had in conjunction with ascorbic gel and use 1 based on crosslinked HA, 4-butanediol diglycidyl ether (BDDE) is as the preparation of cross-linking agent, according to certain embodiments of the present invention, it shows Tyndall effect and reduces and other advantage.In embodiment 19, vitamin C derivatives is ascorbic acid 2-glucoside (AA2G) and in embodiment 20, vitamin C derivatives is ascorbic acid 3-aminopropyl phosphate ester (vitagen).These gels have best rheological characteristic, good syringeability and high HA concentration (25mg/g).Although do not wish to be subject to any particular theory of operation to retrain, but the inventor has been found that and under AA2G or vitagen existence, makes HA and the crosslinked character that has greatly changed gel of BDDE, with respect to the crosslinked commercial HA gel of BDDE, gel has high crosslink density, high HA concentration, low viscosity and low extruding force.Because there is AA2G or vitagen during crosslinked, so the gel forming at present has separately or via BDDE, as the cross-linking agent of side group and bridge joint HA chain and these ascorbic acid derivates of HA chain coupling.The microstructure of gel changes, even if cause gel by the thin pin to 30G, extruding force is also very low.And gel has extremely about 10mol% or the vitamin C of being combined with HA up to about 15mol% of about 3mol%.In the time of injected gel, they for example discharge active vitamin C from fibroblastic alpha-glucosidase or phosphatase by endogenous enzymes.Active vitamin C can cause skin collagen and forms and can play free radical scavenger and suppress gel degradation.

embodiment 19

the preparation of the HA/AA2G gel that Tyndall effect reduces

Adding after the 5wt%NaOH solution of 1764.0mg, make the LMW HA of 400.1mg in syringe and the mixture hydration 60min of 402.3mg AA2G.The AA2G that adds 800.8mg in independent bottle, then adds the 9.1wt%NaOH solution of 1401.1mg and the BDDE of 252.6mg.Make gained solution (pH>12) reaction～20min in 50 DEG C of water-baths, transferred to afterwards in hydration HA.After interpolation, by it is transmitted back and forth mixture is mixed～20 times between two syringes.Then paste is transferred in bottle, be placed on afterwards in 50 DEG C of water-baths～2.5h.After crosslinked, add containing the 12M HCl of 197.0mg and the solution of 9.18g10 × PBS (pH7.4) with neutralization bases, and make the gel 72h that expands on orbital shaker.Force the mesh screen in its pass through～60 μ m apertures to divide gel.By it being transmitted back and forth between two syringes to gel～20 time that nixing sieve is crossed, transferred to afterwards in cellulose esters bag filter (MWCO～20kDa) and with PBS (pH7.4) buffer dialysis 5 days, twice of exchange buffering every day liquid.After dialysis, gel is assigned in 1ml COC syringe, under 5000RPM, centrifugal 5min, to remove bubble, and uses moist steam sterilizing.This gel has the HA ultimate density of 25mg/g, the AA2G mol% of the approximately 10mol% calculating as described in example 2 above and the G ' of about 80Pa.Prepare in a similar manner other gel, G ' value is for about 60Pa is to about 80Pa.

embodiment 19A

the preparation of what Tyndall effect reduced the have HA/AA2G gel of lignocaine

Add a certain amount of lignocaine to generate the HA/AA2G gel with lignocaine to the gel of embodiment 19, it has 0.3%ww lignocaine.By lignocaine HCl is dissolved in PBS buffer (pH～7.4) and prepares lignocaine solution.After dialysis but before sterilizing, add the lignocaine solution of aliquot to the gel in embodiment 19.Then complete mixed gel is to obtain the homogeneous mixture with 0.3%ww lignocaine concentration.

