Hyaluronic acid-antioxidant conjugates for treating hearing loss

Abstract

Disclosed herein are compositions comprising hyaluronic acid-antioxidant conjugates, wherein a low molecular weight hyaluronic acid is used in said conjugates. These compositions can be used to prevent or treat subjects suffering from hearing loss, such as oxidative stress-mediated hearing loss.

Description

Hyaluronic Acid-Antioxidant Conjugates for Treating Hearing LossRELATED APPLICATION INFORMATION

[0001] This application claims priority to U.S. Application No. 63 / 607,463 filed on December 7, 2023, the contents of which are herein incorporated by reference.FIELD

[0002] The present disclosure relates to compositions comprising hyaluronic acid-antioxidant conjugates, wherein a low molecular weight hyaluronic acid is used in said conjugates. The compositions of the present disclosure can be used to prevent or treat subjects suffering from hearing loss, such as oxidative stress-mediated hearing loss.GOVERNMENT FUNDING INFORMATION

[0003] This invention was made with government support under P20 GM 103546 awarded by the National Institutes of Health NIGMS and N00014-20-1-2874 awarded by the Department of the Navy Office of Naval Research. The government has certain rights in the invention.BACKGROUND

[0004] Hearing loss affects over 5% of the global population or roughly 430 million people.1Some causative agents include drugs like chemotherapeutics or aminoglycosides, diseases such as viral or bacterial infections, natural aging processes, loud noises, and repeated ear infections.1,2Based on the ear structures affected, hearing loss can be categorized into three types: conductive, sensorineural (SNHL), or mixed. SNHL is the most common type of hearing loss and involves permanent damage to inner ear structures including the organ of Corti, auditory nerve, or central nervous system.3,4Regardless of the causative agents, the mechanisms of SNHL invariably involve the production of reactive oxygen species (ROS), inflammation, glutamate excitotoxicity, calcium overload and disturbances to energy metabolism.5Therefore, many studies focused on developing therapeutics or prophylactics against SNHL target the reduction of ROS and / or inflammation.5-14Nevertheless, there are still no FDA-approved prophylactics or therapeutics against hearing loss, and the only approved treatment option is a cochlear implant requiring an invasive medical procedure.6,15,16

[0005] Previous animal studies have highlighted the potential value of using antioxidants to mitigate SNHL.13,17-19In these studies, the primary challenges were reaching a high enough therapeutic concentration of antioxidant within the cochlea.6,20In order to reach the cochlea via systemic delivery, a therapeutic would need to cross the blood-labyrinth barrier, which similarly to the blood-brain barrier, poses significant hurdles to adequate drug availability. Conversely, topical drug delivery would require drug passage across the tympanic membrane (TM), a thin tissue that separates the outer ear and middle ear, and the round window membrane (RWM), which separates the middle ear and inner ear.5-7,20The mechanism of ROS production in noise- induced hearing loss (NIHL), a type of SNHL, has been previously investigated.21-25Acoustic trauma leads to mechanical damage, such as rupture and / or displacement of the TM, displacement of the basilar membrane, shearing of the stereocilia of hair cells, and injury to the supporting cochlear cells.26-28In addition to mechanical damage, pro-inflammatory cytokines are released which initiate additional inflammatory and cellular death pathways, as well as ROS generation.22,23,28-33This subsequently leads to permanent damage to cochlear structures including the organ of Corti.29-31Repetitive acoustic trauma, was also shown to lead to swelling of the stria vascularis, resulting in additional generation of ROS. Although animal studies have explored the successful use of antioxidants to target ROS associated with NIHL, and the mechanism of ROS generation is understood, human data lag.21,34,35As previously mentioned, adequate drug delivery of prophylactics or treatments to the cochlea where the ROS production occurs, is challenging. Thus, there is a need for improved therapeutics that can be delivered successfully to the to prevent and / or treat conditions such as NIHL where ROS production occurs.SUMMARY

[0006] In one embodiment, the present disclosure relates to a composition comprising: a hyaluronic acid (HA)-antioxidant conjugate, wherein the antioxidant is not methionine.

[0007] In some aspects, the antioxidant is a nutritional antioxidant, a phytochemical antioxidant, an enzyme, or any combination thereof.

[0008] In other aspects, the antioxidant: (a) contains at least one sulfur containing moiety and at least one primary amine; or (b) is a compound that can be hydrolyzed or reduced to produce compounds containing at least one sulfur atom and at least one primary amine group. In someaspects, the antioxidant is glutathione, taurine, cysteine, or combinations thereof. In still other aspects, the antioxidant is glutathione. In yet further aspects, the antioxidant is cysteine.

[0009] In still further aspects, the conjugate, when administered to a subject, permeates the tympanic membrane and round window membrane in a subject to reach the cochlea.

[0010] In yet other aspects of the above composition, the hyaluronic acid is a low molecular weight hyaluronic acid, a medium molecular weight hyaluronic acid, or a high molecular weight hyaluronic acid. In other aspects, the hyaluronic acid is a low molecular weight hyaluronic acid. In still other aspects, the conjugate has a molecular weight of from about 25,000 Daltons to about 40,000 Daltons, from about 25,000 Daltons to about 38,000 Daltons or from 26,000 Daltons to about 35,000 Daltons.

[0011] In still further aspects, the composition further comprises one or more pharmaceutically acceptable excipients.

[0012] In another embodiment, the present disclosure relates to a method of treating oxidative stress-mediated hearing loss in a subject. The method comprises administering to a subject suffering from oxidative stress-mediated hearing loss and in need of treatment thereof, a therapeutically effective amount of the above composition.

[0013] In some aspects of the above method, the oxidative stress-mediated hearing loss is noise- induced hearing loss (NIHL).

[0014] In some aspects, the composition is administered topically. In other aspects, it is administered parenterally. In still further aspects, it is administered orally.

[0015] In some aspects, the subject being treated according to the method is a human.

[0016] In still other aspects, the subject being treated according to the method is a non-human mammal, a bird, a reptile, or a fish. In yet further aspects, the non-human mammal is a non- human primate, a dog, a cat, a rabbit, a guinea pig, a ferret, a hamster, or a horse.

[0017] In still further aspects of the above method, the oxidative stress-mediated hearing loss is caused by noise, aging, chemotherapy, bacterial infection, viral infection, or any combination thereof.BRIEF DESCRIPTION OF THE FIGURES

[0018] Figure 1 shows HA MW effects on cellular internalization and membrane permeation. Figure 1 A shows internalization of 24 or 240 kDa HA-BODIPY in HEI-OC1 cells over 4 hrs. Cells were grown to confluence in 6-well plates and treated with 250 pg / mL HA-BODIPY. Results were normalized using a BCA assay to correct for technical variability.#pg of HA- BODIPY per mg of total protein lysate, n = 6; Welch’s T-test. Figure IB shows the percent of 24 or 240 kDa HA-BODIPY which permeated across TM and RWM permeation models over 24 hrs. TM and RWM models were placed in permeation devices and HA-BODIPY (0.1 mL of 6.7 mg / mL) was placed on apical surface. After 24 hrs, the basal solution was collected and permeated amount was determined, n = 7; multiple Welch’s T-tests with Holm-Sidak correction. (Figure 1A, Figure IB) *p<0.05, ****p<0.0001, ns = not significant. HA, hyaluronan; RWM, round window membrane; TM, tympanic membrane; MW, molecular weight.

[0019] Figure 2 shows a reaction scheme and cytocompatibility of HA-antioxidant conjugates. Figure 2A shows the reaction scheme used for synthesis of HA-antioxidant conjugates. R, rest of the molecule. Figure 2B shows a MTS cytocompatibility assay of various molecular weights of 1.5 mg / mL CMHA or M-HA applied to HEI-OC1 cells for 24 hrs. n = 5. Figure 2C shows a MTS cytocompatibility assay of 1.5 mg / mL C-HA and G-HA in HEI-OC1 cells treated for 24 hr. n = 6. (Figure 2B, Figure 2C) One-way ANOVA with Dunnett’s correction; *p<0.05, **p<0.01 ; ns = not significant. C, cysteine; CMHA, carboxy methyl HA; G, reduced glutathione; HA, hyaluronan; M, D-methionine.

[0020] Figure 3 shows acellular ROS-scavenging assays. Figure 3 A shows an acellular peroxide scavenging assay presented in ascorbic acid equivalence units. Each conjugate was tested at 1.5 mg / mL and compared to its respective unconjugated antioxidant at the equivalent concentration determined by its conjugation efficiency. Higher values indicate improved peroxide scavenging, n = 9 Figure 3B shows an acellular superoxide assay. Each conjugate was tested at tested 2 mg / mL, its respective unconjugated antioxidant was tested at the equivalent concentration. AA (1.76 mg / mL) was included as a positive control. Lower values indicate improved superoxide scavenging, n = 12-20. (Figure 3 A, Figure 3B) Brown-Forsythe and Welch one-way ANOVA with Dunnett’s T3 correction; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant. AA, ascorbic acid; C, cysteine; G, reduced glutathione; HA, hyaluronan; M, D- methionine.

[0021] Figure 4 shows cellular ROS-scavenging assays. Cellular peroxide scavenging assays performed by treating HEI-OC1 cells stressed by 1 mM hydrogen peroxide (H2O2) with 1.5 mg / mL of C-HA or G-HA. Conjugates were either treated (Figure 4A) simultaneously with the stressor, or (Figure 4B) as a 24 hr pre-treatment. n = 7; Brown-Forsythe and Welch ANOVA with Dunnett’s T3 correction; **p<0.01, ***p<0.001, ns = not significant. Figure 4C shows cellular superoxide sequestering kinetics in HEI-OC1 cells which were induced to produce superoxides with 10 pM menadione and treated with 1.5 mg / mL C-HA or G-HA. SOD was used as a control to verify the observed response from menadione was due to superoxide formation, n = 8. C, cysteine; G, reduced glutathione; HA, hyaluronan; M, D-methionine, SOD, superoxide dismutase.

[0022] Figure 5 shows Conjugate permeation experiments. Figure 5 A shows permeation of 0.1 mL of 20 mg / mL C-HA or G-HA and the equivalent concentration of their respective unconjugated antioxidants across the in vitro TM and RWM permeation models over a 24 hr period, n = 4 - 8; independent t-tests; *p<0.05, ns = not significant. Figure 5B shows the permeation kinetics of C-HA and G-HA across the RWM model, n = 3 - 4. C, cysteine; G, reduced glutathione; HA, hyaluronan; RWM, round window membrane; TM, tympanic membrane.

[0023] Figure 6 shows the internalization kinetics of 24 kDa HA-BODIPY. Internalization kinetics of 24 kDa HA-BODIPY in HEI-OC1 cells over 24 hr period, n = 4.#pg of HA-BODIPY per mg of total protein lysate. HA, hyaluronan.

[0024] Figure 7 shows ’H-NMR spectra of different molecular weight CMHA. Molecular weight of starting materials from low to high going down in the figure. All NMRs performed in deuterated water. The top spectrum is the HA starting material and the bottom five spectra are after the carboxymethyl addition. The shaded box highlights the peaks from the methylene protons of the carboxymethyl moiety. CMHA, carboxymethyl-HA; HA, hyaluronan.

[0025] Figure 8 shows ’H-NMR spectra of M-HA conjugates using HA starting materials of different molecular weights in ascending order. Molecular weight listed represents the molecular weight of the HA starting material used in the initial CMHA reaction. The shaded box highlights the peak of interest that is present only after D-methionine has been conjugated to CMHA. CMHA, carboxymethyl-HA; M-HA, methionine-HA; HA, hyaluronan.

