US009 125840B2

(12) United States Patent (10) Patent No.: US 9,125,840 B2 Lebreton (45) Date of Patent: Sep. 8, 2015

(54) METHODS FOR IMPROVING SKIN 4,716,154 A 12/1987 MalSon et al. CONDITIONS 4,772,419 A 9, 1988 MalSon et al. 4,803,075 A 2f1989 Wallace et al. 4,886,787 A 12/1989 De Belder et al. (71) Applicant: Allergan Industrie SAS, Pringy (FR) 4,896,787 A 1/1990 Delamour et al. 5,009,013 A 4, 1991 Wiklund (72) Inventor: Pierre F. Lebreton, Annecy (FR) 5,087,446 A 2f1992 Suzuki et al. 5,091,171 A 2, 1992 Yu et al. (73) Assignee: Allergan Industrie SAS, Pringy (FR) 5,143,724 A 9, 1992 Leshchiner et al. 5,246,698 A 9, 1993 Leshchiner et al. 5,314,874 A 5/1994 Miyata et al. (*) Notice: Subject to any disclaimer, the term of this 5,328,955 A 7, 1994 Rhee et al. patent is extended or adjusted under 35 5,356,883. A 10, 1994 Kuo et al. U.S.C. 154(b) by 0 days. 5,399,351 A 3, 1995 Leshchiner et al. 5,428,024 A 6/1995 Chu et al. (21) Appl. No.: 14/078,057 5,531,716 A 7/1996 Luzio et al. 5,565,519 A 10, 1996 Rhee et al. 5,571,503 A 11/1996 MauSner (22) Filed: Nov. 12, 2013 5,614,587 A 3, 1997 Rhee et al. 5,616,568 A 4/1997 Pouyani et al. (65) Prior Publication Data 5,616,611 A 4/1997 Yamamoto et al. 5,616,689 A 4/1997 Shenoy et al. US 2014/OO736OO A1 Mar. 13, 2014 5,633,001 A 5/1997 Agerup 5,643,464 A 7/1997 Rhee et al. Related U.S. Application Data 5,676,964 A 10, 1997 Della Valle et al. 5,823,671 A 10, 1998 Mitchell et al. (63) Continuation of application No. 12/777,106, filed on 5,824,333 A 10/1998 Scopelianos et al. May 10, 2010, now Pat. No. 8,586,562. 5,827,529 A 10, 1998 Ono et al. (60) Provisional application No. 61/313,664, filed on Mar. (Continued) 12, 2010. FOREIGN PATENT DOCUMENTS (51) Int. Cl. EP O273823 T 1988 A6 IK3I/728 (2006.01) EP O416250 3, 1991 A 6LX3L/75 (2006.01) A6 IK 8/73 (2006.01) (Continued) A6 IK 8/34 (2006.01) A61O 19/08 (2006.01) OTHER PUBLICATIONS A61O 19/00 (2006.01) Adams, Mark. An Analysis of Clinical Studies of the Use of (52) U.S. Cl. Crosslinked Hyaluronan, Hylan, in the Treatment of Osteoarthritis, CPC. A61K 8/73 (2013.01); A61K 8/345 (2013.01); The Journal of Rheumatology, 1993, 16-18, 20 (39). A61K 8/735 (2013.01); A61O 19/00 (2013.01); Albano, Emanuele et al. Hydroxyethyl Radicals in Ethanol A61O 19/08 (2013.01); A61 K 2800/91 Hepatotoxicity, Frontiers in Bioscience, 1999, 533-540, 4. (2013.01) Allemann, Inja Bogdan, Hyaluronic Acid Gel (Juvederm) Prepara (58) Field of Classification Search tions in the Treatment of Facial Wrinkles and Folds, Clinical Inter CPC ...... A61K 8/345; A61K 8/735; A61K 8/73; ventions in Aging, 2008, 629-634, 3 (4). A61K 2800/91; A61Q 19/08: A61Q 19/00 Antunes, Alberto et al. Efficacy of Intrarectal Lidocaine Hydrochlo USPC ...... 514/54; 536/123.1, 124 ride Gel for Pain Control in Patients Undergoing Transrectal Prostate See application file for complete search history. Biopsy, Clinical Urology, 2004, 380-383, 30. (56) References Cited (Continued) U.S. PATENT DOCUMENTS 2,128,827 A 8, 1938 Killian Primary Examiner — Shaojia Anna Jiang 3,548,056 A 12/1970 Eigen Assistant Examiner — Michael C Henry 3,763,009 A 10, 1973 Suzuki et al. 3,949,073. A 4, 1976 Daniels et al. (74) Attorney, Agent, or Firm — Linda Allyson Nassif 4,060,081 A 11, 1977 Yannas et al. 4,140,537 A 2f1979 Lucket al. 4,233,360 A 11, 1980 Lucket al. (57) ABSTRACT 4,261,521 A * 4, 1981 Ashbrook ...... 241.5 4,273,705 A 6, 1981 Kato 4.279,812 A 7, 1981 Cioca The present specification discloses fluid compositions com 4.424,208 A 1, 1984 Wallace et al. prising a matrix polymerand stabilizing component, methods 4,501.306 A 2f1985 Chu et al. of making Such fluid compositions, and methods of treating 4,582,640 A 4, 1986 SmeStadet al. skin conditions in an individual using Such fluid composi 4,582,865 A 4, 1986 BalaZS et al. 4,605,691 A 8, 1986 Balazs et al. tions. 4,636,524 A 1, 1987 Balazs et al. 4,642,117 A 2, 1987 Nguyen et al. 4,713,448 A 12/1987 Balazs et al. 22 Claims, 3 Drawing Sheets US 9,125,840 B2 Page 2

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(56) References Cited eration, Bioconjugate Chemistry, 2010, 240, 21. American Chemical Society. OTHER PUBLICATIONS Yui. Nobuhiko et al. Inflammation Responsive Degradation of Crosslinked Hyaluronic Acid Gels, J of Controlled Release, 1992, Wahl, Gregor, European Evaluation of a New Hyaluronic Acid Filler 105-116, 26. Incorporating Lidocaine, Journal of Cosmetic Dermatology, 2008, Yui. Nobuhiko et al. Photo-responsive Degradation of Heteroge 298-303, 7. Waraszkiewicz, Sigmund et al. Stability-Indicating High-Perfor neous Hydrogels Comprising Crosslinked Hyaluronic Acid and mance Liquid Chromatographic Analysis of Lidocaine Hydrochlo Lipid Microspheres for Temporal Drug Delivery, J of Controlled ride and Lidocaine Hydrochloride with Epinephrine Injectable Solu Release, 1993, 141-145, 26. tions, J of Pharmaceutical Sciences, 1981, 1215-1218, 70 (11). Yun, Yang H. et al., Hyaluronan Microspheres for Sustained Gene Weidmann, Michael, New Hyaluronic Acid Filler for Subdermal and Delivery and Site-Specific Targeting, Biomaterials, 2004. 147-157. Long-lasting Volume Restoration of the Face, European Dermatol 25, US. ogy, 2009, 65-68. Zulian, F. et al. Triamcinolone Acetonide and Hexacetonide Intra Xia, Yun et al. Comparison of Effects of Lidocaine Hydrochloride, articular Treatment of Symmetrical Joints in Juvenile Idiopathic Buffered Lidocaine, Diphenhydramine, and Normal Saline After Arthritis: A Double-blind Trial, Rheumatology, 2004. 1288-1291, Intradermal Injection, J of Clinical Anesthesia, 2002, 339-343, 14. 43. Yeom, Junseoketal, Effect of Cross-linking Reagents for Hyaluronic Acid Hydrogel Dermal Fillers on Tissue Augmentation and Regen * cited by examiner U.S. Patent Sep. 8, 2015 Sheet 1 of 3 US 9,125,840 B2

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8. H- - - - 8 Siii.36:SGSi3cs -- i. 4: ------arrrrr------ca --- www.*H Timrim () - US 9,125,840 B2 1. 2 METHODS FOR IMPROVING SKIN prevents in vivo degradation of the matrix polymer. Admin CONDITIONS istration of the disclosed fluid compositions improves skin conditions such as, e.g., hydration and the cutaneous elastic This patent application is a divisional of U.S. patent appli ity by compensating for the loss of the endogenous polymer. cation Ser. No. 12/777,106, filed May 10, 2010, which claims 5 Thus, aspects of the present specification provide a fluid priority pursuant to 35 U.S.C. S 119(e) to U.S. Provisional composition comprising a matrix polymer and a stabilizing Patent Application Ser. No. 61/313,664 filed Mar. 12, 2010, component. Matrix polymers useful to make Such fluid com the entire disclosure of each of these applications being incor positions include, without limitation, a glycosaminoglycan porated herein by this reference. (like chondroitin Sulfate, dermatan Sulfate, keratan Sulfate, Dermal fillers are useful in soft tissue and dermal correc 10 hyaluronan) and lubricin. Stabilizing components useful to tion. One common polymer used in dermal filler composi make Such fluid compositions include, without limitation, tions is hyaluronan, also known as hyaluronic acid (HA). polyols and flavonoids. Although exhibiting excellent biocompatibility and affinity Other aspects of the present specification provide a method for water molecules, in its natural state, hyaluronan exhibits of making a fluid composition disclosed in the present speci poor biomechanical properties as a dermal filler. Tezel and 15 fication. In an aspect, a method for making a fluid composi Fredrickson, The Science of Hyaluronic Acid Dermal Fillers, tion comprises the steps of: a) combining a stabilizing com J Cosmet Laser Ther. 10(1): 35-42 (2008); Kablik, et al., ponent with a physiologically-acceptable buffer to make a Comparative Physical Properties of Hyaluronic Acid Dermal stabilizing component-buffered solution; and b) combining a Fillers, Dermatol Surg. 35 Suppl 1:302-312 (2009); Beasley, matrix polymer with the stabilizing component-buffered et al., Hyaluronic Acid Fillers: A Comprehensive Review, Solution to hydrate the matrix polymer. In another aspect, a Facial Plast Surg. 25(2): 86-94 (2009); each of which is method for making a fluid composition comprises the steps hereby incorporated by reference in its entirety. One primary of: a) combining a stabilizing component with a physiologi reason is that this polymer is soluble and is cleared rapidly cally-acceptable buffer to make a stabilizing component when administered into a skin region. Tezel, supra, 2008: buffered solution; b) combining a matrix polymer with the Kablik, Supra, 2009; Beasley, supra, 2009. This in vivo clear 25 stabilizing component-buffered solution to hydrate the ance is primarily achieved by degradation, principally enzy matrix polymer, and; c) sizing the fluid composition. This matic degradation via hyaluronidase and chemical degrada method may, or may not, further comprise a step comprising tion via free-radicals. To minimize the effect of these in vivo titrating a stabilizing component-buffered solution to obtain a degradation pathways, matrix polymers like hyaluronan are desired pH after step (a); a step comprising filtering the sta crosslinked to one another to form a hydrogel. Because 30 bilizing component-buffered solution after step (a); a step (b) hydrogels are more solid Substance that are readily soluble, where combining a matrix polymer with the stabilizing com dermal fillers comprising such crosslinked matrix polymers ponent-buffered solution to hydrate the matrix polymer remain in place at the implant site. Tezel, supra, 2008; Kablik, occurs by mixing the matrix polymer with the stabilizing supra, 2009; Beasley, supra, 2009. A crosslinked matrix poly component-buffered solution at a low speed for a relatively mer like hyaluronan is also more Suitable as a component of 35 long period of time; a step (b) where combining a matrix a dermal filler because its more solid nature improves the polymer with the stabilizing component-buffered solution to mechanical properties of the filler, allowing the filler to better hydrate the matrix polymer occurs by mixing the matrix lift and fill a skin region. Tezel, supra, 2008; Kablik, supra, polymer with the stabilizing component-buffered solution at 2009; Beasley, supra, 2009. a low speed for a relatively long period of time and then Hyaluronan is abundant in the different layers of the skin, 40 followed by a rest for a relative long period of time; a step (b) where it has multiple functions such as to ensure good hydra where combining a matrix polymer with the stabilizing com tion, assist in the organization of the collagen matrix, and act ponent-buffered solution to hydrate the matrix polymer as a filler material assisting the organization of the extracel occurs by mixing the matrix polymer with the stabilizing lular matrix. However, with age, the quantity of hyaluronan component-buffered solution at a low speed for a relatively present in the skin decreases. This loss of hyaluronan results 45 long period of time and then by mixing the matrix polymer in various skin conditions such as, e.g., skin dehydration, lack with the stabilizing component-buffered solution using a of skin elasticity, skin roughness, lack of skin tautness, skin cycle of alternating periods of agitation for a relatively short stretch line and/or marks, skin paleness, skin wrinkles, and period of time followed by periods of rest for a relatively long the like. As such, it would be desirable to have a skin therapy period of time; a step (b) where combining a matrix polymer that can replace the endogenous matrix polymers that are lost 50 with the stabilizing component-buffered solution to hydrate with age in order to treat these skin conditions. However, the matrix polymer occurs by mixing the matrix polymer with current dermal fillers comprising hydrogels of crosslinked the stabilizing component-buffered solution at a low speed matrix polymers like crosslinked hyaluronan cannot be used for a relatively long period of time and then by mixing the to replace the lost endogenous polymers because the matrix polymer with the stabilizing component-buffered crosslinking prevents the ability of these polymers to inte 55 Solution using a cycle of alternating periods of agitation for a grate into the extracellular matrix. However, as discussed relatively short period of time followed by periods of rest for above, although uncrosslinked matrix polymers like hyaluro a relatively long period of time, and then followed by a rest for nan are soluble, and as such, could integrate into the extra a relative long period of time; a step comprising degassing a cellular matrix and replace lost endogenous hyaluronan, fluid composition after step (b) or step (c); a step comprising uncrosslinked matrix polymers are rapidly cleared from the 60 filling a syringe with a fluid composition after step (c); and/or body by in vivo degradation pathways. Thus, what is needed a step comprising sterilizing a syringe filled with a fluid is a fluid composition comprising uncrosslinked matrix poly composition after step (c). mers that include an additional stabilizing component that Yet other aspects of the present specification provide a fluid reduces or prevents matrix polymer degradation. composition disclosed in the present specification made by a The fluid compositions disclosed in the present specifica 65 method disclosed in the present specification. tion achieve this goal. Such fluid compositions comprise a Still other aspects of the present specification provide a matrix polymer and a stabilizing component that reduces or method of improving a condition of skin in an individual in US 9,125,840 B2 3 4 need thereof, the method comprising the steps of administer subjected to such a force. It should be noted, that although the ing a fluid composition disclosed in the present specification compositions disclosed in the present specification are fluid into a dermal region of the individual, wherein the adminis in nature due to the presence of uncross linked matrix poly tration improves the condition. Skin conditions treated by the mers, such fluid compositions, may, although may not, disclosed fluid compositions include, without limitation, skin 5 include cross linked matrix polymers, which, by its nature, is dehydration, a lack of skin elasticity, skin roughness, a lack of a gel or other Solid Substance. As such, certain fluid compo skin tautness, a skin stretch line or mark, skin paleness, and/or sitions disclosed in the present specification exhibit vis skin wrinkles. coelastic properties. Aspects of the present specification provide, in part, a fluid BRIEF DESCRIPTION OF THE DRAWINGS 10 composition comprising a matrix polymer. As used herein, FIG. 1 is a graph showing the results of hyaluronan poly the tem"matrix polymer refers to a polymer that can become mer degradation with and without mannitol. FIG. 1A shows a part of or function as an extracellular matrix polymer and graph plotting G"G" over time. FIG. 1B shows a graph plot pharmaceutically acceptable salts thereof. Non-limiting ting dynamic viscosity over time. SGM181a is a fluid com- 15 examples of a matrix polymer include a glycosaminoglycan position comprising 13.5 mg/mL hyaluronan polymer with like chondroitin Sulfate, dermatan Sulfate, keratan Sulfate, out mannitol (control) and SGM181b is a fluid composition hyaluronan, lubricants; and collagens. Non-limiting comprising 13.5 mg/mL hyaluronan polymer and 5% man examples of a pharmaceutically acceptable salt of a matrix nitol. polymer includes sodium salts, potassium salts, magnesium FIG. 2 is a graph showing the effects of increasing Stabi- 20 salts, calcium salts, and combinations thereof. lizing concentration on the dynamic viscosity of a fluid com Aspects of the present specification provide, in part, a fluid position comprising a matrix polymer. The graph plots composition comprising a glycosaminoglycan. As used dynamic viscosity (Pas) over time (s). SGM173a is a fluid herein, the term 'glycosaminoglycan' is synonymous with composition comprising 13.5 mg/mL hyaluronan polymer “GAG” and “mucopolysaccharide' and refers to long without mannitol (control), SGM173c is a fluid composition 25 unbranched polysaccharides consisting of a repeating disac comprising 13.5 mg/mL hyaluronan polymer and 5% man charide units. The repeating unit consists of a hexose (six nitol and SGM173d is a fluid composition comprising 13.5 carbon Sugar) or a hexuronic acid, linked to a hexosamine mg/mL hyaluronan polymer and 9% mannitol. (six-carbon Sugar containing nitrogen) and pharmaceutically acceptable salts thereof. Members of the GAG family vary in DETAILED DESCRIPTION 30 the type of hexosamine, hexose or hexuronic acid unit they contain, such as, e.g., glucuronic acid, iduronic acid, galac Aspects of the present specification provide, in part, a fluid tose, galactosamine, glucosamine) and may also vary in the composition comprising a matrix polymer and a stabilizing geometry of the glycosidic linkage. Any glycosaminoglycan component. As used herein, the term “fluid refers to a con is useful in the compositions disclosed in the present specifi tinuous, amorphous Substance whose molecules move freely 35 cation with the proviso that the glycosaminoglycan improves past one another. A fluid cannot Sustain a shearing force when a condition of the skin, Such as, e.g., hydration or elasticity. at rest and undergoes a continuous change in shape when Table 1 lists representative GAGs. TABLE 1

Examples of GAGs

Glycosidic Hexuronic linkage Name acid Hexose Hexosamine geometry Unique features

Chondroitin GlcUA or GalNAc or -4GlcUAB1- Most prevalent GAG sulfate GlcUA(2S) GalNAc(4S) or 3GalNAcf.1- GalNAc(6S) or GalNAc(4S,6S) Dermaitan GlcUA or GalNAc or -4Idol JAB1- Distinguished from chondroitin sulfate Idol JA or GalNAc(4S) or 3GalNAcf.1- sulfate by the presence of iduronic Idol JA(2S) GalNAc(6S) or acid, although some hexuronic GalNAc(4S,6S) acid monosaccharides may be glucuronic acid. Keratan Gal or GlcNAc or -3Gal(6S)B1- Keratan Sulfate type II may be sulfate Gal(6S) GlcNAc(6S) 4GlcNAc(6S)B1- fucosylated. Heparin GlcUA or GlcNAc or -4Idol JA(2S)C1- Highest negative charge density of Idol JA(2S) GlcNS or 4GlcNS(6S)C1- any known biological molecule GlcNAc(6S) or GlcNS(6S) Heparan GlcUA or GlcNAc or -4GlcUAB1- Highly similar in structure to sulfate Idol JA or GlcNS or 4GlcNAco.1- heparin, however heparan Sulfates Idol JA(2S) GlcNAc(6S) or disaccharide units are organised GlcNS(6S) into distinct Sulfated and non Sulfated domains. US 9,125,840 B2 5 TABLE 1-continued Examples of GAGs Glycosidic Hexuronic linkage Name acid.Hexose Hexosamine geometry Unique features Hyaluronan GlcUA GlcNAc -4GlcUAB1 The only GAG that is exclusively 3GlcNAcf.1- non-sulfated GlcUA = B-D-glucuronic acid GlcuA(2S) = 2-O-sulfo-f-D-glucuronic acid Idol JA = O-L-iduronic acid Idol JA(2S) = 2-O-sulfo-O-L-iduronic acid Gal = B-D-galactose Gal(6S) = 6-O-sulfo-f-D-galactose GalNAc = B-D-N-acetylgalactosamine GalNAc-4S) = B-D-N-acetylgalactosamine-4-O-sulfate GalNAccGS) = B-D-N-acetylgalactosamine-6-O-sulfate GalNAc?4S,6S) = B-D-N-acetylgalactosamine-4-O, 6-O-sulfate GlcNAc = C-D-N-acetylglucosamine GlcNS = C-D-N-sulfoglucosamine GlcNS(6S) = C-D-N-sulfoglucosamine-6-O-sulfate

