CN108379112A - Dermal augmentation agent composition for microgroove treatment - Google Patents

Abstract

It is particularly advantageous to the amendment of face's microgroove the present invention provides height injectable, the long-acting hydrogel dermal augmentation agent composition based on hyaluronic acid.

Description

Dermal augmentation agent composition for microgroove treatment

The application be the applying date be September in 2012 13, application No. is " 201280055657.7 ", entitled " use In microgroove treatment dermal augmentation agent composition " application for a patent for invention divisional application.

Related application

The priority of the U.S. Provisional Patent Application No.61/534,780 submitted for 14th this application claims September in 2011 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, requires 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 compositions, and relate more specifically to effective to the microgroove on treatment skin Injectable dermal augmentation agent composition.

Skin aging is a progressive phenomenon, occurs and is influenced by Lifestyle factors as time goes by, such as It drinks, smoke and sunshine.The aging of atrophy, relaxation and fat characterization skin of face can be used.Atrophy corresponds to skin histology thickness Greatly reduce.Subcutaneous tissue relaxation causes skin excessive and sagging and leads to cheek and ptosis occur.Obesity refer to because Increase excess weight for face and the swelling of neck bottom.These variation usually to drying, follow the string it is coarse related to skin texture.

Hyaluronic acid (HA), also referred to as hyaluronan are the connective for being distributed widely in entire human body, epithelium and nerve fiber 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 participates 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, being repeated exposure to the (for example) ultraviolet light from the sun causes dermal cell to reduce the generation of its hyaluronan and increase The degradation rate of hyaluronan.This material loss leads to 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 treatment aging skin.Filler can replace the endogenous parent lost poly- The function of closing object or 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 the substance existing for entire human body natural.These fillers are logical Normal well-tolerated, impermanency, and be the treatment to the suitable low-risk of a variety of skins.

Tyndall effect (Tyndall effect) is to applied one of the dermal augmentation agent based on hyaluronic acid (HA) The adverse events occurred in a little patients.Tyndall effect is characterized in that, is gone out in the skin part for having injected dermal augmentation agent Existing blue variable colour, this indicates to see visible hyaluronic acid by translucent epidermis.Clinical report show filler application technique and Skin properties can influence the performance of this adverse events.High rigidity and elastomeric filler are used successfully to correct the upper such as nose of face The regions such as labial groove, cheek and chin, without having any worry to change color, because Material injection is at middle part and deep dermis area In.However, when using these packing materials to correct superficial, fine wrinkles, such as tear ditch, glabella line, canthus line, laugh line or preceding Volume, or in upper part of dermis region too superficial mistake in application, being frequently observed the light blue discoloration of skin.This phenomenon, Be considered as Tyndall effect as a result, cause application site semipermanent change colour, and sometimes only applied degradation It disappears 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, and 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 (for example) 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 among the patients, especially 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 seem 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 It is tested in advance in individual.(calcium hydroxy apetite) is subcutaneous, injectable implant, and 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.

Needs are provided to the noting based on hyaluronic acid for the light blue discoloration that will not show to be attributed to Tyndall effect Penetrate dermal augmentation agent.

It summarizes

The present invention describes preparation and can be applied in the corium of top, does not generate any light blue discoloration of skin, or at least The composition and preparation method of the dermal augmentation agent based on HA of not notable or unconspicuous light blue discoloration.Further, It is found that the gel filled duration in vivo of presently described many present invention is obviously commercially available more solidifying than on Vehicles Collected from Market Glue is longer.In some aspects of the present invention, the optically transparent dermal augmentation agent useful to enhancing skin appearance is provided, is increased Volume and richness are added, and have reduced the appearance of even fine wrinkles, has 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 Relevant negative blue variable colour.

More specifically, in one aspect of the invention, providing long-acting treatment dermal augmentation agent composition, usually wrap 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 cross-linking component 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, by suitable reaction process, such as be etherified, Amidation or esterification and polymer covalent bond.

At one of present invention aspect extensively, dermal augmentation agent composition is provided, the composition includes and crosslinking group Divide crosslinked hyaluronic acid component and the additive in addition to the cross-linking component.Hyaluronic acid component can be with additive chemistry knot It closes.Further, when in the corium area for being administered to patient, relative in addition to no additive, substantially the same group Object is closed, the composition shows 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, the method for providing the microgroove on treatment patient skin.In one embodiment, The method includes being introduced in the skin to patient comprising hyaluronic acid component, be crosslinked the cross-linking component of the hyaluronic acid and removed The step of composition of the mixture of additive other than the cross-linking component, the composition generally optical clear, and Wherein relative in addition to no additive, substantially the same composition, the composition shows Tyndall effect and subtracts It is small.

In another aspect of this invention, providing improves the method for face appearance, and the method 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, crosslinking agent and vitamin C derivatives are made Reaction, it includes covalently bound that the crosslinking agent reacted, which is added in the hyaluronic acid with vitamin C derivatives to be formed, Ascorbic cross-linked-hyaluronic acid composition；And it homogenizes and neutralizes 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, the method for reducing that microgroove occurs in patient's thin skin area, wherein the method are provided It is typically included in the depth no more than about 1mm, dermal augmentation agent composition, a kind of generally optical clear are applied to the patient The dermal augmentation agent composition based on hyaluronic acid comprising vitamin C or vitamin C derivatives.In some embodiments In, the composition described in the deeper injection no more than 0.8mm, no more than 0.6mm or no more than 0.4mm.

In still another aspect of the invention, it provides a kind of dermal augmentation agent composition, generally optical clear and leads to Often comprising with the crosslinked hyaluronic acid component of cross-linking component and with the covalently bound vitamin C derivatives of hyaluronic acid component. In one exemplary implementation scheme, the G ' values of the composition are between about 40Pa and about 100Pa.Further, described group The hyaluronic acid concentration for closing object may be between about 18mg/g and about 30mg/g.In treatment skin, 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 be had Effect.In some embodiments, composition of the invention after introducing skin continue at least three moon, at least six moon or be up to one Year.

It may be easier to understand and recognize these and other aspects of the invention and excellent with reference to following drawings and detailed description Point.

Brief description

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

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

Fig. 3 is the structural schematic diagram of STAY-C 50 (AA2P).

Fig. 4 is the structural schematic diagram of 1,4-butanediol diglycidyl ether (BDDE).

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

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

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

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

Fig. 9 is for 4 kinds of different alpha-glucosaccharase enzyme concentrations, and in terms of the time (Minute), that observes comes 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 of the free AsA from combination dermal augmentation agent according to the present invention (sustained release) (AA2G compared with the reaction time converts mol%).

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

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

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

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

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

Figure 16 is shown implantation is the present invention is based on the dermal augmentation gel of HA and some are commercially available for 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, dermal augmentation agent composition is provided, the composition generally comprises 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 sustained release for the vitamin C of skin collagen hyperplasia and other treatments or beauty interests.Work as introducing When skin, for example, it is intradermal, and the composition is reacted with internal endogenous enzyme, and over time, is enzymatically cleaved off in vivo Generate bioactive vitamin C.Because vitamin C is discharged from the composition in a few weeks or months so that following Interests it is available to body.

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

The polymer can 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 can be selected from hydroxyl, amine With the surface of solids of carboxyl functional group.

The polymer can be polysaccharide, such as 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 implementation scheme 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 is poly- for term Sugar " is synonymous with " GAG " and " mucopolysaccharide " and refers to the long linear polysaccharide of repetition disaccharide unit composition.Repetitive unit by with hexosamine The hexose (hexose) or hexuronic acid composition of (nitrogenous hexose) and its pharmaceutically acceptable salt connection.GAG families at 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, which is 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；With Polysaccharides and Hydrogels thus Obtained, United States Patent (USP) public affairs Cloth 2006/0194758；With Di Napoli, Composition and Method for Intradermal Soft Tissue It is described in hydrogel composition disclosed herein and method in Augmentation, international patent publications WO 2004/073759 In useful glycosaminoglycan and its resulting polymers, be respectively 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 hyaluronan 30、Ultra、Ultra Plus、Ultra XC andUltra Plus XC (Allergan Inc, Irvine, Califomia).Table 1 lists representative GAG.

The aspect of the present invention provides 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 monosaccharide That is the disaccharides of D-Glucose aldehydic acid (GlcA) and 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 known as dermatan sulfate in the case of kind.It is more than 100 individually sugared chains that chondroitin sulfate, which can have, respectively with Variable position and amount sulphation.Chondroitin sulfate polymer is the important feature component of cartilage and provides it and permitted compression Multiresistance.Any chondroitin sulfate polymer is useful in compositions disclosed herein, and condition is chondroitin sulfate polymer Improve skin.The non-limiting examples of chondroitin sulfate pharmaceutically acceptable salt include that sodium chondroitin sulfate, sulfuric acid are soft Ossein potassium, chondroitin sulfate magnesium, calcium chondroitin sulfate and combinations thereof.

The hydrogel composition for including keratan sulfate polymer is provided in part in terms of this specification.Such as this paper institutes It uses, 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 through fucosylation and N-acetyl-neuraminate in the end of chain.Any keratan sulfate polymer is public herein Useful in the composition opened, condition, which is 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 provided 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, 3 glucosides key connections D-Glucose aldehydic acid together and D-N- acetylglucosamine monomers and its pharmaceutically acceptable salt.It can Hyaluronan polymer is purified from animal and non-animal.The polymer sizes range 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 provided in part in terms of this specification.As herein It is used, term " crosslinking " refers to intermolecular linkage, and individual 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 object molecule that is polymerized alone A intermolecular linkage.The crosslinking of Glycosaminoglycan Polymer typically results in hydrogel and is formed.Such water-setting adhesiveness is high and needs phase When big power squeezes out 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 four glycidol ether of pentaerythrite (PETGE), diethyl Bis- (2, the 3- glycidoxy) ethylene (EGDGE) of alkene sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), 1,2-, 1,2, 7,8-- diepoxyoctanes (DEO), (phenylene is double-(ethyl)-carbodiimide and 1,6 hexylidenes bis- (ethyl carbodiimides), oneself Two hydrazides (ADH), bis- (sulfosuccinic base) suberates (BS), hexamethylene diamine (HMDA), 1- (2,3- glycidoxy) -2,3- rings Oxygen hexamethylene, lysine, lysine methyl ester or combinations thereof.In Stroumpoulis and Tezel, Tunably Crosslinked Polysaccharide Compositions are disclosed in the U.S. Patent application 12/910,466 that on October 22nd, 2010 submits Other useful crosslinking agents are integrally incorporated by reference.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 WO 2004/073759 Property example processed, and useful Glycosaminoglycan Polymer in compositions disclosed herein and method, respectively accordingly by drawing With being integrally incorporated.