[0026] Figure 9 shows ’H-NMR spectra of CMHA, HA-Cysteine (C-HA), and HA-glutathione (G-HA) conjugates. NMR performed in deuterated water. The shaded box highlights the peak of interest that is present only after cysteine or glutathione have been conjugated to CMHA. C, cysteine; CMHA, carboxymethyl-HA; G, reduced glutathione; HA, hyaluronan.

[0027] Figure 10 shows LDH cytocompatibility assay of 1.5 mg / mL C-HA and G-HA in HEI- OC1 cells treated for 24 hrs. n = 6, one-way ANOVA with Dunnett’s correction; ns = not significant. C, cysteine, G, reduced glutathione; HA, hyaluronic acid; LDH, lactate dehydrogenase.

[0028] Figure 11 shows tissue viability after conjugate permeation. Tissue viability measured by MTT assay of (Figure 11 A) RWM and (Figure 1 IB) TM permeation models after 24 hr exposure to 20 mg / mL of C-HA, G-HA, or the equivalent concentration of their respective unconjugated antioxidants. 5% SDS was used as a positive control for toxicity, n = 3-6; one-way ANOVA with Dunnett’s correction vs control; **p<0.01; ****p<0.0001; only significant differences from control are indicated. C, cysteine; G, reduced glutathione; HA, hyaluronan; RWM, round window membrane; TM, tympanic membrane.DETAILED DESCRIPTION

[0029] Provided herein are compositions containing at least one hyaluronic acid-antioxidant conjugate, wherein the conjugate comprises a low molecular weight hyaluronic acid. The compositions described herein can be used to prevent or treat subjects suffering from hearing loss, such as oxidative stress-mediated hearing loss.DEFINITIONS

[0030] The following terms are used to describe the invention of the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecularbiology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0032] Compounds and materials are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. The following terms are used to describe the invention of the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

[0033] The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. By way of example, "an element" means one element or more than one element.

[0034] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise. Furthermore, the terms first, second, etc., as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers.

[0035] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and", and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0036] The terms “about” or “approximately,” as used herein, is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined byone of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within ± 10% or 5% of the stated value. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1 -2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0037] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0038] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or "exactlyone of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0039] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0040] The phrase "one or more," as used herein, means at least one, and thus includes individual components as well as mixtures / combinations of the listed components in any combination.

[0041] Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients and / or reaction conditions are to be understood as being modified in all instances by the term "about," meaning within 10% of the indicated number (e.g., "about 10%" means 9%-l 1% and "about 2%" means 1.8%-2.2%).

[0042] All percentages and ratios are calculated by weight unless otherwise indicated. All percentages are calculated based on the total composition unless otherwise indicated. Generally, unless otherwise expressly stated herein, "weight" or "amount" as used herein with respect to the percent amount of an ingredient refers to the amount of the raw material comprising the ingredient, wherein the raw material may be described herein to comprise lessthan and up to 100% activity of the ingredient. Therefore, weight percent of an active in a composition is represented as the amount of raw material containing the active that is used and may or may not reflect the final percentage of the active, wherein the final percentage of the active is dependent on the weight percent of active in the raw material.

[0043] All ranges and amounts given herein are intended to include subranges and amounts using any disclosed point as an end point. Thus, a range of "1% to 10%, such as 2% to 8%, such as 3% to 5%," is intended to encompass ranges of "1% to 8%," "1% to 5%," "2% to 10%," and so on. All numbers, amounts, ranges, etc., are intended to be modified by the term "about," whether or not so expressly stated. Similarly, a range given of "about 1% to 10%" is intended to have the term "about" modifying both the 1% and the 10% endpoints. Further, it is understood that when an amount of a component is given, it is intended to signify the amount of the active material unless otherwise specifically stated.

[0044] As used herein, the term "administering" means either directly administering a compound, composition, or dosage form of the present disclosure or administering a prodrug, derivative or analog which will form an equivalent amount of the active compound(s), active ingredient(s), or substance(s) within the body of a subject or patient.

[0045] As used herein, the term “antioxidant” refers to compounds or substances that neutralize free radicals by donating electrons, to prevent cellular damage and breaking the chain reactions initiated by these unstable molecules. This protective action is vital for maintaining cellular health and preventing oxidative stress. Antioxidants work by stabilizing free radicals through electron donation. Free radicals are highly reactive due to their unpaired electrons; they seek to stabilize themselves by stealing electrons from nearby molecules, which can lead to further cellular damage. By donating an electron to these free radicals, antioxidants effectively neutralize them without becoming harmful themselves. Examples of antioxidants that can be used herein include: (a) nutrient antioxidants (e.g., vitamins such as vitamin C (ascorbic acid) and vitamin E (tocopherol) and / or minerals like selenium, copper, and zinc), (b) phytochemical antioxidants (e.g., plant-derived compounds, such as flavonoids and / or carotenoids); (c) enzymes (e.g., such as superoxide dismutase and / or catalase); or (d) any combinations of (a)-(c). In some aspects, the antioxidant is an antioxidant containing at least one sulfur-containing compound and at least one primary amine.

[0046] As used herein, the phrase “antioxidant containing at least one sulfur-containing moiety and at least one primary amine” refers to a compound that contains both at least one sulfur atom and at least one primary amine group (-NH2) in its molecular structure or a compound that can be hydrolyzed or reduced to provide a compound containing at least one sulfur atom and at least one primary amine group (-NH2) in its molecular structure. Examples of antioxidants containing at least one sulfur-containing moiety and at least one primary amine include glutathione, taurine, cysteine, or any combinations thereof. Glutathione is a tripeptide composed of glutamate, cysteine, and glycine. It contains both a sulfhydryl group from cysteine and a primary amine from glycine. Glutathione is a major cellular antioxidant and plays a critical role in maintaining redox balance. Taurine is a sulfur-containing compound with antioxidant properties. Specifically, taurine contains a sulfonic acid group (-SO3H) and a primary amine. Cysteine contains at least one sulfur atom and at least one primary amine.

[0047] Examples of compounds that can be hydrolyzed or reduced to provide a compound containing at least one sulfur atom and at least one primary amine group (-NH2) in its molecular structure is N-acetylcysteine (NAC), derivatives of lipoic acid and combinations thereof. N-acetylcysteine is a derivative of cysteine with antioxidant properties. N- acetylcysteine does not have at least contain a least one primary amine group, but can be hydrolyzed to release cysteine, which contains at least one primary amine. Derivatives of lipoic acid can be hydrolyzed or reduced to produce compounds containing at least one sulfur atom and at least one primary amine group (-NH2) in their molecular structure. Lipoic acid contains a disulfide bond that can be reduced to form dihydrolipoic acid (DHLA), which has two thiol (-SH) groups. Additionally, lipoic acid is often found covalently bound to lysine residues in proteins, forming lipoyllysine. Enzymatic hydrolysis of these protein-bound lipoic acid molecules can release lipoyllysine, which contains both sulfur atoms from lipoic acid and the primary amine group of lysine. The reduction of lipoic acid to DHLA can occur through various enzymatic pathways, including glutathione reductase, thioredoxin reductase, and lipoamide dehydrogenase. These reduced forms maintain the sulfur atoms while potentially exposing or creating amine groups, depending on the specific derivative and reduction process.

[0048] Furthermore, synthetic derivatives of lipoic acid can be designed to incorporate additional amine groups or to facilitate the formation of amine-containing compounds upon hydrolysis or reduction. These modifications can be tailored to enhance specific properties orfunctions of the lipoic acid molecule while maintaining its core structure, including the sulfur atoms.

[0049] As used herein, the phrase “carboiimide reaction” refers to a chemical process that utilizes compounds containing the functional group RN=C=NR to facilitate the formation of amide bonds between carboxylic acids and amines. This reaction is widely employed in organic synthesis and biochemistry for various purposes, including peptide synthesis, protein modification, and the creation of bioconjugates. Carbodiimide reactions proceed through the following steps: (1) the carbodiimide activates the carboxyl group by forming an unstable O- acylisourea intermediate; (2) this activated intermediate then reacts with a primary amine to form an amide bond; and (3) the reaction yields the desired amide product and a urea derivative as a byproduct. Examples of some commonly used carbodiimides include: (a) EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide): water-soluble, suitable for aqueous reactions; (b) DCC (N,N' -dicyclohexylcarbodiimide): used in solid-phase and non-aqueous reactions; and (c) DIC (N,N'-diisopropylcarbodiimide): used in solid-phase synthesis due to easier purification.

[0050] As used herein, the term “hyaluronic acid” (HA), or “hyaluronan” as used interchangeably herein, refers to an anionic, nonsulfated glycosaminoglycan widely distributed throughout connective, epithelial, and neural tissues. Hyaluronic acid is a linear polymer composed of alternating units of D-glucuronic acid and N-acetyl-D-glucosamine, linked via 0- (1— >4) and 0-(l — >3) glycosidic bonds. Hyaluronic acid is unique among glycosaminoglycans due to its non-sulfated nature and its formation in the plasma membrane rather than the Golgi apparatus. It plays crucial roles in various biological processes, including maintaining tissue hydration, lubrication of joints, and facilitating cell migration during wound healing and embryonic development.

[0051] As used herein, the term “low molecular weight hyaluronic acid” or “LMW-HA” refers to hyaluronic acid having a molecular range from about 20,000 to about 300,000 Daltons (kDa). Low molecular weight hyaluronic acids are known to possess the ability to penetrate into the skin, potentially reaching the bottom of the epidermis of the skin. “Medium molecular weight hyaluronic acid” as used herein, refers to hyaluronic acid having a molecular weight above about 300,000 to about 1,000,000 Daltons (kDa). Medium molecular weight hyaluronic acids are known to penetrate to the center of the epidermis of skin. As used herein,“high molecular weight hyaluronic acid” or “HMW-HA” refers to hyaluronic acids having a molecular weight greater than about 1,000,000 to about 1,400,000 Daltons (kDa). High molecular weight hyaluronic acids are known to primarily act on the surface of the skin creating a film-like barrier.

[0052] As used herein, the phrase “primary amine” refers to an organic compound derived from ammonia (NH3) in which one hydrogen atom has been replaced by an alkyl or aryl group. The general structure of a primary amine can be represented as R-NHi, where R is an alkyl or aryl group, and the amino group (-NH2) is directly bonded to one carbon atom. Key characteristics of primary amines include: (1) they contain one organic substituent attached to the nitrogen atom; (2) the nitrogen atom is bonded to two hydrogen atoms and one carbon atom; and (3) the carbon atom to which the amino group is attached cannot be part of a carbonyl group. Primary amines can be classified into two categories: (1) aliphatic primary amines, which are amines that have an alkyl group attached to the nitrogen atom. Examples include methylamine (CH3NH2) and amino acids; and (2) aromatic primary amines in which a nitrogen atom is directly attached to an aromatic ring. Examples include aniline (C6H5NH2).

[0053] The term “subject” or “patient” is used herein to refer to an animal, such as (a) a mammal, including a primate (such as a human) or a non-human mammal (such as a nonhuman primate, (such as a monkey, an ape, a new world primates, or prosimian), cow, a pig, a camel, a llama, a horse, a donkey, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a ferret, a mouse, and a whale), (b) a bird (e.g., a duck or a goose), (c) a reptile (e.g., snake, lizards, turtles, iguanas, crocodile / alligator, geckos), or (d) a fish (e.g., a shark). In an embodiment, the subject or patient is a human subject or a human patient, such as a human being treated or assessed for a disease, disorder or condition, a human at risk for a disease, disorder or condition, a human having a disease, disorder or condition, and / or human being treated for a disease, disorder or condition as described herein. In one embodiment, the subject is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age. In another embodiment, the subject is about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70- 75, 75-80, 80-85, 85-90, 90-95, 95-100 years of age. Values and ranges intermediate to the above recited ranges are also intended to be part of this invention. In addition, ranges of values using a combination of any of the above-recited values as upper and / or lower limits are intended to be included. As used herein, a subject is “in need of treatment” if such subjectwould benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0054] The phrase, “pharmaceutically acceptable”, whether by itself or in conjunction with another term or terms, indicates that the designated entity such as, for example, a pharmaceutically acceptable excipient, is generally chemically and / or physically compatible with other ingredients in a composition, and / or is generally physiologically compatible with the recipient thereof.