embodiment 20

the preparation of the HA/ vitagen gel that Tyndall effect reduces

Make hydration～45min in the 1wt%NaOH solution of LMW HA 2355.0mg in syringe of 401.0mg.303.8mg BDDE is added in hydration HA and by mixing between syringe, mix 10 times.Make mixture react in advance 15min in 50 DEG C of water-baths.The vitagen of 800.1mg is dissolved in separately in the 15wt%NaOH of 950.6mg, is then dissolved in 510.1Milli-Q water.Use between syringe and mix, vitagen solution is mixed 30 times back and forth with the hydration HA/BDDE mixture of preheating.Mixture is put back in 50 DEG C of water-baths and continued reaction 2h again, afterwards the solution of 10 × PBS (pH7.4) of the 12M HCl that contains 148.1mg and 8523.1mg is added in cross-linking agent.Add HCl-PBS solution with in and gel make its expansion.Make gel neutralization and expand more than 48h on orbital shaker.Pass through～60 μ m sieves sieve gels and pass through and transmit and mix～20 times back and forth between 2 syringes.Gel is put in 20,000MWCO CE bag filter and in PBS (pH7.4) buffer and dialysed.Carry out～197h of dialysis, frequently changes PBS buffer.After dialysis, gel is transferred in 1ml COC syringe, under 5000RPM, centrifugal 5min also uses moist steam sterilizing.The HA ultimate density of gel is 24mg/g.

through 1-ethyl-3-[3-dimethyl aminopropyl] HA of carbodiimide hydrochloride (EDC) chemical crosslinking gel

In embodiment 21 and 22, describe according to certain embodiments of the present invention below, shown Tyndall effect and reduce and the preparation of the gel based on crosslinked HA of other advantage.In embodiment 21, use and in embodiment 20, use as 3-[3-(3-aminopropyl)-2 for the cross-linking agent of hexamethylene diamine (HMDA), two (3-amino-propoxyl group methyl)-propoxyl group of 2-] cross-linking agent of-propylamine (4 arm amine-4AA), through EDC chemical preparation gel.Under temperate condition, for example, under room temperature and for example pH5.4, be cross-linked.Capable of regulating reaction condition has the height network gel of best gelling properties, good syringeability and high HA ultimate density (～24mg/g) with preparation.Inventor has been found that; under low-down hydration or reaction density; with appropriate HMDA or 4AA; together with coupling agent, i.e. 1-ethyl-3-[3-dimethyl aminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxy-succinamide (NHS) or sulfonyl-NHS (sulfo group-NHS) make that HA is crosslinked may be favourable.The crosslinking points of gel will be mutually away from, therefore highly cross-linked material has high damping force.On the contrary, crosslinked under so low hydration or reaction density of HA and BDDE may be infeasible due to cross-linking agent relative nullity.

embodiment 21

the preparation of the HA/HMDA gel that Tyndall effect reduces

The 100mM MES buffer (pH5.2) of 20.0g is added in the syringe of LMW HA that 1000.0mg is housed.By 260.9mg HMDA.HCl being dissolved in the 100mM MES buffer (pH5.2) of 2010.5mg, and the 1M NaOH of interpolation 2 μ l makes pH reach 5.2 preparation HMDA solution.By the EDC of 254.2mg being dissolved in to preparation EDC solution in 1188.4mg100mM MES buffer (pH5.2), and in independent bottle, the NHS of 44.3mg is dissolved in the 100mM MES buffer (pH5.2) of 1341.8mg.After the complete hydration of HA ,～1h, to the HMDA solution that adds 790 μ l in hydration HA.By mixing homogenized mix between syringe 10 times.Then 490 μ l EDC and 490 μ l NHS solution are added in homogenize paste and by mixing and remix 10 times between syringe.Then mixture is transferred in bottle and at the 1 × PBS buffer (pH7.4) that adds 17.9ml before at room temperature crosslinked 5h.Before forcing its net that passes through 60 μ m apertures, gel is expanded 3 days on cylinder.The gel of screening is placed in cellulose ester membrane Dialysis tubing MWCO20KDa and with 1 × PBS dialysis 4 days, twice of exchange buffering every day liquid.Gel is distributed in 1ml COC syringe, centrifugal 5min under 5000RPM, and use moist steam sterilizing.The HA ultimate density of gel is 25mg/g.

