WO2019125166A2

WO2019125166A2 - Hyaluronic acid formulations

Info

Publication number: WO2019125166A2
Application number: PCT/NL2018/050871
Authority: WO
Inventors: John Lokhnauth; Alfred Liang; Venkataramana DINGARI; Crilles LARSEN
Original assignee: Ferring B.V.
Priority date: 2017-12-22
Filing date: 2018-12-21
Publication date: 2019-06-27
Also published as: WO2019125166A3; TW201929872A

Abstract

The present disclosure describes stabilized compositions of hyaluronic acid or pharmaceutically acceptable salt thereof, and methods related to using or producing the compositions.

Description

HYALURONIC ACID FORMULATIONS

1. BACKGROUND

[0001] Hyaluronic acid, or hyaiuronan, is an anionic, non-sulfated giycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues in all vertebrates including humans. Hyaluronic acid serves as a significant constituent in parts of the body, e.g., synovial fluid, articular cartilage, and the skin. For example, hyaluronic acid is a component of synovial fluid, and can increase the viscosity of the fluid. Hence, hyaluronic acid is believed to be an important lubricating component.

[0002] Certain diseases or disorders of the eye, skin, and articular cartilage are believed to be caused by a decreased level of hyaluronic acid. As a result, preparations of hyaluronic acid may be used to enhance the level of hyaluronic acid in the body, thereby treating such a disease or disorder.

[0003] Hyaluronic acid, depending on its source and preparation, may have a half-life of from less than a day to several days in the human body. Methods such as cross-linking the hyaluronic acid are known to be able to stabilize hyaluronic acid formulations in vivo. However, cross-linked hyaluronic acid may suffer from disadvantages such as difficult or complex synthesis, or inferior viscoelastic properties as compared to non-cross-linked hyaluronic acid. Accordingly, there is a need for novel compositions of hyaluronic acid with improved stability and methods of use and production related to the formulations.

2. SUMMARY

[0004] In an aspect, the present application discloses an aqueous composition of hyaluronic acid or pharmaceutically acceptable salt thereof. In some embodiments, aqueous compositions disclosed herein are capable of achieving adequate stability for in vivo pharmaceutical use without the use of cross-linked hyaluronic acid.

[0005] Provided herein is an aqueous composition comprising: hyaluronic acid or a pharmaceutically acceptable salt thereof; an antioxidant; and a saccharide. In certain instances, the aqueous compositions comprise hyaluronic acid at from about 0.1 to about 100 mg/mL, e.g., at 10 mg/mL.

[0006] Suitable antioxidants include ascorbic acid, citric acid, metabisulfite salts (e.g., sodium metabisulfite), tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, monosaccharides (e.g., mannose), sugar alcohols (e.g., mannitol), amino acids (e.g., histidine, methionine, cysteine), peptides (e.g., those comprising 2-5 amino acids such as glutathione), and pharmaceutically acceptable salts thereof. For example, an antioxidant can be histidine or methionine, or pharmaceutically acceptable salts thereof.

[0007] Appropriate saccharides include alginic acid, carrageenan, carboxymethyl cellulose, chitosan, gelatin, arabic gum, guar gum, mannitol, sorbitol, sucrose, xanthan gum, polyethylene glycol, and pharmaceutically acceptable salts thereof. For example, a saccharide can be sodium alginate.

[0008] An example of an aqueous composition of the disclosure comprises: about 10 mg/mL hyaluronic acid; about 20 mg/mL sodium alginate; about 1 mg/mL histidine; a phosphate buffer; and a tonicity agent. The aqueous solution can have a pH of from about 6.5 to about 7.5.

[0009] Another example of an aqueous composition of the disclosure comprises: about 10 mg/mL hyaluronic acid; about 20 mg/mL sodium alginate; about 2 mg/mL methionine; a phosphate buffer; and a tonicity agent. The aqueous solution can have a pH of from about 6.5 to about 7.5.

[0010] Also provided herein is a method of treating a disease or condition exhibiting a reduced level of hyaluronic acid, such as osteoarthritis. Such methods may comprise administering the aqueous composition of the disclosure by intra-articular injection into the patient.

3. BRIEF DESCRIPTION OF THE FIGURES

[0011] FIG. 1 shows viscosity {%) (relative to control)(y-axis) over time (sec)(x-axis) of 2 mg/mL hyaluronic acid test compositions in the presence of different antioxidants - ascorbic acid, histidine, sodium metabisulfite ("NaMBS"), mannose, mannitol, butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT"), butylated hydroxyanisole and butylated hydroxytoluene ("BHA + BHT"), tocopherol, propyl gallate ("P Gallate"), or sorbitol - in the presence of 0.016% hydrogen peroxide. Control composition is 2 mg/mL hyaluronic acid at pH 6.5-7.5 without antioxidant or hydrogen peroxide. Legend: solid circle = ascorbic acid; shaded triangle = histidine; shaded diamond = sodium metabisulfite; short dash = mannose; hollow circle = mannitol; hollow triangle = BHA; hollow circle = BHT; solid square = BHA + BHT; shaded circle = tocopherol; hollow square = propyl gallate; long dash = sorbitol.

[0012] FIG. 2 shows viscosity (in cPaj(y-axis) at a shear rate of 25 sec^-1 and pH 6.5-7.5 over time (sec)(x- axis) of 2 mg/mL hyaluronic acid test compositions in the presence of different antioxidants - histidine, BHA, BHT, tocopherol, or propyl gallate - in the presence of 0.001 mg/mL hyaluronidase. Control composition is 2 mg/mL hyaluronic acid at pH 6.5-7.5 without antioxidant or hyaluronidase; control (stressed) shows the behavior of the control solution in the presence of 0.001 mg/mL hyaluronidase. Legend: shaded circle = control; solid diamond = control (stressed); hollow square = histidine; dash =

BHA; hollow triangle = BHT; plus = tocopherol; solid circle = propyl gallate. [0013] FIGS. 3A-3C show the viscosity {% relative to control)(y-axis) at a shear rate of 25 sec ¹ over time (sec)(x-axis) of 2 mg/mL hyaluronic acid test compositions in the presence of 0.016% hydrogen peroxide and a low, medium, or high concentration of different antioxidants: FIG. 3A shows the viscosity over time of hyaluronic acid test compositions comprising antioxidant histidine at a low (0.1%, hollow circle), medium ("mid”, 0.2%, shaded triangle), or high (0.4%, hollow diamond) concentration as compared with unstressed control (solid square); FIG. 3B shows the viscosity over time of hyaluronic acid test compositions comprising antioxidant tocopherol at a low (0.04%, hollow circle), medium ("mid", 0.07%, shaded triangle), or high (0.1%, hollow diamond) concentration as compared with unstressed control (solid square); FIG. 3C shows the viscosity over time of hyaluronic acid test compositions comprising propyl gallate at a low (0.02%, hollow circle), medium ("mid", 0.1%, shaded triangle), or high (0.2%, hollow diamond) concentration as compared with unstressed control (solid square).

[0014] FIG. 4 shows the viscosity (%) (relative to control)(y-axis) over time (sec)(x-axis) of 2 mg/mL hyaluronic acid test compositions in the presence of 0.016% hydrogen peroxide and 2 mg/mL antioxidant cysteine (solid square), glutathione (hollow circle), or methionine (shaded triangle). Control composition is 2 mg/mL aqueous hyaluronic acid without hydrogen peroxide or antioxidant.

[0015] FIG. 5 shows the viscosity (%) (relative to control)(y-axis) over time (sec)(x-axis) of 2 mg/mL hyaluronic acid test compositions in the presence of 0.016% hydrogen peroxide and varying

concentrations of antioxidant methionine: 1 mg/mL (solid square), 2 mg/mL (hollow circle), or 4 mg/mL (shaded triangle). Control composition is 2 mg/mL aqueous hyaluronic acid without hydrogen peroxide or antioxidant.

[0016] FIG. 6 shows the viscosity ("normalized", i.e., % relative to control)(y-axis) over time (sec)(x-axis) of 2 mg/mL hyaluronic acid test compositions in the presence of 0.001 mg/mL hyaluronidase and different saccharides - polyethylene glycol (PEG) (solid line, solid circle), sodium alginate ("Alginate") (dotted line, dotted triangle), arabic gum ("Arabic") (solid line, dashed square), Carrageenan 109 (dashed line, solid triangle), Carrageenan 209 (solid line, solid square), Carrageenan IMF (solid line, shaded diamond), sodium carboxymethyl cellulose ("CMC") (dashed line, dashed circle), Gelatin (solid line, asterisk), Guar Gum (solid line, solid diamond), Mannitol (dotted line, solid circle), Sucrose (solid line, shaded circle), xanthan gum 180 ("Xanthan 180") (dashed line, shaded circle), or xanthan gum 75 ("Xanthan 75") (solid line, shaded circle).

[0017] FIGS. 7A-7D show the viscosity (%) (relative to control)(y-axis) over time (sec)(x-axis) of 2 mg/mL hyaluronic acid compositions in the presence of 0.001 mg/mL hyaluronidase and different

concentrations of saccharides : FIG. 7A shows viscosity over time with compositions comprising 0.5 mg/mL (shaded triangle), 1 mg/mL (hollow circle), or 5 mg/mL (solid square) carrageenan NF; FIG. 7B shows viscosity over time with formulations comprising 1 mg/mL (shaded triangle), 5 mg/mL (hollow circle), or 10 mg/mL (solid square) sodium alginate; FIG. 7C shows viscosity over time with compositions comprising 1 mg/mL (shaded triangle), 5 mg/mL (hollow circle), or 10 mg/mL (solid square) sodium carboxymethyl cellulose; FIG. 7D shows viscosity over time with compositions comprising 0.5 mg/mL (shaded triangle), 1 mg/mL (hollow circle), or 5 mg/mL (solid square) xanthan gum 75.