[0055] As used herein, the phrase, “pharmaceutically acceptable excipient” refers to a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a vehicle, carrier, or diluent to facilitate administration of an active ingredient or agent and that is compatible therewith. Pharmaceutically acceptable excipients will be known to those of skill in the art, and include, for example, excipients described in treatises such as, for example, Paul J. Sheskey, et al. (eds), Handbook of Pharmaceutical Excipients, Pharmaceutical Press,' 9th Revised edition (October 20, 2020).

[0056] As used herein, whether by itself or in conjunction with another term or terms, it should be understood that the phrases "method of treating" and "method of treatment" may be used interchangeably with the phrase "for use in the treatment of a particular disorder or disease”.

[0057] As used herein, whether used alone or in conjunction with another term or terms, "therapeutic" and "therapeutically effective amount" refer to an amount of a compound or composition that (a) treats a particular condition, symptom, disorder, or disease described herein; (b) attenuates, ameliorates, or eliminates one or more symptoms of a particular condition, disorder, or disease described herein; (c) delays the onset or relapse (reoccurrence) of a particular condition, symptom, disorder, or disease described herein; (d) prevents the onset of a particular condition, symptom, disorder, or disease described herein. It should be understood that the terms "therapeutic" and "therapeutically effective" encompass any one of the aforementioned effects (a)-(d), either alone or in combination with any of the others (a)-(d).

[0058] As used herein, whether by themselves or in conjunction with another term or terms, "treats," "treating," "treated," and "treatment," refer to and include ameliorative, palliative,and / or curative uses and results, or any combination thereof. In other embodiments, the methods described herein can be used prophylactically, that is, preventatively. It should be understood that "prophylaxis" or a prophylactic use or result do not refer to nor require absolute or total prevention (i.e., a 100% preventative or protective use or result). As used herein, prophylaxis or a prophylactic (preventative) use or result refers to uses and results in which administration of a compound or composition diminishes or reduces the severity of a particular condition, symptom, disorder, or disease described herein; diminishes or reduces the likelihood of experiencing a particular condition, symptom, disorder, or disease described herein; or delays the onset or relapse (reoccurrence) of a particular condition, symptom, disorder, or disease described herein; or any combination of the foregoing.

[0059] A used herein, the phrase, “zero-length cross-linker” refers to a type of chemical reagent that is used to directly conjugate two molecules without introducing any additional atoms between the molecules. These cross-linkers facilitate the formation of a bond between two functional groups on different molecules or within the same molecule, effectively creating a "zero-length" spacer. Examples of zero-length cross-linkers include carbodiimides, such as EDC (l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide). EDC functions by activating carboxyl groups (-COOH) to form an intermediate that can then react with primary amines (-NH2), resulting in the formation of an amide bond. This reaction occurs without the cross-linker becoming part of the final linkage, hence the term "zero-length". Some key characteristics of zero-length cross-linkers include: (1) direct conjugation: they enable the formation of a direct bond between two molecules without adding any spacer atoms; (2) specific reactivity: they typically target specific functional groups, such as carboxyls and primary amines in the case of EDC; (3) minimal structural alteration: by not introducing additional atoms, they minimize changes to the overall structure of the conjugated molecules; (4) versatility: zero-length crosslinkers can be used for various applications, including studying protein-protein interactions, creating enzyme-substrate complexes, and investigating protein architecture; and watersolubility: many zero-length cross-linkers, like EDC, are water-soluble, making them suitable for use in aqueous biological systems.

[0060] As used herein, the phrase “zero-length crosslinking reaction” refer to reactions that use zero-length crosslinkers to form direct bonds between molecules without introducing additional atoms, providing precise distance constraints. Examples of zero-length crosslinkingreactions include: (a) carbodiimide-mediated crossing or a carboiimide reaction; (b) Woodward’s Reagent K; (c) solid-state dehydration; or (d) photo-induced cross-linking.I. Hyaluronic Acid-Antioxidant Conjugate containing Compositions

[0061] In one embodiment, the present disclosure relates to compositions comprising at least one hyaluronic acid (HA)-antioxidant conjugate. In some aspects, the composition is a pharmaceutical composition containing a therapeutically effective amount of at least one HA- antioxidant conjugate. The compositions of the present disclosure containing said conjugates can be used to treat subjects suffering from hearing loss, such as, for example, noise-induced hearing loss (NIHL). Specifically, when administered to a subject as described in Section II, the at least one HA-antioxidant conjugate permeates the round window membrane (RWM) and tympanic membrane (TM) in the subject to reach the cochlea and protects cochlear cells against oxidative stress by reducing or neutralizing reactive oxygen species (ROS).

[0062] In some aspects, the hyaluronic acid used in the HA-antioxidant conjugate has an optimized molecular weight which allows effective permeation of the RWM and TM. Specifically, it was found that the molecular weight of HA affected the ability of the conjugate to effectively permeate in vitro permeation models of the TM and RWM41’43as well as its cellular internalization into cochlear cells. Thus, as a result, the HA used in the conjugate of the present disclosure in these aspects, is a low molecular weight hyaluronic acid (e.g., has a molecular weight of from about 20,000 Daltons to about 300,000 Daltons). In some aspects, the HA has a molecular weight of from about 20,000 Daltons to about 275,000 Daltons, from about 20,000 Daltons to about 250,000 Daltons, from about 20,000 Daltons to about 225,000 Daltons, from about 20,000 Daltons to about 200,000 Daltons, from about 20,000 Daltons to about 175,000 Daltons, from about 20,000 Daltons to about 150,000 Daltons, from about 20,000 Daltons to about 125,000 Daltons, from about 20,000 Daltons to about 100,000 Daltons, from about 20,000 Daltons to about 75,000 Daltons, from about 20,000 Daltons to about 50,000 Daltons, or from about 20,000 Daltons to about 40,000 Daltons. In another aspect, the HA has a molecular weight of about 20,000 Daltons, about 21,000 Daltons, about 22,000 Daltons, about 23,000 Daltons, about 24,000 Daltons, about 25,000 Daltons, about 26,000 Daltons, about 27,000 Daltons, about 28,000 Daltons, about 29,000 Daltons, about 30,000 Daltons, about 31,000 Daltons, about 32,000 Daltons, about 33,000 Daltons, about,000 Daltons, about 35,000 Daltons, about 36,000 Daltons, about 37,000 Daltons, about,000 Daltons, about 39,000 Daltons, about 40,000 Daltons, about 41,000 Daltons, about,000 Daltons, about 43,000 Daltons, about 44,000 Daltons, about 45,000 Daltons, about,000 Daltons, about 47,000 Daltons, about 48,000 Daltons, about 49,000 Daltons, about,000 Daltons, about 51,000 Daltons, about 52,000 Daltons, about 53,000 Daltons, about,000 Daltons, about 55,000 Daltons, about 56,000 Daltons, about 57,000 Daltons, about,000 Daltons, about 59,000 Daltons, about 60,000 Daltons, about 61,000 Daltons, about,000 Daltons, about 63,000 Daltons, about 64,000 Daltons, about 65,000 Daltons, about,000 Daltons, about 67,000 Daltons, about 68,000 Daltons, about 69,000 Daltons, about,000 Daltons, about 71,000 Daltons, about 72,000 Daltons, about 73,000 Daltons, about,000 Daltons, about 75,000 Daltons, about 76,000 Daltons, about 77,000 Daltons, about,000 Daltons, about 79,000 Daltons, about 80,000 Daltons, about 81,000 Daltons, about,000 Daltons, about 83,000 Daltons, about 84,000 Daltons, about 85,000 Daltons, about,000 Daltons, about 87,000 Daltons, about 88,000 Daltons, about 89,000 Daltons, about,000 Daltons, about 91,000 Daltons, about 92,000 Daltons, about 93,000 Daltons, about,000 Daltons, about 95,000 Daltons, about 96,000 Daltons, about 97,000 Daltons, about,000 Daltons, about 99,000 Daltons, about 100,000 Daltons, about 101,000 Daltons, about2,000 Daltons, about 103,000 Daltons, about 104,000 Daltons, about 105,000 Daltons, about6,000 Daltons, about 107,000 Daltons, about 108,000 Daltons, about 109,000 Daltons, about0,000 Daltons, about 111,000 Daltons, about 112,000 Daltons, about 113,000 Daltons, about4,000 Daltons, about 115,000 Daltons, about 116,000 Daltons, about 117,000 Daltons, about8,000 Daltons, about 119,000 Daltons, about 120,000 Daltons, about 121,000 Daltons, about2,000 Daltons, about 123,000 Daltons, about 124,000 Daltons, about 125,000 Daltons, about6,000 Daltons, about 127,000 Daltons, about 128,000 Daltons, about 129,000 Daltons, about0,000 Daltons, about 131,000 Daltons, about 132,000 Daltons, about 133,000 Daltons, about4,000 Daltons, about 135,000 Daltons, about 136,000 Daltons, about 137,000 Daltons, about8,000 Daltons, about 139,000 Daltons, about 140,000 Daltons, about 141,000 Daltons, about2,000 Daltons, about 143,000 Daltons, about 144,000 Daltons, about 145,000 Daltons, about6,000 Daltons, about 147,000 Daltons, about 148,000 Daltons, about 149,000 Daltons, about0,000 Daltons, about 151,000 Daltons, about 152,000 Daltons, about 153,000 Daltons, about4,000 Daltons, about 155,000 Daltons, about 156,000 Daltons, about 157,000 Daltons, about,000 Daltons, about 159,000 Daltons, about 160,000 Daltons, about 161,000 Daltons, about,000 Daltons, about 163,000 Daltons, about 164,000 Daltons, about 165,000 Daltons, about,000 Daltons, about 167,000 Daltons, about 168,000 Daltons, about 169,000 Daltons, about,000 Daltons, about 171,000 Daltons, about 172,000 Daltons, about 173,000 Daltons, about,000 Daltons, about 175,000 Daltons, about 176,000 Daltons, about 177,000 Daltons, about,000 Daltons, about 179,000 Daltons, about 180,000 Daltons, about 181,000 Daltons, about,000 Daltons, about 183,000 Daltons, about 184,000 Daltons, about 185,000 Daltons, about,000 Daltons, about 187,000 Daltons, about 188,000 Daltons, about 189,000 Daltons, about,000 Daltons, about 191,000 Daltons, about 192,000 Daltons, about 193,000 Daltons, about,000 Daltons, about 195,000 Daltons, about 196,000 Daltons, about 197,000 Daltons, about,000 Daltons, about 199,000 Daltons, about 200,000 Daltons, about 201,000 Daltons, about,000 Daltons, about 203,000 Daltons, about 204,000 Daltons, about 205,000 Daltons, about,000 Daltons, about 207,000 Daltons, about 208,000 Daltons, about 209,000 Daltons, about,000 Daltons, about 211,000 Daltons, about 212,000 Daltons, about 213,000 Daltons, about,000 Daltons, about 215,000 Daltons, about 216,000 Daltons, about 217,000 Daltons, about,000 Daltons, about 219,000 Daltons, about 220,000 Daltons, about 221,000 Daltons, about,000 Daltons, about 223,000 Daltons, about 224,000 Daltons, about 225,000 Daltons, about,000 Daltons, about 227,000 Daltons, about 228,000 Daltons, about 229,000 Daltons, about,000 Daltons, about 231,000 Daltons, about 232,000 Daltons, about 233,000 Daltons, about,000 Daltons, about 235,000 Daltons, about 236,000 Daltons, about 237,000 Daltons, about,000 Daltons, about 239,000 Daltons, about 240,000 Daltons, about 241,000 Daltons, about,000 Daltons, about 243,000 Daltons, about 244,000 Daltons, about 245,000 Daltons, about,000 Daltons, about 247,000 Daltons, about 248,000 Daltons, about 249,000 Daltons, about,000 Daltons, about 251,000 Daltons, about 252,000 Daltons, about 253,000 Daltons, about,000 Daltons, about 255,000 Daltons, about 256,000 Daltons, about 257,000 Daltons, about,000 Daltons, about 259,000 Daltons, about 260,000 Daltons, about 261,000 Daltons, about,000 Daltons, about 263,000 Daltons, about 264,000 Daltons, about 265,000 Daltons, about,000 Daltons, about 267,000 Daltons, about 268,000 Daltons, about 269,000 Daltons, about,000 Daltons, about 271,000 Daltons, about 272,000 Daltons, about 273,000 Daltons, about,000 Daltons, about 275,000 Daltons, about 276,000 Daltons, about 277,000 Daltons, about,000 Daltons, about 279,000 Daltons, about 280,000 Daltons, about 281,000 Daltons, about282,000 Daltons, about 283,000 Daltons, about 284,000 Daltons, about 285,000 Daltons, about286,000 Daltons, about 287,000 Daltons, about 288,000 Daltons, about 289,000 Daltons, about290,000 Daltons, about 291,000 Daltons, about 292,000 Daltons, about 293,000 Daltons, about294,000 Daltons, about 295,000 Daltons, about 296,000 Daltons, about 297,000 Daltons, about298,000 Daltons, about 299,000 Daltons, about 300,000 Daltons. In some aspects, HA has a molecular weight of about 25,000 Daltons to about 40,000 Daltons. In still other aspects, the HA has a molecular weight of from about 25,000 Daltons to about 38,000 Daltons. In still other aspects, the HA has a molecular weight of about 26,000 Daltons to about 35,000 Daltons. In still other aspects, the HA has a molecular weight of about 26,000 Daltons. In still other aspects, the HA has a molecular weight of 31,000 Daltons. In yet still other aspects, the HA has a molecular weight of about 32,000 Daltons. In yet still other aspects, the HA has a molecular weight of about 33,000 Daltons. In still yet another aspect, the HA has a molecular weight of about 34,000 Daltons. In still yet a further aspect, the HA has a molecular weight of about 35,000 Daltons.