The water for including the crosslinking Glycosaminoglycan Polymer with certain degree of cross linking is provided 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 weight percent of crosslinking agent and glycosaminoglycan.At this Invention certain 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 to be crosslinked Glycosaminoglycan Polymer, such as cross-linked-hyaluronic acid, The concentration of the crosslinking Glycosaminoglycan Polymer is (for example) between about 18mg/g and about 30mg/ present in the wherein described composition Between g.In some embodiments, the hyaluronic acid total concentration of the composition is about 24mg/g or about 25mg/g.

Provided in part in terms of this specification the hyaluronan polymer comprising low molecular weight, high molecular weight it is transparent The hydrogel composition of matter alkane polymer or the hyaluronan polymer of low molecular weight and high molecular weight.As used herein, when Term " high molecular weight " refers to average molecular weight and polymerize for 1,000,000Da or higher hyaluronans when referring to " hyaluronan " Object.The non-limiting examples of high molecular weight 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, The hyaluronan polymer of 000Da.As used herein, when referring to " hyaluronan ", term " low molecular weight " refers to mean molecule Hyaluronan polymer of the amount less than 1,000,000Da.The non-limiting examples of low molecular weight hyaluronan polymer include about 200,000Da, about 300,000Da, about 400,000Da, about 500,000Da, about 600,000Da, about 700,000Da, about 800, The hyaluronan polymer of 000Da and about 900,000Da.

In one embodiment, composition includes the cross-linked transparent matter alkane polymer of low molecular weight.In the embodiment Aspect, composition include average molecular weight be (for example) about 100,000Da, about 200,000Da, about 300,000Da, about 400, The cross-linked transparent of 000Da, about 500,000Da, about 600,000Da, about 700,000Da, about 800,000Da or about 900,000Da Matter alkane polymer.In the other aspects of the embodiment, composition include average molecular weight be (for example) 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 The cross-linked transparent matter alkane polymer of 700,000Da, at most 800,000Da, at most 900,000Da or at most 950,000Da.At this The other aspects of embodiment, composition include that average molecular weight is (for example) 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 The cross-linked transparent matter alkane polymer of 300,000Da to about 800,000Da or about 400,000Da to about 700,000Da.

In another embodiment, composition includes the cross-linked transparent matter alkane polymer of high molecular weight.In the embodiment Aspect, composition include average molecular weight be (for example) 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, The cross-linked transparent matter alkane polymer of 000,000Da.In the other aspects of the embodiment, composition is comprising average molecular weight (for example) 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 aspects of the embodiment, composition includes that average molecular weight is (for example) 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 the cross-linked transparent matter alkane polymer of about 3,000,000Da.

In another embodiment, composition includes cross-linked transparent matter alkane polymer, wherein the cross-linked transparent matter alkane polymerize Object includes the combination of the high molecular weight hyaluronan polymer and low molecular weight 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: 1, about 15: 1, about 10: 1, about 5: 1, about 1: 1, about 1: 5, about 1: 10, about 1: 15 or about 1: 20 high molecular weight hyalomitome The combination of alkane polymer and low molecular weight hyaluronan polymer.

The hydrogel composition for including uncrosslinked Glycosaminoglycan Polymer is provided 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 individual Glycosaminoglycan Polymer molecule or monomer chain.Cause And uncrosslinked Glycosaminoglycan Polymer is not connect 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 Object.Uncrosslinked Glycosaminoglycan Polymer is water-soluble and usually keeps mobility in itself.Thus, uncrosslinked glycosaminoglycan Polymer often mixes with the hydrogel composition based on Glycosaminoglycan Polymer as lubricant thin to promote composition to pass through The extrusion of needle.

In one embodiment, composition includes uncrosslinked Glycosaminoglycan Polymer, wherein the uncrosslinked osamine is poly- A concentration of (for example) about 2mg/g, about 3mg/g, about 4mg/g, about 5mg/g, about 6mg/g, about 7mg/g existing for glycopolymers, 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 aspects of case, composition includes uncrosslinked glycosaminoglycan, wherein a concentration of (example existing for the uncrosslinked glycosaminoglycan 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 aspects of the embodiment, composition includes Uncrosslinked glycosaminoglycan, wherein a concentration of (for example) at most 1mg/g, at most 2mg/g existing for the uncrosslinked glycosaminoglycan, 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 aspects of embodiment, composition includes uncrosslinked glycosaminoglycan, wherein concentration existing for the uncrosslinked glycosaminoglycan For (for example) about 1mg/g to about 60mg/g, about 10mg/g 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 provided in part in terms of this specification. As used herein, term substantially free (or " substantially by ... form "), which refers to, wherein only can detect micro friendship Join the composition of matrix polymer.In the one side of the embodiment, composition includes substantially free of crosslinking chondroitin sulfate The chondroitin sulfate of plain polymer, substantially free of crosslinking dermatan sulfate polymer dermatan sulfate, substantially free of friendship Keratan sulfate, the heparan, substantially substantially free of crosslinking heparan polymer for joining keratan sulfate polymer Heparan sulfate without crosslinking Heparan sulfate polymer or the sulfuric acid substantially free of cross-linked transparent matter alkane polymer Hyaluronan.

The hydrogel composition for being entirely free of crosslinking Glycosaminoglycan Polymer is provided in part in terms of this specification.Such as Used herein, term " being entirely free of " refers to composition within the scope of the instrument or fabrication evaluation used, cannot detect crosslinking Glycosaminoglycan Polymer not can confirm that its presence.In the one side of the embodiment, composition includes to be 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 is provided 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 preparing compositions disclosed herein: fluid ratio is useful, condition are 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 packet 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 Object, wherein gel: fluid ratio be (for example) about 0: 100, about 1: 99, about 2: 98, about 3: 97, about 4: 96, about 5: 95, about 6: 94, About 7: 93, about 8: 92, about 9: 91 or about 10: 90.In the other aspects of the embodiment, composition includes that crosslinking glycosaminoglycan is poly- Close object and uncrosslinked Glycosaminoglycan Polymer, wherein gel: fluid ratio is (for example) at most 1: 99, at most 2: 98, at most 3: 97, At most 4: 96, at most 5: 95, at most 6: 94, at most 7: 93, at most 8: 92, at most 9: 91 or at most 10: 90.In the embodiment Other aspects, composition includes crosslinking Glycosaminoglycan Polymer and uncrosslinked Glycosaminoglycan Polymer, wherein gel: fluid ratio It is (for example) about 0: 100 to about 3: 97, about 0: 100 to about 5: 95 or about 0: 100 to about 10: 90.

In the other aspects of the embodiment, composition includes that crosslinking Glycosaminoglycan Polymer and uncrosslinked glycosaminoglycan are poly- Close object, wherein gel: fluid ratio is (for example) about 15: 85, about 20: 80, about 25: 75, about 30: 70, about 35: 65, about 40: 60, About 45: 55, about 50: 50, about 55: 45, about 60: 40, about 65: 35, about 70: 30, about 75: 25, about 80: 20, about 85: 15, about 90: 10, about 95: 5, about 98: 2 or about 100: 0.In the other aspects of the embodiment, composition includes crosslinking Glycosaminoglycan Polymer With uncrosslinked Glycosaminoglycan Polymer, wherein gel: fluid ratio be (for example) at most 15: 85, at most 20: 80, at most 25: 75, At most 30: 70, at most 35: 65, at most 40: 60, at most 45: 55, at most 50: 50, at most 55: 45, at most 60: 40, at most 65: 35, at most 70: 30, at most 75: 25, at most 80: 20, at most 85: 15, at most 90: 10, at most 95: 5, at most 98: 2 or at most 100∶0.In the other aspects of the embodiment, composition includes crosslinking Glycosaminoglycan Polymer and the polymerization of uncrosslinked glycosaminoglycan Object, wherein gel: fluid ratio be (for example) about 10: 90 to about 70: 30, about 15: 85 to about 70: 30, about 10: 90 to about 55: 45, About 80: 20 to about 95: 5, about 90: 10 to about 100: 0, about 75: 25 to about 100: 0 or about 60: 40 to about 100: 0.

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

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

The aspect of the present invention provides 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 It is effectively measured with the pain experienced after the composition.Thus, the anesthesia that composition disclosed in the present specification includes The amount of agent is between by between general composition weight meter about 0.1% to about 5%.The non-limiting examples of anesthetic include benefit card Cause, ambucaine (ambucaine), amolanone (arnolanone), 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), ***e (***e), 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-pseudo***e (psuedo***e), Pyrrocaine (pyrrocaine), Ropivacaine (ropivacaine), saligenin, totokaine (tetracaine), Tolycaine (toIycaine), trimecaine (trimecaine), zolamine (zolamine), a combination thereof And its salt.Amino ester local anesthetic includes procaine, chloroprocanine, ***e, 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 may 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 aspects of the embodiment, compositions disclosed herein include procaine, chloroprocanine, ***e, cyclomethycaine, Cimethocaine, propoxycaine, procaine, proparacaine, totokaine or its salt or any combination thereof.In the embodiment Other aspects, compositions disclosed herein include Articaine, bupivacaine, cinchocaine, Etidocaine, left Bu Bika Cause, lidocaine, mepivacaine, piperocaine, prilocaine, Ropivacaine, trimecaine or its salt or any combination thereof. The other aspect of the embodiment, compositions disclosed herein include lidocaine/prilocaine combination.

