Hyaluronic acid-nonsteroidal Anti-inflammatory drug conjugates for treating inflammation-mediated pathologies

Abstract

Disclosed herein are compositions comprising hyaluronic acid-nonsteroidal anti-inflammatory drug conjugates and methods for making the same. The compositions of the present disclosure can be used to prevent or treat subjects suffering from inflammation related conditions, such as, inflammation-mediated hearing loss.

Description

Hyaluronic Acid-Nonsteroidal Anti-Inflammatory Drug Conjugates for Treating Inflammation-Mediated PathologiesRELATED APPLICATION INFORMATION

[0001] This application claims priority to U.S. Application No. 63 / 607,469 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-nonsteroidal anti-inflammatory drug conjugates and methods for making the same. The compositions of the present disclosure can be used to prevent or treat subjects suffering from inflammation related conditions, such as, for example, inflammation-mediated hearing loss.GOVERNMENT FUNDING INFORMATION

[0003] This invention was made with government support under P20 GM103546 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] Cochlear damage from acoustic trauma is a multifaceted degenerative phenomenon caused by a cascade of detrimental responses such as oxidative stress, inflammation, and excitotoxicity, which ultimately result in both necrotic and apoptotic cell death.1 6Both reactive oxygen species and inflammation have been identified as major contributors to noise-induced hearing loss (NIHL). Several studies have shown that noise-induced inflammatory responses in the cochlea are triggered by the recruitment of inflammatory cells such as macrophages, and the upregulation of pro-inflammatory cytokines.7 9Macrophages have been identified in the cochlea along the lateral wall and within the spiral limbus, and when activated through a variety of stressors,10 11they migrate to the scala tympani.3,12Various studies have identified some of the inflammatory cytokine and gene expression patterns in noise-induced inflammatory conditions; however, the specific mechanisms of the damage have not been fully elucidated. Recent studieshave reported that excessive noise exposure activates NF-KB signaling, which then upregulates the expression of the pro-inflammatory cytokines IL-6 and TNF-a.13-15Additional studies have shown that acoustic trauma in mice stimulates TNF-a production by the macrophages, leading to hair cell (cochlear sensory cell) death and consequently, hearing loss.16Various antiinflammatory approaches have been investigated for the management of NIHL; for example, anti-inflammatory steroids such as dexamethasone, metformin, and etanercept (an FDA- approved TNF-a inhibitor), have been shown to reduce inflammatory cytokines and protect against hearing loss.16-19

[0005] While systemic treatments against hearing loss have been extensively explored in the past, they can be problematic due to limited drug concentrations reaching the target tissue, premature metabolic deactivation, and increased risk of adverse off-target effects.20To address these shortcomings, localized drug delivery to the target tissue would be expected to improve drug availability, therapeutic efficiency, and safety. However, anatomical membranes such as the tympanic membrane (TM), which separates the outer ear and the middle ear, and the round window membrane (RWM), which separates the middle ear and the inner ear, pose significant challenges to topical drug delivery. Thus, there is a need for improved therapeutics that can be delivered successfully to the to prevent and / or treat conditions associated with inflammation, such as, for example, inflammation-mediated hearing loss.SUMMARY

[0006] In one embodiment, the present disclosure relates to a [composition comprising: a hyaluronic acid (HA)-nonsteroidal anti-inflammatory drug (NSAID) conjugate, wherein toxicity of the NSAID is reduced compared to a NSAID that is not conjugated to hyaluronic acid.

[0007] In some aspects, in the composition, the conjugate reduces inflammation related or induced cytotoxicity in cochlear cells when administered to a subject.

[0008] In still other aspects, the hyaluronic acid in the composition is a low molecular weight hyaluronic acid, a medium molecular weight hyaluronic acid, or a high molecular weight hyaluronic acid. More specifically, in some aspects, the hyaluronic acid is a low molecular weight hyaluronic acid.

[0009] In still further aspects of the composition, the conjugate has a molecular- weight of from about 20,000 Daltons to about 40,000 Daltons or from about 20,000 Daltons to about 30,000 Daltons.

[0010] In yet further aspects of the composition, the NS AID is a propionic acid derivative. More specifically, in some aspects, propionic acid derivative is ibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaproin, or any combinations thereof. Even more specifically, in some aspects, the propionic acid derivative is ibuprofen.

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

[0012] In yet another embodiment, the present disclosure relates to method of treating a subject suffering from inflammation. The method comprises the steps of:

[0013] administering to a subject suffering from at least one type of inflammation and in need of treatment thereof, a therapeutically effective amount of the above-described composition, wherein the inflammation is an inflammation-mediated hearing loss, arthritis, a musculoskeletal disorder, a headache, a migraine, dysmenorrhea, dental pain, post-operative pain, or a soft tissue injury, or any combination thereof.

[0014] In some aspects of the above method, the composition is administered topically.

[0015] In further aspects of the above method, the composition is administered via parenterally.

[0016] In still further aspects of the above method, the composition is administered orally.

[0017] In yet other aspects of the above method, the arthritis is rheumatoid arthritis, osteoarthritis, psoriatic arthritis, gout, or any combinations thereof.

[0018] In still further aspects of the above method, the musculoskeletal disorder is bursitis, tendinitis, a muscle strain, a muscle sprain, back pain, or any combinations thereof.

[0019] In still further aspects of the above method, the subject is a human.

[0020] In still further aspects of the above method, the subject being treated 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.

[0021] In still further aspects of the above method, the inflammation-mediated hearing loss is noise-induced hearing loss. More specifically, the inflammation-mediated hearing loss is caused by an autoimmune inner ear disease, a systemic autoimmune disease, an auto inflammatory disease, infection-related inflammation, or any combination thereof.BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1 shows a reaction scheme for synthesis of HACs. Figure 1A shows an amine functionalization used for ibuprofen. Figure IB shows an amine functionalization of carboxyl containing anti-inflammatories. Figure 1C shows a HA-anti-inflammatory conjugation reaction. BOC, tert-butyloxycarbonyl protecting group; CMHA, carboxymethyl-hyaluronic acid; DCM, dichloromethane; DMAP, 4-dimethylaminopyridine; EDC HCL, l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride; EtOH, ethanol; HA, hyaluronic acid; R, rest of molecule; RT, room temperature.

[0023] Figure 2 shows the structures of aminated anti-inflammatory molecules. HC, hydrocortisone; IBU, ibuprofen; PS, prednisolone.

[0024] Figure 3 shows the normalized cytokine release in LPS-stressed macrophages treated with HACs, or the respective unconjugated anti-inflammatory drugs. L-6 (Figure 3A) and TNF-a (Figure 3B) release from RAW264.7 macrophages treated for 4 hrs with 10 ng / mL LPS (all) and simultaneously, untreated control (LPS Control, black), 1.5 mg / mL of the indicated HAC (HAC + LPS, grey), or the estimated equivalent concentration of unconjugated anti-inflammatory drug (Drug Eq. + LPS, striped grey). IL-6 (Figure 3C) and TNF-a (Figure 3D) release from RAW264.7 macrophages treated for 24 hrs with 10 ng / mL LPS (all) and simultaneously, untreated control (LPS Control, black), 1.5 mg / mL of the indicated HAC (HAC + LPS, grey), or the estimated equivalent concentration of unconjugated anti-inflammatory drug (Drug Eq. + LPS, striped grey). (Figure 3A-Figure 3D) Values shown are normalized to the level of the respective untreated controls, n = 3 (I-HA); n = 2 - 4 (P-HA, HC-HA); two-way ANOVA with Dunnett’s Correction; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant. All graphs show mean ± SD. HAC, hyaluronic acid-anti-inflammatory conjugate; HC-HA, hydrocortisone-HA conjugate; I-HA, ibuprofen-HA conjugate; LPS, lipopolysaccharide; P-HA, prednisolone-HA conjugate.

[0025] Figure 4 shows I-HA cytocompatibility in macrophages and inner ear cells. MTS (Figure 4A) and LDH (Figure 4B) cell viability assay in RAW264.7 macrophages treated for 24 hrs with control, 1.5 mg / mL I-HA, the estimated equivalent concentration (0.18 mg / mL) of unconjugated IBU, or a blend of the estimated equivalent concentration of unconjugated IBU (0.18 mg / mL) and CMHA (1.32 mg / mL). (Figure 4A) n = 12; (Figure 4B) n = 4; (Figure 4A, Figure 4B) one-way ANOVA with Tukey’s Correction; *p<0.05, ****p<0.0001, ns = not significant. MTS (Figure 4C) and LDH (Figure 4D) cell viability assay in HELOC1 cells treated for 24 hrs with control(black) or 1 .5 mg / mL I-HA (red). (C,D) n = 9; T-test; ns = not significant. All graphs show mean ± SD. I-HA, ibuprofcn-HA conjugate; LDH, lactate dehydrogenase.

[0026] Figure 5 shows cytokine release in LPS-stressed macrophages treated with I-HA or IBU. Figure 5A shows cytokine release in RAW264.7 macrophages treated for 24 hr with 10 ng / mL LPS (all) and simultaneously, untreated control (black), 1.5 mg / mL I-HA (grey), or the estimated equivalent concentration (0.18 mg / mL) of unconjugated IBU (striped grey). IL-6 (Figure 5B) and TNF-a (Figure 5C) release, which were retested independently due to exceeding the upper quantification limit in the multiplex assay. (Figure 5A-Figure 5C) Mean ± SD; n = 3-4; an independent one-way ANOVA with Tukey’s Correction was performed for each cytokine, for cytokines with a group below the assay quantification limit (“L”) the valid groups were tested against the value of the quantification limit; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant. LHA, ibuprofen-HA conjugate; L, below lower detection limit; LPS, lipopoly saccharide .

[0027] Figure 6 shows the effect of macrophage cytokine release on cochlear cell viability. Figure 6A shows a mTS viability assay in HELOC1 cells treated with media (Control), media with LPS (LPS Control), or with conditioned media consisting of media collected from macrophages after 24 hrs (CM) or media collected from macrophages treated with LPS for 24 hrs (LPS CM), n = 8. MTS (Figure 6B) and LDH (Figure 6C) viability assays in HELOC1 cells treated with control, a blend of cytokines reflecting the cytokine levels released by LPS-stressed macrophages (“High Inflammation”), or a blend of cytokines reflecting the cytokine levels released by LPS-stressed macrophages treated by 1.5 mg / mL I-HA (“Reduced Inflammation”), n = 16 - 40. (Figure 6A- Figure 6C) Brown-Forsythe and Welch one-way ANOVA test with Dunnett T3 Correction; ****p<0.0001. All graphs show mean ± SD. CM, conditioned media; I- HA, ibuprofen-HA conjugate; LDH, lactate dehydrogenase; LPS, lipopolysaccharide.