[0063] In other aspects, the HA used in the conjugate is a medium molecular weight hyaluronic acid (e.g., has a molecular weight greater than from about 300,000 to about 1,000,000). In some aspects, the HA has a molecular weight of greater than about 300,000 (e.g., a molecular weight of about 303,005) to about 1,000,000. In other aspects, the HA has a molecular weight of greater than about 300,000 to about 900,000. In yet other aspects, the HA has a molecular weight of greater than about 300,000 to about 800,000. In still yet other aspects, the HA has a molecular weight of greater than about 300,000 to about 700,000. In still yet other aspects, the HA has a molecular weight of greater than about 300,000 to about 600,000. In still yet further aspects, the HA has a molecular weight of greater than about 300,000 to about 500,000. In still yet further aspects, the HA has a molecular weight of greater than about 300,000 to about 400,000.

[0064] In still other aspects, the HA used in the conjugate is a high molecular weight hyaluronic acid (e.g., has a molecular weight greater than from about 1,000,000 to about 1,400,000). In some aspects, the HA has a molecular weight of greater than about 1,000,000 (e.g., a molecular weight of about 1,100,005) to about 1,400,000. In other aspects, the HA has a molecular weight of greater than about 1,000,000 to about 1,300,000. In yet other aspects, the HA has a molecular weight of greater than about 1,100,000 to about 1,200,000.

[0065] In some aspects, the at least one antioxidant used in the composition is a nutritional antioxidant, a phytochemical antioxidant, an enzyme, or any combination thereof. Examples of nutritional antioxidants that can be used include vitamins such as vitamin C (ascorbic acid) and / or vitamin E (tocopherol) and / or minerals like selenium, copper, and zinc. Examples of phytochemical antioxidant that can be used include plant-derived compounds, such as flavonoids and / or carotenoids. Examples of enzymes that can be used include superoxide dismutase and / or catalase.

[0066] In some aspects, the at least one antioxidant used in the composition contains at least one sulfur containing moiety and at least one primary amine. In some aspects, the antioxidant is glutathione, taurine, cysteine, or combinations thereof. In yet other aspects, the antioxidant is glutathione. In other aspects, the antioxidant is taurine. In still other aspects, the antioxidant is cysteine. In still yet further aspects, the antioxidant used in the conjugate is not methionine. In still yet further aspects, the antioxidant used in the conjugate is not D-methionine. In still yet further aspects, the antioxidant used in the conjugate is not L-methionine.

[0067] In still other aspects, the at least one antioxidant used is the composition is a compound that has been hydrolyzed or reduced to produce a compound containing at least one sulfur atom and at least one primary amine group. Examples of compounds that can be hydrolyzed or reduced to produce a compound containing at least one sulfur atom and at least one primary amine group is N-acetylcysteine, a derivative of lipoic acid, or a combination thereof.

[0068] In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of from about 20,000 Daltons to about 275,000 Daltons, from about 20,000 Daltons to about 250,000 Daltons, from about 20,000 Daltons to about 225,000 Daltons, from about 20,000 Daltons to about 200,000 Daltons, from about 20,000 Daltons to about 175,000 Daltons, from about 20,000 Daltons to about 150,000 Daltons, from about 20,000 Daltons to about 125,000 Daltons, from about 20,000 Daltons to about 100,000 Daltons, from about 20,000 Daltons to about 75,000 Daltons, from about 20,000 Daltons to about 50,000 Daltons, or from about 20,000 Daltons to about 40,000 Daltons. In another aspect, the at least one HA-antioxidant conjugate has a molecular weight of about 20,000 Daltons, about 21,000 Daltons, about 22,000 Daltons, about 23,000 Daltons, about 24,000 Daltons, about 25,000 Daltons, about 26,000 Daltons, about 27,000 Daltons, about 28,000 Daltons, about 29,000 Daltons, about 30,000 Daltons, about 31,000 Daltons, about 32,000Daltons, about 33,000 Daltons, about 34,000 Daltons, about 35,000 Daltons, about 36,000Daltons, about 37,000 Daltons, about 38,000 Daltons, about 39,000 Daltons, about 40,000Daltons, about 41,000 Daltons, about 42,000 Daltons, about 43,000 Daltons, about 44,000Daltons, about 45,000 Daltons, about 46,000 Daltons, about 47,000 Daltons, about 48,000Daltons, about 49,000 Daltons, about 50,000 Daltons, about 51,000 Daltons, about 52,000Daltons, about 53,000 Daltons, about 54,000 Daltons, about 55,000 Daltons, about 56,000Daltons, about 57,000 Daltons, about 58,000 Daltons, about 59,000 Daltons, about 60,000Daltons, about 61,000 Daltons, about 62,000 Daltons, about 63,000 Daltons, about 64,000Daltons, about 65,000 Daltons, about 66,000 Daltons, about 67,000 Daltons, about 68,000Daltons, about 69,000 Daltons, about 70,000 Daltons, about 71,000 Daltons, about 72,000Daltons, about 73,000 Daltons, about 74,000 Daltons, about 75,000 Daltons, about 76,000Daltons, about 77,000 Daltons, about 78,000 Daltons, about 79,000 Daltons, about 80,000Daltons, about 81,000 Daltons, about 82,000 Daltons, about 83,000 Daltons, about 84,000Daltons, about 85,000 Daltons, about 86,000 Daltons, about 87,000 Daltons, about 88,000Daltons, about 89,000 Daltons, about 90,000 Daltons, about 91,000 Daltons, about 92,000Daltons, about 93,000 Daltons, about 94,000 Daltons, about 95,000 Daltons, about 96,000Daltons, about 97,000 Daltons, about 98,000 Daltons, about 99,000 Daltons, about 100,000Daltons, about 101,000 Daltons, about 102,000 Daltons, about 103,000 Daltons, about 104,000 Daltons, about 105,000 Daltons, about 106,000 Daltons, about 107,000 Daltons, about 108,000 Daltons, about 109,000 Daltons, about 110,000 Daltons, about 111,000 Daltons, about 112,000 Daltons, about 113,000 Daltons, about 114,000 Daltons, about 115,000 Daltons, about 116,000 Daltons, about 117,000 Daltons, about 118,000 Daltons, about 119,000 Daltons, about 120,000 Daltons, about 121,000 Daltons, about 122,000 Daltons, about 123,000 Daltons, about 124,000 Daltons, about 125,000 Daltons, about 126,000 Daltons, about 127,000 Daltons, about 128,000 Daltons, about 129,000 Daltons, about 130,000 Daltons, about 131,000 Daltons, about 132,000 Daltons, about 133,000 Daltons, about 134,000 Daltons, about 135,000 Daltons, about 136,000 Daltons, about 137,000 Daltons, about 138,000 Daltons, about 139,000 Daltons, about 140,000 Daltons, about 141,000 Daltons, about 142,000 Daltons, about 143,000 Daltons, about 144,000 Daltons, about 145,000 Daltons, about 146,000 Daltons, about 147,000 Daltons, about 148,000 Daltons, about 149,000 Daltons, about 150,000 Daltons, about 151,000 Daltons, about 152,000 Daltons, about 153,000 Daltons, about 154,000 Daltons, about 155,000 Daltons, about 156,000Daltons, about 157,000 Daltons, about 158,000 Daltons, about 159,000 Daltons, about 160,000Daltons, about 161,000 Daltons, about 162,000 Daltons, about 163,000 Daltons, about 164,000Daltons, about 165,000 Daltons, about 166,000 Daltons, about 167,000 Daltons, about 168,000Daltons, about 169,000 Daltons, about 170,000 Daltons, about 171,000 Daltons, about 172,000Daltons, about 173,000 Daltons, about 174,000 Daltons, about 175,000 Daltons, about 176,000Daltons, about 177,000 Daltons, about 178,000 Daltons, about 179,000 Daltons, about 180,000Daltons, about 181,000 Daltons, about 182,000 Daltons, about 183,000 Daltons, about 184,000Daltons, about 185,000 Daltons, about 186,000 Daltons, about 187,000 Daltons, about 188,000Daltons, about 189,000 Daltons, about 190,000 Daltons, about 191,000 Daltons, about 192,000Daltons, about 193,000 Daltons, about 194,000 Daltons, about 195,000 Daltons, about 196,000Daltons, about 197,000 Daltons, about 198,000 Daltons, about 199,000 Daltons, about 200,000Daltons, about 201,000 Daltons, about 202,000 Daltons, about 203,000 Daltons, about 204,000Daltons, about 205,000 Daltons, about 206,000 Daltons, about 207,000 Daltons, about 208,000Daltons, about 209,000 Daltons, about 210,000 Daltons, about 211,000 Daltons, about 212,000Daltons, about 213,000 Daltons, about 214,000 Daltons, about 215,000 Daltons, about 216,000Daltons, about 217,000 Daltons, about 218,000 Daltons, about 219,000 Daltons, about 220,000Daltons, about 221,000 Daltons, about 222,000 Daltons, about 223,000 Daltons, about 224,000Daltons, about 225,000 Daltons, about 226,000 Daltons, about 227,000 Daltons, about 228,000Daltons, about 229,000 Daltons, about 230,000 Daltons, about 231,000 Daltons, about 232,000Daltons, about 233,000 Daltons, about 234,000 Daltons, about 235,000 Daltons, about 236,000Daltons, about 237,000 Daltons, about 238,000 Daltons, about 239,000 Daltons, about 240,000Daltons, about 241,000 Daltons, about 242,000 Daltons, about 243,000 Daltons, about 244,000Daltons, about 245,000 Daltons, about 246,000 Daltons, about 247,000 Daltons, about 248,000Daltons, about 249,000 Daltons, about 250,000 Daltons, about 251,000 Daltons, about 252,000Daltons, about 253,000 Daltons, about 254,000 Daltons, about 255,000 Daltons, about 256,000Daltons, about 257,000 Daltons, about 258,000 Daltons, about 259,000 Daltons, about 260,000Daltons, about 261,000 Daltons, about 262,000 Daltons, about 263,000 Daltons, about 264,000Daltons, about 265,000 Daltons, about 266,000 Daltons, about 267,000 Daltons, about 268,000Daltons, about 269,000 Daltons, about 270,000 Daltons, about 271,000 Daltons, about 272,000Daltons, about 273,000 Daltons, about 274,000 Daltons, about 275,000 Daltons, about 276,000Daltons, about 277,000 Daltons, about 278,000 Daltons, about 279,000 Daltons, about 280,000Daltons, about 281,000 Daltons, about 282,000 Daltons, about 283,000 Daltons, about 284,000 Daltons, about 285,000 Daltons, about 286,000 Daltons, about 287,000 Daltons, about 288,000 Daltons, about 289,000 Daltons, about 290,000 Daltons, about 291,000 Daltons, about 292,000 Daltons, about 293,000 Daltons, about 294,000 Daltons, about 295,000 Daltons, about 296,000 Daltons, about 297,000 Daltons, about 298,000 Daltons, about 299,000 Daltons, about 300,000 Daltons. In further aspects, HA-antioxidant conjugate has a molecular weight of about 25,000 Daltons to about 40,000 Daltons. In still other aspects, the HA-antioxidant conjugate has a molecular weight of from about 25,000 Daltons to about 38,000 Daltons. In still other aspects, the HA-antioxidant conjugate has a molecular weight of about 26,000 Daltons to about 35,000 Daltons. In still other aspects, the HA-antioxidant conjugate has a molecular weight of about 26,000 Daltons. In still other aspects, the HA-antioxidant conjugate has a molecular weight of about 26,400 Daltons. In still other aspects, the HA-antioxidant conjugate has a molecular weight of 31,000 Daltons. In yet still other aspects, the HA-antioxidant conjugate has a molecular weight of about 32,000 Daltons. In yet still other aspects, the HA-antioxidant conjugate has a molecular weight of about 33,000 Daltons. In still yet another aspect, the HA- antioxidant conjugate has a molecular weight of about 34,000 Daltons. In still yet a further aspect, the HA-antioxidant conjugate has a molecular weight of about 35,000 Daltons.