[0018] FIG. 8 shows the viscosity (mPa-sec)(y-axis) vs. shear rate (sec^_1)(x-axis) of 2 mg/mL hyaluronic acid test compositions comprising different sodium alginate concentrations: 0 ("control", solid line, solid circle), 1 mg/mL (dotted line, dotted diamond), 5 mg/mL (dotted line, hollow square), 10 mg/mL (solid line, solid triangle), or 20 mg/mL (solid line, solid square).

[0019] FIG. 9 shows the viscosity (%) (relative to control)(y-axis) over time (sec)(x-axis) of 2 mg/mL hyaluronic acid test compositions in the presence of 0.001 mg/mL hyaluronidase and sodium alginate having different physicochemical profiles. Sodium alginate preparations tested: "LVG" = Pronova™ UP LVG (solid square); "VLVG" = Pronova™ UP VLVG (hollow circle); "LVM" = Pronova™ UP LVM (shaded triangle); "VLVM" = Pronova™ UP VLVM (hollow diamond); control without sodium alginate (dash).

[0020] FIG. 10A shows the viscosity (%) (relative to control)(y-axis) over time (sec)(x-axis) of exemplary hyaluronic acid compositions comprising 5 mg/mL (solid square), 10 mg/mL (hollow circle), or 20 mg/mL (shaded triangle) Pronova™ UP VLVM sodium alginate.

[0021] FIG. 10B is an exploded view of FIG. 10A that further illustrates the results from the tested hyaluronic acid compositions.

[0022] FIG. 11 shows the viscosity (%) (relative to control)(y-axis) over time (sec)(x-axis) of exemplary hyaluronic acid composition Prototype A when subjected to 0.016% hydrogen peroxide or 0.001 mg/mL hyaluronidase. The composition was tested twice in the presence of hydrogen peroxide ("Oxidative 1", shaded triangle; Oxidative 2", hollow diamond) and twice in the presence of hyaluronidase ("Enzymatic 1", solid square; "Enzymatic 2", hollow circle).

[0023] FIG. 12 shows the viscosity (%) (relative to control)(y-axis) over time (sec)(x-axis) of exemplary hyaluronic acid composition Prototype B when subjected to 0.016% hydrogen peroxide or 0.001 mg/mL hyaluronidase. The composition was tested twice in the presence of hydrogen peroxide ("Oxidative 1", shaded triangle; "Oxidative 2", hollow diamond) and twice in the presence of hyaluronidase ("Enzymatic 1", solid square; "Enzymatic 2", hollow circle).

[0024] FIG. 13 shows the viscosity (%) (relative to control)(y-axis) over time (sec)(x-axis) of a comparative hyaluronic acid composition not comprising antioxidant or saccharide when subjected to 0.016% hydrogen peroxide or 0.001 mg/mL hyaluronidase. The composition was tested twice in the presence of hydrogen peroxide ("Oxidative 1", shaded triangle; "Oxidative 2", hollow diamond) and twice in the presence of hyaluronidase ("Enzymatic 1", solid square; "Enzymatic 2" , hollow circle).

[0025] FIG. 14A shows the results of the long term viscosity at 0.1 s ¹ (mPa-sec)(y-axis) over time (months)(x-axis) of composition Prototype A at 5 sc (14.1), 25 ⁹C/60% RH (14.2), and 40 sc/75% RH (14.3).

[0026] FIG. 14B shows the results of long term viscosity at 1000 s ¹ (mPa-sec)(y-axis) over time

(months)(x-axis) of composition Prototype A at 5 ^ec (14.1), 25 ⁹C/60% RH (14.2), and 40 ⁹C/75% RH (14.3).

[0027] FIG. 14C shows the results of long term viscosity at 0.1 s ¹ (mPa-sec)(y-axis) over time

(months)(x-axis) of composition Prototype B at 5 ⁹C (14.4), 25 ⁹C/60% RH (14.5), and 40 ⁹C/75% RH (14.6).

[0028] FIG. 14D shows the results of long term viscosity at 1000 s ¹ (mPa-sec)(y-axis) over time

[0029] FIG. 15A shows the results of long term pH (y-axis) over time (months)(x-axis) of Prototype A at 5 ⁹C (15.1), 25 ⁹C/60% RH (15.2), and 40 ⁹C/75% RH (15.3).

[0030] FIG. 15B shows the results of long term pH (y-axis) over time (months)(x-axis) of Prototype B at 5 ⁹C (15.4), 25 ⁹C/60% RH (15.5), and 40 ⁹C/75% RH (15.6).

[0031] FIG. 16A shows the results of the incapacitance test measured using mean ±SE weight bearing difference (left-right)(y-axis) over time (study days)(x-axis) of composition Prototype B (SI), 1% HA in PBS (S2), 1.5% HA in PBS (S3), Tramadol (S4), control (S5), and sham surgery (S6).

[0032] FIG. 16B shows the incapacitance AUC calculation measured using mean ±SE weight bearing difference (g)*Days (y-axis) for composition Prototype B (SI), 1% HA in PBS (S2), 1.5% HA in PBS (S3), Tramadol (S4), control (S5), and sham surgery (S6) treatment group (x-axis).

[0033] FIG. 17A is a photomicrograph at 16x magnification of a right rat knee joint treated with PBS (0.5 mg/rat) after 30 days residence time showing lateral femoral condyle (L), medial femoral condyle (M), normal lateral synovium (arrow), and normal cruciate insertion (arrowhead).

[0034] FIG. 17B is a photomicrograph at 16x magnification of a right rat knee joint treated with Prototype B (0.5 mg/rat) after 30 days residence time showing lateral femoral condyle (L), medial femoral condyle (M), Prototype B at edge of synovium (arrows), and Prototype B in bone recess at cruciate insertion (arrowhead). 4. DETAILED DESCRIPTION

4.1. Definitions

[0035] As used herein, the following terms are intended to have the following meanings:

[0036] "About" refers to an approximate value. For example, "about" can include a value ± 10% of the recited value.

[0037] "Amino acid" may include any of the alpha amino acids, such as alanine, arginine, asparagine, aspartic acid, cysteine, methionine, homocysteine, homoserine, glutamic acid, glutamine, glycine, phenylglycine, histidine, isoleucine, leucine, lysine, ornithine, methionine, norleucine, norvaline, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, and pharmaceutically acceptable salts thereof. In other embodiments, "amino acid" also includes or is limited to non-naturally occurring amino acids, such as non-naturally occurring alpha amino acids; beta amino acids, e.g., b- alanine, b-phenylalanine, 3-aminobutanoic acid, 3-aminoisobutyric acid, 3-amino-3-methylpropionic acid; and gamma amino acids. Examples of non-naturally occurring amino acids contemplated herein include modified naturally occurring amino acids, such as homologs of naturally occurring amino acids (e.g., homocysteine, which includes an additional methylene group in the sidechain), N-substituted variants of naturally occurring amino acids (e.g., N-methyl variants), ring-substituted naturally occurring amino acids (e.g., ring-substituted phenylalanine and tyrosine derivatives), and dimeric amino acids (e.g., cysteine and lanthionine). Examples of non-natural amino acids include halogenated amino acids such as 4-fluorophenylalanine, 3,5-difluorophenylalanine; cyclic amino acids such 2- aminocycloheptanecarboxylic acid, 3-aminobicyclo[2.2.1]heptane-2-carboxylic acid, and 3- morpholineacetic acid; and heterocyclic amino acids such as b-2-thienylalanine and b-(4-ίM3zoIgI)- alanine. The amino acid generally has a L stereochemistry, though racemic (DL) or D amino acid stereochemistry can also be used.

[0038] "Antioxidant" refers to a molecule that inhibits and/or prevents oxidation of another molecule, e.g., by being oxidized itself. Antioxidants may be water-soluble, e.g., ascorbic acid, glutathione, lipoic acid, uric acid, or lipid-soluble, e.g., carotenes, tocopherol, ubiquinol. Suitable antioxidants include ascorbic acid, histidine, metabisulfite, mannose, mannitol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tocopherol, propyl gallate, sorbitol, and pharmaceutically acceptable salts thereof, and combinations of any of the foregoing. [0039] "Patient" refers to a mammal such as a human, a chimpanzee, a cynomolgus monkey, a horse, a dog, a cat, and a rabbit.

[0040] "Peptide" refers to any one of a chain of from 2 to 10 amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, that can be attached either in a linear or branched fashion, and pharmaceutically acceptable salts thereof.

[0041] "Pharmaceutically acceptable salt" refers to the relatively non-toxic, inorganic and organic acid addition salts. These salts can be prepared in situ during the final isolation and purification of the compound, or by separately admixing, e.g., reacting, a purified compound in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts of acidic compounds include alkali salts such as sodium and potassium salts; alkaline earth salts such as magnesium or calcium salts; and quaternary ammonium salts such as tetramethylammonium, carnitine, choline, tris(hydroxymethyl)aminomethane. Representative salts of alkaline compounds include the bromide, chloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulfonate salts, and amino acid salts, and the like. See, for example, Berge et al. 1977, "Pharmaceutical Salts," J. Pharm. Sci. 66: 1-19.

[0042] "Saccharide" refers to sugar alcohols such as mannitol, sorbitol, and xylitol; glycols such as ethylene glycol and propylene glycol; disaccharides such as sucrose; and polysaccharides such as guar gum, arabic gum, xanthan gum (e.g., xanthan gum 75, xanthan gum 180), chitosan, carrageenan (e.g., carrageenan 109, carrageenan 209, carrageenan NF), carboxymethylcellulose, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), gelatin, and polyethylene glycol (PEG), and pharmaceutically acceptable salts thereof.

[0043] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.

