CN104105474B - Dermal augmentation agent composition for microgroove treatment - Google Patents

Abstract

The invention provides height injectable, the long-acting hydrogel dermal augmentation agent composition based on hyaluronic acid, its amendment to face's microgroove are particularly advantageous.

Description

Dermal augmentation agent composition for microgroove treatment

Related application

The U.S. Provisional Patent Application No.61/534,780 submitted for 14th this application claims September in 2011 priority and Interests, and be the part continuation application for the U.S. Patent Application Serial Number 13/593,313 that August in 2012 is submitted on the 23rd, the latter It is the part continuation application for the U.S. Patent Application Serial Number 13/486,754 submitted on June 1st, 2012, it require that 2011 The U.S. Provisional Patent Application No.61/493 submitted June 3,309 priority and interests, each in these application cases Complete disclosure is integrally incorporated herein by this specific reference.

Background

The present invention relates generally to dermal augmentation agent composition, and relate more specifically to effective to the microgroove on treatment skin Injectable dermal augmentation agent composition.

Skin aging is a progressive phenomenon, elapses over time and occurs and influenceed by Lifestyle factors, such as Drink, smoking and sunshine.Atrophy, relaxation and the fat aging for characterizing skin of face can be used.Atrophy corresponds to skin histology thickness Greatly reduce.Hypodermis relaxation causes skin excessive and sagging and causes cheek and ptosis occur.Obesity refer to because Increase excess weight for face and the swelling of neck bottom.These change generally to drying, follow the string it is coarse related to skin texture.

Hyaluronic acid (HA), also referred to as hyaluronan, it is connective, epithelium and the nerve fiber for being distributed widely in whole human body Non sulphate glycosaminoglycan.Hyaluronic acid is all very abundant in different skin layer, and wherein hyaluronic acid has multiple functions, example Such as ensure good aquation, help the composition of extracellular matrix, play packing material and participate in tissue repair mechanisms. However, the amount of other matrix polymers present in hyaluronic acid, collagen, elastin laminin and skin is reduced with the age.Example Such as, be repeated exposure to (such as) ultraviolet light from the sun causes dermal cell to reduce the generation of its hyaluronan and add The degradation rate of hyaluronan.The loss of this material causes various skins, such as corrugates, recess, water loss and has Help other undesirable conditions of aging appearance.

Injectable dermal augmentation agent has been used successfully to treat aging skin.The endogenous parent that filler can replace losing gathers Compound, or the function of the existing matrix polymer of enhancing/promotion, to treat these skins.Corium based on hyaluronic acid Filler has become to become more and more popular, because hyaluronic acid is in material existing for whole human body natural.These fillers lead to Normal well-tolerated, impermanency, and be the treatment to the suitable low-risk of a variety of skins.

Tyndall effect (Tyndall effect) is to application of one of the dermal augmentation agent based on hyaluronic acid (HA) The adverse events occurred in a little patients.Tyndall effect is characterised by, is gone out in the skin part for having injected dermal augmentation agent Existing blue variable colour, this represents to see visible hyaluronic acid by translucent epidermis.Clinical report shows, filler application technique and Skin properties can influence the performance of this adverse events.It is upper such as nose that high rigidity and elastomeric filler are used successfully to amendment face The regions such as labial groove, cheek and chin, without there is any worry to change color, because Material injection is at middle part and deep dermis area In.These packing material amendment superficial, fine wrinkles, such as tear ditch, glabella line, canthus line, laugh line or preceding are used however, working as Volume, or in upper part of dermis region too superficial mistake application when, be frequently observed the light blue discoloration of skin.This phenomenon, It is considered as the result of Tyndall effect, causes application site semipermanent to change colour, and is sometimes only application of degraded Disappeared after the hyaluronidase of packing material.Therefore, Tyndall effect is more conventional in the patient for the treatment of superficial fine wrinkles.Only Gel is wanted to maintain in skin, Tyndall effect performance extends, usual some months, is an original of principal concern in patient Cause.

The dermal augmentation gel based on HA has especially been prepared to treat around tear ditch, forehead, canthus line, glabella line etc. It was found that " fine wrinkles ".Commercially available HA " microgroove " gel includes 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 (such as) by making linear HA chains It is slightly cross-linked and/or reduce the final HA concentration of these gels with a small amount of crosslinking agent, these gels are configured to low elasticity Modulus, but most of commercially available " microgroove " gel still shows Tyndall effect in some patients, particularly When superficial is injected, such as in the depth less than about 1mm.

The gel based on collagen can be used in the treatment of superficial wrinkle and seems that Tyndall effect will not be caused. Gel based on collagen, which is not affected by height, to be favored, because their duration in skin are relatively poor and need Tested in advance in individual.(calcium hydroxy apetite) is subcutaneous, injectable implant, and its key component is synthesis hydroxyl Base apatite calcium, rather than hyaluronic acid.Different from the dermal augmentation agent based on hyaluronic acid, calcium hydroxy apetite is opaque, because This avoids the complication of Tyndall effect.However, if placement is too shallow, this filler can be considered as under skin Whiteness.In addition, compared with the filler based on hyaluronic acid,Longer syringe needle is needed to inject and lead to Often do not recommend to be used for ocular.

It will need to provide the noting based on hyaluronic acid that will not show the light blue discoloration for being attributed to Tyndall effect Penetrate dermal augmentation agent.

General introduction

The present invention describes preparation and can applied in the corium of top, does not produce any light blue discoloration of skin, or at least The composition and compound method of the dermal augmentation agent based on HA of not notable or unconspicuous light blue discoloration.Further, It is found that presently described many gel filled duration in vivo of the invention are substantially commercially available more solidifying than on Vehicles Collected from Market Glue is longer.In some aspects of the present invention, there is provided the optically transparent dermal augmentation agent useful to enhancing skin appearance, it increases Volume and richness are added, and have reduced the appearance of even fine wrinkles, do not had " Tyndall effect ".This composition can introduce In the microgroove of skin, or even thin skin region and suitable superficial, the dermal augmentation agent transparent with many normal opticals will not be caused Related negative blue variable colour.

More specifically, in one aspect of the invention, there is provided long-acting treatment dermal augmentation agent composition, it is generally wrapped Containing biocompatible polymer, such as cross-linked-hyaluronic acid component and the additive that merges with hyaluronic acid component.

In one embodiment, polymer is polysaccharide, such as hyaluronic acid.Hyaluronic acid includes linked and can Further comprise non-crosslinked components.Additive may include vitamin, for example, vitamin C (for example, vitamin C of stable form) or Vitamin C derivatives are (for example, L-AA 2- glucosides (AA2G), ascorbic acid 3- aminopropyl phosphate (vitagens (Vitagen)) or STAY-C 50 (AA2P)).

In one aspect of the invention, additive is vitamin derivative, its by be adapted to course of reaction, such as etherificate, Amidatioon or esterification and polymer covalent bond.

At the extensive aspect of the present invention, there is provided dermal augmentation agent composition, the composition includes and crosslinking group Divide the hyaluronic acid component of crosslinking and the additive in addition to the linked.Hyaluronic acid component can be with additive chemistry knot Close.Further, when in the corium area for being administered to patient, relative in addition to no additive, substantially the same group Compound, the composition show Tyndall effect reduction.The composition can further include other additives, such as anaesthetize Agent (such as lidocaine).In one embodiment, additive is vitamin C derivatives, such as AA2G.In another embodiment party In case, additive is vitagen.

In one embodiment, hyaluronic acid component is combined with additive chemistry, and conjugation is between about 3mol% peace treaties Between 40mol, such as between about 3mol% and about 10mol%.

The composition may generally optical clear.G ' the values of the composition are generally between about 40Pa and about 100Pa Between, such as no more than about 100Pa, such as not less than about 40Pa.

In another aspect of this invention, there is provided the method for the microgroove on treatment patient skin.In one embodiment, Methods described includes introducing into the skin of patient comprising hyaluronic acid component, is crosslinked the linked of the hyaluronic acid and removes The step of composition of the mixture of additive beyond the linked, the composition generally optical clear, and Wherein relative in addition to no additive, substantially the same composition, the composition shows Tyndall effect and subtracted It is small.

In another aspect of this invention, there is provided improve the method for face appearance, methods described generally includes the following steps： The generally optically transparent corium for not showing or showing unconspicuous Tyndall effect is applied to the corium area of patient Bulking agent compositions.The composition is made through the following steps：Hyaluronic acid is provided, makes crosslinking agent and vitamin C derivatives Reaction, the crosslinking agent reacted and vitamin C derivatives are added in the hyaluronic acid included with formation it is covalently bound Ascorbic cross-linked-hyaluronic acid composition；And homogenize and neutralize the cross-linked-hyaluronic acid composition to obtain injectable Dermal augmentation agent composition.In some embodiments, vitamin C derivatives are AA2G.In other embodiments, dimension life Plain C derivatives are vitagens.

In still another aspect of the invention, there is provided reduce the method that microgroove occurs in patient's thin skin area, wherein methods described The depth no more than about 1mm is typically included in, dermal augmentation agent composition, a kind of generally optical clear are applied to the patient The dermal augmentation agent composition based on hyaluronic acid, it includes vitamin C or vitamin C derivatives.In some embodiments In, no more than 0.8mm, no more than 0.6mm or no more than composition described in 0.4mm deeper injection.

In still another aspect of the invention, there is provided a kind of dermal augmentation agent composition, it generally optical clear and leads to Often include with linked crosslinking hyaluronic acid component and with the covalently bound vitamin C derivatives of hyaluronic acid component. In one exemplary, the G ' values of the composition are between about 40Pa and about 100Pa.Further, described group The hyaluronic acid concentration of compound may be between about 18mg/g and about 30mg/g.Skin is being treated, for example, even very thin In microgroove or superficial gauffer on skin, such as skin of the thickness no more than about 1mm, these compositions may be particularly useful and had Effect.In some embodiments, composition of the invention continues at least three moon after skin is introduced, at least six moon or up to one Year.

It may be easier to understand with reference to drawings below and being described in detail and recognize these and other aspects of the invention and excellent Point.

Brief description

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

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

Fig. 3 is STAY-C 50 (AA2P) structural representation.

Fig. 4 is 1,4- butanediol diglycidyl ethers (BDDE) structural representation.

Fig. 5 is the structural representation of pentaerythrite glycidol ether (Star-PEG epoxides).

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

Fig. 7 is the table for showing conjugation and G ' values according to various dermal augmentation agent compositions of the present invention.

Fig. 8 be show it is dense according to the conjugation, HA of HA-AA2G of the present invention (BDDE) dermal augmentation agent composition The table of degree and G ' values.

Fig. 9 be for 4 kinds of different alpha-glucosaccharase enzyme concentrations, in terms of the time (Minute), it was observed that come from The pictorial diagram of the percentage of the AsA releases of solution of the AA2G in PBS.

Figure 10 shows the release profiles schematic diagram from the free AsA according to combination dermal augmentation agent of the present invention (sustained release) (the AA2G conversions mol% compared with the reaction time).

Figure 11 A and 11B show the additional release data according to various dermal augmentation agent of the present invention.

Figure 12 is shown in dermal augmentation gel of the superficial injection present invention based on HA and some commercially available confessions After the gel of microgroove application, the image of skin.

Figure 13 shows dermal augmentation gel of the present invention based on HA and some commercially available for microgroove application The vision Tyndall score of gel.

Figure 14 shows dermal augmentation gel of the present invention based on HA and some are commercially available for microgroove application The blue light % that gel is emitted from skin.

Figure 15 show implantation dermal augmentation gel of the present invention based on HA and some it is commercially available should for microgroove After gel 1 week, total % of remaining gel.

Figure 16 show implantation dermal augmentation gel of the present invention based on HA and some commercially available supply microgroove The gel of application the 0th week, the 12nd week, the 24th week and the 40th week, total % of remaining gel.

It is described in detail

In one aspect of the invention, there is provided dermal augmentation agent composition, the composition generally comprise bio-compatible Property polymer, for example, polysaccharide (such as cross-linked-hyaluronic acid) and with the covalently bound vitamin C derivatives of the polymer.It is described Composition provides vitamin C and other treatment or beauty interests of the sustained release for skin collagen hyperplasia.Work as introducing During skin, for example, it is intracutaneous, and the composition reacts with internal endogenous enzyme, and over time, is enzymatically cleaved off in vivo Generate bioactive vitamin C.Because vitamin C discharges from the composition in a few weeks or months so that following Interests body can use.

The polymer may be selected from protein, peptide and polypeptide, polylysine, collagen, procollagen, elastin laminin And laminin.

The polymer may be selected from the synthetic polymer with hydroxyl, amine and carboxyl functional group：Poly- (vinyl alcohol), poly- second two Alcohol, polyvinylamine, polyallylamine, deacetylate polyacrylamide, polyacrylic acid and polymethylacrylic acid.The polymer is optional From dendroid or branched polymer, including dendrimeric polyols and dendroid polyamine.The polymer may be selected from hydroxyl, amine With the surface of solids of carboxyl functional group.

The polymer can be polysaccharide, be selected from following group of polysaccharide, including starch and its derivative；Glucan and its Derivative；Cellulose and its derivates；Chitin, chitosan and alginates and its derivative.

In the exemplary of the present invention, the polymer is glycosaminoglycan.Hydrogel disclosed herein Composition can further include two or more different Glycosaminoglycan Polymers.As used herein, " osamine gathers term Sugar " is synonymous with " GAG " and " mucopolysaccharide " and refers to the long linear polysaccharide of repetition disaccharide unit composition.Repeat unit by with hexosamine The hexose (hexose) or hexuronic acid composition of (nitrogenous hexose) and its pharmaceutically acceptable salt connection.GAG families into It is different in the type of hexosamine of the member contained by it, hexose or hexuronic acid unit, for example, glucuronic acid, iduronic acid, Galactolipin, galactosamine, gucosamine and may also be different in the geometric shape of glycosidic inkage.Any Glycosaminoglycan Polymer Useful in hydrogel composition disclosed herein, condition is that Glycosaminoglycan Polymer improves skin.Glycosaminoglycan Non-limiting examples include chondroitin sulfate, dermatan sulfate, keratan sulfate, hyaluronan.The acceptable salt of glycosaminoglycan Non-limiting examples include sodium salt, sylvite, magnesium salts, calcium salt and combinations thereof.For example, in 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, United States Patent (USP) Announce 2008/0089918；Lebreton, Hyaluronic Acid-Based Gels Including Lidocaine, the U.S. Patent disclosure 2010/0028438；It is public with Polysaccharides and Hydrogels thus Obtained, United States Patent (USP) Cloth 2006/0194758；With Di Napoli, Composition and Method for Intradermal Soft Tissue Described in Augmentation, international patent publications WO2004/073759 in hydrogel composition disclosed herein and method In useful glycosaminoglycan and its resulting polymers, it is each integrally incorporated accordingly by reference.In hydrogel disclosed herein Useful GAG is commercially available in composition and method, such as the dermal augmentation agent based on hyaluronan30、Ultra、Ultra Plus、Ultra XC andUltra Plus XC(Allergan Inc,Irvine, California).Table 1 lists representative GAG.

The aspect of the present invention provide in part the hydrogel composition for including chondroitin sulfate polymer.As made herein With term " chondroitin sulfate polymer " refers to unbranched, the sulfated polymers of variable-length, and it includes two alternating monose That is D-Glucose aldehydic acid (GlcA) and the disaccharides of N-ACETYL-D- GALACTOSAMINE (GalNAc) and its pharmaceutically acceptable salt. Chondroitin sulfate polymer may also comprise the D-Glucose aldehydic acid residue that epimerism turns to L- iduronic acids (IdoA), at this Gained disaccharides is referred to as dermatan sulfate in the case of kind.Chondroitin sulfate can have more than 100 individually sugar chains, its each with Variable position and amount sulphation.Chondroitin sulfate polymer is the important feature component of cartilage and provides it compression is permitted Multiresistance.Any chondroitin sulfate polymer is useful in compositions disclosed herein, and condition is chondroitin sulfate polymer Improve skin.It is soft that the non-limiting examples of chondroitin sulfate pharmaceutically acceptable salt include sodium chondroitin sulfate, sulfuric acid Ossein potassium, chondroitin sulfate magnesium, calcium chondroitin sulfate and combinations thereof.

The hydrogel composition for including keratan sulfate polymer is provide in part in terms of this specification.Such as this paper institutes Use, term " keratan sulfate polymer " refers to the polymer of the variable-length comprising disaccharide unit, itself includes β-D- half Lactose and N-ACETYL-D- GALACTOSAMINE (GalNAc) and its pharmaceutically acceptable salt.Two in keratan sulfate duplicate block Sugar may be covered in the end of chain through fucosylation and N-acetyl-neuraminate.Any keratan sulfate polymer is public herein Useful in the composition opened, condition is that keratan sulfate polymer improves skin.Keratan sulfate is pharmaceutically acceptable Salt non-limiting examples include keratan sulfate sodium, keratan sulfate potassium, keratan sulfate magnesium, keratan sulfate calcium and its Combination.