[0069] In other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight greater than 300,000 to about 1,000,000 (e.g., has a molecular weight greater than from about 303,005 to about 1,000,000). In other aspects, the at least one HA- antioxidant conjugate used in the composition has a molecular weight greater than 300,000 to about 900,000. In yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight greater than about 300,000 to about 800,000. In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight greater than about 300,000 to about 700,000. In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight greater than about 300,000 to about 600,000. In still yet further aspects, the at least one HA- antioxidant conjugate used in the composition has a molecular weight greater than about 300,000 to about 500,000. In still yet further aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight greater than about 300,000 to about 400,000.

[0070] In other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight greater than 1,000,000 to about 1,400,000 (e.g., has a molecular weight greater than from about 1,100,005 to about 1,400,000). In other aspects, the at least one HA- antioxidant conjugate used in the composition has a molecular weight greater than 1 ,000,000 to about 1,300,000. In yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight greater than about 1,000,000 to about 1,200,000.

[0071] In some aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of about 25,000 Daltons to about 40,000 Daltons and the antioxidant in the conjugate is cysteine or glutathione. In yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of about 25,000 Daltons to about 38,000 Daltons and the antioxidant in the conjugate is cysteine or glutathione. In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of about 26,000 Daltons to about 35,000 Daltons and the antioxidant in the conjugate is cysteine or glutathione. In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of about 26,000 Daltons and the antioxidant in the conjugate is cysteine or glutathione. In still yet other aspects, the at least one HA- antioxidant conjugate used in the composition has a molecular weight of about 26,400 Daltons and the antioxidant in the conjugate is cysteine or glutathione. In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of about 31,000 Daltons and the antioxidant in the conjugate is cysteine or glutathione. In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of about 32,000 Daltons and the antioxidant in the conjugate is cysteine or glutathione. In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of about 33,000 Daltons and the antioxidant in the conjugate is cysteine or glutathione. In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of about 34,000 Daltons and the antioxidant in the conjugate is cysteine or glutathione. In still yet other aspects, the at least one HA-antioxidant conjugate used in the composition has a molecular weight of about 35,000 Daltons and the antioxidant in the conjugate is cysteine or glutathione.

[0072] The composition containing at least one HA-antioxidant conjugate can also contain one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptableexcipients that can be used in the composition include, for example, diluents / fillers (such as lactose, microcrystalline cellulose, mannitol, sorbitol, starch, calcium phosphate, calcium carbonate, dextrose, sucrose, and maltodextrin), binders (including povidone, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, gelatin, acacia gum, tragacanth, sodium alginate, and polyethylene glycol), disintegrants (like croscarmellose sodium, sodium starch glycolate, crospovidone, low-substituted hydroxypropyl cellulose, alginic acid, and calcium silicate), lubricants (such as magnesium stearate, stearic acid, sodium stearyl fumarate, talc, polyethylene glycol, and glyceryl behenate), glidants (e.g., colloidal silicon dioxide, talc, and magnesium silicate), surfactants (including sodium lauryl sulfate, polysorbates, sorbitan esters, poloxamers, docusate sodium, and sodium dodecyl sulfate), pH adjusters (such as citric acid, tartaric acid, fumaric acid, sodium citrate, tromethamine, sodium bicarbonate, and potassium bicarbonate), preservatives (like methylparaben, propylparaben, benzyl alcohol, sodium benzoate, potassium sorbate, and benzalkonium chloride), additional antioxidants (e.g., butylated hydroxyanisole, butylated hydroxytoluene, ascorbic acid, tocopherols, and propyl gallate), flavoring agents (including natural and artificial flavors, and sweeteners like sucralose and aspartame), coloring agents (such as FD&C dyes, iron oxides, and titanium dioxide), viscosity modifiers (including xanthan gum, carbomer, carboxymethylcellulose, hydroxypropyl cellulose, and polyvinyl alcohol), emulsifiers (like lecithin, glyceryl monostearate, cetyl alcohol, and stearyl alcohol), penetration enhancers (such as propylene glycol, dimethyl sulfoxide, isopropyl myristate, and oleic acid), mucoadhesive polymers (e.g., carbopol, chitosan, and sodium carboxymethylcellulose), solubilizers (including cyclodextrins and polyvinylpyrrolidone), tonicity agents (like sodium chloride, glycerin, and dextrose), chelating agents (such as disodium EDTA and citric acid), film-forming agents (e.g., hydroxypropyl methylcellulose, polyvinylpyrrolidone, and ethylcellulose), plasticizers (including triethyl citrate, dibutyl sebacate, and propylene glycol), sustained release polymers (such as ethylcellulose, Eudragit polymers, and cellulose acetate), and bioadhesive polymers (like carbopol, polycarbophil, and sodium alginate). These excipients are used in various combinations and proportions depending on the specific dosage form requirements, route of administration, and desired product characteristics.

[0073] More specifically, for topical administration, excipients commonly used include a variety of functional components for effective drug delivery and product stability. These include, forexample, polymers for viscosity control, such as xanthan gum, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), and carbomer, play a crucial role in maintaining the desired consistency and flow properties of the formulation. Surface active agents like polyglyceryl-3 oleate assist in emulsification and solubilization of active ingredients. Preservatives, including various antimicrobial agents, are incorporated to extend shelf life and ensure microbiological stability. Penetration enhancers, such as Transcutol®, facilitate the absorption of active ingredients through the skin barrier. Emollients and oils, exemplified by MIGLYOL® 812 N (caprylic / capric triglyceride) and IMWITOR® 742 (glyceryl mono and dicaprylocaprate), contribute to the formulation's moisturizing properties and improve its sensory characteristics. Emulsifiers like Pemulen® (an acrylic-based viscosifying agent) aid in creating stable emulsions. Volatile solvents, which evaporate after application, are often included to modulate the formulation's properties during and after application. These excipients collectively contribute to controlling viscosity, enhancing drug solubility and absorption, extending shelflife, promoting skin penetration, providing desirable sensory properties, creating appropriate vehicles for drug delivery, and ensuring overall stability and efficacy of topical formulations.

[0074] The at least one HA-antioxidant conjugates of the present disclosure can be prepared using techniques known in the art. In some aspects, the HA-antioxidant conjugates described herein can be synthesized by generating a carboxymethyl-HA (CMHA) reaction intermediate prior to conjugation of the antioxidant. In some aspects, the HA used as starting material to synthesize the conjugate is a low molecular weight HA as described above. In other aspects, the HA used as starting material is a medium molecular weight HA as described above. In still other aspects, the HA used as starting material is a high molecular weight HA as described above. In some aspects, the molecular weight of the HA used as starting material to synthesize the conjugate is from about 30,000 Daltons to about 50,000 Daltons. In other aspects, the molecular weight of the HA used as starting material to synthesize the conjugate is from about 30,000 Daltons to about 40,000 Daltons. In yet further aspects, the molecular weight of the HA used as starting material to synthesize the conjugate is about 31,000 Daltons.

[0075] In some aspects, the HA-antioxidant conjugates can be made by chemically functionalizing the HA to add one or more primary amine groups. In some aspects, the addition of one or more primary amine groups can be achieved by adding a BOC-protectedhydrazine derivative, such as, tert-butyl carbazate, which can be subsequently deprotected to provide a reactive amine. When BOC-coupling is employed, a catalyst, such as DMAP, can be used, which can result in the addition of one or more primary amine groups to the HA.However, in some aspects, the use of a catalyst, such as DMAP, is not required. Once the HA is functionalized to add at least one primary amine, it is conjugated to the antioxidant provided it contains one or more carboxyl functionalities, using routine techniques known in the art, such as by using a zero-length crosslinking reaction (e.g., using a zero-length cross-linker). In some aspects, the zero-length crosslinking reaction is a carbodiimide reaction.

[0076] In still other aspects, the conjugates can be prepared by utilizing a primary amine on an antioxidant. In some aspects, for antioxidants that do not contain a primary amine (e.g., like glutathione or cysteine), one or more primary amines can be added in a similar manner as to HA as described above. The primary amine on the antioxidant can be covalently attached to a low, medium, or high molecular weight HA (adding additional carboxyl functionalities if needed) using a zero-length crosslinking reaction (e.g., using a zero-length cross-linker). In some aspects, the zero-length crosslinking reaction is a carbodiimide reaction, which is well known in the art.

[0077] The compositions containing the HA-antioxidant conjugates described above were found to possess antioxidant properties both in cellular oxidative stress assays (e.g., in vitro acellular and cellular assays such as, acellular superoxide scavenging assay, acellular hydrogen peroxide scavenging assay, and / or cellular superoxide / peroxide assay) and permeate in vitro models of RWM and TM.II. Methods of Treating Oxidative Stress-Mediated Hearing Loss

[0078] In yet another embodiment, the present disclosure relates to a method of treating hearing loss in a subject in need of treatment. In some aspects, the hearing loss is oxidative stress- mediated hearing loss. In some aspects, the oxidative stress -mediated hearing loss is noise- induced hearing loss (NIHL). In still further aspects, the oxidative stress-mediated hearing loss is caused by noise, aging, chemotherapy, bacterial infection, viral infection, or any combination thereof. In some aspects, the subject being treated is a human. In other aspects, the subject being treated is a non-human mammal, a bird, a reptile, or a fish. In some aspects, the subject is a monkey, an ape, a cow, a pig, a camel, a llama, a horse, a donkey a goat, a rabbit, a sheep, ahamster, a guinea pig, a cat, a dog, a rat, a mouse, a ferret, a whale, a duck, a goose, a chicken, a snake, a lizard, a turtle, an iguana, a crocodile, an alligator, a geckos, a gecko, or a shark.