embodiment 22

the preparation of the HA/4AA gel that Tyndall effect reduces

The 100mM MES buffer (pH5.2) of 32.55g is added in the syringe of LMW HA that 1000.4mg is housed.By 256.3mg4AA being dissolved in the 100mM MES buffer (pH5.2) of 1039.8mg, and the 6M HCl of interpolation 380 μ l makes pH reach 5.2 preparation 4AA solution.By the EDC of 251.2mg being dissolved in to preparation EDC solution in 1013.8mg100mMMES buffer (pH5.2), and in independent bottle, the NHS of 74.7mg is dissolved in the 100mM MES buffer (pH5.2) of 2020.0mg.After the complete hydration of HA ,～1h, to the 4AA solution that adds 260 μ l in hydration HA.By mixing homogenized mix between syringe 10 times.Then 277 μ l EDC and 273 μ l NHS solution are added in homogenize paste and by mixing and remix 10 times between syringe.Then mixture is transferred in bottle and at the 10 × PBS buffer (pH7.4) that adds 6.4ml before at room temperature crosslinked 5h.Before forcing its net that passes through 60 μ m apertures, gel is expanded 3 days on cylinder.The gel of screening is placed in cellulose ester membrane Dialysis tubing MWCO20KDa and with 1 × PBS dialysis 4 days, twice of exchange buffering every day liquid.Gel is distributed in 1ml COC syringe, centrifugal 5min under 5000RPM, and use moist steam sterilizing.The HA ultimate density of gel is 23mg/g.

embodiment 23

the mensuration of the gel rheology of embodiment 19-22

Use vibration parallel-plate rheometer, Anton Paar Physica MCR301, the rheological characteristic of measurement gel.In the time that being 1mm, clearance height uses the board diameter of 25mm.Under 25 DEG C of constant temperature, measure.Each measurement is made up of the frequency scanning of 1-10Hz under 2% constant strain and frequency logarithmic growth, is then with strain logarithmic growth, the strain sweep of 1-300% under 5Hz constant frequency.Under 1% strain, obtain storage modulus (G ') and viscous modulus (G ") by strain sweep.

the storage modulus of the gel being obtained by embodiment 19-22 and viscous modulus

Sample ID	Storage modulus (G ') Pa	Viscous modulus (G ") Pa
			Embodiment 19	84	25
Embodiment 20	83	33.7
			Embodiment 21	67	42
Embodiment 22	41	29.5

embodiment 24

the extruding force of the gel of embodiment 19-22 is measured

Use Instron5564 and Bluehill2 software measurement that gel is extruded to the required power of 30G pin.Gel is extruded to 30G1/2TSK pin from 1ml COC syringe.The speed of pressing 100mm/min promotes piston 11.35min, and records extruding force.

the extruding force of the gel being obtained by embodiment 19-22

Sample ID	Extruding force (N)
		Embodiment 19	25
Embodiment 20	24
		Embodiment 21	22
Embodiment 22	19.5

embodiment 25

the biocompatibility test of the gel of embodiment 19-22

Implant the gel of 50 μ l bolus injections at the back side Intradermal of Shi-Dao Er Shi rat (Sprague Dawley rat).In the time of 1 week, take out implant and with h and E (H & E) dye and be monokaryon inflammatory cell mark CD68 dye, by histologic analysis implant.3 20 × image scoring 0-4 that are CD68 based on dye levels.Then average these are worth to provide sample score.4 samples of every kind of gel analysis.

the average CD68 score of embodiment 19-22

Sample ID	Score
		Embodiment 1	1.8

Embodiment 2	1.6
		Embodiment 3	2.7
Embodiment 4	1.3

embodiment 26

the cell toxicity test of the gel of embodiment 19-22, ISO10993-5.

According to the agarose cladding process of ISO10993-5, carry out the vitro cytotoxicity test of gel by NAMSA: " BiologicalEvaluationofMedicalDevice "-5th part: vitro cytotoxicity test.Answer holes interpolation 0.1ml to three and be placed on the test specimen on filter disc, and 0.9%NaCl solution, the high density polyethylene (HDPE) of growing as the 1cm of negative control and 1 × 1cm as positive control ²latex part.Each is placed on to the agarose surface of direct covering monolayer L929 l cell.At 37 DEG C at 5%CO ₂in hatch after 24h, check any abnormal cell form and the lysis of culture from both macro and micro.Based on the cracking zone near sample, be sample scoring 0-4.Because specimen sample does not demonstrate the sign that causes any lysis or toxicity, so must be divided into 0 from the test material of embodiment 1,3 and 4.

the quantitative analysis of Tyndall effect

For the further comparative advantage analysis of supporting visual observation and carrying out HA filler, think and must carry out the quantitative analysis of Tyndall effect.Corium filler there is not equally in the literature the quantitative technique of specific Tyndall effect.But, based on to light scattering and light and the interactional existing understanding of science of skin, adopt based on (a) colorimetry and (b) two kinds of distinct methods of spectroscopy quantize the Tyndall effect on skins.Based on these technology, define 3 kinds of different quantitative parameters (as follows) and measured Tyndall effect in body.