In the other aspects of the embodiment, compositions disclosed herein includes to be measured as (example based on the weight of total composition 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%, 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 includes to be measured as (for example) at least based on the weight of total composition 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, At least 0.9%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0% or at least 10% anesthetic.In also other aspects, compositions disclosed herein packet Containing based on the weight of total composition, measure as (for example) at most 0.1%, at most 0.2%, at most 0.3%, at most 0.4%, at most 0.5%, at most 0.6%, at most 0.7%, at most 0.8%, at most 0.9%, at most 1.0%, at most 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 aspects, compositions disclosed herein includes and is measured as (for example) about 0.1% to about based on the weight of total composition 0.5%, 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 about 0.5% to about 2.0% anesthetic.

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

In one aspect of the invention, injectable dermal augmentation agent is provided, it includes polymer, such as glycosaminoglycan are poly- Close object, such as hyaluronic acid polymer, for example, at least a part of crosslinked hyaluronic acid and additive or with the polymer group The beneficial agent of conjunction.

Beneficial agent with the combination of polymers includes vitamin, such as vitamin C.It is suitble to the ascorbic of form Non-limiting examples include the fat of sodium salt, sylvite and the calcium salt of ascorbic acid and ascorbic acid, ascorbic acid and long chain fatty acids Solubleness ester (ascorbyl palmitate or ascorbyl stearate), magnesium ascorbyl phosphate (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, with the polymer covalent bond and based on the weight of total composition, in group Close object present in 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, include following at least one 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 may include single dimension life Plain C reagents or multivitamin C reagents.

In another embodiment of the present invention, dermal augmentation agent is provided, wherein hyaluronic acid is crosslinked with BDDE.At this In a 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.The mankind are being introduced for example, providing When in skin, be released effectively ascorbic acid or other vitamins at least about 1 month to the mankind or it is long of about 20 months or longer when Between dermal augmentation agent.

The aspect of the present invention provides in part display complex modulus, elasticity modulus, viscous modulus and tan disclosed herein The hydrogel composition of δ.When applied force (stress, deformation), compositions disclosed herein has viscoplasticity, because composition has (liquid for example uncrosslinked glycosaminoglycan is poly- for elastic component (solid-like is for example crosslinked Glycosaminoglycan Polymer) and sticky ingredient Close object or carrier phase).The rheological attributes for describing the property are complex modulus (G*), define composition to the total anti-of deformation Property.Complex modulus is the plural number for having real and imaginary parts：G*=G '+iG ".The absolute value of G* is Abs (G*)=Sqrt (G '²+G ″²).Complex modulus can be defined as to the summation of elasticity modulus (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 respectively integrally incorporated accordingly by reference.

Elasticity modulus or modulus of elasticity refer to the ability of hydrogel material resistance to deformation, or on the contrary, when to its applied force Tendency of the object to impermanency deformation.Elasticity modulus characterizes the fastness of composition, and because it is described from group The energy storage for closing object movement, so also referred to as storage modulus.Elasticity modulus 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 elasticity modulus 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 Elasticity modulus in (Pascal ' s)；Stress is transmutative force divided by the area of applied force；And it is that stress causes to strain Variation and the ratio between object original state.Although depending on the speed of applied force, composition is harder, and the elasticity modulus having is got over It is high and so that material is deformed in distance to a declared goal the power of flower bigger, such as inject.It illustrates how to measure stress, including direction, permit Perhaps the elasticity modulus 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 elasticity modulus, tan δ=G "/G '.Falcone, ibid, 2009.For tan δ disclosed in the present specification 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 elasticity modulus.In the embodiment Aspect, hydrogel composition show (for example) about 25Pa, about 50Pa, about 75Pa, about 100Pa, about 125Pa, about 150Pa, about 175Pa, about 200Pa, about 250Pa, about 300Pa, about 350Pa, about 400Pa, about 450Pa, about 500Pa, about 550Pa, about 600Pa, about 650Pa, about 700Pa, about 750Pa, about 800Pa, about 850Pa, about 900Pa, about 950Pa, 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, The elasticity modulus of 000Pa, about 2,100Pa, about 2,200Pa, about 2,300Pa, about 2,400Pa or about 2,500Pa.In the embodiment party The other aspects of case, hydrogel composition show (for example) 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, The elasticity modulus of 000Pa, at least 2,100Pa, at least 2,200Pa, at least 2,300Pa, at least 2,400Pa or at least 2,500Pa. At the other aspect of the embodiment, hydrogel composition shows (for example) 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, The elasticity modulus of 200Pa, at most 1,300Pa, at most 1,400Pa, at most 1,500Pa or at most 1,600Pa.The embodiment Other aspect, hydrogel composition show (for example) 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 (for example) about 10Pa, about 20Pa, about 30Pa, about 40Pa, about 50Pa, about 60Pa, about 70Pa, about 80Pa, about 90Pa, about 100Pa, about 150Pa, about 200Pa, about 250Pa, about 300Pa, about 350Pa, about 400Pa, about The viscous modulus of 450Pa, about 500Pa, about 550Pa, about 600Pa, about 650Pa or about 700Pa.In other sides of the embodiment Face, hydrogel composition show (for example) 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 The at most viscous modulus of 700Pa.At the other aspect of the embodiment, hydrogel composition shows (for example) 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 shows (for example) about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, About 2.2, about 2.3, about 2.4 or about 2.5 tan δ.In the other aspects 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 1.9, at most 2.0, at most 2.1, at most 2.2, at most 2.3, at most 2.4 or at most 2.5 tan δ.The embodiment in addition Other aspects, hydrogel composition show (for example) about 0.1 to about 0.3, about 0.3 to about 0.5, about 0.5 to about 0.8, about 1.1 To about 1.4, about 1.4 to about 1.7, about 0.3 to about 0.6, about 0.1 to about 0.5, about 0.5 to about 0.9, about 0.1 to about 0.6, about 0.1 to about 1.0, about 0.5 to about 1.5, about 1.0 to about 2.0 or about 1.5 to about 2.5 tan δ.

The hydrogel group with the transparency and/or translucence disclosed herein is provided in part in terms of this specification Close object.Optical transparence is to allow physical property of the visible light by material, and translucence is (also referred to as translucent or translucent Degree) only light diffusion is allowed to pass through.Opposite property is opacity.Transparent material is limpid, and trnaslucent materials cannot 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 conducts (for example) about 75% light, about 80% light, about 85% light, about 90% light, about 95% Light or about 100% light.In the other aspects of the embodiment, hydrogel combination diffusion conducts (for example) 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 the diffusion (for example) 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 light of conduction 100%.

Hydrogel composition disclosed herein and optionally and carrier can be further processed 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 diameter, for example, 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 provides in part the composition of injectable disclosed herein.As used herein, term " can Injection ", which refers to material, has injection device of the use with fine needle to property necessary to composition described in individual's skin Zoned application. As used herein, term " fine needle " refers to 27G or smaller needles.It can be by determining hydrogel particle as discussed above Size realize compositions disclosed herein syringeability.

In terms of the embodiment, hydrogel composition disclosed herein can be injected by fine needle.In the embodiment Other aspects, hydrogel composition disclosed herein can be injected by the needle of (for example) about 27G, about 30G or about 32G.At this The other aspect of embodiment, hydrogel composition disclosed herein can by (for example) 22G or smaller, 27G or smaller, 30G or smaller or 32G or smaller needle are injected.In the other aspect of the embodiment, hydrogel combination disclosed herein Object can be by (for example) about 22G to about 35G, about 22G to about 34G, about 22G to about 33G, about 22G to about 32G, about 22G to about The needle of 27G or about 27G to about 32G are injected.

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

The aspect of the present invention provides in part the hydrogel composition disclosed herein for showing cohesiveness.Cohesiveness, Also referred to as cohesion adhesion gravitation, bonding force or compressing force, are played between similar molecules in material by the work of molecule synthesis one The physical property of material caused by intermolecular attraction.Cohesiveness is indicated with fors (gmf).Among other things, caking property By initial free Glycosaminoglycan Polymer molecular weight than remaining free sugar 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 application 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 keeping functional when cycle.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 It holds positioned at using 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 provides in part, disclosed herein shows the infiltration of physiologically acceptable volume molecule The hydrogel composition of concentration (osmolarity).As used herein, term " volume molecule osmotic concentration " refers in solution The concentration of osmotically active solute.As used herein, term " physiologically acceptable volume molecule osmotic concentration " refers to and lives The normal function of organism is consistent or its distinctive volume molecule osmotic concentration.Thus, when being administered to mammal, using this Hydrogel composition disclosed in text shows the volume molecule osmotic concentration generally without long-term or permanent adverse effect.Hold Product gram molecule osmotic concentration is indicated 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 the molal quantity of osmotically active solute particle rather than The molal quantity of solute.Because certain compounds can dissociate in the solution, and others cannot, so there is difference.It can be under Row expression formula calculates the volume molecule osmotic concentration of solution：WhereinFor infiltration coefficient, say The Non Ideal Degree of solution is illustrated；η be molecular dissociation at particle (such as ion) quantity；And C is the mole dense of solute Degree；And i is the index for indicating specific solute homogeneity.The conventional method for measuring solution can be used to measure hydrogel disclosed herein The volume molecule osmotic concentration of composition.

In one embodiment, hydrogel composition disclosed herein shows physiologically acceptable volume molecule 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 living 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 indicated 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.Osmometer can be used to measure the weight molar concentration infiltration of solution Concentration.Most common instrument, which is freezing point, in modern laboratory reduces osmometer.The apparatus measures are oozed with weight molar concentration Saturating concentration increases the variation (freezing point reduction osmometer) that freezing point occurs in solution or increases solution with osmolality The variation (the low osmometer of steam drop) that middle vapour pressure occurs.