[0028] Figure 7 shows I-HA tissue compatibility and permeation across RWM and TM models. Figure 7A shows tissue viability after 24 hr exposure to control (DPBS), cytotoxic control (5% SDS), I-HA (20 mg / mL), or the estimated equivalent concentration (2.4 mg / mL) of unconjugated IBU. Mean ± SD; n = 3-6; two-way ANOVA with Sldak Correction; ***p<0.001, ****p<0.0001, ns = not significant. Figure 7B shows 24 hr permeation of LHA across in vitro RWM permeation model, n = 4 - 7; Welch’s T-test; *p<0.05. Figure 7C shows 24 hr permeation of I-HA across in vitro TM permeation model. “LOD” dotted line indicates the approximate limit of detection of I-HA which was not detected in this experiment, n = 4. (Figure 7B, Figure 7C) Show percent of total drug permeated following application of 0.1 mL of I-HA (20 mg / mL) or estimated equivalent concentration (2.4 mg / mL) of unconjugated IBU. Mean ± SD. I-HA, ibuprofen-HA conjugate; RWM, round window membrane; TM, tympanic membrane.

[0029] Figure 8 shows the cytotoxicity of IBU in TM tissue models. Figure 8A shows TEER TM tissue models after 24 hr exposure to control (DPBS), cytotoxic control (5% SDS), I-HA (20 mg / mL), or the estimated equivalent concentration (2.4 mg / mL) of unconjugated IBU. n = 3; two- way ANOVA with Sldak Correction; ***p<0.001, ****p<0.0001, ns = not significant. Figure 8B is a representative micrograph of H&E stained TM tissues exposed to control (DPBS), I-HA (20 mg / mL), or estimated equivalent concentration (2.4 mg / mL) of unconjugated IBU for 24 hrs. Scale bar applies to all panels. I-HA, ibuprofen-HA conjugate; SDS, sodium dodecyl sulfate; TEER, transepithelial electrical resistance; TM, tympanic membrane.

[0030] Figure 9 shows anti-inflammatory molecules utilized in Example 1.

[0031] Figure 10A shows1H-NMR spectra for the amine modification of ibuprofen. All run in D6- DMSO. The top spectrum is the ibuprofen starting material. The middle spectrum is the amine modified ibuprofen with a BOC functionality protecting the amine. The bottom spectrum is the deprotected amine modified ibuprofen. The shaded box highlights the area of interest. Peaks at 8=1.34 ppm originate from the BOC moiety. BOC, tert-butyloxycarbonyl. Figure 10B shows1H- NMR spectra for the amine modification of prednisolone 21 -hemisuccinate sodium salt. All run in D6-DMSO. The top spectrum is the prednisolone starting material. The middle is the amine modified prednisolone with a BOC functionality protecting the amine. The bottom spectrum is after deprotection of the amine by removing the BOC protecting group. Shaded box highlights area of interest (8-1.5 ppm) from protons originating from the BOC moiety. BOC, tertbutyloxycarbonyl. Figure 10C shows ’I I-NMR spectra for the amine modification of hydrocortisone 21 -hemisuccinate sodium salt. All run in D6-DMSO. The top spectrum is the hydrocortisone starting material. The middle is the amine modified hydrocortisone with a BOC protecting the amine. The bottom is after removing the BOC functionality from the amine. Shaded box highlights area of interest (8-1.5 ppm) from protons originating from the BOC moiety. BOC, tert-butyloxycarbonyl.

[0032] Figure HA shows ’H-NMR spectra for the conjugation of the amine modified ibuprofen and CMHA to form LHA. All run in deuterated water. The top is the CMHA starting material andthe bottom spectrum is after the conjugation. The shaded boxes highlight the peaks of interest that arc present only after amine modified ibuprofen has been conjugated to CMHA. CMHA, carboxymethyl-hyaluronic acid; I-HA, Ibuprofen-CMHA conjugate. Figure 11B shows ’ H-NMR spectra for the conjugation of the amine modified prednisolone and CMHA to form P-HA. All run in deuterated water. The top is the CMHA starting material and the bottom spectrum is after the conjugation. The shaded boxes highlight the peaks of interest that are present only after amine modified prednisolone has been conjugated to CMHA. CMHA, carboxymethyl-hyaluronic acid; P-HA, prednisolone-CMHA conjugate. Figure 11C shows ’ H-NMR spectra for the conjugation of the amine modified hydrocortisone and CMHA to form HC-HA. All run in deuterated water. The top is the CMHA starting material and the bottom spectrum is after the conjugation. The shaded boxes highlight the peaks of interest that are present only after amine modified hydrocortisone has been conjugated to CMHA. CMHA, carboxymethyl-hyaluronic acid; HC-HA, hydrocortisone-CMHA conjugate.

[0033] Figure 12 shows a LPS dose-response in macrophages. (A) IL-6 and (B) TNF-a release from RAW264.7 macrophages treated for 4 (white) or 24 (grey) hrs with various concentrations of LPS. n = 2 per group per dose. (C) Cell viability by MTS assay of macrophages treated for 4 (white) or 24 (grey) hrs with various concentrations of LPS. n = 4 per group per dose. All graphs show mean ± SD. N, not detected; LPS, lipopolysaccharide.

[0034] Figure 13 shows a cytokine release in LPS-stressed macrophages. RAW264.7 macrophages were treated for 24 hrs with 10 ng / mL LPS, then the supernatant was analyzed for cytokine release in a panel of inflammatory cytokines. Mean ± SD; n = 2-3. N, not detected; L, below lower quantification limit; LPS, lipopolysaccharide.

[0035] Figure 14 shows a dose-response cell viability (MTS assay) curves of RAW264.7 macrophages (Figure 14A) or HEI-OC1 (Figure 14B) cells treated with IBU for 24 hrs. Maximum dose tested was at IBU’s solubility limit in cell media (0.3 mg / mL). n = 7 - 8. Variable slope nonlinear fit shown.

[0036] Figure 15 shows the effect of individual cytokines from defined cytokine blend on cochlear cell viability. HELOC1 cells were treated with either TNF-a, IL-6, IL-10, or IL-12p70 at 42, 16, 1.3, and 2.1 ng / mL, respectively, for 24 hrs before measuring cell viability by MTS assay. Mean ± SD; n = 4; Brown-Forsythe and Welch one-way ANOVA test with Dunnett T3 Correction; *p<0.05, ns = not significant.

[0037] Figure 16 shows the kinetics of T-HA permeation across in vitro RWM permeation model, n = 3 - 4.DETAILED DESCRIPTION

[0038] Provided herein are compositions containing at least one hyaluronic acid-nonsteroidal anti-inflammatory drug conjugate and methods for making the same. The compositions described herein can be used to prevent or treat subjects suffering from inflammation, such as, for example, inflammation-mediated hearing loss, arthritis, a musculoskeletal disorder, a headache, a migraine, dysmenorrhea, dental pain, post-operative pain, or a soft tissue injury, or any combination thereof.DEFINITIONS

[0039] 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.

[0040] 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, molecular biology, 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.

[0041] 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.

[0042] The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) arc 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.

[0043] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0044] 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.

[0045] 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.

[0046] 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 by one 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 unlessotherwise claimed. No language in the specification should be construed as indicating any non- claimcd element as essential to the practice of the invention as used herein.

[0047] 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.

[0048] 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 "exactly one 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."

[0049] 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, inone 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.

[0050] 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.

[0051] 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%).

[0052] 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 less than 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.

[0053] 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.

[0054] 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 ingrcdicnt(s), or substancc(s) within the body of a subject or patient.

[0055] 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- dimethylaminopropyljcarbodiimide): 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): Preferred for use in solid-phase synthesis due to easier purification.

[0056] 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 - (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.

[0057] 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 molecularweight 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 bander.

[0058] As used herein, the phrase “inflammation-mediated hearing loss” or “inflammation- mediated pathology” as used interchangeably herein, refers to sensorineural hearing loss (SNHL) or any other type of hearing loss caused by one or more inflammatory processes within the inner ear of a subject. This type of hearing loss occurs when the body's immune response, intended to protect against pathogens or cellular’ debris, inadvertently damages the delicate structures of the cochlea. Some key aspects of inflammation-mediated hearing loss include: (1) autoantibody- mediated damage in which autoantibodies attack inner ear structures like the cochlea or vestibular system; (2) immune complex deposition in which antibody -antigen complexes trigger inflammatory responses in the inner ear; (3) T cell-mediated inflammation in which activated T cells infiltrate the inner ear, initiating an inflammatory cascade; and / or (4) cytokine imbalance in which dysregulation of pro-inflammatory and anti-inflammatory cytokines leads to chronic inflammation. Some inflammatory mediators include: (1) proinflammatory cytokines (e.g., TNF-a, IL-ip, IL-6); (2) chemokines (e.g., MCP-1, MIP-2); (3) adhesion molecules (e.g., ICAM-1); and (4) growth factors (e.g., VEGF). The consequences of inflammation in the ear include: (1) tissue damage as inflammation can cause irreversible damage to cochlear structures, leading to permanent hearing loss; (2) vascular effects as inflammation may induce vasospasm, resulting in ischemia and subsequent hearing loss; and / or (3) oxidative stress as excessive formation of reactive oxygen species contributes to cell death pathways in the cochlea.

[0059] Noise-induced hearing loss (NIHL) is an example of a type of inflammation-mediated hearing loss. When the inner ear is exposed to excessive noise, it undergoes a series of damaging processes that include both oxidative stress and inflammation. Another example of inflammation-mediated hearing loss is autoimmune inner ear disease (AIED). Autoimmune inner ear disease is an inflammatory condition caused by an uncontrolled immune system response that attacks the inner ear, resulting in progressive sensorineural hearing loss. Yet another example of inflammation-mediated hearing loss is hearing loss resulting from a systemic autoimmune disease such as, systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, Cogan’s syndrome, and / or relapsing polychondritis. Still yet another example of inflammation-mediated hearing loss is hearing loss resulting from autoinflammatory diseasessuch as Cogan's syndrome and / or Behcet's disease. Tn still yet another example of inflammation- mediated hearing loss is hearing loss resulting from infection-related inflammation such as bacterial meningitis.