A) tyndall effect range estimation score: scale range is 1-5, and increment is 0.5.Normal and do not have the injection portion of blue variable colour to give score 1 to the colour of skin.Give top score 5 to thick and obvious blue variable colour (conventionally relevant to Restylane or Juv é derm Ultra Plus).Before being the blind scoring of sample, be that 3 independent observation persons train scale.

B) blue color component-" b " of skin color: use colorimeter (CM2600D, Konica Minolta, NJ) to quantize the blue color component of the light of the skin part outgoing from having injected different filleies.This realizes by " b " component that uses L-a-b colour code.

C) from " the blue light % " of skin outgoing: use portable spectrophotometer (CM2600D, Konica Minolta, NJ) to quantize the blue light % from skin outgoing in total visible-range.This can be by asking the integration of the area under 400-490nm visible spectrum and its standardization being realized with the gross area under spectrum (400-700nm).

embodiment 27

the Tyndall assessment of gel

Use straight line injection, by 27G1/2TSK pin to the thigh intradermal injection gel of 2 monthly ages without hair rat.Shallow table implanted gel is to simulate clinical microgroove program.After implanting, gel carries out Tyndall test 48h.Carry out after Tyndall test, make animal euthanasia to improve owing to lacking hemoglobin, the contrast of Tyndall effect.

In Figure 12, illustrate and implanted after 2 days, from the image of the gel of embodiment 19 and 21.Also show the image of commercial Juv é derm Refine and Restylane Touch so that comparison.In the image of commercial Juv é derm Refine and Restylane Touch, blue line (Tyndall effect) is high-visible.Gel from embodiment 19,19A (not shown) and 21 does not show Tyndall effect.

Using the range estimation score 1-5 that increment is 0.5, is injection site scoring.Must be divided into 1 injection site and not demonstrate dyschromasia, demonstrate the serious blue variable colour of skin and must be divided into 5 injection site.Also by means of colorimeter (CM2600D, Konica Minolta, NJ), injection site is carried out to spectrum analysis.The blue color component " b " of skin color and from blue light % (400-700nm) independent measurement of skin outgoing.Figure 13 and 14 shows range estimation Tyndall score and outgoing blue light %.Do not show Tyndall effect and there is lower range estimation Tyndall score and outgoing blue light value % from the gel of embodiment 19 and 21.For Juv é derm Refine and Restylane Touch, Tyndall score and outgoing blue light value % are higher.Belotero Soft does not demonstrate Tyndall effect and value and is comparable to the gel of embodiment 19 and 21.See Figure 13 and 14.

embodiment 28

by histology, the persistent period in the body of gel is assessed

Implant gel of the present invention and the commercial gel of 50 μ l bolus injections at the back side Intradermal of Shi-Dao Er Shi rat.In the time of 1 week, take out implant and dye and pass through histologic analysis with h and E (H & E).Just get section in injection portion.Cut two sections and use from each tissue sample and sew up indicator stitching H & E stained.Then by sample grouping and as follows according to the amount scoring of surplus material: do not have (0%), low (25%), in (50%) and height (100%).See Figure 15.

embodiment 28A

by MRI, the persistent period in the body of gel is assessed

Use nuclear magnetic resonance (MRI) research to be evaluated in female Shi-Dao Er Shi rat after intradermal injection, the volume of 40 weeks interior gels of the present invention and commercial gel and surface area are over time.By every kind of implant 150 μ l target volume injected gel.Make implant at slightly caudal two the offside positions of shoulder, two offside positions between end to end away from two offside positions of knee kiss side and mid point slightly.Can be in the enterprising line scanning of 7Tesla70/30Bruker Biospec MRI scanner.Implanting the same day (the 0th week) and implanting and within latter 12,24,40 weeks, collect image.Gel absolute volume and the figure of time have been shown in Figure 16 below.High persistency gel has high absolute volume in the time implanting 40 weeks.

embodiment 29

be used for the treatment of the present composition of socket of the eye Zhou Wen

There is tiny wrinkle and require the treatment of corium filler in the modest woman Kuang Zhou district of 40 years old.Use 30G pin, doctor for example introduces 0.6ml, according to the gel (gel of describing in embodiment 19) based on HA of the present invention to the shallow table of microgroove in her He Leigou district, every eyes below.Although introduce gel at shallow table, do not observe blue variable colour and patient satisfied to result.