Aspects of the present specification provide, in part, a fluid from animal and non-animal sources. Polymers of hyaluro composition comprising a chondroitin Sulfate. As used nan can range in size from about 5,000 Da to about 20,000, herein, the term “chondroitin sulfate” refers to an 000 Da. Any hyaluronan is useful in the compositions dis unbranched, sulfated GAG of variable length comprising dis 25 closed in the present specification with the proviso that the accharides of two alternating monosaccharides of D-glucu hyaluronan improves a condition of the skin, Such as, e.g., ronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) hydration or elasticity. Non-limiting examples of pharmaceu and pharmaceutically acceptable salts thereof. A chondroitin tically acceptable salts of hyaluronan include Sodium hyalu Sulfate may also include D-glucuronic acid residues that are roman, potassium hyaluronan, magnesium hyaluronan, cal epimerized into L-iduronic acid (IdoA), in which case the 30 cium hyaluronan, and combinations thereof. resulting disaccharide is referred to as dermatan Sulfate. A Aspects of the present specification provide, in part, a fluid chondroitin sulfate polymer can have a chain of over 100 individual sugars, each of which can be sulfated in variable composition comprising a lubricin. As used herein, the term positions and quantities. Chondroitin Sulfate is an important “lubricin” refers to a large, water soluble glycoprotein structural component of cartilage and provides much of its 35 encoded by the PRG4 gene and pharmaceutically acceptable resistance to compression. Any chondroitin Sulfate is useful salts thereof. It has a molecular weight of 206,000 Da and in the compositions disclosed in the present specification with comprises approximately equal proportions of protein and the proviso that the chondroitin sulfate improves a condition glycosaminoglycans. The structure of lubricin molecule is of the skin, such as, e.g., hydration or elasticity. Non-limiting that of a partially extended flexible rod and, in solution, examples of pharmaceutically acceptable salts of chondroitin 40 occupies a smaller spatial domain than would be expected Sulfate include Sodium chondroitin Sulfate, potassium chon from structural predictions. This characteristic may aid in the droitin Sulfate, magnesium chondroitin Sulfate, calcium molecule's boundary lubricating ability. Lubricin is present chondroitin Sulfate, and combinations thereof. in synovial fluid and on the surface (superficial layer) of Aspects of the present specification provide, in part, a fluid articular cartilage and therefore plays an important role in composition comprising a keratan Sulfate. As used herein, the 45 joint lubrication and synovial homeostasis. Any lubricin is term “keratan sulfate' refers to a GAG of variable length useful in the compositions disclosed in the present specifica comprising disaccharide units, which themselves include tion with the proviso that the lubricin improves a condition of B-D-galactose and N-acetyl-D-galactosamine (GalNAc) and the skin, such as, e.g., hydration or elasticity. Non-limiting pharmaceutically acceptable salts thereof. Disaccharides examples of pharmaceutically acceptable salts of lubricin within the repeating region of keratan Sulfate may be fucosy 50 include Sodium lubricin, potassium lubricin, magnesium lated and N-Acetylneuraminic acid caps the end of the chains. lubricin, calcium lubricin, and combinations thereof. Any keratan Sulfate is useful in the compositions disclosed in Aspects of the present specification provide, in part, a fluid the present specification with the proviso that the keratan composition comprising a crosslinked matrix polymer. As Sulfate improves a condition of the skin, such as, e.g., hydra sued herein, the term "crosslinked’ refers to the intermolecu tion or elasticity. Non-limiting examples of pharmaceutically 55 lar bonds joining the individual polymer molecules, or mono acceptable salts of keratan Sulfate include Sodium keratan merchains, into a more stable structure like a gel. As such, a Sulfate, potassium keratan Sulfate, magnesium keratan Sul crosslinked matrix polymer has at least one intermolecular fate, calcium keratan Sulfate, and combinations thereof. bond joining at least one individual polymer molecule to Aspects of the present specification provide, in part, a fluid another one. Matrix polymers disclosed in the present speci composition comprising a hyaluronan. As used herein, the 60 fication may be crosslinked using dialdehydes and disulfides term "hyaluronic acid' is synonymous with “HA'. “hyalu crosslinking agents including, without limitation, divinyl Sul ronic acid', and "hyaluronate” refers to an anionic, non fones, diglycidyl ethers, and bis-epoxides. Non-limiting Sulfated glycosaminoglycan polymer comprising disaccha examples of hyaluronan crosslinking agents include divinyl ride units, which themselves include D-glucuronic acid and sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), 1,2- D-N-acetylglucosamine monomers, linked together via alter 65 bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxy nating 3-1.4 and 3-1.3 glycosidic bonds and pharmaceuti octane (DEO), biscarbodiimide (BCD), adipic dihydrazide cally acceptable salts thereof. Hyaluronan can be purified (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethyl US 9,125,840 B2 7 8 enediamine (NMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclo uncrosslinked matrix polymer where the uncrosslinked hexane, or combinations thereof. matrix polymer represents, e.g., about 90% by weight, about Aspects of the present specification provide, in part, a fluid 91% by weight, about 92% by weight, about 93% by weight, composition comprising acrosslinked matrix polymer having about 94% by weight, about 95% by weight, about 96% by a degree of crosslinking. As used herein, the term “degree of 5 weight, about 97% by weight, about 98% by weight, or about crosslinking” refers to the percentage of matrix polymer 99%, or about 100% by weight, of the total matrix polymer monomeric units that are bound to a cross-linking agent, Such present in the composition. In yet other aspects of this as, e.g., the disaccharide monomer units of hyaluronan. Thus, embodiment, a fluid composition comprises an uncrosslinked a fluid composition that that has a crosslinked matrix polymer matrix polymer where the uncrosslinked matrix polymer rep with a 4% degree of crosslinking means that on average there 10 resents, e.g., at least 90% by weight, at least 91% by weight, are four crosslinking molecules for every 100 monomeric at least 92% by weight, at least 93% by weight, at least 94% units. Every other parameter being equal, the greater the by weight, at least 95% by weight, at least 96% by weight, at degree of crosslinking, the harder the gel becomes. Non least 97% by weight, at least 98% by weight, or at least 99% limiting examples of a degree of crosslinking include about by weight, of the total matrix polymer present in the compo 1% to about 15%. 15 sition. In still other aspects of this embodiment, a fluid com Aspects of the present specification provide, in part, a fluid position comprises an uncrosslinked matrix polymer where composition comprising a uncrosslinked matrix polymer. As the uncrosslinked matrix polymer represents, e.g., about 90% used herein, the term "uncrosslinked’ refers to a lack of to about 100% by weight, about 93% to about 100% by intermolecular bonds joining the individual matrix polymer weight, about 95% to about 100% by weight, or about 97% to molecules, or monomer chains. As such, an uncrosslinked about 100% by weight, of the total matrix polymer present in matrix polymer is not linked to any other matrix polymer by the composition. an intermolecular bond. In another embodiment, a fluid composition comprises a Aspects of the present specification provide, in part, a fluid Substantially uncrosslinked glycosaminoglycan. In aspects of composition comprising a substantially uncrosslinked matrix this embodiment, a fluid composition comprises a Substan polymer. As sued herein, the term “substantially 25 tially uncrosslinked chondroitin Sulfate polymer, a Substan uncrosslinked’ refers to the presence of uncrosslinked matrix tially uncrosslinked chondroitin Sulfate polymer, or a Sub polymers in a fluid composition disclosed in the present stantially uncrosslinked hyaluronan polymer. In other aspects specification at a level of at least 90% by weight of the of this embodiment, a fluid composition comprises an composition, with the remaining at most 10% by weight of uncrosslinked glycosaminoglycan where the uncrosslinked the composition being comprised of other components 30 glycosaminoglycan represents, e.g., about 90% by weight, including crosslinked matrix polymers. The matrix polymer about 91% by weight, about 92% by weight, about 93% by included in a fluid composition disclosed in the present speci weight, about 94% by weight, about 95% by weight, about fication exhibit a low degree of cross-linking in order to 96% by weight, about 97% by weight, about 98% by weight, remain water soluble. or about 99%, or about 100% by weight, of the total gly Aspects of the present specification provide, in part, a fluid 35 cosaminoglycan present in the composition. In yet other composition that is essentially free of a crosslinked matrix aspects of this embodiment, a fluid composition comprises an polymer. As used herein, the term “essentially free” (or “con uncrosslinked glycosaminoglycan where the uncrosslinked sisting essentially of) refers to a fluid composition where glycosaminoglycan represents, e.g., at least 90% by weight, only trace amounts of cross-linked matrix polymers can be at least 91% by weight, at least 92% by weight, at least 93% detected. 40 by weight, at least 94% by weight, at least 95% by weight, at Aspects of the present specification provide, in part, a fluid least 96% by weight, at least 97% by weight, at least 98% by composition that is entirely free of a crosslinked matrix poly weight, or at least 99% by weight, of the total glycosami mer. As used herein, the term “entirely free” refers to a fluid noglycan present in the composition. In still other aspects of composition that within the detection range of the instrument this embodiment, a fluid composition comprises an or process being used, crosslinked matrix polymers cannot be 45 uncrosslinked glycosaminoglycan where the uncrosslinked detected or its presence cannot be confirmed. glycosaminoglycan represents, e.g., about 90% to about Aspects of the present specification provide, in part, a fluid 100% by weight, about 93% to about 100% by weight, about composition comprising a ratio of crosslinked matrix poly 95% to about 100% by weight, or about 97% to about 100% merand uncrosslinked polymer. This ratio of crosslinked and by weight, of the total glycosaminoglycan present in the uncrosslinked matrix polymer is also known as the gel: fluid 50 composition. ratio. Any gel: fluid ratio is useful in making the fluid compo In yet another embodiment, a fluid composition comprises sitions disclosed in the present specification with the proviso a Substantially uncrosslinked lubricin. In aspects of this that Such ratio produces a fluid composition disclosed in the embodiment, a fluid composition comprises an uncrosslinked present specification that improves a skin condition as dis lubricin where the uncrosslinked lubricin represents, e.g., closed in the present specification. Non-limiting examples of 55 about 90% by weight, about 91% by weight, about 92% by gel: fluid ratios include 100:0, 98:2, 90:10, 75:25, 70:30, weight, about 93% by weight, about 94% by weight, about 60:40, 50:50, 40:60, 30.70, 25:75, 10:90: 2:98, and 0:100. 95% by weight, about 96% by weight, about 97% by weight, Aspects of the present specification provide, in part, a fluid about 98% by weight, or about 99%, or about 100% by composition comprising a matrix polymer having a mean weight, of the total lubricin present in the composition. In molecular weight. As used herein, the term “molecular 60 other aspects of this embodiment, a fluid composition com weight” refers to the sum of the atomic weights of the atoms prises an uncrosslinked lubricin where the uncrosslinked in a molecule. For example, that of methane (CH) is 16.043 lubricin represents, e.g., at least 90% by weight, at least 91% g/mol, the atomic weights being carbon-12.011 g/mol. by weight, at least 92% by weight, at least 93% by weight, at hydrogen-1.008 g/mol. least 94% by weight, at least 95% by weight, at least 96% by Thus, in an embodiment, a fluid composition comprises a 65 weight, at least 97% by weight, at least 98% by weight, or at Substantially uncrosslinked matrix polymer. In other aspects least 99% by weight, of the total lubricin present in the com of this embodiment, a fluid composition comprises an position. In yet other aspects of this embodiment, a fluid US 9,125,840 B2 10 composition comprises an uncrosslinked lubricin where the about 5%, about 6%, about 7%, about 8%, about 9%, about uncrosslinked lubricin represents, e.g., about 90% to about 10%, about 11%, about 12%, about 13%, about 14%, or about 100% by weight, about 93% to about 100% by weight, about 15%. In yet other aspects of this embodiment, a fluid compo 95% to about 100% by weight, or about 97% to about 100% sition comprises a crosslinked matrix polymer where the by weight, of the total lubricin present in the composition. degree of crosslinking is at most 1%, at most 2%, at most 3%, In another embodiment, a fluid composition comprises an at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, uncrosslinked matrix polymer that is entirely free of a at most 9%, at most 10%, at most 11%, at most 12%, at most crosslinked matrix polymer. 13%, at most 14%, or at most 15%. In still other aspects of this In yet another embodiment, a fluid composition comprises embodiment, a fluid composition comprises a crosslinked an uncrosslinked glycosaminoglycan that is entirely free of a 10 matrix polymer where the degree of crosslinking is about 1% crosslinked glycosaminoglycan. In an aspect of this embodi to about 15%, about 2% to about 11%, about 3% to about ment, a fluid composition comprises an uncrosslinked chon 10%, about 1% to about 5%, about 10% to about 15%, about droitin sulfate polymer that is entirely free of a crosslinked 11% to about 15%, about 6% to about 10%, or about 6% to chondroitin sulfate polymer. In another aspect of this embodi about 8%. ment, a fluid composition comprises an uncrosslinked keratan 15 In yet another embodiment, a fluid composition comprises sulfate polymer that is entirely free of a crosslinked keratan a crosslinked glycosaminoglycan. In aspect of this embodi Sulfate polymer. In yet another aspect of this embodiment, a ment, a fluid composition comprises a crosslinked chon fluid composition comprises an uncrosslinked hyaluronan droitin Sulfate polymer, a crosslinked keratan Sulfate poly polymer that is entirely free of a crosslinked hyaluronan mer, or a crosslinked hyaluronan polymer. In other aspects of polymer. this embodiment, a fluid composition comprises a In still another embodiment, a fluid composition comprises crosslinked glycosaminoglycan where the crosslinked gly an uncrosslinked lubricin that is entirely free of a crosslinked cosaminoglycan represents, e.g., about 1% by weight, about lubricin. 2% by weight, about 3% by weight, about 4% by weight, In another embodiment, a fluid composition comprises an about 5% by weight, about 6% by weight, about 7% by uncrosslinked matrix polymer that is essentially free of a 25 weight, about 8% by weight, or about 9%, or about 10% by crosslinked matrix polymer. weight, of the total glycosaminoglycan present in the com In yet another embodiment, a fluid composition comprises position. In yet other aspects of this embodiment, a fluid an uncrosslinked glycosaminoglycan that is essentially free composition comprises a crosslinked glycosaminoglycan of a crosslinked glycosaminoglycan. In an aspect of this where the crosslinked glycosaminoglycan represents, e.g., at embodiment, a fluid composition comprises an uncrosslinked 30 most 1% by weight, at most 2% by weight, at most 3% by chondroitin sulfate polymer that is essentially free of a weight, at most 4% by weight, at most 5% by weight, at most crosslinked chondroitin sulfate polymer. In another aspect of 6% by weight, at most 7% by weight, at most 8% by weight, this embodiment, a fluid composition comprises an at most 9% by weight, or at most 10% by weight, of the total uncrosslinked keratan Sulfate polymer that is essentially free glycosaminoglycan present in the composition. In still other of a crosslinked keratan Sulfate polymer. In yet another aspect 35 aspects of this embodiment, a fluid composition comprises a of this embodiment, a fluid composition comprises an crosslinked glycosaminoglycan where the crosslinked gly uncrosslinked hyaluronan polymer that is essentially free of a cosaminoglycan represents, e.g., about 0% to about 10% by crosslinked hyaluronan polymer. weight, about 1% to about 10% by weight, about 3% to about In still another embodiment, a fluid composition comprises 10% by weight, or about 5% to about 10% by weight, of the an uncrosslinked lubricin that is essentially free of a 40 total glycosaminoglycan present in the composition. crosslinked lubricin. In other aspects of this embodiment, a fluid composition In another embodiment, a fluid composition comprises a comprises a crosslinked glycosaminoglycan where the crosslinked matrix polymer. In other aspects of this embodi degree of crosslinking is about 1%, about 2%, about 3%, ment, a fluid composition comprises a crosslinked matrix about 4%, about 5%, about 6%, about 7%, about 8%, about polymer where the partially crosslinked matrix polymer rep 45 9%, about 10%, about 11%, about 12%, about 13%, about resents, e.g., about 1% by weight, about 2% by weight, about 14%, or about 15%. In yet other aspects of this embodiment, 3% by weight, about 4% by weight, about 5% by weight, a fluid composition comprises a crosslinked glycosaminogly about 6% by weight, about 7% by weight, about 8% by can where the degree of crosslinking is at most 1%, at most weight, or about 9%, or about 10% by weight, of the total 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most matrix polymer present in the composition. In yet other 50 7%, at most 8%, at most 9%, at most 10%, at most 11%, at aspects of this embodiment, a fluid composition comprises a most 12%, at most 13%, at most 14%, or at most 15%. In still crosslinked matrix polymer where the partially crosslinked other aspects of this embodiment, a fluid composition com matrix polymer represents, e.g., at most 1% by weight, at prises a crosslinked glycosaminoglycan where the degree of most 2% by weight, at most 3% by weight, at most 4% by crosslinking is about 1% to about 15%, about 2% to about weight, at most 5% by weight, at most 6% by weight, at most 55 11%, about 3% to about 10%, about 1% to about 5%, about 7% by weight, at most 8% by weight, at most 9% by weight, 10% to about 15%, about 11% to about 15%, about 6% to or at most 10% by weight, of the total matrix polymer present about 10%, or about 6% to about 8%. in the composition. In still other aspects of this embodiment, In still another embodiment, a fluid composition comprises a fluid composition comprises a crosslinked matrix polymer a crosslinked lubricin. In aspects of this embodiment, a fluid where the partially crosslinked matrix polymer represents, 60 composition comprises a crosslinked lubricin where the e.g., about 0% to about 10% by weight, about 1% to about crosslinked lubricin represents, e.g., about 1% by weight, 10% by weight, about 3% to about 10% by weight, or about about 2% by weight, about 3% by weight, about 4% by 5% to about 10% by weight, of the total matrix polymer weight, about 5% by weight, about 6% by weight, about 7% present in the composition. by weight, about 8% by weight, or about 9%, or about 10% by In other aspects of this embodiment, a fluid composition 65 weight, of the total lubricin present in the composition. In comprises a crosslinked matrix polymer where the degree of other aspects of this embodiment, a fluid composition com crosslinking is about 1%, about 2%, about 3%, about 4%, prises a crosslinked lubricin where the crosslinked lubricin US 9,125,840 B2 11 12 represents, e.g., at most 1% by weight, at most 2% by weight, mg/mL, about 12 mg/mL, about 13 mg/mL, about 13.5 at most 3% by weight, at most 4% by weight, at most 5% by mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL, weight, at most 6% by weight, at most 7% by weight, at most about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, or about 8% by weight, at most 9% by weight, or at most 10% by 20 mg/mL. In other aspects of this embodiment, a fluid com weight, of the total lubricin present in the composition. In yet position comprises an uncrosslinked glycosaminoglycan other aspects of this embodiment, a fluid composition com where the uncrosslinked glycosaminoglycan is present at a prises a crosslinked lubricin where the crosslinked lubricin concentration of e.g., at least 1 mg/mL, at least 2 mg/mL, at represents, e.g., about 0% to about 10% by weight, about 1% least 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 10 to about 10% by weight, about 3% to about 10% by weight, or mg/mL, at least 15 mg/mL, at least 20 mg/mL, or at least 25 about 5% to about 10% by weight, of the total lubricin present 10 mg/mL. In yet other aspects of this embodiment, a fluid in the composition. composition comprises an uncrosslinked glycosaminoglycan In other aspects of this embodiment, a fluid composition where the uncrosslinked glycosaminoglycan is present at a comprises a crosslinked lubricin where the degree of concentration of, e.g., at most 1 mg/mL, at most 2 mg/mL, at crosslinking is about 1%, about 2%, about 3%, about 4%, most 3 mg/mL, at most 4 mg/mL, at most 5 mg/mL, at most about 5%, about 6%, about 7%, about 8%, about 9%, about 15 10 mg/mL, at most 15 mg/mL, at most 20 mg/mL, or at most 10%, about 11%, about 12%, about 13%, about 14%, or about 25 mg/mL. In still other aspects of this embodiment, a fluid 15%. In yet other aspects of this embodiment, a fluid compo composition comprises an uncrosslinked glycosaminoglycan sition comprises a crosslinked lubricin where the degree of where the uncrosslinked glycosaminoglycan is present at a crosslinking is at most 1%, at most 2%, at most 3%, at most concentration of, e.g., about 7.5 mg/mL to about 19.5 mg/mL, 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most about 8.5 mg/mL to about 18.5 mg/mL, about 9.5 mg/mL to 9%, at most 10%, at most 11%, at most 12%, at most 13%, at about 17.5 mg/mL, about 10.5 mg/mL to about 16.5 mg/mL, most 14%, or at most 15%. In still other aspects of this about 11.5 mg/mL to about 15.5 mg/mL, or about 12.5 embodiment, a fluid composition comprises a crosslinked mg/mL to about 14.5 mg/mL. lubricin where the degree of crosslinking is about 1% to about In still another embodiment, a fluid composition comprises 15%, about 2% to about 11%, about 3% to about 10%, about 25 an uncrosslinked lubricin where the uncrosslinked lubricin is 1% to about 5%, about 10% to about 15%, about 11% to about present in an amount Sufficient to improve a condition of the 15%, about 6% to about 10%, or about 6% to about 8%. skin, such as, e.g., hydration or elasticity. In aspects of this In another embodiment, a fluid composition comprises an embodiment, a fluid composition comprises an uncrosslinked uncrosslinked matrix polymer where the uncrosslinked lubricin where the uncrosslinked lubricin is present at a con matrix polymer is present in an amount Sufficient to improve 30 centration of, e.g., about 5 mg/mL, about 6 mg/mL, about 7 a condition of the skin, such as, e.g., hydration or elasticity. In mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, aspects of this embodiment, a fluid composition comprises an about 11 mg/mL, about 12 mg/mL, about 13 mg/mL, about uncrosslinked matrix polymer where the uncrosslinked 13.5 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 matrix polymer is present at a concentration of, e.g., about 5 mg/mL, about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, 35 or about 20 mg/mL. In other aspects of this embodiment, a about 9 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 fluid composition comprises an uncrosslinked lubricin where mg/mL, about 13 mg/mL, about 13.5 mg/mL, about 14 the uncrosslinked lubricin is present at a concentration of mg/mL, about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, e.g., at least 1 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at about 18 mg/mL, about 19 mg/mL, or about 20 mg/mL. In least 15 mg/mL, at least 20 mg/mL, or at least 25 mg/mL. In other aspects of this embodiment, a fluid composition com 40 yet other aspects of this embodiment, a fluid composition prises an uncrosslinked matrix polymer where the comprises an uncrosslinked lubricin where the uncrosslinked uncrosslinked matrix polymer is presentata concentration of lubricin is present at a concentration of e.g., at most 1 e.g., at least 1 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at mg/mL, at most 5 mg/mL, at most 10 mg/mL, at most 15 least 15 mg/mL, at least 20 mg/mL, or at least 25 mg/mL. In mg/mL, at most 20 mg/mL, or at most 25 mg/mL. In still other yet other aspects of this embodiment, a fluid composition 45 aspects of this embodiment, a fluid composition comprises an comprises an uncrosslinked matrix polymer where the uncrosslinked lubricin where the uncrosslinked lubricin is uncrosslinked matrix polymer is presentata concentration of present at a concentration of e.g., about 7.5 mg/mL to about e.g., at most 1 mg/mL, at most 5 mg/mL, at most 10 mg/mL, 19.5 mg/mL, about 8.5 mg/mL to about 18.5 mg/mL, about at most 15 mg/mL, at most 20 mg/mL, or at most 25 mg/mL. 9.5 mg/mL to about 17.5 mg/mL, about 10.5 mg/mL to about In still other aspects of this embodiment, a fluid composition 50 16.5 mg/mL, about 11.5 mg/mL to about 15.5 mg/mL, or comprises an uncrosslinked matrix polymer where the about 12.5 mg/mL to about 14.5 mg/mL. uncrosslinked matrix polymer is presentata concentration of In a further embodiment, a fluid composition comprises a e.g., about 7.5 mg/mL to about 19.5 mg/mL, about 8.5 mg/mL crosslinked matrix polymer and an uncrosslinked matrix to about 18.5 mg/mL, about 9.5 mg/mL to about 17.5 mg/mL, polymer. In another aspect of this embodiment, a fluid com about 10.5 mg/mL to about 16.5 mg/mL, about 11.5 mg/mL 55 position comprises a crosslinked matrix polymer and an to about 15.5 mg/mL, or about 12.5 mg/mL to about 14.5 uncrosslinked matrix polymer where the gel: fluid ratio is mg/mL. sufficient to form a fluid. In other aspects of this embodiment, In yet another embodiment, a fluid composition comprises a fluid composition comprises a crosslinked matrix polymer an uncrosslinked glycosaminoglycan where the and an uncrosslinked matrix polymer where the gel: fluid ratio uncrosslinked glycosaminoglycan is present in an amount 60 is, e.g., about 0:100, about 1:99, about 2:98, about 3:97, about Sufficient to improve a condition of the skin, such as, e.g., 4:96, about 5:95, about 6:94, about 7:93, about 8:92, about hydration or elasticity. In aspects of this embodiment, a fluid 9:91, or about 10:90. In yet other aspects of this embodiment, composition comprises an uncrosslinked glycosaminoglycan a fluid composition comprises a crosslinked matrix polymer where the uncrosslinked glycosaminoglycan is present at a and an uncrosslinked matrix polymer where the gel: fluid ratio concentration of, e.g., about 2 mg/mL, about 3 mg/mL, about 65 is, e.g., at most 1:99, at most 2:98, at most 3:97, at most 4:96, 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, at most 5:95, at most 6:94, at most 7:93, at most 8:92, at most about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11 9:91, or at most 10:90. In still other aspects of this embodi US 9,125,840 B2 13 14 ment, a fluid composition comprises a crosslinked matrix a concentration of, e.g., at most 1 mg/mL, at most 5 mg/mL, polymer and an uncrosslinked matrix polymer where the at most 10 mg/mL, at most 15 mg/mL, at most 20 mg/mL, or gel: fluid ratio is, e.g., about 0:100 to about 3:97, about 0:100 at most 25 mg/mL. In still other aspects of this embodiment, to about 5:95, or about 0:100 to about 10:90. a fluid composition comprises an uncrosslinked hyaluronan In other aspects of this embodiment, a fluid composition where the uncrosslinked hyaluronan is present at a concen comprises acrosslinked matrix polymerand an uncrosslinked tration of e.g., about 7.5 mg/mL to about 19.5 mg/mL, about matrix polymer where the gel: fluid ratio is, e.g., about 15:85, 8.5 mg/mL to about 18.5 mg/mL, about 9.5 mg/mL to about about 20:80, about 25:75, about 30:70, about 35:65, about 17.5 mg/mL, about 10.5 mg/mL to about 16.5 mg/mL, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, 11.5 mg/mL to about 15.5 mg/mL, or about 12.5 mg/mL to about 65:35, about 70:30, about 75:25, about 80:20, about 10 about 14.5 mg/mL. 85:15, about 90:10, about 95:5, about 98:2, or about 100:0. In In other aspects of this embodiment, a fluid composition yet other aspects of this embodiment, a fluid composition comprises an uncrosslinked hyaluronan where the comprises acrosslinked matrix polymerand an uncrosslinked uncrosslinked hyaluronan has a mean molecular weight of matrix polymer where the gel: fluid ratio is, e.g., at most e.g., about 1,000,000 Da, about 1,500,000 Da, about 2,000, 15:85, at most 20:80, at most 25:75, at most 30:70, at most 15 000 Da, about 2,500,000 Da, about 3,000,000 Da, about 35:65, at most 40:60, at most 45:55, at most 50:50, at most 3,500,000 Da, about 4,000,000 Da, about 4,500,000 Da, or 55:45, at most 60:40, at most 65:35, at most 70:30, at most about 5,000,000 Da. In yet other aspects of this embodiment, 75:25, at most 80:20, at most 85:15, at most 90:10, at most a fluid composition comprises an uncrosslinked hyaluronan 95:5, at most 98:2, or at most 100:0. In still other aspects of where the uncrosslinked hyaluronan has a mean molecular this embodiment, a fluid composition comprises a weight of, e.g., at least 1,000,000 Da, at least 1,500,000 Da, at crosslinked matrix polymer and an uncrosslinked matrix least 2,000,000 Da, at least 2,500,000 Da, at least 3,000,000 polymer where the gel:fluid ratio is, e.g., about 10:90 to about Da, at least 3,500,000 Da, at least 4,000,000 Da, at least 70:30, about 15:85 to about 70:30, about 10:90 to about 4,500,000 Da, or at least 5,000,000 Da. In still other aspects of 55:45, about 80:20 to about 95:5, about 90:10 to about 100:0, this embodiment, a fluid composition comprises an about 75:25 to about 100:0, or about 60:40 to about 100:0. 25 uncrosslinked hyaluronan where the uncrosslinked hyaluro In another embodiment, a fluid composition comprises a nan has a mean molecular weight of, e.g., about 1,000,000 Da Substantially uncrosslinked hyaluronan. In aspects of this to about 5,000,000 Da, about 1,500,000 Da to about 5,000, embodiment, a fluid composition comprises an uncrosslinked 000 Da, about 2,000,000 Da to about 5,000,000 Da, about hyaluronan where the uncrosslinked hyaluronan represents, 2,500,000 Da to about 5,000,000 Da, about 2,000,000 Da to e.g., about 90% by weight, about 91% by weight, about 92% 30 about 3,000,000 Da, about 2,500,000 Da to about 3,500,000 by weight, about 93% by weight, about 94% by weight, about Da, or about 2,000,000 Da to about 4,000,000 Da. In further 95% by weight, about 96% by weight, about 97% by weight, aspects, a fluid composition comprises an uncrosslinked about 98% by weight, or about 99%, or about 100% by hyaluronan where the uncrosslinked hyaluronan has a mean weight, of the total hyaluronan present in the composition. In molecular weight of, e.g., greater than 2,000,000 Da and less other aspects of this embodiment, a fluid composition com 35 than about 3,000,000 Da, greater than 2,000,000 Da and less prises an uncrosslinked hyaluronan where the uncrosslinked than about 3,500,000 Da, greater than 2,000,000 Da and less hyaluronan represents, e.g., at least 90% by weight, at least than about 4,000,000 Da, greater than 2,000,000 Da and less 91% by weight, at least 92% by weight, at least 93% by than about 4,500,000 Da, greater than 2,000,000 Da and less weight, at least 94% by weight, at least 95% by weight, at than about 5,000,000 Da. least 96% by weight, at least 97% by weight, at least 98% by 40 In another embodiment, a fluid composition comprises an weight, or at least 99% by weight, of the total hyaluronan uncrosslinked hyaluronan where the uncrosslinked hyaluro present in the composition. In yet other aspects of this nan comprises a combination of both high molecular weight embodiment, a fluid composition comprises an uncrosslinked hyaluronan and low molecular weight hyaluronan, in various hyaluronan where the uncrosslinked hyaluronan represents, ratios. As used herein, the term “high molecular weight e.g., about 90% to about 100% by weight, about 93% to about 45 hyaluronan’ refers to a hyaluronan polymer that has a 100% by weight, about 95% to about 100% by weight, or molecular weight of 1,000,000 Da or greater. Non-limiting about 97% to about 100% by weight, of the total hyaluronan examples of a high molecular weight hyaluronan include a present in the composition. hyaluronan of about 1,500,000 Da, a hyaluronan of about In yet another embodiment, a fluid composition comprises 2,000,000 Da, a hyaluronan of about 2,500,000 Da, a hyalu an uncrosslinked hyaluronan where the uncrosslinked hyalu 50 roman of about 3,000,000 Da, a hyaluronan of about 3,500, roman is present in an amount Sufficient to improve a condi 000 Da, a hyaluronan of about 4,000,000 Da, a hyaluronan of tion of the skin, Such as, e.g., hydration or elasticity. In aspects about 4,500,000 Da, and a hyaluronan of about 5,000,000 Da. of this embodiment, a fluid composition comprises an As used herein, the term “low molecular weight hyaluronan' uncrosslinked hyaluronan where the uncrosslinked hyaluro refers to a hyaluronan polymer that has a molecular weight of nan is present at a concentration of, e.g., about 5 mg/mL, 55 less than 1,000,000 Da. Non-limiting examples of a low about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 molecular weight hyaluronan include a hyaluronan of about mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, 200,000 Da, a hyaluronan of about 300,000 Da, a hyaluronan about 13 mg/mL, about 13.5 mg/mL, about 14 mg/mL, about of about 400,000 Da, a hyaluronan of about 500,000 Da, a 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18 hyaluronan of about 600,000 Da, a hyaluronan of about 700, mg/mL, about 19 mg/mL, or about 20 mg/mL. In other 60 000 Da, a hyaluronan of about 800,000 Da, and a hyaluronan aspects of this embodiment, a fluid composition comprises an of about 900,000 Da. uncrosslinked hyaluronan where the uncrosslinked hyaluro Thus, in an embodiment, a fluid composition comprises an nan is present at a concentration of e.g., at least 1 mg/mL, at uncrosslinked hyaluronan where the uncrosslinked hyaluro least 5 mg/mL, at least 10 mg/mL, at least 15 mg/mL, at least nan comprises a combination of both high molecular weight 20 mg/mL, or at least 25 mg/mL. In yet other aspects of this 65 hyaluronan and low molecular weighthyaluronan in a ratio of embodiment, a fluid composition comprises an uncrosslinked about 20:1, about 15:1, about 10:1, about 5:1, about 1:1, about hyaluronan where the uncrosslinked hyaluronan is present at 1:5 about 1:10, about 1:15, or about 1:20. US 9,125,840 B2 15 16 In still another embodiment, a fluid composition comprises where the gel: fluid ratio is, e.g., about 0:100, about 1:99, a crosslinked hyaluronan. In aspects of this embodiment, a about 2.98, about 3:97, about 4:96, about 5:95, about 6:94, fluid composition comprises a crosslinked hyaluronan where about 7.93, about 8:92, about 9:91, or about 10:90. In yet the crosslinked hyaluronan represents, e.g., about 1% by other aspects of this embodiment, a fluid composition com weight, about 2% by weight, about 3% by weight, about 4% prises a crosslinked hyaluronan and an uncrosslinked hyalu by weight, about 5% by weight, about 6% by weight, about roman where the gel: fluid ratio is, e.g., at most 1:99, at most 7% by weight, about 8% by weight, or about 9%, or about 2:98, at most 3:97, at most 4:96, at most 5:95, at most 6:94, at 10% by weight, of the total hyaluronan present in the com most 7:93, at most 8:92, at most 9:91, or at most 10:90. In still position. In other aspects of this embodiment, a fluid compo other aspects of this embodiment, a fluid composition com sition comprises a crosslinked hyaluronan where the 10 prises a crosslinked hyaluronan and an uncrosslinked hyalu crosslinked hyaluronan represents, e.g., at most 1% by roman where the gel: fluid ratio is, e.g., about 0:100 to about weight, at most 2% by weight, at most 3% by weight, at most 3:97, about 0:100 to about 5:95, or about 0:100 to about 4% by weight, at most 5% by weight, at most 6% by weight, 10:90. at most 7% by weight, at most 8% by weight, at most 9% by In other aspects of this embodiment, a fluid composition weight, or at most 10% by weight, of the total hyaluronan 15 comprises a crosslinked hyaluronan and an uncrosslinked present in the composition. In yet other aspects of this hyaluronan where the gel: fluid ratio is, e.g., about 15:85, embodiment, a fluid composition comprises a crosslinked about 20:80, about 25:75, about 30:70, about 35:65, about hyaluronan where the crosslinked hyaluronan represents, 40:60, about 45:55, about 50:50, about 55:45, about 60:40, e.g., about 0% to about 10% by weight, about 1% to about about 65:35, about 70:30, about 75:25, about 80:20, about 10% by weight, about 3% to about 10% by weight, or about 85:15, about 90:10, about 95:5, about 98:2, or about 100:0. In 5% to about 10% by weight, of the total hyaluronan present in yet other aspects of this embodiment, a fluid composition the composition. comprises a crosslinked hyaluronan and an uncrosslinked In other aspects of this embodiment, a fluid composition hyaluronan where the gel:fluid ratio is, e.g., at most 15:85, at comprises a crosslinked hyaluronan where the degree of most 20:80, at most 25:75, at most 30:70, at most 35:65, at crosslinking is about 1%, about 2%, about 3%, about 4%, 25 most 40:60, at most 45:55, at most 50:50, at most 55:45, at about 5%, about 6%, about 7%, about 8%, about 9%, about most 60:40, at most 65:35, at most 70:30, at most 75:25, at 10%, about 11%, about 12%, about 13%, about 14%, or about most 80:20, at most 85:15, at most 90:10, at most 95:5, at 15%. In yet other aspects of this embodiment, a fluid compo most 98:2, or at most 100:0. In still other aspects of this sition comprises a crosslinked hyaluronan where the degree embodiment, a fluid composition comprises a crosslinked of crosslinking is at most 1%, at most 2%, at most 3%, at most 30 hyaluronan and an uncrosslinked hyaluronan where the gel: 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most fluid ratio is, e.g., about 10:90 to about 70:30, about 15:85 to 9%, at most 10%, at most 11%, at most 12%, at most 13%, at about 70:30, about 10:90 to about 55:45, about 80:20 to about most 14%, or at most 15%. In still other aspects of this 95:5, about 90:10 to about 100:0, about 75:25 to about 100:0, embodiment, a fluid composition comprises a crosslinked or about 60:40 to about 100:0. hyaluronan where the degree of crosslinking is about 1% to 35 Aspects of the present specification provide, in part, a fluid about 15%, about 2% to about 11%, about 3% to about 10%, composition comprising a stabilizing component. As used about 1% to about 5%, about 10% to about 15%, about 11% herein, the term “stabilizing component” refers to a molecule to about 15%, about 6% to about 10%, or about 6% to about that reduces or prevents the degradation of a matrix polymer 8%. disclosed in the present specification. The stabilizing compo In other aspects of this embodiment, a fluid composition 40 nent can reduce or prevent enzymatic degradation and/or comprises a crosslinked hyaluronan where the crosslinked reduce or prevent chemical degradation. Non-limiting hyaluronan has a mean molecular weight of, e.g., about examples of Stabilizing components include a polyol and a 1,000,000 Da, about 1,500,000 Da, about 2,000,000 Da, flavonoid. about 2,500,000 Da, about 3,000,000 Da, about 3,500,000 Aspects of the present specification provide, in part, a fluid Da, about 4,000,000 Da, about 4,500,000 Da, or about 5,000, 45 composition comprising a polyol. As used herein, the term 000 Da. In yet other aspects of this embodiment, a fluid "polyol is synonymous with “sugar alcohol.” “polyhydric composition comprises a crosslinked hyaluronan where the alcohol.” and “polyalcohol and refers to a hydrogenated crosslinked hyaluronan has a mean molecular weight of, e.g., form of carbohydrate, whose carbonyl group (aldehyde or at least 1,000,000 Da, at least 1,500,000 Da, at least 2,000,000 ketone, reducing Sugar) has been reduced to a primary or Da, at least 2,500,000 Da, at least 3,000,000 Da, at least 50 secondary hydroxyl group (hence the alcohol). Such as, e.g., 3,500,000 Da, at least 4,000,000 Da, at least 4,500,000 Da, or mannitol from mannose, Xylitol from Xylose, and lactitol at least 5,000,000 Da. In still other aspects of this embodi from lactulose. Polyols have the general formula H(HCHO) ment, a fluid composition comprises a crosslinked hyaluro +1 H. Both monosaccharides and disaccharides can form nan where the crosslinked hyaluronan has a mean molecular polyols; however, polyols derived from disaccharides are not weight of, e.g., about 1,000,000 Da to about 5,000,000 Da. 55 entirely hydrogenated because only one aldehyde group is about 1,500,000 Da to about 5,000,000 Da, about 2,000,000 available for reduction. A fluid composition disclosed in the Da to about 5,000,000 Da, about 2,500,000 Da to about present specification may comprise a single polyol, or a plu 5,000,000 Da, about 2,000,000 Da to about 3,000,000 Da, rality of polyols. about 2,500,000 Da to about 3,500,000 Da, or about 2,000, A fluid composition disclosed in the present specification 000 Da to about 4,000,000 Da. 60 comprises a polyol that is pharmaceutically acceptable. As In a further embodiment, a fluid composition comprises a used herein, the term “pharmaceutically acceptable” means crosslinked hyaluronan and an uncrosslinked hyaluronan. In any molecular entity or composition that does not produce an an aspect of this embodiment, a fluid composition comprises adverse, allergic or other untoward or unwanted reaction a crosslinked hyaluronan and an uncrosslinked hyaluronan when administered to a mammal. It is known in the art that the where the gel: fluid ratio is sufficient to form a fluid. In other 65 two-carbon polyol, glycol, is not pharmaceutically accept aspects of this embodiment, a fluid composition comprises a able because this polyol is toxic to mammals. It is also known crosslinked hyaluronan and an uncrosslinked hyaluronan in the art that polyols comprising three or more carbonatoms US 9,125,840 B2 17 18 are typically pharmaceutically acceptable. As such, polyols leads to the formation of the hydroxyl radical (OH.). A free comprising three or more carbon atoms are generally useful radical formed in this way can give rise to a series of reactions in making the compositions disclosed in the present specifi leading to the formation of different species of active oxygen. cation. Table 2 describes the principal active species of oxygen. TABLE 2 Principal Species of active oxygen