[0060] As used herein, the phrase a “musculoskeletal disorder” or “MSD” refers injuries and conditions that affect the musculoskeletal system, including joints, muscles, tendons, ligaments, cartilage, and occasionally, nerves of a subject. These disorders can result from overexertion, repetitive movements, or excessive strain on soft tissues when the demands of work or daily activities exceed the body's capacity to adapt. Musculoskeletal disorders include a wide range of conditions, including: (a) bursitis: inflammation of the bursae, which are small fluid-filled sacs that cushion joints; (b) tendinitis: inflammation of tendons, often caused by repetitive use of a joint; (c) muscle strains: injuries to muscles or tendons, typically resulting from overuse or sudden force; (d) muscle sprains: injuries to ligaments, often caused by stretching or tearing due to sudden or abnormal movements; (e) back pain: a common MSD that can affect the lower back (lumbar region) and may involve muscles, nerves, or spinal discs; or (f) any combination of (a)- (3). Musculoskeletal disorders can develop gradually or suddenly, depending on the cause and intensity of the strain placed on the affected tissues. Musculoskeletal disorders can lead to various symptoms, including pain, stiffness, swelling, reduced range of motion, and in some cases, numbness or tingling sensations.

[0061] As used herein, the phrase “nonsteroidal anti-inflammatory drug” or “NSAID” refers to a class of anti-inflammatory drugs which structurally generally comprise: (a) an acidic moiety (e g., a carboxylic acid group); and (b) a planar aromatic functional group. This structural arrangement is a key component of the mechanism of action of NSAIDs, which is to inhibit cyclooxygenase enzymes, such as COX-1 and COX-2. Cyclooxygenase enzymes are responsible for the production of prostaglandins, which are key mediators of inflammation, pain and fever. The structural similarity of NSAIDs allow these drugs to interact with the COX enzymes in a comparable manner, leading to their anti-inflammatory, analgesic, and antipyretic effects. In some aspects, the NSAID is a propionic acid derivative. Propionic acid derivatives are compounds comprise a substituted phenyl ring connected to a propionic acid moiety (e.g., a 2- arylpropionic acid structure). Example of propionic acid derivatives include ibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaproin, or any combinations thereof.

[0062] 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-NH2, 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).

[0063] 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 non-human 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 subject would 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.

[0064] The phrase, “pharmaceutically acceptable”, whether by itself or in conjunction with another term or terms, indicates that the designated entity such as, for example, apharmaceutically 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.

[0065] As used herein, the phrase, “pharmaceutically acceptable excipient” refers to a substance that is non-loxic, 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 ail, and include, lor 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).

[0066] As used herein “soft tissue injury” refers damage to muscles, ligaments, and tendons throughout the body in a subject. These injuries can occur due to various causes, such as sudden trauma (e.g., a fall, twist, or blow to the body), overuse or repetitive motions, and / or excessive strain on soft tissues. Examples of soft issue injuries include sprains (e.g., the stretching or tearing of ligaments, often affecting wrists, ankles, and knees), strains (injuries to muscles or tendons, frequently occurring in areas like the foot, back of leg (e.g., hamstring), or back), contusions (which result from blunt force trauma, causing discoloration of the skin due to underlying muscle fibers and connective tissue being crushed), and / or bursitis (which involves the inflammation of the bursae, small fluid-filled sacs that cushion the joints).

[0067] 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”.

[0068] 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), cither alone or in combination with any of the others (a)-(d).

[0069] 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.

[0070] 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 crosslinkers 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 cross-linkers can be used for various applications, including studying protein-protein interactions, creating enzyme-substrate complexes, and investigating protein architecture; and water- solubility: many zero-length crosslinkers, like EDC, are water-soluble, making them suitable for use in aqueous biological systems.

[0071] 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 crosslinking reactions 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. Compositions Containing Hyaluronic Acid-Nonsteroidal Anti-Inflammatory Drug Conjugates

[0072] In one embodiment, the present disclosure relates to compositions comprising at least one hyaluronic acid (HA)-nonsteroidal anti-inflammatory drug (NSAID) conjugate. In some aspects, the composition is a pharmaceutical composition containing a therapeutically effective amount of at least one HA-NSAID conjugate. The compositions of the present disclosure containing said conjugates can be used to treat subjects suffering from inflammation-mediated hearing loss, such as, for example, noise-induced hearing loss (NIHL). Specifically, when administered to a subject as described in Section III, the at least one HA-NSAID conjugate reduces macrophage- mediated inflammatory cytotoxicity in cochlear cells.

[0073] In some aspects, the hyaluronic acid used in the HA-NSAID conjugate has a molecular weight which allows for the effective permeation of the round window membrane (RWM) and tympanic membrane (TM) of the ear. Specifically, the HA used in the conjugate of the present disclosure 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,000 Daltons, about 33,000 Daltons, about 34,000 Daltons, about 35,000 Daltons, about,000 Daltons, about 37,000 Daltons, about 38,000 Daltons, about 39,000 Daltons, about,000 Daltons, about 41,000 Daltons, about 42,000 Daltons, about 43,000 Daltons, about,000 Daltons, about 45,000 Daltons, about 46,000 Daltons, about 47,000 Daltons, about,000 Daltons, about 49,000 Daltons, about 50,000 Daltons, about 51,000 Daltons, about,000 Daltons, about 53,000 Daltons, about 54,000 Daltons, about 55,000 Daltons, about,000 Daltons, about 57,000 Daltons, about 58,000 Daltons, about 59,000 Daltons, about,000 Daltons, about 61,000 Daltons, about 62,000 Daltons, about 63,000 Daltons, about,000 Daltons, about 65,000 Daltons, about 66,000 Daltons, about 67,000 Daltons, about,000 Daltons, about 69,000 Daltons, about 70,000 Daltons, about 71,000 Daltons, about,000 Daltons, about 73,000 Daltons, about 74,000 Daltons, about 75,000 Daltons, about,000 Daltons, about 77,000 Daltons, about 78,000 Daltons, about 79,000 Daltons, about,000 Daltons, about 81,000 Daltons, about 82,000 Daltons, about 83,000 Daltons, about,000 Daltons, about 85,000 Daltons, about 86,000 Daltons, about 87,000 Daltons, about,000 Daltons, about 89,000 Daltons, about 90,000 Daltons, about 91,000 Daltons, about,000 Daltons, about 93,000 Daltons, about 94,000 Daltons, about 95,000 Daltons, about,000 Daltons, about 97,000 Daltons, about 98,000 Daltons, about 99,000 Daltons, about0,000 Daltons, about 101,000 Daltons, about 102,000 Daltons, about 103,000 Daltons, about4,000 Daltons, about 105,000 Daltons, about 106,000 Daltons, about 107,000 Daltons, about8,000 Daltons, about 109,000 Daltons, about 110,000 Daltons, about 111,000 Daltons, about2,000 Daltons, about 113,000 Daltons, about 114,000 Daltons, about 115,000 Daltons, about6,000 Daltons, about 117,000 Daltons, about 118,000 Daltons, about 119,000 Daltons, about0,000 Daltons, about 121,000 Daltons, about 122,000 Daltons, about 123,000 Daltons, about4,000 Daltons, about 125,000 Daltons, about 126,000 Daltons, about 127,000 Daltons, about8,000 Daltons, about 129,000 Daltons, about 130,000 Daltons, about 131,000 Daltons, about2,000 Daltons, about 133,000 Daltons, about 134,000 Daltons, about 135,000 Daltons, about6,000 Daltons, about 137,000 Daltons, about 138,000 Daltons, about 139,000 Daltons, about0,000 Daltons, about 141,000 Daltons, about 142,000 Daltons, about 143,000 Daltons, about4,000 Daltons, about 145,000 Daltons, about 146,000 Daltons, about 147,000 Daltons, about8,000 Daltons, about 149,000 Daltons, about 150,000 Daltons, about 151,000 Daltons, about2,000 Daltons, about 153,000 Daltons, about 154,000 Daltons, about 155,000 Daltons, about,000 Daltons, about 157,000 Daltons, about 158,000 Daltons, about 159,000 Daltons, about,000 Daltons, about 161,000 Daltons, about 162,000 Daltons, about 163,000 Daltons, about,000 Daltons, about 165,000 Daltons, about 166,000 Daltons, about 167,000 Daltons, about,000 Daltons, about 169,000 Daltons, about 170,000 Daltons, about 171,000 Daltons, about,000 Daltons, about 173,000 Daltons, about 174,000 Daltons, about 175,000 Daltons, about,000 Daltons, about 177,000 Daltons, about 178,000 Daltons, about 179,000 Daltons, about,000 Daltons, about 181 ,000 Daltons, about 182,000 Daltons, about 183,000 Daltons, about,000 Daltons, about 185,000 Daltons, about 186,000 Daltons, about 187,000 Daltons, about,000 Daltons, about 189,000 Daltons, about 190,000 Daltons, about 191,000 Daltons, about,000 Daltons, about 193,000 Daltons, about 194,000 Daltons, about 195,000 Daltons, about,000 Daltons, about 197,000 Daltons, about 198,000 Daltons, about 199,000 Daltons, about,000 Daltons, about 201,000 Daltons, about 202,000 Daltons, about 203,000 Daltons, about,000 Daltons, about 205,000 Daltons, about 206,000 Daltons, about 207,000 Daltons, about,000 Daltons, about 209,000 Daltons, about 210,000 Daltons, about 211,000 Daltons, about,000 Daltons, about 213,000 Daltons, about 214,000 Daltons, about 215,000 Daltons, about,000 Daltons, about 217,000 Daltons, about 218,000 Daltons, about 219,000 Daltons, about,000 Daltons, about 221,000 Daltons, about 222,000 Daltons, about 223,000 Daltons, about,000 Daltons, about 225,000 Daltons, about 226,000 Daltons, about 227,000 Daltons, about,000 Daltons, about 229,000 Daltons, about 230,000 Daltons, about 231,000 Daltons, about,000 Daltons, about 233,000 Daltons, about 234,000 Daltons, about 235,000 Daltons, about,000 Daltons, about 237,000 Daltons, about 238,000 Daltons, about 239,000 Daltons, about,000 Daltons, about 241,000 Daltons, about 242,000 Daltons, about 243,000 Daltons, about,000 Daltons, about 245,000 Daltons, about 246,000 Daltons, about 247,000 Daltons, about,000 Daltons, about 249,000 Daltons, about 250,000 Daltons, about 251,000 Daltons, about,000 Daltons, about 253,000 Daltons, about 254,000 Daltons, about 255,000 Daltons, about,000 Daltons, about 257,000 Daltons, about 258,000 Daltons, about 259,000 Daltons, about,000 Daltons, about 261,000 Daltons, about 262,000 Daltons, about 263,000 Daltons, about,000 Daltons, about 265,000 Daltons, about 266,000 Daltons, about 267,000 Daltons, about,000 Daltons, about 269,000 Daltons, about 270,000 Daltons, about 271,000 Daltons, about,000 Daltons, about 273,000 Daltons, about 274,000 Daltons, about 275,000 Daltons, about,000 Daltons, about 277,000 Daltons, about 278,000 Daltons, about 279,000 Daltons, about280,000 Daltons, about 281 ,000 Daltons, about 282,000 Daltons, about 283,000 Daltons, about284,000 Daltons, about 285,000 Daltons, about 286,000 Daltons, about 287,000 Daltons, about288,000 Daltons, about 289,000 Daltons, about 290,000 Daltons, about 291,000 Daltons, about292,000 Daltons, about 293,000 Daltons, about 294,000 Daltons, about 295,000 Daltons, about296,000 Daltons, about 297,000 Daltons, about 298,000 Daltons, about 299,000 Daltons, about300,000 Daltons. In some aspects, the HA has a molecular weight of about 20,000 Daltons to about 40,000 Daltons. In still other aspects, the HA has a molecular weight of from about 20,000 Daltons to about 30,000 Daltons. In still other aspects, the HA has a molecular weight of about 25,000 Daltons. In still other aspects, the HA has a molecular weight of 26,000 Daltons. In yet still other aspects, the HA has a molecular weight of about 27,000 Daltons. In yet still other aspects, the HA has a molecular weight of about 28,000 Daltons. In still yet another aspect, the HA has a molecular weight of about 29,000 Daltons. In still yet a further aspect, the HA has a molecular weight of about 30,000 Daltons.