4.2. Aqueous Compositions

[0044] The present disclosure generally describes compositions of hyaluronic acid or a pharmaceutically acceptable salt thereof. Such compositions typically exhibit improved stability and are useful for pharmaceutical purposes, e.g., for injection into a patient, e.g., a human patient, to treat a disease or condition. [0045] In an aspect, described herein is an aqueous composition comprising hyaluronic acid or a pharmaceutically acceptable salt thereof; an antioxidant; and a saccharide. In some embodiments, the composition is a solution, e.g., a solution that is freely flowing. In some embodiments, the composition is a solution with all or substantially all (e.g., greater than about 90%, 93%, 95%, 97%, or 99% by weight) of the solid components dissolved.

[0046] In some embodiments, the aqueous compositions described herein are neutral, or

approximately neutral, in pH to allow for pharmaceutical use in a patient, e.g., a human patient. In some embodiments, the aqueous composition has a pH of from about 6.5 to about 7.5, e.g., from about 6.8 to about 7.2, from about 6.9 to about 7.3, from about 6.7 to about 7.4, from about 6.5, 6.6, 6.7, 6.8, or 6.9 to about 7.1, 7.2, 7.3, 7.4, or 7.5, such as about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.

4.3. Hyaluronic acid or pharmaceutically acceptable salt

[0047] A hyaluronic acid or pharmaceutically acceptable salt thereof used in the present disclosure is generally highly purified. In some embodiments, hyaluronic acid or pharmaceutically acceptable salt thereof has a molecular weight of from about 8,000 to about 6,000,000, e.g., from about 8,000; 10,000; 100,000; 250,000; 500,000; 1,000,000; 1,500,000; 2,000,000; or 2,400,000 to about 3,000,000;

3,600,000; 4,000,000; 4,500,000; 5,000,000; 5,500,000; or 6,000,000 Daltons. In certain embodiments, hyaluronic acid or pharmaceutically acceptable salt thereof has a molecular weight of from about 2,400,000 to about 3,600,000 Daltons. Hyaluronic acid or pharmaceutically acceptable salt thereof can be either cross-linked or non-cross-linked, but, in certain embodiments, is non-cross-linked. When cross- linked, the hyaluronic acid may have a degree of crosslinking ("CrD" - defined, e.g., in Wende et al., Carbohydrate Polymers, 10 (157), 1525-1530 (2017)) from about 0.5% to about 6%, such as from about 0.5% to about 1.5%, from about 1% to about 2%, from about 1% to about 3%, from about 2% to about 3%, from about 4% to about 6%, from about 0.5, 1, 1.5, 2, 2.5, 3, or 3.5% to about 4, 4.5, 5, 5.5, or 6%. Degree of cross-linking may be determined by any number of methods known in the art, e.g., nuclear magnetic resonance (NMR) or size exclusion chromatography with multi-angle light scattering (SEC- MALS).

[0048] In some embodiments, the pharmaceutically acceptable salt is selected from sodium, potassium, magnesium, calcium, tetramethylammonium, carnitine, choline, and tris(hydroxymethyl)aminomethane salts. In certain embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0049] Hyaluronic acid is widely available in vertebrates and in some bacteria. Accordingly, the hyaluronic acid or a pharmaceutically acceptable salt thereof used in the present disclosure can be sourced from any one of a number of animal sources. In some embodiments, hyaluronic acid or pharmaceutically acceptable salt thereof is derived from chicken, e.g., produced from chicken combs. In some embodiments, hyaluronic acid or pharmaceutically acceptable salt thereof is derived from bacteria, e.g., by streptococcal fermentation.

[0050] Generally, the concentration of the hyaluronic acid or pharmaceutically acceptable salt thereof allows for a freely flowing aqueous composition that can be injected, e.g., via syringe, for pharmaceutical use. In some embodiments, the hyaluronic acid or pharmaceutically acceptable salt thereof is present in a range of from about 0.01% to about 10%, e.g., from about 0.5% to about 5%, from about 0.8% to about 1.2%, from about 0.5% to about 2%, from about 0.1% to about 3%, from about 0.2% to about 2%, from about 0.5% to about 1.5%, by weight, such as about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by weight. In some embodiments, the hyaluronic acid or pharmaceutically acceptable salt thereof is present in a range of from about 0.1 to about 100 mg/mL, e.g., from about 5 to about 50, from about 8 to about 12, from about 2 to about 20, from about 5 to about 20, from about 1 to about 30, from about 5 to about 15 mg/mL, such as about 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg/mL.

[0051] In some embodiments, the aqueous composition of hyaluronic acid or pharmaceutically acceptable salt thereof has a viscosity allowing for administration by injection. In some embodiments, the composition has a viscosity in a range of from about 10 to about 400 cPa, e.g., from about 10, 20, 30, 50, 70, 90, or 100 to about 150, 200, 250, 300, 350, or 400 cPa, such as about 10, 20, 30, 50, 70, 90, 100, 150, 200, 250, 300, 350, or 400 cPa (optionally +/- 5, 10, 20, or 25%), at a shear rate of about 1000 sec ¹. In some embodiments, the composition has a viscosity in a range of from about 8,000 to about 18,000 cPa, e.g., from about 8,000; 8,500; 9,000; 9,500; 10,000; 10,500; 11,000, 11,500; or 12,000 to about 14,000; 14,500; 15,000; 15,500; 16,000; 16,500; 17,000; 17,500; or 18,000 cPa, such as about 9,000; 9,500; 10,000; 10,500; 11,000; 11,500; 12,000; 13,000; 13,500; 14,000; 14,500; 15,000; 15,500; 16,000; or 16,500 cPa (optionally +/- 5, 10, 20, or 25%), at a shear rate of about 0.1 sec ¹.

[0052] To maintain a viscosity that is acceptable for injectable administration, in some embodiments, the aqueous composition of hyaluronic acid or pharmaceutically acceptable salt thereof in the presence of antioxidant and saccharide has a viscosity similar to that of a comparable hyaluronic acid composition without antioxidant and saccharide. Hence, such an aqueous composition of the disclosure does not have a significantly increased viscosity compared to a reference hyaluronic acid composition without the antioxidant and saccharide but otherwise comprising the same ingredients. [0053] Accordingly, in some embodiments, an aqueous composition comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, an antioxidant, and a saccharide has a viscosity within about 50%, e.g., within about 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, of the viscosity of a reference composition of hyaluronic acid or pharmaceutically acceptable salt thereof without the antioxidant and the saccharide. In some embodiments, an aqueous composition comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, an antioxidant, and a saccharide has a viscosity of from about 0.5 to about 1.5 times, e.g., from about 0.5, 0.6, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95 to about 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.4, or 1.5 times, such as about 0.5, 0.6, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15,

1.2, 1.25, 1.3, 1.4, or 1.5 times, the viscosity of a reference composition of hyaluronic acid or pharmaceutically acceptable salt thereof without the antioxidant and the saccharide. In some embodiments, an aqueous composition comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, an antioxidant, and a saccharide has a viscosity of no more than about 50%, e.g., no more than about 50%, 40%, 30%, 25%, 20%, 15%, 10%, or 5%, higher than the viscosity of a reference composition of hyaluronic acid or pharmaceutically acceptable salt thereof without the antioxidant and the saccharide. In some embodiments, an aqueous composition comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, an antioxidant, and a saccharide has a viscosity of no more than about 1.5 times, e.g., no more than about 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.4, or 1.5 times, the viscosity of a reference composition of hyaluronic acid or pharmaceutically acceptable salt thereof without the antioxidant and the saccharide.

[0054] In some embodiments, an aqueous composition comprising hyaluronic acid or a

pharmaceutically acceptable salt thereof, an antioxidant, and a saccharide, when subjected to a shear force, such as experienced when injected with a syringe, exhibits a change in viscosity (e.g., a change of at least about 5%, 10%, 25%, 50%, 75%, 100%, or 200%). For instance, in some embodiments, the aqueous composition, when subjected to a shear force, exhibits a decrease in viscosity (e.g., a decrease of at least about 5%, 10%, 25%, 50%, 75%, 100%, or 200%). In other embodiments, the aqueous composition, when subjected to a shear force, exhibits an increase in viscosity (e.g., an increase of at least about 5%, 10%, 25%, or 50%) In certain embodiments, the change (e.g., decrease or increase) in viscosity is unrelated to the presence of the saccharide (e.g., sodium alginate). For example, the presence of the saccharide does not impact the magnitude of the change (e.g., decrease or increase) in viscosity, i.e., the change (e.g., decrease or increase) in viscosity is independent of the saccharide. In yet other embodiments, the aqueous composition, when subjected to a shear force, exhibits little or no change in viscosity (e.g., a change - e.g., increase or decrease - of about 0.5%, 1%, 2%, 5%, 7%, or 10% or less).

[0055] In certain embodiments, giycosaminoglycan-based therapeutic agents generally can be used in the compositions described herein, i.e., the present compositions are not limited to hyaluronic acid and pharmaceutically acceptable salts thereof. Other glycosaminoglycans, besides hyaluronic acid, that are contemplated for use herein include, for instance, heparin, chondroitin, and keratin sulfates.

4.4. Antioxidant

[0056] An antioxidant can improve the stability of an aqueous solution of hyaluronic acid or pharmaceutically acceptable salt thereof. In some embodiments, the antioxidant is selected from ascorbic acid, citric acid, metabisulfite (e.g., sodium metabisulfite), tocopherol (including salts, e.g., tocopherol acetate; derivatized versions, e.g., tocopherol propylene glycol, and salts thereof, e.g., tocopherol propylene glycol succinate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, mannose, mannitol, an amino acid, a peptide, and pharmaceutically acceptable salts thereof. In some embodiments, the antioxidant is selected from propyl gallate, histidine, methionine, tocopherol, and pharmaceutically acceptable salts thereof. In certain embodiments, the antioxidant is an amino acid, e.g., histidine, or a thiol-containing amino acid, such as cysteine or methionine, or a pharmaceutically acceptable salt of any of these. In certain embodiments, the antioxidant is a peptide, e.g., a peptide that consists of from 2 to 5 amino acids, e.g., 2, 3, 4, or 5 amino acids. In certain embodiments the peptide contains one or more amino acid residues bearing a thiol group, such as a peptide including one or more methionine or cysteine residues, such as glutathione. In certain embodiments, the antioxidant is a hydroxylated benzene compound, such as BHA, BHT, tocopherol, or propyl gallate. In certain embodiments, the antioxidant is histidine or a pharmaceutically acceptable salt thereof. In certain embodiments, the antioxidant is methionine or a pharmaceutically acceptable salt thereof. In certain embodiments, the antioxidant is tocopherol. In some embodiments the antioxidant comprises a combination of any of the above mentioned antioxidants, e.g., a combination of BHA and BHT.