Chemical Name Symbol Comments Superoxide O Superoxide anions are formed by reduction of molecular anion oxygen. They are minimally reactive in aqueous media, which enables them to migrate quite a long way from their site of production. Superoxide anions have a weak oxidative action but are capable of generating more reactive radicals. Hydrogen H2O2 Hydrogen peroxides are formed either by bivalent reduction peroxide of molecular oxygen, or by dismutation of the Superoxide anion. The absence of electric charges on their Surface makes them very lipophilic and minimally reactive in aqueous media. The degradation of hydrogen peroxides (Fenton's reaction) produces very reactive radicals called hydroxyl radicals. Hydroxyl radical OH Hydroxyl radicals are formed by degradation of hydrogen peroxide in the presence of transition metals in their reduced form (Fenton's reaction). Hydroxyl radicals are very reactive. Their half-life is on the order of 10's. Peroxyl radical ROO Peroxyl radicals are formed by addition of molecular oxygen to free carbon radicals. They are minimally reactive. Organic ROOH Organic hydroperoxides are the protonated form of peroxyl hydroperoxide radicals. They are very reactive and decompose anew into peroxyl radicals and alcoxyl radicals. Alcoxyl radical RO Alcoxyl radicals are formed during the degradation of organic peroxides. They are very reactive. Their half-life is on the order of 10s. Nitric oxide NO Nitric oxides are synthesized from arginine (an amino acid) via the action of nitric oxide synthetase. They interact with hydroxyl radicals to form peroxynitrites. Nitric oxide is a neuromediator and can also be used by immune cells to destroy microbes or dangerous cells. Peroxynitrite ONOO Peroxynitrites are capable of oxidizing certain Substances Such as methionine (an amino acid serving as a constituent of proteins and enzymes) or of reacting with SOD (Cf. S 1.3.2.a) thereby “nitrating tyrosine (another very important amino acid) Nitrosyl radical ONOOH Degradation of nitrosyl radicals leads to the formation of hydroxyl radicals. Oxidizing power: OH >RO > ROO > NO

A fluid composition disclosed in the present specification However, because of their reactivity, these same free radi comprises a polyol in an amount Sufficient to protect cals can participate in unwanted side reactions resulting in uncrosslinked glycosaminoglycans, like hyaluronan, from cell damage. For purposes of the present specification, OH. degradation, such as enzymatic degradation and chemical radicals interact with the hydrogen located on the carbon degradation. One primary means of Such glycosaminoglycan adjacent to the carboxyl group in the glucuronic ring of hyalu degradation is chemical breakdown from exposure to free 50 roman and other glycosaminoglycans, thereby removing it. radicals, such as, e.g., OH. radicals. Free radicals are atoms, This removal causes splitting of the glycoside bond and hence molecules, or ions with unpaired electrons on an open shell depolymerization of the polymer. configuration. The unpaired electrons cause them to be highly chemically reactive. Free radicals play an important role in a number of biologi 55 cal processes, some of which are necessary for life. Such as OH / the intracellular killing of bacteria by phagocytic cells Such as H HOC granulocytes and macrophages. Free radicals have also been implicated in certain cell signaling processes, called redox HO O O- O O signaling. The two most important oxygen-centered free radi 60 O HO cals are superoxide and hydroxyl radical. They are derived NH OH from molecular oxygen under reducing conditions. For example, the Superoxide anion (O) can capture another O ={ pi electron to form the peroxide ion (O.), which in turn may CH react with two H-protons to form hydrogen peroxide (HO). 65 Site of OHo attach The degradation of hydrogen peroxide in the presence of transition metals in their reduced form (Fenton's reaction) US 9,125,840 B2 19 20 In general, the crosslinking observed in the hyaluronan and fluid composition comprises a polyol in an amount of e.g., other matrix polymers used in dermal fillers protect these about 0.1% (w/v) of the composition, about 0.2% (w/v) of the polymers against the chemical breakdown of free radical composition, about 0.3% (w/v) of the composition, about species of oxygen. This is because the bonds formed during 0.4% (w/v) of the composition, about 0.5% (w/v) of the crosslinking mask the hydrogen attached by the free radicals. 5 composition, about 0.6% (w/v) of the composition, about However, the uncrosslinked glycosaminoglycans disclosed in 0.7% (w/v) of the composition, about 0.8% (w/v) of the the present specification is afforded no such protection. The composition, about 0.9% (w/v) of the composition, about present specification discloses an alternative means of pro 1.0% (w/v) of the composition, about 2.0% (w/v) of the tecting matrix polymers from oxidative degradation of free composition, about 3.0% (w/v) of the composition, about radicals. It has been determined that polyols disclosed in the 10 4.0% (w/v) of the composition, about 5.0% (w/v) of the present specification act as stabilizing agents that can neu composition, about 6.0% (w/v) of the composition, about tralize free radicals of active oxygen. As a stabilizing, a polyol 7.0% (w/v) of the composition, about 8.0% (w/v) of the is stabilizing component that protects matrix polymers like composition, about 9.0% (w/v) of the composition, or about hyaluronan against the effects of oxidative stress and limiting 10% (w/v) of the composition. In other aspects, a fluid com the degradation of the fluid compositions disclosed in the 15 position comprises a polyol in an amount of, e.g., at least present specification. 0.1% (w/v) of the composition, at least 0.2% (w/v) of the Thus, any polyol is useful in making the compositions composition, at least 0.3% (w/v) of the composition, at least disclosed in the present specification, with the proviso that the 0.4% (w/v) of the composition, at least 0.5% (w/v) of the polyol is non-toxic to a mammal and the polyol protects composition, at least 0.6% (w/v) of the composition, at least uncrosslinked matrix polymers like hyaluronan from degra 0.7% (w/v) of the composition, at least 0.8% (w/v) of the dation. Non-limiting examples of polyols include, glycerol, composition, at least 0.9% (w/v) of the composition, at least erythritol, threitol, arabitol, erythritol, ribitol, xylitol, galac 1.0% (w/v) of the composition, at least 2.0% (w/v) of the titol (or dulcitol), glucitol (or sorbitol), iditol, inositol, man composition, at least 3.0% (w/v) of the composition, at least nitol, isomalt, lactitol, maltitol, and polyglycitol. Other non 4.0% (w/v) of the composition, at least 5.0% (w/v) of the limiting examples of polyols can be found in, e.g., 25 composition, at least 6.0% (w/v) of the composition, at least Pharmaceutical Dosage Forms and Drug Delivery Systems 7.0% (w/v) of the composition, at least 8.0% (w/v) of the (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins composition, at least 9.0% (w/v) of the composition, or at Publishers, 7" ed. 1999); Remington: The Science and Prac least 10% (w/v) of the composition. In yet other aspects, a tice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Will fluid composition comprises a polyol in an amount of, e.g., at iams & Wilkins, 20' ed. 2000); Goodman & Gilman's The 30 most 0.1% (w/v) of the composition, at most 0.2% (w/v) of Pharmacological Basis of Therapeutics (Joel G. Hardman et the composition, at most 0.3% (w/v) of the composition, at al., eds., McGraw-Hill Professional, 10" ed. 2001); and most 0.4% (w/v) of the composition, at most 0.5% (w/v) of Handbook of Pharmaceutical Excipients (Raymond C. Rowe the composition, at most 0.6% (w/v) of the composition, at et al., APhA Publications, 4" edition 2003), each of which is most 0.7% (w/v) of the composition, at most 0.8% (w/v) of hereby incorporated by reference in its entirety. 35 the composition, at most 0.9% (w/v) of the composition, at Thus in an embodiment, a fluid composition comprises a most 1.0% (w/v) of the composition, at most 2.0% (w/v) of pharmaceutically acceptable polyol that can reduce or pre the composition, at most 3.0% (w/v) of the composition, at vent degradation of a matrix polymer. In aspects of this most 4.0% (w/v) of the composition, at most 5.0% (w/v) of embodiment, a fluid composition comprises a pharmaceuti the composition, at most 6.0% (w/v) of the composition, at cally acceptable three-carbon polyol, a pharmaceutically 40 most 7.0% (w/v) of the composition, at most 8.0% (w/v) of acceptable four-carbon polyol, a pharmaceutically accept the composition, at most 9.0% (w/v) of the composition, or at able five-carbon polyol, a pharmaceutically acceptable six most 10% (w/v) of the composition. In still other aspects, a carbon polyol, a pharmaceutically acceptable seven-carbon fluid composition comprises a polyol in an amount of e.g., polyol, a pharmaceutically acceptable eight-carbon polyol, a about 0.1% (w/v) to about 1.0% (w/v) of the composition, pharmaceutically acceptable nine-carbon polyol, a pharma 45 about 0.1% (w/v) to about 2.0% (w/v) of the composition, ceutically acceptable ten-carbon polyol, a pharmaceutically about 0.1% (w/v) to about 3.0% (w/v) of the composition, acceptable eleven-carbon polyol, or a pharmaceutically about 0.1% (w/v) to about 4.0% (w/v) of the composition, acceptable twelve-carbon polyol. In other aspects of this about 0.1% (w/v) to about 5.0% (w/v) of the composition, embodiment, a fluid composition comprises glycerol, eryth about 0.2% (w/v) to about 0.9% (w/v) of the composition, ritol, threitol, arabitol, erythritol, ribitol, xylitol, galactitol (or 50 about 0.2% (w/v) to about 1.0% (w/v) of the composition, dulcitol), glucitol (or Sorbitol), iditol, inositol, mannitol, iso about 0.2% (w/v) to about 2.0% (w/v) of the composition, malt, lactitol, maltitol, or polyglycitol. about 0.5% (w/v) to about 1.0% (w/v) of the composition, or In another embodiment, a fluid composition comprises a about 0.5% (w/v) to about 2.0% (w/v) of the composition. single polyol that can reduce or prevent degradation of a In other aspects of this embodiment, a fluid composition matrix polymer. In yet another embodiment, a fluid compo 55 comprises a polyol is presentata concentration of, e.g., about sition comprises a plurality of polyols, each of which can 0.01 mg/mL, about 0.02 mg/mL, about 0.03 mg/mL, about reduce or prevent the degradation of a matrix polymer. In 0.04 mg/mL, about 0.05 mg/mL, about 0.06 mg/mL, about aspects of this embodiment, a fluid composition comprises 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about one or more polyols, two or more polyols, three or more 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 polyols, four or more polyols, or five or more polyols. In other 60 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 aspects of this embodiment, a fluid composition comprises mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1.0 one to five polyols, two to five polyols, three to five polyols, mg/mL, about 2.0 mg/mL, about 3.0 mg/mL, about 4.0 two to four polyols, two to five polyols, or three to five mg/mL, about 5.0 mg/mL, about 6.0 mg/mL, about 7.0 polyols. mg/mL, about 8.0 mg/mL, about 9.0 mg/mL, or about 10 In yet another embodiment, a fluid composition comprises 65 mg/mL. In yet other aspects of this embodiment, a fluid a polyol in an amount Sufficient to reduce or prevent degra composition comprises a polyol presentata concentration of dation of a matrix polymer. In aspects of this embodiment, a e.g., at least 0.01 mg/mL, at least 0.02 mg/mL, at least 0.03 US 9,125,840 B2 21 22 mg/mL, at least 0.04 mg/mL, at least 0.05 mg/mL, at least dihydro-2-phenylchromen-4-one have the 2,3-dihydro 0.06 mg/mL, at least 0.07 mg/mL, at least 0.08 mg/mL, at group, but lack the 3-hydroxyl group; and flavanonols (3-hy least 0.09 mg/mL, at least 0.1 mg/mL, at least 0.2 mg/mL, at droxyflavanone or 2,3-dihydroflavonol), compounds derived least 0.3 mg/mL, at least 0.4 mg/mL, at least 0.5 mg/mL, at from 3-hydroxy-2,3-dihydro-2-phenylchromen-4-one have least 0.6 mg/mL, at least 0.7 mg/mL, at least 0.8 mg/mL, at both functional groups. least 0.9 mg/mL, at least 1.0 mg/mL, at least 2.0 mg/mL, at Non-limiting examples of flavones include acacetin, apiin, least 3.0 mg/mL, at least 4.0 mg/mL, at least 5.0 mg/mL, at apigenin, apigetirin, artoindonesianin P. baicalein, baicalin, least 6.0 mg/mL, at least 7.0 mg/mL, at least 8.0 mg/mL, at chrysin, cynaroside, dioSmetin, dioSmin, eupatilin, flavoxate, least 9.0 mg/mL, or at least 10 mg/mL. In still other aspects of 6-hydroxyflavone, genkwanin, hidrosmin, luteolin, nepetin, this embodiment, a fluid composition comprises a polyol 10 nepitrin (nepetin 7-glucoside), nobiletin, orientin (isoorien present at a concentration of, e.g., at most 0.01 mg/mL, at tin), oroXindin, oroxylin A. rhoifolin, Scutellarein, Scutel most 0.02 mg/mL, at most 0.03 mg/mL, at most 0.04 mg/mL, larin, tangeritin, techtochrysin, tetuin, tricin, Veronicastro at most 0.05 mg/mL, at most 0.06 mg/mL, at most 0.07 side, Vitexin (isoVitexin), and wogonin. Non-limiting mg/mL, at most 0.08 mg/mL, at most 0.09 mg/mL, at most 0.1 examples of flavonols include 3-hydroxyflavone, azaleatin, mg/mL, at most 0.2 mg/mL, at most 0.3 mg/mL, at most 0.4 15 fisetin, galangin, gossypetin, kaempferide, kaempferol, mg/mL, at most 0.5 mg/mL, at most 0.6 mg/mL, at most 0.7 isorhamnetin, morin, myricetin, natsudaidain, pachypodol. mg/mL, at most 0.8 mg/mL, at most 0.9 mg/mL, at most 1.0 quercetin, rhamnazin, rhamnetin, and Sophorin. Non-limiting mg/mL, at most 2.0 mg/mL, at most 3.0 mg/mL, at most 4.0 examples offlavanones includebutin, eriodictyol. hesperetin, mg/mL, at most 5.0 mg/mL, at most 6.0 mg/mL, at most 7.0 hesperidin, homoeriodictyol, isosakuranetin, maringenin, mg/mL, at most 8.0 mg/mL, at most 9.0 mg/mL, or at most 10 naringin, pinocembrin, poncirin, Sakuranetin, Sakuranin, and mg/mL. In further aspects, a fluid composition comprises a Sterubin. Non-limiting examples of flavanonols include taxi polyol present at a concentration of e.g., about 0.01 mg/mL folin (dihydroquercetin), and aromadedrin (dihydro to about 0.7 mg/mL, about 0.06 mg/mL to about 0.7 mg/mL, kaempferol). about 0.01 mg/mL to about 1.0 mg/mL, about 0.05 mg/mL to Isoflavonoids include isoflavones and isoflavanes (Table about 1.0 mg/mL, about 0.06 mg/mL to about 1.0 mg/mL, 25 3). Non-limiting examples of isoflavonoids include alpinumi about 0.1 mg/mL to about 1.0 mg/mL, about 0.1 mg/mL to Soflavone, anagyroidisoflavone A and B, calycosin, daidzein, about 2.0 mg/mL, about 0.1 mg/mL to about 3.0 mg/mL, daidzin, derrubone, di-O-methylalpinumisoflavone, for about 0.1 mg/mL to about 4.0 mg/mL, about 0.1 mg/mL to mononetin, genistein, genistin, glycitein, ipriflavone, irige about 5.0 mg/mL, about 0.2 mg/mL to about 0.9 mg/mL, nin, iridin, irilone, 4'-methyl-alpinumisoflavone, 5-O-meth about 0.2 mg/mL to about 1.0 mg/mL, about 0.2 mg/mL to 30 ylgenistein, luteone, ononin, orobol, pratensein, prunetin, about 2.0 mg/mL, about 0.5 mg/mL to about 1.0 mg/mL, or pseudobaptigenin, psi-tectorigenin, puerarin, retusin, tectori about 0.5 mg/mL to about 2.0 mg/mL. din, tectorigenin, and wighteone. Aspects of the present specification provide, in part, a fluid Neoflavonoids include 4-arylcoumarins (neoflavones), composition that can optionally comprise or not comprise a 4-arylchromanes, dalbergiones and dalbergiquinols (Table flavonoid (Table 3). A flavonoid (or bioflavonoid) refers to the 35 3). Neoflavones are compounds derived from 4-phenylcou class of polyphenolic ketone-containing and non-ketone-con marin (or 4-Aryl-coumarin); neoflavenes compounds derived taining secondary metabolites found in plants that are well from 4-phenylchromen. Non-limiting examples of neofla known to have diverse beneficial biochemical and antioxidant vonoids include calophyllolide, coutareagenin, dalber effects. Non-limiting examples of flavonoids include C-me gichromene, dalbergin, and nivetin. thylated flavonoids, O-methylated flavonoids, isoflavonoids, 40 Non-ketone-containing flavonoids, include flavan-3-ols neoflavonoids, flavonolignans, furanoflavonoids, pyranofla and catechins. Flavan-3-ols (flavanols) are a class of fla vonoids, methylenedioxyflavonoids, prenylated flavonoids, vonoids derived from 2-phenyl-3,4-dihydro-2H-chromen-3- aurones, flavones, flavonols, flavanones, flavanonols, flavan ol skeleton. Catechin possesses two benzene rings (called the 3-ols, flavan-4-ols, leucoanthocyanidin (flavan-3,4-diols), A- and B-rings) and a dihydropyran heterocycle (the C-ring) anthocyanidins, and tannins. It is understood that these and 45 with an hydroxyl group on carbon 3. The A ring is similar to other Substances known in the art of pharmacology can be a resorcinol moiety while the Bring is similar to a catechol included in a fluid composition disclosed in the present speci moiety. There are two chiral centers on the molecule on fication. See for example, Remington’s Pharmaceutical Sci carbons 2 and 3. It has therefore four diastereoisomers. Two ences Mac Publishing Company, Easton, Pa. 16' Edition of the isomers are in trans configuration and are called cat 1980. 50 echin and the other two are in cis configuration and are called Aurones are compounds derived from 2-benzylidene-1- epicatechin. Non-limiting examples of non-ketone-contain benzofuran-3-one. Non-limiting examples of aurones include ing flavonoids include afzelechin, arthromerinA, arthromerin 4.5,6-trihydroxy-aurone, aureusidin, hispidol, leptosidin, B, catechin, epicatechin, epigallocatechin, epicatechin gal maritimetin, and Sulfuretin. late, epigallocatechingallate, epigallocatechin gallate, epiaf Three major classes of ketone-containing flavonoids are 55 Zelechin, fisetinidol, gallocatechin, gallocatechin gallate, flavones, compounds derived from 2-phenylchromen-4-one guibourtinidol, meciadanol (3-O-methylcatechin), mesqui (2-phenyl-1,4-benzopyrone); isoflavones, compounds tol, robinetinidol, and thearubigin. derived from 3-phenylchromen-4-one (3-phenyl-1,4-ben Flavan-4-ols (3-deoxyflavonoids) are flavone-derived Zopyrone); and neoflavones, compounds derived from 4-phe alcohols derived from 2-phenylchroman-4-ol. Non-limiting nylcoumarin (4-phenyl-1,2-benzopyrone) (Table 3). Fla 60 examples of flavan-4-ols include apiforoland luteoforol. vones are themselves divided into four groups based on the Leucoanthocyanidin (flavan-3,4-diols) are compounds presence or absence of 3-hydroxyl 2,3-dihydro functional derived from 2-phenyl-3,4-dihydro-2H-chromene-3,4-diol. groups: flavones, compounds derived from 2-phenyl Non-limiting examples of flavan-3,4-diols include leucocya chromen-4-one lack both functional groups; flavonols (3-hy nidin, leucodelphinidin, leucomalvidin, leucopelargonidin, droxyflavone), compounds derived from 3-hydroxy-2-phe 65 leucopeonidin, leucorobinetinidin, and melacacidin. nylchromen-4-one have the 3-hydroxyl group, but lack the Anthocyanidins are compounds derived from 2-phenyl 2,3-dihydro group; flavanones, compounds derived from 2,3- chromenylium. Non-limiting examples of anthocyanidins US 9,125,840 B2 23 24 include antirrhinin, apigeninidin, aurantinidin, capensinidin, include castalagin (vescalagin), castalin, casuarictin, chrysanthenin, columnidin, commelinin, cyanidin, 6-hy casuarin, casuarinin, cornusin E. grandinin, pedunculagin, droxycyanidin, cyanidin-3-(di-p-coumarylglucoside)-5-glu punicacortein C, punigluconin, punicalagin, punicalagin coside, cyanoSalvian in, delphinid in, dioSmetinidin, euro alpha, punicalin, 2-O-galloyl-punicalin, stachyurin, stricti pinidin, fisetinidin, gesneridin, guibourtinidin, hirsutidin, nin, and tellimagrandin II. Non-limiting examples of gallo luteolinidin, malvidin, 5-desoxy-malvidin, malvin, myrtillin, tannins include corilagin, galloyl glucose, digalloyl glucose, oenin, peonidin, 5-desoxy-peonidin, pelargonidin, petunidin, trigalloyl glucose, tetragalloylglucose, pentagalloyl glucose, primulin, protocyanin, protodelphin, pulchellidin, pulchelli hexagalloyl glucose, heptagalloyl glucose, octagalloyl glu din 3-glucoside, pulchellidin 3-rhamnoside, robinetinidin, cose, and tannic acid. Non-limiting examples of unclassified rosinidin, tricetinidin, tulipanin, and violdelphin. 10 tannins include acutissimin A, acutissimin B, chebulagic Tannins are compounds derived from 2-phenylchromeny acid, chebulinic acid, cinnamtannin B1, combreglutinin, lium. There are three major classes of tannins: hydrolyzable geraniin, granatin B, roburin A, roburin B, roburin C. roburin tannins; non-hydrolyzable tannins (condensed tannins; D, roburin E, stachyurin, tercatin, terflavins A, terflavins B, proanthocyanidins); and pseudotannins. tergallagin, Vescalin, 1.3,4-tri-O-galloylquinic acid, 3,5-di Hydrolyzable tannins are themselves divided into four 15 O-galloyl-shikimic acid, and 3,4,5-tri-O-galloylshikimic groups: oligomer tannins including aglycone tannins and gly acid. coside tannins; elagitannins; gallotannins, and unclassified Condensed tannins (proanthocyanidins) are essentially tannins. Non-limiting examples of aglycone tannins include polymer chains of flavonoids Such as catechins. Non-limiting ellagic acid, gallagic acid, and gallic acid. Non-limiting examples of condensed tannins include proanthocyanidin, examples of glycoside tannins include glucose, quinic acid, prodelphinidin, profisetinidin, proguibourtinidin, and pro and shikimic acid. Non-limiting examples of elagitannins robinetidin. TABLE 3