The hydrogel composition for including hyaluronan polymer is provide in part in terms of this specification.As made herein With, term " hyaluronan polymer " is synonymous with " HA polymer ", " hyaluronic acid polymer " and " hyaluronate polymers ", Refer to anion, the non sulphate Glycosaminoglycan Polymer for including disaccharide unit, itself include passing through alternate β-Isosorbide-5-Nitrae and β -1, The D-Glucose aldehydic acid and D-N- acetylglucosamine monomers and its pharmaceutically acceptable salt that 3 glycosidic bonds link together.Can From animal and non-animal purifying hyaluronan polymer.The polymer sizes scope of hyaluronan can from about 5,000Da to About 20,000,000Da.Any hyaluronan polymer is useful in compositions disclosed herein, and condition is that hyaluronan changes Kind skin.The non-limiting examples of hyaluronan pharmaceutically acceptable salt include hyaluronan sodium, hyaluronan potassium, thoroughly Bright matter alkane magnesium, hyaluronan calcium and combinations thereof.

The hydrogel composition for including crosslinking Glycosaminoglycan Polymer is provide in part in terms of this specification.As herein Used, term " crosslinking " refers to intermolecular linkage, and single polymer molecule or monomer chain link imaging gel is equally more stable Structure.Thus, crosslinking Glycosaminoglycan Polymer has is connected to another at least one by least one thing molecule that is polymerized alone Individual intermolecular linkage.The crosslinking of Glycosaminoglycan Polymer typically results in hydrogel and formed.Such water-setting adhesiveness is high and needs phase When big power extrudes fine needle.Dialdehyde and disulphide cross-linking agents Glycosaminoglycan Polymer disclosed herein can be used, including But it is not limited to multi-functional PEG group crosslinking agent, divinyl sulfone, glycidol ether and di-epoxide, dual-carbodiimide.Hyaluronan The non-limiting examples of crosslinking agent include multi-functional PEG group crosslinking agent, such as the glycidol ether of pentaerythrite four (PETGE), diethyl Alkene sulfone (DVS), 1,4- butanediol diglycidyl ethers (BDDE), 1,2- double (2,3- glycidoxies) ethene (EGDGE), 1,2, 7,8- diepoxyoctanes (DEO), (phenylene is double-(ethyl)-carbodiimide and 1,6 hexylidenes double (ethyl carbodiimides), oneself two Hydrazides (ADH), double (sulfosuccinic base) suberates (BS), hexamethylene diamine (HMDA), 1- (2,3- glycidoxies) -2,3- epoxies Hexamethylene, lysine, lysine methyl ester or its combination.In Stroumpoulis and Tezel, Tunably Crosslinked Polysaccharide Compositions, the U.S. Patent application 12/910 that on October 22nd, 2010 submits, disclosed in 466 Other useful crosslinking agents, it is integrally incorporated by quoting.For example, in 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 The non-limit of the method for crosslinking Glycosaminoglycan Polymer is described in Augmentation, international patent publications WO2004/073759 Property example processed, and useful Glycosaminoglycan Polymer in compositions disclosed herein and method, it is each accordingly by drawing With being integrally incorporated.

The water for including the crosslinking Glycosaminoglycan Polymer with certain degree of cross linking is provide in part in terms of this specification Gel combination.As used herein, term " degree of cross linking " refers to Glycosaminoglycan Polymer monomeric unit, such as is combined with crosslinking agent Hyaluronan disaccharides monomeric unit percentage.The degree of cross linking is expressed as the percentage by weight of crosslinking agent and glycosaminoglycan.At this Invention some Favourable implementations in, the degree of cross linking between about 3% and about 12%, such as between about 5% and about 10% it Between.

In one embodiment, hydrogel composition includes crosslinking Glycosaminoglycan Polymer, such as cross-linked-hyaluronic acid, Present in wherein described composition it is described crosslinking Glycosaminoglycan Polymer concentration (such as) between about 18mg/g and about 30mg/ Between g.In some embodiments, the hyaluronic acid total concentration of the composition is about 24mg/g or about 25mg/g.

Provide in part in terms of this specification the hyaluronan polymer comprising low molecule amount, HMW it is transparent The hydrogel composition of the hyaluronan polymer of matter alkane polymer or low molecule amount and HMW.As used herein, when Term " HMW " refers to the hyaluronan that mean molecule quantity is 1,000,000Da or higher and polymerize when referring to " hyaluronan " Thing.The non-limiting examples of HMW hyaluronan polymer include about 1,500,000Da, about 2,000,000Da, about 2, 500,000Da, about 3,000,000Da, about 3,500,000Da, about 4,000,000Da, about 4,500,000Da and about 5,000, 000Da hyaluronan polymer.As used herein, when referring to " hyaluronan ", term " low molecule amount " refers to mean molecule Hyaluronan polymer of the amount less than 1,000,000Da.The non-limiting examples of low molecule amount hyaluronan polymer are included about 200,000Da, about 300,000Da, about 400,000Da, about 500,000Da, about 600,000Da, about 700,000Da, about 800, 000Da and about 900,000Da hyaluronan polymer.

In one embodiment, composition includes the cross-linked transparent matter alkane polymer of low molecule amount.In the embodiment Aspect, composition include mean molecule quantity for (such as) about 100,000Da, about 200,000Da, about 300,000Da, about 400, 000Da, about 500,000Da, about 600,000Da, about 700,000Da, about 800,000Da or about 900,000Da cross-linked transparent Matter alkane polymer.In the other side of the embodiment, composition include mean molecule quantity for (such as) at most 100,000Da, At most 200,000Da, at most 300,000Da, at most 400,000Da, at most 500,000Da, at most 600,000Da, at most 700,000Da, at most 800,000Da, at most 900,000Da or at most 950,000Da cross-linked transparent matter alkane polymer.At this The other side of embodiment, composition include mean molecule quantity for (such as) about 100,000Da to about 500,000Da, about 200,000Da to about 500,000Da, about 300,000Da to about 500,000Da, about 400,000Da to about 500,000Da, about 500,000Da to about 950,000Da, about 600,000Da to about 950,000Da, about 700,000Da to about 950,000Da, about 800,000Da to about 950,000Da, about 300,000Da to about 600,000Da, about 300,000Da to about 700,000Da, about 300,000Da to about 800,000Da or about 400,000Da to about 700,000Da cross-linked transparent matter alkane polymer.

In another embodiment, composition includes the cross-linked transparent matter alkane polymer of HMW.In the embodiment Aspect, composition include mean molecule quantity for (such as) about 1,000,000Da, about 1,500,000Da, about 2,000,000Da, About 2,500,000Da, about 3,000,000Da, about 3,500,000Da, about 4,000,000Da, about 4,500,000Da or about 5, 000,000Da cross-linked transparent matter alkane polymer.In the other side of the embodiment, composition is comprising mean molecule quantity (such as) 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 5,000,000Da's Cross-linked transparent matter alkane polymer.In the other side of the embodiment, composition include mean molecule quantity for (such as) about 1, 000,000Da to about 5,000,000Da, about 1,500,000Da to about 5,000,000Da, about 2,000,000Da to about 5,000, 000Da, about 2,500,000Da are to about 5,000,000Da, about 2,000,000Da to about 3,000,000Da, about 2,500,000Da To about 3,000,000Da cross-linked transparent matter alkane polymer.

In another embodiment, composition includes cross-linked transparent matter alkane polymer, wherein the cross-linked transparent matter alkane polymerize Thing includes the combination of the HMW hyaluronan polymer and low molecule amount hyaluronan polymer of different proportion.In the implementation The aspect of scheme, composition includes cross-linked transparent matter alkane polymer, wherein the cross-linked transparent matter alkane polymer is comprising ratio About 20:1st, about 15:1st, about 10:1st, about 5:1st, about 1:1st, about 1:5th, about 1:10th, about 1:15 or about 1:20 HMW hyalomitome The combination of alkane polymer and low molecule amount hyaluronan polymer.

The hydrogel composition for including uncrosslinked Glycosaminoglycan Polymer is provide in part in terms of this specification.Such as this Text is used, and term " uncrosslinked " refers to the intermolecular linkage for lacking and connecting single Glycosaminoglycan Polymer molecule or monomer chain.Cause And uncrosslinked Glycosaminoglycan Polymer is not connected by intermolecular linkage with any other Glycosaminoglycan Polymer.In the embodiment party The aspect of case, composition include uncrosslinked chondroitin sulfate polymer, uncrosslinked dermatan sulfate polymer, uncrosslinked sulfuric acid angle Quality polymer, uncrosslinked heparan polymer, uncrosslinked Heparan sulfate polymer or the polymerization of uncrosslinked hyaluronan Thing.Uncrosslinked Glycosaminoglycan Polymer is water-soluble and generally keeps mobility in itself.Thus, uncrosslinked glycosaminoglycan Polymer often mixes thin to promote composition to pass through with as the hydrogel composition based on Glycosaminoglycan Polymer of lubricant The extrusion of pin.

In one embodiment, composition includes uncrosslinked Glycosaminoglycan Polymer, wherein the uncrosslinked osamine gathers Concentration existing for glycopolymers for (such as) 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 embodiment party The other side of case, composition include uncrosslinked glycosaminoglycan, wherein concentration existing for the uncrosslinked glycosaminoglycan is (example As) 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, extremely Few 20mg/g, at least 25mg/g, at least 35mg/g or at least 40mg/g.In the other side of the embodiment, composition includes Uncrosslinked glycosaminoglycan, wherein concentration existing for the uncrosslinked glycosaminoglycan for (such as) at most 1mg/g, at most 2mg/g, extremely More 3mg/g, at most 4mg/g, at most 5mg/g, at most 10mg/g, at most 15mg/g, at most 20mg/g or at most 25mg/g.At this The other side of embodiment, composition includes uncrosslinked glycosaminoglycan, wherein concentration existing for the uncrosslinked glycosaminoglycan For (such as) about 1mg/g to about 60mg/g, about 10mg/g be to about 40mg/g, about 7.5mg/g to about 19.5mg/g, about 8.5mg/g To about 18.5mg/g, about 9.5mg/g to about 17.5mg/g, about 10.5mg/g to about 16.5mg/g, about 11.5mg/g to about 15.5mg/g or about 12.5mg/g are to about 14.5mg/g.

The hydrogel composition substantially free of crosslinking Glycosaminoglycan Polymer is provide in part in terms of this specification. As used herein, term substantially free (or " substantially by ... form ") refers to wherein only detectable micro crosslinking The composition of matrix polymer.In the one side of the embodiment, composition is included substantially free of crosslinking chondroitin sulfate The chondroitin sulfate of polymer, substantially free of crosslinking dermatan sulfate polymer dermatan sulfate, substantially free of crosslinking The keratan sulfate of keratan sulfate polymer, the heparan, substantially not substantially free of crosslinking heparan polymer The Heparan sulfate of the Heparan sulfate polymer containing crosslinking or the sulfuric acid substantially free of cross-linked transparent matter alkane polymer are saturating Bright matter alkane.

The hydrogel composition for being entirely free of crosslinking Glycosaminoglycan Polymer is provide in part in terms of this specification.Such as Used herein, term " being entirely free of " refers to composition in the range of the instrument or fabrication evaluation used, it is impossible to detects crosslinking Glycosaminoglycan Polymer not can confirm that its presence.In the one side of the embodiment, composition includes and is entirely free of crosslinking The chondroitin sulfate of chondroitin sulfate polymer, the dermatan sulfate, completely not for being entirely free of crosslinking dermatan sulfate polymer Containing crosslinking keratan sulfate polymer keratan sulfate, be entirely free of crosslinking heparan polymer heparan, completely It is saturating without the Heparan sulfate for being crosslinked Heparan sulfate polymer or the sulfuric acid for being entirely free of cross-linked transparent matter alkane polymer Bright matter alkane.

It provide in part in terms of this specification comprising a certain proportion of crosslinking Glycosaminoglycan Polymer and uncrosslinked sugar The hydrogel composition of amine chitosan polymer.This ratio of crosslinking and uncrosslinked Glycosaminoglycan Polymer is alternatively referred to as gel: Fluid ratio.Any gel in compositions disclosed herein is prepared:Fluid ratio is useful, and condition is that the generation of this ratio is disclosed herein Improvement skin as disclosed herein composition.Gel in the present composition:Fluid than non-limiting examples bag Include 100:0、98:2、90:10、75:25、70:30、60:40、50:50、40:60、30:70、25:75、10:90；2:98 and 0: 100。

In terms of the embodiment, composition includes crosslinking Glycosaminoglycan Polymer and the polymerization of uncrosslinked glycosaminoglycan Thing, wherein gel:Fluid ratio for (such as) about 0:100th, about 1:99th, about 2:98th, about 3:97th, about 4:96th, about 5:95th, about 6:94、 About 7:93rd, about 8:92nd, about 9:91 or about 10:90.In the other side of the embodiment, composition includes crosslinking glycosaminoglycan and gathered Compound and uncrosslinked Glycosaminoglycan Polymer, wherein gel:Fluid ratio for (such as) at most 1:99th, at most 2:98th, at most 3:97、 At most 4:96th, at most 5:95th, at most 6:94th, at most 7:93rd, at most 8:92nd, at most 9:91 or at most 10:90.In the embodiment Other side, composition includes crosslinking Glycosaminoglycan Polymer and uncrosslinked Glycosaminoglycan Polymer, wherein gel:Fluid ratio For (such as) about 0:100 to about 3:97th, about 0:100 to about 5:95 or about 0:100 to about 10:90.

In the other side of the embodiment, composition includes crosslinking Glycosaminoglycan Polymer and uncrosslinked glycosaminoglycan gathers Compound, wherein gel:Fluid ratio for (such as) about 15:85th, about 20:80th, about 25:75th, about 30:70th, about 35:65th, about 40:60、 About 45:55th, about 50:50th, about 55:45th, about 60:40th, about 65:35th, about 70:30th, about 75:25th, about 80:20th, about 85:15th, about 90: 10th, about 95:5th, about 98:2 or about 100:0.In the other side of the embodiment, composition includes crosslinking Glycosaminoglycan Polymer With uncrosslinked Glycosaminoglycan Polymer, wherein gel:Fluid ratio for (such as) at most 15:85th, at most 20:80th, at most 25:75、 At most 30:70th, at most 35:65th, at most 40:60th, at most 45:55th, at most 50:50th, at most 55:45th, at most 60:40th, at most 65: 35th, at most 70:30th, at most 75:25th, at most 80:20th, at most 85:15th, at most 90:10th, at most 95:5th, at most 98:2 or at most 100:0.In the other side of the embodiment, composition includes crosslinking Glycosaminoglycan Polymer and the polymerization of uncrosslinked glycosaminoglycan Thing, wherein gel:Fluid ratio for (such as) about 10:90 to about 70:30th, about 15:85 to about 70:30th, about 10:90 to about 55:45、 About 80:20 to about 95:5th, about 90:10 to about 100:0th, about 75:25 to about 100:0 or about 60:40 to about 100:0.

Hydrogel composition disclosed herein can further include to be provided beneficial to work when to individual applying said compositions Another reagent or agent combination.Such beneficial agent includes but is not limited to antioxidant, antipruritic, the fat agent that disappears, anti-scar (such as styptic or anti-fiber are molten for trace agent, antiinflammatory, anesthetic, counter-stimulus, vasoconstrictor, vasodilator, antihaemorrhagics agent Solve protein agent), remover, tensioning agent, anti-acne agent, pigmentation agent, anti-pigmentation agent or NMF.

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

The aspect of the present invention provide in part the hydrogel composition disclosed herein that can optionally include anesthetic.Anesthesia Agent is preferably local anesthetic, that is, causes invertibity local anaesthesia and lose the anesthetic of nociception, such as amino amides office Anesthetic and amino ester local anesthetic.The amount for the anesthetic that compositions disclosed herein includes is being applied to mitigating individual Effectively measured with the pain experienced after the composition.Thus, the anesthesia that composition disclosed in this manual includes The amount of agent is arrived between about 5% between by general composition weight meter about 0.1%.The non-limiting examples of anesthetic include benefit card Cause, ambucaine (ambucaine), amolanone (amolanone), amylocaine (amylocaine), Oxybuprocaine Because of (benoxinate), benzocainum (benzocaine), betoxycaine (betoxycaine), xenysalate (biphenamine), bupivacaine (bupivacaine), butacaine (butacaine), butamben (butamben), cloth Smooth cacaine (butanilicaine), butethamine (butethamine), butoxycaine (butoxycaine), carticaine (carticaine), chloroprocanine (chloroprocaine), hexyl benzoic acid Ai Kangyin (cocaethylene), cocaine (cocaine), cyclomethycaine (cyclomethycaine), cinchocaine (dibucaine), quotane (dimethysoquin), dimethocaine (dimethocaine), the piperazine winter (diperodon), bentyl (dycyclonine), Water sprout is gone to subtract (ecgonidine), bud subtracts (ecgonine), chloroethanes, Etidocaine (etidocaine), betaeucaine (beta-eucaine), Euprocin (euprocin), fenalcomine (fenalcomine), formocaine, Hexylcaine (hexylcaine), hydroxytetracaine (hydroxy teracaine), cycloform, leucinocaine mesylate (leucinocaine mesylate), Levoxadrol (Ievoxadrol), lidocaine, mepivacaine (mepivacaine), Meprylcaine (meprylcaine), metabutoxycaine (metabutoxycaine), chloromethanes, myrtecaine (myrtecaine), Receive her cacaine (naepaine), Octacaine (octacaine), Orthocaine (orthocaine), Mucaine (oxethazaine), parethoxycaine (parethoxycaine), phenacaine hydrochloride (phenacaine), phenol, piperocaine (piperocaine), Piridocaine (piridocaine), polidocanol (polidocanol), pramoxine (pramoxine), prilocaine (prilocaine), procaine (procaine), Propanocaine (propanocaine), third U.S. cacaine (proparacaine), Bing Veins cacaines (propipocaine), propoxycaine (propoxycaine), d-pseudococaine (psuedococaine), Pyrrocaine (pyrrocaine), Ropivacaine (ropivacaine), saligenin, totokaine (tetracaine), Tolycaine (toIycaine), trimecaine (trimecaine), zolamine (zolamine), its combination And its salt.Amino ester local anesthetic includes procaine, chloroprocanine, cocaine, cyclomethycaine, cimethocaine (larocaine (larocaine)), propoxycaine, procaine (procaine hydrochloride (novocaine)), proparacaine, totokaine (amethocaine (amethocaine)).The non-limiting examples of amino amides local anesthetic include Articaine (articaine), bupivacaine, cinchocaine (cinchocaine) (cinchocaine), Etidocaine, chirocaine (levobupivacaine), lidocaine (lignocaine (lignocaine)), mepivacaine, piperocaine, prilocaine, Ropivacaine and trimecaine.Compositions disclosed herein can include single anesthesia agent or a variety of anesthetic.Combine local anaesthesia The non-limiting examples of medicine are lidocaine/prilocaine (EMLA).