In terms of the embodiment, hydrogel composition shows (for example) about 100mOsm/L, about 150mOsm/L, about 200mOsm/L, about 250mOsm/L, about 300mOsm/L, about 350mOsm/L, about 400mOsm/L, about 450mOsm/L or about The volume molecule osmotic concentration of 500mOsm/L.In the other aspects of the embodiment, hydrogel composition is shown (for example) At least 100mOsm/L, at least 150mOsm/L, at least 200mOsm/L, at least 250mOsm/L, at least 300mOsm/L, at least The volume molecule osmotic concentration of 350mOsm/L, at least 400mOsm/L, at least 450mOsm/L or at least 500mOsm/L.At this The other aspect of embodiment, hydrogel composition show (for example) 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 The volume molecule osmotic concentration of 450mOsm/L or at most 500mOsm/L.In the other aspect of the embodiment, water-setting Glue composition show (for example) about 100mOsm/L to about 500mOsm/L, about 200mOsm/L to about 500mOsm/L, about 200mOsm/L to about 400mOsm/L, about 300mOsm/L to about 400mOsm/L, about 270mOsm/L to about 390mOsm/L, about 225mOsm/L to about 350mOsm/L, about 250mOsm/L are to about 325mOsm/L, about 275mOsm/L to about 300mOsm/L or about The volume molecule osmotic concentration of 285mOsm/L to about 290mOsm/L.

The aspect of the present invention provides 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 thermal stability ", " generally thermostabilization ", " autoclave stabilization " or " steam sterilizing stabilization " refer to water disclosed herein Gel combination is generally stablized when by heat treatment as disclosed herein.

It can be by making hydrogel composition heat-treated, such as under normal pressure or pressurization (for example, high pressure sterilization) Steam sterilizing measures 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) it surveys The extruding force variation (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 variation of extruding force is less than 2N Show that hydrogel composition is generally stablized；And/or 2) measure the rheological characteristic change of hydrogel composition disclosed herein after sterilizing Change, is surveyed wherein subtracting (the not tan δ 1Hz of the gel preparation of additive) by (the tan δ 1Hz for having the gel preparation of additive) The variation of amount, tan δ 1Hz shows that hydrogel composition is generally stablized less than 0.1.Thus, generally stabilization disclosed herein 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, volume molecule osmotic concentration, pH or heat treatment needed for hydrogel.

In one embodiment, the use of maintain the heat treatment process of required hydrogel properties disclosed herein include osamine 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 aspects of the embodiment, make With (for example) at least 100 DEG C, at least 105 DEG C, at least 110 DEG C, at least 115 DEG C, at least 120 DEG C, at least 125 DEG C or at least 130 DEG C heat treatment process include the hydrogel composition of Glycosaminoglycan Polymer and at least one reagent disclosed herein.In the reality The other aspect for applying scheme, using (for example) 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, it measures 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) estimate the clarity and color of hydrogel composition after 45 DEG C of heat treatments, hydrogel composition is limpid and uncolored table Subject hydrogel compositions are generally stablized；2) extruding force for measuring hydrogel composition disclosed herein after being heat-treated at 45 DEG C 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) it measures, the variation of extruding force shows water-setting less than 2N Glue composition is generally stablized；And/or 3) the rheological characteristic variation of hydrogel composition disclosed herein after sterilizing is measured, 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) it measures, the variation of tan δ 1Hz 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 is generally stablized (for example) about 3 months, about 6 at room temperature 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 aspects of the embodiment, hydrogel composition is generally stablized (for example) at room temperature At least three moon, at least six moon, at least nine moon, at least 12 months, at least 15 months, at least 18 months, at least 21 months, extremely It is 24 months, at least 27 months, at least 30 months, at least 33 months or at least 36 months few.In the other aspects of the embodiment, Hydrogel composition is generally stablized (for example) about 3 months to about 12 months, about 3 months to about 18 months, about 3 at room temperature 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 A 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 not limited to other pharmaceutically acceptable components, including but not limited to buffer, Preservative, tension regulator, salt, antioxidant, osmolality modifier, emulsifier, 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, phosphate buffer and phosphate buffered saline (PBS).It can will be any The pharmaceutically acceptable buffer of concentration is using the slow of effective concentration for preparing pharmaceutical composition disclosed herein, condition Electuary resumes treatment a effective amount of active constituent.The non-limiting examples of physiologically acceptable buffer concentration are present in about Within the scope of 0.1mM to about 900mM.The pH of pharmaceutically acceptable buffer is adjusted, condition is that gained preparation is pharmaceutically It is acceptable.It should be understood that acid or alkali can be used to adjust the pH of pharmaceutical composition as needed.The pH levels of any buffering can be used for Compounding pharmaceutical composition, condition are to resume treatment a effective amount of matrix polymer using effective pH levels.Physiologically it is subjected to The non-limiting examples of pH be present in the range of about pH 5.0 to about pH 8.5.For example, hydrogel combination disclosed herein The pH of object 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 not limited to sodium pyrosulfite, sodium thiosulfate, acetylcysteine, fourth Hydroxyl anisole and Butylated Hydroxytoluene.Pharmaceutically acceptable preservative includes but not limited to benzalkonium chloride, methaform, thimerosal, vinegar Sour benzene mercury, phenylmercuric nitrate stabilize oxygen chlorine composition, such as(Allergan, Inc.Irvine, CA) and chelating Agent, such as DTPA or DTPA- bisamides, DTPA calcium and CaNaDTPA- bisamides.

Include but 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 together be formed with many acid, including But it is not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic 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 may include in pharmaceutical composition disclosed herein in pharmaceutical field These and other substance known.Other non-limiting examples of pharmaceutically acceptable component can in such as Ansel, ibid, (1999)；Gennaro, ibid, (2000)；Hardman, ibid, (2001)；And Rowe is ibid found, thus in (2003) It will wherein each be integrally incorporated by reference.

The aspect of the present invention provides in part soft group by application hydrogel composition disclosed herein treatment individual The method for knitting situation.As used herein, term " treatment " refers to be mitigated or eliminated in individual and is characterized in that soft tissue defect, lacks The beauty of the soft tissue condition of damage, disease and/or illness or clinical symptoms；Or it is characterized in that 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 mitigate 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%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%.Can by observe one or more beauty, It clinical symptoms and/or is characterized in that in treatment with the relevant physiological index determining of situation hydrogel composition disclosed herein Effect in the situation of soft tissue defects, disease and/or illness.It also can be by showing that soft tissue lacks to the needs reduction 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 treated with compound or composition disclosed herein Candidate.

Hydrogel composition according to the present invention is applied to individual.Individual is typically any age, gender or race The mankind.In general, any individual for being the candidate of conventional program treatment soft tissue condition is also the time of method disclosed herein It chooses.Although the subject for experiencing skin aging sign is adult, the premature aging or other for being suitble 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 Gel combination and method can be applied to seek body part or the small/moderate in region is expanded, change in shape or profile change Individual, this technically can not possibly or cannot aesthetically be received with existing soft tissue implanted prosthetics.Public affairs are assented except informing comprehensively It opens 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 increases, breast reconstruction, breast is fixed, breast is too small, breast development is not complete, Poland's 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 increases, face reproduces, U.S. rope therapy, parry-Romberg syndrome (Parry-Romberg Syndrome), lupus erythematosus profundus, corium point, 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 abdomen), buttocks, big The increase of leg, shank (including calf), foot (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 of drop application.

Usually 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 determine together with any method disclosed herein The amount of the hydrogel composition used.The effect of composition application can use following one or more clinical and/or beauty measurement marks Standard indicates：Soft tissue shape changes and/or improves, soft tissue size changes and/or improves, soft tissue profile changes and/or changes Kind, function of organization changes and/or improves, tissue ingrowth is supported and/or new collagen deposition, composition continuous infusion, Patient satisfaction and/or quality of life improve 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 wrinkle, fold or the microgroove on skin Resistance；Skin moisturizing；Skin elasticity increases；Pachylosis is mitigated or eliminated；Skin tautness increases and/or improves；Drag line Or striae of pregnancy reduces or eliminates；The colour of skin, gloss, brightness and/or honorable enhancing and/or improvement；Skin color enhances and/or changes 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 effect for composition and method that sphincter is supported Fruit can use following one or more clinical measures canonical representations：The reduction of incontinence frequency, continuous infusion, patient satisfaction and/or life Quality advance living and the use that can plant external filler are reduced.

In terms of the embodiment, the amount of the hydrogel composition of application is (for example) about 0.01g, about 0.05g, about 0.1g, about 0.5g, about 1g, about 5g, about 10g, about 20g, about 30g, about 40g, about 50g, about 60g, about 70g, about 80g, about 90g, About 100g, about 150g or about 200g.It is (for example) about in the amount of the other aspects of the embodiment, the hydrogel composition of application 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 application is (for example) about 0.01mL, about 0.05mL, about 0.1mL, about 0.5mL, about 1mL, about 5mL, about 10mL, about 20mL, about 30mL, about 40mL, about 50mL, about 60mL, about 70g, about 80mL, about 90mL, about 100mL, about 150mL or about 200mL.It is in the amount of the other aspects of the embodiment, the hydrogel composition of application (for example) about 0.01mL to about 0.1mL, about 0.1mL are to about 1mL, about 1mL to about 10mL, about 10mL to about 100mL or about 50mL To about 200mL.

Usually by body part or region based on beauty and/or clinical effectiveness and treatment needed for individual and/or doctor Measure the duration for the treatment of.In terms of the embodiment, soft tissue can be treated using hydrogel composition disclosed herein Situation, for example, 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 aspects 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 A 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 aspects 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 is provided in part using hydrogel composition disclosed herein.As used herein, term " application " be directed toward individual provide compositions disclosed herein any delivery mechanism, 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 not limited to below because usually determining：The type of skin, the position of skin, skin original 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 drug effect of the excretion rate, the particular composition used of determining composition, the particular composition used include Property, particular route of administration, specific feature, individual medical history and risk factors (such as age, weight, health status etc.) or its What is combined.In the one side of the embodiment, by injecting the skin area to individual using compositions disclosed herein.