Flavonoids Flavonoids Base compound Examples Aurones 2-benzylidene-1-benzofuran-3-one 4,5,6-trihydroxy-aurone, aureusidin, hispidol, leptosidin, maritimetin, and Sulfuretin Flavones 2-phenylchromen-4-one acacetin, apilin, apigenin, apigetirin, artoindonesianin P. baicalein, baicalin, chrysin, cynaroside, dioSmetin, dioSmin, eupatilin, flavoxate, 6-hydroxyflavone, genkwanin, hidrosmin, luteolin, nepetin, nepitrin, nobiletin, orientin, orOXindin, oroxylin A, rhoifolin, scutellarein, Scutellarin, tangeritin, techtochrysin, tetuin, tricin, Veronicastroside, Vitexin, wogonin Flavonols 3-hydroxy-2-phenylchromen-4-one 3-hydroxyflavone, azaleatin, fisetin, galangin, gossypetin, kaempferide, kaempferol, isorhamnetin, morin, myricetin, natsudaidain, pachypodol, quercetin, rhamnazin, rhamnetin, Sophorin Flavanones 2,3-dihydro-2-phenylchromen-4-one butin, eriodictyol, hesperetin, hesperidin, homoeriodictyol, isosakuranetin, maringenin, naringin, pinocembrin, poncirin, Sakura.netin, Sakuranin, sterubin Flavanonols 3-hydroxy-2,3-dihydro-2- aromadedrin, taxifolin phenylchromen-4-one Isoflavones 3-phenylchromen-4-one alpinumisoflavone, anagyroidisoflavone A and B, calycosin, daidzein, daidzin, derrubone, di-O- methylalpinumisoflavone, formononetin, genistein, genistin, glycitein, ipriflavone, irigenin, iridin, irilone, 4'-methyl alpinumisoflavone, 5-O- methylgenistein, luteone, Ononin, orobol, pratensein, prunetin, pseudobaptigenin, psi-tectorigenin, puerarin, retusin, tectoridin, tectorigenin, wighteone Isoflavenes 3-phenylchroman lonchocarpane, laxiflorane Neoflavones 4-phenylcoumarine calophyllolide Neoflavenes 4-phenylchromen dalbergichromene Flavan-3-ols 2-phenyl-3,4-dihydro-2H-chromen arthromerin A, arthromerin B, 3-ol fisetinidol, guibourtinidol, meciadanol (3-O-methylcatechin), mesquitol, robinetinidol, thearubigin. US 9,125,840 B2 25 26 TABLE 3-continued

Flavonoids Flavonoids Base compound Examples Catechins (2R,3S)-2-(3,4-dihydroxyphenyl)- (+)-catechin (2R-3S), (-)-catechin 3,4-dihydro-2H-chromene-3,5,7-triol (2S-3R), (-)-Epicatechin (2R-3R), (+)-epicatechin (2S-3S) Flavan-4-ols 2-phenylchroman-4-ol apiforol, luteoforol Flavan-3,4- 2-phenyl-3,4-dihydro-2H-chromene leucocyanidin, leucodelphinidin, diols 3,4-diol leucomalvidin, leucopelargonidin, leucopeonidin, leucorobinetinidin, melacacidin Anthocyanidins 2-phenylchromenylium antirrhinin, apigeninidin, aurantinidin, capensinidin, chrysanthenin, columnidin, commelinin, cyanidin, 6– hydroxycyanidin, cyanidin-3-(di-p- coumarylglucoside)-5-glucoside, cyanoSalvianin, delphinidin, dioSmetinidin, europinidin, fisetinidin, gesneridin, guibourtinidin, hirsutidin, luteolinidin, malvidin, 5-desoxy malvidin, malvin, myrtillin, oenin, peonidin, 5-desoxy-peonidin, pelargonidin, petunidin, primulin, protocyanin, protodelphin, pulchellidin, pulchellidin 3 glucoside, pulchellidin 3 rhamnoside, robinetinidin, rosinidin, tricetinidin, tulipanin, violdelphin Hydrolyzable gallic acid or elagic acid castalagin, castalin, casuarictin, tannins casuariin, casuarinin, corilagin, cornusiin E, grandinin, galloyl glucose, digalloyl glucose, trigalloyl glucose, tetragalloylglucose, pentagalloyl glucose, hexagalloyl glucose, heptagalloyl glucose, octagalloyl glucose, pedunculagin, punicacortein C, punigluconin, punicalagin, punicalagin alpha, punicalin, 2-O-galloyl-punicalin, stachyurin, strictinin, tannic acid, tellimagrandin II Condensed polymer chains of flavonoid units proanthocyanidin, prodelphinidin, tannins profisetinidin, proguibourtinidin, prorobinetidin