[0079] The method of treating oxidative stress-mediated hearing loss involves administering to a subject in need of treatment, a therapeutically effective amount of the composition described in Section I.

[0080] In some aspects, the composition is administered to the subject topically (including ophthalmic and to mucous membranes including vaginal and / or rectal delivery or via patches).

[0081] In yet other aspects, the composition is administered to the subject parenterally. Parenteral administration includes intravenous, intraarterial, subcutaneous, intra-peritoneal, or intramuscular injection or infusion, or intracranial (e.g., intrathecal or intraventricular) injection, using routine techniques known in the art.

[0082] In still other aspects, the composition is administered orally (e.g., by tablet, capsule, gel, and / or liquid).

[0083] In still other aspects, the composition may be administered in conjunction or in combination with a medical device (e.g., microneedle patch) or tube (e.g., a tympanostomy tube).

[0084] The amount of antioxidant that can be administered or delivered to the subject is from about 0.001 mg / kg to about 2000 mg / kg. Methods for determining the amount of antioxidant to be administered or delivered to a patient are well known to those skilled in the art, such as a skilled clinician and / or physician.

[0085] In another aspects, the present disclosure relates to a method of preventing hearing loss, such as oxidative stress-mediated hearing loss, specifically noise-induced hearing loss, by administering to a subject at risk of hearing loss, a therapeutically effective amount of the composition described in Section I. Subjects at risk of hearing loss, such as oxidative stress- mediated hearing loss can be identified or determined using audiometry tests known in the art.

[0086] In some aspects, the composition is administered to the subject topically (including ophthalmic and to mucous membranes including vaginal and / or rectal delivery or via patches).

[0087] In yet other aspects, the composition is administered to the subject parenterally. Parenteral administration includes intravenous, intraarterial, subcutaneous, intra-peritoneal, or intramuscular injection or infusion, or intracranial (e.g., intrathecal or intraventricular) injection, using routine techniques known in the art.

[0088] In still other aspects, the composition is administered orally (e.g., by tablet, capsule, gel, and / or liquid).

[0089] The amount of antioxidant that can be administered or delivered to the subject is from about 0.001 mg / kg to about 2000 mg / kg.

[0090] Certain aspects of the presently disclosed subject matter having been stated herein above, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying examples.

[0091] EXAMPLE 1

[0092] Materials

[0093] The following reagents and consumables have been used in this example: HA of five molecular weights (31 kDa, 146 kDa, 285 kDa, 608 kDa, and 1030 kDa (Lifecore Biomedical Chaska, MN)), bis-tris buffer pH 6.0 (Thermo Scientific, Waltham, MA), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC, Thermo Scientific, Waltham, MA), sodium chloride (NaCl, Thermo Scientific, Waltham, MA), iodoacetic acid (Thermo Scientific, Waltham, MA), ethanol absolute (EtOH, VWR, Radnor, PA), Dulbecco’s phosphate buffered- saline (DPBS, Coming, Coming, NY), #2 Whatman paper (Thermo Scientific, Waltham, MA), deuterated water (D2O, Thermo Scientific, Waltham, MA), 70 ml 3500 kDa MW cut-off (MWCO) dialysis cassettes (Thermo Scientific, Waltham, MA), HA-BODIPY of two molecular weights (24 kDa and 240 kDa) (Echelon Biosciences, Salt Lake City, UT), D-methionine (Thermo Scientific, Waltham, MA), cysteine (Thermo Scientific, Waltham, MA), glutathione- reduced (Thermo Scientific, Waltham, MA), glutathione-oxidized (Thermo Scientific, Waltham, MA), N-hydroxysuccinimide (NHS, Thermo Scientific, Waltham, MA), dithiothreitol (DTT, Thermo Scientific, Waltham, MA), 12 N hydrochloric acid (HCL, Thermo Scientific, Waltham, MA), sodium hydroxide (NaOH, Thermo Scientific, Waltham, MA), potassium phosphate buffer (Thermo Scientific, Waltham, MA), boric acid (Thermo Scientific, Waltham, MA), potassium hydroxide (Thermo Scientific, Waltham, MA), mercaptoacetic acid (Thermo Scientific, Waltham, MA), o-phthalaldehyde (OPA, Thermo Scientific, Waltham, MA), isopropanol (IP A, VWR, Radnor, PA), Gemini 3 pm Cl 8 110A column (Phenomenex, Torrance, CA), nitro blue tetrazolium chloride (NBT, Thermo Scientific, Waltham, MA), phenazine methosulfate (PMS, Thermo Scientific, Waltham, MA), P-nicotinamide adenine dinucleotidereduced disodium salt (NADH, Thermo Scientific, Waltham, MA), L (+)- ascorbic acid (Thermo Scientific, Waltham, MA), hydrogen peroxide (H2O2, Millipore Sigma, St. Louis, MO), Superoxide anion assay kit (Millipore Sigma, St. Louis, MO), UV -transparent microplates (Coming, Coming, NY), Fisherbrand 96-well white opaque plates (12566619, Thermo Scientific, Waltham, MA) , Coming Spin-X centrifuge tube filter (0.22 pm cellulose acetate, Coming, Coming, NY), Measure-iT thiol assay kit (Thermo Scientific, Waltham, MA) PL Aquagel OH mixed-H SEC column (Agilent, Santa Clara, CA), micro bicinchonic acid protein assay kit (BCA, Thermo Scientific, Waltham, MA), and Pierce immobilized TCEP disulfide reducing gel (Thermo Scientific, Waltham, MA).

[0094] The following cell lines and cell culture reagents have been used in this example: House Ear Institute-Organ of Corti (HEI-OC1, Kalinec lab, UCLA, Los Angeles, CA), fetal bovine serum (FBS, Coming, Coming, NY), Dulbecco's modified Eagle medium (DMEM, Coming, Coming, NY), CellTiter 96 AQueous One Solution cell proliferation assay (MTS assay, Promega, Madison, WI), CyQUANT lactate dehydrogenase (LDH) cytotoxicity assay (Invitrogen, Waltham, MA), chloromethyl derivative of 2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Thermo Scientific, Waltham, MA), hydrogen peroxide (H2O2, Millipore Sigma, St. Louis, MO), trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA, Coming, Coming, NY), human primary neonatal epidermal keratinocytes (Gibco, Burlington, MA), Epilife media (Gibco, Burlington, MA), human keratinocyte growth supplement (HKGS, Gibco, Burlington, MA), calcium chloride (Thermo Scientific, Waltham, MA), 2-phospho-L- ascorbic acid trisodium salt (Sigma, St. Louis, MO), human keratinocyte growth factor (Sigma, St. Louis, MO), primary small airway epithelial cells (ATCC, Manassas, VA), airway epithelial cell basal medium (ATCC, Manassas, VA), bronchial epithelial cell growth kit (ATCC, Manassas, VA), Pneumacult-ALI medium (Stemcell, Vancouver, Canada), and Hank’s balanced salt solution (HBSS, Thermo Scientific, Waltham, MA).

[0095] The following instruments have been used for in this example: Bruker 400 with BBO broadband probe and 60 sample auto express autosampler (Bruker, Billerica, MA), Agilent Biotek Cytation 5 imaging multi-mode microplate reader (Santa Clara, CA), Agilent 1260 Infinity II HPLC instrument with a UV-VIS detector (Agilent, Santa Clara, CA) equipped with an OptiLab differential refractive index (RI) detector (Wyatt Technologies, Santa Barbara, CA), and a miniDawn 3-angle / multi-angle light scattering (MALS) detector (Wyatt Technologies,Santa Barbara, CA), and Malvern Zetasizer Ultra advanced light scattering system with folded capillary zeta cells (Malvern Panalytical, Westborough, MA).

[0096] Synthesis of Various Molecular Weight Carboxymethylated HA (CMHA)

[0097] CMHA was synthesized as previously described36, using five different molecular weights of HA (31 kDa, 146 kDa, 285 kDa, 608 kDa, and 1030 kDa). Briefly, hyaluronan (1000 mg) was added to 45% W / V NaOH (10 mL) and allowed to activate for 2 hrs. Prior to the end of the 2 hrs, iodoacetic acid (1.98 g) was dissolved in IPA (25 mL). The iodoacetic acid solution was added to the HA / NaOH solution and then IPA (75 mL) was added. The reaction mixture was stirred for 2 hrs. After reacting, the solution was filtered with filter paper and the precipitate was solubilized in water (100 mL) and neutralized to a pH of 7.0. The reaction solution was then loaded into a dialysis cassette and dialyzed against H2O with 3 changes per 24 hrs for a total of 72 hrs. The percent of carboxymethylation was determined using the previously established efficiency protocol via]H-NMR.36

[0098] Synthesis of HA-antioxidant Conjugates (M-HA, C-HA, G-HA)

[0099] M-HA was synthesized following a previously established protocol36using five different MW HA starting materials for CMHA (31 kDa, 146 kDa, 285 kDa, 608 kDa, and 1030 kDa). The stoichiometry of the reaction was 8:1 moles of methionine to moles of CMHA carboxy, and 3 : 1 moles of EDC to moles of CMHA carboxy. Briefly, CMHA (200 mg) was dissolved in MES buffer (40 mL) and D-met (880 mg) and EDC (400 mg) were then added to the reaction mixture and allowed to react for 24 hrs. After 24 hrs, the reaction solution was neutralized and loaded into a dialysis cassette and dialyzed against H2O for 72 hrs with 3 water changes a day.

[0100] Synthesis of C-HA was performed based on a previously published protocol,36but with modified reaction stoichiometry and addition of NHS for stabilization. A 1.65:1 ratio of moles of cysteine to moles of CMHA carboxy, 6.5:1 moles of EDC to moles of CMHA carboxy, and 3:1 moles of EDC to moles of NHS was used for this reaction. Preliminary reactions consisted of 100 mg of CMHA, which was scaled up to 500 mg of CMHA in subsequent batches. Briefly, 100 mg of CMHA (31 kDa HA starting material) was solubilized in 20 mL of water and the pH was adjusted to 5.5. The reaction mix was stirred until fully solubilized (~ 5 min). EDC (466 mg) and NHS (92 mg) were added to the CMHA solution and stirred vigorously for 15 min. Cysteine (100 mg) was solubilized in water (2 mL) and then it was added to the CMHA / EDC / NHS mixture. The reaction stirred for 6 hrs at RT. After 6 hrs, the solution wasplaced in dialysis cassetes with a MWCO of 3500 and dialyzed against water with 1 % w / v NaCl and 0.2 mM HC1 for 48 hrs, with 3 water changes per day. After 48 hrs the solution was dialyzed against water with 0.2 mM HC1 for another 48 hrs with 3 changes per day. Subsequently, the solution was dialyzed against water for 1 hr before removing from dialysis cassetes, transferring to a suitable container, and placing in freezer prior to lyophilization. The reaction yielded 94.53 mg of C-HA. ’H-NMR was performed as described above.