Therefore in one embodiment, compositions disclosed herein includes anesthetic and its salt.In the embodiment Aspect, compositions disclosed herein include amino amides local anesthetic and its salt or amino ester local anesthetic and its salt. The other side of the embodiment, compositions disclosed herein include procaine, chloroprocanine, cocaine, cyclomethycaine, Cimethocaine, propoxycaine, procaine, proparacaine, totokaine or its salt or its any combinations.In the embodiment Other side, compositions disclosed herein include Articaine, bupivacaine, cinchocaine, Etidocaine, left Bu Bika Cause, lidocaine, mepivacaine, piperocaine, prilocaine, Ropivacaine, trimecaine or its salt or its any combinations. The other aspect of the embodiment, compositions disclosed herein include lidocaine/prilocaine combination.

In the other side of the embodiment, compositions disclosed herein is included based on the weight of total composition, measured as (example As) about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%th, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0% or about 10% Anesthetic.At other aspect, compositions disclosed herein is included based on the weight of total composition, measure for (such as) at least 0.1%th, 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%th, at least 8.0%, at least 9.0% or at least 10% anesthetic.In also other side, compositions disclosed herein bag Containing based on the weight of total composition, measure for (such as) at most 0.1%, at most 0.2%, at most 0.3%, at most 0.4%, at most 0.5%th, at most 0.6%, at most 0.7%, at most 0.8%, at most 0.9%, at most 1.0%, at most 2.0%, at most 3.0%, At most 4.0%, at most 5.0%, at most 6.0%, at most 7.0%, at most 8.0%, at most 9.0% or at most 10% anesthesia Agent.In other side, compositions disclosed herein is included based on the weight of total composition, measure for (such as) about 0.1% to about 0.5%th, about 0.1% to about 1.0%, about 0.1% to about 2.0%, about 0.1% to about 3.0%, about 0.1% to about 4.0%, about 0.1% to about 5.0%, about 0.2% to about 0.9%, about 0.2% to about 1.0%, about 0.2% to about 2.0%, about 0.5% to about 1.0% or the anesthetic of about 0.5% to about 2.0%.

In another embodiment, compositions disclosed herein does not include anesthetic.

In one aspect of the invention, there is provided injectable dermal augmentation agent, it includes polymer, such as glycosaminoglycan gathers Compound, such as hyaluronic acid polymer, for example, at least a part crosslinking hyaluronic acid, and additive or with the polymer group The beneficial agent of conjunction.

Vitamin, such as vitamin C are included with the beneficial agent of the combination of polymers.It is adapted to the ascorbic of form The fat of sodium salt, sylvite and calcium salt of the non-limiting examples including ascorbic acid and ascorbic acid, ascorbic acid and long chain fatty acids Solubleness ester (ascorbyl palmitate or ascorbyl stearate), MAP (MAP), ascorbic acid phosphoric acid Sodium (SAP) and ascorbic acid 2- glucosides (AA2G^TM), STAY-C 50 (AA2P), ascorbic acid disodium sulfate and anti- Bad hematic acid 3- aminopropyls phosphate (vitagen).

In a particularly advantageous embodiment, beneficial agent and the polymer covalent bond.For example, beneficial agent Can be vitamin C or vitamin C derivatives, it is with the polymer covalent bond and based on the weight of total composition, in group Present in compound amount between about 0.04% to about 5.0%, such as based on the weight of total composition between about 0.1% to about Between 4.0%, such as based on the weight of total composition between about 0.2% to about 2.0%.In one embodiment, originally The ascorbic amount that composition disclosed in text includes is based on the weight of total composition between about 0.3% to about 1.2%.

Preferably, following at least one is included with the covalently bound vitamin C of the composition：Ascorbic acid, L- are anti-bad Hematic acid, L-AA 2- sulfuric esters (AA-2S) and L-AA 2- phosphates (AA-2P), ascorbic acid 2-O- glucose Glycosides (AA-2G), 6-O- acyl group -2-O- α-D- glucopyranosyls-L-AA (6- acyl groups-AA-2G), (ascorbic acid 3- Aminopropyl phosphate, ascorbyl palmitate), its derivative and combination.Compositions disclosed herein can include single dimension life Plain C reagents or multivitamin C reagents.

In another embodiment of the present invention, there is provided dermal augmentation agent, wherein hyaluronic acid are crosslinked with BDDE.At this In individual embodiment, conjugation can be between about 3mol% and about 10mol%, between about 15mol% to about 40mol%.

In some embodiments, dermal augmentation agent has lasting bioavailability.Such as, there is provided introducing the mankind When in skin, ascorbic acid or other vitamins at least about 1 month are effectively discharged to the mankind or it is long of about 20 months or longer when Between dermal augmentation agent.

The aspect of the present invention provide in part display complex modulus, modulus of elasticity, viscous modulus and tan disclosed herein δ hydrogel composition.When applying power (stress, deformation), compositions disclosed herein tool viscoplasticity, because composition has (liquid for example uncrosslinked glycosaminoglycan gathers for elastic component (solid-like is for example crosslinked Glycosaminoglycan Polymer) and sticky ingredient Compound or carrier phase).The rheological attributes for describing the property are complex modulus (G*), and its combinations of definitions thing is to the total anti-of deformation Property.Complex modulus is the plural number for having real and imaginary parts：G*=G'+iG''.G* absolute value is Abs (G*)=Sqrt (G'²+ G"²).Complex modulus can be defined as to the summation of modulus of elasticity (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, ibid, 2008；Kablik, ibid, 2009；Beasley, ibid, 2009；It is each integrally incorporated accordingly by reference.

Modulus of elasticity or modulus of elasticity refer to the ability of hydrogel material resistance to deformation, or on the contrary, when to its applying power Tendency of the object to impermanency deformation.Modulus of elasticity characterizes the fastness of composition, and because it is described from group The energy storage of compound motion, so also referred to as storage modulus.Modulus of elasticity describes the phase interaction between elasticity and intensity With (G '=stress/strain), and thus provide the quantitative measurment to composition hardness or pliability.By the modulus of elasticity of object It is defined as the slope of its load-deformation curve in elastic deformation area：λ=stress/strain, wherein λ are Pascal's theorems Modulus of elasticity in (Pascal ' s)；Stress is transmutative force divided by the area of applying power；And it is that stress causes to strain Change and the ratio between object original state.Although depending on the speed of applying power, composition is harder, and the modulus of elasticity having is got over It is high and material is deformed in distance to a declared goal Hua Geng great power, such as inject.Illustrate how to measure stress, including direction, permit Perhaps the modulus of elasticity of many types is defined.3 primary resilient modulus are stretch modulus, modulus of shearing and bulk modulus.

Viscous modulus is also referred to as loss modulus, because it describes the energy being lost with viscous dissipation.Tan δ are viscosity The ratio between modulus and modulus of elasticity, tan δ=G "/G '.Falcone, ibid, 2009.For tan δ disclosed in this manual Value, tan δ are obtained by the dynamic modulus under 1Hz frequencies.Lower tan δ correspond to harder, firmer or more flexible Composition.

In another embodiment, hydrogel composition disclosed herein shows modulus of elasticity.In the embodiment Aspect, hydrogel composition show (such as) 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, about 1,000Pa, about 1, 200Pa, about 1,300Pa, about 1,400Pa, about 1,500Pa, about 1,600Pa, about 1700Pa, about 1800Pa, about 1900Pa, about 2, 000Pa, about 2,100Pa, about 2,200Pa, about 2,300Pa, about 2,400Pa or about 2,500Pa modulus of elasticity.In the embodiment party The other side of case, hydrogel composition show (such as) at least 25Pa, at least 50Pa, at least 75Pa, at least 100Pa, extremely Few 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 2,500Pa modulus of elasticity. At the other aspect of the embodiment, hydrogel composition show (such as) at most 25Pa, at most 50Pa, at most 75Pa, at most 100Pa, at most 125Pa, at most 150Pa, at most 175Pa, at most 200Pa, at most 250Pa, at most 300Pa, extremely More 350Pa, at most 400Pa, at most 450Pa, at most 500Pa, at most 550Pa, at most 600Pa, at most 650Pa, at most 700Pa, at most 750Pa, at most 800Pa, at most 850Pa, at most 900Pa, at most 950Pa, at most 1,000Pa, at most 1, 200Pa, at most 1,300Pa, at most 1,400Pa, at most 1,500Pa or at most 1,600Pa modulus of elasticity.The embodiment Other aspect, hydrogel composition show (such as) about 25Pa to about 150Pa, about 25Pa to about 300Pa, about 25Pa extremely About 500Pa, about 25Pa to about 800Pa, about 125Pa to about 300Pa, about 125Pa to about 500Pa, about 125Pa to about 800Pa, about 500Pa to about 1,600Pa, about 600Pa to about 1,600Pa, about 700Pa to about 1,600Pa, about 800Pa to about 1,600Pa, about 900Pa to about 1,600Pa, about 1,000Pa to about 1,600Pa, about 1,100Pa to about 1,600Pa, about 1,200Pa to about 1, 600Pa, about 500Pa are to about 2,500Pa, about 1,000Pa to about 2,500Pa, about 1,500Pa to about 2,500Pa, about 2,000Pa To about 2,500Pa, about 1,300Pa to about 1,600Pa, about 1,400Pa to about 1,700Pa, about 1,500Pa to about 1,800Pa, about 1,600Pa to about 1,900Pa, about 1,700Pa to about 2,000Pa, about 1,800Pa to about 2,100Pa, about 1,900Pa to about 2, The springform of 200Pa, about 2,000Pa to about 2,300Pa, about 2,100Pa to about 2,400Pa or about 2,200Pa to about 2,500Pa Amount.

In another embodiment, hydrogel composition disclosed herein shows viscous modulus.In the embodiment Aspect, hydrogel composition show (such as) 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 viscous modulus.In other sides of the embodiment Face, hydrogel composition show (such as) at most 10Pa, at most 20Pa, at most 30Pa, at most 40Pa, at most 50Pa, at most 60Pa, at most 70Pa, at most 80Pa, at most 90Pa, at most 100Pa, at most 150Pa, at most 200Pa, at most 250Pa, at most 300Pa, at most 350Pa, at most 400Pa, at most 450Pa, at most 500Pa, at most 550Pa, at most 600Pa, at most 650Pa or At most 700Pa viscous modulus.At the other aspect of the embodiment, hydrogel composition show (such as) about 10Pa To about 30Pa, about 10Pa to about 50Pa, about 10Pa to about 100Pa, about 10Pa to about 150Pa, about 70Pa to about 100Pa, about 50Pa to about 350Pa, about 150Pa are to about 450Pa, about 250Pa to about 550Pa, about 350Pa to about 700Pa, about 50Pa to about 150Pa, about 100Pa to about 200Pa, about 150Pa to about 250Pa, about 200Pa to about 300Pa, about 250Pa to about 350Pa, about 300Pa to about 400Pa, about 350Pa are to about 450Pa, about 400Pa to about 500Pa, about 450Pa to about 550Pa, about 500Pa to about The viscous modulus of 600Pa, about 550Pa to about 650Pa or about 600Pa to about 700Pa.

In another embodiment, hydrogel composition disclosed herein shows tan δ.In terms of the embodiment, Hydrogel composition show (such as) about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9th, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, About 2.2, about 2.3, about 2.4 or about 2.5 tan δ.In the other side of the embodiment, hydrogel composition shows (example As) at most 0.1, at most 0.2, at most 0.3, at most 0.4, at most 0.5, at most 0.6, at most 0.7, at most 0.8, at most 0.9, extremely More 1.0, at most 1.1, at most 1.2, at most 1.3, at most 1.4, at most 1.5, at most 1.6, at most 1.7, at most 1.8, at most 1.9th, at most 2.0, at most 2.1, at most 2.2, at most 2.3, at most 2.4 or at most 2.5 tan δ.In the embodiment in addition Other side, hydrogel composition show (such as) about 0.1 to about 0.3, about 0.3 to about 0.5, about 0.5 to about 0.8, about 1.1 To about 1.4, about 1.4 to about 1.7, about 0.3 to about 0.6, about 0.1 to about 0.5, about 0.5 to about 0.9, about 0.1 to about 0.6, about 0.1 to about 1.0, about 0.5 to about 1.5, about 1.0 to the about 2.0 or tan δ of about 1.5 to about 2.5.

The hydrogel group with the transparency and/or translucence disclosed herein is provide in part in terms of this specification Compound.Optical transparence is to allow physical property of the visible ray by material, and translucence is (also referred to as translucent or translucent Degree) only allow light diffusion to pass through.Relative property is opacity.Transparent material is limpid, and trnaslucent materials can not be clearly Understand thoroughly.Hydrogel disclosed herein is preferably optical clear or at least translucent.

In one embodiment, hydrogel composition optical clear disclosed herein.In terms of the embodiment, water Gel Compositions diffusion conduction (such as) about 75% light, about 80% light, about 85% light, about 90% light, about 95% Light or about 100% light.In the other side of the embodiment, the conduction of hydrogel combination diffusion (such as) at least 75% Light, at least 80% light, at least 85% light, at least 90% light or at least 95% light.The embodiment it is other its Its aspect, the conduction of hydrogel combination diffusion (such as) light of about 75% to about 100%, the light of about 80% to about 100%, about The light of the light of 85% to about 100%, the light of about 90% to about 100% or about 95% to about 100%.In one embodiment, Hydrogel composition optical clear disclosed herein and the visible ray of conduction 100%.

Hydrogel composition disclosed herein and optionally and carrier can be processed further by the way that hydrogel is worn into particle Mutually such as water or saline solution mixing are to form injectable or topical substance, such as solution, oil, washing lotion, gel, ointment, emulsifiable paste, slurry Material, ointment or paste.Thus, disclosed hydrogel composition can be single-phase or heterogeneous compositions.Hydrogel can be milled into directly The granularity that about 10 μm to about 1000 μm of footpath, e.g., from about 15 μm to about 30 μm, about 50 μm to about 75 μm, about 100 μm to about 150 μm, About 200 μm to about 300 μm, about 450 μm to about 550 μm, about 600 μm to about 700 μm, about 750 μm to about 850 μm or about 900 μm To about 1,000 μm.

The aspect of the present invention provide in part the composition of injectable disclosed herein.As used herein, term " can Injection ", which refers to material, to be had using property necessary to composition described in the injection device with fine needle to individual's skin Zoned application. As used herein, term " fine needle " refers to 27G or smaller pin.Can be by determining hydrogel particle as discussed above Size realize the syringeability of compositions disclosed herein.

In terms of the embodiment, hydrogel composition disclosed herein can be injected by fine needle.In the embodiment Other side, hydrogel composition disclosed herein can by (such as) about 27G, about 30G or about 32G pin injects.At this The other aspect of embodiment, hydrogel composition disclosed herein can by (such as) 22G or smaller, 27G or smaller, 30G or smaller or 32G or the injection of smaller pin.In the other aspect of the embodiment, hydrogel combination disclosed herein Thing can by (such as) about 22G to about 35G, about 22G be to about 34G, about 22G to about 33G, about 22G to about 32G, about 22G to about 27G or about 27G to about 32G pin injection.