The amount of a flavonoid included in a fluid composition able flavanone, a pharmaceutically acceptable flavanonol, a disclosed in the present specification is an amount effective to pharmaceutically acceptable flavan-3-ol, a pharmaceutically reduce or prevent degradation of a matrix polymer disclosed 45 acceptable flavan-4-ol, a pharmaceutically acceptable leu in the present specification. As such, the amount of a fla coanthocyanidin, a pharmaceutically acceptable anthocyani vonoid included in a fluid composition disclosed in the din, and a pharmaceutically acceptable tannin. present specification is between about 0.1% to about 10% by In another embodiment, a fluid composition comprises a weight of the total composition. In addition, a fluid compo single flavonoid that can reduce or prevent degradation of a sition disclosed in the present specification may comprise a 50 matrix polymer. In yet another embodiment, a fluid compo single flavonoid, or a plurality of flavonoid. Further, a fluid sition comprises a plurality of flavonoids, each of which can composition disclosed in the present specification comprises reduce or prevent the degradation of a matrix polymer. In a flavonoid that is pharmaceutically acceptable. aspects of this embodiment, a fluid composition comprises Thus in an embodiment, a fluid composition comprises a one or more flavonoids, two or more flavonoids, three or more pharmaceutically acceptable flavonoid that can reduce or pre 55 flavonoids, four or more flavonoids, or five or more fla vent degradation of a matrix polymer. In aspects of this vonoids. In other aspects of this embodiment, a fluid compo embodiment, a fluid composition comprises a pharmaceuti sition comprises one to five flavonoids, two to five flavonoids, cally acceptable C-methylated flavonoid, a pharmaceutically three to five flavonoids, two to four flavonoids, two to five acceptable O-methylated flavonoid, a pharmaceutically flavonoids, or three to five flavonoids. acceptable isoflavonoid, a pharmaceutically acceptable 60 In yet another embodiment, a fluid composition comprises neoflavonoid, a pharmaceutically acceptable flavonolignan, a a flavonoid in an amount Sufficient to reduce or prevent deg pharmaceutically acceptable furanoflavonoid, a pharmaceu radation of a matrix polymer. In aspects of this embodiment, tically acceptable pyranoflavonoid, a pharmaceutically a fluid composition comprises a flavonoid in an amount of acceptable methylenedioxyflavonoid, a pharmaceutically e.g., about 0.1% (w/v) of the composition, about 0.2% (w/v) acceptable prenylated flavonoid, a pharmaceutically accept 65 of the composition, about 0.3% (w/v) of the composition, able aurone, a pharmaceutically acceptable flavone, a phar about 0.4% (w/v) of the composition, about 0.5% (w/v) of the maceutically acceptable flavonol, a pharmaceutically accept composition, about 0.6% (w/v) of the composition, about US 9,125,840 B2 27 28 0.7% (w/v) of the composition, about 0.8% (w/v) of the Thus in an embodiment, a fluid composition comprises a composition, about 0.9% (w/v) of the composition, about matrix polymer, a stabilizing component, and an anti-itch 1.0% (w/v) of the composition, about 2.0% (w/v) of the agent. In aspects of this embodiment, a fluid composition composition, about 3.0% (w/v) of the composition, about comprises a matrix polymer, a stabilizing component, and an 4.0% (w/v) of the composition, about 5.0% (w/v) of the anti-itch agent where the amount of anti-itch agent present is composition, about 6.0% (w/v) of the composition, about about 0.1% (w/v) to about 5% (w/v) of the total composition, 7.0% (w/v) of the composition, about 8.0% (w/v) of the about 0.1% (w/v) to about 1% (w/v) of the total composition, composition, about 9.0% (w/v) of the composition, or about or about 0.1% (w/v) to about 0.5% (w/v) of the total compo 10% (w/v) of the composition. In other aspects, a fluid com sition. In other aspects of this embodiment, a fluid composi position comprises a flavonoid in an amount of e.g., at least 10 tion comprises a matrix polymer, a stabilizing component, 0.1% (w/v) of the composition, at least 0.2% (w/v) of the and an anti-itch agent where the amount of anti-itch agent composition, at least 0.3% (w/v) of the composition, at least present is about 0.3%. In another aspect of this embodiment, 0.4% (w/v) of the composition, at least 0.5% (w/v) of the a fluid composition comprises a matrix polymer, a stabilizing composition, at least 0.6% (w/v) of the composition, at least component, and methyl Sulphonyl methane, sodium bicar 0.7% (w/v) of the composition, at least 0.8% (w/v) of the 15 bonate, calamine, allantoin, kaolin, peppermint, tea tree oil, composition, at least 0.9% (w/v) of the composition, at least or combinations thereof. In aspects of this embodiment, a 1.0% (w/v) of the composition, at least 2.0% (w/v) of the fluid composition comprises a matrix polymer, a stabilizing composition, at least 3.0% (w/v) of the composition, at least component, and a methyl Sulphonyl methane, Sodium bicar 4.0% (w/v) of the composition, at least 5.0% (w/v) of the bonate, calamine, allantoin, kaolin, peppermint, tea tree oil, composition, at least 6.0% (w/v) of the composition, at least or combinations thereof where the amount of methylsulpho 7.0% (w/v) of the composition, at least 8.0% (w/v) of the nyl methane, Sodium bicarbonate, calamine, allantoin, composition, at least 9.0% (w/v) of the composition, or at kaolin, peppermint, or tea tree oil present is about 0.1% (w/v) least 10% (w/v) of the composition. In yet other aspects, a to about 5% (w/v) of the total composition, about 0.1% (w/v) fluid composition comprises a flavonoid in an amount of, e.g., to about 1% (w/v) of the total composition, or about 0.1% at most 0.1% (w/v) of the composition, at most 0.2% (w/v) of 25 (w/v) to about 0.5% (w/v) of the total composition. In other the composition, at most 0.3% (w/v) of the composition, at aspects of this embodiment, a fluid composition comprises a most 0.4% (w/v) of the composition, at most 0.5% (w/v) of matrix polymer, a stabilizing component, and a methyl Sul the composition, at most 0.6% (w/v) of the composition, at phonyl methane, sodium bicarbonate, calamine, allantoin, most 0.7% (w/v) of the composition, at most 0.8% (w/v) of kaolin, peppermint, tea tree oil, or combinations thereof the composition, at most 0.9% (w/v) of the composition, at 30 where the amount of methyl Sulphonyl methane, Sodium most 1.0% (w/v) of the composition, at most 2.0% (w/v) of bicarbonate, calamine, allantoin, kaolin, peppermint, or tea the composition, at most 3.0% (w/v) of the composition, at tree oil present is about 0.3%. In another embodiment, a fluid most 4.0% (w/v) of the composition, at most 5.0% (w/v) of composition comprises a matrix polymer, a stabilizing com the composition, at most 6.0% (w/v) of the composition, at ponent, but not an anti-itch agent. most 7.0% (w/v) of the composition, at most 8.0% (w/v) of 35 Aspects of the present specification provide, in part, a fluid the composition, at most 9.0% (w/v) of the composition, or at composition that can optionally comprise or not comprise an most 10% (w/v) of the composition. In still other aspects, a anti-cellulite agent. The amount of an anti-cellulite agent fluid composition comprises a flavonoid in an amount of, e.g., included in a fluid composition disclosed in the present speci about 0.1% (w/v) to about 1.0% (w/v) of the composition, fication is an amount effective to mitigate a fatty deposit about 0.1% (w/v) to about 2.0% (w/v) of the composition, 40 experienced by an individual upon administration of the com about 0.1% (w/v) to about 3.0% (w/v) of the composition, position. As such, the amount of an anti-cellulite agent about 0.1% (w/v) to about 4.0% (w/v) of the composition, included in a fluid composition disclosed in the present speci about 0.1% (w/v) to about 5.0% (w/v) of the composition, fication is between about 0.1% to about 5% by weight of the about 0.2% (w/v) to about 0.9% (w/v) of the composition, total composition. Non-limiting examples of anti-cellulite about 0.2% (w/v) to about 1.0% (w/v) of the composition, 45 agents include forskolin, Xanthine compounds such as, but about 0.2% (w/v) to about 2.0% (w/v) of the composition, not limited to, caffeine, theophylline, theobromine, and ami about 0.5% (w/v) to about 1.0% (w/v) of the composition, or nophylline, and combinations thereof. about 0.5% (w/v) to about 2.0% (w/v) of the composition. Thus in an embodiment, a fluid composition comprises a Aspects of the present specification provide, in part, a fluid matrix polymer, a stabilizing component, and an anti-cellulite composition that can optionally comprise or not comprise 50 agent. In aspects of this embodiment, a fluid composition another active ingredient. As used herein, the term “active comprises a matrix polymer, a stabilizing component, and an ingredient' includes but is not limited to a drug. A drug can anti-cellulite agent where the amount of anti-cellulite agent generally be defined as a chemical Substance used in the present is about 0.1% (w/v) to about 5% (w/v) of the total treatment, cure, prevention, or diagnosis of disease or used to composition, about 0.1% (w/v) to about 1% (w/v) of the total otherwise enhance physical or mental well-being. 55 composition, or about 0.1% (w/v) to about 0.5% (w/v) of the Aspects of the present specification provide, in part, a fluid total composition. In other aspects of this embodiment, a fluid composition that can optionally comprise or not comprise an composition comprises a matrix polymer, a stabilizing com anti-itch agent. The amount of an anti-itch agent included in ponent, and an anti-cellulite agent where the amount of anti a fluid composition disclosed in the present specification is an cellulite agent present is about 0.3%. In another aspect of this amount effective to mitigate an itch response experienced by 60 embodiment, a fluid composition comprises a matrix poly an individual upon administration of the composition. As mer, a stabilizing component, and forskolin, a Xanthine com Such, the amount of an anti-itch agent included in a fluid pound, or combinations thereof. In aspects of this embodi composition disclosed in the present specification is between ment, a fluid composition comprises a matrix polymer, a about 0.1% to about 5% by weight of the total composition. stabilizing component, and a forskolin, a Xanthine com Non-limiting examples of anti-itch agents include methyl 65 pound, or combinations thereof where the amount of forsko Sulphonyl methane, Sodium bicarbonate, calamine, allantoin, lin or a xanthine compound present is about 0.1% (w/v) to kaolin, peppermint, tea tree oil and combinations thereof. about 5% (w/v) of the total composition, about 0.1% (w/v) to US 9,125,840 B2 29 30 about 1% (w/v) of the total composition, or about 0.1% (w/v) about 5% by weight of the total composition. Non-limiting to about 0.5% (w/v) of the total composition. In other aspects examples of anti-inflammatory agents include dexametha of this embodiment, a fluid composition comprises a matrix Sone, prednisolone, corticosterone, budesonide, estrogen, polymer, a stabilizing component, and a forskolin, a Xanthine Sulfasalazine, mesalamine and combinations thereof. compound, or combinations thereof where the amount of 5 Thus in an embodiment, a fluid composition comprises a forskolin or a xanthine compound present is about 0.3%. In matrix polymer, a stabilizing component, and an anti-inflam another embodiment, a fluid composition comprises a matrix matory agent. In aspects of this embodiment, a fluid compo polymer, a stabilizing component, but not an anti-cellulite sition comprises a matrix polymer, a stabilizing component, agent. and an anti-inflammatory agent where the amount of anti Aspects of the present specification provide, in part, a fluid 10 inflammatory agent present is about 0.1% (w/v) to about 5% composition that can optionally comprise or not comprise an (w/v) of the total composition, about 0.1% (w/v) to about 1% anti-scarring agent. The amount of an anti-scarring agent (w/v) of the total composition, or about 0.1% (w/v) to about included in a fluid composition disclosed in the present speci 0.5% (w/v) of the total composition. In other aspects of this fication is an amount effective to mitigate a scaring response embodiment, a fluid composition comprises a matrix poly experienced by an individual upon administration of the com 15 mer, a stabilizing component, and an anti-inflammatory agent position. As such, the amount of an anti-scarring agent where the amount of anti-inflammatory agent present is about included in a fluid composition disclosed in the present speci 0.3%. In another aspect of this embodiment, a fluid compo fication is between about 0.1% to about 5% by weight of the sition comprises a matrix polymer, a stabilizing component, total composition. Non-limiting examples of anti-scarring and dexamethasone, prednisolone, corticosterone, budes agents include IFN-Y, fluorouracil, poly(lactic-co-glycolic onide, estrogen, SulfaSalazine, mesalamine, or combinations acid), methylated polyethylene glycol, polylactic acid, poly thereof. In aspects of this embodiment, a fluid composition ethylene glycol and combinations thereof. comprises a matrix polymer, a stabilizing component, and a Thus in an embodiment, a fluid composition comprises a dexamethasone, prednisolone, corticosterone, budesonide, matrix polymer, a stabilizing component, and an anti-scarring estrogen, Sulfasalazine, mesalamine, or combinations thereof agent. In aspects of this embodiment, a fluid composition 25 where the amount of dexamethasone, prednisolone, corticos comprises a matrix polymer, a stabilizing component, and an terone, budesonide, estrogen, SulfaSalazine, or mesalamine anti-scarring agent where the amount of anti-scarring agent present is about 0.1% (w/v) to about 5% (w/v) of the total present is about 0.1% (w/v) to about 5% (w/v) of the total composition, about 0.1% (w/v) to about 1% (w/v) of the total composition, about 0.1% (w/v) to about 1% (w/v) of the total composition, or about 0.1% (w/v) to about 0.5% (w/v) of the composition, or about 0.1% (w/v) to about 0.5% (w/v) of the 30 total composition. In other aspects of this embodiment, a fluid total composition. In other aspects of this embodiment, a fluid composition comprises a matrix polymer, a stabilizing com composition comprises a matrix polymer, a stabilizing com ponent, and a dexamethasone, prednisolone, corticosterone, ponent, and an anti-scarring agent where the amount of anti budesonide, estrogen, SulfaSalazine, mesalamine, or combi scarring agent present is about 0.3%. In another aspect of this nations thereof where the amount of dexamethasone, pred embodiment, a fluid composition comprises a matrix poly 35 nisolone, corticosterone, budesonide, estrogen, Sulfasalazine, mer, a stabilizing component, and IFN-Y, fluorouracil, poly or mesalamine present is about 0.3%. In another embodiment, (lactic-co-glycolic acid), methylated polyethylene glycol, a fluid composition comprises a matrix polymer, a stabilizing polylactic acid, polyethylene glycol, or combinations thereof. component, but not an anesthetic agent. In aspects of this embodiment, a fluid composition comprises Aspects of the present specification provide, in part, a fluid a matrix polymer, a stabilizing component, and a IFN-Y, fluo 40 composition that can optionally comprise or not comprise an rouracil, poly(lactic-co-glycolic acid), methylated polyethyl anesthetic agent. An anesthetic agent is preferably a local ene glycol, polylactic acid, polyethylene glycol, or combina anesthetic agent, i.e., an anesthetic agent that causes a revers tions thereof where the amount of IFN-y, fluorouracil, poly ible local anesthesia and a loss of nociception, Such as, e.g., (lactic-co-glycolic acid), methylated polyethylene glycol, aminoamide local anesthetics and aminoester local anesthet polylactic acid, or polyethylene glycol present is about 0.1% 45 ics. The amount of an anesthetic agent included in a fluid (w/v) to about 5% (w/v) of the total composition, about 0.1% composition disclosed in the present specification is an (w/v) to about 1% (w/v) of the total composition, or about amount effective to mitigate pain experienced by an indi 0.1% (w/v) to about 0.5% (w/v) of the total composition. In vidual upon administration of the composition. As such, the other aspects of this embodiment, a fluid composition com amount of an anesthetic agent included in a fluid composition prises a matrix polymer, a stabilizing component, and a IFN 50 disclosed in the present specification is between about 0.1% Y, fluorouracil, poly(lactic-co-glycolic acid), methylated to about 5% by weight of the total composition. Non-limiting polyethylene glycol, polylactic acid, polyethylene glycol, or examples of anesthetic agents include lidocaine, ambucaine, combinations thereof where the amount of IFN-Y, fluorou amolanone, amylocalne, benoximate, benzocaine, betox racil, poly(lactic-co-glycolic acid), methylated polyethylene ycaine, biphenamine, bupivacaine, butacaine, butamben, glycol, polylactic acid, or polyethylene glycol present is 55 butanilicaine, butethamine, butoxycaine, carticaine, chlorop about 0.3%. In another embodiment, a fluid composition rocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, comprises a matrix polymer, a stabilizing component, but not dimethisoquin, dimethocaine, diperodon, dicyclonine, an anti-scarring agent. ecgonidine, ecgonine, ethyl chloride, etidocaine, beta Aspects of the present specification provide, in part, a fluid eucaine, euprocin, fenalcomine, formocaine, hexylcaine, composition that can optionally comprise or not comprise an 60 hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine anti-inflammatory agent. The amount of an anti-inflamma mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, tory agent included in a fluid composition disclosed in the metabutoxycaine, methyl chloride, myrtecaine, naepaine, present specification is an amount effective to mitigate an octacaine, orthocaine, oxethazaine, parethoxycaine, phen inflammatory response experienced by an individual upon acaine, phenol, piperocaine, piridocaine, polidocanol, administration of the composition. As such, the amount of an 65 pramoxine, prilocaine, procaine, propanocaine, propara anti-inflammatory agent included in a fluid composition dis caine, propipocaine, propoxycaine, pseudococaine, pyr closed in the present specification is between about 0.1% to rocaine, ropivacaine, Salicyl alcohol, tetracaine, tolycaine, US 9,125,840 B2 31 32 trimecaine, Zolamine, combinations thereof, and salts Aspects of the present specification provide, in part, a fluid thereof. Non-limiting examples of aminoester local anesthet composition disclosed in the present specification exhibiting ics include procaine, chloroprocaine, cocaine, cyclom a complex modulus, an elastic modulus, a viscous modulus ethycaine, dimethocaine (larocaine), propoxycaine, procaine and a tan Ö. Matrix polymers disclosed in the present speci (novocaine), proparacaine, tetracaine (amethocaine). Non fication are viscoelastic in that the composition has an elastic limiting examples of aminoamide local anesthetics include component (solid-like Such as, e.g., crosslinked matrix poly articaine, bupivacaine, cinchocaine (dibucaine), etidocaine, mer) and a viscous component (liquid-like Such as, e.g., levobupivacaine, lidocaine (lignocaine), mepivacaine, piper uncrosslinked matrix polymer) when a force is applied ocaine, prilocaine, ropivacaine, and trimecaine. A non-limit (stress, deformation). The rheological attribute that described ing example of a combination local anesthetic is lidocaine/ 10 this property is the complex modulus (G), which defines a prilocaine (EMLA). fluid compositions total resistance to deformation. The com Thus in an embodiment, a fluid composition comprises a plex modulus can be defined as the Sum of the elastic modulus matrix polymer, a stabilizing component, and an anesthetic (G') and the viscous modulus (G"). Falcone, et al., Temporary agent and salts thereof. In aspects of this embodiment, a fluid Polysaccharide Dermal Fillers: A Model for Persistence composition comprises a matrix polymer, a stabilizing com 15 Based on Physical Properties, Dermatol Surg. 35.(8): 1238 ponent, and an aminoamide local anesthetic and salts thereof 1243 (2009); Tezel, supra, 2008; Kablik, supra, 2009; Beas or an aminoester local anesthetic and salts thereof. In other ley, supra, 2009; each of which is hereby incorporated by aspects of this embodiment, a fluid composition comprises a reference in its entirety. Elastic modulus characterizes the matrix polymer, a stabilizing component, and procaine, chlo firmness of a composition and is also known as the storage roprocaine, cocaine, cyclomethycaine, dimethocaine, pro modulus because it describes the storage of energy from the poxycaine, procaine, proparacaine, tetracaine, or salts motion of the composition. The elastic modulus describes the thereof. In yet other aspects of this embodiment, a fluid com interaction between elasticity and strength (G'=stress/strain) position comprises a matrix polymer, a stabilizing compo and, as Such, provides a quantitative measurement of a com nent, and articaine, bupivacaine, cinchocaine, etidocaine, positions hardness or softness. Although depending on the levobupivacaine, lidocaine, mepivacaine, piperocaine, 25 speed at which the force is applied, a stiffer composition will prilocaine, ropivacaine, trimecaine, or salts thereof. In still have a higher elastic modulus and it will take a greater force other aspects of this embodiment, a fluid composition com to deform the material a given distance, Such as, e.g., an prises a matrix polymer, a stabilizing component, and injection. lidocaine/prilocaine combination. Viscous modulus is also known as the loss modulus In other aspects of this embodiment, a fluid composition 30 because it describes the energy that is lost as Viscous dissipa comprises a matrix polymer, a stabilizing component, and an tion. Tan Ö is the ratio of the viscous modulus and the elastic anesthetic agent where the amount of anesthetic agent present modulus, tan 8–G'/G". Falcone, supra, 2009. Fortan & values is about 0.1% (w/v) to about 5% (w/v) of the total composi disclosed in the present specification, a tan Ö is obtained from tion, about 0.1% (w/v) to about 1% (w/v) of the total compo the dynamic modulus at a frequency of 0.628 rad/s. A lower sition, or about 0.1% (w/v) to about 0.5% (w/v) of the total 35 tan Ö corresponds to a stiffer, harder, or more elastic compo composition. In other aspects of this embodiment, a fluid sition. composition comprises a matrix polymer, a stabilizing com Thus, in an embodiment, a fluid composition exhibits a ponent, and an anesthetic agent where the amount of anes complex modulus. In aspects of this embodiment, a fluid thetic agent present is about 0.3%. In other aspects of this composition exhibits a complex modulus of, e.g., about 25 Pa, embodiment, a fluid composition comprises a matrix poly 40 about 50 Pa, about 75 Pa, about 100 Pa, about 125 Pa, about mer, a stabilizing component, and an aminoamide local anes 150 Pa, about 175 Pa, about 200 Pa, about 250 Pa, about 300 thetic and salts thereof oran aminoester local anesthetic and Pa, about 350 Pa, about 400 Pa, about 450 Pa, about 500 Pa, salts thereof where the amount of the local anesthetic present about 550 Pa, about 600 Pa, about 650 Pa, about 700 Pa, about is about 0.1% (w/v) to about 5% (w/v) of the total composi 750 Pa, or about 800 Pa. In other aspects of this embodiment, tion, about 0.1% (w/v) to about 1% (w/v) of the total compo 45 a fluid composition exhibits a complex modulus of, e.g., at sition, or about 0.1% (w/v) to about 0.5% (w/v) of the total most 25 Pa, at most 50 Pa, at most 75 Pa, at most 100 Pa, at composition. In other aspects of this embodiment, a fluid most 125 Pa, at most 150 Pa, at most 175 Pa, at most 200 Pa, composition comprises a matrix polymer, a stabilizing com at most 250 Pa, at most 300 Pa, at most 350 Pa, at most 400 Pa, ponent, and an aminoamide local anesthetic and salts thereof at most 450 Pa, at most 500 Pa, at most 550 Pa, at most 600 Pa, or an aminoester local anesthetic and salts thereof where the 50 at most 650 Pa, at most 700 Pa, at most 750 Pa, or at most 800 amount of the local anesthetic agent present is about 0.3%. Pa. In yet other aspects of this embodiment, a fluid composi In another aspect of this embodiment, a fluid composition tion exhibits a complex modulus of, e.g., about 25 Pato about comprises a matrix polymer, a stabilizing component, and 150 Pa, about 25 Pato about 300 Pa, about 25 Pato about 500 lidocaine or a lidocaine salt. In aspects of this embodiment, a Pa, about 25 Pato about 800 Pa, about 125 Pato about 300 Pa, fluid composition comprises a matrix polymer, a stabilizing 55 about 125 Pato about 500 Pa, or about 125 Pato about 800 Pa. component, and a lidocaine or a lidocaine Salt where the In another embodiment, a fluid composition exhibits an amount of lidocaine or a lidocaine salt present is about 0.1% elastic modulus. In aspects of this embodiment, a fluid com (w/v) to about 5% (w/v) of the total composition, about 0.1 position exhibits an elastic modulus of, e.g., about 25 Pa, (w/v) to about 1% (w/v) of the total composition, or about about 50 Pa, about 75 Pa, about 100 Pa, about 125 Pa, about 0.1% (w/v) to about 0.5% (w/v) of the total composition. In 60 150 Pa, about 175 Pa, about 200 Pa, about 250 Pa, about 300 other aspects of this embodiment, a fluid composition com Pa, about 350 Pa, about 400 Pa, about 450 Pa, about 500 Pa, prises a matrix polymer, a stabilizing component, and a about 550 Pa, about 600 Pa, about 650 Pa, about 700 Pa, about lidocaine or a lidocaine salt where the amount of lidocaine or 750 Pa, or about 800 Pa. In other aspects of this embodiment, a lidocaine salt present is about 0.3%. a fluid composition exhibits an elastic modulus of, e.g., at In another embodiment, a fluid composition comprises a 65 most 25 Pa, at most 50 Pa, at most 75 Pa, at most 100 Pa, at matrix polymer, a stabilizing component, but not an anes most 125 Pa, at most 150 Pa, at most 175 Pa, at most 200 Pa, thetic agent. at most 250 Pa, at most 300 Pa, at most 350 Pa, at most 400 Pa, US 9,125,840 B2 33 34 at most 450 Pa, at most 500 Pa, at most 550 Pa, at most 600 Pa, density with the SI unit of kg/m. Kinematic viscosity is at most 650 Pa, at most 700 Pa, at most 750 Pa, or at most 800 usually measured by a glass capillary viscometer as has an SI Pa. In yet other aspects of this embodiment, a fluid composi unit of m/s. tion exhibits an elastic modulus of, e.g., about 25 Pa to about The viscosity of a fluid is highly temperature dependent 150 Pa, about 25 Pato about 300 Pa, about 25 Pato about 500 and for either dynamic or kinematic viscosity to be meaning Pa, about 25 Pato about 800 Pa, about 125 Pato about 300 Pa, ful, the reference temperature must be quoted. For the viscos about 125 Pato about 500 Pa, or about 125 Pato about 800 Pa. ity values disclosed in the present specification, a dynamic In another embodiment, a fluid composition exhibits a Viscosity is measured at 1 Pa with a cone/plane geometry Viscous modulus. In aspects of this embodiment, a fluid com 2/40 cm and a temperature of 20° C. Examples of the 10 dynamic viscosity of various fluids at 20° C. is as follows: position exhibits a viscous modulus of, e.g., about 10 Pa, water is about 1.0x10 Pas, blood is about 3-4x10 Pa's, about 20 Pa, about 30 Pa, about 40 Pa, about 50 Pa, about 60 vegetable oil is about 60-85x10 Pas, motor oil SE 30 is Pa, about 70 Pa, about 80 Pa, about 90 Pa, about 100 Pa, about about 0.2 Pas, glycerin is about 1.4 Pas, maple syrup is about 110 Pa, about 120 Pa, about 130 Pa, about 140 Pa, or about 2-3 Pas, honey is about 10 Pas, chocolate syrup is about 150 Pa. In other aspects of this embodiment, a fluid compo 15 10-25 Pa's, peanut butter is about 150-250 Pa's, lard is about sition exhibits a viscous modulus of, e.g., at most 10 Pa, at 1,000 Pas, vegetable shortening is about 1,200 Pas, and taris most 20 Pa, at most 30 Pa, at most 40 Pa, at most 50 Pa, at most about 30,000 Pas. 60 Pa, at most 70 Pa, at most 80 Pa, at most 90 Pa, at most 100 Thus, in an embodiment, a fluid composition comprising a Pa, at most 110 Pa, at most 120 Pa, at most 130 Pa, at most 140 matrix polymer and a stabilizing component exhibits a Pa, or at most 150 Pa. In yet other aspects of this embodiment, dynamic viscosity. In aspects of this embodiment, a fluid a fluid composition exhibits a viscous modulus of, e.g., about composition comprising a matrix polymer and a stabilizing 10 Pa to about 30 Pa, about 10 Pa to about 50 Pa, about 10 Pa component exhibits a dynamic viscosity of e.g., about 10 to about 100 Pa, about 10 Pa to about 150 Pa, or about 70 Pa Pa's, about 20 Pas, about 30 Pas, about 40 Pas, about 50 to about 100 Pa. Pa's, about 60 Pas, about 70 Pas, about 80 Pas, about 90 In another embodiment, a fluid composition disclosed in 25 Pa's, about 100 Pas, about 125 Pas, about 150 Pas, about the present specification exhibiting a tan Ö. In aspects of this 175 Pas, about 200 Pas, about 225 Pas, about 250 Pas, embodiment, a fluid composition exhibits a tan Ö of e.g., about 275 Pas, about 300 Pas, about 400 Pas, about 500 about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, Pa's, about 600 Pas, about 700 Pas, about 750 Pas, about about 0.7, about 0.8, about 0.9, or about 1.0. In other aspects 800 Pas, about 900 Pas, about 1,000 Pas, about 1,100 Pas, 30 or about 1,200 Pas. In other aspects of this embodiment, a of this embodiment, a fluid composition exhibits a tan 8 of fluid composition comprising a matrix polymer and a stabi e.g., at most 0.1, at most 0.2, at most 0.3, at most 0.4, at most lizing component exhibits a dynamic viscosity of, e.g., at 0.5, at most 0.6, at most 0.7, at most 0.8, at most 0.9, or at most most 10 Pas, at most 20 Pas, at most 30 Pas, at most 40 Pas, 1.0. In yet other aspects of this embodiment, a fluid compo at most 50 Pas, at most 60 Pas, at most 70 Pas, at most 80 sition exhibits a tan 8 of, e.g., about 0.1 to about 0.3, about 0.3 35 Pa's, at most 90 Pas, at most 100 Pas, at most 125 Pas, at to about 0.5, about 0.3 to about 0.6, about 0.1 to about 0.5, or most 150 Pas, at most 175 Pas, at most 200 Pas, at most 225 about 0.1 to about 0.6. Pa's, at most 250 Pas, at most 275 Pas, at most 300 Pas, at Aspects of the present specification provide, in part, a fluid most 400 Pas, at most 500 Pas, at most 600 Pas, at most 700 composition disclosed in the present specification exhibiting Pa's, at most 750 Pas, at most 800 Pas, at most 900 Pas, or a dynamic viscosity. Viscosity is resistance of a fluid to shear 40 at most 1000 Pas. In yet other aspects of this embodiment, a or flow caused by either shear stress or tensile stress. Viscosity fluid composition comprising a matrix polymer and a stabi describes a fluids internal resistance to flow caused by inter lizing component exhibits a dynamic viscosity of e.g., about molecular friction exerted when layers of fluids attempt to 10 Pa's to about 100 Pas, about 10 Pars to about 150 Pas, slide by one another and may be thought of as a measure of about 10 Pa's to about 250 Pas, about 50 Pa's to about 100 fluid friction. The less viscous the fluid, the greater its ease of 45 Pa's, about 50 Pa's to about 150 Pas, about 50 Pa's to about movement (fluidity). 250 Pas, about 100 Pa's to about 500 Pas, about 100 Pars to Viscosity can be defined in two ways; dynamic viscosity about 750 Pas, about 100 Pa's to about 1,000 Pas, about 100 (LL, although m is sometimes used) or kinematic viscosity (V). Pa's to about 1,200 Pas, about 300 Pa's to about 500 Pas, Dynamic viscosity, also known as absolute or complex vis about 300 Pa's to about 750 Pas, about 300 Pa's to about cosity, is the tangential force per unit area required to move 50 1,000 Pas, or about 300 Pa's to about 1,200 Pars. one horizontal plane with respect to the other at unit velocity Aspects of the present specification provide, in part, a fluid when maintained a unit distance apart by the fluid. The SI composition disclosed in the present specification that is physical unit of dynamic viscosity is the Pascal-second (Pas), injectable. As used herein, the term “injectable” refers to a which is identical to N-m-2's. Dynamic viscosity can be fluid composition disclosed in the present specification hav expressed as TudvX/dz, where T-shearing stress, L dynamic 55 ing the properties necessary to administer the composition viscosity, and dvX/dz is the velocity gradient over time. For into a dermal region of an individual using an injection device example, if a fluid with a viscosity of one Pa's is placed with a fine needle. As used herein, the term “fine needle’ between two plates, and one plate is pushed sideways with a refers to a needle that is 27 gauge or smaller. Injectability of shear stress of one Pascal, it moves a distance equal to the a fluid composition disclosed in the present specification can thickness of the layer between the plates in one second. 60 be accomplished by sizing the fluid composition, as discussed Dynamic viscosity symbolize by is also used, is measured below. with various types of rheometers, devices used to measure the Thus, in an embodiment, a fluid composition comprising a way in which a liquid, Suspension or slurry flows in response matrix polymer and a stabilizing component, wherein the to applied forces. composition is injectable. In aspect of this embodiment, a Kinematic viscosity (V) is the ratio of dynamic viscosity to 65 fluid composition comprising a matrix polymer and a stabi density, a quantity in which no force is involved and is defined lizing component is injectable through a fine needle. In other as follows: VL/p, where L is the dynamic viscosity p is aspects of this embodiment, a fluid composition comprising a US 9,125,840 B2 35 36 matrix polymer and a stabilizing component is injectable manent detrimental effect when administered to mammal. through a needle of, e.g., about 27 gauge, about 30 gauge, or Osmolality refers to the concentration of osmotically active about 32 gauge. In yet other aspects of this embodiment, a solutes per kilo of water in the body and is expressed in terms fluid composition comprising a matrix polymer and a stabi of osmoles of osmotically active solute per kilogram of Sol lizing component is injectable through a needle of, e.g., 27 vent (Osmol/kg or Osm/kg) and is equal to the Sum of the gauge or Smaller, 30 gauge or Smaller, or 32 gauge or Smaller. molalities of all the solutes present in that solution. The osmo In still other aspects of this embodiment, a fluid composition lality of a solution can be measured using an osmometer. The comprising a matrix polymer and a stabilizing component is most commonly used instrument in modern laboratories is a injectable through a needle of, e.g., about 27 gauge to about freezing point depression osmometer. This instruments mea 32 gauge. 10 Sure the change in freezing point that occurs in a solution with In other aspects of this embodiment, a fluid composition increasing osmolality (freezing point depression osmometer) comprising a crosslinked matrix polymer where the mean or the change in vapor pressure that occurs in a solution with particle size of the crosslinked matrix polymer is, e.g., about increasing osmolality (vapor pressure depression osmom 200 um, about 250 um, about 300 um, about 350 lum, about eter). 