[0057] Generally, an antioxidant is present in an amount in the aqueous formulation sufficient to improve the stability of the hyaluronic acid or pharmaceutical salt thereof while allowing for a freely flowing aqueous solution that can be injected, e.g., via syringe, for pharmaceutical use. In some embodiments, the antioxidant is present in a range of from about 0.01% to about 10% by weight, e.g., from about 0.01% to about 1%, from about 0.01% to about 5%, from about 0.02% to about 2%, from about 0.1% to about 2%, or from about 0.1% to about 0.5% by weight, such as from about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.8%, 1%, or 1.2% to about 1.5%, 1.8% , 2%, 3%, 4%, 5%, 6%, 7%, or 8% by weight, including about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8% , 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 10% by weight. In certain embodiments, the antioxidant is propyl gallate, which is present in a range of from about 0.01% to about 0.1% by weight.

[0058] In some embodiments, the antioxidant is present in a range of from about 0.1 to about 100 mg/mL, e.g., from about 0.1 to about 5, from about 0.1 to about 10, from about 0.1 to about 50, from about 0.2 to about 20, from about 1 to about 20, or from about 1 to about 5 mg/mL, such as from about 0.1, 0.2, 0.5, 1, or 2 mg/mL to about 2.5, 5, 8, 10, 12, 15, 18, 20, 30, 40, 50, 60, 70, 80, or 100 mg/mL, including about 0.1, 0.2, 0.5, 1, 2, 5, 8, 10, 12, 15, 18, 20, 30, 40, 50, 60, 70, 80, or 100 mg/mL.

[0059] In certain embodiments, the antioxidant is histidine or a pharmaceutically acceptable salt thereof, which is present in a range of from about 0.01% to about 5%, e.g., from about 0.01%, 0.02%, 0.05%, 0.07%, 0.1%, 0.2%, 0.4%, or 0.5% to about 0.8%, 1%, 1.5%, 2%, 3%, or 5%, such as from about 0.1% to about 0.5%, by weight. In certain embodiments, when the antioxidant is histidine or a pharmaceutically acceptable salt thereof, the antioxidant is present in a range of from about 0.1 to about 50 mg/mL, e.g., from about 0.1, 0.2, 0.5, or 1 mg/mL to about 5, 10, 20, or 30 mg/mL. Further as an example, in some embodiments, histidine is present in an amount of about 0.5 mg/mL, 1 mg/mL, 2 mg/mL, or 4 mg/mL.

[0060] In certain embodiments, the antioxidant is methionine or a pharmaceutically acceptable salt thereof, which is present in a range of from about 0.01% to about 5%, e.g., from about 0.01%, 0.02%, 0.05%, 0.07%, 0.1%, 0.2%, or 0.5% to about 0.8%, 1%, 1.5%, 2%, 3%, or 5%, such as from about 0.1% to about 0.5%, by weight. In certain embodiments, when the antioxidant is methionine or a

pharmaceutically acceptable salt thereof, the antioxidant is present in a range of from about 0.1 to about 50 mg/mL, e.g., from about 0.1, 0.2, 0.5, or 1 mg/mL to about 5, 10, 20, or 30 mg/mL. Further as an example, in some embodiments, methionine is present in an amount of about 1 mg/mL, 2 mg/mL, or 4 mg/mL.

4.5. Saccharide

[0061] A saccharide can improve the stability of an aqueous solution of hyaluronic acid or

pharmaceutically acceptable salt thereof. In some embodiments, the saccharide is selected from alginic acid, carrageenan, carboxymethyl cellulose, hydroxypropyl methylcellulose (HPMC),

hydroxypropylcellulose (HPC), chitosan, gelatin, arabic gum, guar gum, mannitol, sorbitol, sucrose, xanthan gum, polyethylene glycol, and pharmaceutically acceptable salts thereof. In some

embodiments, the saccharide is selected from alginic acid, carrageenan, carboxymethyl cellulose, chitosan, gelatin, arabic gum, guar gum, mannitol, sorbitol, sucrose, xanthan gum, polyethylene glycol, and pharmaceutically acceptable salts thereof. In some embodiments, the saccharide is selected from alginic acid, carrageenan, carboxymethyl cellulose, xanthan gum, and pharmaceutically acceptable salts thereof. In certain embodiments, the saccharide is alginic acid or a pharmaceutically acceptable salt thereof, such as sodium alginate. In certain embodiments, the saccharide is carrageenan. In certain embodiments, the saccharide is sodium carboxymethyl cellulose. In certain embodiments, the saccharide is xanthan gum.

[0062] Generally, a saccharide is present in an amount in the aqueous formulation sufficient to improve the stability of the hyaluronic acid or pharmaceutical salt thereof while allowing for a freely flowing aqueous solution that can be injected, e.g., via syringe, for therapeutic treatment of a patient in need thereof. In some embodiments, the saccharide is present in a range of from about 0.005% to about 5% by weight, such as from about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, or 1% to about 2%, 3%, or 5%, e.g., from about 0.01% to about 5%, from about 0.01% to about 2%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, from about 0.01% to about 0.1%, from about 0.01% to about 0.05%, from about 0.05% to about 5%, from about 0.05% to about 2%, from about 0.05% to about 1%, from about 0.05% to about 0.5%, from about 0.05% to about 0.1%, from about 0.1% to about 5%, from about 0.1% to about 2%, from about 0.1% to about 1%, from about 0.1% to about 0.5%, from about 0.2% to about 5%, from about 0.2% to about 2%, from about 0.2% to about 1%, or from about 0.2% to about 0.5% by weight. In certain embodiments, the saccharide is hydroxypropyl methylcellulose (HPMC), which is present in a range of from about 0.01% to about 1% by weight. In certain embodiments, the saccharide is hydroxypropyl cellulose (HPC), which is present in a range of from about 0.01% to about 1% by weight. In certain embodiments, the saccharide is carrageenan, which is present in a range of from about 0.01% to about 1% by weight. In certain embodiments, the saccharide is sodium carboxymethyl cellulose, which is present in a range of from about 0.1% to about 1% by weight. In certain

embodiments, the saccharide is xanthan gum, which is present in a range of from about 0.1% to about 1% by weight.

[0063] In some embodiments, the saccharide is present in a range of from about 0.05 to about 50 mg/mL, e.g., from about 0.1 to about 50 mg/mL, from about 0.1 to about 20 mg/mL, from about 0.1 to about 10 mg/mL, from about 0.1 to about 5 mg/mL, from about 0.1 to about 1 mg/mL, from about 0.1 to about 0.5 mg/mL, from about 0.5 to about 50 mg/mL, from about 0.5 to about 20 mg/mL, from about 0.5 to about 10 mg/mL, from about 0.5 to about 5 mg/mL, from about 0.5 to about 1 mg/mL, from about 1 to about 50 mg/mL, from about 1 to about 20 mg/mL, from about 1 to about 10 mg/mL, from about 1 to about 5 mg/mL, from about 2 to about 50 mg/mL, from about 2 to about 20 mg/mL, from about 2 to about 10 mg/mL, or from about 2 to about 5 mg/mL. In certain embodiments, the saccharide is carrageenan, which is present in a range of from about 0.1 to about 10 mg/mL. in certain embodiments, the saccharide is sodium carboxymethyl cellulose, which is present in at least about 1 mg/mL, e.g., in a range of from about 1 to about 10 mg/mL. In certain embodiments, the saccharide is xanthan gum, which is present in at least about 1 mg/mL, e.g., in a range of from about 1 to about 10 mg/mL.

[0064] In certain embodiments, sodium alginate can serve as the saccharide sufficient to stabilize the aqueous hyaluronic acid or pharmaceutically acceptable salt thereof. The sodium alginate can be present in a range of from about 0.02% to about 5% by weight, such as from about 0.02%, 0.05%, 0.1%, 0.2%, or 0.5% to about 0.7%, 1%, 1.5%, 2%, 3%, or 5% by weight. In some embodiments, the sodium alginate is present in a range of from about 0.2 to about 50 mg/mL, e.g., from about 0.2, 0.5, 1, 2, or 3 mg/mL to about 5, 7, 10, 15, 20, 30, or 50 mg/mL, including from about 0.2 to about 20, from about 0.2 to about 10, from about 0.5 to about 50, from about 0.5 to about 20, from about 0.5 to about 10, or from about 1 to about 10 mg/mL. In certain embodiments, the sodium alginate is present in an amount of about 0.1, 0.5, 1, 5, 10, 12, 15, 17, 20, 23, 25, 30, 35, 40, or 50 mg/mL.

[0065] In some embodiments, sodium alginate that has low viscosity more effectively stabilizes hyaluronic acid or pharmaceutically acceptable salt thereof. In certain embodiments, the sodium alginate has a viscosity of no more than about 10, 20, 30, 40, or 50 mPa-sec, particularly no more than about 20 mPa-sec. In certain embodiments, the sodium alginate has a viscosity in a range of from about 1 to about 200 mPa-sec, such as from about 1 to about 175 mPa-sec, from about 1 to about 150 mPa- sec, from about 1 to about 125 mPa-sec, from about 1 to about 100 mPa-sec, from about 1 to about 75 mPa-sec, or from about 1 to about 50 mPa-sec.