In terms of the embodiment, hydrogel composition disclosed herein can use about 60N, about 55N, about 50N, about 45N, About 40N, about 35N, about 30N, about 25N, about 20N or about 15N extruding force, with 100mm/min speed injection.In the embodiment party The other side of case, hydrogel composition disclosed herein can be by 27G pins, with about 60N or smaller, about 55N or smaller, about 50N or smaller, about 45N or smaller, about 40N or smaller, about 35N or smaller, about 30N or smaller, about 25N or smaller, about 20N or Smaller, about 15N or smaller, about 10N or smaller or about 5N or the injection of smaller extruding force.In the other side of the embodiment Face, hydrogel composition disclosed herein can be by 30G pins, with about 60N or smaller, about 55N or smaller, about 50N or smaller, about 45N or smaller, about 40N or smaller, about 35N or smaller, about 30N or smaller, about 25N or smaller, about 20N or smaller, about 15N or Smaller, about 10N or smaller or about 5N or the injection of smaller extruding force.At the other aspect of the embodiment, it is disclosed herein Hydrogel composition can by 32G pins, with about 60N or smaller, about 55N or smaller, about 50N or smaller, about 45N or smaller, About 40N or smaller, about 35N or smaller, about 30N or smaller, about 25N or smaller, about 20N or smaller, about 15N or smaller, about 10N Or smaller or about 5N or the injection of smaller extruding force.

The aspect of the present invention provide in part the hydrogel composition disclosed herein for showing cohesiveness.Cohesiveness, Also referred to as cohesion adhesion gravitation, bonding force or compression stress, played the work of molecule synthesis one in material between similar molecules The physical property of material caused by intermolecular attraction.Cohesiveness is represented with fors (gmf).Among other things, caking property By initial free Glycosaminoglycan Polymer molecular weight than the free sugar that is remained after, the degree of cross linking of Glycosaminoglycan Polymer, crosslinking The amount of amine chitosan polymer and the pH of hydrogel composition influence.Composition should have enough caking property, to remain in administration Part.In addition, in some applications, enough caking property keeps its shape to composition, and mechanical load therefore is occurring It is critically important for holding feature during circulation.Thus, in one embodiment, hydrogel composition disclosed herein is shown Cohesiveness, with water peer-level.In yet another embodiment, gel combination disclosed herein shows enough cohesiveness to protect Hold to be located at and apply part.In yet another embodiment, gel combination disclosed herein shows enough cohesiveness to keep it Shape.In yet another embodiment, gel combination disclosed herein shows enough cohesiveness to keep its shape and function Property.

The aspect of the present invention, which provide in part, disclosed herein shows the infiltration of physiologically acceptable M The hydrogel composition of concentration (osmolarity).As used herein, term " M osmotic concentration " refers in solution The concentration of osmotically active solute.As used herein, term " physiologically acceptable M osmotic concentration " refers to living The normal function of organism is consistent or its distinctive M osmotic concentration.Thus, when being administered to mammal, using this Hydrogel composition disclosed in text shows the M osmotic concentration generally without long-term or permanent adverse effect.Hold Product gram molecule osmotic concentration is represented with the osmol(e) (Osmol/L or Osm/L) of osmotically active solute in every liter of solvent.Volume Gram molecule osmotic concentration is different from molar concentration because it measure osmotically active solute particle molal quantity rather than The molal quantity of solute.Because some compounds can dissociate in the solution, and others can not, so there is difference.Can be under Row expression formula calculates the M osmotic concentration of solution：, whereinFor infiltration coefficient, it is said Understand the Non Ideal Degree of solution；η be molecular dissociation into particle (such as ion) quantity；And C is the mole dense of solute Degree；And i is the index for representing specific solute homogeneity.The conventional method of measurement solution can be used to measure hydrogel disclosed herein The M osmotic concentration of composition.

In one embodiment, hydrogel composition disclosed herein shows physiologically acceptable M Osmotic concentration.As used herein, term " osmolality " refers to osmotically active solute in internal every kilogram of solvent Concentration.As used herein, term " physiologically acceptable osmolality " refers to and the normal machine of live organism It can be consistent or its distinctive osmolality.Thus, when being administered to mammal, using water-setting disclosed herein Glue composition shows the osmolality generally without long-term or permanent adverse effect.Weight molar concentration is permeated Concentration is represented with the osmol(e) (osmol/kg or Osm/kg) of osmotically active solute in every kilogram of solvent and molten equal to this The summation of the weight-molality of all solutes present in liquid.The weight molar concentration infiltration of osmometer measurement solution can be used Concentration.The most frequently used instrument is that freezing point reduces osmometer in modern laboratory.The apparatus measures are oozed with weight molar concentration The change (freezing point reduction osmometer) or increase solution with osmolality that freezing point occurs in saturating concentration increase solution The change (the low osmometer of steam drop) that middle vapour pressure occurs.

In terms of the embodiment, hydrogel composition show (such as) 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 M osmotic concentration.In the other side of the embodiment, hydrogel composition show (such as) 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 at least 500mOsm/L M osmotic concentration.At this The other aspect of embodiment, hydrogel composition show (such as) at most 100mOsm/L, at most 150mOsm/L, extremely More 200mOsm/L, at most 250mOsm/L, at most 300mOsm/L, at most 350mOsm/L, at most 400mOsm/L, at most 450mOsm/L or at most 500mOsm/L M osmotic concentration.In the other aspect of the embodiment, water-setting Glue composition show (such as) 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 are to about 325mOsm/L, about 275mOsm/L to about 300mOsm/L or about 285mOsm/L to about 290mOsm/L M osmotic concentration.

The aspect of the present invention provide in part the hydrogel composition disclosed herein for showing substantial equalization.Such as this Text is used, and term " stability " or " stabilization " refer to composition and are being administered to when referring to hydrogel composition disclosed herein Before individual, be not easy to degrade, decompose or be crushed in storage it is any generally or significant degree.As used herein, Term " substantially heat endurance ", " generally thermostabilization ", " autoclave is stable " or " steam sterilizing is stable " refer to water disclosed herein Gel combination is generally stable when by heat treatment as disclosed herein.

Can be by making hydrogel composition heat-treated, such as under normal pressure or pressurization (for example, autoclaving) Steam sterilizing, determine the stability of hydrogel composition disclosed herein.Hot place is carried out at a temperature of preferably at least about 100 DEG C Reason about 1 minute to about 10 minutes.The substantial equalization of hydrogel composition disclosed herein can be assessed in the following manner：1) survey The extruding force change (Δ F) of hydrogel composition disclosed herein after fixed sterilizing, wherein by (having the hydrogel of specified additive The extruding force of composition) (extruding force of not additivated hydrogel composition) measurement is subtracted, the change of extruding force is less than 2N Show that hydrogel composition is generally stable；And/or 2) determine the rheological characteristic change of hydrogel composition disclosed herein after sterilizing Change, surveyed wherein subtracting (without the tan δ 1Hz of the gel preparation of additive) by (the tan δ 1Hz for having the gel preparation of additive) Amount, tan δ 1Hz change show that hydrogel composition is generally stable less than 0.1.Thus, it is disclosed herein generally stable Hydrogel composition maintains following one or more characteristics after sterilization：Uniformity, extruding force, caking property, hyaluronan are dense Other rheological behaviors before degree, reagent concentration, M osmotic concentration, pH or heat treatment needed for hydrogel.

In one embodiment, osamine is included using the heat treatment process of maintenance required hydrogel properties disclosed herein The hydrogel composition of chitosan polymer and at least one reagent disclosed herein.In terms of the embodiment, (example is used Such as) about 100 DEG C, about 105 DEG C, about 110 DEG C, about 115 DEG C, about 120 DEG C, about 125 DEG C or about 130 DEG C of heat treatment process includes sugar The hydrogel composition of amine chitosan polymer and at least one reagent disclosed herein.In the other side of the embodiment, make With (such as) 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 heat treatment process include the hydrogel composition of Glycosaminoglycan Polymer and at least one reagent disclosed herein.In the reality Apply the other aspect of scheme, use (such as) about 100 DEG C to about 120 DEG C, about 100 DEG C to about 125 DEG C, about 100 DEG C to about 130 DEG C, about 100 DEG C to about 135 DEG C, about 110 DEG C to about 120 DEG C, about 110 DEG C to about 125 DEG C, about 110 DEG C to about 130 DEG C, about 110 DEG C to about 135 DEG C, about 120 DEG C to about 125 DEG C, about 120 DEG C to about 130 DEG C, about 120 DEG C to about 135 DEG C, about 125 DEG C to about 130 DEG C or about 125 DEG C to about 135 DEG C of heat treatment process includes Glycosaminoglycan Polymer and at least one reagent disclosed herein Hydrogel composition.

Can be by making hydrogel composition heat-treated, such as be stored in about 45 DEG C of environment about 60 days, measure is public herein The long-time stability for the hydrogel composition opened.The long-term steady of hydrogel composition disclosed herein can be assessed in the following manner It is qualitative：1) clarity and color of hydrogel composition after 45 DEG C of heat treatments are estimated, hydrogel composition is limpid and uncolored table Subject hydrogel compositions are generally stable；2) extruding force of measure hydrogel composition disclosed herein after 45 DEG C of heat treatment becomes Change (Δ F), wherein subtracting (45 DEG C by (before 45 DEG C of heat treatments, there is the extruding force of the hydrogel composition of specified additive) After heat treatment, there is the extruding force of the hydrogel composition of specified additive) measure, the change of extruding force shows water-setting less than 2N Glue composition is generally stable；And/or 3) the rheological characteristic change of hydrogel composition disclosed herein after sterilizing is determined, wherein logical Crossing and (before 45 DEG C of heat treatments, there is the tan δ 1Hz of the gel preparation of specified additive) to subtract (after 45 DEG C of heat treatments, has specified The tan δ 1Hz of the gel preparation of additive) measure, tan δ 1Hz change shows that hydrogel composition is generally steady less than 0.1 It is fixed.Thus, maintain following one or more feature evaluations hydrogel composition disclosed herein after 45 DEG C of heat treatment Long-time stability：Clarity (transparent and translucent), uniformity and caking property.

In terms of the embodiment, hydrogel composition at room temperature generally it is stable (such as) about 3 months, about 6 Month, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, about 24 months, about 27 months, about 30 months, about 33 months or about 36 months.In the other side of the embodiment, hydrogel composition at room temperature generally it is stable (such as) At least three moon, at least six moon, at least nine moon, at least at least 12 months, at least 15 months, at least 18 months, 21 months, extremely It is few 24 months, at least 27 months, at least 30 months, at least 33 months or at least 36 months.In the other side of the embodiment, Hydrogel composition at room temperature generally it is stable (such as) about 3 months to about 12 months, about 3 months to about 18 months, about 3 Month to about 24 months, about 3 months to about 30 months, about 3 months to about 36 months, about 6 months to about 12 months, about 6 months extremely About 18 months, about 6 months to about 24 months, about 6 months to about 30 months, about 6 months to about 36 months, about 9 months to about 12 Individual month, about 9 months to about 18 months, about 9 months to about 24 months, about 9 months to about 30 months, about 9 months to about 36 months, About 12 months to about 18 months, about 12 months to about 24 months, about 12 months to about 30 months, about 12 months to about 36 months, About 18 months to about 24 months, about 18 months to about 30 months or about 18 months to about 36 months.

This composition can optionally include but is not limited to other pharmaceutically acceptable components, including but not limited to buffer, Preservative, tension regulator, salt, antioxidant, osmolality modifier, emulsifying agent, wetting agent etc..

Pharmaceutically acceptable buffer is the buffer that can be used for preparing hydrogel composition disclosed herein, and condition is Gained preparation is pharmaceutically subjected to.The non-limiting examples of pharmaceutically acceptable buffer include acetate buffer, boron Hydrochlorate buffer, citrate buffer agent, neutral buffered saline, PB and phosphate buffered saline (PBS).Can will be any The pharmaceutically acceptable buffer of concentration is used to prepare pharmaceutical composition disclosed herein, and condition is using the slow of valid density Electuary resumes treatment the active component of effective dose.The non-limiting examples of physiologically acceptable buffer concentration are present in about 0.1mM is to about in the range of 900mM.The pH of pharmaceutically acceptable buffer is can adjust, condition is that gained preparation is pharmaceutically It is acceptable.It should be understood that can be as needed using acid or the pH of alkali regulating drug composition.The pH levels of any buffering can be used for Compounding pharmaceutical composition, condition are that the matrix polymer of effective dose is resumed treatment using effective pH levels.Physiologically it is subjected to PH non-limiting examples be present in the range of about pH5.0 to about pH8.5.For example, hydrogel composition disclosed herein PH can be about 5.0 to about 8.0 or about 6.5 to about 7.5, about 7.0 to about 7.4 or about 7.1 to about 7.3.

Pharmaceutically acceptable preservative includes but is not limited to sodium pyrosulfite, sodium thiosulfate, acetylcysteine, fourth Hydroxyl anisole and Butylated Hydroxytoluene.Pharmaceutically acceptable preservative includes but is not limited to benzalkonium chloride, methaform, thimerosal, vinegar Sour benzene mercury, phenylmercuric nitrate, oxygen chlorine composition is stabilized, such as(Allergan, Inc.Irvine, CA) and chelating Agent, such as DTPA or DTPA- bisamides, DTPA calcium and CaNaDTPA- bisamides.

Include but is not limited to salt for the pharmaceutically acceptable tension regulator in hydrogel composition disclosed herein Such as sodium chloride and potassium chloride；And glycerine.The composition can be provided as salt and can be formed together with many acid, including But it is not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, butanedioic acid etc..Salt tends to than corresponding free alkali shape Formula is more soluble in water or other proton solvents.It should be understood that it may include in pharmaceutical composition disclosed herein in pharmaceutical field These and other material known.Other non-limiting examples of pharmaceutically acceptable component can be same as above in such as Ansel, (1999)；Gennaro, ibid, (2000)；Hardman, ibid, (2001)；And Rowe, ibid, found in (2003), thus Each of which is integrally incorporated by reference.

The aspect of the present invention provide in part soft group individual by applying hydrogel composition disclosed herein treatment The method for knitting situation.As used herein, term " treatment " refers to be mitigated or eliminated in individual and is characterised by soft tissue defect, lacks The beauty of the soft tissue condition of damage, disease and/or illness or clinical symptoms；Or it is characterised by soft tissue in delay or prevention individual Defect, defect, the beauty of the situation of disease and/or illness or onset of clinical symptoms.For example, term " treatment " can refer to mitigation feature Be the symptom of the situation of soft tissue defects, disease and/or illness, for example, at least 20%, at least 30%, at least 40%, at least 50%th, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%.Can by observe one or more beauty, Clinical symptoms and/or the physiological index determining related to situation hydrogel composition disclosed herein are characterised by treatment Effect in the situation of soft tissue defects, disease and/or illness.Also it can show that soft tissue lacks by being reduced to the needs of synchronous therapy Damage, disease and/or illness improve.Those skilled in the art will be appreciated that related to specific soft tissue defects, disease and/or illness Appropriate symptom or index and will appreciate how determine individual whether be with compound disclosed herein or composition treatment Candidate.

Applied to individual according to hydrogel composition of the present invention.Individual is typically any age, sex or race The mankind.Generally, it is any individual of candidate and the time of method disclosed herein of conventional program treatment soft tissue condition Choose.Although the subject for experiencing skin aging sign is adult, the premature aging or other for being adapted to treatment is experienced The subject of skin (for example, scar) can also be treated with hydrogel composition disclosed herein.In addition, presently disclosed water Small/moderate that gel combination and method can be applied to seek body part or region expands, the change of change in shape or profile Individual, this technically can not possibly or can not aesthetically be received with existing soft tissue implanted prosthetics.Public affairs are assented except informing comprehensively Open outside all relevant risks and interests of described program, preoperative evaluation generally includes conventional history and physical examination.

Hydrogel composition disclosed herein and method are useful to treatment soft tissue condition.Soft tissue condition includes but unlimited In soft tissue defect, defect, disease and/or illness.The non-limiting examples of soft tissue condition include breast defect, defect, disease Disease and/or illness, such as breast increase, breast reconstruction, breast is fixed, breast is too small, breast development is not complete, Polish Cotard (Poland ' s syndrome), the defect caused by complication of implant such as pouch are shunk and/or rupture；Facial defects, lack Damage, disease or illness, such as face increase, facial reconstruction, U.S. rope therapy, parry-Romberg syndrome (Parry-Romberg Syndrome), lupus erythematosus profundus, corium pit, scar, have sunken cheeks, after thin lip, nose defect or defect, socket of the eye defect or Defect, facial fold, microgroove and/or wrinkle, such as glabella line, muffle line, mouth week line and/or corners of the mouth line, and/or face is other Profile deformity or defect；Neck defect, defect, disease or/or illness；Skin defect, defect, disease or/or illness；It is other soft Tissue defects, defect, disease or/or illness, for example, it is upper arm, underarm, hand, shoulder, back, trunk (including belly), buttocks, big The increase of leg, shank (including calf), pin (including vola fat pad), eyes, genitals or other body parts, region or area Or reconstruction, or influence the disease or illness of these body parts, region or area；Aconuresis, incontinence of faces, other forms Incontinence；With gastroesophageal reflux disease (GERD).As used herein, term " U.S. rope therapy " refers to the No operation beauty therapeutic of skin Technology, involve to epidermis, dermal-epidermal junction and/or corium through in epidermis, intradermal and/or be subcutaneously injected in multiple small The reagent that drop is applied.