As illustrated, compositions of the present invention, for example compositions of embodiment 19 and 21, Tyndall effect reduces or is not obvious, and the commercial gel with respect to some based on HA, for example Juvederm Refine/Surgiderm18 and Belotero Soft, the persistent period is in vivo long a lot.For example, with respect to commercial " microgroove " preparation Belotero Soft, embodiments of the invention 19 not only have at least the Tyndall score equally favourable with this commercial gel, and advantageously show the persistent period in high a lot of body.

embodiment 30

the Injectable composition of the present invention occurring for improving microgroove

Separately and the additive of combination, for example vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and derivant thereof so that the mode of multiple optical clear substantially, the injectable gel based on HA that generates be combined with cross-linked hyaluronic acid gel.As other local definition of this paper, HA component at least 90% by weight, for example, be entirely LMW HA substantially, or approximately 100% LMW HA.Use any applicable mode that these additives are combined with HA hydrogel.The gel that sieves and process combination to be to generate injectable, pH neutrality, cementitious compositions, and its HA concentration is at least about 20mg/g, for example approximately 23, about 24mg/g, about 25mg/g, up to about 30mg/g and be applicable to injecting by thin gage needle.The G ' of gel is worth at least about 50PA, about 60Pa, about 70Pa, about 80Pa, up to or be not more than about 100Pa.Packaging gel also uses pressure cooker, ultraviolet light or other applicable mode sterilizing.

Every kind of gel is all useful to shallow table injection, for example, arrive patient's wrinkle in the deeper injection that is not more than about 1.0mm, and for example Kuang Zhou district, nasolabial fold region, Lei Gou district, neck regions maybe will be benefited from the skin of any other facial zone of corium filling.Although shallow table is introduced gel, do not observe variable color and patient satisfaction result due to Tyndall effect.

Finally, although should be understood that the every aspect of having described this description with reference to each embodiment, those skilled in the art will readily recognize that, disclosed specific embodiment is only that theme principle disclosed herein has been described.Therefore, should be understood that disclosed theme is never limited to specific process described herein, scheme and/or reagent etc.Thereby under the prerequisite of spirit that does not deviate from this description, those skilled in the art can make many different amendments or variation or alternate configuration to disclosed theme according to instruction herein.Under the prerequisite of the spirit of the present invention defining, can do detailed variation in not deviating from claims.Finally, term used herein is only used to describe particular, but not is intended to limit the scope of the invention, and this scope is only defined by claim.In addition, it is intended that all material shown in contained in above description or accompanying drawing and should be only interpreted as illustrative and non-limiting.Therefore, the invention is not restricted to accurately to show and describe those.

This paper describes certain embodiments of the present invention, comprise that inventor becomes known for carrying out optimal mode of the present invention.Certainly, the modification of these embodiments of description is being read after aforementioned description and will become apparent for those of ordinary skill in the art.Inventor expects that technical staff optionally adopts this type of modification, and inventor is intended to make the present invention to implement to be different from specifically described method herein.Therefore,, as applicable law license, the present invention includes all modifications and the equivalents of the theme of narrating in claims.And, unless otherwise indicated or in addition and the obvious contradiction of context, otherwise the present invention contain its likely any combination of the above-mentioned element in modification.

The grouping of substitute element of the present invention disclosed herein or embodiment should be interpreted as to restriction.The member of each group can be separately or to mention with claimed with any combination of other member of this group or other element of finding herein.Estimate, one or more members of group can delete because the reason of convenient and/or patentability is included in group or therefrom.In the time that any this type of comprises or delete generation, description is all regarded as containing the group being modified, thereby has completed the written description for all Ma Kuxi groups (Markush group) of claims.