It is usually 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, including but not limited to band needle injection, pistol (for example, hydropneumatic compression pistol), conduit, part Or it is implanted by Direct Surgery.Hydrogel composition disclosed herein can be applied to skin area, such as corium area or rim surface zone. For example, injecting water disclosed herein from about 4mm to the needle of about 14mm using diameter about 0.26mm to about 0.4mm and length range Gel combination.Optionally, needle can be 21-32G and length is about 4mm to about 70mm.Preferably, needle is disposable Needle.The needle can be combined 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 be applied a primary or point several periods, 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.It is applied to individual With hydrogel composition disclosed herein can one month or two months it is primary every 3,6,9 or application in 12 months it is primary.

The aspect of the present invention provides in part corium area.As used herein, term " corium area " refers to true comprising epidermis- 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 provides water proofing property and as the barrier of infection；Corium is used as the position of cutaneous appendage； With subcutaneous tissue (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 of connective tissue under epidermis and is giving of body stress and strain.Corium is logical with epidermis Basement membrane is crossed to be tightly coupled.It also includes 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 the cell from own And remove waste from the basal layer of epidermis.Corium is divided into the areas Liang Ge in structure：Neighbouring epidermis, the superficial area for being known as mamillary region The thicker area in deep of referred to as webbed region.

Mamillary region is made of the areolar connective tissue that relaxes.It is referred to as nipple because of its finger-like projection object, prolongs to epidermis It stretches.Nipple provides " rugged " surface to intermesh with epidermis for corium, strengthens the connection between two layers of skin. Webbed region is located at mamillary region depths, and usually thick very much.It is made of fine and close irregular connective tissue, 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.The striae of pregnancy generated by pregnancy also is located in corium.

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

In the one side of the embodiment, is 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 (for example) epidermal-dermal junctional area, nipple Area, webbed region or any combination thereof injection, to individual corium area apply hydrogel composition disclosed herein.

Advantageously, some compositions of the present invention it is particularly useful to the appearance for reducing microgroove (for example) in patient's thin skin area and Effectively.For example, the method for providing microgroove treatment, is included in the depth no more than about 1mm, elsewhere herein to patient's application The step of dermal augmentation agent composition of description.

The method that the other aspects of this specification partly disclose treatment skin, includes to skin The step of individual application hydrogel composition disclosed herein, wherein applying said compositions improve skin, 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, To treat skin dehydration.In the another aspect of the embodiment, the treatment inelastic method of skin includes to skin The step of inelastic individual of skin applies hydrogel composition disclosed herein, wherein applying said compositions increase skin Elasticity lacks flexibility to treat skin.At the another aspect of the embodiment, the method for treating pachylosis includes to trouble There is the step of individual of pachylosis applies hydrogel composition disclosed herein, wherein applying said compositions reduce skin It is coarse, to treat pachylosis.At the another aspect of the embodiment, method that treatment skin lacks tautness include to Lack the step of individual of tautness is using hydrogel composition disclosed herein with skin, wherein applying said compositions make Skin is tighter, lacks tautness to treat skin.

At the another aspect of the embodiment, the method for treating skin stretch line or striae of pregnancy includes 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, to having treated skin stretch line or striae of pregnancy.In the another aspect of the embodiment, treatment The method of ochrodermia includes the steps that applying hydrogel composition disclosed herein to the individual with ochrodermia, wherein applying The colour of skin or brilliance are enhanced with the composition, to treat ochrodermia.In the another aspect of the embodiment, skin is treated The method of skin wrinkle includes the steps that applying hydrogel composition disclosed herein to the individual with wrinkle of skin, wherein applying The composition reduce or eliminates wrinkle of skin, to treat wrinkle of skin.In the another aspect of the embodiment, treatment The method of wrinkle of skin includes the steps that applying hydrogel composition disclosed herein to individual, and wherein applying said compositions make The anti-wrinkle of skin of skin, to treat wrinkle of skin.

In some embodiments, dermal augmentation agent has lasting bioavailability.The mankind are being introduced for example, providing When in skin, (for example, through intradermal or subcutaneous introducing mankind to correct the soft tissue hole defect on face) are anti-to mankind's release 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 assessment is 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).Since alpha-glucosidase is attached on cell membrane, institute Occurred in filler/Cellular interfaces with the release of AsA.Further, AsA will degrade from the release of HA-AA2G with HA so that It is available to fibroblast to obtain AA2G.Therefore AA2G conjugation and duration of the release of AsA depending on HA.Conjugation is about The gel of 5mol% can be at least up to 1 month, such as active vitamin C is discharged within about 3~5 month periods；10mol% The gel of conjugation can at least be up to release active vitamin C in 6~8 months periods；The gel of 15mol% conjugations can be At least it is up to release active vitamin C in 10~a month period；30mol% is 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.

The effective concentration of AsA is 0.05mM and keeps 2 days 2.13*2/ (0.05*30)=2.8 (a month) of effective ＞

In one embodiment of the invention, provide a kind of dermal augmentation agent, it includes with Star-PEG epoxidations The crosslinked hyaluronic acid of object 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 suitble to obtain containing by 4- armlets The composition of the capped AA2G of oxide (AA2G-4 armlets oxide), unreacted 4 armlet oxide and free AA2G.4- arms The capped AA2G of epoxides (AA2G-4 armlets oxide) is combined through epoxy group with hyaluronic acid.Unreacted 4 armlet oxidation Object 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, a kind of dermal augmentation agent is provided, it includes crosslinked transparent with BDDE Matter is sour and derives with the vitamin C combined with hyaluronic acid with the conjugation between about 3mol% and about 10mol% Object 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).

The method for preparing this dermal augmentation agent includes making BDDE with ascorbic acid 2- glucosides (AA2G) by certain ratio Example reaction, reaction temperature and reaction time are suitble to obtain containing by the capped AA2G of BDDE (AA2G-BDDE), unreacted BDDE With the composition of free AA2G.The capped AA2G of BDDE (AA2G-BDDE) are combined through epoxy group 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 a concentration of 6.3 units/g gels of alpha-glucosidase, AA2G is several in 15min It is fully converted to AsA.When a concentration of 4.7 units/g gels of alpha-glucosidase, 30min is needed to be fully converted to AA2G AsA.Further decreasing alpha-glucosidase concentration causes AA2G slow to the conversion of AsA.

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

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

In one aspect of the invention, dermal augmentation agent is provided, it is especially effective to the appearance for treating and eliminating microgroove, Such as with respect to the gauffer of superficial on skin, such as, but not limited to eyes, the areas Lei Gou, 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.It has ground Embodiment of the present invention is sent out, providing 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 generated by Tyndall effect.Microgroove or superficial Wrinkle be generally understood as it is usually most thin in skin, i.e., the dermis thickness of skin be less than 1mm facial area (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 needles (needle for being commonly used in microgroove gel application) Mean outside diameter is about 0.30 and about 0.24mm.

The present invention provides the dermal augmentation agent groups that will not lead to Tyndall effect being for example described elsewhere herein Close object.For example, the composition of the present invention includes and the hyaluronic acid component of cross-linking agents, the additive in addition to cross-linking component； When in the corium area for being administered to patient, relative in addition to no additive, substantially the same composition, the combination Object 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, cross-linking component is BDDE and conjugation between about 3mol% and about 10mol%, Or up to 15mol% or higher.In some embodiments, the composition is further included suitable for being carried after injection for patient For the anesthetic comfortably measured, such as lidocaine.

The method for additionally providing the microgroove on treatment patient skin.The method generally includes to introduce example into patient skin The step of composition as described herein.Such as the composition includes the crosslinking group of hyaluronic acid component, cross-linked-hyaluronic acid Point and additive in addition to cross-linking component 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 includes using EDC chemistry and diamines or polyamine crosslinkers Crosslinked hyaluronic acid component.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 repetitive unit (for example, HA dimers).Therefore, 10mol% conjugations mean every 100 HA repetitive units The AA2G combined containing 10.The method illustrated in following example 2 or other methods well known by persons skilled in the art can be used Calculations incorporated degree.

Embodiment

Embodiment 1：It is combined using BDDE as crosslinking agent AA2G with crosslinked HA gels

The low-molecular-weight hyaluronic acid (LMW HA) of 400.6mg is set to be hydrated in the 1wt%NaOH of 1802mg in syringe ~30min.The AA2G of 800.7mg is put into bottle, the 10%NaOH of the BDDE and 1416.8mg of 713.7mg are subsequently placed into. Before being added in hydration HA, make the above solution (pH ＞ 12) reaction~20min in 50 DEG C of water-baths.After addition, pass through Being transmitted back and forth between 2 syringes makes mixture mix~20 times.Mixed paste is put into bottle and 50 DEG C of water-baths~ 2.5h.The 12M HCl of 223.5mg are added in 9.05g PBS (pH7.4).After~2.5h, HA-AA2G gels are formed.It will coagulate Glue is cut into slices, and adds HCl-PBS solution thereto.So that gel is neutralized and is 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 15,000 It dialyses in MWCO RC bag filters and in PBS (pH7.4) buffer solution.Dialyse progress~185h, frequently replaces PBS buffer solution. After dialysis, gel is put into syringe and is stored in 4 DEG C of refrigerators.

Embodiment 2：The measurement 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.Per ＞ 8h in 1L PBS, do not occur stopping dialysis when obvious AA2G.It is divided light using UV/Vis Degree meter (Nanodrop 2000C, ThermoScientific) measures AA2G at 260nm.Use the difference of AA2G in 2%HA Concentration (A@260nm=1.4838 [AA2G (mM)]) calculates the calibration curve of AA2G.

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

The mmol of AA2G after dialysis：It will be inhaled in equation (A@260nm=1.4838 [AA2G (mM)]) after dialysis It receives and is set under 260nm.