Generally by based on required change and/or improvement, the mitigation of required soft tissue condition symptom and/or elimination, individual and/ Or the body part or region of clinical and/or cosmetic result and treatment needed for doctor are determined together with any method disclosed herein The amount of the hydrogel composition used.The effect that composition is applied can use following one or more clinical and/or beauty measurement marks Standard represents：Soft tissue alteration of form and/or improvement, soft tissue size change and/or improved, soft tissue profile changes and/or changed Kind, function of organization changes and/or improved, tissue ingrowth is supported and/or new collagen deposition, composition continuous infusion, Patient satisfaction and/or quality of life are improved and the use of implantable foreign matter is reduced.

The effect of the composition and method treatment facial soft tissue can use following one or more clinical and/or beauty degree Measure canonical representation：Size, shape and/or the profile of facial characteristics improve, size, shape such as lip, cheek or ocular And/or profile improves；Size, shape and/or the profile of facial characteristics change, size, shape such as lip, cheek or ocular Shape and/or profile change；Wrinkle, fold or microgroove on skin reduce or eliminate；Have to the wrinkle on skin, fold or microgroove Resistance；Skin moisturizing；Skin elasticity increase；Pachylosis is mitigated or eliminated；Skin tautness increases and/or improved；Drag line Or striae of pregnancy reduces or eliminates；The colour of skin, gloss, brightness and/or honorable enhancing and/or improvement；Skin color strengthens and/or changed Kind, ochrodermia is mitigated or eliminated；Composition continuous infusion；Side effect is reduced；Patient satisfaction and/or quality of life improve.

As another example, for aconuresis operation, the composition and the effect of method supported for sphincter Fruit can use following one or more clinical measures canonical representations：The reduction of incontinence frequency, continuous infusion, patient satisfaction and/or life The use of Quality advance living and implantable external filler is reduced.

In terms of the embodiment, the amount of the hydrogel composition of administration for (such as) 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 the embodiment, the amount of the hydrogel composition of administration for (such as) about 0.01g to about 0.1g, about 0.1g are to about 1g, about 1g to about 10g, about 10g to about 100g or about 50g to about 200g.In the implementation The other aspect of scheme, the amount of the hydrogel composition of administration for (such as) 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 the embodiment, the amount of the hydrogel composition of administration is (such as) about 0.01mL to about 0.1mL, about 0.1mL be to about 1mL, about 1mL to about 10mL, about 10mL to about 100mL or about 50mL To about 200mL.

Generally by based on the beauty needed for individual and/or doctor and/or the body part or region of clinical effectiveness and treatment Determine the duration for the treatment of.In terms of the embodiment, soft tissue can be treated using hydrogel composition disclosed herein Situation, e.g., from about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, About 14 months, about 15 months, about 18 months or about 24 months.In the other side of the embodiment, using water disclosed herein Gel combination can treat soft tissue condition, for example, at least 6 months, at least seven moon, at least eight moon, at least nine moon, at least 10 Individual month, 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 the embodiment, soft tissue condition can be treated using hydrogel composition disclosed herein, such as About 6 months to about 12 months, about 6 months to about 15 months, about 6 months to about 18 months, about 6 months to about 21 months, about 6 Month to about 24 months, about 9 months to about 12 months, about 9 months to about 15 months, about 9 months to about 18 months, about 9 months extremely About 21 months, about 6 months to about 24 months, about 12 months to about 15 months, about 12 months to about 18 months, about 12 months to about 21 months, about 12 months to about 24 months, about 15 months to about 18 months, about 15 months to about 21 months, about 15 months to about 24 months, about 18 months to about 21 months, about 18 months to about 24 months or about 21 months to about 24 months.

The aspect of the present invention provide in part using hydrogel composition disclosed herein.As used herein, term " administration " point to individual provide compositions disclosed herein any delivery mechanism, its potentially resulted in clinically, treating Upper or experimentally beneficial result.Actual delivery mechanism for applying from composition to individual can pass through ordinary skill people Member considers to include but is not limited to following factor to determine：The type of skin, the position of skin, the original of skin Because the seriousness of, skin, needed for alleviate degree, needed for the duration alleviated, the particular composition used, the spy that uses Other compounds that the excretion rate of determining composition, the drug effect of the particular composition used, the particular composition that uses include Property, particular route of administration, particular characteristics, individual medical history and risk factors (such as age, body weight, health status etc.) or its What is combined.In the one side of the embodiment, compositions disclosed herein is applied by the skin area injected to individual.

It is generally that body part or region based on beauty and/or clinical effectiveness and treatment needed for individual and/or doctor is true Orient the approach that individual patient applies hydrogel composition.Compositions disclosed herein can be by those of ordinary skill in the art Any mode known is applied, and includes but is not limited to band needle injection, pistol (for example, hydropneumatic compression pistol), conduit, part Or it is implanted into by Direct Surgery.Hydrogel composition disclosed herein can be applied to skin area, such as corium area or rim surface zone. For example, inject water disclosed herein from about 4mm to about 14mm pin using diameter about 0.26mm to about 0.4mm and length range Gel combination.Alternatively, pin can be 21-32G and length is about 4mm to about 70mm.Preferably, pin is disposable Pin.The pin can combine with syringe, conduit and/or pistol.

In addition, compositions disclosed herein can be applied once or several times.Finally, the timing used will be according to nursing quality Standard.For example, hydrogel composition disclosed herein can once or a point several periods apply, period interval a few days or a few weeks.Example Such as, individual can apply hydrogel composition disclosed herein in every 1,2,3,4,5,6 or 7 day or every 1,2,3 or 4 week.Applied to individual With hydrogel composition disclosed herein can one month or two months once every 3,6,9 or 12 months apply once.

The aspect of the present invention provide in part corium area.As used herein, term " corium area " refers to comprising epidermis-true The region of the skin of skin intersection and corium including superficial corium (mamillary region) and deep dermis (webbed region).Skin is by three Main layer composition：Epidermis, it provides water proofing property and as the barrier of infection；Corium, it is used as the position of cutaneous appendage； With hypodermis (subcutaneous layer of fat).Epidermis is free of blood vessel, and by being nourished from the diffusion of corium.Form the main of epidermis Cell type is keratinocyte, melanocyte, Langerhans cell (Langerhans cell) and Merkel cell (Merkels cell)。

Corium is the skin layer being made up of under epidermis connective tissue and is giving of body stress and strain.Corium leads to epidermis Basement membrane is crossed to be tightly coupled.It is also comprising many mechanoreceptor/nerve endings for providing sense of touch and thermal sensation.It contains hairiness Capsule, sweat gland, sebaceous glands, apocrine gland, lymphatic vessel and blood vessel.Blood vessel in the dermis provides nutrition and from the cell of its own And remove waste from the basalis of epidermis.Corium is divided into Liang Ge areas in structure：Neighbouring epidermis, the superficial area for being referred to as mamillary region The thicker area in deep of referred to as webbed region.

Mamillary region is made up of the areolar connective tissue that relaxes.It is referred to as nipple because of its finger-like projection thing, and it prolongs to epidermis Stretch.Nipple provides " rugged " surface to be intermeshed with epidermis for corium, strengthens the connection between two layers of skin. Webbed region is located at mamillary region depths, and generally thick a lot.It is made up of the irregular connective tissue of densification, and by weaving its Collagen, elasticity and the reticular fibre of dense concentrations are gained the name.These azelons give corium intensity, ductility and elastic property. It is root of hair, sebaceous glands, sweat gland, receptor, nail and blood vessel also to be located within webbed region.Tattoo ink is retained in corium In.Striae of pregnancy also is located in corium as caused by pregnancy.

Hypodermis is located at below corium.It is that connection dermis of skin area carries with following bone and muscle and for it that it, which is acted on, Feeder vessels and nerve.It is made up of loose connective tissue and elastin laminin.Main cell type is fibroblast, macrophage is thin Born of the same parents and lipocyte (hypodermis contains 50% body fat).Fat is used as the bedding and padding (padding) and insulation material of body.

In the one side of the embodiment, injected by the corium area in subcutaneously region, applied to the skin area of individual With hydrogel composition disclosed herein.In terms of the embodiment, by (such as) epidermal-dermal junctional area, nipple Area, webbed region or the injection of its any combinations, hydrogel composition disclosed herein is applied to the corium area of individual.

Advantageously, some compositions of the present invention to reduce (such as) in patient's thin skin area the appearance of microgroove it is particularly useful and Effectively.Such as, there is provided the method for microgroove treatment, it is included in the depth no more than about 1mm, is applied elsewhere herein to patient The step of dermal augmentation agent composition of description.

The method that the other side of this specification partly discloses treatment skin, including to skin Individual applies the step of hydrogel composition disclosed herein, and wherein applying said compositions improve skin, so as to control Skin is treated.In the one side of the embodiment, the method for (skin is) treatment skin dehydration include to The individual of skin dehydration is applied the step of hydrogel composition disclosed herein, and wherein applying said compositions are skin moisturizing, So as to treat skin dehydration.In the another aspect of the embodiment, the inelastic method for the treatment of skin is included to skin The step of inelastic individual of skin applies hydrogel composition disclosed herein, wherein applying said compositions add skin Elasticity, lacked flexibility so as to treat skin.At the another aspect of the embodiment, treating the method for pachylosis is included to trouble The step of individual for having pachylosis applies hydrogel composition disclosed herein, wherein applying said compositions reduce skin It is coarse, so as to treat pachylosis.At the another aspect of the embodiment, treatment skin lack tautness method include to The step of individual with skin shortage tautness applies hydrogel composition disclosed herein, wherein applying said compositions make Skin is tighter, lacks tautness so as to treat skin.

At the another aspect of the embodiment, treating the method for skin stretch line or striae of pregnancy is included to skin stretch The individual of line or striae of pregnancy applies the step of hydrogel composition disclosed herein, and wherein applying said compositions reduce or eliminate Skin stretch line or striae of pregnancy, so as to having treated skin stretch line or striae of pregnancy.In the another aspect of the embodiment, treatment The step of method of ochrodermia to the individual with ochrodermia including applying hydrogel composition disclosed herein, wherein applying The colour of skin or brilliance are enhanced with the composition, so as to treat ochrodermia.In the another aspect of the embodiment, skin is treated The step of method of skin wrinkle to the individual with wrinkle of skin including applying hydrogel composition disclosed herein, wherein applying The composition reduce or eliminates wrinkle of skin, so as to treat wrinkle of skin.In the another aspect of the embodiment, treatment The step of method of wrinkle of skin is included to individual administration hydrogel composition disclosed herein, wherein applying said compositions make The anti-wrinkle of skin of skin, so as to treat wrinkle of skin.

In some embodiments, dermal augmentation agent has lasting bioavailability.Such as, there is provided introducing the mankind When in skin, (for example, through intracutaneous or subcutaneous introducing mankind to correct the soft tissue hole defect on face), discharged to the mankind anti- Bad hematic acid (or other vitamins) at least about 1 month or the long dermal augmentation agent of about 20 months or longer time.

For example, to predict that the vitamin C consistent with the filler duration continues effect, conjugation is assessed.This Kind is assessed based on the etherified preparations combined with HA of AA2G.The preparation is stablized in physiological conditions, but starts by being attached to Alpha-glucosidase on cell membrane starts to discharge ascorbic acid (AsA).Because alpha-glucosidase is attached on cell membrane, institute Occurred with AsA release in filler/Cellular interfaces.Further, AsA will be degraded from HA-AA2G release with HA so that AA2G is obtained to can use fibroblast.Therefore AA2G conjugation and duration of the AsA release depending on HA.Conjugation is about 5mol% gel at least can be up to 1 month, such as active vitamin C is discharged within about 3~5 month periods；10mol% The gel of conjugation can discharge active vitamin C being at least up in 6~8 months periods；The gel of 15mol% conjugations can be At least up to release active vitamin C in 10~individual month period；30mol%, up to 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)

* assumes：

AsA is discharged with constant rate of speed.

AsA valid density is 0.05mM and keeps 2 days 2.13*2/ (0.05*30)=2.8 (individual month) of effective ＞

In one embodiment of the invention, there is provided a kind of dermal augmentation agent, it is included and Star-PEG epoxidations The hyaluronic acid of thing crosslinking and with being combined with hyaluronic acid with the conjugation between about 3mol% and about 40mol% Vitamin C derivatives are for example, AA2G (ascorbic acid 2- glucosides), vitagen (3- aminopropyls-L-AA phosphoric acid One of ester) and SAP (STAY-C 50).

Prepare this dermal augmentation agent method include make pentaerythrite glycidol ether (Star-PEG epoxides) with Ascorbic acid 2- glucosides (AA2G) react by a certain percentage, and reaction temperature and reaction time are adapted to obtain containing by 4- armlets AA2G (AA2G-4 armlets oxide), unreacted 4 armlet oxide and free AA2G that oxide caps composition.4- arms The AA2G (AA2G-4 armlets oxide) that epoxides caps is combined through epoxy radicals with hyaluronic acid.Unreacted 4 armlet oxidation Thing is used as crosslinking agent with cross-linked-hyaluronic acid and as bonding agent with further combined with AA2G.

In another embodiment of the present invention, there is provided a kind of dermal augmentation agent, it includes transparent with BDDE crosslinkings Matter is sour and derives with the vitamin C combined with the conjugation between about 3mol% and about 10mol% with hyaluronic acid Thing is for example, AA2G (ascorbic acid 2- glucosides), vitagen (3- aminopropyls-L-AA phosphate) and SAP are (anti-bad One of hematic acid sodium phosphate).

Preparing the method for this dermal augmentation agent includes making BDDE with ascorbic acid 2- glucosides (AA2G) by certain ratio Example reaction, reaction temperature and reaction time are adapted to obtain AA2G (AA2G-BDDE), unreacted BDDE containing being capped by BDDE With free AA2G composition.The AA2G (AA2G-BDDE) that BDDE is capped is combined through epoxy radicals with hyaluronic acid.It is unreacted BDDE is used as crosslinking agent with cross-linked-hyaluronic acid and as bonding agent with further combined with AA2G.

Fig. 9 is the table for showing influence of the alpha-glucosaccharase enzyme concentration to discharging AsA from AA2G-PBS solution.AA2G is to AsA Conversion depending on alpha-glucosidase concentration.When alpha-glucosidase concentration is 6.3 units/g gels, AA2G is several in 15min It is fully converted to AsA.When alpha-glucosidase concentration is 4.7 units/g gels, it is necessary to which AA2G is fully converted to by 30min AsA.Further reducing alpha-glucosidase concentration causes conversions of the AA2G to AsA slow.

Figure 10 shows the release profiles schematic diagram from the free AsA according to combination dermal augmentation agent of the present invention (sustained release) (the AA2G conversions mol% compared with the reaction time).AA2G is complete in 40min in AA2G/HA mixtures It is converted into AsA.AA2H/HA conjugates show that AA2G is converted into AsA time dependence.

Figure 11 A and 11B show the additional release data according to various dermal augmentation agent of the present invention.More specifically Ground, in HA-AA2G gels conversions of the AA2G to AsA depend on alpha-glucosidase concentration.High alpha-glucosidase concentration cause AA2G to AsA rapid conversions.For specifying alpha-glucosidase concentration, different preparations show the heterogeneity that AA2G converts to AsA.

In one aspect of the invention, there is provided dermal augmentation agent, its appearance to treating and eliminating microgroove is especially effective, Such as with respect to the gauffer of superficial on skin, such as, but not limited to eyes, Lei Gou areas, the microgroove near forehead, canthus line, glabella line Deng.

It is that some corium are filled out blue variable colour (Tyndall effect) occur in the skin part for having injected dermal augmentation agent Fill the great adverse events of agent patient experience.Tyndall effect is more conventional in the patient for the treatment of superficial fine wrinkles.Grind Embodiment of the present invention is sent out, it, which is provided, can inject in superficial to treat microgroove and wrinkle, or even in relatively thin skin Skin is injected in region, without long-acting, the translucent filler of any blue variable colour as caused by Tyndall effect.Microgroove or superficial Wrinkle be generally understood as it is generally most thin in skin, i.e., the dermis thickness of skin be less than 1mm facial zone (forehead, outer canthus, lip Red edge/mouth contour) in find skin on wrinkle or gauffer.In forehead, average dermis thickness is for normal skin About 0.95mm and be about 0.81mmm for the skin that wrinkles.Corium around outer canthus is even more thin (such as normal skin For about 0.61mm and for the skin that wrinkles about 0.41mm).30 or 32G pins (pin for being generally used for microgroove gel application) Mean outside diameter is about 0.30 and about 0.24mm.

The invention provides the dermal augmentation agent group that will not cause Tyndall effect being for example described elsewhere herein Compound.For example, the composition of the present invention includes and the hyaluronic acid component of cross-linking agents, the additive in addition to linked； When in the corium area for being administered to patient, relative in addition to no additive, substantially the same composition, the combination Thing shows Tyndall effect reduction.The composition can generally optical clear.

In one embodiment, additive is vitamin C derivatives, for example, can with it is described elsewhere herein transparent The chemically combined AA2G of matter acid.

In some embodiments, linked be BDDE and conjugation between about 3mol% and about 10mol%, Or up to 15mol% or higher.In some embodiments, the composition, which further includes, is suitable to carry for patient after injection For the anesthetic comfortably measured, such as lidocaine.