[0101] G-HA synthesis was conducted as described for C-HA, substituting the molar equivalency of glutathione-oxidized and glutathione-reduced in place of cysteine. For the glutathione-oxidized reaction product, the intrinsic disulfide bonds were reduced with DTT. DTT (92.5 mg) was added to the reaction solution and the pH was adjusted to 8.5 with NaOH. The reaction was stirred overnight at RT. The next morning, the pH was decreased to 3.5 with HC1, and the solution was then dialyzed as described for C-HA. GO-HA reaction yielded 80.2 mg and GR-HA reaction yielded 119.99 mg. ’H-NMR was performed for both reaction products as outlined above.

[0102] ’H-NMR Analysis of Compounds

[0103] To confirm the structures and purity of CMHA and conjugates, ’H-NMR spectral data were obtained using a Bruker 400 at 20 °C. For the analyses, samples were dissolved in deuterium oxide at a concentration of 5 mg / mL and all spectra were referenced to the residual solvent peak (D2O) at 3 = 4.65 ppm.

[0104] Determination of Conjugation Efficiency

[0105] The methionine content for each batch of M-HA was determined according to a previously published protocol.44

[0106] The quantity of thiol containing antioxidant in C-HA or G-HA was determined using the Measure-iT Thiol Assay kit. Two (2) volumes (relative to volume of sample to be added) of immobilized TCEP disulfide reducing resin were added to a spin-X centrifuge tube filter (0.22 pm cellulose acetate) and centrifuged (2 min, 100*rcf) to remove liquid. The filters were transferred to fresh collection tubes and one volume of samples / standards was added to the reducing resin. These were placed onto a shaker for 75 min at room temperature to allow complete sample disulfide reduction. Samples were then centrifuged (2 min, 100*rcf) and the flow-through containing the reduced sample was collected. Thiol quantification was then determined using a Measure-iT Thiol Assay Kit per manufacturers protocol. Briefly, 100 pL ofkit quantification buffer was added to the well of a 96 well plate. Sample (10 pL) was then dispensed into the well and mixed via pipetting. After 30 min sample florescence was measured by plate reader (excitation / emission: 494 / 517 nm).

[0107] Molecular Weight of Conjugates

[0108] The molecular weight and polydispersity of CMHA and CMHA-antioxidant conjugates were determined by size exclusion chromatography with multi-angle light scattering as described in a previously established protocol.44

[0109] Zeta-Potential of Conjugates

[0110] The zeta-potentials of the conjugates C-HA, G-HA, and M-HA were determined using the Zetasizer Ultra. A 5 mg / mL solution was loaded into cuvettes using between 700-800 pL for each run.

[0111] Cellular Internalization of HA-BODIPY

[0112] Mouse cochlear HEI-OC1 cells were seeded in 6-well plates and allowed to proliferate until confluent. The cells were then treated with 24 kDa or 240 kDa HA-BODIPY at 250 pg / mL in 0.9 ml growth media for the indicated amount of time. Once treated, the cells were rinsed with 1.5 ml chilled DPBS, then released with 0.05% trypsin / EDTA. The cells were then transferred to a 1.5 mL microfuge tube, centrifuged for 5 min (500*rcf, 4°C) and the pellet was washed three times with 1.5 mL chilled DPBS. The pellet was then resuspended in 0.25 mL nanopure water and mechanically lysed over three repetitions of the following: Freeze cells at - 80 °C; thaw then centrifuge the cells for 15 min (16,000*rcf, 4°C); use sonicating water bath to disrupt and homogenize pellet, briefly vortex; repeat. Once lysed, the solution was stored at -20 °C until use. Fluorescence was read on a plate reader (excitation / emission: 485 / 530 nm) and HA- BODIPY content was determined against a standard curve of each HA-BODIPY. A BCA assay was used to determine lysed protein concentration and normalize the results to total cell lysate protein.

[0113] Permeation and Cytocompatibility of HA-conjugates in TM and RWM Models

[0114] In vitro RWM and TM 3D tissue permeation models were cultured and compound permeation was evaluated as previously described.41,42For permeation testing, the tissues were placed into 3D-printed permeation devices42, transepithelial electrical resistance was measured to confirm tissue integrity using a Millicell ERS-2 voltohmmeter (Millipore, MERS00002), then they were placed into a 12-well plate containing DPBS (0.75 mL / well, receiver solution).Treatments (0.1 mL in DPBS) were placed onto the apical side of the tissue and the tissues were placed in a humidified incubator (5% CO2, 37 °C) until collection. HA-BODIPY was used at 6.67 mg / mL, C-HA and G-HA were treated at 20 mg / mL, cysteine (C) and reduced glutathione (G) were treated at the equivalent concentration of the respective C-HA and G-HA batches based on conjugation efficiency. At indicated timepoints, receiver solutions were collected and analyzed. HA-BODIPY concentrations were determined via fluorescence measurements as outlined above (Methods section: Cellular Internalization of HA-BODIPY). C-HA, C, G-HA and G concentrations were quantified by thiol detection as outlined in Section 2.5. SEC-MALS was used to confirm that the drug cargo did not detach during tissue permeation.

[0115] Cellular Cytocompatibility

[0116] HEI-OC1 cells were seeded in a 96-well plate at 1.5 x 104cells / well in 100 L of DMEM media with 10% FBS and incubated at 33°C / 1O% CO2 overnight. Subsequently the media were aspirated, replaced with sterile filtered media containing HA conjugate and controls, respectively, and plates were incubated overnight. For the LDH assay, 45 min prior to end of treatment, lysis buffer (10 pL / well) was added to the lysis control wells and sterile water (10 pL / well) was added to all other wells. The plate was returned to incubator for 45 min, then supernatant (50 pL) was transferred to a separate 96-well plate and mixed with LDH reagent (50 pL). This was incubated for 30 min at RT before stop solution (50 pL) was added, mixed, and absorbance was read in a plate reader (490 nm, ref. 680 nm). Absorbance was reference and blank (no cell wells) corrected, then normalized to lysis control (= 100%). For the MTS viability assay, treatments were removed and replaced with MTS reagent (20 pL / well) and media (100 pL / well), then returned to incubator for 1.5 hrs. Absorbance was then read at 450 nm with a plate reader, blank (no cell wells) corrected, then normalized to untreated controls (100% viability).

[0117] Acellular Superoxide Scavenging Assay

[0118] Initially, reagent stocks were prepared: NADH (1 mM in 0.01 M Tris-HCl buffer, pH8.5), NBT (1 mM in water), PMS (1 mM in water), which were then diluted to the final assay concentrations (with 16 mM Tris-HCl buffer, pH 8.0). NADH (500 pM, 50 pL), NBT (300 pM, 50 pL), and vehicle control (DPBS, 100 pL) or HA -drug conjugate (2 mg / mL in DPBS, 100 pL) were each added to a 96-well plate and mixed. PMS (25 pM, 50pL) was then added to initiate the reaction, mixed, and incubated for 10 min at room temperature. Sample absorbance was then read at 560 nm (reflective of superoxide-mediated formazan production). Blank wells had PMSreplaced with 50pL of nanopure water. Ascorbic acid (1.76 mg / mL in DPBS) in place of HA- drug conjugate was used as a positive control.45,46

[0119] Acellular Hydrogen Peroxide Scavenging Assay

[0120] HA-drug conjugates (20 pL, 2 mg / mL in DPBS) or control (20 pL, DPBS) were loaded into a UV-transparent 96-well plates. Hydrogen peroxide (180 pL, 40 mM in water) was added to the sample wells, 180 pL of PBS was added to the blank wells. Absorbance at 230 nm (reflective of the amount of hydrogen peroxide present) was measured every 30 min for 3 hrs, only 3 hr data is presented.47

[0121] Cellular Superoxide Anion Assay

[0122] A Millipore Sigma Superoxide Anion Assay Kit was used for this assay with slight modifications from manufacturer’s protocol. Menadione stock (1 mM) was prepared in nanopure water + 10 % DMSO, a 100 pM working solution was then prepared by diluting menadione stock in assay buffer. The following respective volumes (in pL) of each kit component (assay buffer, luminol, enhancer, SOD, conjugate (15 mg / mL in DPBS), DPBS) were added to respective wells of an opaque 96-well plate and mixed: Control - 68, 5, 5, 0, 0, 20; Menadione - 68, 5, 5, 0, 0, 20; SOD + Menadione - 67, 5, 5, 1, 0, 20; Conjugate + Menadione - 68, 5, 5, 0, 20, 0. HEI-OC1 cells (4*105cells / well , 100 pL / well) was then added to each well and mixed. Menadione working solution (100 pM, 2 pL) was quickly added to all well except the control which received the vehicle (1% DMSO in assay buffer, 2 pL). The plate was immediately placed in plate reader, shaken for 10 sec, then luminescence was read every 3 min for 30 min.

[0123] Cellular Peroxide Assay

[0124] HEI-OC1 cells were plated in a 96-well plate (8xl03cells / well, 100 pL / well) in DMEM media + 10% FBS and allowed to adhere overnight. The next day CM-H2DCFDA (1 mM stock solution) was diluted with cell culture media to a final concentration of 1.5 pM. The dye (100 pL) was then added to all wells except the no dye control and incubated for 45 min. The dye was then aspirated and replaced with 100 pL of treatment and stressor in HBSS + 2% FBS. The stressor and / or treatment were then incubated for 45 min. After incubation the fluorescent signal was read with the plate reader. The wells were then aspirated and washed with 150 pL of HBSS + 2% FBS. Fresh HBSS + 2% FBS (100 pL) was added to all wells and the fluorescent signal was read a second time.

[0125] Statistics

[0126] Normality testing and statistics were performed using the tests outlined in reach respective figure caption using Prism version 10 (GraphPad, San Diego, CA, USA). Statistical significance was defined as a p-value of <0.05.

[0127] Results

[0128] Evaluation of HA MW Effects on Tissue Permeation and Cellular Internalization

[0129] As first step in the development of HA-antioxidant conjugates, it was sought to understand the effects of HA MW on tissue permeability and cellular internalization. For this, fluorescently labelled HA derivatives (HA-BODIPY) of two MWs (24 kDa and 240 kDa) were tested in vitro for cellular internalization by using mouse cochlear cells (HEI-OC1) (Figure 1 A). The results indicate that over 20-times more 24 kDa HA-BODIPY was internalized in HEI-OC1 cells compared to the 240 kDa counterpart. It was next sought to understand the internalization kinetics of the 24 kDa HA-BODIPY. The data indicate that the internalization process was rapid, with 45% of the total amount detected internalized within 15 min, and the maximum amount internalized by 2 hrs (Figure 6).

[0130] Next, the tissue permeation capability of the two HA-BODIPY molecules was investigated in in vitro TM and RWM tissue permeation models. Similar to the cellular data, the permeation experiments indicated a clear interdependence between permeation efficiency and MW, with the lower MW molecule showing significantly higher permeation its higher MW counterpart (Figure IB).

[0131] Synthesis of HA-Antioxidant Conjugates

[0132] With an understanding of the effects of MW on tissue permeation and cellular internalization, it was next sought to understand the synthesis parameters that would yield conjugates of the desired MW. Therefore, the correlation between the MW of the starting HA and the MW of the final conjugate was investigated. For this, five different MW of HA (31 , 146, 285, 608, and 1030 kDa) were employed for intermediate CMHA syntheses. The high viscosity of the 1030 kDa HA sample presented a significant challenge to solubilization in the required reaction conditions, and was therefore excluded from subsequent evaluations. The structures of the four different MW CMHA intermediates were confirmed by ’H-NMR (Figure 7), and the obtained conjugation efficiencies were determined to be 51-57% (additional -COOH moieties). Moreover, the MW of all the obtained intermediates (Table 1) were determined. All obtained CMHA intermediates were readily water soluble, and therefore suitable for subsequentconjugation to primary amine-containing small molecules (i.e. antioxidant drugs) using previously outlined carbodiimide chemistry.36Table 1

[0133] Building on previous work36, M-HA was then synthesized with the four different MW CMHAs and conjugate structures were confirmed via ’H-NMR (Figure 8). A general reaction scheme for the syntheses of all conjugates is presented in Figure 2A. The four resulting different MW M-HAs were then tested in HEI-OC1 cells to assess the potential impact of different MW on cytocompatibility (Figure 2B). No statistically significant differences in cell viability were observed between the conjugates tested, indicating that that the MW of the antioxidant carrier is not affecting cellular compatibility. Based on these results, the 31 kDa HA starting material was selected for all subsequent conjugates.