400 um, about 450 um, about 500 um, about 550 lum, about 15 Thus, in an embodiment, a fluid composition comprising a 600 um, about 650 um, about 700 um, about 750 um, or about matrix polymer and a stabilizing component exhibit a physi 800 um. In yet other aspects of this embodiment, a fluid ologically-acceptable osmolarity. In aspects of this embodi composition comprising a crosslinked matrix polymer where ment, a fluid composition comprising a matrix polymer and a the mean particle size of the crosslinked matrix polymer is, stabilizing component exhibit an osmolarity of, e.g., about e.g., at most 200 um, at most 250 um, at most 300 um, at most 100 mOsm/L, about 150 mOsm/L, about 200 mOsm/L, about 350 Lum, at most 400 um, at most 450 Lim, at most 500 um, at 250 mOsm/L, about 300 mOsm/L, about 350 mOsm/L, about most 550 um, at most 600 um, at most 650 um, at most 700 400 mOsm/L, about 450 mOsm/L, or about 500 mOsm/L. In um, at most 750 um, or at most 800 um. In still other aspects other aspects of this embodiment, a fluid composition com of this embodiment, a fluid composition comprising a prising a matrix polymer and a stabilizing component exhibit crosslinked matrix polymer where the mean particle size of 25 an osmolarity of, e.g., at least 100 mOsm/L. at least 150 the crosslinked matrix polymeris, e.g., about 200um to about mOsm/L, at least 200 mOsm/L, at least 250 mOsm/L, at least 300 um, about 300 um to about 400 um, about 400 um to 300 mOsm/L, at least 350 mOsm/L, at least 400 mOsm/L, at about 500 um, about 500 um to about 600 um, about 600 um least 450 mOsm/L, or at least 500 mOsm/L. In yet other to about 700 um, about 700 um to about 800 um, about 200 aspects of this embodiment, a fluid composition comprising a um to about 400 um, about 200 um to about 500 um, about 30 matrix polymer and a stabilizing component exhibit an osmo 200 um to about 600 um, about 200 um to about 700 um, larity of, e.g., at most 100 mOsm/L. at most 150 mOsm/L. at about 200 um to about 800 um, about 300 um to about 500 most 200 mOsm/L at most 250 mOsm/L, at most 300 mOsm/ um, about 300 um to about 600 um, about 300 um to about L., at most 350 mOsm/L, at most 400 mOsm/L, at most 450 700 um, or about 300 um to about 800 um. mOsm/L, or at most 500 mOsm/L. In still other aspects of this Aspects of the present specification provide, in part, a fluid 35 embodiment, a fluid composition comprising a matrix poly composition disclosed in the present specification exhibiting mer and a stabilizing component exhibit an osmolarity of a physiologically-acceptable osmolarity. As used herein, the e.g., about 100 mOsm/L to about 500 mOsm/L, about 200 term “a physiologically-acceptable osmolarity refers to an mOsm/L to about 500 mOsm/L, about 200 mOsm/L to about osmolarity in accord with, or characteristic of the normal 400 mOsm/L, about 300 mOsm/L to about 400 mOsm/L. functioning of a living organism. As such, administration of a 40 about 270 mOsm/L to about 390 mOsm/L, about 225 fluid composition disclosed in the present composition exhib mOsm/L to about 350 mOsm/L, about 250 mOsm/L to about its an osmolarity that has substantially no long term or per 325 mOsm/L, about 275 mOsm/L to about 300 mOsm/L, or manent detrimental effect when administered to mammal. about 285 mC)sm/L to about 290 mOsm/L. Osmolarity refers to the concentration of osmotically active In another embodiment, a fluid composition comprising a Solutes in Solution. Osmolarity is expressed in terms of 45 matrix polymer and a stabilizing component exhibit a physi osmoles of osmotically active solute per liter of solvent (Os ologically-acceptable osmolality. In aspects of this embodi mol/L or Osm/L). Osmolarity is distinct from molarity ment, a fluid composition comprising a matrix polymer and a because it measures moles of osmotically active solute par stabilizing component exhibit an osmolality of, e.g., about ticles rather than moles of solute. The distinction arises 100 mOsm/kg, about 150 mOsm/kg, about 200 mOsm/kg, because some compounds can dissociate in solution, whereas 50 about 250 mOsm/kg, about 300 mOsm/kg, about 350 mOsm/ others cannot. The osmolarity of a solution can be calculated kg, about 400 mOsm/kg, about 450 mOsm/kg, or about 500 from the following expression: Osmol/L Xp.m.C., where p is mOsm/kg. In other aspects of this embodiment, a fluid com the osmotic coefficient, which accounts for the degree of position comprising a matrix polymer and a stabilizing com non-ideality of the solution; r, is the number of particles (e.g. ponent exhibit an osmolality of, e.g., at least 100 mOsm/kg, at ions) into which a molecule dissociates; and C is the molar 55 least 150 mOsm/kg, at least 200 mOsm/kg, at least 250 concentration of the solute; and i is the index representing the mOsm/kg, at least 300 mOsm/kg, at least 350 mCSm/kg, at identity of a particular solute. The osmolarity of a composi least 400 mOsm/kg, at least 450 mOsm/kg, or at least 500 tion disclosed in the present specification can be measured mOsm/kg. In yet other aspects of this embodiment, a fluid using a conventional method that measures solutions. composition comprising a matrix polymer and a stabilizing Aspects of the present specification provide, in part, a fluid 60 component exhibit an osmolality of e.g., at most 100 mOsm/ composition disclosed in the present specification exhibiting kg, at most 150 mOsm/kg, at most 200 mOsm/kg, at most 250 a physiologically-acceptable osmolality. As used herein, the mOsm/kg, at most 300 mOsm/kg, at most 350 mOsm/kg, at term “a physiologically-acceptable osmolality” refers to an most 400 mOsm/kg, at most 450 mOsm/kg, or at most 500 osmolality in accord with, or characteristic of the normal mOsm/kg. In still other aspects of this embodiment, a fluid functioning of a living organism. As such, administration of a 65 composition comprising a matrix polymer and a stabilizing fluid composition disclosed in the present composition exhib component exhibit an osmolality of, e.g., about 100 mOsm/ its an osmolality that has substantially no long term or per kg to about 500 mOsm/kg, about 200 mOsm/kg to about 500 US 9,125,840 B2 37 38 mOsm/kg, about 200 mOsm/kg to about 400 mOsm/kg, about useful in formulating a pharmaceutical composition dis 300 mOsm/kg to about 400 mOsm/kg, about 270 mOsm/kg to closed in the present specification, with the proviso that a about 390 mOsm/kg, about 225 mOsm/kg to about 350 therapeutically effective amount of the matrix polymeractive mOsm/kg, about 250 mOsm/kg to about 325 mCSm/kg, about ingredient is recovered using this effective concentration of 275 mOsm/kg to about 300 mOsm/kg, or about 285 mCsm/kg buffer. Non-limiting examples of concentrations of physi to about 290 mOsm/kg. ologically-acceptable buffers occur within the range of about Aspects of the present specification provide, in part, a fluid 0.1 mM to about 900 mM. The pH of pharmaceutically composition disclosed in the present specification that is a acceptable buffers may be adjusted, provided that the result pharmaceutical composition. As used herein, the term “phar ing preparation is pharmaceutically acceptable. It is under maceutical composition' is synonymous with “pharmaceuti 10 stood that acids or bases can be used to adjust the pH of a cally-acceptable composition' and refers to a therapeutically pharmaceutical composition as needed. Any buffered pH effective concentration of an active ingredient, such as, e.g., level can be useful in formulating a pharmaceutical compo any of the matrix polymers disclosed in the present specifi sition, with the proviso that atherapeutically effective amount cation. A pharmaceutical composition comprising a matrix of the matrix polymeractive ingredientis recovered using this polymeractive ingredient is useful for medical and Veterinary 15 effective pH level. Non-limiting examples of physiologi applications. A pharmaceutical composition may be admin cally-acceptable pH occur within the range of about pH 5.5 to istered to a patientalone, or in combination with other Supple about pH 8.5. mentary active ingredients, agents, drugs or hormones. Pharmaceutically acceptable antioxidants include, without Aspects of the present specification provide, in part, a fluid limitation, Sodium metabisulfite, Sodium thiosulfate, acetyl composition disclosed in the present specification that is a cysteine, butylated hydroxyanisole and butylated hydroxy pharmaceutical composition comprising a pharmacologi toluene. Pharmaceutically acceptable preservatives include, cally acceptable excipient. As used herein, the term “pharma without limitation, benzalkonium chloride, chlorobutanol, cologically acceptable excipient' is synonymous with “phar thimerosal, phenylmercuric acetate, phenylmercuric nitrate, macological excipient’ or “excipient' and refers to any a stabilized oxychloro composition, such as, e.g., PURITE(R) excipient that has substantially no long term or permanent 25 (Allergan, Inc. Irvine, Calif.) and chelants. Such as, e.g., detrimental effect when administered to mammal and encom DTPA or DTPA-bisamide, calcium DTPA, and CaNaDTPA passes compounds such as, e.g., stabilizing agent, a bulking bisamide. agent, a cryo-protectant, a lyo-protectant, an additive, a Tonicity adjustors useful in a pharmaceutical composition vehicle, a carrier, a diluent, or an auxiliary. An excipient include, without limitation, salts such as, e.g., Sodium chlo generally is mixed with an active ingredient, or permitted to 30 ride and potassium chloride; and glycerin. The pharmaceuti dilute or enclose the active ingredient and can be a solid, cal composition may be provided as a salt and can be formed semi-solid, or liquid agent. It is also envisioned that a phar with many acids, including but not limited to, hydrochloric, maceutical composition comprising a matrix polymer active Sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend ingredient can include one or more pharmaceutically accept to be more soluble in aqueous or other protonic solvents than able excipients that facilitate processing of an active ingredi 35 are the corresponding free base forms. It is understood that ent into pharmaceutically acceptable compositions. Insofar these and other Substances known in the art of pharmacology as any pharmacologically acceptable excipient is not incom can be included in a pharmaceutical composition useful in the patible with the matrix polymer active ingredient, its use in invention. Other non-limiting examples of pharmacologi pharmaceutically acceptable compositions is contemplated. cally acceptable components can be found in, e.g., Ansel, Non-limiting examples of pharmacologically acceptable 40 supra, (1999); Gennaro, supra, (2000); Hardman, supra, excipients can be found in, e.g., Pharmaceutical Dosage (2001); and Rowe, supra, (2003), each of which is hereby Forms and Drug Delivery Systems (Howard C. Ansel et al., incorporated by reference in its entirety. eds., Lippincott Williams & Wilkins Publishers, 7" ed. 1999); A pharmaceutical compositions disclosed in the present Remington: The Science and Practice of Pharmacy (Alfonso specification generally is administered as a pharmaceutical R. Gennaro ed., Lippincott, Williams & Wilkins, 20" ed. 45 acceptable composition comprising a matrix polymer active 2000); Goodman & Gilman's The Pharmacological Basis of ingredient. As used herein, the term "pharmaceutically Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill acceptable” means any molecular entity or composition that Professional, 10" ed. 2001); and Handbook of Pharmaceuti does not produce an adverse, allergic or other untoward or cal Excipients (Raymond C. Rowe et al., APhA Publications, unwanted reaction when administered to an individual. 4" edition 2003), each of which is hereby incorporated by 50 Aspects of the present specification provide, in part, a reference in its entirety. method of making a fluid composition disclosed in the It is further envisioned that a pharmaceutical composition present specification. In an aspect, a method for making a disclosed in the present specification may optionally include fluid composition, the method comprising the steps of: a) or not include, without limitation, other pharmaceutically combining a stabilizing component with a physiologically acceptable components (or pharmaceutical components), 55 acceptable buffer to make a stabilizing component-buffered including, without limitation, buffers, preservatives, tonicity Solution; b) combining a matrix polymer with the stabilizing adjusters, salts, antioxidants, osmolality adjusting agents, component-buffered solution to hydrate the matrix polymer, emulsifying agents, wetting agents, Sweetening or flavoring and; c) sizing the fluid composition. This method may, or may agents, and the like. not, further comprise a step comprising titrating a stabilizing Pharmaceutically acceptable buffer is any buffer that can 60 component-buffered solution to obtain a desired pH after step be used to prepare a pharmaceutical composition disclosed in (a); a step comprising filtering the stabilizing component the present specification, provided that the resulting prepara buffered solution after step (a); a step (b) where combining a tion is pharmaceutically acceptable. Non-limiting examples matrix polymer with the stabilizing component-buffered of pharmaceutically acceptable buffers include acetate buff Solution to hydrate the matrix polymer occurs by mixing the ers, borate buffers, citrate buffers, neutral buffered salines, 65 matrix polymer with the stabilizing component-buffered phosphate buffers, and phosphate buffered salines. Any con Solution at a low speed for a relatively long period of time; a centration of a pharmaceutically acceptable buffer can be step (b) where combining a matrix polymer with the stabiliz US 9,125,840 B2 39 40 ing component-buffered solution to hydrate the matrix poly a relative long period of time; a step comprising degassing a mer occurs by mixing the matrix polymer with the stabilizing fluid composition after step (b) or step (c); a step comprising component-buffered solution at a low speed for a relatively filling a syringe with a fluid composition after step (c); and/or long period of time and then followed by a rest for a relative a step comprising sterilizing a syringe filled with a fluid long period of time; a step (b) where combining a matrix 5 composition after step (c). polymer with the stabilizing component-buffered solution to Aspects of the present specification provide, in part, a hydrate the matrix polymer occurs by mixing the matrix method having a step of combining a stabilizing component polymer with the stabilizing component-buffered solution at with a physiologically-acceptable buffer to make a stabilizing a low speed for a relatively long period of time and then by component-buffered solution. A stabilizing component can mixing the matrix polymer with the stabilizing component 10 be any one of the stabilizing components disclosed in the buffered solution using a cycle of alternating periods of agi present specification. As used herein, the term “a physiologi tation for a relatively short period of time followed by periods cally-acceptable buffer refers to a buffer in accord with, or of rest for a relatively long period of time; a step (b) where characteristic of the normal functioning of a living organism. combining a matrix polymer with the stabilizing component AS Such, a buffer used to make a fluid composition disclosed buffered solution to hydrate the matrix polymer occurs by 15 in the present specification exhibits a buffering capacity that mixing the matrix polymer with the stabilizing component has substantially no long term or permanent detrimental buffered solution at a low speed for a relatively long period of effect when administered to mammal. Physiologically-ac time and then by mixing the matrix polymer with the stabi ceptable buffers include, without limitation, acetate buffers, lizing component-buffered solution using a cycle of alternat borate buffers, citrate buffers, neutral buffered salines, phos ing periods of agitation for a relatively short period of time phate buffers, and phosphate buffered salines. In addition, the followed by periods of rest for a relatively long period of time, physiologically-acceptable buffer is at a concentration to and then followed by a rest for a relative long period of time: achieve an effective buffering capacity. Non-limiting a step comprising degassing a fluid composition after step (b) examples of a concentration to a physiologically-acceptable or step (c); a step comprising filling a syringe with a fluid buffer to achieve an effective buffering capacity is from composition after step (c); and/or a step comprising steriliz 25 between about 0.1 mM to about 900 mM. ing a syringe filled with a fluid composition after step (c). Thus, in an embodiment, a stabilizing component is com Aspects of the present specification provide, in part, a fluid bined with a physiologically-acceptable buffer to make a composition disclosed in the present specification made by a stabilizing component-buffered solution. In aspects of this method disclosed in the present specification. In an aspect, a embodiment, a stabilizing component is combined with an fluid composition comprises a matrix polymer and a stabiliz 30 acetate buffer, a borate buffers, a citrate buffer, a neutral ing component wherein the fluid composition is made by a buffered saline, a phosphate buffer, or a phosphate buffered method comprising the steps of: a) combining a stabilizing saline to make a stabilizing-buffered solution. In other an component with a physiologically-acceptable buffer to make stabilizing component is combined with sodium chloride, a stabilizing component-buffered solution; b) combining a Sodium phosphate, or both. matrix polymer with the stabilizing component-buffered 35 In another embodiment, a physiologically-acceptable Solution to hydrate the matrix polymer, and; c) sizing the fluid buffer is at a concentration necessary to achieve an effective composition. This method may, or may not, further comprise buffering capacity. In aspects of this embodiment, a physi a step comprising titrating a stabilizing component-buffered ologically-acceptable buffer is at a concentration of, e.g., at Solution to obtain a desired pH after step (a); a step compris least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.4 ing filtering the stabilizing component-buffered solution after 40 mM, at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least step (a); a step (b) where combining a matrix polymer with the 0.8 mM, or at least 0.9 mM. In other aspects of this embodi stabilizing component-buffered solution to hydrate the ment, a physiologically-acceptable buffer is at a concentra matrix polymer occurs by mixing the matrix polymer with the tion of, e.g., at least 1.0 mM, at least 2.0 mM, at least 3.0 mM, stabilizing component-buffered solution at a low speed for a at least 4.0 mM, at least 5.0 mM, at least 6.0 mM, at least 7.0 relatively long period of time; a step (b) where combining a 45 mM, at least 8.0 mM, or at least 9.0 mM. In yet other aspects matrix polymer with the stabilizing component-buffered of this embodiment, a physiologically-acceptable buffer is at Solution to hydrate the matrix polymer occurs by mixing the a concentration of e.g., at least 10 mM, at least 20 mM, at matrix polymer with the stabilizing component-buffered least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, Solution at a low speed for a relatively long period of time and at least 70 mM, at least 80 mM, or at least 90 mM. In still other then followed by a rest for a relative long period of time; a step 50 aspects of this embodiment, a physiologically-acceptable (b) where combining a matrix polymer with the stabilizing buffer is at a concentration of, e.g., at least 100 mM, at least component-buffered solution to hydrate the matrix polymer 200 mM, at least 300 mM, at least 400 mM, at least 500 mM, occurs by mixing the matrix polymer with the stabilizing at least 600 mM, at least 700 mM, at least 800 mM, or at least component-buffered solution at a low speed for a relatively 900 mM. long period of time and then by mixing the matrix polymer 55 In further aspects of this embodiment, a physiologically with the stabilizing component-buffered solution using a acceptable buffer is at a concentration of, e.g., at most 0.1 cycle of alternating periods of agitation for a relatively short mM, at most 0.2 mM, at most 0.3 mM, at most 0.4 mM, at period of time followed by periods of rest for a relatively long most 0.5 mM, at most 0.6 mM, at most 0.7 mM, at most 0.8 period of time; a step (b) where combining a matrix polymer mM, or at most 0.9 mM. In still other aspects of this embodi with the stabilizing component-buffered solution to hydrate 60 ment, a physiologically-acceptable buffer is at a concentra the matrix polymer occurs by mixing the matrix polymer with tion of, e.g., at most 1.0 mM, at most 2.0 mM, at most 3.0 mM, the stabilizing component-buffered solution at a low speed at most 4.0 mM, at most 5.0 mM, at most 6.0 mM, at most 7.0 for a relatively long period of time and then by mixing the mM, at most 8.0 mM, or at most 9.0 mM. In yet other aspects matrix polymer with the stabilizing component-buffered of this embodiment, a physiologically-acceptable buffer is at Solution using a cycle of alternating periods of agitation for a 65 a concentration of, e.g., at most 10 mM, at most 20 mM, at relatively short period of time followed by periods of rest for most 30 mM, at most 40 mM, at most 50 mM, at most 60 mM, a relatively long period of time, and then followed by a rest for at most 70 mM, at most 80 mM, or at most 90 mM. In still US 9,125,840 B2 41 42 other aspects of this embodiment, a physiologically-accept matrix polymers disclosed in the present specification, its able buffer is at a concentration of, e.g., at most 100 mM, at salts, and/or mixtures thereof. A matrix polymer can be a most 200 mM, at most 300 mM, at most 400 mM, at most 500 partially crosslinked matrix polymer, a Substantially mM, at most 600 mM, at most 700 mM, at most 800 mM, or uncrosslinked matrix polymer, a matrix polymer that is essen at most 900 mM. In still further aspects of this embodiment, tially free of a crosslinked matrix polymer, or a matrix poly a physiologically-acceptable buffer is at a concentration of mer that is entirely free of a crosslinked matrix polymer as e.g., about 0.1 mM to about 900 mM, 0.1 mM to about 500 disclosed in the present specification. The source of the mM, 0.1 mM to about 100 mM, 0.1 mM to about 90 mM, 0.1 matrix polymer can be from a bacterial source or an animal mM to about 50 mM, 1.0 mM to about 900 mM, 1.0 mM to SOUC. about 500 mM, 1.0 mM to about 100 mM, 1.0 mM to about 90 10 Combining a matrix polymer with a stabilizing compo mM, or 1.0 mM to about 50 mM. nent-buffered solution can be accomplished by any method Aspects of the present specification provide, in part, a with the proviso that the method used is sufficient to hydrate method having an optional step of titrating a stabilizing com the matrix polymer in a manner that produces a fluid compo ponent-buffered solution to obtain a desired pH. A stabilizing sition disclosed in the present specification. It is also under component-buffered solution can be titrated to any physi 15 stood that any method employed does not result in Substantial ologically-acceptable pH desired. As used herein, the term “a degradation of matrix polymer as this is inconsistent with a physiologically-acceptable pH refers to a pH in accord with, fluid composition disclosed in the present specification. For or characteristic of the normal functioning of a living organ example, the step of combining a matrix polymer with a ism. As such, a pH used to make a fluid composition disclosed stabilizing component-buffered solution can comprises mix in the present specification is a pH that has substantially no ing a matrix polymer with a stabilizing component-buffered long term or permanent detrimental effect when administered Solution at a low speed for a relatively long period of time. to mammal. Non-limiting examples of physiologically-ac Non-limiting examples of a speed used for mixing is from ceptable pH occur within the range of about pH 5.5 to about about 50 rpm to about 500 rpm. The relatively long time pH 8.5. It is understood that acids or bases can be used to period for agitation is a time period sufficient to effectively adjust the pH of a stabilizing component-buffered solution. 25 combine a matrix polymer with a stabilizing component Thus, in an embodiment, a stabilizing component-buffered buffered solution to allow the matrix polymer to hydrate. Solution is titrated to a physiologically-acceptable pH. In an Non-limiting examples of time periods for mixing at a rela aspect of this embodiment, a stabilizing component-buffered tive low speed include from about 4 hours to about 16 hours. solution is titrated to pH of, e.g., at least about pH 5.0, at least This combining step is performed at cool ambient tempera about pH 5.5, at least about pH 6.0, at least about pH 6.5, at 30 ture. Non-limiting examples of a cool ambient temperature least about pH 7.0 or at about pH 7.5. In another aspect of this include a temperature not exceeding about 25°C. Such as, a embodiment, a stabilizing component-buffered solution is temperature not exceeding about 20° C., a temperature not titrated to pH of, e.g., at most about pH 5.0, at most about pH exceeding about 15°C., or a temperature not exceeding about 5.5, at most about pH 6.0, at most about pH 6.5, at most about 10°C. As another non-limiting example, a cool ambient tem pH 7.0 or at most about pH 7.5. In yet another aspect of this 35 perature is a temperature from about 2°C. and about 8°C. embodiment, a stabilizing component-buffered solution is As another non-limiting example, the step of combining a titrated to pH of, e.g., about pH 5.0 to about pH 8.0, an matrix polymer with a stabilizing component-buffered solu effective pH level is about pH 5.0 to about pH 7.0, an effective tion may, or may not, involve a cycle of alternating periods of pH level is about pH 5.0 to about pH 6.0, is about pH 5.5 to agitation for a relatively short period of time followed by about pH 8.0, an effective pH level is about pH 5.5 to about pH 40 periods of rest for a relatively long period of time. This 7.0, an effective pH level is about pH 5.5 to about pH 5.0, is agitation/rest step may be performed once in order to com about pH 5.5 to about pH 7.5, an effective pH level is about pH bine a matrix polymer with a stabilizing component-buffered 5.5 to about pH 6.5. Solution composition, or may be performed for a plurality of Aspects of the present specification provide, in part, a times. For example, the agitation/rest step may be performed method having an optional step of filtering a stabilizing com 45 two or more times, five or more times, or ten or more times. ponent-buffered solution to remove particulates and impuri Agitation of a matrix polymer with a stabilizing component ties. For example, a stabilizing component-buffered solution buffered solution can be accomplished by any method suffi may, or may not, be filtered to remove particulates and impu cient to agitate the composition including, without limitation, rities from the stabilizing component-buffered solution. The mechanical shaking, manual shaking, ultrasound, vibration, filters used should be of a pore size that sufficiently removes 50 and the like, and combinations thereof. The relatively short the particulates and impurities desired to be removed from a time period for agitation is a time period Sufficient to effec stabilizing component-buffered solution. Non-limiting tively combine a matrix polymer with a stabilizing compo examples of pore sizes are of the range of 5.0 um or less. nent-buffered solution to create a fluid composition disclosed Thus, in an embodiment, a stabilizing component-buffered in the present specification. Non-limiting examples of time Solution is filtered to remove particulates and impurities. In an 55 periods for agitation include from about 1 minute to about 15 aspect of this embodiment, a stabilizing component-buffered minutes. Similarly, the relatively long time period for rest is a solution is filtered through a pore size sufficient to remove time period sufficient to effectively combine a matrix poly particulates and impurities. In other aspects of this embodi mer with a stabilizing component-buffered solution to create ment, a stabilizing component-buffered solution is filtered a fluid composition disclosed in the present specification. through a pore size of, e.g., about 0.1 um or less, about 0.25 60 Non-limiting examples of time periods for rest include from um or less, about 0.5um or less, about 0.75 um or less, about about 15 minute to about 180 minutes. For example, a matrix 1 Lum or less, about 2 umorless, about 3 um or less, about 4 um polymer with a stabilizing component-buffered solution or less, or about 5 um or less. composition can be agitated for about 1 minute and then Aspects of the present specification provide, in part, a allowed to rest for about 30 minutes. If such an agitation/rest method having a step of combining a matrix polymer with a 65 step is used, it typically follows the combining step compris stabilizing component-buffered solution to hydrate the ing a low speed for a relatively long period of time described matrix polymer. A matrix polymer can be any one of the above. US 9,125,840 B2 43 44 After combining a matrix polymer with a stabilizing com minutes, about 165 minutes, or about 180 minutes. In other ponent-buffered solution, this composition is allowed to rest aspects of this embodiment, a matrix polymer and a stabiliz for a relative long period of time. A relatively longtime period ing component-buffered solution is agitated for about 1 for rest is a time period sufficient to effectively combine a minute or more, about 5 minutes or more, about 10 minutes or matrix polymer with a stabilizing component-buffered solu more, or about 15 minutes or more, and then allowed to rest tion to create a fluid composition disclosed in the present for about 15 minutes or more, about 30 minutes or more, specification. Non-limiting examples of time periods for rest about 45 minutes or more, about 60 minutes or more, about 75 ing after the combining step include from about 4 hours to minutes or more, about 90 minutes or more, about 105 min about 16 hours. utes, about 120 minutes or more, about 135 minutes or more, Thus, in an embodiment, a matrix polymer is combined 10 about 150 minutes or more, about 165 minutes or more, or with a stabilizing component-buffered solution in order to about 180 minutes or more. In yet other aspects of this hydrate the matrix polymer. In an aspect of this embodiment, embodiment, a matrix polymer and a stabilizing component a matrix polymer is combined with a stabilizing component buffered solution is agitated for at most 1 minute, at most 5 buffered solution in order to hydrate the matrix polymer and minutes, at most 10 minutes, or at most 15 minutes, and then achieve a Smooth consistency of the composition. In other 15 allowed to rest for at most 15 minutes, at most 30 minutes, at aspects of this embodiment, a stabilizing component-buff most 45 minutes, at most 60 minutes, at most 75 minutes, at ered solution is combined with a partially crosslinked matrix most 90 minutes, at most 105 minutes, at most 120 minutes, polymer, a substantially uncrosslinked matrix polymer, a at most 135 minutes, at most 150 minutes, at most 165 min matrix polymer that is essentially free of a crosslinked matrix utes, or at most 180 minutes. In still other aspects of this polymer, or a matrix polymer that is entirely free of a embodiment, a matrix polymer and a stabilizing component crosslinked matrix polymer. buffered solution is agitated for at most 1 minute, at most 5 In another embodiment, a matrix polymer is combined minutes, at most 10 minutes, or at most 15 minutes, and then with a stabilizing component-buffered solution by mixing the allowed to rest for about 15 minutes or more, about 30 min matrix polymer with the stabilizing component-buffered utes or more, about 45 minutes or more, about 60 minutes or Solution at a low speed for a relatively long period of time. In 25 more, about 75 minutes or more, about 90 minutes or more, aspects of this embodiment, a matrix polymer is combined about 105 minutes, about 120 minutes or more, about 135 with a stabilizing component-buffered solution by mixing the minutes or more, about 150 minutes or more, about 165 matrix polymer with the stabilizing component-buffered minutes or more, or about 180 minutes or more. In further solutionat, e.g., about 50 rpm, about 100 rpm, about 150 rpm, aspects of this embodiment, a matrix polymer and a stabiliz about 200 rpm, about 250 rpm, about 300 rpm, about 350 rpm, 30 ing component-buffered solution is agitated for about 1 about 400 rpm, about 450 rpm, or about 500 rpm for about 6 minute to about 15 minutes, and then allowed to rest for about hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 30 minutes to about 60 minutes. 13 hours, or 14 hours. In other aspects of this embodiment, a Aspects of the present specification provide, in part, a matrix polymer is combined with a stabilizing component method having a step of sizing the fluid composition. Sizing buffered solution by mixing the matrix polymer with the 35 the fluid composition is particularly important if crosslinked stabilizing component-buffered solution at, e.g., about 50 matrix polymers are present because this step 1) produces a rpm, about 100 rpm, about 150 rpm, about 200 rpm, about 250 composition with a particular mean sized of gel particle, rpm, about 300 rpm, about 350 rpm, about 400 rpm, about 450 and/or 2) produces a composition with a smooth consistency rpm, or about 500 rpm for about 6 hours or more, 7 hours or as opposed to a granular consistency. In fluid compositions more, 8 hours or more, 9 hours or more, 10 hours or more, 11 40 comprising a crosslinked matrix polymer, the initial hours or more, 12 hours or more, 13 hours or more, or 14 crosslinking process produces a large gel mass that must be hours or more. In yet other aspects of this embodiment, a sized down in order to produce a composition that can be matrix polymer is combined with a stabilizing component properly administered to an individual. Such as, e.g., by injec buffered solution by mixing the matrix polymer with the tion. Sizing of gel particles can be accomplished by any stabilizing component-buffered solution at, e.g., at most 50 45 method suitable to produce a fluid composition that can be rpm, at most 100 rpm, at most 150 rpm, at most 200 rpm, at properly administered into an individual. Non-limiting most 250 rpm, at most 300 rpm, at most 350 rpm, at most 400 examples include sieving and homogenization. In a sieving rpm, at most 450 rpm, or at most 500 rpm for about 6 hours or method, a large gel mass included in a fluid composition is more, 7 hours or more, 8 hours or more, 9 hours or more, 10 broken down by passing through a series of sieves or screens hours or more, 11 hours or more, 12 hours or more, 13 hours 50 in order to size the gel particles. This method produces gel or more, or 14 hours or more. In still other aspects of this particles that have a well-defined average size. In a homog embodiment, a matrix polymer is combined with a stabilizing enization method, a large gel mass included in a fluid com component-buffered solution by mixing the matrix polymer position is broken down by recirculating the fluid composi with the stabilizing component-buffered solution at about 50 tion between a first vessel and a second vessel through a rpm to about 500 rpm for about 8 hours to about 12 hours. 