The method for additionally providing the microgroove on treatment patient skin.Methods described generally includes to introduce example into patient skin The step of composition as described herein.Such as the composition includes hyaluronic acid component, the crosslinking group of cross-linked-hyaluronic acid Point and additive in addition to linked mixture, the composition generally optical clear；And wherein relative to except not having Have outside the additive, substantially the same composition, dermal augmentation agent composition shows Tyndall effect reduction.

In some embodiments of the present invention, the composition is included using EDC chemistry and diamines or polyamine crosslinkers The hyaluronic acid component of crosslinking.For example, the crosslinking agent can be HMDA.

In certain embodiments of the present invention that crosslinking agent is HMDA, the G ' of the composition is up to about 70Pa, G "/G ' Between about 0.65 and about 0.75, extruding force about 24N or smaller, and final HA concentration is between about 24mg/g and about 25mg/g Between.

In certain embodiments of the present invention that additive is HA-AA2G conjugates or HA- vitagen conjugates, knot It is right between about 3mol% and about 10mol%, or up to about 15mol%, or up to about 40mol%.These compositions G ' is from least about 30Pa, and more preferably at least about 40Pa to about 100Pa, G "/G ' are between about 0.30 and about 0.50, and extruding force is about 27N or smaller, 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 combination, such as AA2G and hyaluronic acid repeat unit (for example, HA dimers).Therefore, 10mol% conjugations mean every 100 HA repeat units AA2G containing 10 combinations.The method illustrated in example below 2, or other methods well known by persons skilled in the art can be used Calculations incorporated degree.

Embodiment

Embodiment 1：Combined using BDDE as crosslinking agent AA2G with the HA gels being crosslinked

400.6mg low-molecular-weight hyaluronic acid (LMW HA) is set to be hydrated in syringe in 1802mg 1wt%NaOH ~30min.800.7mg AA2G is put into bottle, is subsequently placed into 713.7mg BDDE and 1416.8mg 10%NaOH. Before being added in hydration HA, make above solution (pH>12) reaction~20min in 50 DEG C of water-baths.After addition, by Being transmitted back and forth between 2 syringes makes mixture mix~20 times.The paste of mixing is put into bottle and 50 DEG C of water-baths~ 2.5h.223.5mg 12M HCl are added in 9.05g PBS (pH7.4).After~2.5h, HA-AA2G gels are formed.Will be solidifying Glue is cut into slices, and adds HCl-PBS solution thereto.Neutralize gel and expanded whole night on orbital shaker.Pass through ~60 μm of sieves sieve gel and by transmitting mixing~20 times back and forth between 2 syringes.Gel is put into 15, Dialysed in 000MWCO RC bag filters and in PBS (pH7.4) buffer solution.Dialyse progress~185h, frequently changes PBS bufferings Liquid.After dialysis, gel is put into syringe and is stored in 4 DEG C of refrigerators.

Embodiment 2：The measure that AA2G is combined

The weight of gel as described in Example 1 is correctly recorded before dialysis and after dialysis.Assuming that gel after dialysis For~1g/mL.It is every in 1L PBS>8h, do not occur stopping dialysis during obvious AA2G.Use UV/Vis spectrophotometrics Meter (Nanodrop2000C, ThermoScientific) measures AA2G under 260nm.Use the various concentrations of AA2G in 2%HA (A@260nm=1.4838 [AA2G (mM)]) calculates AA2G calibration curve.

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

AA2G mmol after dialysis：It will be inhaled in equation (A@260nm=1.4838 [AA2G (mM)]) after dialysis Receive and be located under 260nm.

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

The AA2G conjugations in gel are 14.7mol% as described in example 1 above.

Embodiment 3：The measure of gel rheology

It is solidifying using being obtained in vibration parallel-plate rheometer (Anton Paar, Physica MCR301) measurement embodiment 1 The property of glue.A diameter of 25mm of plate used.Inter-plate gap is set to 1mm.For measurement every time, strained under fixed frequency Before scanning, the frequency scanning under constant strain is carried out first.G ' (energy storage is obtained by strain sweep curve under 1% strain Modulus).Value is 1450Pa for gel.

Embodiment 4：Using BDDE as crosslinking agent, with adjustable conjugation and gel rheology AA2G and the HA gels of crosslinking With reference to

Program is similar to described in embodiment 1.By adjusting crosslinking agent conjugation is changed with HA and AA2G mol ratios.Such as Gelling properties are measured described in embodiment 3.Details as Follows：

Make 400.8mg LMW HA hydration~30min in 1752.1mg 1%NaOH in syringe.By 800.3mg's AA2G is put into bottle, is subsequently placed into 354.1mg BDDE and 1402.0mg 10%NaOH.It in HA is hydrated being added to Before, make above solution (pH>12) reaction~20min in 50 DEG C of water-baths.After addition, by between 2 syringes back and forth Transmission makes mixture mix~20 times.The paste of mixing is put into bottle and 50 DEG C of water-baths~2.5h.By 140.9mg 12M HCl is added in 9.0053g PBS (pH7.4).After~2.5h, HA-AA2G gels are formed.Gel is cut into slices, and to its Middle addition HCl-PBS solution.Neutralize gel and expanded whole night on orbital shaker.Gel is sieved by~60 μm of sieves And by transmitting mixing~20 times back and forth between 2 syringes.Gel is put into 15,000MWCO RC bag filters and Dialysed in PBS (pH7.4) buffer solution.Dialyse progress~164.5h, frequently changes PBS.After dialysis, gel is put into In syringe and it is stored in 4 DEG C of refrigerators.Conjugation is 13%.Gel storage modulus (G ') is 803Pa.

Embodiment 5：Using BDDE as crosslinking agent, AA2G is combined with the HA gels being crosslinked, conjugation 5.3%, and G ' is ~300Pa.

Make 400.3mg LMW HA hydration~30min in 3002.0mg 1%NaOH in syringe.By 800.5mg's AA2G is put into bottle, is subsequently placed into 264.3mg BDDE and 1100.0mg 10%NaOH.It in HA is hydrated being added to Before, make above solution (pH>12) reaction~20min in 50 DEG C of water-baths.After addition, by between 2 syringes back and forth Transmission makes mixture mix~20 times.The paste of mixing is put into bottle and 50 DEG C of water-baths~2.5h.By 104.2mg 12M HCl is added in 8.5128g PBS (pH7.4).After~2.5h, HA-AA2G gels are formed, and add HCl-PBS thereto Solution.Neutralize gel and whole weekend (~55h) is expanded on orbital shaker.Gel is sieved by~60 μm of sieves simultaneously And by transmitting mixing~20 times back and forth between 2 syringes.By gel be put into 15,000MWCO RC bag filters and Dialysed in PBS (pH7.4) buffer solution.Dialyse progress~114h, frequently changes PBS.After dialysis, gel is put into injection In device and it is stored in 4 DEG C of refrigerators.By the program measurement conjugation and gel rheology described in embodiment 2 and 3.Conjugation For 5.3%.Gel storage modulus is~300Pa.

Embodiment 6：Using star-PEG epoxides as crosslinking agent, AA2G is combined with the HA gels being crosslinked, conjugation It is~235Pa for 29.4%, G '.

Make 200.4mg LMW HA hydration~30min in 2000mg 1%NaOH in syringe.By 400mg AA2G It is put into bottle, is subsequently placed into 312.7mg star-PEG epoxides and 1026.5mg 10%NaOH.It is being added to hydration Before in HA, make above solution reaction~20min in 50 DEG C of water-baths.After addition, by being returned between 2 syringes Passing makes mixture mix~20 times.The paste of mixing is put into bottle and 50 DEG C of water-baths~2.5h.By 187.4mg 12M HCl is added in 3.034g PBS (pH7.4).After~2.5h, HA-AA2G gels are formed, and it is molten to add HCl-PBS thereto Liquid.Neutralize gel and whole weekend (~68h) is expanded on orbital shaker.By~60 μm of sieves sieve gels and By transmitting mixing~20 times back and forth between 2 syringes.By gel be put into 15,000MWCO RC bag filters and Dialysed in PBS (pH7.4) buffer solution.Dialyse progress~95h, frequently changes PBS.After dialysis, gel is put into injection In device and it is stored in 4 DEG C of refrigerators.By the program measurement conjugation and gel rheology described in embodiment 2 and 3.Conjugation For 29.4%.Gel storage modulus is~235Pa.

Embodiment 7：Using star-PEG epoxides as crosslinking agent, AA2G is combined with the HA gels being crosslinked, conjugation It is~363Pa for 27.8%, G '.

Make 200.3mg LMW HA hydration~30min in 2000mg 1%NaOH in syringe.By 400.2mg's AA2G is put into bottle, is subsequently placed into 313.4mg star-PEG epoxides and 1022.6mg 10%NaOH.Will be molten above Liquid is added in hydration HA.After addition, mixture is set to mix~20 times by being transmitted back and forth between 2 syringes.Will be mixed The paste of conjunction is put into bottle and 50 DEG C of water-baths~2.5h.196.5mg 12M HCl are added to 3.016g PBS (pH7.4) In.After~2.5h, HA-AA2G gels are formed, and add HCl-PBS solution thereto.Gel is set to neutralize and be vibrated in orbit determination Expanded on device (~24h) whole night.Gel is sieved by~60 μm of sieves and is mixed by being transmitted back and forth between 2 syringes ~20 times.Gel is put into 15,000MWCO RC bag filters and dialysed in PBS (pH7.4) buffer solution.Dialysis progress~ 98.5h, frequently change PBS.After dialysis, gel is put into syringe and is stored in 4 DEG C of refrigerators.By embodiment Program measurement conjugation and gel rheology described in 2 and 3.Conjugation is 27.8%.Gel storage modulus is~363Pa.

Embodiment 8：Using BDDE as crosslinking agent, AA2G is combined with the HMW HA gels being crosslinked, and conjugation is about 10mol%, G ' it is about 240Pa.

Make 400.3mg HMW HA hydration~30min in 2501.3mg 4wt%NaOH in syringe.By 1200mg AA2G be put into bottle, be subsequently placed into 304.7mg BDDE and 1178.6mg 16wt%NaOH.Be added to hydration HA it Before, make above solution (pH>12) reaction~20min in 50 DEG C of water-baths.After addition, by between 2 syringes back and forth Transmission makes mixture mix~20 times.The paste of mixing is put into 20cc bottles and 50 DEG C of water-baths~2.5h.After~2.5h, shape Into HA-AA2G gels.226.6mg 12M HCl are added in 8492.2mg10 × PBS (pH7.4) and obtain HCl-PBS solution And HCl-PBS solution is added so that gel neutralizes and expansion.Make gel neutralize and on orbital shaker expand 48h with On.Gel is sieved by~60 μm of sieves and by transmitting mixing~20 times back and forth between 2 syringes.Gel is put into Dialysed in 20,000MWCO CE bag filters and in PBS (pH7.4) buffer solution.Dialyse progress~114h, frequently changes PBS Buffer solution.After dialysis, gel is put into syringe and is stored in 4 DEG C of refrigerators.By the program described in embodiment 2 and 3 Measure conjugation and gel rheology.Conjugation is 10mol%.Gel storage modulus is about 240Pa.

Embodiment 9：Using BDDE as crosslinking agent, vitagen is combined with the LMW HA gels being crosslinked, and conjugation is 15mol%, G ' it is about 365Pa.

Make 398.2mg LMW HA hydration~40min in 1753.24mg 1wt%NaOH in syringe.To expansion BDDE (311.7mg) is added in HA and continues to allow HA reflation 80min.Make the HA/BDDE mixtures of expansion pre- at 50 DEG C First react 20min.

801.9mg vitagen is individually dissolved in 1459.7mg 10wt%NaOH and and reacted in advance with BDDE HA mixing.Mixture is set to continue to react 2.5h again at 50 DEG C.After~2.5h, HA- vitagens are formed.By 195mg 12M HCl, which is added in 9004.0mg 10 × PBS (pH7.4), to be obtained HCl-PBS solution and adds HCl-PBS solution so that gel Neutralize and expand.Neutralize gel and more than 48h is expanded on orbital shaker.By~60 μm of sieves sieve gels and By transmitting mixing~20 times back and forth between 2 syringes.By gel be put into 20,000MWCO CE bag filters and Dialysed in PBS (pH7.4) buffer solution.Dialyse progress~120h, frequently changes PBS.After dialysis, gel is put into injection In device and it is stored in 4 DEG C of refrigerators.Gel rheology is measured by the program described in embodiment 3.Using with being retouched in embodiment 2 It is about 15mol% that the AA2G stated, which determines similar method measure conjugation,.Gel storage modulus is about 365Pa.

Embodiment 10：Vitagen is combined through acid amides is chemical with linear HA

200.3mg HMW HA are made to be hydrated in 60cc syringes in 10ml water.500mg vitagen is dissolved in In 0.5ml water and solution is neutralized and arrive pH4.8.197.7mg EDC and 149mg NHS is individually dissolved in 6ml water.Will be with Upper solution (solution and EDC/NHS solution) is added in another 60cc syringes equipped with 23.5ml water.By in 2 syringes Between back and forth transmit mixing 20 times.Mixture is stored in a syringe and immerses 4h in 37 DEG C of baths.Finally use PBS (pH7.4) buffer solution dialysis solution, until obvious vitagen is not observed.Pass through the class as described in embodiment 3 Conjugation is determined like method.Conjugation is about 10mol%.

Embodiment 11：AA2P and the HA gels of crosslinking combination

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

Embodiment 12

For the preparation for the HA/BDDE dermal augmentation agent products with AA2G for reducing microgroove appearance

To any gel described in above example, after dialysis, added into gel and dissociate HA gels in right amount to carry Height changes gel cohesiveness and/or syringeability.For example, free HA fibers are made to be expanded in phosphate buffer, to obtain Uniform viscoelastic gel (" free " HA gels).Before step of dialysing, this uncrosslinked gel is added in embodiment 1 In the HA/BDDE cross-linked gels of acquisition (for example, to obtain the composition with about 1% to about 5%w/w free HA).Then will The gel-filled high pressure steam sterilization into the asepsis injector for being easy to fill and under sufficient temp and pressure of gained is at least about 1min.After high pressure steam sterilization, pack HA/AA2G final products and be distributed to doctor and injected for use as dermal augmentation agent superficial, Improve the appearance of microgroove in periorbit or other facial zones.

Embodiment 13

The preparation of HA-AA2G dermal augmentation agent including lidocaine

According to the program of embodiment 12, but after step of dialysing and before the free HA gels of addition, into mixture Add lidocaine hydrochloride (lidocaine HCl).(the lidocaine HCl) of powder type is dissolved in WFI first and Filtered by 0.2 μm of filter.NaOH weak solutions are added into sticky HA/AA2G gels to reach alkalescent pH (for example, being situated between PH between about 7.5 and about 8).Then lidocaine HCl solution is added in alkalescent gel to reach final required dense Degree, e.g., from about 0.3% (w/w) concentration.Then the gained pH of HA/AA2G/ lidocaine mixtures is about 7 and HA concentration is about 24mg/g.Mechanical mixture is carried out in the standard reaction device equipped with appropriate mixer to obtain suitably homogenous property.

Embodiment 14

The combination of additive and HA hydrogels containing carboxyl functional group

Additive, such as retinoic acid (AKA, vitamin A acid), Adapalene (adapalence) and alpha-lipoic acid contain carboxyl Functional group (- COOH).Using EDC chemistry through esterification, these additives are made to be combined with HA hydrogels.The following describe basis The example of combination described in one embodiment of the invention：

200mg HMW HA are made to be hydrated in 60cc syringes in 10ml pH4.8MES buffer solutions.In another injection In device, 200mg retinoic acid is dissolved in 5ml boiling mixture (water/acetone volume ratio 1:3).Pass through syringe connector Mix two above syringe about 20 times.Then 197.7mg EDC and 149mg NHS is dissolved in independent syringe respectively In 6ml water.Syringe equipped with EDC and NHS is connected with the syringe equipped with HA and retinoic acid, with by 2 syringes it Between back and forth transmit make reactant mix at least 20 times.Mixture is stored in a syringe and immerses 4h in 37 DEG C of baths. Gel is dialysed to remove uncombined retinoic acid with isopropanol, is then aseptically dialysed with PBS.By gel bag It is attached in asepsis injector and is stored at 4 DEG C.

Embodiment 15

The combination of the additive and HA hydrogels of hydroxy functional groups.

Additive, such as retinol (AKA, vitamin A acid), catalase, DMAE and g- tocopherols contain hydroxyl Base functional group (- OH).Using EDC chemistry through esterification, these additives are made to be combined with HA hydrogels.The following describe combination Representative instance：

Make 200mg HMW HA 10ml in 60cc syringes 2- (N- morpholines) ethyl sulfonic acid (MES) buffer solution (pH4.8) Middle hydration.In another syringe, 200mg retinol is dissolved in 5ml boiling mixture (water/acetone volume ratio 1: 3).Two above syringe is mixed about 20 times by syringe connector.Then by 197.7mg EDC and 149mg NHS points It is not dissolved in the 6ml water in independent syringe.Syringe equipped with EDC and NHS is connected with the syringe equipped with HA and retinol, To make reactant mix at least 20 times by being transmitted back and forth between 2 syringes.Mixture is stored in a syringe And immerse 4h in 37 DEG C of baths.Gel is dialysed to remove uncombined retinol with isopropanol, then aseptically uses PBS Buffer solution is dialysed.Gel pack is stored into asepsis injector and at 4 DEG C.