Unless otherwise noted, all numerals of expressing the amount, character molecular weight, the reaction condition etc. of description and claim (for example for) of composition all will be understood to be modified by term " about " in all cases.As used herein, term " about " refer to like this quantitative project, parameter or term be encompassed in the value of gainer, parameter or term upper and lower ± 10% scope.Therefore, unless point out on the contrary, the digital parameters of listing in description and claims is approximation, and its required character that can try hard to according to the present invention obtain changes.At least, and unlike attempting, the application of doctrine of equivalents is limited to the scope of claim, each digital parameters should be at least according to the numeral of the significant digit of report and understand by applying common approximate number technology.Although listing numerical range and the parameter of broad range of the present invention is approximation, the numerical value of listing is in a particular embodiment as far as possible accurately report all.But any numerical value contains some error that must be caused by the standard deviation of finding in its thermometrically separately inherently.

Unless otherwise indicated or with the obvious contradiction of context, with otherwise be understood to include odd number and plural number in describing term " ", " one ", " described " and the similar indicant of (especially below in the context of claim) in context of the present invention.The narration that is worth scope herein is only intended to as the stenography method of mentioning separately the each independent values that belongs to this scope.Unless otherwise indicated, otherwise each independent value being just incorporated in description as in this article it being narrated separately.Unless otherwise indicated or in addition and the obvious contradiction of context, otherwise all methods described herein can any applicable order be carried out.Use any and all embodiment provided herein or exemplary wording (for example, " for example ") to be only intended to the present invention is described better, and do not form the restriction to the claimed in addition scope of the invention.Wording in description must not be interpreted as and point out implementing the requisite any element that does not require protection of the present invention.

Can use by wording and form or substantially formed by wording, particular disclosed herein is further limited in claim.When for claim, be no matter as the correction of filing or the correction adding at every turn, transitional term " by ... composition " has all been got rid of unspecified any element, step or composition in the claims.Transitional term " substantially by ... composition " is limited to specific material or step by the scope of claim and can affect in fact material or the step of fundamental characteristics and new features.Intrinsic or describe clearly and enabled claimed embodiment of the present invention like this herein.

All patents of quoting in this manual and identify, patent are announced and other announces that (for example) describe in this type of is announced can combine with the present invention the compositions of use and the object of method in order to describe and disclose, and separately and clearly entirety is incorporated to this paper by reference.Before the application's date of application, these are announced only for the open of them provides.Should not be construed as on this point and admit, due to previous invention or any other reason, to have no right to make inventor open prior to this type of.About all statements on date or about the statement of these document contents based on the available information of applicant, do not form about date or any of content correctness of these documents and admit.

Claims (42)

1. a corium bulking agent compositions, comprises:

With the crosslinked hyaluronic acid component of crosslinking component;

Additive except described crosslinking component;

Described compositions optical clear substantially; And

When in the corium district that is administered to patient, with respect to except not having described additive, identical compositions substantially, described compositions table reveals Tyndall effect and reduces.

2. compositions according to claim 1, wherein said additive is vitamin C derivatives.

3. compositions according to claim 1, wherein said additive is AA2G.

4. compositions according to claim 1, wherein said additive is vitagen.

5. compositions according to claim 1, wherein said hyaluronic acid component is combined with described additive chemistry.

6. compositions according to claim 1, wherein said hyaluronic acid component is combined with described additive chemistry, and conjugation is between about 3mol% and about 40mol%.

7. compositions according to claim 1, wherein said hyaluronic acid component is combined with described additive chemistry, and conjugation is between about 3mol% and about 10mol%.

8. compositions according to claim 1, wherein said crosslinking component is BDDE.

9. compositions according to claim 1, further comprises anesthetis.

10. compositions according to claim 1, further comprises lignocaine.

11. compositionss according to claim 1, its G ' is worth between about 40Pa and about 100Pa.

12. compositionss according to claim 1, its G ' value is not more than about 100Pa.

13. compositionss according to claim 1, its G ' value is not less than about 40Pa.

Treat the method for the microgroove on patient skin for 14. 1 kinds, described method comprises:

In patient's skin, introduce the compositions that comprises hyaluronic acid component, is cross-linked the mixture of the crosslinking component of described hyaluronic acid component and the additive except described crosslinking component;

Described compositions optical clear substantially; And

Wherein with respect to except not having described additive, identical compositions substantially, described corium bulking agent compositions shows Tyndall effect and reduces.