[0134] The resulting conjugates were water-soluble, and the successful covalent conjugation of the antioxidants was confirmed by ’H-NMR (Figure 9). The obtained conjugates were extensively characterized for MW, conjugation efficiency, polydispersity index (D), refractive index increment and zeta (Q potential (Table 2).Table 2

[0135] The M-HA conjugate had a weight-average MW (Mw) ranging from 26.0-26.4 kDa, a D of 1.262-1.382, and a conjugation efficiency of 7.85-7.90% w / w (antioxidant mass to total conjugate mass). The C-HA conjugate had a Mwranging from 31.9-32.0 kDa, a D of 1.230- 1.349, and a conjugation efficiency of 5.96-5.97% w / w. G-HA had a Mwranging from 31.5-34.4 kDa, a D of 1.287, and a conjugation efficiency of 22.26-26.12% w / w. C-HA and G-HA were also assessed in HEI-OC1 cells, with our data indicating adequate cytocompatibility for both conjugates (Figure 2C, Figure 10).

[0136] Acellular Oxidative Protection Screenings

[0137] It was next sought to assess the effect of conjugation on the antioxidant activity of M, C, and G. For this, acellular assays were employed to unequivocally, without any background interference, assess the conjugates’ inherent antioxidant activity. The data indicate that all three conjugates reduced peroxide radical levels (Figure 3A, reported in ascorbic acid equivalence units) with C-HA and G-HA showing statistically higher protective efficiencies than M-HA. Additionally, all three conjugates performed significantly better than their respective unconjugated antioxidants. In the superoxide radical scavenging assay, all three conjugates were comparably effective at reducing superoxide levels (Figure 3B). C-HA and M-HA were able to scavenge superoxide species better than their respective unconjugated drugs, whereas G-HA scavenged at a level equivalent to unconjugated G. Based on their performance in these assays, which indicate higher overall scavenging potential, C-HA and G-HA were selected as lead compounds for further evaluation.

[0138] Cellular Oxidative Protection Screening

[0139] The two lead conjugates were then tested for antioxidant activity in cell-based assays. First, conjugates and stressor (1 mM H2O2) were simultaneously added to cells for 45 min, thenthe total peroxide level was measured (Figure 4A). Both conjugates significantly reduced peroxide levels relative to the stressed control. Next, cells were first pre-treated with conjugates for 24 hrs, then challenged with stressor for 45 min (Figure 4B). Unlike in the simultaneous treatment, pre-treatment appeared to have marginal protective effects, which were not statistically significant compared to the stressed control. A superoxide assay was also performed, in which conjugates were applied to cells in conjunction with menadione, which was used to induce cellular superoxide generation (Figure 4C). In this assay, both C-HA and G-HA drastically and rapidly reduced cellular superoxide levels, with G-HA showing an immediate reduction to nearly baseline levels.

[0140] Conjugates In Vitro Tissue Permeation

[0141] To test the conjugates’ potential as topical therapeutics, we assessed their permeability across physiologically-representative in vitro TM and RWM permeation models previously developed by our group41,42. The conjugates, as well as corresponding equivalent amounts unconjugated antioxidants, were placed on top of the tissues and the amount of each permeated at various time points was quantified (Figure 5A, Figure 5B). For TM, there were no significant differences in the permeated amounts of conjugates versus unconjugated drugs, and overall the permeated amount at 24 hrs was just above 1% of total compound applied. Our data also indicate that C-HA and G-HA can readily permeate the RWM model, with 16 and 21%, respectively, of the total applied drug permeating within 4 hrs and over 30 % permeating within 24 hrs. Finally, we also assessed the viability of the RWM and TM models after a 24 hr exposure to the conjugates (Figure 11 A, Figure 1 IB). As seen in the cochlear cell cytocompatibility assays, both treatments were well tolerated in the tissue models, with no cytotoxic effects.

[0142] Overall, this example demonstrates the successful synthesis and characterization of novel HA-antioxidant conjugates that show promise as potential therapeutics against oxidative stress-induced NIHL. Specifically, this example shows that there is a MW-dependent effect on HA cellular internalization and tissue permeation, which informed the selection of lower MW HA starting materials for the syntheses of HA-antioxidant conjugates. Moreover, it was demonstrated that the conjugates were cytocompatible and showed adequate antioxidant activity in two acellular assays. The outcomes of these assays informed the selection of the two lead compounds, C-HA and G-HA, that were then shown to be effective antioxidants in cell-basedassays. Additionally, it was shown that both conjugates were able to permeate RWM and TM permeation models supporting their potential as topical therapeutics.INCORPORATION BY REFERENCE

[0143] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In case of conflict, the present application, including any definitions herein, will control.

[0144] The present disclosure has multiple aspects, illustrated by the non-limiting examples described herein.

[0145] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.

[0146] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.

[0147] For reasons of completeness, various aspects of the disclosure are set out in the following numbered clauses:

[0148] Clause 1. A composition comprising: a hyaluronic acid (HA)-antioxidant conjugate, wherein the antioxidant is not methionine.

[0149] Clause 2. The composition of clause 1, wherein the antioxidant is a nutritional antioxidant, a phytochemical antioxidant, an enzyme, or any combination thereof.

[0150] Clause 3. The composition of any of clause 1 or clause 2, wherein the antioxidant: (a) contains at least one sulfur containing moiety and at least one primary amine; or (b) is a compound that can be hydrolyzed or reduced to produce compounds containing at least one sulfur atom and at least one primary amine group.

[0151] Clause 4. The composition of any of clauses 1-3, wherein the conjugate, when administered to a subject, permeates the tympanic membrane and round window membrane in a subject to reach the cochlea.

[0152] Clause 5. The composition of any of clauses 1-4, wherein the hyaluronic acid is a low molecular weight hyaluronic acid, a medium molecular weight hyaluronic acid, or a high molecular weight hyaluronic acid.

[0153] Clause 6. The composition of any of clauses 1-5, wherein the hyaluronic acid is a low molecular weight hyaluronic acid.

[0154] Clause 7. The composition of any of clauses 1 -6, wherein the conjugate has a molecular weight of from about 25,000 Daltons to about 40,000 Daltons, from about 25,000 Daltons to about 38,000 Daltons or from 26,000 Daltons to about 35,000 Daltons.

[0155] Clause 8. The composition of clause 3, wherein the antioxidant is glutathione, taurine, cysteine, or combinations thereof.

[0156] Clause 9. The composition of clause 8, wherein the antioxidant is glutathione.

[0157] Clause 10. The composition of clause 8, wherein the antioxidant is cysteine.

[0158] Clause 11. The composition of any of clauses 1-10, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

[0159] Clause 12. A method of treating oxidative stress-mediated hearing loss in a subject, the method comprising:

[0160] administering to a subject suffering from oxidative stress-mediated hearing loss and in need of treatment thereof, a therapeutically effective amount of the composition of any of clauses 1-11.

[0161] Clause 13. The method of clause 12, wherein the oxidative stress-mediated hearing loss is noise-induced hearing loss (NIHL).

[0162] Clause 14. The method of clause 12, wherein the composition is administered topically.

[0163] Clause 15. The method of clause 12, wherein the composition is administered parenterally.

[0164] Clause 16. The method of clause 12, wherein the composition is administered orally.

[0165] Clause 17. The method of any of clauses 12-16, wherein the subject is a human, a non-human mammal, a bird, a reptile, or a fish.

[0166] Clause 18. The method of claim 17, wherein the non-human mammal is a non-human primate, a dog, a cat, a rabbit, a guinea pig, a ferret, a hamster, or a horse.

[0167] Clause 19. The method of any of clauses 12-18, wherein the oxidative stress-mediated hearing loss is caused by noise, aging, chemotherapy, bacterial infection, viral infection, or any combination thereof.References1) WHO. Deafness and hearing loss, https: / / www.who.int / news-room / fact- sheets / detail / deafiness-and-hearing-loss (accessed 2023-08-16).(2) Mayo Clinic. Hearing loss - Symptoms and causes. Mayo Clinic. https: / / www.mayoclinic.org / diseases-conditions / hearing-loss / symptoms-causes / syc-20373072 (accessed 2023-08-16).(3) Alexander, T. H.; Harris, J. P. Incidence of Sudden Sensorineural Hearing Loss. Otol. Neurotol. Off. Publ. Am. Otol. Soc. Am. Neurotol. Soc. Eur. Acad. Otol. Neurotol. 2013, 34 (9), 1586-1589. https: / / doi.org / 10.1097 / MA0.0000000000000222.(4) Tanna, R. J.; Lin, J. W; De Jesus, O. Sensorineural Hearing Loss. In StatPearls,' StatPearls Publishing: Treasure Island (FL), 2023.(5) Mao, H.; Chen, Y. 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Claims

WHAT IS CLAIMED IS:

1. A composition comprising: a hyaluronic acid (HA)-antioxidant conjugate, wherein the antioxidant is not methionine.

2. The composition of claim 1, wherein the antioxidant is a nutritional antioxidant, a phytochemical antioxidant, an enzyme, or any combination thereof.

3. The composition of any of claim 1 or claim 2, wherein the antioxidant: (a) contains at least one sulfur containing moiety and at least one primary amine; or (b) is a compound that can be hydrolyzed or reduced to produce compounds containing at least one sulfur atom and at least one primary amine group.

4. The composition of any of claims 1-3, wherein the conjugate, when administered to a subject, permeates the tympanic membrane and round window membrane in a subject to reach the cochlea.

5. The composition of any of claims 1 -4, wherein the hyaluronic acid is a low molecular weight hyaluronic acid, a medium molecular weight hyaluronic acid, or a high molecular weight hyaluronic acid.

6. The composition of any of claims 1 -5, wherein the hyaluronic acid is a low molecular weight hyaluronic acid.

7. The composition of any of claims 1-6, wherein the conjugate has a molecular weight of from about 25,000 Daltons to about 40,000 Daltons, from about 25,000 Daltons to about 38,000 Daltons or from 26,000 Daltons to about 35,000 Daltons.

8. The composition of claim 3, wherein the antioxidant is glutathione, taurine, cysteine, or combinations thereof.

9. The composition of claim 8, wherein the antioxidant is glutathione.

10. The composition of claim 8, wherein the antioxidant is cysteine.

11. The composition of any of claims 1-10, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

12. A method of treating oxidative stress-mediated hearing loss in a subject, the method comprising: administering to a subject suffering from oxidative stress-mediated hearing loss and in need of treatment thereof, a therapeutically effective amount of the composition of any of claims13. The method of claim 12, wherein the oxidative stress-mediated hearing loss is noise-induced hearing loss (NIHL).

14. The method of claim 12, wherein the composition is administered topically.

15. The method of claim 12, wherein the composition is administered parenterally.

16. The method of claim 12, wherein the composition is administered orally.

17. The method of any of claims 12-16, wherein the subject is a human, a non-human mammal, a bird, a reptile, or a fish.

18. The method of claim 17, wherein the non-human mammal is a non-human primate, a dog, a cat, a rabbit, a guinea pig, a ferret, a hamster, or a horse.

19. The method of any of claims 12-18, wherein the oxidative stress-mediated hearing loss is caused by noise, aging, chemotherapy, bacterial infection, viral infection, or any combination thereof.

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