[0074] In other aspects, where effective permeation of the round window membrane (RWM) and tympanic membrane (TM) of the ear is not required, 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.

[0075] In other aspects, where effective permeation of the round window membrane (RWM) and tympanic membrane (TM) of the ear is not required, 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. Inyet other aspects, the HA has a molecular weight of greater than about 1 ,100,000 to about 1,200,000.

[0076] Any NSAID can be used in the conjugate of the present disclosure. In some aspects, the NSAID is a propionic acid derivative. Examples of priopionic acid derivatives that can be used in the conjugate are ibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaproin, or any combinations thereof. In some aspects, the priopionic acid derivative is ibuprofen. In some aspects, it was unexpectedly discovered that when an NSAID (e.g., ibuprofen), was conjugated to HA, that the toxicity of the NSAID (e.g., ibuprofen) was reduced compared to when the NSAID was unconjugated. More specifically, it was unexpectedly discovered that the tissue toxicity of an NSAID (e.g., such as ibuprofen) conjugated to HA was less than the tissue toxicity of an NSAID that was not conjugated to HA.

[0077] In still yet other aspects, the at least one HA-NSAID conjugate used in the composition has a molecular weight of from about 20,000 Daltons to about 300,000 Daltons, 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-NSAID 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 32,000 Daltons, about 33,000 Daltons, about 34,000 Daltons, about 35,000 Daltons, about 36,000 Daltons, about 37,000 Daltons, about 38,000 Daltons, about 39,000 Daltons, about 40,000 Daltons, about 41,000 Daltons, about 42,000 Daltons, about 43,000 Daltons, about 44,000 Daltons, about 45,000 Daltons, about 46,000 Daltons, about 47,000 Daltons, about 48,000 Daltons, about 49,000 Daltons, about 50,000 Daltons, about 51,000 Daltons, about 52,000 Daltons, about 53,000 Daltons, about 54,000 Daltons, about 55,000 Daltons, about 56,000 Daltons, about 57,000 Daltons, about 58,000 Daltons, about 59,000 Daltons, about 60,000 Daltons, about 61,000 Daltons, about 62,000 Daltons, about 63,000Daltons, about 64,000 Daltons, about 65,000 Daltons, about 66,000 Daltons, about 67,000Daltons, about 68,000 Daltons, about 69,000 Daltons, about 70,000 Daltons, about 71,000Daltons, about 72,000 Daltons, about 73,000 Daltons, about 74,000 Daltons, about 75,000Daltons, about 76,000 Daltons, about 77,000 Daltons, about 78,000 Daltons, about 79,000Daltons, about 80,000 Daltons, about 81,000 Daltons, about 82,000 Daltons, about 83,000Daltons, about 84,000 Daltons, about 85,000 Daltons, about 86,000 Daltons, about 87,000Daltons, about 88,000 Daltons, about 89,000 Daltons, about 90,000 Daltons, about 91,000Daltons, about 92,000 Daltons, about 93,000 Daltons, about 94,000 Daltons, about 95,000Daltons, about 96,000 Daltons, about 97,000 Daltons, about 98,000 Daltons, about 99,000Daltons, about 100,000 Daltons, about 101,000 Daltons, about 102,000 Daltons, about 103,000Daltons, about 104,000 Daltons, about 105,000 Daltons, about 106,000 Daltons, about 107,000Daltons, about 108,000 Daltons, about 109,000 Daltons, about 110,000 Daltons, about 111 ,000Daltons, about 112,000 Daltons, about 113,000 Daltons, about 114,000 Daltons, about 115,000Daltons, about 116,000 Daltons, about 117,000 Daltons, about 118,000 Daltons, about 119,000Daltons, about 120,000 Daltons, about 121,000 Daltons, about 122,000 Daltons, about 123,000Daltons, about 124,000 Daltons, about 125,000 Daltons, about 126,000 Daltons, about 127,000Daltons, about 128,000 Daltons, about 129,000 Daltons, about 130,000 Daltons, about 131,000Daltons, about 132,000 Daltons, about 133,000 Daltons, about 134,000 Daltons, about 135,000Daltons, about 136,000 Daltons, about 137,000 Daltons, about 138,000 Daltons, about 139,000Daltons, about 140,000 Daltons, about 141,000 Daltons, about 142,000 Daltons, about 143,000Daltons, about 144,000 Daltons, about 145,000 Daltons, about 146,000 Daltons, about 147,000Daltons, about 148,000 Daltons, about 149,000 Daltons, about 150,000 Daltons, about 151,000Daltons, about 152,000 Daltons, about 153,000 Daltons, about 154,000 Daltons, about 155,000Daltons, about 156,000 Daltons, about 157,000 Daltons, about 158,000 Daltons, about 159,000Daltons, about 160,000 Daltons, about 161,000 Daltons, about 162,000 Daltons, about 163,000Daltons, about 164,000 Daltons, about 165,000 Daltons, about 166,000 Daltons, about 167,000Daltons, about 168,000 Daltons, about 169,000 Daltons, about 170,000 Daltons, about 171,000Daltons, about 172,000 Daltons, about 173,000 Daltons, about 174,000 Daltons, about 175,000Daltons, about 176,000 Daltons, about 177,000 Daltons, about 178,000 Daltons, about 179,000Daltons, about 180,000 Daltons, about 181,000 Daltons, about 182,000 Daltons, about 183,000Daltons, about 184,000 Daltons, about 185,000 Daltons, about 186,000 Daltons, about 187,000Daltons, about 188,000 Daltons, about 189,000 Daltons, about 190,000 Daltons, about 191 ,000Daltons, about 192,000 Daltons, about 193,000 Daltons, about 194,000 Daltons, about 195,000Daltons, about 196,000 Daltons, about 197,000 Daltons, about 198,000 Daltons, about 199,000Daltons, about 200,000 Daltons, about 201,000 Daltons, about 202,000 Daltons, about 203,000Daltons, about 204,000 Daltons, about 205,000 Daltons, about 206,000 Daltons, about 207,000Daltons, about 208,000 Daltons, about 209,000 Daltons, about 210,000 Daltons, about 211,000Daltons, about 212,000 Daltons, about 213,000 Daltons, about 214,000 Daltons, about 215,000Daltons, about 216,000 Daltons, about 217,000 Daltons, about 218,000 Daltons, about 219,000Daltons, about 220,000 Daltons, about 221,000 Daltons, about 222,000 Daltons, about 223,000Daltons, about 224,000 Daltons, about 225,000 Daltons, about 226,000 Daltons, about 227,000Daltons, about 228,000 Daltons, about 229,000 Daltons, about 230,000 Daltons, about 231,000Daltons, about 232,000 Daltons, about 233,000 Daltons, about 234,000 Daltons, about 235,000Daltons, about 236,000 Daltons, about 237,000 Daltons, about 238,000 Daltons, about 239,000Daltons, about 240,000 Daltons, about 241,000 Daltons, about 242,000 Daltons, about 243,000Daltons, about 244,000 Daltons, about 245,000 Daltons, about 246,000 Daltons, about 247,000Daltons, about 248,000 Daltons, about 249,000 Daltons, about 250,000 Daltons, about 251,000Daltons, about 252,000 Daltons, about 253,000 Daltons, about 254,000 Daltons, about 255,000Daltons, about 256,000 Daltons, about 257,000 Daltons, about 258,000 Daltons, about 259,000Daltons, about 260,000 Daltons, about 261,000 Daltons, about 262,000 Daltons, about 263,000Daltons, about 264,000 Daltons, about 265,000 Daltons, about 266,000 Daltons, about 267,000Daltons, about 268,000 Daltons, about 269,000 Daltons, about 270,000 Daltons, about 271,000Daltons, about 272,000 Daltons, about 273,000 Daltons, about 274,000 Daltons, about 275,000Daltons, about 276,000 Daltons, about 277,000 Daltons, about 278,000 Daltons, about 279,000Daltons, about 280,000 Daltons, about 281,000 Daltons, about 282,000 Daltons, about 283,000Daltons, about 284,000 Daltons, about 285,000 Daltons, about 286,000 Daltons, about 287,000Daltons, about 288,000 Daltons, about 289,000 Daltons, about 290,000 Daltons, about 291,000Daltons, about 292,000 Daltons, about 293,000 Daltons, about 294,000 Daltons, about 295,000Daltons, about 296,000 Daltons, about 297,000 Daltons, about 298,000 Daltons, about 299,000Daltons, about 300,000 Daltons. In further aspects, the HA-NSAID conjugate has a molecular weight of about 20,000 Daltons to about 40,000 Daltons. In still other aspects, the HA-NSAID conjugate has a molecular weight of from about 20,000 Daltons to about 30,000 Daltons. In stillother aspects, the HA-NSAID conjugate has a molecular weight of about 25,000 Daltons. In still other aspects, the HA-NSAID conjugate has a molecular weight of 26,000 Daltons. In yet still other aspects, the HA-NSAID conjugate has a molecular weight of about 27,000 Daltons. In yet still other aspects, the HA-NSAID conjugate has a molecular weight of about 28,000 Daltons. In still yet another aspect, the HA-NSAID conjugate has a molecular weight of about 29,000 Daltons. In still yet a further aspect, the HA-NSAID conjugate has a molecular weight of about 30,000 Daltons.

[0078] In other aspects, the at least one HA-NSAID 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-NSAID 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-NSAID 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-NSAID 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-NSAID 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-NSAID 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-NSAID conjugate used in the composition has a molecular weight greater than about 300,000 to about 400,000.

[0079] In other aspects, the at least one HA-NSAID 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-NSAID 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-NSAID conjugate used in the composition has a molecular weight greater than about 1,000,000 to about 1,200,000.