[0066] In some embodiments, sodium alginate that has lower average molecular weight more effectively stabilizes hyaluronic acid or pharmaceutically acceptable salt thereof. In certain

embodiments, sodium alginate has a molecular weight of no more than about 200,000 Daltons, such as no more than about 175,000, 150,000, 100,000, 75,000, or 50,000 Daltons, particularly no more than about 75,000 Da. In certain embodiments, sodium alginate has a molecular weight of from about 1,000 to about 200,000 Daltons, e.g., from about 1,000 to about 150,000 Da or from about 1,000 to about 75,000 Da, such as from about 5,000 to about 75,000 Da, from about 10,000 to about 75,000 Da, from about 15,000 to about 75,000 Da, from about 20,000 to about 75,000 Da, from about 25,000 to about 75,000 Da, from about 30,000 to about 75,000 Da, from about 40,000 to about 75,000 Da, or from about 50,000 to about 75,000 Da.

[0067] The percentage of monomeric constituents in the sodium alginate may affect its ability to stabilize hyaluronic acid. In some embodiments, sodium alginate that has a higher level of mannuronate monomers (M) as compared with guluronate monomers (G) more effectively stabilizes hyaluronic acid or pharmaceutically acceptable salt thereof. In certain embodiments, sodium alginate comprises a ratio of G to M of no more than about 1.5:1, e.g., no more than about 1:1, such as from about 0.5:1 to about 1.5:1, e.g., from about 0.5:1 to about 1:1, from about 0.6:1 to about 1:1, from about 0.7:1 to about 1:1, from about 0.8:1 to about 1:1, from about 0.8:1 to about 1.1:1, or from about 0.8:1 to about 1.2:1. In certain embodiments, sodium alginate comprises a ratio of G to M of about 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1, or 0.5:1.

[0068] Sodium alginate preparations are known in the art, and specific preparations are commercially available. Exemplary sodium alginate preparations include ultrapure sodium alginate from brown algae, FEMA Number 2015 (Sigma-Aldrich) and Pronova™ UP ultrapure sodium alginate (Novamatrix^® from DuPont). For example, as shown in Table A below, the commercial Pronova™ UP ultrapure sodium alginate series offers several preparations that vary in viscosity {low, e.g., <20 mPa-sec; medium, e.g., 20- 200 mPa-sec; or high, e.g., >200 mPa-sec), molecular weight (low, e.g., <75,000 Da; medium, e.g., 75,000-200,000 Da; or high, e.g., >200,000 Da), and relative ratio of guluronate and mannuronate monomers (lower, e.g., < 50% guluronate monomer; or higher, e.g., > 60% guluronate monomer). In certain embodiments, the sodium alginate is Pronova™ UP VLVM.

[0069] Table A: Exemplary Physicochemical Properties of Pronova™ UP Sodium Alginates

[0070] In some embodiments, specific ratios of hyaluronic acid or pharmaceutically acceptable salt thereof to antioxidant and/or saccharide provide greater stability within the compositions disclosed herein. In some embodiments, an aqueous composition comprises a ratio of hyaluronic acid or pharmaceutically acceptable salt thereof to antioxidant of from about 2:1 to about 50:1, such as from about 2:1, 4:1, 5:1, 6:1, or 8:1 to about 12:1, 15:1, 20:1, 25:1, 30:1, 40:1, or 50:1, e.g., about 2:1, 4:1, 5:1, 6:1, 8:1, 10:1, 12:1, 15:1, 20:1, 25:1, 30:1, 40:1, or 50:1. In some embodiments, an aqueous composition comprises a ratio of hyaluronic acid or pharmaceutically acceptable salt thereof to saccharide of from about 1:5 to about 2:1, such as from about 1:5, 1:4, or 1:3 to about 1:1 or 2:1, e.g., about 1:5, 1:4, 1:3, 1:2, 1:1.5, 1:1, 1.5:1, or 2:1. In some embodiments, an aqueous composition comprises a ratio of antioxidant to saccharide of from about 1:50 to about 1:4, such as from about 1:50, 1:40, or 1:30 to about 1:8, 1:6, 1:5, or 1:4, e.g., about 1:50, 1:40, 1:30, 1:20, 1:15, 1:10, 1:8, 1:6, 1:5, or 1:4.

4.6. Excipients

[0071] The aqueous compositions disclosed herein, in addition to water, may include one or more pharmaceutically acceptable excipients. Examples of such excipients include buffer solutions and tonicity agents.

[0072] Pharmaceutically acceptable buffers suitable for injection are known in the art. Buffers that can be used in the aqueous compositions of the present disclosure include those comprising maleate, tartrate, lactate, citrate, acetate, carbonate, or phosphate salts. In some embodiments, the aqueous composition comprises a phosphate buffer. In certain embodiments the buffer, such as a phosphate buffer, are use in the present compositions in an amount to provide a desired pH level or range discussed above.

[0073] For tonicity agents, examples include ionic tonicity agents, such as halide salts {including calcium chloride, potassium bromide, potassium chloride, lithium chloride, sodium iodide, sodium bromide, and sodium chloride), EDTA, citric acid, and sodium citrate. Tonicity agents further include nonionic tonicity agents, such as glycerol, mannitol, sorbitol, propylene glycol, dextrose, and urea. In a particular embodiment, the tonicity agent is sodium chloride.

4.7. Specific embodiments

[0074] In a specific embodiment, an aqueous composition described herein comprises about 10 mg/mL sodium hyaluronate; about 20 mg/mL sodium alginate; about 1 mg/mL histidine; a phosphate buffer, e.g., about 0.18 mg/mL of phosphate buffer; and a tonicity agent, e.g., about 7.5 mg/mL sodium chloride; wherein the pH of the composition is from about 6.5 to about 7.5. In another specific embodiment, an aqueous composition described herein comprises about 10 mg/mL sodium hyaluronate; about 20 mg/mL sodium alginate; about 2 mg/mL methionine; a phosphate buffer, e.g., about 0.18 mg/mL of phosphate buffer; and a tonicity agent, e.g., about 7.5 mg/mL sodium chloride; wherein the pH of the composition is from about 6.5 to about 7.5.

[0075] In certain aspects, the sodium alginate in the above specific embodiments has a G/M ratio of < 1. In further such aspects, the sodium alginate in the above specific embodiments has a molecular weight of no more than about 75,000 Daltons.

4.8. Test degradation conditions

[0076] A degradation condition can be used to determine the ability of an aqueous composition of the present disclosure comprising hyaluronic acid or pharmaceutically acceptable salt thereof to resist degrading as compared with an equivalent amount of a standard composition of hyaluronic acid or pharmaceutically acceptable salt thereof. Degradation conditions may comprise oxidative conditions, e.g., by subjecting the disclosed composition to a reactive oxygen species (such as hydrogen peroxide), or enzymatic conditions, e.g., by subjecting the disclosed composition to a physiologically relevant enzyme, e.g., hyaluronidase.

[0077] Stability of hyaluronic acid or pharmaceutically acceptable salt thereof can be assessed by any number of physical methods. In some embodiments, in vitro testing can serve as a surrogate for in vivo hyaluronic acid stability. In vitro measurement of a change, e.g., decrease, in physical measures such as viscosity, molecular weight, or particle size of hyaluronic acid molecules in the composition can be used to determine relative composition stability. For example, in vitro decrease in viscosity can be used to determine hyaluronic acid composition stability, particularly in response to oxidative and/or enzymatic stresses. A viscosity of a composition can be measured by a number of methods, e.g., by rheometer or viscometer.

[0078] In some embodiments, an aqueous composition comprising hyaluronic acid or pharmaceutically acceptable salt thereof, after being subjected to oxidative stress conditions, has at least about 10%, e.g., at least about 15%, 20%, 30%, 40%, 50%, or 60%, higher viscosity, e.g., from about 10% to about 90% higher, such as from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, higher as compared with an equivalent amount of a comparative hyaluronic acid composition that does not comprise antioxidant and/or saccharide. Oxidative stress conditions include those known in the art for modeling stability of hyaluronic acid aqueous compositions. In one embodiment, the oxidative stress conditions comprise incubating the aqueous composition with an amount of an oxidant, e.g., a reactive oxygen species, e.g., hydrogen peroxide, such as about 0.016% hydrogen peroxide for 1 hour at room temperature.

[0079] In some embodiments, an aqueous composition comprising hyaluronic acid or pharmaceutically acceptable salt thereof, after being subjected to enzymatic stress conditions, has at least about 10%, e.g., at least about 15%, 20%, 30%, 40%, 50%, or 60%, higher viscosity, e.g., from about 10% to about 90% higher, such as from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, higher as compared with an equivalent amount of a comparative hyaluronic acid composition that does not comprise antioxidant or saccharide. Enzymatic stress conditions include those known in the art for modeling in vivo stability of hyaluronic acid aqueous compositions. In one embodiment, the enzymatic stress conditions comprise incubating the aqueous composition with an amount of a physiologically relevant enzyme, e.g., hyaluronidase, such as about 0.001 mg/mL hyaluronidase, for 1 hour at room temperature.

4.9. Methods of use

[0080] Disclosed herein is a method of treating a disease or condition exhibiting decreased hyaluronic acid levels, comprising administering to a patient in need thereof a therapeutically effective amount of an aqueous composition described herein, e.g., comprising: hyaluronic acid or a pharmaceutically acceptable salt thereof, an antioxidant, and a saccharide.

[0081] Hyaluronic acid is known in the art to be used for treatment of conditions of the joint, eye conditions, and skin conditions. Accordingly, in some embodiments, the disease or disorder is joint condition. In some embodiments, the disease or condition is osteoarthritis. In some embodiments, the disease or condition is an eye condition. In some embodiments, the disease or condition is a skin condition.

[0082] In some embodiments, the patient is a human patient.