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

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

Embodiment 3：The measurement of gel rheology

It is solidifying using being obtained in oscillation parallel-plate rheometer (Anton Paar, Physica MCR 301) measurement embodiment 1 The property of glue.A diameter of 25mm of plate used.Inter-plate gap is set as 1mm.For measuring 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 crosslinked HA gels In conjunction with

Program to it is described in embodiment 1 similar.Conjugation is changed by adjusting crosslinking agent and HA and AA2G molar ratios.Such as Gelling properties are measured described in embodiment 3.Details as Follows：

Make the LMW HA of 400.8mg hydration~30min in the 1%NaOH of 1752.1mg in syringe.By 800.3mg's AA2G is put into bottle, is subsequently placed into the 10%NaOH of the BDDE and 1402.0mg of 354.1mg.It is hydrated it in HA being added to Before, make the 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.Mixed paste is put into bottle and 50 DEG C of water-baths~2.5h.By the 12M of 140.9mg 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.So that gel is neutralized and is 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 It dialyses in PBS (pH7.4) buffer solution.Dialyse progress~164.5h, frequently replaces PBS buffer solution.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 crosslinked HA gels, conjugation 5.3%, and G ' is ~300Pa.

Make the LMW HA of 400.3mg hydration~30min in the 1%NaOH of 3002.0mg in syringe.By 800.5mg's AA2G is put into bottle, is subsequently placed into the 10%NaOH of the BDDE and 1100.0mg of 264.3mg.It is hydrated it in HA being added to Before, make the 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.Mixed paste is put into bottle and 50 DEG C of water-baths~2.5h.By the 12M of 104.2mg HCl is added in 8.5128g PBS (pH7.4).After~2.5h, HA-AA2G gels are formed, and add HCl-PBS thereto Solution.So that gel is neutralized and expands entire weekend (~55h) on orbital shaker.Gel is sieved by~60 μm of sieves simultaneously And by transmitting mixing~20 times back and forth between 2 syringes.Gel is put into 15,000 MWCO RC bag filters and It dialyses in PBS (pH7.4) buffer solution.Dialyse progress~114h, frequently replaces PBS buffer solution.After dialysis, gel is put into note In emitter and it is stored in 4 DEG C of refrigerators.Conjugation and gel rheology are measured by the program described in embodiment 2 and 3.In conjunction with Degree is 5.3%.Gel storage modulus is~300Pa.

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

Make the LMW HA of 200.4mg hydration~30min in the 1%NaOH of 2000mg in syringe.By the AA2G of 400mg It is put into bottle, is subsequently placed into the star-PEG epoxides of 312.7mg and the 10%NaOH of 1026.5mg.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.Mixed paste is put into bottle and 50 DEG C of water-baths~2.5h.By the 12M of 187.4mg HCl is added in 3.034g PBS (pH7.4).After~2.5h, HA-AA2G gels are formed, and addition HCl-PBS is molten thereto Liquid.So that gel is neutralized and expands entire weekend (~68h) 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 It dialyses in PBS (pH7.4) buffer solution.Dialyse progress~95h, frequently replaces PBS buffer solution.After dialysis, gel is put into injection In device and it is stored in 4 DEG C of refrigerators.Conjugation and gel rheology are measured by the program described in embodiment 2 and 3.Conjugation It is 29.4%.Gel storage modulus is~235Pa.

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

Make the LMW HA of 200.3mg hydration~30min in the 1%NaOH of 2000mg in syringe.By 400.2mg's AA2G is put into bottle, is subsequently placed into the star-PEG epoxides of 313.4mg and the 10%NaOH of 1022.6mg.It will be above molten Liquid is added in hydration HA.After addition, mixture is set to mix~20 times by being transmitted back and forth between 2 syringes.It will mix The paste of conjunction is put into bottle and 50 DEG C of water-baths~2.5h.The 12M HCl of 196.5mg 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 It is expanded on device (~for 24 hours) 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 15,000 MWCO RC bag filters and is dialysed in PBS (pH7.4) buffer solution.Dialysis carries out ~98.5h, frequently replaces PBS buffer solution.After dialysis, gel is put into syringe and is stored in 4 DEG C of refrigerators.By implementation Program described in example 2 and 3 measures conjugation and gel rheology.Conjugation is 27.8%.Gel storage modulus be~ 363Pa。

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

Make the HMW HA of 400.3mg hydration~30min in the 4wt%NaOH of 2501.3mg in syringe.By 1200mg AA2G be put into bottle, be subsequently placed into the 16wt%NaOH of the BDDE and 1178.6mg of 304.7mg.Be added to hydration HA it Before, make the 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.Mixed paste is put into 20cc bottles and 50 DEG C of water-baths~2.5h.After~2.5h, shape At HA-AA2G gels.It is molten that the 12M HCl of 226.6mg are added to acquisition HCl-PBS in 10 × PBS of 8492.2mg (pH7.4) Liquid and add HCl-PBS solution so that gel neutralize 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 It dialyses in 20,000 MWCO CE bag filters and in PBS (pH7.4) buffer solution.Dialyse progress~114h, frequently replaces 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 crosslinked LMW HA gels, and conjugation is 15mol%, G ' it is about 365Pa.

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

The vitagen of 801.9mg is individually dissolved in the 10wt%NaOH of 1459.7mg and and is reacted in advance with BDDE HA mixing.Mixture continuation is set to react 2.5h again at 50 DEG C.After~2.5h, HA- vitagens are formed.By the 12M of 195mg HCl, which is added in 10 × PBS (pH7.4) of 9004.0mg, to be obtained HCl-PBS solution and adds HCl-PBS solution so that gel It neutralizes and expands.So that gel is neutralized and expands 48h or more 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 It dialyses in PBS (pH7.4) buffer solution.Dialyse progress~120h, frequently replaces PBS buffer solution.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.It is retouched using with embodiment 2 It is about 15mol% that the AA2G stated, which measures similar method and measures conjugation,.Gel storage modulus is about 365Pa.

Embodiment 10：Vitagen is combined through amide chemistry with linear HA

The HMW HA of 200.3mg are made to be hydrated in the water of 10ml in 60cc syringes.The vitagen of 500mg is dissolved in It is neutralized to pH 4.8 in 0.5ml water and by solution.The NHS of the EDC of 197.7mg and 149mg is individually dissolved in 6ml water.It will The above solution (solution and EDC/NHS solution) is added in another 60cc syringes equipped with 23.5ml water.By being injected at 2 Mixing 20 times is transmitted between device back and forth.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 measured like method.Conjugation is about 10mol%.

Embodiment 11：The combination of AA2P and crosslinked HA gels

Make hydration~30min in 5.2 buffer solutions of MES of LMW HA 1000mg in syringe of 200.4mg.By 292mg AA2P be put into bottle, then add 300mg star-PEG amine.The above solution is set to react at room temperature whole night.It is buffered with PBS Liquid makes gel hydration and is dialysed with PBS buffer solution to remove unreacted AA2P.As described in embodiment 2 and 3, characterize last Gel is to measure conjugation and gel rheology.Conjugation is about 20mol%.Storage modulus (G ') is about 500Pa.

Embodiment 12

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

To any gel described in above example, after dialysis, the HA gels that dissociate in right amount are added into gel to carry Height changes gel cohesiveness and/or syringeability.For example, making free HA fibers 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 Gained is gel-filled to being easy in the asepsis injector of filling and high pressure steam sterilization is at least about under sufficient temp and pressure 1min.After high pressure steam sterilization, packs HA/AA2G final products and is distributed to doctor and injected for use as dermal augmentation agent superficial, Improve the appearance of microgroove in periorbit or other facial areas.

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 It is 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, the concentration of for example, about 0.3% (w/w).Then the gained pH of HA/AA2G/ lidocaine mixtures is about 7 and HA is a concentration of about 24mg/g.Mechanical mixture is carried out in the standard reaction device equipped with appropriate blender 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 is made to be combined with HA hydrogels.The following describe bases The example of combination described in one embodiment of the invention：

The HMW HA of 200mg are made to be hydrated in the pH 4.8MES buffer solutions of 10ml in 60cc syringes.In another injection In device, the retinoic acid of 200mg is dissolved in the boiling mixture (water/acetone volume ratio 1: 3) of 5ml.Pass through syringe connector Mix two above syringe about 20 times.Then the NHS of the EDC of 197.7mg and 149mg is dissolved in respectively in independent syringe In 6ml water.Syringe equipped with EDC and NHS is connect 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 unbonded retinoic acid with isopropanol, is then aseptically dialysed with PBS buffer solution.By gel packet 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, dimethylaminoethanol and g- tocopherols contain hydroxyl Base functional group (- OH).Using EDC chemistry through esterification, these additives is made to be combined with HA hydrogels.The following describe combinations Representative instance：

Make 2- (N- morpholines) ethanesulfonic acid (MES) buffer solution (pH of HMW HA 10ml in 60cc syringes of 200mg 4.8) hydration in.In another syringe, the retinol of 200mg is dissolved in the boiling mixture (water/acetone volume of 5ml Than 1: 3).Two above syringe is mixed by syringe connector about 20 times.Then by the EDC's of 197.7mg and 149mg NHS is dissolved in respectively in the 6ml water in independent syringe.Syringe equipped with EDC and NHS and the syringe equipped with HA and retinol Connection, to make reactant mix at least 20 times by being transmitted back and forth between 2 syringes.Mixture is stored in an injection In device and immerse 4h in 37 DEG C of baths.Gel is dialysed with isopropanol to remove unbonded retinol, then aseptically It is dialysed with PBS buffer solution.It will be stored in gel pack to 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.) with activation The carboxyl of HA.