[0080] In still other aspects, the at least one HA-NSAID conjugate used in the composition has a molecular weight of from about 20,000 Daltons to about 30,000 Daltons and the NSAID is ibuprofen. In still other aspects, the at least one HA-NSAID conjugate used in the composition has a molecular weight of about 25,000 Daltons and the NSAID is ibuprofen. In still other aspects, the at least one HA-NSAID conjugate used in the composition has a molecular weight ofabout 26,000 Daltons and the NSAID is ibuprofen. Tn yet still other aspects, the at least one HA- NSAID conjugate used in the composition has a molecular weight of about 27,000 Daltons and the NSAID is ibuprofen. In yet still other aspects, the at least one HA-NSAID conjugate used in the composition has a molecular weight of about 28,000 Daltons and the NSAID is ibuprofen. In still yet another aspect, the at least one HA-NSAID conjugate used in the composition has a molecular weight of about 29,000 Daltons and the NSAID is ibuprofen. In still yet a further aspect, the at least one HA-NSAID conjugate used in the composition has a molecular weight of about 30,000 Daltons and the NSAID is ibuprofen.

[0081] The composition containing at least one HA-NSAID conjugate can also contain one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients 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 NSAIDs (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 (c.g., hydroxypropyl mcthylccllulosc, 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.

[0082] More specifically, for topical administration, excipients commonly used include a variety of functional components for effective drug delivery and product stability. These include, for example, 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 shelf life, promoting skin penetration, providing desirable sensory properties, creating appropriate vehicles for drug delivery, and ensuring overall stability and efficacy of topical formulations.

[0083] The compositions containing at least one HA-NSAID conjugate as described above were found to be well tolerated by cochlear’ cells in contrast to unconjugated NSAIDs (e.g., ibuprofen), which demonstrated cytotoxic effects. Additionally, it was found that compositions containing at least one HA-NSAID conjugate were able to reduce macrophage-mediated inflammatory cytotoxicity in HEI-OC1 cells. Moreover, the HA-NSAID conjugates described above were found to permeate in vitro models of RWM and TM. 1II. Methods for Making Hyaluronic Acid-NSAID Conjugates

[0084] In another embodiment, the present disclosure relates to methods of making HA-NSAID conjugates. In one aspect, the HA-NSAID conjugates can be made by enriching HA with one or more carboxy functionalities to increase the number of potential conjugation sites for attachment of one or more NSAIDs (e.g., such as ibuprofen) using routine techniques known in the art. The number of carboxy functionalities added to the HA is not critical. 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 to synthesize the conjugate is a medium molecular weight HA as described above. In yet other aspects, the HA used as starting material to synthesize the conjugate is high molecular weight HA as described above. In still further aspects, when HA is enriched with carboxy functionalities, the intermediate carboxymethyl-HA (CMHA) is produced.

[0085] After the HA is enriched with one or more carboxy functionalities, the NSAID (e.g., ibuprofen) is chemically functionalized 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- protected hydrazine 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 NSAID. However, in some aspects, the use of a catalyst, such as DMAP, is not required. Once the NSAID is functionalized to add at least one primary amine it is conjugated to the HA that has been enriched with one or more carboxy 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.).

[0086] In another aspect, the HA-NSAID 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-protected hydrazine 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 NSAID, provided it contains one more carboxyfunctionalities, using routine techniques known in the art, such as by using a zero-length crosslinking reaction (c.g., using a zero-length cross -linker). In some aspects, the zero-length crosslinking reaction is a carbodiimide reaction.III. Methods of Treating Inflammation-Mediated Pathologies

[0087] In yet another embodiment, the present disclosure relates to a method of treating a subject suffering from inflammation and in need of treatment. In some aspects, the inflammation is inflammation-mediated hearing loss is noise-induced hearing loss (NIHL). In other aspects, the inflammation-mediated hearing loss is caused by an autoimmune inner ear disease, a systemic autoimmune disease, an autoinflammatory disease, infection-related inflammation, or any combination thereof. In some aspects, the subject 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, a hamster, 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.

[0088] In other aspects, the inflammation is inflammation caused by one or more types of arthritis. In some aspects, the arthritis is rheumatoid arthritis, osteoarthritis, psoriatic arthritis, gout, or any combinations thereof.

[0089] In another aspect, the inflammation is inflammation is caused by one or more types of musculoskeletal disorders. In some aspects, the musculoskeletal disorder is one or more of bursitis, tendinitis, a muscle strain, a muscle sprain, back pain, or any combinations thereof.

[0090] In still other aspects, the inflammation is caused by a headache and / or migraine.

[0091] In still other aspect, the inflammation is caused by dysmenorrhea.

[0092] In still yet other aspects, the inflammation is caused by dental pain.

[0093] In still yet other aspects, the inflammation is caused by post-operative pain.

[0094] In still other aspects, the inflammation is caused by a soft tissue injury

[0095] The method of treating the subject suffering from inflammation loss involves administering to a subject in need of treatment, a therapeutically effective amount of the composition described in Section I.

[0096] 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).

[0097] In yet other aspects, the composition is administered to the subject parenterally. Parenteral administration includes intravenous, intraarterial, subcutaneous, intra-pcritoncal, or intramuscular injection or infusion, or intracranial (e.g., intrathecal or intraventricular) injection, using routine techniques known in the art.

[0098] In still other aspects, the composition is administered orally (e.g., by tablet, capsule, gel, and / or liquid).

[0099] 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).

[0100] The amount of the NSAID that can be administered or delivered to the subject is from about 100 mg about 3200 mg, depending on the NSAID employed. Methods for determining the amount of NSAID 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.

[0101] In another aspects, the present disclosure relates to a method of preventing inflammation- mediated herein loss in a subject by administering to a subject at risk of inflammation-mediated hearing loss, a therapeutically effective amount of the composition described in Section I. In some aspects, a subject at risk of inflammation-mediated hearing loss can be identified or determined using audiometry tests known in the art.

[0102] 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).

[0103] 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.

[0104] In still other aspects, the composition is administered orally (e.g., by tablet, capsule, gel, and / or liquid).

[0105] 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).

[0106] The amount of the NSAID that can be administered or delivered to the subject is from about 100 mg about 3200 mg, depending on the NSAID employed. Methods for determining the amount of NSAID to be administered or delivered to a patient are well known to those skilled in the art.

[0107] Certain aspects of the presently disclosed subject matter having been stated herein above, which arc 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.

[0108] EXAMPLE 1

[0109] 1.1 Materials

[0110] The following cell lines, cell culture reagents and assay kits have been used in this example; House Ear Institute-organ of Corti (HEI-OC1, Kalinec Lab, UCLA, Los Angeles, CA), RAW264.7 ( TIB-71, ATCC, Manassas, VA), 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), Dulbecco’s phosphate buffered- saline (DPBS, Corning, Corning, NY), trypsin-ethylenediaminetetraacetic acid (trypsin- EDTA, Corning, Corning, NY), TNF-a mouse instant ELISA kit (#BMS607-2INST, Invitrogen, Waltham, MA), IL-6 mouse ELISA kit (#KMC0061, Invitrogen, Waltham, MA), V-PLEX proinflammatory panel 1 mouse kit (#K15048D Meso Scale Discovery (MSD), Rockville, MD), lipopolysaccharide (LPS, E. Coli O111:B4, Millipore Sigma, St. Louis, MO), mouse IL-10 recombinant protein (550070, BD Biosciences, Franklin Lakes, NI, USA), mouse IL-12p70 recombinant protein (Thermo Scientific, Waltham, MA), mouse TNF-a recombinant protein (#RMTNFAI Invitrogen, Waltham, MA), mouse IL-6 recombinant protein (#RMIL6I Invitrogen, Waltham, MA).

[0111] The following reagents and consumables have been used for this example: HA 20 kDa (Lifecore Biomedical, Chaska, MN), l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Thermo Scientific, Waltham, MA), sodium chloride (NaCl, Thermo Scientific, Waltham, MA), sodium hydroxide (NaOH, Thermo Scientific, Waltham, MA), ethanol (EtOH, VWR, Radnor, PA), iodoacetic acid (Thermo Scientific, Waltham, MA), isopropanol (VWR, Radnor, PA), #2 Whatman paper (Thermo Scientific, Waltham, MA), deuterated water (D2O, Thermo Scientific, Waltham, MA), Dimethyl Sulfoxide (DMSO, Thermo Scientific, Waltham, MA,) De-DMSO (Thermo Scientific, Waltham, MA), 70 mL 3500 kDa MW cut-off (MWCO) dialysis cassettes (Thermo Scientific, Waltham, MA), t-butyl carbazate (Thermo Scientific, Waltham, MA), 4-(dimethylamino)pyridine (DMAP, TCI Chemicals,Philadelphia, PA), N,N-diisopropylethylamine (TCI Chemicals, Philadelphia, PA), sodium sulfate (TCI Chemicals, Philadelphia, PA), ammonium chloride (NH4CL, TCI Chemicals, Philadelphia, PA), sodium bicarbonate (NaHCOa. TCI Chemicals, Philadelphia, PA), dichloromethane (DCM, Thermo Scientific, Waltham, MA), ethyl acetate (Thermo Scientific, Waltham, MA), trifluoro acetic acid (TFA, Thermo Scientific, Waltham, MA), toluene (Thermo Scientific, Waltham, MA), methanol (MeOH, Thermo Scientific, Waltham, MA), ibuprofen (Thermo Scientific, Waltham, MA), SNAP-50 cartridges (Biotage, Uppsala, Sweden), thin layer chromatography paper (Thermo Scientific, Waltham, MA), prednisolone 21 -hemisuccinate sodium salt (Sigma, St. Louis, MO), and hydrocortisone 21 -hemisuccinate sodium salt (Sigma, St. Louis, MO).

[0112] The following instruments have been used for this example: Bruker 400 with BBO broadband probe and 60 sample auto express autosampler (Bruker, Billerica, MA), MESO QuickPlex SQ 120 MESO QuickPlex SQ 120 (Meso Scale Discovery, Rockville, MD), Agilent Cytation 5 cell imaging multimode reader (Agilent Technologies Inc., Santa Clara, CA), Agilent 1260 Infinity II HPLC system (Agilent Technologies Inc., Santa Clara, CA) with UV-Vis DAD, paired with Wyatt miniDAWN MALS and Optilab dRI detectors (Wyatt Technologies, Santa Barbara, CA), Malvern Zetasizer Ultra zeta-potential analyzer with folded capillary zeta cells (Malvern Panalytical, Westborough, MA), Agilent Cary 100 UV-Vis spectrophotometer (Agilent Technologies Inc., Santa Clara, CA), and Millicell ERS-2 voltohmmeter (MilliporeSigma, Burlington, MA).