[0083] In certain embodiments, the methods of treating described herein involve administration of one or more unit doses of the aqueous compositions described herein. Unit doses may include about 0.25 to 25 mL quantities of an aqueous composition described herein, such as from about 0.25, 0.5, 1, 2, 3, or 4 mL to about 5, 7, 10, 15, 20, or 25 mL, including about 0.25, 0.5, 1, 2, 3, 4, 5, 7, 10, 15, 20, or 25 mL. [0084] In some embodiments, the method comprises intra-articular injection of one or more of the aqueous compositions described herein into the patient. Sites of injection into the patient may include the knee, ankle, foot, elbow, wrist, hand, shoulder, or neck of the patient.

[0085] The treatment methods described herein may be performed according to treatment regimen, and may involve a single injection regimen; semi-weekly, weekly, or bi-weekly injections; semi-monthly, monthly, bi-monthly injections; or semi-annual or annual injections. In some embodiments, the treatment methods comprise a treatment regimen involving administering multiple unit doses at periodic intervals, such as two doses (one semi-weekly) over one week, two doses (one per week) over two weeks, two doses (one per bi-week) over four weeks, three doses (one per week) over three weeks, three doses (one per bi-week) over six weeks, four doses (one per week) over four weeks, four doses (one per bi-week) over eight weeks, particularly three doses (one per week) over three weeks.

5. EXAMPLES

[0086] The following Examples, which highlight certain features and properties of embodiments of the hyaluronic acid or pharmaceutically acceptable salt described herein are provided for purposes of illustration, and not limitation.

[0087] The comparative aqueous composition of hyaluronic acid or pharmaceutically acceptable salt thereof used in the Examples described herein comprised 1% non-cross-linked sodium hyaluronate in phosphate-buffered saline.

Example 1: Degradation Measurement Methods

[0088] The following degradation measurements use the loss of hyaluronic acid viscosity as a measure to assess the stability of hyaluronic acid or pharmaceutically acceptable salt under stress conditions.

5.1. Oxidative Degradation

[0089] A sample of an aqueous composition comprising 10 mg/mL hyaluronic acid was diluted by 1:5 to a test solution at about 2 mg/mL hyaluronic acid. Hydrogen peroxide (25 pL of 10% solution) was added to 16 mL of the test composition, and the mixture was spun by vortex for 30 seconds. Test mixture viscosity was measured immediately thereafter in a Malvern Kinexus Pro+ Rheometer (Malvern Instruments Ltd., Malvern, Worcestershire, United Kingdom) with a Cup & Bob geometry (analysis temperature = 25 °C; shear rate = 25 sec ¹ for 1 hr; reported results in mPa-sec). 5.2. Enzymatic Degradation

[0090] A sample of an aqueous composition comprising 10 mg/mL hyaluronic acid was diluted by 1:5 to a test solution at about 2 mg/mL hyaluronic acid. Hyaluronidase (32 pL of 0.5 mg/mL solution) was added to 16 mL of the test composition, and the mixture was spun by vortex for 30 seconds. Test mixture viscosity was measured immediately thereafter in a Malvern Kinexus Pro+ Rheometer (Malvern Instruments Ltd., Malvern, Worcestershire, United Kingdom) with a Cup & Bob geometry (analysis temperature = 25 °C; shear rate = 25 sec ¹ for 1 hr; reported results in mPa-sec).

Example 2: Screening of Antioxidants

[0091] The antioxidants were screened to determine those that exhibited the highest ability to prevent oxidative degradation of hyaluronic acid or pharmaceutically acceptable salt thereof.

[0092] FIG. 1 shows the results of antioxidant screening. Antioxidant concentrations were based on FDA Inactive Ingredient Database guidance as follows.

Formulations comprising antioxidant ascorbic acid, histidine, sodium metabisulfite, mannose, mannitol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), a combination of BHA and BHT ("BHA + BHT”), tocopherol, propyl gallate ("P gallate"), or sorbitol were evaluated in the presence of hydrogen peroxide under the experimental protocol described in Section 5.1. As the graph demonstrates, the stability of hyaluronic acid compositions vary widely depending on antioxidant selected, with histidine, tocopherol, and propyl gallate exhibiting the highest antioxidant effect under the conditions tested.

[0093] A summary of the results shown in FIG. 1 can also be found in Table 1 below.

[0094] Table 1: Hyaluronic Acid Composition Stabilization with Variable Antioxidant

% stabilization = {viscosity with H₂0₂)/(viscosity without H₂0₂) x 100

[0095] Despite the promising screening results shown in Table 1, when the compositions of hyaluronic acid or pharmaceutically acceptable salt and antioxidant evaluated in the presence of the enzyme hyaluronidase under the experimental protocol described in Section 5.2, degradation was observed. FIG. 2 depicts the effect of hyaluronidase on the viscosity of hyaluronic acid formulations comprising antioxidant histidine, BHA, BHT, tocopherol, or propyl gallate. In each composition, significant loss of viscosity was observed over the course of 1 hr.

Example 3: Concentration Dependence of Selected Antioxidants

[0096] The antioxidants that performed the best in the screening experiment described in Example 2 were further evaluated to determine any concentration dependence of their observed stability effect on compositions of hyaluronic acid or pharmaceutically acceptable salt. Histidine, propyl gallate, and tocopherol were evaluated at low, medium, or high concentrations in hyaluronic acid compositions under the oxidative degradation conditions described in Section 5.1. The results of the oxidative stress tests are shown in FIGS. 3A-3C.

[0097] Histidine performed the best of the three antioxidants evaluated. FIG. 3A shows that histidine at

0.1%, 0.2%, or 0.4% concentration in the hyaluronic acid formulations preserved hyaluronic acid composition viscosity. While 0.02% or 0.10% propyl gallate also effectively preserved composition viscosity, at a higher level of 0.20% propyl gallate, significant hyaluronic acid viscosity was lost (FIG. 3B). Additionally, at all concentrations of tocopherol evaluated (0.04%, 0.07%, or 0.10%), hyaluronic acid composition viscosity was preserved, but there was some attenuation of viscosity (FIG. 3C), which was not observed in the compositions comprising histidine.

Example 4: Evaluation of Amino Acid Antioxidants

[0098] Due to the promising results of experiments of Example 3 with the compositions comprising histidine, other amino acids and peptides were evaluated for stabilization effect on hyaluronic acid compositions. Antioxidants cysteine, glutathione, or methionine, in hyaluronic acid compositions under the oxidative degradation conditions described in Section 5.1. FIG. 4 shows that methionine preserved hyaluronic acid composition viscosity, while cysteine or glutathione was not able to effect a stabilized composition.

[0099] Methionine was further evaluated for any concentration dependence on its stability effect on compositions of hyaluronic acid or pharmaceutically acceptable salt. FIG. 5 shows that, at all concentrations tested (1, 2, or 4 mg/mL), methionine was able to preserve hyaluronic acid composition viscosity under the oxidative degradation conditions described in Section 5.1.

Example 5: Screening of Saccharides

[0100] As a result of the antioxidant experiments described in Examples 2-4, histidine or methionine was selected as antioxidant for further hyaluronic acid composition evaluation. However, as depicted in FIG. 2, addition of antioxidant alone may not be sufficient to stabilize hyaluronic acid composition in vivo. Accordingly, saccharides were evaluated for stabilization effect of hyaluronic acid compositions to enzymatic degradation, e.g., degradation by hyaluronidase.

[0101] Different saccharides, including sugar alcohols such as mannitol, disaccharides such as sucrose, and polymers derived from saccharides or sugar alcohols, e.g., sodium alginate, PEG, arabic gum, xanthan gum, carrageenan, were screened for effectiveness against hyaluronidase degradation of viscosity under the enzymatic degradation conditions described in Section 5.2. As shown in FIG. 6, the saccharides sodium alginate, carrageenan 109, xanthan gum 75, and xanthan gum 180 each preserved viscosity of hyaluronic acid compositions effectively. Also preserving viscosity were the compositions comprising carrageenan 209, carrageenan NF, or sodium carboxymethyl cellulose, though with slight attenuation of viscosity in these experiments. By contrast, hyaluronic acid compositions comprising PEG, arabic gum, gelatin, guar gum, mannitol, or sucrose did not effectively preserve viscosity over 0.5 hr when subjected to in vitro enzymatic stress conditions. Example 6: Concentration Dependence of Selected Saccharides

[0102] The saccharides that performed the best in the screening experiment described in Example 5 were further evaluated to determine any concentration dependence of their observed stability effect on compositions of hyaluronic acid or pharmaceutically acceptable salt.

[0103] Hyaluronic acid compositions comprising carrageenan, sodium alginate, sodium carboxymethyl cellulose, or xanthan gum 75 were tested for their ability to preserve viscosity under the enzymatic degradation conditions described in Section 5.2. As depicted in FIGS. 7A-7D, carrageenan NF (at 0.5, 1, or 5 mg/mL) (FIG. 7A) and sodium alginate (at 1, 5, or 10 mg/mL) (FIG. 7B) did not show concentration dependence for the concentrations tested, while sodium carboxymethyl cellulose (at 1, 5, or 10 mg/mL) (FIG. 7C) and xanthan gum 75 (at 0.5, 1, or 5 mg/mL) (FIG. 7D) showed decreased viscosity at lower concentrations tested.

Example 7: Sodium Alginate Level Optimization for Viscosity

[0104] Owing to the positive results for sodium alginate in the experiments described in Examples 5 and 6, different concentrations of sodium alginate were screened to optimize for viscosity. As higher levels of sodium alginate in the hyaluronic acid compositions were determined to afford greater stability, higher sodium alginate concentrations were preferred for maintenance of hyaluronic acid viscosity. However, overall composition viscosity was evaluated with varying sodium alginate concentrations to ensure that viscosity of the hyaluronic acid compositions across shear rates was compatible with in vivo administration, e.g., intra-articular administration via syringe.

[0105] FIG. 8 depicts the viscosity of different concentrations of hyaluronic acid compositions comprising sodium alginate vs. shear rate. From the results, 20 mg/mL sodium alginate was selected for evaluation in further composition tests.