Embodiment 16

The combination that the additive and HA hydrogels of hydroxy functional groups pass through post-modification.

This is a two step process.

Step 1：With EDC/NHS handle crosslinking HA gels, such as based on HA commercial dermal augmentation agent (for example,Allergan, Irvine CA orMedicis Aesthetics, Inc.) to activate HA Carboxyl.

Step 2：HA hydrogels are activated with the additive treating of hydroxyl.The additive of hydroxyl is retinol, peroxidating Hydrogen enzyme, DMAE and g- tocopherol hydroxy functional groups (- OH).

The representative instance that additive is combined with crosslinking HA gels is as follows：

At room temperature mix 2gm Juvederm gels and 200gm EDC and 150mg NHS.Then 200mg is added Retinol in 3ml acetone-water mixtures.Thing mixed above is set to react 4h at 37 DEG C.Gel is dialysed with isopropanol to go Except uncombined retinol, then aseptically dialysed with PBS.By gel pack into asepsis injector and in Stored at 4 DEG C.

Embodiment 17

The combination of growth factor, peptide or elastin laminin and HA hydrogels

Additive containing amine functional group, such as EGF (EGF), TGF (TGF) and peptide, can be with HA is combined to be formed beneficial to dermal augmentation agent.These additives are combined by amidatioon chemistry with HA.The following describe combination Representative instance：

200.3mg HMW HA are made to be hydrated in 10ml MES pH5.4 buffer solutions.It is molten in 100mg MES to add 20mg EGF in liquid.To thing mixed above, 197.7mg EDC and 149mg are added.Gained reactant mixture is set to be reacted at 37 DEG C 4h.After the completion of reaction, further dialysed gel with isopropanol, then aseptically dialysed with PBS.By gel bag It is attached in asepsis injector and is stored at 4 DEG C.

Invention further provides the method for enhancing transplant fat regular organization activity power.Methods described may generally include to The step of patient is adjacent to the skin introducing composition of transplant fat tissue, the composition is combination described elsewhere herein Thing.For example, the composition can include hyaluronic acid and with the covalently bound vitamin C derivatives of hyaluronic acid, wherein combining Degree is between about 3mol% and about 40mol%.In the other side of the present invention, treating the method for skin includes drawing into skin The step of entering comprising adipose tissue, hyaluronic acid and the ascorbic composition combined with hyaluronic acid.

Embodiment 18

The combination of growth factor, peptide or elastin laminin and HA hydrogels

Make to assess the mitogenesis of vitamin C and its derivative to the stem cell (hASC) of Human Adipose Tissue-derived With hASC is not supplemented in supplement or in vitamin C (ascorbic acid) of free form or derivatives thereof in tissue culturing plastic Culture 4 days in the complete MesenPro culture mediums (Invitrogen, Carlsbad, CA) of (vitagen or AA2G).By such as MTT determination methods estimation propagation described by manufacturer (ATCC, Manassas, VA).After 4 days, ascorbic acid 0.25,0.5 is found Propagation is set (to be converted into purple first by by dehydrogenase with 1mM concentrationYellow tetrazolium MTT measurement) (purple first Washed agent dissolving) respectively than the control raising 60%, 80% and 96% of shortage ascorbic acid.Use AA2G points of same concentrations The propagation that control 70%, 60% and 50% Huo get not exceeded improves.Analog result is obtained with vitagen, is shown respectively than control Increase by 70%, 60% and 30%.In a word, vitamin C and its derivative, AA2G and vitagen are in the culture medium containing growth factor In the presence of improve in cell culture hASC propagation.

With with reference to ascorbic crosslinking HA gels

Described in embodiment 19 and 20 below ascorbic based on the gel for being crosslinked HA and using 1,4- with combining Preparation of the butanediol diglycidyl ether (BDDE) as crosslinking agent, according to certain embodiments of the present invention, it shows the court of a feudal ruler Dare effect reduces and further advantage.In embodiment 19, vitamin C derivatives be ascorbic acid 2- glucosides (AA2G) and In embodiment 20, vitamin C derivatives are ascorbic acid 3- aminopropyls phosphates (vitagen).These gels have optimal Rheological characteristic, excellent syringeability and high HA concentration (25mg/g).While not wishing to constrained by any particular theory of operation, still The inventors have discovered that HA and BDDE is set to be crosslinked the property for changing gel significantly in the presence of AA2G or vitagen, relatively In the commercial HA gels being crosslinked with BDDE, gel has high crosslink density, high HA concentration, low sticky and low extruding force.Because hand over AA2G or vitagen during connection be present, so the gel currently formed has individually or via BDDE, as side base and bridge joint The crosslinking agent of HA chains and these ascorbic acid derivates of HA chains coupling.The microstructure of gel has changed, and causes gel i.e. Make by carefully also very low to 30G pin, extruding force.Moreover, gel is with about 3mol% to about 10mol% or up to about The 15mol% vitamin C combined with HA.In injected gel, they by endogenous enzymes, such as from fibroblastic α- Glucuroide or phosphoric acid enzyme r e lease active vitamin C.Active vitamin C can trigger skin collagen to be formed and can rise certainly Gel degradation is suppressed by the effect of base scavenger.

Embodiment 19

The preparation for the HA/AA2G gels that Tyndall effect reduces

After addition 1764.0mg 5wt%NaOH solution, make the LMW HA and 402.3mg of 400.1mg in syringe AA2G mixture hydration 60min.800.8mg AA2G is added in independent bottle, then adds 1401.1mg 9.1wt% The BDDE of NaOH solution and 252.6mg.Make resulting solution (pH>12) reaction~20min in 50 DEG C of water-baths, is shifted afterwards Into hydration HA.After addition, mixture is set to mix~20 times by the way that it is transmitted back and forth between two syringes.Then will Paste is transferred in bottle, is placed in 50 DEG C of water-baths~2.5h afterwards.After crosslinking, add 12M HCl containing 197.0mg and 9.18g10 × PBS (pH7.4) solution makes gel expand 72h on orbital shaker to neutralize alkali.It is forced through The mesh screen fractional condensation glue in~60 μm of apertures.By the way that it is transmitted into sieved gel~20 time of mixing back and forth between two syringes, It is transferred into cellulose esters bag filter (MWCO~20kDa) and is dialysed 5 days with PBS (pH7.4) buffer solution afterwards, daily Change buffer solution twice.After dialysis, gel is assigned in 1ml COC syringes, 5min is centrifuged under 5000RPM to go degasification Bubble, and sterilized with moist steam.The gel has 25mg/g HA ultimate densities, the pact calculated as described in example 2 above 10mol% AA2G mol% and about 80Pa G '.Other gels are prepared in a similar manner, and G ' values are about 60Pa to about 80Pa.

Embodiment 19A

The preparation for the HA/AA2G gels with lidocaine that Tyndall effect reduces

A certain amount of lidocaine is added to generate the HA/AA2G gels with lidocaine to the gel of embodiment 19, It has 0.3%ww lidocaines.By the way that lidocaine HCl is dissolved in PBS (pH~7.4), to prepare lidocaine molten Liquid.After dialysis but before sterilizing, the lidocaine solution of the gel addition aliquot into embodiment 19.Then it is complete Full mixed gel is to obtain the homogeneous mixture with 0.3%ww lidocaine concentrations.

Embodiment 20

The preparation for the HA/ vitagen gels that Tyndall effect reduces

Make 401.0mg LMW HA hydration~45min in 2355.0mg 1wt%NaOH solution in syringe.Will 303.8mg BDDE are added in hydration HA and by being mixed between syringe, mix 10 times.Make mixture in 50 DEG C of water-baths Reaction 15min in advance.800.1mg vitagen is individually dissolved in 950.6mg 15wt%NaOH, is then dissolved in In 510.1Milli-Q water.Mixed using between syringe, make vitagen solution and the hydration HA/BDDE mixtures of preheating back and forth Mixing 30 times.Mixture is put back in 50 DEG C of water-baths and is further continued for reacting 2h, afterwards by the 12M HCl containing 148.1mg and 8523.1mg 10 × PBS (pH7.4) solution is added in crosslinking agent.HCl-PBS solution is added to neutralize gel and make it Expansion.Neutralize gel and more than 48h is expanded on orbital shaker.By~60 μm of sieves sieve gels and by Mixing~20 times is transmitted between 2 syringes back and forth.Gel is put into 20,000MWCO CE bag filters and in PBS (pH7.4) dialysed in buffer solution.Dialyse progress~197h, frequently changes PBS.After dialysis, gel is transferred to 1ml In COC syringes, 5min is centrifuged under 5000RPM and is sterilized with moist steam.The HA ultimate densities of gel are 24mg/g.

The HA gels being chemically crosslinked through 1- ethyls -3- [3- dimethyl aminopropyls] carbodiimide hydrochlorides (EDC)

Described in embodiment 21 and 22 below according to certain embodiments of the present invention, show Tyndall effect and subtract The preparation of small and further advantage the gel based on crosslinking HA.In embodiment 21, the crosslinking agent for hexamethylene diamine (HMDA) is used And used in embodiment 20 as 3- [double (3- the amino-propoxymethyls)-propoxyl group of 3- (3- aminopropyls) -2,2-]-propylamine (4 Arm amine -4AA) crosslinking agent, through EDC chemical preparation gels.In a mild condition, such as under room temperature and such as pH5.4 handed over Connection.Reaction condition be can adjust to prepare with optimal gelling properties, excellent syringeability and high HA ultimate densities (~24mg/g) Height network gel.It was found by the inventors that under low-down hydration or reaction density, with appropriate HMDA or 4AA, together with Coupling agent, i.e. 1- ethyls -3- [3- dimethyl aminopropyls] carbodiimide hydrochloride (EDC) and n-hydroxysuccinimide (NHS) Or sulfonyl-NHS (sulfo group-NHS) makes HA crosslinkings favourable.The crosslinking points of gel will be located remotely from each other, therefore highly cross-linked Material has high damping force.On the contrary, crosslinkings of the HA and BDDE under so low hydration or reaction density may be due to crosslinking agent Relative nullity and it is infeasible.

Embodiment 21

The preparation for the HA/HMDA gels that Tyndall effect reduces

20.0g 100mM MES buffer solutions (pH5.2) are added in the syringe of the LMW HA equipped with 1000.0mg. By 260.9mg HMDA.HCl being dissolved in 2010.5mg 100mM MES buffer solutions (pH5.2), and add 2 μ l 1M NaOH makes pH reach 5.2 preparation HMDA solution.By the way that 254.2mg EDC is dissolved in into 1188.4mg100mM MES buffer solutions (pH5.2) EDC solution is prepared in, and in independent bottle, the 100mM MES that 44.3mg NHS is dissolved in 1341.8mg delay In fliud flushing (pH5.2).After HA is fully hydrated ,~1h, 790 μ l HMDA solution is added into hydration HA.By being mixed between syringe Close homogenized mix 10 times.Then 490 μ l EDC and 490 μ l NHS solution are added to and homogenize in paste and pass through syringe Between mix and remix 10 times.Then mixture is transferred in bottle and in addition 17.9ml 1 × PBS (pH7.4) before, 5h is crosslinked at room temperature.Before the net in 60 μm of apertures is forced through, gel is set to expand 3 on roller My god.The gel of screening is placed in cellulose ester membrane dialysis tubing MWCO20KDa and with 1 × PBS 4 days, changed daily slow Fliud flushing is twice.By gel distribution in 1ml COC syringes, 5min is centrifuged under 5000RPM, and sterilized with moist steam.Gel HA ultimate densities be 25mg/g.

Embodiment 22

The preparation for the HA/4AA gels that Tyndall effect reduces

32.55g 100mM MES buffer solutions (pH5.2) are added in the syringe of the LMW HA equipped with 1000.4mg. By 256.3mg4AA being dissolved in 1039.8mg 100mM MES buffer solutions (pH5.2), and add 380 μ l 6M HCl PH is set to reach 5.2 preparation 4AA solution.By the way that 251.2mg EDC is dissolved in 1013.8mg100mMMES buffer solutions (pH5.2) EDC solution is prepared, and in independent bottle, 74.7mg NHS is dissolved in 2020.0mg 100mM MES buffer solutions (pH5.2) in.After HA is fully hydrated ,~1h, 260 μ l 4AA solution is added into hydration HA.Homogenized by being mixed between syringe Mixture 10 times.Then 277 μ l EDC and 273 μ l NHS solution are added to and homogenized in paste and by being mixed between syringe Remix 10 times.Then mixture is transferred in bottle and before addition 6.4ml 10 × PBS (pH7.4), 5h is crosslinked at room temperature.Before the net in 60 μm of apertures is forced through, gel is set to be expanded on roller 3 days.By the solidifying of screening Glue is placed in cellulose ester membrane dialysis tubing MWCO20KDa and with 1 × PBS 4 days, changes buffer solution twice daily.Will be solidifying Glue is distributed in 1ml COC syringes, 5min is centrifuged under 5000RPM, and sterilized with moist steam.The HA ultimate densities of gel are 23mg/g。

Embodiment 23

The measure of embodiment 19-22 gel rheology

Using vibration parallel-plate rheometer, Anton Paar Physica MCR301, the rheological characteristic of gel is measured. 25mm board diameter is used when gap is highly 1mm.Measured under 25 DEG C of constant temperature.Measurement is by 2% constant strain and frequency every time Rate logarithm increases lower 1-10Hz frequency scanning composition, increases followed by with strain logarithm, 1-300% under 5Hz constant frequencies Strain sweep.Storage modulus (G ') and viscous modulus (G ") are obtained by strain sweep under 1% strain.

By the storage modulus and viscous modulus of the embodiment 19-22 gels obtained

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 measurement of embodiment 19-22 gel

Measure power gel extruded needed for 30G pins using Instron5564 and Bluehill2 softwares.By gel from 1ml COC syringes extrude 30G1/2TSK pins.Piston 11.35min is promoted by 100mm/min speed, and records extruding force.

By the extruding force of the embodiment 19-22 gels obtained

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

Embodiment 25

The biocompatibility test of embodiment 19-22 gel

In the gel of 50 μ l bolus injections of the intracutaneous implantation in the back side of Shi-road Er Shi rats (Sprague Dawley rat). Implant was taken out at 1 week and dyes and be that the CD68 of monokaryon inflammatory cell mark dyes with h and E (H＆E), is led to Cross histologic analysis implant.Based on 3 20 × images scoring 0-4 that dye levels are CD68.Then be averaged these values with to Go out sample score.Every kind of 4 samples of gel analysis.

Embodiment 19-22 average CD68 scores

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 embodiment 19-22 gel, ISO10993-5.

According to ISO10993-5 agarose cladding process, pass through the vitro cytotoxicity test of NAMSA progress gels：《Doctor Treat apparatus biological assessment》- the 5 part：Vitro cytotoxicity test.The examination being placed on to three multiple holes addition 0.1ml on filter disc Test sample, and the high density polyethylene (HDPE) grown of 0.9%NaCl solution, the 1cm as negative control and as positive control 1 × 1cm²Latex part.Each is placed on to the agarose surface of directly covering individual layer L929 l cells.At 37 DEG C 5%CO₂After middle incubation 24h, any abnormal cell form and the cell cracking of culture are checked from both macro and micro.Based on by The cracking zone of nearly sample, for sample scoring 0-4.Because specimen sample does not show the mark for causing any cell cracking or toxicity As so the test material from embodiment 1,3 and 4 is scored at 0.

The quantitative analysis of Tyndall effect

In order to further support visual observation and carry out the comparative advantage analysis of HA fillers, it is believed that Tyndall must be carried out The quantitative analysis of effect.There is not the quantitative technique of specific Tyndall effect to dermal augmentation agent in the literature equally.So And based on the existing understanding of science to light scattering and the interaction of light and skin, using based on (a) colorimetric method and (b) spectroscopy Two kinds of distinct methods quantify skin on Tyndall effect.Based on these technologies, 3 kinds of different quantitative parameters are defined (such as Shown in lower) measure internal Tyndall effect.

a)Tyndall effect visual score：Scale range is 1-5, increment 0.5.It is normal to the colour of skin and there is no blue variable colour Injection portion give score 1.To thick and obvious blue variable colour (generally with Restylane or Juv é derm Ultra Plus It is related) give top score 5.It is that 3 independent observers train scale before for the blind scoring of sample.

b)The blue color component of skin color-" b "：Using colorimeter (CM2600D, Konica Minolta, NJ) quantify from The blue color component of the light of the skin part outgoing of different fillers is injected.This is real by using " b " component of L-a-b colour codes It is existing.

c)From " the blue light % " of skin outgoing：Using portable spectrophotometer (CM2600D, Konica Minolta, NJ) quantify in total visible-range from the blue light % of skin outgoing.This can be by seeking the area under 400-490nm visible spectrums Integrate and standardized and realized with the gross area under spectrum (400-700nm).

Embodiment 27

The Tyndall of gel is assessed

Using straight line injection, the thigh intracutaneous injection gel by 27G1/2TSK pins to 2 monthly ages hairless rat.Superficial Implanted gel is to simulate clinical microgroove program.Tyndall experiment 48h is carried out after gel implantation.After carrying out Tyndall experiment, make Animal euthanasia with improve due to lack hemoglobin, the contrast of Tyndall effect.