[0113] 1.2 Synthesis of Carboxymethylated HA (CMHA)

[0114] HA was derivatized with carboxymethyl functionalities to increase the conjugation sites available for the covalent attachment of molecules containing primary amines as previously published.6Briefly, HA (2 g) was added to NaOH (20 mL, 45% w / v) and allowed to activate at room temperature (RT) for 2 hrs. In parallel, iodoacetic acid (2 g) was dissolved in isopropanol (50 mL). The viscous HA solution was added to isopropanol (150 mL), then the iodoacetic acid solution was added to the activated HA / isopropanol solution. The reaction mixture was allowed to react for 2 hrs and was then filtered using a Buchner funnel with a #2 Whatman filter paper. The white filter cake obtained after filtration was dissolved in deionized water (200 mL) and the pH of the resulting solution was neutralized using HC1 (6 N). The resulting CMHA solution was then loaded into 3500 MWCO dialysis cassettes and dialyzed for 72 hrs with a minimum of threewater changes every 24 hr to remove residual reagent and salts. After dialysis, the CMHA solution was removed from the cassettes, frozen in a -80 DC freezer for a minimum of 4 hr, and subsequently lyophilized. The reaction yielded 1.580 g of CMHA. Carboxymethylation was confirmed by proton nuclear magnetic resonance (1H-NMR).

[0115] 1.3 Synthesis of Amine Modified Ibuprofen

[0116] IBU (Figure 9) was functionalized with a primary amine in order to undergo a future carbodiimide reaction with CMHA (Figure 1A). IBU (3.4 g), EDC (3.84 g), and t-butyl carbazate (2.6 g), were added to a 200 mL round bottom flask. EtOH (50 mL) was added and the reaction was covered and allowed to stir at RT for 24 hrs. Then the reaction was concentrated in vacuo at 40 °C to remove the EtOH. A ' H-NMR in De DMSO was performed on the protected amine modified IBU. The following day a deprotection of the amine was performed with the 5:1 ratio of DCM to TFA quenching after 2.5 hrs with a 1:1 ratio of toluene to MeOH and concentrated in vacuo. After drying, a ' H-NMR in De DMSO was performed.

[0117] 1.4 Synthesis of Amine Modified Hydrocortisone

[0118] Hydrocortisone 21 -hemisuccinate sodium salt (Figure 9) was functionalized with a primary amine in order to undergo subsequent conjugation with CMHA (Figure IB). Hydrocortisone 21 -hemisuccinate sodium salt (1.5 g), EDC (768 mg), DMAP (480 mg), a catalyst, and t-butyl carbazate (530 mg), a hydrazine derivative, were added to a 200 mL round bottom flask. DCM (25 mL) was then added followed by N,N-diisopropylethylamine (1.1 mL). The reaction was then covered and allowed to stir at RT for 72 hr. Thin layer chromatography was performed to ensure the reaction was complete. Ethyl acetate was used for extraction after neutralizing with ammonium chloride. The organic and aqueous phases were then separated with a separatory funnel and sodium sulfate was added to the organic phase prior to vacuum filtration. The filtrate was placed in a clean round bottom flask and concentrated in vacuo at 40 °C to remove ethyl acetate. The following day deprotection of the amine was performed with the 5:1 ratio of DCM to TFA quenching after 2.5 hr with a 1:1 ratio of toluene to MeOH and concentrated in vacuo. After drying, purification was performed using flash chromatography. A Biotage SNAP 50 g cartridge with an initial gradient of 1% MeOH and 99% DCM and ending gradient of 10% MeOH was used for purification. To determine the final product a combination of thin layer chromatography and 'H-NMR were performed.

[0119] 1.5 Synthesis of Amine Modified Prednisolone

[0120] The same synthesis reaction was followed as outlined in Section 1 .4 using the stoichiometric equivalent amount of prednisolone 21-hcmiscuccinatc sodium salt (Figure 9) in place of hydrocortisone 21 -hemisuccinate sodium salt.

[0121] 1.6 Synthesis of HA Conjugates

[0122] Conjugation of the anti-inflammatory molecules to CMHA were carried out in a similar manner as the previously published protocols.6Briefly, CMHA (275 mg) was dissolved in nanopure water (30 mL). After CMHA was fully solubilized, EDC (183 mg) was added. While EDC solubilized, the amine modified small molecules (275 mg) were solubilized in EtOH (0.5 mL each) with sonication. The solubilized small molecule was added dropwise with a hypodermic needle very slowly. The water volume was increased stepwise to 90 mL followed by 150 mL, and the stir plate was increased to 400 rpm to prevent precipitation. The reaction was allowed to stir for 24 hrs at RT. After 24 hrs, the solution was filtered using a Buchner filter with # 2 Whatman paper. The filtrate was then neutralized to a pH of 7.0 and placed in 70 mL dialysis cassettes with a MWCO of 3500 kDa. The solutions were dialyzed for 72 hrs with a minimum of three water changes per day before being placed in the freezer for 4 hrs and lyophilized. IBU- CMHA conjugate (I-HA) yielded 277.38 mg, PS-CMHA conjugate (P-HA) yielded 239.24 mg and HC-CMHA conjugate (HC-HA) yielded 278.5 mg of lyophilized product. The structure of the product was then assessed via1H-NMR solubilized in D2O.

[0123] 1.7 Determination of HAC Physicochemical Properties

[0124] Refractive index increment (dn / dc) and size exclusion chromatography multi-angle light scattering (SEC-MALS) were used to determine I-HA molecular weight and polydispersity index using methods previously described in detail.28Potassium phosphate buffer (0.01 M, pH 7.58) was used as the mobile phase. For dn / dc, five concentrations (0.1 - 1.0 mg / mL) were injected into a Wyatt Opitlab RI detector and dn / dc was determined using manufacturer’s software analysis. SEC-MALS used Wyatt miniDAWN and Optilab detectors connected to an Agilent 1260 Infinity II HPLC. I-HA was injected (50 pL, 0.4 mg / mL) at a flow of 1.0 mL / min through a 6.0 - 10,000 kDa 8 pm PL Aquagel-OH Mixed-H 7.5 x 300 mm SEC column (Agilent, PL1149- 6800). Molecular weight and polydispersity were computed and reported by Wyatt ASTRA software.

[0125] Zeta-potential of the conjugates (5 mg / mL in nanopure water) was determined using a Malvern Zetasizer Red Ultra zeta-potential analyzer according to manufacturer’s instructions.

[0126] Estimated conjugation efficiency of HACs was determined using the relative absorbance contribution of individual conjugated components to the final conjugate. Specifically, the absorbance spectra of the HACs and each individual component of the conjugates (CMHA, antiinflammatories, amine modified anti-inflammatories) at various concentrations was determined.Unique absorbance maxima with minimal crossover between components (I-HA, 222 nm; P-HA, 278 nm; HC-HA 280 nm) were used to determine the extinction coefficients for each component and the HACs. Where A = Absorbance, C = concentration, s = extinction coefficient, H = HAC, i= conjugate component 1, and ii = conjugate component 2; and the following assumptions are true: A = C x E (assuming path length is consistent), CH= + Cit. and AH= At+ A^, the following equation can be derived and was used to estimate the concentration of a component in a known concentration of conjugate: CuUsing this method, the estimated conjugation efficiency of I-HA is 12.0-12.9% w / w (mass of anti-inflammatory to total conjugate mass), P-HA is 9.0-13.9%, and HC-HA is 7.6-18.0%. While this method does rely on certain fallible assumptions, we currently lack the capability of a more certain process in determining conjugation efficiency for these conjugates and believe the method to provide a general approximation of conjugation efficiency. Further study into these HACs would require the development of a more robust method conjugation efficiency. Absorbance was measured in 1 cm cuvettes on a Cary 100 UV / Vis spectrophotometer all samples were solubilized in a solution of 10 % methanol and 90 % phosphate buffer (0.01 M, pH 7.58) which permitted all components to remain soluble in tested linear absorbance range.

[0127] 1.8 Cell Culture

[0128] RAW 264.7 mouse macrophage cells were cultured in DMEM high glucose w / L- glutamine w / o sodium pyruvate with 10% FBS in a 37 °C, 5% CO2, humidified incubator. Cells were grown in T75 flasks and media was replaced every 2-3 days until they reached 80% confluence. Cells were scraped gently from the flask using a sterile cell scraper and counted with an Invitrogen Countess II automated cell counter before plating.

[0129] House Ear Institute-organ of Corti 1 (HEI-OC1) cells were cultured in T75 flasks using DMEM supplemented with 10% FBS. The cells were grown until they reached approximately 80% confluence. HEI-OC1 cells were grown in a 33 °C, 10% CO2, humidified incubator to support optimal cell growth. Cells were released with trypsin / EDTA and counted for plating.

[0130] After passing, HEI-OC1 cells (6xl03cells, 100 pL / well) and RAW264.7 macrophages(1 ,5xl04cells, 100 pL per well) were seeded in 96-well plates and incubated overnight before treatments were started according to specific experimental conditions. The seeding densities and incubation conditions of both cell types were kept the same across all the experiments in this study unless specified otherwise.

[0131] 1.9 Cell Viability Assays

[0132] After the indicated treatment for each experiment, MTS and LDH cytotoxicity assays were performed per the manufacturer’s protocols. Cells were treated with the indicated treatment in growth media (100 uL / wcll). For the LDH assay, 45 min prior to the end of treatment, lysis buffer (10 pL) was added to the lysis control wells, and all other groups were given sterile water (10 pL). The plate was tapped to mix then returned to the incubator for 45 min. Cell supernatant (50 pL) from all wells was then collected and, in a separate 96-well plate, added to LDH detection buffer (50 pL). After incubating at room temperature (30 min), stop solution (50 pL) was added, mixed, and the absorbance (490 nm, ref. 680 nm) was read on a microplate reader. All values were reference absorbance and blank (no cells) corrected, then normalized to the lysis control group (= 100% lysis). For the MTS assay, treatment was removed, then MTS reagent (20 pL) in media (100 pL) was added to each well and the cells were returned to the incubator for 1.5 hrs. Absorbance was read then at 450 nm. All values were blank (no cells) corrected then normalized to the untreated controls.

[0133] 1.10 Lipopolysaccharide Stimulation of Macrophages

[0134] Macrophages were plated as outlined above in Section 1.8. To determine optimal lipopolysaccharide (LPS) concentration for robust release of pro-inflammatory cytokines, macrophages were treated with a range of LPS concentrations (between 0-50 ng / mL) for 4 or 24 hrs. After treatment, culture supernatant was gently collected and analyzed for TNF-a and IL-6 release in the previously detailed ELISA kits according to the manufacturer’s instructions. The remaining cell supernatant was gently removed and cell viability assays were performed.