Example 8: Evaluation of Pronova™ Sodium Alginate Preparations

[0106] As the present disclosure describes stabilized compositions of hyaluronic acid or

pharmaceutically acceptable salt thereof as measured by preservation of viscosity, different preparations of sodium alginate were evaluated to determine whether the physicochemical properties of the sodium alginate affected its hyaluronic acid composition stabilization ability.

[0107] Sodium alginate is available from a number of different commercial sources, such as Sigma- Aldrich. Different preparations of sodium alginate in the Pronova™ UP ultrapure sodium alginate series (Novamatrix^® from DuPont) were evaluated as saccharide. The commercial Pronova™ UP ultrapure sodium alginate varied in viscosity (low, e.g., <20 mPa-sec; medium, e.g., 20-200 mPa-sec; or high, e.g., >200 mPa-sec), molecular weight (low, e.g., <75,000 Da; medium, e.g., 75,000-200,000 Da; or high, e.g., >200,000 Da), and relative ratio of guluronate and mannuronate monomers (lower, e.g., < 50% guluronate monomer; or higher, e.g., > 60% guluronate monomer). These differing physicochemical properties gave rise to different viscosities of the hyaluronic acid compositions, as depicted in Table 2 below.

[0108] Table 2: Viscosity Measurements of Exemplary Hyaluronic Acid Compositions Comprising

Different Sodium Alginate Preparations*

* Hyaluronic acid compositions: 10 mg/mL hyaluronic acid, 7.5 mg/mL sodium chloride, 0.56 mg/mL disodium hydrogen phosphate dodecahydrate, 0.05 mg/mL sodium dihydrogen phosphate dihydrate, 1 mg/mL L-histidine, 20 mg/mL sodium alginate (as indicated), water for injection.

[0109] Different sodium alginate preparations in hyaluronic acid compositions were tested for their ability to preserve viscosity under the enzymatic degradation conditions described in Section 5.2. The results of the stability are depicted in FIG. 9, which shows that all preparations comprising sodium alginate exhibited a higher level of viscosity of the hyaluronic acid formulation after 0.5 hr under the enzymatic degradation conditions described in Section 5.2 as compared with control hyaluronic acid composition that did not comprise sodium alginate. Among the Pronova™ sodium alginate-comprising hyaluronic acid compositions tested, the VLVM preparation preserved viscosity most efficiently, followed by LVM, and the LVG and VLVG, which performed similarly.

[0110] As depicted in FIG. 10A, hyaluronic acid compositions comprising Pronova™ UP VLVM sodium alginate exhibited a high level of stability at concentrations of 5, 10, or 20 mg/mL. Detailed comparison of the concentrations (FIG. 10B) showed that at 20 mg/mL, the hyaluronic acid composition may exhibit slightly higher stability than 5 or 10 mg/mL Pronova™ UP VLVM sodium alginate under the Section 5.2 enzymatic stress conditions.

Example 9: Tonicity Optimization

[0111] Sodium chloride concentrations were screened to determine a composition of hyaluronic acid, or pharmaceutically acceptable salt thereof, that is about isotonic with intra-articular fluid. Table 3 below summarizes the results of tonicity optimization studies. Two preparations of each concentration of sodium chloride were evaluated, with osmolality of the samples measured three times. The osmolality was corrected for dilution, and the mean osmolality was calculated by an average of all measurements across the two samples. Accordingly, 6.5 mg/mL to 7.5 mg/mL sodium chloride was selected as salt concentration for use in further formulations.

[0112] Table 3: Tonicity Measurement of 10 mg/mL Hyaluronic Acid Compositions Comprising Different Sodium Chloride Concentrations

Example 10: Evaluation of Exemplary Hyaluronic Acid Compositions Comprising

Antioxidant and Saccharide

[0113] As noted in Example 2, while hyaluronic acid compositions comprising an antioxidant such as histidine or methionine exhibited higher stability against oxidative stress conditions, such compositions comprising an antioxidant alone still exhibited significant loss of viscosity under enzymatic stress conditions. Accordingly, exemplary hyaluronic acid compositions comprising both an antioxidant and a saccharide were evaluated for stability against both oxidative and enzymatic degradation stress.

[0114] Exemplary compositions of the disclosure comprising hyaluronic acid or pharmaceutically acceptable salt thereof:

*Water for injection was used as needed to dilute each composition to the indicated concentration.

[0115] Each composition - Prototype A or Prototype B - was subjected to the oxidative stress conditions in Section 5.1 or the enzymatic stress conditions described in Section 5.2. The compositions were each tested twice under each condition. The results are summarized in FIG. 11 for Prototype A and FIG. 12 for Prototype B.

[0116] Prototype A exhibited good stability against the oxidative conditions described in Section 5.1, exhibiting 99.3% viscosity after 1 hr (FIG. 11) as compared with a hyaluronic acid composition in the absence of hydrogen peroxide. Prototype A exhibited good stability against the enzymatic conditions described in Section 5.2, exhibiting 79.0% viscosity after 1 hr as compared with a hyaluronic acid composition in the absence of hydrogen peroxide.

[0117] Prototype B exhibited good stability against the oxidative conditions described in Section 5.1, exhibiting 98% viscosity after 1 hr (FIG. 12) as compared with a hyaluronic acid composition in the absence of hyaluronidase. Prototype B exhibited good stability against the enzymatic conditions described in Section 5.2, exhibiting 89.7% viscosity after 1 hr as compared with a hyaluronic acid composition in the absence of hyaluronidase.

[0118] Each of the compositions Prototype A and Prototype B exhibited a higher stability against either oxidative or enzymatic stress as compared to a control hyaluronic acid composition. The control composition comprised the same concentration of hyaluronic acid, phosphate, and sodium chloride as Prototype A or Prototype B but without antioxidant or sodium alginate. As shown in FIG. 13, the control composition exhibited 41.4% viscosity after 1 hr against the oxidative conditions described in Section 5.1 as compared with a hyaluronic acid composition in the absence of hydrogen peroxide. The control composition exhibited 41.6% viscosity after 1 hr against the enzymatic conditions described in Section 5.2 as compared with a hyaluronic acid composition in the absence of hyaluronidase. Example 11: Long Term Viscosity and pH Testing

[0119] Compositions Prototype A and Prototype B were filled into syringes to a volume of 5 mL in 10 mL BD clear glass syringes to represent a ready-for-injection composition. The plunger stoppers were placed to minimize air access into the syringe. The compositions were not sterilized and filling of the syringes was performed under non-sterile conditions. The syringe and plunger stopper used are listed in Table 4.

[0120] Table 4.

[0121] Long term viscosity measurements were taken for Prototype A and Prototype B to monitor any shortening, breaking, or complete chemical degradation of the HA polymer chains through chemical or biological activity that would result in decreased viscosity. The samples were stored in syringes under three conditions: 5 ⁹C, 25 ⁹C/60% relative humidity (RH), and 40 ⁹C/75% RH. Stability samples were tested "as-is" without dilution using a rheometer and viscosity data was collected at shear rates of 0.1 s ¹ and 1000 s ¹ using the cone-plate geometry (40 mm plate diameter and a 2 degree cone angle).

[0122] FIG. 14A-B and FIG. 14C-D show the long term viscosity of compositions Prototype A and Prototype B, respectively, under the three different storage conditions.

[0123] Results show an expected trend of decreasing viscosity at 0.1 s ¹ shear rate with increased temperature for both compositions across all time points up to 6 months. Both compositions experience a decrease in stability at the 40 ⁹C/75% RH condition beginning as early as the 2 weeks. Prototype B appears to be more stable than Prototype A at 25 ⁹C/60% RH up to the 6 month time point, however, all results at 0.1 s ¹ are lower for 25 ⁹C/60% RH than for 5 ⁹C indicating some degradation. Prototype B appears to be more stable than Prototype A at 5 ⁹C across all time points up to 6 months.

[0124] Long term pH of compositions Prototype A and Prototype B were taken potentiometrically after dilution (1:1) in water.

[0125] FIG. 15A and 15B show the long term pH of compositions Prototype A and Prototype B.

[0126] All pH measurements were in the range of 7.1 to 7.7 and exhibited a decrease over 6 months (< 0.4) for both Prototype A and Prototype B. The decrease was more evident at 40 ⁹C/75% RH. Example 12: In vivo Assessment

[0127] An incapacitance analysis was performed to evaluate the preclinical efficacy of composition Prototype B when injected into the knee joint of rodents.

[0128] Male Lewis rats (250-275 g, 9 weeks) were selected for the in vivo assessments and were habituated to an animal colony for one week and handled four times for five minutes each after a week of habituation. Animals were habituated to the testing rack three times during this process. After one week of conditioning, the rodents where given a medial meniscal tear on the right hind knee on day 1 and at day 7, 14, and 21 were treated with composition Prototype B, 1% HA in PBS, 1.5% HA in PBS, or control vehicle (PBS) by intra-articular injection, or oral Tramadol. Control vehicle (PBS) was also administered to rats having a sham surgery to account for incision trauma.

[0129] Changes in hind paw weight distribution between the right (inflamed/diseased) and left (contralateral control) limbs can be utilized as an index of joint discomfort. An incapacitance tester (Linton Instrumentation, Norfolk, UK) is employed for determination of hind paw weight distribution.

See Boye, S.E. et al., Osteoarthritis and Cartilage, 11(11): 821-830.

[0130] A rat was placed with its hind paws in the center of the force plates and its upper body inside of the modified restraint tube and the rat was allowed to acclimate to the tube for thirty seconds. The rat must be standing straight and square and not leaning on the sides of the tube and may be lightly manipulated to encourage proper posture prior to recording data. Once the animal had achieved proper posture a reading was taken by pressing the "enter" button and the process was repeated in triplicate for several rats. The results were recorded for left and right limbs.

[0131] FIG. 16A and 16B show the results of the incapacitance test using compositions described in Table 5.