55 narrow aperture. The recirculation of a fluid composition may In another embodiment, a matrix polymer is combined comprise passing the composition from a first vessel into a with a stabilizing component-buffered solution by mixing the second vessel through an orifice having any diameter suffi matrix polymer with the stabilizing component-buffered cient to achieve a smooth fluid composition. Non-limiting Solution using a cycle of alternating periods of agitation for a examples of orifice diameters include about 2 mm to about 10 relatively short period of time followed by periods of rest for 60 mm. The recirculating step can be performed once or a plu a relatively long period of time. In aspects of this embodi rality of times, such as, e.g., from 2 to 10 times. ment, a matrix polymer and a stabilizing component-buffered Thus, in an embodiment, a fluid composition is sized. In Solution is agitated for about 1 minute, about 5 minutes, about other aspects of this embodiment, a fluid composition is sized 10 minutes, or about 15 minutes, and then allowed to rest for by sieving wherein after sizing the mean particle size of the about 15 minutes, about 30 minutes, about 45 minutes, about 65 crosslinked matrix polymer is, e.g., about 200 um, about 250 60 minutes, about 75 minutes, about 90 minutes, about 105 um, about 300 um, about 350 um, about 400 um, about 450 minutes, about 120 minutes, about 135 minutes, about 150 um, about 500 um, about 550 um, about 600 um, about 650 US 9,125,840 B2 45 46 um, about 700 um, about 750 um, or about 800 um. In yet about 200 um to about 500 um, about 200 um to about 600 other aspects of this embodiment, a fluid composition is sized um, about 200 um to about 700 um, about 200 um to about by sieving wherein after sizing the mean particle size of the 800 um, about 300 um to about 500 um, about 300 um to crosslinked matrix polymer is, e.g., at most 200 um, at most about 600 um, about 300 um to about 700 um, or about 300 250 um, at most 300 um, at most 350 Lim, at most 400 um, at um to about 800 um. most 450 um, at most 500 um, at most 550 um, at most 600 In other aspects of this embodiment, a fluid composition um, at most 650 um, at most 700 um, at most 750 um, or at comprising a crosslinked matrix polymer is sized by recircu most 800 um. In still other aspects of this embodiment, a fluid lating between a first vessel and a second vessel through a composition is sized by sieving whereinafter sizing the mean narrow aperture wherein after recirculation the mean particle particle size of the crosslinked matrix polymer is, e.g., about 10 size of the crosslinked matrix polymer is, e.g., about 200 um, 200 um to about 300 um, about 300 um to about 400 um, about 250 um, about 300 um, about 350 m, about 400 um, about 400 um to about 500 um, about 500 um to about 600 about 450 Lim, about 500 um, about 550 um, about 600 um, um, about 600 um to about 700 um, about 700 um to about about 650 um, about 700 um, about 750 um, or about 800 um. 800 um, about 200 um to about 400 um, about 200 um to In yet other aspects of this embodiment, a fluid composition about 500 um, about 200 um to about 600 um, about 200 um 15 comprising a crosslinked matrix polymer is sized by recircu to about 700 um, about 200 um to about 800 um, about 300 lating between a first vessel and a second vessel through a um to about 500 um, about 300 um to about 600 um, about narrow aperture wherein after recirculation the mean particle 300 um to about 700 um, or about 300 um to about 800 um. size of the crosslinked matrix polymer is, e.g., at most 200 In other aspects of this embodiment, a fluid composition um, at most 250 um, at most 300 um, at most 350 Lim, at most comprising a crosslinked matrix polymer is sized by sieving 400 um, at most 450 um, at most 500 um, at most 550 um, at wherein after sizing the mean particle size of the crosslinked most 600 um, at most 650 um, at most 700 um, at most 750 matrix polymer is, e.g., about 200 um, about 250 um, about um, or at most 800 um. In still other aspects of this embodi 300 um, about 350 um, about 400 um, about 450 Lim, about ment, a fluid composition comprising a crosslinked matrix 500 um, about 550 um, about 600 um, about 650 um, about polymer is sized by recirculating between a first vessel and a 700 um, about 750 um, or about 800 um. In yet other aspects 25 second vessel through a narrow aperture wherein after recir of this embodiment, a fluid composition comprising a culation the mean particle size of the crosslinked matrix poly crosslinked matrix polymer is sized by sieving wherein after meris, e.g., about 200 um to about 300 um, about 300 um to sizing the mean particle size of the crosslinked matrix poly about 400 um, about 400 um to about 500 um, about 500 um meris, e.g., at most 200 um, at most 250 um, at most 300 um, to about 600 um, about 600 um to about 700 um, about 700 at most 350 lum, at most 400 um, at most 450 um, at most 500 30 um to about 800 um, about 200 um to about 400 um, about um, at most 550 um, at most 600 um, at most 650 Lim, at most 200 um to about 500 um, about 200 um to about 600 um, 700 um, at most 750 um, or at most 800 um. In still other about 200 um to about 700 um, about 200 um to about 800 aspects of this embodiment, a fluid composition comprising a um, about 300 um to about 500 um, about 300 um to about crosslinked matrix polymer is sized by sieving wherein after 600 um, about 300 um to about 700 um, or about 300 um to sizing the mean particle size of the crosslinked matrix poly 35 about 800 um. meris, e.g., about 200 um to about 300 um, about 300 um to In aspects of this embodiment, a fluid composition is sized about 400 um, about 400 um to about 500 um, about 500 um by recirculating between a first vessel and a second vessel to about 600 um, about 600 um to about 700 um, about 700 through a narrow aperture once. In aspects of this embodi um to about 800 um, about 200 um to about 400 um, about ment, a fluid composition is sized recirculating between a first 200 um to about 500 um, about 200 um to about 600 um, 40 vessel and a second vessel through a narrow aperture, e.g., about 200 um to about 700 um, about 200 um to about 800 twice, three times, four times, five times, six times, seven um, about 300 um to about 500 um, about 300 um to about times, eight times, nine times, or ten times. In aspects of this 600 um, about 300 um to about 700 um, or about 300 um to embodiment, a fluid composition is sized by recirculating about 800 um. between a first vessel and a second vessel through a narrow In other aspects of this embodiment, a fluid composition is 45 aperture, e.g., twice, three times, four times, five times, six sized by recirculating between a first vessel and a second times, seven times, eight times, nine times, or ten times. In vessel through a narrow aperture wherein after recirculation other aspects of this embodiment, a fluid composition is sized the mean particle size of the crosslinked matrix polymer is, by recirculating between a first vessel and a second vessel e.g., about 200 um, about 250 um, about 300 um, about 350 through a narrow aperture, e.g., twice to ten times, twice to um, about 400 um, about 450 um, about 500 um, about 550 50 eight times, twice to six times, twice to four times, three times um, about 600 um, about 650 um, about 700 um, about 750 to five times, three times to six times, or three times to seven um, or about 800 um. In yet other aspects of this embodiment, times. a fluid composition is sized by recirculating between a first In other aspects of this embodiment, a fluid composition is vessel and a second vessel through a narrow aperture once recirculated between a first vessel and a second vessel wherein after recirculation the mean particle size of the 55 through a narrow aperture having a diameter of, e.g., about 2 crosslinked matrix polymer is, e.g., at most 200 um, at most mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, 250 um, at most 300 um, at most 350 Lim, at most 400 um, at about 7 mm, about 8 mm, about 9 mm, or about 10 mm. In yet most 450 um, at most 500 um, at most 550 um, at most 600 other aspects of this embodiment, a fluid composition is recir um, at most 650 um, at most 700 um, at most 750 um, or at culated between a first vessel and a second vessel through a most 800 um. In still other aspects of this embodiment, a fluid 60 narrow aperture having a diameter of, e.g., at least 2 mm, at composition is sized by recirculating between a first vessel least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least and a second vessel through a narrow aperture wherein after 7 mm, at least 8 mm, at least 9 mm, or at least 10 mm. In still recirculation the mean particle size of the crosslinked matrix other aspects of this embodiment, a fluid composition is recir polymer is, e.g., about 200 um to about 300 um, about 300 um culated between a first vessel and a second vessel through a to about 400 um, about 400 um to about 500 um, about 500 65 narrow aperture having a diameter of e.g., at most 2 mm, at um to about 600 um, about 600 um to about 700 um, about most 3 mm, at most 4 mm, at most 5 mm, at most 6 mm, at 700 um to about 800 um, about 200 um to about 400 um, most 7 mm, at most 8 mm, at most 9 mm, or at most 10 mm. US 9,125,840 B2 47 48 In further aspects of this embodiment, a fluid composition is fluid composition can remain stable for about 3 months to recirculated between a first vessel and a second vessel about 3 years. One method of sterilization of a filled syringe through a narrow aperture having a diameter of, e.g., about 2 is by autoclave. Autoclaving can be accomplished by apply mm to about 4 mm, about 2 mm to about 6 mm, about 2 mm ing a mixture of heat, pressure and moisture to a sample in to about 8 mm, or about 2 mm to about 10 mm. need of sterilization. Many different sterilization tempera Additional method steps in accordance with making a fluid tures, pressures and cycle times can be used for this step. For composition disclosed in the present specification may, or example, the filled Syringes may be sterilized at a temperature may not, include degassing the composition, filling Syringes of at least about 120° C. to about 130° C. or greater. Moisture with the composition, and sterilizing the composition. may or may not be utilized. The pressure applied is in some Aspects of the present specification provide, in part, a 10 embodiments depending on the temperature used in the ster method having an optional step of degassing a fluid compo ilization process. The sterilization cycle may be at least about sition disclosed in the present specification. Degassing a fluid 1 minute to about 20 minutes or more. composition disclosed in the present specification can be Another method of sterilization incorporates the use of a accomplished using a standard device based on conventional gaseous species which is known to kill or eliminate transmis techniques and may be done under vacuum. Degassing is 15 sible agents. Preferably, ethylene oxide is used as the steril performed for a time period sufficient to remove the desired ization gas and is known in the art to be useful in sterilizing amount of gases from a fluid composition. Non-liming medical devices and products. examples of Sufficient time period for degassing include A further method of sterilization incorporates the use of an about 2 hours to about 8 hours. irradiation source which is known in theart to kill or eliminate Aspects of the present specification provide, in part, a transmissible agents. A beam of irradiation is targeted at the method having an optional step of filling a syringe with a fluid Syringe containing the HA composition, and the wavelength composition disclosed in the present specification. Syringes of energy kills or eliminates the unwanted transmissible useful according to the present description include any agents. Preferable energy useful include, but is not limited to Syringe known in the art for administering a therapeutically ultraviolet (UV) light, gamma irradiation, visible light, effective amounta fluid composition disclosed in the present 25 microwaves, or any other wavelength or band of wavelengths specification into a dermal region, Such as, e.g., a Syringe which kills or eliminates the unwanted transmissible agents, having an internal volume of about 0.4 mL to about 3.0 mL. preferably without substantially altering of degrading the HA In addition the type of needle used in conjunction with the composition. syringe is a needle sufficient to effectively administer athera Thus, in an embodiment, a syringe filled with a fluid com peutically effective amount of a fluid composition disclosed 30 position is sterilized. In aspects of this embodiment, a Syringe in the present specification into a dermal region, Such as, e.g., filled with a fluid composition is sterilized by autoclaving, gas a diameter of between about 18 Gandabout 40G and a needle sterilization, or irradiation. In other aspects of this embodi length of at about 2 mm or more in length. ment, a Syringe filled with a fluid composition can remain Thus, in an embodiment, a fluid composition is filled into a stable after sterilization for about 3 months, about 6 months, Syringe. In aspects of this embodiment, a fluid composition is 35 about 9 months, about 12 months, about 18 months, about 24 filled into a syringe having an internal Volume of about 0.4 months, about 30 months, or about 36 months. In yet other mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, aspects of this embodiment, a syringe filled with a fluid com about 0.9 mL, about 1 mL, about 1 mL, about 1.5 mL, about position can remain stable after sterilization for at least 3 2 mL, about 2.5 mL, or about 3 mL. In other aspects of this months, at least 6 months, at least 9 months, at least 12 embodiment, a fluid composition is filled into a syringe hav 40 months, at least 18 months, at least 24 months, at least 30 ing an internal Volume of at least 0.4 mL, at least 0.5 mL, at months, or at least 36 months. In still other aspects of this least 0.6 mL, at least 0.7 mL, at least 0.8 mL, at least 0.9 mL, embodiment, a Syringe filled with a fluid composition can at least 1 mL, at least 1 mL, at least 1.5 mL, at least 2 mL, at remainstable after sterilization for about 3 months to about 12 least 2.5 mL, or at least 3 mL. In yet other aspects of this months, about 3 months to about 24 months, about 3 months embodiment, a fluid composition is filled into a syringe hav 45 to about 36 months, about 6 months to about 12 months, about ing an internal volume of about 0.4 mL to about 3 mL, about 6 months to about 24 months, or about 6 months to about 36 0.5 mL to about 1.5 mL, or about 0.8 mL to about 2.5 mL. In months. another aspect, a syringe filled with a fluid composition is Aspects of the present specification provide, in part, a combined with a needle. In aspects of this embodiment, a method of improving a condition of skin in an individual in syringe filled with a fluid composition is combined with a 50 need thereof, the method comprising the steps of administer needle having a diameter of about 18 G, about 22 G, about 25 ing a fluid composition disclosed in the present specification G, about 28 G, about 30 G, about 33 G, or about 40G. In other into a dermal region of the individual, wherein the adminis aspects of this embodiment, a syringe filled with a fluid com tration improves the condition. position is combined with a needlehaving a diameter of about Aspects of the present invention provide, in part, a condi 18 G or smaller, about 22 G or smaller, about 25G or smaller, 55 tion of skin. Non-limiting examples of a skin condition about 28 G or smaller, about 30 G or smaller, about 33 G or include dehydration, lack of skin elasticity, roughness, lack of smaller, or about 40 G or smaller. In yet other aspects of this skin tautness, skin stretch line and/or marks, skin paleness, embodiment, a Syringe filled with a fluid composition is com skin wrinkles, and the like. bined with a needle having a diameter of about 18 G to about Aspects of the present invention provide, in part, improv 40 G, about 22 G to about 33 G, or about 26 G to about 40 G. 60 ing a skin condition. Non-limiting examples of improving a Aspects of the present specification provide, in part, a skin condition include rehydrating the skin, providing method having an optional step of sterilizing a syringe filled increased elasticity to the skin, reducing skin roughness, with a fluid composition disclosed in the present specifica making the skin tauter, reducing or eliminating stretch lines or tion. As used herein, the term “sterilizing refers to any marks, giving the skin better tone, shine, brightness and/or method known in the art to effectively kill or eliminate trans 65 radiance to reduce paleness, reducing or eliminating wrinkles missible agents without Substantially altering of degrading a in the skin, providing wrinkle resistance to the skin, and the fluid composition disclosed in the specification. A sterilized like. US 9,125,840 B2 49 50 Thus, in an embodiment, a method of treating a skin con The dermis is the layer of skin beneath the epidermis that dition comprises the step of administering to an individual consists of connective tissue and cushions the body from Suffering from a skin condition a fluid composition compris stress and strain. The dermis is tightly connected to the epi ing a matrix polymer and a stabilizing component, wherein dermis by a basement membrane. It also harbors many the administration of the composition improves the skin con Mechanoreceptor/nerve endings that provide the sense of dition, thereby treating the skin condition. In an aspect of this touch and heat. It contains the hair follicles, Sweat glands, embodiment, a method of treating skin dehydration com sebaceous glands, apocrine glands, lymphatic vessels and prises the step of administering to an individual Suffering blood vessels. The blood vessels in the dermis provide nour from skin dehydration a fluid composition comprising a ishment and waste removal from its own cells as well as from matrix polymer and a stabilizing component, wherein the 10 the Stratum basale of the epidermis. The dermis is structurally administration of the composition rehydrates the skin, divided into two areas: a Superficial area adjacent to the thereby treating skin dehydration. In another aspect of this epidermis, called the papillary region, and a deep thicker area embodiment, a method of treating a lack of skin elasticity known as the reticular region. comprises the step of administering to an individual Suffering The papillary region is composed of loose areolar connec from a lack of skin elasticity a fluid composition comprising 15 tive tissue. It is named for its fingerlike projections called a matrix polymer and a stabilizing component, wherein the papillae that extend toward the epidermis. The papillae pro administration of the composition increases the elasticity of vide the dermis with a "bumpy' surface that interdigitates the skin, thereby treating a lack of skin elasticity. In yet with the epidermis, strengthening the connection between the another aspect of this embodiment, a method of treating skin two layers of skin. The reticular region lies deep in the pap roughness comprises the step of administering to an indi illary region and is usually much thicker. It is composed of vidual Suffering from skin roughness a fluid composition dense irregular connective tissue, and receives its name from comprising a matrix polymer and a stabilizing component, the dense concentration of collagenous, elastic, and reticular wherein the administration of the composition decreases skin fibers that weave throughout it. These protein fibers give the roughness, thereby treating skin roughness. In still another dermis its properties of strength, extensibility, and elasticity. aspect of this embodiment, a method of treating a lack of skin 25 Also located within the reticular region are the roots of the tautness comprises the step of administering to an individual hair, sebaceous glands, Sweat glands, receptors, nails, and Suffering from a lack of skin tautness a fluid composition blood vessels. Tattoo ink is held in the dermis. Stretch marks comprising a matrix polymer and a stabilizing component, from pregnancy are also located in the dermis. wherein the administration of the composition makes the skin The hypodermis is not part of the skin, and lies below the tauter, thereby treating a lack of skin tautness. 30 dermis. Its purpose is to attach the skin to underlying bone In a further aspect of this embodiment, a method of treating and muscle as well as Supplying it with blood vessels and a skin stretchline or mark comprises the step of administering nerves. It consists of loose connective tissue and elastin. The to an individual Suffering from a skin stretch line or mark a main cell types are fibroblasts, macrophages and adipocytes fluid composition comprising a matrix polymer and a stabi (the hypodermis contains 50% of body fat). Fat serves as lizing component, wherein the administration of the compo 35 padding and insulation for the body. sition reduces or eliminates the skin stretch line or mark, Aspects of the present invention provide, in part, an indi thereby treating a skin stretch line or mark. In another aspect vidual. As used herein, the term “individual' refers to any of this embodiment, a method of treating skin paleness com mammal including a human being. prises the step of administering to an individual Suffering Aspects of the present invention provide, in part, adminis from skin paleness a fluid composition comprising a matrix 40 tering a fluid composition disclosed in the present specifica polymer and a stabilizing component, wherein the adminis tion. As used herein, the term “administering means any tration of the composition increases skin tone or radiance, delivery mechanism that provides a fluid composition com thereby treating skin paleness. In another aspect of this prising a matrix polymer and a stabilizing component to an embodiment, a method of treating skin wrinkles comprises individual that potentially results in a clinically, therapeuti the step of administering to an individual Suffering from skin 45 cally, or experimentally beneficial result. The actual delivery wrinkles a fluid composition comprising a matrix polymer mechanism used to administer a fluid composition disclosed and a stabilizing component, wherein the administration of in the present specification to an individual can be determined the composition reduces or eliminates skin wrinkles, thereby by a person of ordinary skill in the art by taking into account treating skin wrinkles. In yet another aspect of this embodi factors, including, without limitation, the type of skin condi ment, a method of treating skin wrinkles comprises the step of 50 tion, the location of the skin condition, the cause of the skin administering to an individual a fluid composition compris condition, the severity of the skin condition, the degree of ing a matrix polymer and a stabilizing component, wherein relief desired, the duration of relief desired, the particular the administration of the composition makes the skin resistant fluid composition used, the rate of excretion of the particular to skin wrinkles, thereby treating skin wrinkles. fluid composition used, the pharmacodynamics of the par Aspects of the present invention provide, in part, a dermal 55 ticular fluid composition used, the nature of the other com region. As used herein, the term "dermal region” refers to the pounds included in the particular fluid composition used, the region of skin comprising the epidermal-dermal junction and particular route of administration, the particular characteris the dermis including the Superficial dermis (papillary region) tics, history and risk factors of the individual. Such as, e.g., and the deep dermis (reticular region). The skin is composed age, weight, general health and the like, or any combination of three primary layers: the epidermis, which provides water 60 thereof. proofing and serves as a barrier to infection; the dermis, Thus, in an embodiment, a fluid composition comprising a which serves as a location for the appendages of skin; and the matrix polymer and a stabilizing component is administered hypodermis (Subcutaneous adipose layer). The epidermis to a skin region of an individual. In an aspect of this embodi contains no blood vessels, and is nourished by diffusion from ment, a fluid composition comprising a matrix polymer and a the dermis. The main type of cells which make up the epider 65 stabilizing component is administered to a skin region of an mis are keratinocytes, melanocytes, Langerhans cells and individual by injection. In another aspect of this embodiment, Merkels cells. a fluid composition comprising a matrix polymer and a sta US 9,125,840 B2 51 52 bilizing component is administered to a skin region of an Fluid compositions made for testing were prepared as individual by injection into a dermal region. In aspects of this described in Example 1, except that 1) 1 mL of each fluid embodiment, a fluid composition comprising a matrix poly composition was made and the amount of mannitol added to mer and a stabilizing component is administered to a skin the test fluid compositions was 0% (w/v) for control, 0.5% region of an individual by injection into, e.g., an epidermal (w/v) and 5% (w/v). dermal junction region, a papillary region, a reticular region, Degradation of hyaluronan polymer included in the fluid or any combination thereof. compositions and rheological processing of the results were EXAMPLES performed according to conventional methods. Resistance of hyaluronan polymer to degradation was assessed in fluid The following examples illustrate representative embodi 10 composition with and without mannitol and these composi ments now contemplated, but should not be construed to limit tions were compared by analyzing the time it took for the the disclosed fluid compositions, methods of forming Such composition to reach a dynamic viscosity of 5 Pa's T(m=5 fluid compositions, and methods of treating skin conditions Pas). The degradation test was repeated twice and a mean using Such fluid compositions. dynamic viscosity T(m=5 Pas) was calculated from 15 the 2 tests (Table 4). In general, the shorter the time to reach Example 1 a dynamic viscosity of 5 Pa's T(m=5 Pas), the lower the resistance of hyaluronan polymer to degradation. Thus, to A Method of Making a Fluid Composition show that addition of mannitol to a fluid composition had a This example illustrates how to make a fluid composition beneficial effect on resistance of hyaluronan polymer to disclosed in the present specification. breakdown, it was shown that T(m=5 Pas), , PT To combine a stabilizing component with a physiologi (m-5 Pars), i.e., ii. For each fluid composition the cally-acceptable buffer to make a stabilizing component results of the rheological test (values of G', G", tan 8, m*=f buffered solution, 0.9 g of mannitol (Cooper Pharmaceutical, (time)+mathematical model of curve m=f(time)) were deter Inc.) was added to 1 L of a phosphate buffer solution (com mined and the curves obtained (FIG. 1). prising 8.5 g NaCl, 0.563 g NaH2PO, 0.045g Na HPO, and 25 water) in a 10 liter bottle, and mixed for 15 minutes. This step TABLE 4 produces a stabilizing component-buffered solution compris ing a phosphate buffer solution (pH 7.2) containing 0.9 Breakdown Test mg/mL of mannitol. 30 Fluid T(m = Mean T(m = Std. CV To filter the stabilizing component-buffered solution, the Composition Test 5 Pas) 5 Pas) deviation (%) mannitol-phosphate buffer solution made above was filtered on line with a 0.2 um filter under pressure. Hyaluronan 1 747 1099 498 45 (13.5 mg/L) 2 1452 To combining a matrix polymer with the stabilizing com Hyaluronan 1 3351 3127 317 10 ponent-buffered solution to hydrate the matrix polymer, (13.5 mg/L) 2 2903 about 60% of the mannitol-phosphate buffer solution was 35 5% mannitol poured in a new bottle and uncrosslinked sodium hyaluronan Hyaluronan 1 2933 2355 818 35 polymer having a molecular weight between about 2.5 MDa (13.5 mg/L) 2 1776 and about 3.0 MDa was added to the mannitol-phosphate 0.5% mannitol buffer solution. This was completed with the remaining 40% of the mannitol-phosphate buffer Solution, giving a final con 40 As shown in Table 4, hyaluronan polymer was more resis centration of uncrosslinked hyaluronan of about 13.5 mg/L. tant to degradation when mannitol was included in the fluid This composition was then gently, mechanically mixed at a composition as compared to the resistance hyaluronan poly speed of about 100 rpm to about 200 rpm for about 8 hours to mer in composition without mannitol (Table 4, FIG. 1). In about 12 hours at a cool ambient temperature of about 2°C. to fact, the higher the mannitol concentration in a fluid compo about 8°C. The composition was then subjected to agitation 45 sition, the longer the time required for the hyaluronan poly by manually shaking the bottle containing the composition mer to degrade. As such, incorporating a stabilizing compo for about 1 minute, followed by a period of rest for about 30 nent like mannitol into a fluid composition allowed minutes. This mixing step was repeated three additional times uncrosslinked hyaluronan polymer remained intact longer before the composition was then allowed to rest about 8 to because the stabilizing component increased the resistance of about 12 hours. 50 hyaluronan polymer to degradation (FIG. 1). Thus, the addi To size a fluid composition, the fluid composition above tion of a stabilizing component to a fluid composition pro was recirculated four times from one bottle into a second vided protection to a matrix polymer to degradation. bottle thorough a narrow orifice having a diameter of about 3 mm and about 5 mm. Example 2B To degas a fluid composition, the composition described 55 above was degassed for about four hours using standard Effects of Stabilizing Component on Various device based on conventional techniques. Properties of Fluid Composition Example 2A This example illustrates the effects of a stabilizing compo 60 nent on various properties of a fluid composition disclosed in Stabilizing Component Reduces or Prevents the present specification including fluid appearance, pH, Degradation of Matrix Polymer osmolarity, and dynamic viscosity. Fluid compositions made for testing were prepared as This example illustrates that the addition of a stabilizing described in Example 1, except that 1) 1 mL of each fluid component reduces or prevents degradation of a matrix poly 65 composition was made and the amount of mannitol added to mer included in a fluid composition disclosed in the present the test fluid compositions was 0% (w/v) for control, 0.5% specification. (w/v), 5% (w/v), 9% (w/v), and 17% (w/v). US 9,125,840 B2 53 54 The appearance of the fluid compositions was visually 162 Pars; compositions comprising 5% (w/v) mannitol had a examined. Test compositions comprising 0% (w/v), 0.5% mean dynamic viscosity of 152 Pas; compositions compris (w/v), 5% (w/v), and 9% (w/v) mannitol all appeared color ing 9% (w/v) mannitol had a mean dynamic viscosity of 148 less and transparent, while the fluid composition comprising Pas. These results indicate that the effect of a stabilizing 17% (w/v) mannitol appeared white and cloudy (Table 5). 5 component was negligible because the addition of mannitol in These results indicate that mannitol was soluble in all fluid an amount less than or equal to about 9% only resulted in a compositions tested, except for the composition comprising change in dynamic viscosity of about 20 Pa's at 1 Pa. 17% (w/v) mannitol. Furthermore, it is known that mannitol Example 3 solubility in water is 1 g mannitol/5.5 ml water, i.e. mass solubility of 15.4%. Taken together, these results indicate that 10 Effects of Sterilization on Fluid Compositions fluid compositions comprising a matrix polymer can include Comprising a Stabilizing Component about 15.4% mannitol before this polyol begins to precipitate out of Solution. This example illustrates the effects of sterilization on vari The pH of the fluid compositions was measured using a pH ous properties of a fluid composition disclosed in the present 15 specification including pH, dynamic viscosity and stability of meter. Test compositions comprising 0% (w/v), 0.5% (w/v), stabilizing component. 5% (w/v), and 9% (w/v) mannitol all had a pH of 6.9, while Fluid compositions made for testing were prepared as the fluid composition comprising 17% (w/v) had a pH of 6.8 described in Example 1, except that 1) 1 mL of each fluid (Table 5). These results indicate that mannitol has no effect on composition was made and the amount of mannitol added to the pH of a fluid composition because all pH values were the test fluid compositions was 0% (w/v) and 1.1% (w/v). within the measurement error range of the pH meter (+0.2). Fluid compositions prepared above were filled in a glass The osmolarity of the fluid compositions was measured syringe and sterilized by autoclaving at 130°C. for 3 minutes. using an osmometer. Test compositions exhibited a wide The pH of the fluid compositions was measured using a pH range of osmolarity that was correlated with the amount of meter. These results indicate that sterilization had no effect on mannitol added to the composition (Table 5). In general, the 25 the pH of a fluid composition because all pH values were osmolarity of a fluid composition increase as the amount of within the measurement error range of the pH meter (+0.2) mannitol increased. The results indicated that a fluid compo (Table 6). sition comprising a matrix polymer and 0.5% mannitol pre The dynamic viscosity of the fluid compositions was mea served the required osmolarity for hyaluronan, namely an sured before and after sterilization using a rheometer. The osmolarity range of about 270 mOsm/L to about 390 mOsm/ 30 difference in viscosity before and after sterilization was 54 L. It general, an osmolarity range of about 200 mOsm/L to Pa's for fluid compositions without mannitol and 61 Pa's for about 400 mOsm/L is the range approved for viscoelastic fluid compositions comprising 1.1% (w/v) mannitol (Table ophthalmic devices. 6). These results indicate that the addition of a stabilizing The data obtained from the osmolarity test were further component had a negligible effect on dynamic viscosity analyzed by linear regression. This analysis revealed that 35 because the difference between the two compositions was y=68.576x-17.118 and that R=0.9888. From this formula, it within the measurementerror range of the rheometer (10%). was calculated that the addition of 1% mannitol to a fluid As such, there was no significant difference in dynamic vis composition comprising hyaluronan would increase the cosity of the fluid compositions before and after sterilization. osmolarity of the composition by 52 mOsm/L to approxi The chemical and physical stability of mannitol after ster mately 351 mOsm/L. As such, a fluid composition compris 40 ilization was determined by autoclaving 25% Mannitol (In ing a matrix polymer and 1% mannitol was an effective com venex Pharmaceuticals, Itasca, Ill.) five times at 121°C. for bination that balanced the opposite effects of improving a 15 minutes. The results indicate that mannitol was chemically fluid composition’s resistance to hyaluronan degradation and and physically stable after this sterilization regime. the increase in osmolarity upon addition of mannitol to the composition. 45 TABLE 6 TABLE 5 Effects of Sterilization on Properties of Fluid Compositions Mannitol Effects on Properties of Fluid Compositions Fluid Composition Hyaluronan Appearance of Fluid Osmolarity 50 Fluid Composition Composition pH (mCosm/L) Composition Hyaluronan (13.5 mg/L) Sterilization Property (13.5 mg/L) Mannitol (1.1%) Hyaluronan (13.5 mg/L) Colorless and transparent 6.9 299 Hyaluronan (13.5 mg/L) Colorless and transparent 6.9 333 Before Dynamic 102 Pa. S 118 Pa. S Mannitol (0.5%) sterilization viscosity at 1 Pa Hyaluronan (13.5 mg/L) Colorless and transparent 6.9 589 pH 7.1 7.0 Mannitol (5%) 55 After Dynamic 48 Pa. S S7 Pa. S Hyaluronan (13.5 mg/L) Colorless and transparent 6.9 918 sterilization viscosity at 1 Pa Mannitol (9%) pH 7.1 7.0 Hyaluronan (13.5 mg/L) White and cloudy 6.8 ND Mannitol (17%) 60 Example 4 The dynamic viscosity of the fluid compositions was mea Sured using a rheometer. Test compositions comprising 0% Determining the Stability of Fluid (w/v), 5% (w/v), and 9% (w/v) mannitol exhibited a consis Composition-Filled Syringe tent dynamic viscosity overtime and all within 20Pas of each other at 1 Pa (FIG. 2). The mean dynamic viscosity of these 65 This example illustrates how to determine the long-term three fluid compositions was as follows: compositions com stability of a fluid composition disclosed in the present speci prising 0% (w/v) mannitol had a mean dynamic viscosity of fication. US 9,125,840 B2 55 56 Fluid compositions were prepared as described in Example Skin measurements were assessed at each of the four visits 1, except that the amount of mannitol added to the test fluid from day 0 to day 60, on different areas, by Skin Evidence for compositions was about 1% (w/v). The degassed fluid com IOMATM (IntuiSkin, Research Triangle Park, N.C.). Skin position was then filled into a glass Syringe and sterilized by Evidence for IOMA is a probe-based system that measures autoclaving at 130° C. for 3 minutes. The resulting composition-filled syringes will be stored at the physical and visual parameters of the skin. The Visio about 2°C. to about 8° C. and at about 18°C. to about 22°C. Probe uses its high-resolution sensor to capture with an to determine shelf life of the fluid compositions. The stored extreme precision the skin images including wrinkles, sebum, compositions will be tested for various properties including hairiness, dark spots, and clogged pores/bacterial infection. dynamic viscosity, pH appearance and osmolarity at 3 The Physio Probe contains high-technology sensors and months, 6 months, 9 months, 12 months, 18 months, 24 10 extracts in Vivo the key characteristics of the skin including months, 30 months, and 36 months. These tests will deter hydration, trans-epidermal water loss (TEWL), and the skin mine the stability of fluid composition-filled Syringes and as temperature. such the shelf life of such products. For each individual administered the fluid composition using a depot injection method, the cheek, neck, peri-oral and Example 5 15 eye regions were measured for hydration (%) at each visit from D0 to D60 by Skin Evidence for IOMATM and a mean Safety and Efficacy Study of Fluid Composition hydration was determined at each visit (Table 7). Coefficient of anisotropy (%), arithmetical roughness (Ra), and lumi This example illustrates the safety and efficacy of a fluid nance (L) were also measured for each individual at each visit composition disclosed in the present specification. from D0 to D60 by Skin Evidence for IOMATM and mean Fluid compositions were prepared as described in Example values calculated. Statistical analyses were performed using 1, except that the amount of mannitol added to the test fluid SASR software version 9.1 (SAS Institute, Inc. Cary, N.C.). compositions was about 1% (w/v). The degassed fluid com The results indicate that overall skin hydration of the cheek position was then filled into a glass Syringe and sterilized by (p=0.0036), neck (p=0.0346) and peri-oral (p=0.0024) autoclaving at 130° C. for 3 minutes. 25 regions was significantly increased by the treatment with the The study included 39 individuals and comprised men and fluid composition. In particular, skin hydration of the cheek women between the ages of 30 and 45 years. All baseline data region was significantly increased by D15 of treatment (mean including consent, demographics, adverse events and skin of 55.8%) as compared to D0 (mean of 50.9%) and this evidence was collected from each individual before the study improvement in skin condition was maintain through D30 of began. After the baseline visit, each individual was subder 30 treatment (mean of 56.4%) as compared to D0 (mean of mally injected with the fluid composition described above 50.9%, p=0.0262) and D60 of treatment (mean of 59.3%) as and this was considered day 0 (DO). Follow-up visits took compared to D0 (mean of 50.9%, p=0.0021). In addition, skin place at day 15 (D15+7 days) for the second injection, at day hydration of the neck region was significantly increased by 30 (D30+7 days) for the third injection and finally at day 60 D15 of treatment (mean of 66.0%) as compared to D0 (mean (D60+7 days) for final assessment. Dermal administration 35 of 59.0%) and this improvement in skin condition was main was performed using either a depot injection method orpico tain through D30 of treatment (mean of 66.5%) of as com tage injection method. At each study visit, individuals were pared to D0 (mean of 59.0%, p=0.0022), and D60 of treat examined for adverse events and skin evidence, and a ques ment (mean of 65.3%) as compared to D0 (mean of 59.0%, tionnaire was given to assess physician and Subject satisfac p=0.0448). Furthermore, skin hydration of the peri-oral tion. The total study duration was 60 days. 40 region was significantly increased by D15 of treatment (mean The study evaluated efficacy of the compositions on hydra of 58.9%) as compared to D0 (mean of 52.4%) and this tion and elasticity of the skin after 60 days of use of the fluid improvement in skin condition was maintain through D30 of composition on different skin areas including the region treatment (mean of 61.2%) as compared to D0 (mean of around the eye, the cheek region, the peri-oral region, and the 52.4%, p=0.0041) and D60 of treatment (mean of 59.3%) as neck region. compared to D0 (mean of 52.4%, p=0.0467). TABLE 7 Assessment of Fluid Composition on Hydration of Skin Regions (Depot Method)