After implantation being shown in Figure 12 2 days, the image of the gel from embodiment 19 and 21.It also show commercial Juv é Derm Refine and Restylane Touch image are to compare.In commercial Juv é derm Refine and Restylane Blue line (Tyndall effect) is high-visible in Touch image.From embodiment 19,19A (not shown) and 21 gel not Show Tyndall effect.

Using the visual score 1-5 that increment is 0.5, scored for injection site.The injection site for being scored at 1 does not show skin Skin changes colour, and is scored at 5 injection site and shows the serious blue variable colour of skin.Also by means of colorimeter (CM2600D, Konica Minolta, NJ) spectrum analysis has been carried out to injection site.The blue color component " b " of skin color and from skin be emitted Blue light % (400-700nm) independently measure.Figure 13 and 14 shows range estimation Tyndall score and outgoing blue light %.From implementation The gel of example 19 and 21 does not show Tyndall effect and with relatively low range estimation Tyndall score and outgoing blue light value %.It is right For Juv é derm Refine and Restylane Touch, Tyndall score and outgoing blue light value % are higher.Belotero Soft does not show Tyndall effect and value is comparable to the gel of embodiment 19 and 21.See Figure 13 and 14.

Embodiment 28

The duration inside gel is assessed by histology

Gel of the invention and commercial gel in 50 μ l bolus injections of the intracutaneous implantation in the back side of Shi-road Er Shi rats.1 Implant is taken out when all and passes through histologic analysis with h and E (H＆E) dyeing.Just section is taken in injection portion.From every Individual tissue sample is cut two and cut into slices and using suture indicator suture H＆E stained slices.Then by sample be grouped and according to The amount scoring of surplus material is as follows：Do not have (0%), low (25%), in it is (50%) and high (100%).See Figure 15.

Embodiment 28A

The duration inside gel is assessed by MRI

Assessed using magnetic resonance imaging (MRI) research in female Shi-road Er Shi rats after intracutaneous injection, 40 weeks Nei Benfa The volume and surface area of bright gel and commercial gel change with time.By every kind of μ l target volume injected gels of implant 150.Make Implant is located at the slightly caudal two offside positions of shoulder, two offside positions for being slightly away from knee kiss side and midpoint between head Two offside positions between tail.It can be scanned in 7Tesla70/30Bruker Biospec MRI scanners.It is being implanted into Collect image within 12,24,40 weeks after the same day (the 0th week) and implantation.Gel absolute volume and the figure of time are shown in FIG. 16 below. High persistent gel has high absolute volume when being implanted into 40 weeks.

Embodiment 29

For treating the present composition of periorbit line

There are fine wrinkles in periorbit area and requires that dermal augmentation agent is treated in the modest woman of 40 years old.Use 30G pins, doctor Microgroove superficial of the teacher into below her each eye and Lei Gou areas introduces 0.6ml according to the gel (example of the present invention based on HA Such as the gel described in embodiment 19).Although introducing gel in superficial, blue variable colour is not observed and patient is to result It is satisfied.

As illustrated, composition of the invention, such as the composition of embodiment 19 and 21, Tyndall effect reduce or failed to understand It is aobvious, and relative to some commercial gels based on HA, such as Juvederm Refine/Surgiderm18 and Belotero Soft, duration length in vivo are a lot.For example, relative to commercialization " microgroove " preparation Belotero Soft, reality of the invention Applying example 19 not only has Tyndall score favourable at least as this commercial gel, and advantageously shows much higher The internal duration.

Embodiment 30

For improving the Injectable composition of the invention of microgroove appearance

Individually and combination additive, such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and its spread out Biology in order to generate a variety of generally optical clears, injectable the gel based on HA mode and cross-linked hyaluronic acid gel With reference to.As defined elsewhere herein, HA components at least 90%, such as be generally entirely LMW HA by weight, Or about 100% LMW HA.These additives are made to be combined with HA hydrogels using any suitable mode.Sieve and process combination Gel injectable, pH be neutral, cementitious compositions to generate, its HA concentration at least about 20mg/g, e.g., from about 23, about 24mg/g, About 25mg/g, it is up to about 30mg/g and fits through thin gage needle injection.G ' the values at least about 50PA of gel, about 60Pa, about 70Pa, about 80Pa, up to or no more than about 100Pa.Packaging gel is simultaneously gone out using pressure cooker, ultraviolet light or other suitable modes Bacterium.

Every kind of gel is useful to superficial injection, such as in the deeper injection no more than about 1.0mm to patient's wrinkle, such as Periorbit area, nasolabial fold region, Lei Gou areas, neck regions will benefit from the skin of any other facial zone of dermal augmentation.To the greatest extent Pipe superficial introduces gel, but does not observe discoloration and the patient satisfaction result due to Tyndall effect.

Although finally, it is to be understood that describe the every aspect of this specification with reference to each embodiment, this area Technical staff will readily recognize that disclosed specific embodiment is only to illustrate subject matter disclosed herein principle.Therefore, Ying Li Solution, disclosed theme are never limited to specific process, scheme and/or reagent described herein etc..Thus, without departing substantially from this specification Spirit on the premise of, those skilled in the art can make many different modifications according to teaching herein or become to disclosed theme Change or alternate configuration.On the premise of without departing substantially from the present invention spirit defined in appended claims, detailed change can be done.Most Afterwards, terms used herein is just for the sake of description particular, and is not intended to be limiting the scope of the present invention, and the scope is only It is defined by the claims.Should only it be explained in addition, it is intended that all material that is contained or being shown in accompanying drawing in above description To be illustrative and not restrictive.Therefore, the invention is not restricted to those being accurately shown and described.

This document describes certain embodiments of the present invention, including inventor to become known for carrying out the optimal mould of the present invention Formula.Certainly, the modification of these embodiments of description will after reading is described above for one of ordinary skill in the art Become apparent.Inventor it is expected that technical staff optionally uses such modification, and inventor is intended to make the present invention with not The method for being same as specifically describing herein is implemented.Therefore, as applicable law license, the present invention include what is described in appended claims The all modifications and equivalents of theme.Moreover, unless otherwise indicated or otherwise clearly contradicted in addition, otherwise this hair The bright any combinations for covering its above-mentioned element being possible in modification.

The packet of the substitute element of present invention disclosed herein or embodiment should not be interpreted as limiting.Each organize into Member can refer to and be claimed individually or with any combinations of members other with the group or the other elements found herein., it is expected that One or more members of group can be included in group or be removed from it because of the reason for convenient and/or patentability.When any such bag When including or delete generation, specification is considered as containing the group changed, so as to complete for all of appended claims The written description of Markush group (Markush group).

Unless otherwise noted, the amount of composition, property (such as molecular weight for description and claims, reaction are expressed Condition etc.) all numerals all will be understood as being modified by term " about " in all cases.As used herein, term " about " Refer to the scope that so quantitative project, parameter or term cover above and below the value of gainer, parameter or term ± 10%.Cause This, unless pointing out that it can be according to this on the contrary, the digital parameters otherwise listed in specification and appended are approximation The required property that invention is tried hard to obtain changes.At least, it is not as attempting the scope that the application of doctrine of equivalents is limited to claim Equally, each digital parameters should be according at least to the digital of the significant digit of report and by understanding using common rounding techniques. Although it is approximation to list the broad range of number range of the present invention and parameter, the numerical value listed in a particular embodiment is all most Report to possible accuracy.However, any numerical value is inherently containing must be by the standard deviation that finds in its respective test measurement Some errors caused by difference.

It is unless otherwise indicated or otherwise clearly contradicted, with otherwise in description the present invention context in (especially Be claim below context in) term "one", " one kind ", " described " and similar indicant be understood to include Odd number and plural number.The narration of value scope is intended only to serve as individually referring to the shorthand side for each independent values for belonging to the scope herein Method.Unless otherwise indicated, otherwise each individually value is incorporated in specification as the same to its independent narration herein. Unless otherwise indicated or otherwise clearly contradicted in addition, otherwise all methods described herein can be with any suitable order Carry out.Using provided herein is any and all embodiment or exemplary wording (for example, " such as ") be only intended to preferably say The bright present invention, without forming to the limitation for the scope of the invention for requiring in addition that protection.Wording in specification must not be interpreted as Point out any not claimed element essential to implementing the present invention.

It can be used and be made up of wording or be substantially made up of wording, particular disclosed herein is further limited In claim.When for claim, either as the amendment achieved or the amendment added every time, transitional term " by ... form " all eliminate unspecified any element, step or composition in the claims.Transitional term is " substantially By ... form " it the scope of claim is limited to specific material or step and will not substantially influence fundamental characteristics and Xin Te The material or step of property.Embodiment that is intrinsic herein or clearly describing and enable such claimed invention.

All patents, patent disclosure and the other announcements quoted and identified in this manual are in order to describe and open (example As) described in such announcement can with the composition that be used in combination of the present invention and the purpose of method, individually and explicitly by Reference is integrally incorporated herein.Before the date of application of the application, these, which are announced, is only their disclosure and provides.With regard to this point Say and should not be construed as recognizing due to previous invention or any other reason, have no right to make inventor prior to such disclosure.On day All statements of phase or statement on these document contents are based on the available information of applicant, do not form on these documents Date or any of content correctness recognize.

Claims (26)

1. a kind of dermal augmentation agent composition, comprising：

With the hyaluronic acid component of linked crosslinking；

Additive in addition to the linked；

Wherein described linked is 1,4- butanediol diglycidyl ethers；

Wherein described additive is L-AA 2- glucosides, and the hyaluronic acid component and the additive chemistry With reference to conjugation is between 3mol% and 40mol%；

The composition optical clear；And

When in the corium area for being administered to patient, relative in addition to no additive, identical composition, the composition Show Tyndall effect reduction；

Wherein described hyaluronic acid component is entirely LMW HA, the mean molecule quantity of the HA between 100KD and 500KD it Between.

2. composition according to claim 1, wherein the hyaluronic acid component is combined with the additive chemistry, with reference to Degree is between 3mol% and 10mol%.

3. composition according to claim 1, further comprising anesthetic.

4. composition according to claim 1, further comprising lidocaine.

5. composition according to claim 1, its storage modulus G ' values are at most 200Pa；

G ' values are using vibration parallel-plate rheometer Anton Paar Physica MCR 301, are made when clearance height is 1mm With 25mm board diameter, measured under 25 DEG C of constant temperature；Wherein, under measurement is increased by 2% constant strain and frequency logarithm every time 1-10Hz frequency scanning composition, increases followed by with strain logarithm, 1-300% strain sweep under 5Hz constant frequencies, G ' is obtained by strain sweep under 1% strain.

6. composition according to claim 1, its storage modulus G ' values are at least 25Pa and are at most 200Pa；

G ' values are using vibration parallel-plate rheometer Anton Paar Physica MCR 301, are made when clearance height is 1mm With 25mm board diameter, measured under 25 DEG C of constant temperature；Wherein, under measurement is increased by 2% constant strain and frequency logarithm every time 1-10Hz frequency scanning composition, increases followed by with strain logarithm, 1-300% strain sweep under 5Hz constant frequencies, Storage modulus G ' is obtained by strain sweep under 1% strain.

7. composition according to claim 1, its storage modulus G ' values are between 40Pa and 100Pa；

G ' values are using vibration parallel-plate rheometer Anton Paar Physica MCR 301, are made when clearance height is 1mm With 25mm board diameter, measured under 25 DEG C of constant temperature；Wherein, under measurement is increased by 2% constant strain and frequency logarithm every time 1-10Hz frequency scanning composition, increases followed by with strain logarithm, 1-300% strain sweep under 5Hz constant frequencies, Storage modulus G ' is obtained by strain sweep under 1% strain.

8. composition according to claim 1, its storage modulus G ' values are not more than 100Pa；

G ' values are using vibration parallel-plate rheometer Anton Paar Physica MCR 301, are made when clearance height is 1mm With 25mm board diameter, measured under 25 DEG C of constant temperature；Wherein, under measurement is increased by 2% constant strain and frequency logarithm every time 1-10Hz frequency scanning composition, increases followed by with strain logarithm, 1-300% strain sweep under 5Hz constant frequencies, Storage modulus G ' is obtained by strain sweep under 1% strain.

9. composition according to claim 1, its storage modulus G ' values are not less than 40Pa；

G ' values are using vibration parallel-plate rheometer Anton Paar Physica MCR 301, are made when clearance height is 1mm With 25mm board diameter, measured under 25 DEG C of constant temperature；Wherein, under measurement is increased by 2% constant strain and frequency logarithm every time 1-10Hz frequency scanning composition, increases followed by with strain logarithm, 1-300% strain sweep under 5Hz constant frequencies, Storage modulus G ' is obtained by strain sweep under 1% strain.

10. dermal augmentation agent composition according to claim 1 is used for the medicament for preparing the microgroove on treatment patient skin Purposes, wherein：

The medicament is introduced in the skin of patient；

The medicament optical clear；And

Wherein relative in addition to no additive, identical composition, the medicament shows Tyndall effect reduction.

11. purposes according to claim 10, wherein the hyaluronic acid component is combined with the additive chemistry, with reference to Degree is between 3mol% and 10mol%.

12. a kind of optically transparent dermal augmentation agent composition for not showing or showing unconspicuous Tyndall effect is used In the purposes for preparing the medicament for improving face appearance, wherein：

By the corium area of the pharmacy application to patient；And

The composition is made through the following steps：

Hyaluronic acid is provided；

Make crosslinking agent and additive reaction, the crosslinking agent is BDDE, and the additive resists for L- Bad hematic acid 2- glucosides；

The crosslinking agent reacted and L-AA 2- glucosides are added in the hyaluronic acid to form cross-linked transparent Matter acid composition, wherein the cross-linked-hyaluronic acid composition and the L-AA 2- glucoside covalent bonds；Wherein The hyaluronic acid component is combined with the additive chemistry, and conjugation is between 3mol% and 40mol%；And

Homogenize and neutralize the cross-linked-hyaluronic acid composition to obtain the dermal augmentation agent composition of injectable；

Wherein described hyaluronic acid component is entirely LMW HA, the mean molecule quantity of the HA between 100KD and 500KD it Between.

13. purposes according to claim 12, wherein the hyaluronic acid component is combined with the additive chemistry, with reference to Degree is between 3mol% and 10mol%.

It is thin that 14. the optically transparent dermal augmentation agent composition based on hyaluronic acid of claim 1 is used for preparation reduction patient The purposes for the medicament that microgroove occurs in dermatotome, wherein the medicament is applied into the patient in the depth no more than 1mm.

15. purposes according to claim 14, wherein in composition described in the deeper injection no more than 0.8mm.

16. purposes according to claim 14, wherein in composition described in the deeper injection no more than 0.6mm.

17. purposes according to claim 14, wherein in composition described in the deeper injection no more than 0.4mm.

18. a kind of dermal augmentation agent composition, comprising：

The hyaluronic acid component being crosslinked with linked, wherein the linked is BDDE；With

With the covalently bound L-AA 2- glucosides of the hyaluronic acid component；Wherein described hyaluronic acid component and institute State L-AA 2- glucosides chemistry to combine, conjugation is between 3mol% and 40mol%；

The composition optical clear and storage modulus G ' values are between 40Pa and 100Pa；

G ' values are using vibration parallel-plate rheometer Anton Paar Physica MCR 301, are made when clearance height is 1mm With 25mm board diameter, measured under 25 DEG C of constant temperature；Wherein, under measurement is increased by 2% constant strain and frequency logarithm every time 1-10Hz frequency scanning composition, increases followed by with strain logarithm, 1-300% strain sweep under 5Hz constant frequencies, Storage modulus G ' is obtained by strain sweep under 1% strain；

Wherein described hyaluronic acid component is entirely LMW HA, the mean molecule quantity of the HA between 100KD and 500KD it Between.

19. composition according to claim 18, its hyaluronic acid concentration is between 18mg/g and 30mg/g.

20. composition according to claim 18, its hyaluronic acid concentration is between 12mg/g and 30mg/g.

21. composition according to claim 18, wherein the conjugation is 10mol%.

22. a kind of Injectable composition for being used to reduce facial lines appearance, the composition include：

The low-molecular-weight hyaluronic acid (HA) of BDDE (BDDE) crosslinking, the mean molecule quantity of the HA Between 100KD and 500KD；With

With the covalently bound L-AA 2- glucosides of the hyaluronic acid, conjugation 10mol%；

Storage modulus G ' the values of the composition are 60Pa to 80Pa；

G ' values are using vibration parallel-plate rheometer Anton Paar Physica MCR 301, are made when clearance height is 1mm With 25mm board diameter, measured under 25 DEG C of constant temperature；Wherein, under measurement is increased by 2% constant strain and frequency logarithm every time 1-10Hz frequency scanning composition, increases followed by with strain logarithm, 1-300% strain sweep under 5Hz constant frequencies, Storage modulus G ' is obtained by strain sweep under 1% strain.

23. composition according to claim 22, wherein the mean molecule quantity of the HA is between 300KD and 500KD.

24. composition according to claim 23 the, wherein G ' values of the composition are 80Pa.

25. composition according to claim 22, its optical clear.

26. composition according to claim 22, its HA concentration is 25mg/g.