[0132] Table 5. In vivo Test Formulations

[0133] A Dunnett Test was performed to analyze whether results obtained in the incapacitance test were statistically significant for formulation SI to S4 in comparison to control S5. The results of the Dunnett Test are reported in Table 6.

[0134] Table 6. Dunnett Test

[0135] Table 6 shows that the incapacitance test results of injected formulation SI (Prototype B) is statistically significant (p-value < 0.05) in comparison to control S5.

Example 13: Histopathology Analysis

[0136] Histopathological examinations were performed to assess histological scoring and also analyze the residence time of HA formulations after injection into the right knee joint of rats. Histological sections of the treated knee joints were obtained after 30 days and examined under microscope at 16x magnification.

[0137] FIG. 17A and 17B show photomicrographs of PBS (0.5 mg/rat) and Prototype B (0.5 mg/ rat) treated knee joints, respectively.

[0138] In FIG. 17B, Prototype B can still be observed on the synovium membrane showing residence time of greater than 30 days.

[0139] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0140] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).

Claims

1. An aqueous composition comprising:

hyaluronic acid or a pharmaceutically acceptable salt thereof;

an antioxidant; and

a saccharide.

2. The composition of claim 1, wherein the pH of the solution is from about 6.5 to about 7.5.

3. The composition of claim 1 or 2, wherein the pharmaceutically acceptable salt comprises a sodium salt.

4. The composition of any one of claims 1-3, wherein the hyaluronic acid or pharmaceutically acceptable salt thereof is present in a range of from about 0.1% to about 10% by weight.

5. The composition of any one of claims 1-4, wherein the hyaluronic acid or pharmaceutically acceptable salt thereof is present in about 1% by weight.

6. The composition of any one of claims 1-5, wherein the hyaluronic acid or pharmaceutically acceptable salt thereof is present in a range of from about 1 to about 100 mg/mL.

7. The composition of any one of claims 1-6, wherein the hyaluronic acid or pharmaceutically acceptable salt thereof is present in about 10 mg/mL.

8. The composition of any one of claims 1-7, wherein the antioxidant is selected from ascorbic acid, citric acid, metabisulfite, tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, mannose, mannitol, an amino acid, a peptide, and pharmaceutically acceptable salts thereof.

9. The composition of any one of claims 1-8, wherein the antioxidant is an amino acid or a

pharmaceutically acceptable salt thereof.

10. The composition of claim 9, wherein the amino acid is selected from histidine, cysteine, methionine, and pharmaceutically acceptable salts thereof.

11. The composition of any one of claims 1-8, wherein the antioxidant is a peptide.

12. The composition of claim 11, wherein the peptide consists of from 2 to 5 amino acids.

13. The composition of claim 12, wherein the peptide is glutathione or a pharmaceutically acceptable salt thereof.

14. The composition of any one of claims 1-7, wherein the antioxidant is selected from propyl gallate, histidine, methionine, tocopherol, and pharmaceutically acceptable salts thereof.

15. The composition of claim 14, wherein the antioxidant is propyl gallate.

16. The composition of claim 15, wherein the propyl gallate is present in a range of from about 0.01% to about 0.1% by weight.

17. The composition of claim 14, wherein the antioxidant is histidine or a pharmaceutically acceptable salt thereof.

18. The composition of claim 17, wherein the histidine or pharmaceutically acceptable salt thereof is present in a range of from about 0.01% to about 5% by weight.

19. The composition of claim 18, wherein the histidine or pharmaceutically acceptable salt thereof is present in a range of from about 0.05% to about 0.5% by weight.

20. The composition of claim 17, wherein the histidine or pharmaceutically acceptable salt thereof is present in a range of from about 0.5 to about 5 mg/mL.

21. The composition of claim 14, wherein the antioxidant is methionine or a pharmaceutically acceptable salt thereof.

22. The composition of claim 21, wherein the methionine or pharmaceutically acceptable salt thereof is present in a range of from about 0.01% to about 5% by weight.

23. The composition of claim 22, wherein the methionine or pharmaceutically acceptable salt thereof is present in a range of from about 0.1% to about 0.5% by weight.

24. The composition of claim 21, wherein the methionine or pharmaceutically acceptable salt thereof is present in a range of from about 1 to about 5 mg/mL.

25. The composition of claim 14, wherein the antioxidant is tocopherol.

26. The composition of any one of claims 1-7, wherein the antioxidant is present in a range of from about 0.01% to about 1% by weight.

27. The composition of any one of claims 1-7, wherein the antioxidant is present in a range of from about 0.1 to about 10 mg/mL.

28. The composition of any one of claims 1-7, wherein the antioxidant is present in about 2 mg/mL.

29. The composition of any one of claims 1-28, wherein the saccharide is selected from alginic acid, carrageenan, carboxymethyl cellulose, chitosan, gelatin, arabic gum, guar gum, mannitol, sorbitol, sucrose, xanthan gum, polyethylene glycol, and pharmaceutically acceptable salts thereof.

30. The composition of claim 29, wherein the saccharide is selected from alginic acid, carrageenan, carboxymethyl cellulose, xanthan gum, and pharmaceutically acceptable salts thereof.

31. The composition of any one of claims 1-30, wherein the saccharide is present in a range of from about 0.01% to about 5% by weight.

32. The composition of any one of claims 1-30, wherein the saccharide is present in a range of from about 0.1 to about 50 mg/rnL

33. The composition of claim 32, wherein the saccharide is present in a range of from about 1 to about 50 mg/mL.

34. The composition of any one of claims 1-30, wherein the saccharide is carrageenan.

35. The composition of claim 34, wherein the carrageenan is present in a range of from about 0.01% to about 1% by weight.

36. The composition of claim 34, wherein the carrageenan is present in a range of from about 0.1 to about 10 mg/mL.

37. The composition of any one of claims 1-30, wherein the saccharide is sodium carboxymethyl cellulose.

38. The composition of claim 37, wherein the sodium carboxymethyl cellulose is present in at least about 1 mg/mL.

39. The composition of claim 37, wherein the sodium carboxymethyl cellulose is present in a range of from about 0.1% to about 2% by weight.

40. The composition of claim 37, wherein the sodium carboxymethyl cellulose is present in a range of from about 1 to about 20 mg/mL.

41. The composition of any one of claims 1-30, wherein the saccharide is xanthan gum.

42. The composition of claim 41, wherein the xanthan gum is present in a range of from about 0.1% to about 1% by weight.

43. The composition of claim 41, wherein the xanthan gum is present in at least about 1 mg/mL.

44. The composition of claim 41, wherein the xanthan gum is present in a range of from about 1 to about 10 mg/mL.

45. The composition of any one of claims 1-30, wherein the saccharide is sodium alginate.

46. The composition of claim 45, wherein the sodium alginate is present in a range of from about 0.02% to about 5% by weight.

47. The composition of claim 45, wherein the sodium alginate is present in a range of from about 0.2 to about 50 mg/mL.

48. The composition of claim 47, wherein the sodium alginate is present in a range of from about 10 to about 30 mg/mL.

49. The composition of claim 45, wherein the sodium alginate has a viscosity in a range of from about 1 to about 200 mPa-sec.

50. The composition of claim 44, wherein the sodium alginate has a viscosity of no more than about 20 mPa-sec.

51. The composition of claim 45, wherein the sodium alginate has a molecular weight in a range of from about 1,000 to about 200,000 Daltons.

52. The composition of claim 45, wherein the sodium alginate has a molecular weight of no more than about 75,000 Daltons.

53. The composition of claim 45, wherein the sodium alginate comprises a ratio of guluronate to mannuronate monomers of less than about 1.5:1.

54. The composition of claim 45, wherein the sodium alginate comprises a ratio of guluronate to mannuronate monomers of no more than about 1:1.

55. The composition of any one of claims 1-54, wherein the composition has a viscosity in a range of from about 10 to about 400 cPa at a shear rate of about 1000 sec ¹.

56. The composition of any one of claims 1-54, wherein the composition has a viscosity of about 200 cPa at a shear rate of about 1000 sec ¹.

57. An aqueous composition comprising:

from about 5 mg/mL to about 15 mg/mL hyaluronic acid or pharmaceutically acceptable salt thereof;

from about 10 mg/mL to about 30 mg/mL sodium alginate;

from about 0.5 mg/mL to about 5 mg/mL histidine;

a phosphate buffer; and

a tonicity agent;

wherein the pH of the solution is from about 6.5 to about 7.5.

58. The composition of claim 57, wherein the sodium alginate comprises a ratio of guluronate to mannuronate monomers of less than about 1:1, and has a viscosity of no more than about 20 mPa-sec.

59. An aqueous composition comprising:

from about 10 mg/mL to about 30 mg/mL sodium alginate;

from about 0.5 mg/mL to about 5 mg/mL methionine;

a phosphate buffer; and

a tonicity agent;

wherein the pH of the solution is from about 6.5 to about 7.5.

60. The composition of claim 59, wherein the sodium alginate comprises a ratio of guluronate to mannuronate monomers of less than about 1:1, and has a viscosity of no more than about 20 mPa-sec.

61. The composition of any one of claims 1-60 that, after being subject to oxidative stress conditions, has at least about 10% higher viscosity as compared to an equivalent amount of a comparative hyaluronic acid composition that does not comprise antioxidant or saccharide;

wherein oxidative stress conditions comprise subjecting the composition to about 0.02% hydrogen peroxide for about an hour at room temperature.

62. The composition of any one of claims 1-60 that, after being subject to enzymatic stress conditions, has at least about 10% higher viscosity as compared to an equivalent amount of a comparative hyaluronic acid composition that does not comprise antioxidant or saccharide;

wherein enzymatic stress conditions comprise subjecting the composition to about 0.001 mg/mL hyaluronidase for about an hour at room temperature.

63. A method of treating osteoarthritis, the method comprising administering to a human patient in need thereof a therapeutically effective amount of an aqueous composition according to any one of claims 1-62.

64. The method of claim 63, wherein the method comprises intra-articular injection of the composition into the patient.