15. methods according to claim 14, wherein said additive is vitamin C derivatives.

16. methods according to claim 14, wherein said additive is AA2G.

17. methods according to claim 14, wherein said additive is vitagen.

18. methods according to claim 14, wherein said hyaluronic acid component is combined with described additive chemistry.

19. methods according to claim 14, wherein said hyaluronic acid component is combined with described additive chemistry, and conjugation is between about 3mol% and about 10mol%.

20. methods according to claim 14, wherein said hyaluronic acid component is combined with described additive chemistry, and conjugation is between about 3mol% and about 40mol%.

21. 1 kinds are improved the method for face appearance, and described method comprises the following steps:

Use the optically transparent corium bulking agent compositions that does not show or show unconspicuous Tyndall effect substantially to patient's corium district, described compositions is made through the following steps:

Hyaluronic acid is provided;

Cross-linking agent is reacted with vitamin C derivatives;

The cross-linking agent having reacted and vitamin C derivatives are added in described hyaluronic acid and comprise covalently bound ascorbic cross-linked-hyaluronic acid compositions to form; And

Homogenize also neutralizes described cross-linked-hyaluronic acid compositions to obtain injectable corium bulking agent compositions.

22. methods according to claim 21, wherein said additive is vitamin C derivatives.

23. methods according to claim 21, wherein said additive is AA2G.

24. methods according to claim 21, wherein said additive is vitagen.

25. methods according to claim 21, wherein said hyaluronic acid component is combined with described additive chemistry.

26. methods according to claim 21, wherein said hyaluronic acid component is combined with described additive chemistry, and conjugation is between about 3mol% and about 40mol%.

27. methods according to claim 21, wherein said hyaluronic acid component is combined with described additive chemistry, and conjugation is between about 3mol% and about 10mol%.

28. 1 kinds are reduced the method that in patient's thin skin district, microgroove occurs, described method comprises:

Being not more than the degree of depth of about 1mm, use corium bulking agent compositions to described patient, one is optically transparent based on hyaluronic corium bulking agent compositions substantially, and it comprises vitamin C or vitamin C derivatives.

29. methods according to claim 28, are wherein being not more than compositions described in the deeper injection of about 0.8mm.

30. methods according to claim 28, are wherein being not more than compositions described in the deeper injection of about 0.6mm.

31. methods according to claim 28, are wherein being not more than compositions described in the deeper injection of about 0.4mm.

32. 1 kinds of corium bulking agent compositions, comprise:

With the crosslinked hyaluronic acid component of crosslinking component; With

With the conjugation at least about 3mol% and the covalently bound vitamin C of described hyaluronic acid component or vitamin C derivatives;

Described compositions substantially optical clear and G ' is worth between about 40Pa and about 100Pa.

33. compositionss according to claim 32, its hyaluronic acid concentration is between about 18mg/g and about 30mg/g.

34. compositionss according to claim 32, its hyaluronic acid concentration is between about 12mg/g and about 30mg/g.

35. compositionss according to claim 32, wherein said conjugation is about 10mol%.

36. compositionss according to claim 32, wherein said hyaluronic acid at least 90% is LMW HA.

37. compositionss according to claim 32, wherein said hyaluronic acid is entirely LMW HA substantially.

38. 1 kinds of Injectable compositions that occur for reducing facial microgroove, described compositions comprises:

The low-molecular-weight hyaluronic acid (HA) that BDDE (BDDE) is crosslinked, the mean molecule quantity of described HA is between about 300KD and about 900KD; With

With the covalently bound vitamin C derivatives of described hyaluronic acid, conjugation is about 10mol%;

G ' the value of described compositions is for about 60Pa is to about 80Pa.

39. according to the compositions described in claim 38, and the mean molecule quantity of wherein said HA is between about 300KD and about 500KD.

40. according to the compositions described in claim 39, and the G ' value of wherein said compositions is about 80Pa.

41. according to the compositions described in claim 38, its optical clear.

42. according to the compositions described in claim 38, and its HA concentration is about 25mg/g.