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

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

The Juvederm gels of 2gm are made to be mixed with the NHS of the EDC of 200gm and 150mg at room temperature.Then 200mg is added Retinol in 3ml acetone-water mixtures.The above mixture is set to react 4h at 37 DEG C.Gel is dialysed to go with isopropanol Except unbonded retinol, then aseptically dialysed with PBS buffer solution.By in gel pack to asepsis injector and in It is 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 epidermal growth factor (EGF), transforming growth factor (TGF) and peptide, can be with HA is combined to be formed beneficial to dermal augmentation agent.These additives are combined by amidation chemistry with HA.The following describe combinations Representative instance：

The HMW HA of 200.3mg are made to be hydrated in 5.4 buffer solutions of MES pH of 10ml.It is molten in 100mg MES to add 20mg EGF in liquid.To the above mixture, the EDC and 149mg of 197.7mg are added.Gained reaction mixture is set to be reacted at 37 DEG C 4h.After the completion of reaction, is further dialysed gel with isopropanol, then aseptically dialysed with PBS buffer solution.By gel packet It is attached in asepsis injector and is stored at 4 DEG C.

Invention further provides the methods of enhancing transplant fat regular organization activity power.The method may generally include to The step of patient introduces composition adjacent to the skin of transplant fat tissue, the composition is combination described elsewhere herein Object.For example, the composition may 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 aspects of the present invention, the method for treating 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

The mitogenesis of the stem cell (hASC) of Human Adipose Tissue-derived is made for assessment vitamin C and its derivative With, hASC in tissue culturing plastic, vitamin C (ascorbic acid) or derivatives thereof in free form is not supplemented in supplement or Culture 4 days in the complete MesenPro culture mediums (Invitrogen, Carlsbad, CA) of (vitagen or AA2G).By such as MTT measuring methods estimation proliferation described in manufacturer (ATCC, Manassas, VA).After 4 days, ascorbic acid 0.25,0.5 is found Proliferation is set (to be converted into purple first by by dehydrogenase with the concentration of 1mMYellow tetrazolium MTT measurement) (purple firstQuilt Detergent dissolves) respectively 60%, 80% and 96% is improved than lacking the control of ascorbic acid.Distinguished using the AA2G of same concentrations It is more than the proliferation raising for compareing 70%, 60% and 50% to obtain.Analog result is obtained with vitagen, shows increase than control respectively Add 70%, 60% and 30%.In short, vitamin C and its derivative, AA2G and vitagen are deposited in the culture medium containing growth factor The hASC proliferation in cell culture is improved under.

With in conjunction with ascorbic crosslinking HA gels

It describes to have in embodiment 19 and 20 below and combines the ascorbic gel based on crosslinking HA and use Isosorbide-5-Nitrae- Preparation of the butanediol diglycidyl ether (BDDE) as crosslinking agent, certain embodiments according to the present invention show 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 best Rheological characteristic, excellent syringeability and high HA concentration (25mg/g).While not wishing to be constrained by any particular theory of operation, still The inventors have discovered that making HA and BDDE be crosslinked the property for changing gel significantly in the presence of AA2G or vitagen, relatively In with the crosslinked commercialization HA gels of BDDE, gel has high crosslink density, high HA concentration, low viscosity and low extruding force.Because handing over There are AA2G or vitagens during connection, so the gel currently formed has individually or via BDDE, as side group and bridge joint These ascorbic acid derivates of the crosslinking agent of HA chains and the coupling of HA chains.The microstructure of gel has changed, and leads to gel i.e. Make by the way that carefully to the needle of 30G, extruding force is also very low.Moreover, gel is with about 3mol% to about 10mol% or up to about The vitamin C of 15mol% combined with HA.In injected gel, they are by endogenous enzymes, such as from fibroblastic α- Glucuroide or phosphoric acid enzyme r e lease active vitamin C.Active vitamin C can cause skin collagen and be formed and can be risen certainly Gel degradation is inhibited by the effect of base scavenger.

Embodiment 19

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

After the 5wt%NaOH solution of addition 1764.0mg, make the LMW HA and 402.3mg of 400.1mg in syringe The mixture of AA2G is hydrated 60min.The AA2G that 800.8mg is added in independent bottle, then adds the 9.1wt% of 1401.1mg The BDDE of NaOH solution and 252.6mg.Make acquired solution (pH ＞ 12) reaction~20min in 50 DEG C of water-baths, later by its turn It moves on in hydration HA.After addition, mixture is set to mix~20 times by transmitting it back and forth between two syringes.Then Paste is transferred in bottle, places it in 50 DEG C of water-baths~2.5h later.After crosslinking, the 12M HCl containing 197.0mg are added Solution with 9.18g10 × PBS (pH 7.4) makes gel expand 72h on orbital shaker to neutralize alkali.Force it logical Cross the mesh screen fractional condensation glue in~60 μm of apertures.By the way that it to be transmitted to sieved gel~20 of mixing back and forth between two syringes It is secondary, it is transferred into cellulose esters bag filter (MWCO~20kDa) and is dialysed 5 days with PBS (pH 7.4) buffer solution later, Replace buffer solution twice daily.After dialysis, gel is assigned in 1ml COC syringes, 5min is centrifuged at 5000RPM to go Bubble removing, and sterilized with moist steam.The gel has the HA ultimate densities of 25mg/g, the pact calculated as described in example 2 above The G ' of the AA2G mol% and about 80Pa of 10mol%.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 the gel of embodiment 19 to generate the HA/AA2G gels with lidocaine, It is with 0.3%ww lidocaines.By the way that lidocaine HCl to be dissolved in PBS buffer solution (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 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 the LMW HA of 401.0mg hydration~45min in the 1wt%NaOH solution of 2355.0mg in syringe.It 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.The vitagen of 800.1mg is individually dissolved in the 15wt%NaOH of 950.6mg, is then dissolved in In 510.1Milli-Q water.It is mixed using between syringe, makes 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 reaction 2h, later by the 12M HCl containing 148.1mg and The solution of 10 × PBS (pH7.4) of 8523.1mg is added in crosslinking agent.HCl-PBS solution is added to neutralize gel and make it Expansion.So that gel is neutralized and expands 48h or more 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 20,000 MWCO CE bag filters and in PBS (pH7.4) it dialyses in buffer solution.Dialyse progress~197h, frequently replaces PBS buffer solution.After dialysis, gel is transferred to 1ml In COC syringes, 5min is centrifuged at 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)

Certain embodiments according to the present invention are described in embodiment 21 and 22 below, Tyndall effect is shown and subtracts 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 it is 3- [bis- (3- the amino-propoxymethyls)-propoxyl group of 3- (3- aminopropyls) -2,2-]-propylamine (4 to be used in embodiment 20 Arm amine -4AA) crosslinking agent, through EDC chemical preparation gels.In a mild condition, such as under room temperature and such as pH 5.4 it is handed over Connection.Adjustable reaction condition is to prepare with best 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) keeps HA crosslinkings advantageous.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

The 100mM MES buffer solutions (pH 5.2) of 20.0g are added in the syringe of the LMW HA equipped with 1000.0mg. By being dissolved in 260.9mg HMDA.HCl in the 100mM MES buffer solutions (pH 5.2) of 2010.5mg, and add the 1M of 2 μ l NaOH makes pH reach 5.2 preparation HMDA solution.By the way that the EDC of 254.2mg is dissolved in 1188.4mg100mM MES buffer solutions (pH 5.2) EDC solution is prepared in, and in independent bottle, the NHS of 44.3mg is dissolved in the 100mM MES buffer solutions of 1341.8mg In (pH 5.2).After HA is fully hydrated ,~1h adds the HMDA solution of 790 μ l into hydration HA.It is even by being mixed between syringe Change mixture 10 times.Then 490 μ l EDC and 490 μ l NHS solution are added to and are homogenized in paste and by being mixed between syringe Conjunction remixes 10 times.Then by mixture be transferred in bottle and 1 × PBS buffer solution (pH 7.4) of addition 17.9ml it Before, it is crosslinked 5h at room temperature.Before the net for being forced through 60 μm of apertures, gel is made to be expanded on roller 3 days.By screening Gel is placed in cellulose ester membrane dialysis tubing MWCO 20KDa and is dialysed 4 days with 1 × PBS, replaces buffer solution twice daily.It will Gel distributes in 1ml COC syringes, centrifuges 5min at 5000RPM, moist steam is used in combination to sterilize.The HA ultimate densities of gel For 25mg/g.

Embodiment 22

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

The 100mM MES buffer solutions (pH 5.2) of 32.55g are added to the syringe of the LMW HA equipped with 1000.4mg In.By being dissolved in 256.3mg 4AA in the 100mM MES buffer solutions (pH 5.2) of 1039.8mg, and add the 6M of 380 μ l HCl makes pH reach 5.2 preparation 4AA solution.By the way that the EDC of 251.2mg is dissolved in 1013.8mg 100mM MES buffer solutions (pH 5.2) EDC solution is prepared in, and in independent bottle, the NHS of 74.7mg is dissolved in the 100mM MES buffer solutions of 2020.0mg In (pH 5.2).After HA is fully hydrated ,~1h adds the 4AA solution of 260 μ l into hydration HA.It is even by being mixed between syringe Change mixture 10 times.Then 277 μ l EDC and 273 μ l NHS solution are added to and are homogenized in paste and by being mixed between syringe Conjunction remixes 10 times.Then by mixture be transferred in bottle and 10 × PBS buffer solution (pH 7.4) of addition 6.4ml it Before, it is crosslinked 5h at room temperature.Before the net for being forced through 60 μm of apertures, gel is made to be expanded on roller 3 days.By screening Gel is placed in cellulose ester membrane dialysis tubing MWCO 20KDa and is dialysed 4 days with 1 × PBS, replaces buffer solution twice daily.It will Gel distributes in 1ml COC syringes, centrifuges 5min at 5000RPM, moist steam is used in combination to sterilize.The HA ultimate densities of gel For 23mg/g.