[0135] After selecting 10 ng / mL LPS as the standard LPS-stressed treatment for all subsequent experiments herein, the effect of this dose on the expression on ten inflammatory cytokines was evaluated using an MSD V-Plex Proinllammatory Panel per manufacturer’s protocols.

[0136] 1.11 Screening of Anti-inflammatory Conjugates

[0137] Macrophages were prepared as outlined above in Section 1.8. The growth media was removed, and the cells were treated with HACs (1.5 mg / mL, 100 pL / well) or the estimatedequivalent concentrations of unconjugated anti-inflammatory drugs for 4 hrs and 24 hrs. All groups, including controls, received 10 ng / mL LPS. After treatment, cell supernatant was collected and stored at -20 °C until use. The most prominently released inflammatory cytokines (TNF-a and IL-6) were analyzed using the previously detailed ELISA kits according to manufacturer protocols.

[0138] 1.12 Effect of I-HA Treatment on Macrophage Cytokine Release

[0139] Macrophages were cultured as described (Section 1.8). Growth media was aspirated, followed by the addition of a 100 pL of each treatment: LPS at 10 ng / mL; I-HA at 1.5 mg / mL with LPS at 10 ng / mL; or the estimated equivalent concentration of unconjugated IBU (0.18 mg / mL) with LPS at 10 ng / mL. After 24 hrs, 50 pL of cell supernatant was collected stored at - 20 °C until use. The samples were assayed using the MSD V-Plex proinflammatory panel according to manufacturer’s instructions.

[0140] 1.13 Conditioned Media Preparation and HEI-OC1 Treatment

[0141] T-75 tissue culture treated flasks were seeded with 106macrophages and allowed to incubate for 48 hrs before starting treatments, receiving fresh media after 24 hrs. All treatment flasks were given 15 mL of treatment in growth media. The conditioned media (CM) group received media alone. The LPS CM group was treated with 10 ng / mL LPS. Additionally, two control flasks were maintained without cells, one with media alone (control) and one with 10 ng / mL LPS (LPS control). After incubating for 24 hrs, the media from all the groups was collected, filtered with 0.22 pm syringe filters, and stored at -20 °C.

[0142] HEI-OC1 cells were then prepared in 96-well plates (see Section 1.8). After discarding the growth media, the cells were treated with 100 pL of the treatments prepared above (control, LPS control, CM, and LPS CM). Following a 24 hr treatment, LDH and MTS assays were performed (see Section 1.9).

[0143] 1.14 Defined Cytokine Media Treatment on HEI-OC1 Cells

[0144] The four cytokines with the highest release from LPS-stressed macrophages were used to prepa e defined cytokine blends. TNF-a, IL-6, IL- 10, and IL-12p70 at 42, 16, 1.3, and 2.1 ng / mL, respectively, were denoted as the “High Inflammation” blend, which reflects their release concentrations in LPS-stressed macrophages (from Section 1.13). The “Reduced Inflammation” blend (17.8, 3.4, 0.2, and 2.1 ng / mL of each cytokine, respectively) reflects the decreased cytokine levels seen in LPS-stressed macrophages treated with I-HA (Section 1.12). HEI-OC1cells were cultured as outlined (Section 1 .8) and treated with 100 pL / well of the defined cytokine blends described above. After 24 hrs, MTS and LDH assays were performed as described (Section 1.9).

[0145] 1.15 TM and RWM Model Compatibility and Permeation Testing

[0146] In vitro TM and RWM permeation models were previously developed by our group,26,27and grown as described in detail herein. TM models were grown with primary neonatal human keratinocytes cultured at an air-liquid interface for 11 days before testing. RWM models were grown with primary human small airway epithelial cells cultured at an air-liquid interface for 14 days. Permeation testing was also performed as described herein. Briefly, tissues were mounted in custom 3D printed permeation devices and transepithelial electrical resistance (TEER) was measured with a Millicell ERS-2 voltohmmeter to ensure tissue integrity. Tissues were placed in a 12- well plate containing 0.75 mL DPBS (receiver solution) and 0.1 mL of treatment in DPBS was placed onto the apical surface of the tissue. I-HA were treated at 20 mg / mL and IBU was treated at the estimated equivalent concentration of unconjugated IBU (2.4 mg / mL, applied as suspension due to solubility limit of ~0.3 mg / mL). After being allowed to incubate in a 37 °C humidified incubator for the indicated treatment time, receiver solution was collected and stored at -20 °C until analysis.

[0147] LHA concentration was determined using SEC-MALS / RI as detailed above (Section 1.7) against a standard curve. IBU concentration was determined using the same run conditions as SEC-MALS, but rather than use RI, the AUC of UV absorbance at 223 nm was used and compared to an IBU standard curve. The lack of this UV signal in the I-HA samples was also used to confirm that IBU had not noticeably detached from I-HA following tissue permeation.

[0148] Tissue viability was performed by placing 0.1 mL of the indicated treatments in DPBS onto the surface of each tissue. A 5% w / v SDS solution was used as a cytotoxic control. The tissues were then placed in fresh growth media in a 37 °C, 5% CO2, humidified incubator for 24 hrs. After treatment, the tissue was thoroughly washed with DPBS then placed into 0.3 mL media containing MTT (1 mg / mL), which is reduced to a formazan by living cells, for 3 hr in the incubator. The tissues are then placed into a new plate, submerged in 2 mL isopropanol to dissolve the formazan, sealed, and placed on a shaker for 2 hr. Absorbance at 570 nm is then measured in technical duplicates on a Cytation 5 plate reader. TEER and histological preparation / analysis of treated tissues was performed as previously described in detail26,27.

[0149] 1.16 Statistical Analysis

[0150] Prism version 10 (GraphPad, San Diego, CA) was used for normality testing and statistical analysis. Each figure caption details the specific tests utilized for each dataset. A p- value of <0.05 was considered statistically significant.

[0151] 2. Results

[0152] 2.1 HAC Synthesis

[0153] Three anti-inflammatory drugs; IBU, PS, and HC, were selected for conjugation to HA (Figure 9). The initial step of the synthesis process was to enrich HA with carboxyl moieties to increase drug conjugation sites. The structure of this intermediate (CMHA) was confirmed with 'H-NMR. which also revealed a carboxymethylation efficiency of 52-57% (% of possible binding sites). Separately, the anti-inflammatory drugs were each functionalized with a primary amine to facilitate subsequent conjugation to HA (Figure 2). Successful modification was confirmed via 'H-NMR (Figure lOA-Figure 10C). The aminated anti-inflammatory drugs were then covalently conjugated to HA, and the successful syntheses of the three resulting HACs, denoted as I-HA, P-HA, and HC-HA (IBU, PS, and HC, respectively), were confirmed via 'H- NMR (Figure l lA-Figure 11C). All three conjugates were also found to be significantly more water soluble (greater than 20 mg / mL in DPBS) than their unconjugated equivalents.

[0154] 2.2 Anti-inflammatory Screening of HACs

[0155] To assess the anti-inflammatory properties of the conjugates, cytokine release was evaluated in LPS- stimulated macrophages with and without treatments. First, the optimal LPS dose that elicited a robust release of TNF-a and IL-6 (Figure 12A, Figure 12B), without affecting cell viability (Figure 12C), was determined. Based on these results, LPS was dosed at 10 ng / mL in all subsequent assays. A panel of additional inflammatory cytokines was then evaluated to profile the cytokine release patterns of macrophages in response to LPS-stress. (Figure 13). This found that TNF-a and IL-6 were the predominant cytokines released, and showed that LPS induced a very strong response relative to control in every other cytokine tested, with the exception of IFN-y, which was below the quantification limit in both groups.

[0156] Based on these findings, the anti-inflammatory potential of the three HACs was evaluated in the two most responsive cytokines (TNF-a and IL-6). While unconjugated PS and HC reduced IL-6 release in both the 4 and 24 hr treatment condition, reduction of TNF-a was only observed in the 4 hr condition (Figure 3). Similarly, P-HA and HC-HA also showed areduction in TNF-a in the 4 hr condition; however, this was to a lesser extent than the unconjugatcd drugs. Unlike the unconjugatcd drugs, neither P-HA nor HC-HA reduced IL-6 release in either condition. Conversely, I-HA significantly decreased IL-6 and TNF-a release in both the 4 and 24 hr treatment conditions. In this case, unconjugated drug (IBU) only decreased TNF-a release in the 4 hr condition, and did so to a lesser extent than the conjugate (I-HA). Based on these results, P-HA and HC-HA were not further investigated, as they significantly underperformed relative to unconjugated drug. Therefore, I-HA was selected as the lead candidate for further investigation.

[0157] 2.3 I-HA Characterization

[0158] 2.3.1 Physicochemical Properties

[0159] Various physicochemical properties of I-HA were then evaluated. The analyses showed that I-HA has an average molecular weight of 27.8 kDa, a polydispersity index of 1.399, a refractive index increment of 0.1495 mL / g, and a (^-potential of -26.77 mV (Table 1). The estimated conjugation efficiency of I-HA was determined to be 12.0-12.9% w / w (mass I to total mass LHA).Table 1

[0160] 2.3.2 Cytocompatibility

[0161] To confirm I-HA has a minimal risk of cytotoxicity, cytocompatibility assays were performed in macrophages and HELOC1 cochlear cells. In macrophages, 24 hr LHA treatment showed no signs of decreased cell viability (Figure 4A) or membrane integrity (Figure 4B) relative to untreated control. Conversely, unconjugated IBU, or a blend of CMHA and unconjugated IBU (at the estimated equivalent concentrations), severely reduced cell viability (55% and 59%, respectively) and membrane integrity (20% and 19% cell lysis, respectively). A dose-response assay showed that IBU had an IC50 (concentration leading to 50% cell viability) of 0.135 mg / mL (Figure 14A), despite the higher equivalent concentration of LHA showing no loss in viability.

[0162] Similarly, in HEI-OC1 cells, no cell viability (Figure 4C) or membrane integrity (Figure 4D) issues were observed with I-HA treatment. A dose-response assay (Figure 14B) showed that HEI-OC1 cells are less affected by IBU than macrophages, with an IC50 above IBU’s solubility limit (0.3 mg / mE), which resulted in only a 28% reduction in cell viability.

[0163] 2.3.3 I-HA Inflammatory Cytokine Panel

[0164] An inflammatory cytokine panel was then used to evaluate the direct effects of I-HA on macrophage cytokine release. Relative to the EPS-stressed control, I-HA treatment reduced the levels of IL-10, IL-10, IL-2, IL-5, KC / GRO (CXCL1), IL-6, and TNF-a (Figure 5A-C). IL- 12p70 was not reduced by I-HA and IFN-y was below the assay quantification limit in both LHA and the control. LHA did reduce IL-4 levels below the assay quantification limit; however, the control and IBU groups were not significantly different from the quantification limit. Conversely, IBU only reduced the release of IL-10 and IL-10, and IL-10 was the only cytokine IBU reduced more than I-HA treatment. Additionally, IBU also significantly increased the levels of IL-5 and IL-6 relative to control.