Skin Visit

Region DO D 15 D 30 D 60 P

Cheek N 23 2O 17 18 Mixe Mean 50.9% + 11.0% 55.8% + 10.5% 56.4% 13.9% 59.3% + 11.4% mode Median 54.3% 57.70% 55.6% 58.5% P = 0.0036 MiniMax 29.1%. 68.2%. 36.5%72.7%. 28.0%.83.1%. 41.0% 76.1% Neck N 2O 19 17 16 Mixe Mean 59.0% H 9.4% 66.0% 9.6% 66.5% + 11.9% 65.3% 9.9% mode Median 58.7% 66.5% 70.0% 61.4% P = 0.0346 MiniMax 41.4%, 74.7% 48.4%81.2% 43.5% 83.2% 47.4%82.9% Peri-oral N 2O 16 15 15 Mixe Mean 52.4% 13.5% 58.9% - 12.1%. 61.2% 14.8% 59.3% + 11.0% mode Median SO.1% 58.4% 62.6% 61.1% P = 0.0024 MiniMax 27.4%.79.6%. 37.1%81.7%. 31.0%,80.6%. 37.6%, 79.4% Eyes N 21 16 16 14 Mixe Mean 59.6% + 12.2% 62.9% + 10.7%. 55.8% 14.3% 61.4% + 10.7% mode US 9,125,840 B2 57 58 TABLE 7-continued Assessment of Fluid Composition on Hydration of Skin Regions (Depot Method Skin Visit

Region DO D 15 D 30 D 60 P

Median 56.3% 60.6% 53.7% 60.9% P = 0.28O8 MiniMax 42.6%, 81.4% 45.4%. 82.6%. 19.8%. 80.8% 42.4%. 78.3%

For each individual administered the fluid composition fluid composition using the depot injection method assessed using a picotage injection method, the cheek, neck, peri-oral skin texture (roughness) as “improved' or “very improved in and eye regions were measured for hydration (%) at each visit 72.0% of individuals administered the fluid composition at from D0 to D60 by Skin Evidence for IOMATM and a mean 15 D15, in 94.7% of individuals administered the fluid compo hydration was determined at each visit. The results indicated sition at D30, and in 80.9% of individuals administered the that there was no statistically significant difference in the fluid composition at D60. Individuals administered the fluid hydration of the cheek, neck, peri-oral, or eye regions of composition using the depot injection method assessed skin individuals administered the fluid compositions using the brightness as “improved' or “very improved in 84.0% of picotage injection method. individuals administered the fluid composition at D15, in With respect to anisotropy, the results indicate an overall 84.3% of individuals administered the fluid composition at improvement of skin condition from the neck and peri-oral D30, and in 85.7% of individuals administered the fluid com regions when the fluid composition was administered using a position at D60. Individuals administered the fluid composi depot injection method. No statistically significant difference tion using the depot injection method assessed skin hydration was observed in anisotropy when the fluid compositions were 25 as “improved' or “very improved in 84.0% of individuals administered using a picotage injection method. administered the fluid composition at D15, in 94.8% of indi With respect to skin roughness, the results indicate an viduals administered the fluid composition at D30, and in overall improvement of skin condition from the neck region. 95.0% of individuals administered the fluid composition at In particular, skin roughness of the neck region was signifi D60. Individuals administered the fluid composition using cantly decreased by D30 of treatment (median of 16.7%) as 30 the depot injection method assessed skin appearance (color) compared to D0 (median of 13.9%, p=0.0001). No statisti as “improved' or “very improved in 36.0% of individuals cally significant difference was observed in skin roughness administered the fluid composition at D15, in 42.1% of indi when the fluid compositions were administered using a pico viduals administered the fluid composition at D30, and in tage injection method. 42.9% of individuals administered the fluid composition at No statistically significant differences in skin luminance 35 D60. were detected for any of the four regions examined using Regarding questionnaires designed to evaluate individu either the depot injection method or the picotage injection als assessment of injection method, 100% of individuals method. administered the fluid composition using the depot injection Treatment was well-tolerated with all adverse events method assessed global aesthetic effect at day 60 of treatment related to injection technique rather than to the product. All 40 as “improved' or “very improved', whereas only 14.3% of adverse events were transient, with a mean duration of 4 days, individuals administered the fluid composition using the with no sequels. picotage injection method assessed global aesthetic effect at Regarding questionnaires designed to evaluate physicians day 60 of treatment as “improved' or “very improved.” Like assessment of treatment, physicians using the depot injection wise, 100% of individuals administered the fluid composition method assessed skin texture (roughness) as “improved' or 45 using the depot injection method assessed revitalization of “very improved in 88.9% of individuals administered the skin at day 60 of treatment as “improved' or “very improved', fluid composition at D15, in 100% of individuals adminis whereas only 14.3% of individuals administered the fluid tered the fluid composition at D30, and in 95.7% of individu composition using the picotage injection method assessed als administered the fluid composition at D60. Physicians revitalization of skin at day 60 of treatment as “improved' or using the depot injection method assessed skin brightness as 50 “very improved.” Similarly, 78.9% of individuals adminis “improved” or “very improved” in 74.1% of individuals tered the fluid composition using the depot injection method administered the fluid composition at D15, in 87.5% of indi assessed facefullness at day 60 of treatment as “improved' or viduals administered the fluid composition at D30, and in “very improved', whereas none of individuals administered 91.3% of individuals administered the fluid composition at the fluid composition using the picotage injection method D60. Physicians using the depot injection method assessed 55 assessed face fullness as “improved' or “very improved.” All skin hydration as “improved' or “very improved in 88.9% of told, 95% of individuals administered the fluid composition individuals administered the fluid composition at D15, in using the depot injection method were delighted with the 100% of individuals administered the fluid composition at treatment at day 60, whereas only 14.3% of individuals D30, and in 95.7% of individuals administered the fluid com administered the fluid composition using the picotage injec position at D60. Physicians using the depot injection method 60 tion method were delighted with the treatment at day 60. assessed skin appearance (color) as “improved' or “very Overall, the fluid compositions disclosed in the present improved in 48.1% of individuals administered the fluid specification have significant efficacy on skin hydration on composition at D15, in 91.7% of individuals administered the the cheek, neck, and peri-oral regions; significant efficacy on fluid composition at D30, and in 91.3% of individuals admin skin anisotropy on the neck and peri-oral regions; and signifi istered the fluid composition at D60. 65 cant efficacy on skin roughness on the neck region. In addi Regarding questionnaires designed to evaluate individu tion evaluation of questionnaires indicate physician satisfac als assessment of treatment, individuals administered the tion of aesthetic results at each visit showed that skin texture, US 9,125,840 B2 59 60 skin brightness, skin hydration and overall skin condition approximations, the numerical values set forth in the specific were “improved' or very improved for greater than 80% of examples are reported as precisely as possible. Any numerical subjects at Day 60. Similarly, subject satisfaction indicated value, however, inherently contains certain errors necessarily that skin roughness, skin brightness, skin hydration and num resulting from the standard deviation found in their respective ber of fine wrinkles were “improved' or very improved for 5 testing measurements. greater than 80% of subjects. The terms “a,” “an,” “the' and similar referents used in the In closing, it is to be understood that although aspects of the context of describing the invention (especially in the context present specification have been described with reference to of the following claims) are to be construed to cover both the the various embodiments, one skilled in the art will readily singular and the plural, unless otherwise indicated herein or appreciate that the specific examples disclosed are only illus 10 clearly contradicted by context. Recitation of ranges of values trative of the principles of the subject matter disclosed in the herein is merely intended to serve as a shorthand method of present specification. Therefore, it should be understood that referring individually to each separate value falling within the the disclosed subject matter is in no way limited to aparticular range. Unless otherwise indicated herein, each individual methodology, protocol, and/or reagent, etc., described herein. value is incorporated into the specification as if it were indi AS Such, various modifications or changes to or alternative 15 vidually recited herein. All methods described herein can be configurations of the disclosed subject matter can be made in performed in any suitable order unless otherwise indicated accordance with the teachings herein without departing from herein or otherwise clearly contradicted by context. The use the spirit of the present specification. Lastly, the terminology of any and all examples, or exemplary language (e.g., “such used herein is for the purpose of describing particular as') provided herein is intended merely to better illuminate embodiments only, and is not intended to limit the scope of the invention and does not pose a limitation on the scope of the present invention, which is defined solely by the claims. the invention otherwise claimed. No language in the specifi Accordingly, the present invention is not limited to that pre cation should be construed as indicating any non-claimed cisely as shown and described. element essential to the practice of the invention. Certain embodiments of this invention are described Specific embodiments disclosed herein may be further lim herein, including the best mode known to the inventors for 25 ited in the claims using consisting of or consisting essentially carrying out the invention. Of course, variations on these of language. When used in the claims, whether as filed or described embodiments will become apparent to those of added per amendment, the transition term "consisting of ordinary skill in the art upon reading the foregoing descrip excludes any element, step, or ingredient not specified in the tion. The inventor expects skilled artisans to employ Such claims. The transition term “consisting essentially of limits variations as appropriate, and the inventors intend for the 30 the scope of a claim to the specified materials or steps and invention to be practiced otherwise than specifically those that do not materially affect the basic and novel char described herein. Accordingly, this invention includes all acteristic(s). Embodiments of the invention so claimed are modifications and equivalents of the Subject matter recited in inherently or expressly described and enabled herein. the claims appended hereto as permitted by applicable law. All patents, patent publications, and other publications Moreover, any combination of the above-described elements 35 referenced and identified in the present specification are indi in all possible variations thereof is encompassed by the inven vidually and expressly incorporated herein by reference in tion unless otherwise indicated herein or otherwise clearly their entirety for the purpose of describing and disclosing, for contradicted by context. example, the methodologies described in Such publications Groupings of alternative elements or embodiments of the that might be used in connection with the present invention. invention disclosed herein are not to be construed as limita 40 These publications are provided solely for their disclosure tions. Each group member may be referred to and claimed prior to the filing date of the present application. Nothing in individually or in any combination with other members of the this regard should be construed as an admission that the group or other elements found herein. It is anticipated that one inventors are not entitled to antedate such disclosure by virtue or more members of a group may be included in, or deleted of prior invention or for any other reason. All statements as to from, a group for reasons of convenience and/or patentability. 45 the date or representation as to the contents of these docu When any Such inclusion or deletion occurs, the specification ments is based on the information available to the applicants is deemed to contain the group as modified thus fulfilling the and does not constitute any admission as to the correctness of written description of all Markush groups used in the the dates or contents of these documents. appended claims. Unless otherwise indicated, all numbers expressing quan 50 The invention claimed is: tities of ingredients, properties such as molecular weight, 1. A method of treating a skin condition of an individual, reaction conditions, and so forth used in the specification and the method comprising the steps of claims are to be understood as being modified in all instances administering a dermal filler into a dermal region of the by the term “about.” As used herein, the term “about” means individual, wherein the dermal filler comprises a hyalu that the item, parameter or term so qualified encompasses a 55 roman polymer composition consisting essentially of range of plus or minus ten percent above and below the value uncrosslinked hyaluronic acid and mannitol; of the stated item, parameter or term. Accordingly, unless wherein the mannitol is present in an amount of about 0.5% indicated to the contrary, the numerical parameters set forthin (w/v) to about 5% (w/v) of the composition; the specification and attached claims are approximations that wherein the uncrosslinked hyaluronic acid contains at least may vary depending upon the desired properties sought to be 60 95% by weight of a hyaluronic acid having a mean obtained by the present invention. At the very least, and not as molecular weight of at least about 2,500,000 Da; an attempt to limit the application of the doctrine of equiva wherein the skin condition treated is selected from the lents to the scope of the claims, each numerical parameter group consisting of skin dehydration, a lack of skin should at least be construed in light of the number of reported elasticity, skin roughness, a lack of skin tautness, a skin significant digits and by applying ordinary rounding tech 65 stretch line, a skin stretch mark, skin paleness, and skin niques. Notwithstanding that the numerical ranges and wrinkles; and parameters setting forth the broad scope of the invention are wherein the administration treats the skin condition. US 9,125,840 B2 61 62 2. The method of claim 1 wherein the dermal filler has a wherein the uncrosslinked hyaluronic acid contains at least dynamic viscosity of about 50 Pa's to about 150 Pas. 95% by weight of a hyaluronic acid having a mean 3. The method of claim 1 wherein the dermal filler has an molecular weight of at least about 2,500,000 Da: osmolarity of about 200 mOsm/L to about 400 mOsm/L. wherein the skin condition treated is selected from the 4. The method of claim 1 wherein the uncrosslinked hyalu 5 group consisting of skin dehydration, a lack of skin ronic acid is present in the composition at a concentration of elasticity, skin roughness, a lack of skin tautness, a skin about 7.5 mg/mL to about 19.5 mg/mL. stretch line, a stretch mark, skin paleness, and skin 5. The method of claim 1 wherein the uncrosslinked hyalu wrinkles; and ronic acid is present in the composition at a concentration of wherein the administration improves treats the skin condi about 8.5 mg/mL to about 18.5 mg/mL. 10 6. The method of claim 1 wherein the uncrosslinked hyalu t1On. ronic acid is present in the composition at a concentration of 13. The method of claim 12 wherein the uncrosslinked about 9.5 mg/mL to about 17.5 mg/mL. hyaluronic acid is present in the composition at a concentra 7. The method of claim 1 wherein the uncrosslinked hyalu tion of about 7.5 mg/mL to about 19.5 mg/mL. ronic acid is present in the composition at a concentration of 14. The method of claim 12 wherein the uncrosslinked about 10.5 mg/mL to about 16.5 mg/mL. 15 hyaluronic acid is present in the composition at a concentra 8. The method of claim 1 wherein the uncrosslinked hyalu tion of about 11.5 mg/mL to about 15.5 mg/mL. ronic acid is present in the composition at a concentration of 15. The method of claim 12 wherein the uncrosslinked about 11.5 mg/mL to about 15.5 mg/mL. hyaluronic acid is present in the composition at a concentra 9. The method of claim 1 wherein the uncrosslinked hyalu tion of about 14 mg/mL. ronic acid is present in the composition at a concentration of 16. The method of claim 12 wherein the uncrosslinked about 14 mg/mL. hyaluronic acid is present in the composition at a concentra 10. The method of claim 1 wherein the uncrosslinked tion of about 13.5 mg/mL. hyaluronic acid is present in the composition at a concentra 17. The method of claim 12 wherein the mannitol is present at a concentration of about 1% (w/v) of the composition. tion of about 13.5 mg/mL. 25 11. The method of claim 1 wherein the mannitol is present 18. The method of claim 12 wherein the mannitol is present at a concentration of about 0.9% (w/v) of the composition. at a concentration of about 0.9% (w/v) of the composition. 12. A method of treating-askin condition of an individual, 19. The method of claim 12 wherein the dermal region is an the method comprising the steps of: epidermal-dermal junction region. administering a composition into a dermal region of the 20. The method of claim 12 wherein the dermal region is a individual by injection, wherein the composition com 30 papillary region. prises a hyaluronan polymer composition consisting 21. The method of claim 12 wherein the dermal region is a essentially of uncrosslinked hyaluronic acid and manni reticular region. tol; 22. The method of claim 1 wherein the mannitol is present wherein the mannitol is present in an amount of about 0.5% at a concentration of about 1% (w/v) of the composition. (w/v) to about 5% (w/v) of the composition; ck ck ck ck *k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 9,125,840 B2 Page 1 of 2 APPLICATION NO. : 14/078057 DATED : September 8, 2015 INVENTOR(S) : Pierre F. Lebreton It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the title page item 56 On the Page 4, in column 1, under “Other Publications, line 5, delete “Mannitrol, and insert -- Mannitol, --, therefor. On the Page 4, in column 1, under “Other Publications, line 48, delete “Slerotomy and insert -- Sclerotomy --, therefor. On the Page 4, in column 2, under “Other Publications, line 64, delete “Tetracrylates, and insert -- Tetraacrylates, --, therefor. On the Page 4, in column 2, under “Other Publications, line 70, delete “Viscoelstic and insert - Viscoelastic --, therefor. In the specification In column 4, line 11, delete “tem and insert -- term --, therefor. In column 6, line 66, delete “(BCD), and insert -- (BCDI), --, therefor. In column 7, line 1, delete “(NMDA), and insert -- (HMDA), --, therefor. In column 7, line 9, delete “that that and insert -- that --, therefor. In columns 17-18, line 10 (Table 2), delete “Superoxide and insert -- superoxide --, therefor. In columns 17-18, line 25 (Table 2), delete “alcoxyl and insert -- alkoxyl --, therefor. In columns 17-18, line 26 (Table 2), delete “Alcoxyl and insert -- Alkoxyl --, therefor. In columns 17-18, line 26 (Table 2), delete “Alcoxyl and insert -- Alkoxyl --, therefor. In column 23, line 2, delete "chrysanthenin, and insert -- chrysanthemin, --, therefor. In column 23, line 3, delete “cyanidin' and insert -- cyanidin --, therefor. In column 23, line 4, delete “cyanosalvian in, and insert -- cyanosalvianin, --, therefor. In column 23, line 4, delete “delphinid in, and insert -- delphinidin, --, therefor. In column 23, line 6, delete “5-desoxy and insert -- 5-deoxy--, therefor. Signed and Sealed this Twentieth Day of September, 2016 74-4-04- 2% 4 Michelle K. Lee Director of the United States Patent and Trademark Office CERTIFICATE OF CORRECTION (continued) Page 2 of 2 U.S. Pat. No. 9,125,840 B2 In the specification In column 23, line 7, delete “5-desoxy and insert -- 5-deoxy--, therefor. In columns 23-24, line 44 (Table 3), delete “4-phenylcoumarine' and insert -- 4-phenylcoumarin --, therefor. In columns 25-26, line 13 (Table 3-continued), delete "chrysanthenin, and insert -- chrysanthemin, --, therefor. In columns 25-26, line 21 (Table 3-continued), delete “5-desoxy and insert -- 5-deoxy--, therefor. In columns 25-26, line 23 (Table 3-continued), delete “5-desoxy and insert -- 5-deoxy--, therefor. In column 30, line 53, delete "amylocalne, and insert -- amylocaine, --, therefor. In column 30, line 57, delete “dicyclonine, and insert -- dicyclomine, --, therefor. In column 30, line 59, delete “formocaine, and insert -- fomocaine, --, therefor. In column 30, line 63, delete "octacaine, and insert -- octocaine, --, therefor. In column 31, line 58, delete “0.1 and insert -- 0.1% --, therefor. In column 35, line 54, delete “r, and insert -- m --, therefor. In the claims In column 61, line 28, in claim 12, delete “treating-a and insert -- treating a --, therefor. In column 62, line 9, in claim 12, delete “improves treats and insert -- treats --, therefor.