Embodiment 23

The measurement of the gel rheology of embodiment 19-22

Using oscillation parallel-plate rheometer, Anton Paar Physica MCR 301 measure the rheological characteristic of gel. The board diameter of 25mm is used when gap height is 1mm.It is measured under 25 DEG C of constant temperature.It measures every time by 2% constant strain and frequency Rate logarithm increases the frequency scanning composition of lower 1-10Hz, 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 of the gel of embodiment 19-22 measures

Measure power gel squeezed out needed for 30G needles using Instron 5564 and 2 softwares of Bluehill.By gel from 1ml COC syringes squeeze out 30G1/2 TSK needles.Piston 11.35min is pushed by the speed of 100mm/min, 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 the gel of embodiment 19-22

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

The average CD68 scores of embodiment 19-22

Embodiment 26

The cell toxicity test of the gel of embodiment 19-22, ISO 10993-5.

According to the agarose cladding process of ISO 10993-5, the vitro cytotoxicity test of gel is carried out by NAMSA：《Doctor Treat instrument biological assessment》- the 5 part：Vitro cytotoxicity test.It is placed on the examination on filter disc to three multiple holes addition 0.1ml Test sample and 0.9%NaCl solution, as negative control 1cm long high density polyethylene (HDPE) and as positive control 1 × 1cm²Latex part.Each is placed on to the agarose surface of directly covering single layer L929 l cells.At 37 DEG C 5%CO₂After middle incubation for 24 hours, any abnormal cell form and 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 the Tyndall effect of specificity 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 quantization 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 do not have blue variable colour Injection portion give score 1.To thick and apparent blue variable colour (usually 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-" b " of skin color：Using colorimeter (CM2600D, Konica Minolta, NJ) quantization 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)" the blue light % " being emitted from skin：Using portable spectrophotometer (CM2600D, Konica Minolta, NJ) quantify the blue light % being emitted from skin in total visible-range.This can be by seeking the area under 400-490nm visible spectrums It integrates and is standardized and realized with the gross area under spectrum (400-700nm).

Embodiment 27

The Tyndall of gel is assessed

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

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

The visual score 1-5 for the use of increment being 0.5 scores 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 lower 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 % higher.Belotero Soft does not show that Tyndall effect and value are comparable to the gel of embodiment 19 and 21.See Figure 13 and 14.

Embodiment 28

The internal duration of gel is assessed by histology

Gel in the present invention of 50 μ l bolus injections of the intradermal implantation in the back side of Shi-road Er Shi rats and commercial gel.1 Implantation material is taken out when all and passes through histologic analysis with h and E (H＆E) dyeing.Just slice is taken in injection portion.From every A tissue sample is cut two and is sliced 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 internal duration of gel is assessed by MRI

It is assessed in female Shi-road Er Shi rats after intracutaneous injection using magnetic resonance imaging (MRI) research, 40 weeks Nei Benfa The volume and surface area of bright gel and commercial gel change with time.By each 150 μ l target volume injected gel of implantation material.Make Implantation material 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 70/30 Bruker Biospec MRI scanners of 7Tesla.It is planting Image is collected after entering the same day (the 0th week) and implantation within 12,24,40 weeks.Gel absolute volume and time are shown in following FIG. 16 Figure.High persistent gel has high absolute volume when being implanted into 40 weeks.

Embodiment 29

The present composition for treating periorbit line

There are fine wrinkles in periorbit area and dermal augmentation agent are required to treat in 40 years old modest woman.Use 30G needles, doctor Microgroove superficial of the teacher into below her each eye and areas Lei Gou introduces 0.6ml according to the gel (example of the present invention based on HA Gel as 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, for example, embodiment 19 and 21 composition, Tyndall effect reduce or it is unknown It is aobvious, and relative to certain commercial gels based on HA, such as Juvederm Refine/Surgiderm 18 and Belotero There are many Soft, duration length in vivo.For example, relative to commercialization " microgroove " preparation Belotero Soft, reality of the invention Applying example 19 not only has the advantageous Tyndall score at least as this commercial gel, and advantageously shows much higher The internal duration.

Embodiment 30

Injectable composition for the present invention for improving microgroove appearance

Individually and the additive of combination, such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and its spread out Biology is in order to generating the mode and cross-linked hyaluronic acid gel of the gel based on HA of a variety of generally optical clears, injectable In conjunction with.As defined elsewhere herein, HA components at least 90% by weight, such as be generally entirely LMW HA, Or about 100% LMW HA.These additives are made to be combined with HA hydrogels using any suitable mode.It sieves and processes combination Gel injectable, pH be neutral, cementitious compositions to generate, HA concentration at least about 20mg/g, for example, 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.

Each 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, the areas Lei Gou, neck regions will benefit from the skin of any other facial area of dermal augmentation.To the greatest extent Pipe superficial introduces gel, but does not observe the discoloration due to Tyndall effect and patient satisfaction result.

Although finally, it is to be understood that referred to the every aspect that each embodiment describes this specification, this field 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 under the premise of, those skilled in the art can according to the teaching of this article make disclosed theme and many different change or become Change or alternate configuration.Under the premise of without departing substantially from spirit of that invention defined in appended claims, detailed variation can be done.Most Afterwards, terms used herein are just for the sake of description particular embodiment, and the range being not intended to limit the invention, the range is only It is defined by the claims.In addition, it is intended that contained in above description or all material shown in the accompanying drawings should be explained only It is illustrative and not restrictive.Therefore, the present invention is not limited to those of accurately be shown and described.

This document describes certain embodiments of the present invention, including inventor to become known for carrying out the best mould of the present invention Formula.Certainly, the modification of these embodiments of description for those of ordinary skill in the art will after reading foregoing description It becomes apparent.Inventor it is expected that technical staff optionally uses such modification, and inventor is intended to make the present invention with not It is same as the method specifically described herein implementation.Therefore, as applicable law permits that the present invention includes being described in appended claims The all modifications and equivalent form of theme.Moreover, be apparently contradicted in the context unless otherwise indicated or in addition, otherwise this hair The bright any combinations for covering the above-mentioned element in its be possible to modification.

The grouping of the substitute element of present invention disclosed herein or embodiment should not be interpreted as limiting.Each organize at Member can refer to and be claimed individually or with any combinations with the other members of the group or the other elements found herein., it is expected that One or more members of group can include in the group or being removed from it because of the reason of convenient and/or patentability.When any such packet 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 ingredient, property (such as the molecular weight for description and claims, reaction are expressed Condition etc.) all numbers all will be understood as being modified by term " about " in all cases.As used herein, term " about " Refer to the range that so quantitative project, parameter or term cover above and below the value of gainer, parameter or term ± 10%.Cause This, unless point out on the contrary, the digital parameters otherwise listed in the description and the appended claims be approximation, can according to this The required property tried hard to is invented to change.At least, unlike the application for attempting doctrine of equivalents is limited to the scope of the claims Equally, each digital parameters should according at least to the significant digit of report number and understood by the common rounding techniques of application. Although it is approximation to list the broad range of numberical range of the present invention and parameter, the numerical value listed in a particular embodiment is all most Report to possible accuracy.However, inherently contain must be by the standard deviation that finds in being measured in its respective test for any numerical value Certain errors caused by difference.

Unless otherwise indicated or be apparently contradicted in the context, with otherwise in description the present invention context in (especially Be claim below context in) term "one", "an", " described " and similar indicant be understood to include Odd number and plural number.The narration of value range herein is intended only to serve as individually referring to the shorthand side for each independent values for belonging to the range Method.Unless otherwise indicated, otherwise each individual value is incorporated in specification as the same to its independent narration herein. Unless otherwise indicated or in addition it is apparently contradicted in the context, otherwise all methods described herein can be with any suitable sequence It carries out.It is only intended to preferably say using any and all embodiments provided herein or illustrative wording (for example, " such as ") The bright present invention, without constituting the limitation to the scope of the invention for requiring in addition that protection.Wording in specification must not be interpreted as It points out to implementing the essential any not claimed element of the present invention.

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

All patents, patent disclosure and the other announcements quoted and identified in the present specification are for descriptions and disclosures (example As) purpose of composition and method that can be used in combination with the present invention described in such announcement, 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 Speech should not be construed as recognizing previous invention or it is any other due to, have no right to make inventor prior to such disclosure.About day All statements of phase or statement information based on the applicant's availability about these document contents, are not constituted about these documents Date or any of content correctness recognize.

Claims (16)

1. a kind of hydrogel composition of injectable is used to prepare the purposes of the medicament of the wrinkle on treatment people patient skin, wherein The composition includes to be selected from lysine, lysine methyl ester and combinations thereof, and institute with the crosslinked hyaluronic acid of diamines, the diamines State the skin that composition is injected into patient.

2. purposes according to claim 1, wherein the diamines is lysine.

3. purposes according to claim 1 or 2, wherein the diamines is lysine methyl ester.

4. purposes according to claim 1,2 or 3 is not greater than about wherein the composition is injected into patient skin The depth of 1.0mm.

5. purposes according to claim 4, wherein the composition is injected into the depth that patient skin is not greater than about 0.8mm Degree.

6. purposes according to claim 5, wherein the composition is injected into the depth that patient skin is not greater than about 0.6mm Degree.

7. purposes according to claim 1,2 or 3, wherein the composition has the at most storage modulus of 200Pa.

8. purposes according to claim 7, wherein the composition has at least storage modulus of 25Pa.

9. purposes according to claim 7, wherein the composition has the energy storage between about 25Pa and about 150Pa Modulus.

10. purposes according to claim 9, wherein the composition has the storage between about 40Pa and about 100Pa It can modulus.

11. purposes according to claim 7, wherein the composition has the storage modulus no more than about 100Pa.

12. purposes according to claim 7, wherein the composition has the storage modulus of no less than about 40Pa.

13. purposes according to claim 1, wherein the wrinkle is superficial wrinkle or microgroove.

14. purposes according to claim 1, wherein the composition to be injected into periorbit area, the nasolabial groove of patient skin Area, the areas Lei Gou or neck regions.

15. purposes according to claim 1,2 or 3, wherein the hydrogel has been further processed to provide about 10 μm To about 1000 μm of granularity.

16. according to the purposes described in claim 1,2,3 or 15, wherein the composition also includes that uncrosslinked hyaluronic acid is poly- Close object.