[0165] 2.3.4 Inflammatory Cytotoxicity in Cochlear Cells

[0166] To assess the effect of macrophage-mediated inflammation on cochlear cell survival, conditioned media (CM) and LPS CM were produced by treating macrophages for 24 hrs with media alone or media with LPS, respectively. Cochlear cell viability was significantly reduced by treatment with LPS CM compared to control (media only) and LPS control (media with LPS) (Figure 6A). However, CM also significantly reduced cell viability, although to a lesser extent than LPS CM. Since the CM (from unstressed macrophages) also resulted in cell death, it is unclear if the increased cytokine levels in LPS CM are solely responsible for the observed effect. To further understand this, defined cytokine blends were created with the four most prominent cytokines (IL- 10, IL-12p70, IL-6, and TNF-a) released by LPS-stressed macrophages. One blend reflected the previously quantified cytokine concentrations found in LPS-stressed macrophages (denoted as “High Inflammation”), and the other reflected the reduced cytokine concentrations found in LPS-stressed macrophages that were treated with I-HA (denoted “Reduced Inflammation”). These blends were then applied to HEI-OC1 cells for 24 hrs, and the cell viability was determined. As expected, the “High Inflammation” blend significantly reduced cell viability (down to 64%) and membrane integrity (36% cell lysis) (Figure 6B, Figure 6C). The “Reduced Inflammation” blend, reflective of LHA treatment, showed a significant improvementin cell viability (82%) and membrane integrity (30% cell lysis) relative to the “High Inflammation” group. Moreover, investigating the effects of individual cytokines at the “High Inflammation” concentration did not show any negative effects on cell viability (Figure 15), suggesting that the interaction between multiple cytokine pathways is critical to the cellular damage.

[0167] 2.3.5 TM and RWM Models: I- HA Permeation and Tissue Compatibility

[0168] To evaluate I-HA’s potential as a topical therapeutic, tissue compatibility was assessed in in vitro TM and RWM permeation models. As seen in the cytocompatibility assays, unconjugated IBU showed significant toxicity in both tissue models, while I-HA caused no apparent toxicity (Figure 7A).

[0169] I-HA permeability across the RWM and TM models was then evaluated. After 24 hr, 41% of the applied LHA permeated the RWM model, while only 28% of an equivalent concentration of unconjugated IBU permeated (Figure 7B). The kinetics of this permeation was also evaluated to provide additional insight into I-HA flux over time (Figure 16). When tested in the TM model, total I-HA permeated was below the method detection limit (Figure 7C). Lower TM permeability is expected, as it is known to be significantly less permeable than the RWM. Intriguingly, IBU was able to readily permeate the TM at levels comparative to the RWM model. To understand this, a loss of tissue integrity was then investigated as a possible explanation to the higher-than-expected permeability of IBU across the TM. For this, transepithelial electrical resistance (TEER), a measure of barrier integrity, was evaluated. The TEER values were significantly decreased by treatment with IBU, but not I-HA, confirming that IBU treatment resulted of loss of barrier integrity. (Figure 8A). This was further confirmed this histologically, with our analyses revealing that IBU treatment resulted in drastic changes to tissue stratification, keratinocyte differentiation (evidenced by the persistent nuclei within the granular and corneal layers), and basilar keratinocytes detachment (Figure 8B). In contrast, I-HA did not noticeably change tissue morphology.

[0170] The data herein revealed that I-HA was well tolerated by cells despite unconjugated drug showing cytotoxic effects. Additionally, I-HA was also able to reduce macrophage- mediated inflammatory cytotoxicity in HELOC1 cells. Finally, I-HA permeation and tissue viability studies were conducted using in vitro TM and RWM permeation models previously developed.26,27These permeation studies further highlighted the potential of I-HA as a topicaltherapeutic for inflammation-mediated hearing loss.INCORPORATION BY REFERENCE

[0171] 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.

[0172] The present disclosure has multiple aspects, illustrated by the non-limiting examples described herein.

[0173] 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.

[0174] 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.

[0175] For reasons of completeness, various aspects of the disclosure are set out in the following numbered clauses:

[0176] Clause 1. A composition comprising: a hyaluronic acid (HA)-nonsteroidal antiinflammatory drug (NSAID) conjugate, wherein toxicity of the NSAID is reduced compared to a NSAID that is not conjugated to hyaluronic acid.

[0177] Clause 2. The composition of clause 1, wherein the conjugate reduces inflammation related or induced cytotoxicity in cochlear cells when administered to a subject.

[0178] Clause 3. The composition of clause 1 or clause 2, wherein the hyaluronic acid is a low molecular weight hyaluronic acid, a medium molecular weight hyaluronic acid, or a high molecular weight hyaluronic acid.

[0179] Clause 4. The composition of any of clauses 1-3, wherein the hyaluronic acid is a low molecular weight hyaluronic acid.

[0180] Clause 5. The composition of any of clauses 1-4, wherein the conjugate has a molecular weight of from about 20,000 Daltons to about 40,000 Daltons or from about 20,000 Daltons to about 30,000 Daltons.

[0181] Clause 6. The composition of any of clauses 1-5, wherein the NSAID is a propionic acid derivative.

[0182] Clause 7. The composition of clause 6, wherein the propionic acid derivative is ibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaproin, or any combinations thereof.

[0183] Clause 8. The composition of clause 7, wherein the propionic acid derivative is ibuprofen.

[0184] Clause 9. The composition of any of clauses 1-8, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

[0185] Clause 10. A method of treating a subject suffering from inflammation, the method comprising:

[0186] administering to a subject suffering from at least one type of inflammation and in need of treatment thereof, a therapeutically effective amount of the composition of any of clauses 1-9, wherein the inflammation is an inflammation-mediated hearing loss, arthritis, a musculoskeletal disorder, a headache, a migraine, dysmenorrhea, dental pain, post-operative pain, or a soft tissue injury, or any combination thereof.

[0187] Clause 11. The method of clause 10, wherein the composition is administered topically.

[0188] Clause 12. The method of clause 10, wherein the composition is administered via parenterally.

[0189] Clause 13. The method of clause 10, wherein the composition is administered orally.

[0190] Clause 14. The method of any of clauses 10-13, wherein the arthritis is rheumatoid arthritis, osteoarthritis, psoriatic arthritis, gout, or any combinations thereof.

[0191] Clause 15. The method of any of clauses 10-14, wherein the musculoskeletal disorder is bursitis, tendinitis, a muscle strain, a muscle sprain, back pain, or any combinations thereof.

[0192] Clause 16. The method of any of clauses 10-15, wherein the subject is a human a nonhuman mammal, a bird, a reptile, or a fish.

[0193] Clause 17. The method of clause 16, 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.

[0194] Clause 18. The method of any of clauses 10-17, wherein the inflammation-mediated hearing loss is noise-induced hearing loss.

[0195] Clause 19. The method of any of clauses 10-18, wherein the inflammation-mediated hearing loss is caused by an autoimmune inner ear disease, a systemic autoimmune disease, an autoinflammatory disease, infection-related inflammation, or any combination thereof.References1. Paik, C. B., Pei, M. & Oghalai, J. S. Review of blast noise and the auditory system. Hear. Res. 425, 108459 (2022).2. Bohne, B. A., Harding, G. W. & Lee, S. C. Death pathways in noise-damaged outer hair cells. Hear. Res. 223, 61-70 (2007).3. Kalinec, G. M., Lomberk, G., Urrutia, R. A. & Kalinec, F. Resolution of Cochlear Inflammation: Novel Target for Preventing or Ameliorating Drug-, Noise- and Age-related Healing Loss. Front. Cell. Neurosci. 11, 192 (2017).4. Xu, K. et al. Intrinsic mechanism and pharmacologic treatments of noise-induced hearing loss. Theranostics 13, 3524-3549 (2023).5. Yang, S. et al. 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Claims

WHAT IS CLAIMED IS:

1. A composition comprising: a hyaluronic acid (HA)-nonsteroidal antiinflammatory drug (NSAID) conjugate, wherein toxicity of the NSAID is reduced compared to a NSAID that is not conjugated to hyaluronic acid.

2. The composition of claim 1, wherein the conjugate reduces inflammation related or induced cytotoxicity in cochlear cells when administered to a subject.

3. The composition of claim 1 or claim 2, wherein the hyaluronic acid is a low molecular weight hyaluronic acid, a medium molecular weight hyaluronic acid, or a high molecular weight hyaluronic acid.

4. The composition of any of claims 1-3, wherein the hyaluronic acid is a low molecular weight hyaluronic acid.

5. The composition of any of claims 1-4, wherein the conjugate has a molecular weight of from about 20,000 Daltons to about 40,000 Daltons or from about 20,000 Daltons to about 30,000 Daltons.

6. The composition of any of claims 1-5, wherein the NSAID is a propionic acid derivative.

7. The composition of claim 6, wherein the propionic acid derivative is ibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaproin, or any combinations thereof.

8. The composition of claim 7, wherein the propionic acid derivative is ibuprofen.

9. The composition of any of claims 1-8, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

10. A method of treating a subject suffering from inflammation, the method comprising: administering to a subject suffering from at least one type of inflammation and in need of treatment thereof, a therapeutically effective amount of the composition of any of claims 1-9, wherein the inflammation is an inflammation-mediated hearing loss, arthritis, a musculoskeletal disorder, a headache, a migraine, dysmenorrhea, dental pain, post-operative pain, or a soft tissue injury, or any combination thereof.

11. The method of claim 10, wherein the composition is administered topically.

12. The method of claim 10, wherein the composition is administered parenterally.

13. The method of claim 10, wherein the composition is administered orally.

14. The method of any of claims 10-13, wherein the arthritis is rheumatoid arthritis, osteoarthritis, psoriatic arthritis, gout, or any combinations thereof.

15. The method of any of claims 10-14, wherein the musculoskeletal disorder is bursitis, tendinitis, a muscle strain, a muscle sprain, back pain, or any combinations thereof.

16. The method of any of claims 10-15, wherein the subject is a human, a non-human mammal, a bird, a reptile, or a fish.

17. The method of claim 16, 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.

18. The method of any of claims 10-17, wherein the inflammation-mediated hearing loss is noise-induced hearing loss.

19. The method of any of claims 10-18, wherein the inflammation-mediated hearing loss is caused by an autoimmune inner ear disease, a systemic autoimmune disease, an autoinflammatory disease, infection-related inflammation, or any combination thereof.

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