WO2021127275A1

WO2021127275A1 - Gsk-3 modulator otic formulations

Info

Publication number: WO2021127275A1
Application number: PCT/US2020/065749
Authority: WO
Inventors: Alan Foster; Bonnie Elizabeth JACQUES; Fabrice Piu; Stephanie Ann SZOBOTA; Phillip Michael Uribe; Pranav Dinesh MATHUR
Original assignee: Otonony, Inc.
Priority date: 2019-12-17
Filing date: 2020-12-17
Publication date: 2021-06-24

Abstract

Disclosed herein are otic formulations and compositions comprising GSK-3 modulators. These otic formulations and compositions allow for the delivery of the GSK-3 modulator to inner ear to: increase SGN density and/or branching; increase number of Schwann cells; increase myelin protein expression; increase number of neural/glial precursor cells; increase neuron survival upon SGN dissociation; preserve hair cell and SGN density upon cisplatin-induced ototoxcity; or combinations thereof.

Description

GSK-3 MODULATOR OTIC FORMULATIONS

BACKGROUND OF THE DISCLOSURE

[0001] Vertebrates have a pair of ears, placed symmetrically on opposite sides of the head. The ear serves as both the sense organ that detects sound and the organ that maintains balance and body position. The ear is generally divided into three portions: the outer ear, auris media (or middle ear), and the auris interna (or inner ear).

[0002] GSK-3 (Glycogen synthase kinase 3) is a serine/threonine protein kinase that plays a central role in a diverse range of signaling pathways, including those activated by Wnts, hedgehog, growth factors, cytokines, and G protein-coupled ligands. Mammals express GSK-3 in two isoforms: GSK-3 alpha (GSK-3 a) and GSK-3 beta (GSK-3 b). GSK-3 signaling has been implicated in a variety of pathological conditions, including otic diseases and disorders

[0003] Described herein are otic formulations comprising GSK-3 modulators for drag delivery into the outer, middle, and/or inner ear, including the cochlea and vestibular labyrinth.

SUMMARY OF THE DISCLOSURE

[0004] Provided in one aspect is an otic formulation comprising a therapeutically effective amount of a GSK-3 inhibitor and an auris-acceptabie vehicle. In some embodiments, the amount of the GSK-3 inhibitor released into the inner ear is sufficient to: increase hair cell numbers; increase SGN density and/or branching; increase the number of Schwann cells; increase myelin protein expression; preserve hair cells upon drug-induced ototoxcity; or combinations thereof, and wherein the amount of the GSK-3 inhibitor released into the inner ear is below' a toxicity exposure limit.

[0005] In some embodiments, the toxicity exposure limit would result in at least one of decreased SON density and/or branching; decreased number of Schwann cells; decreased myelin protein expression; and decreased hair cell preservation upon drug-induced ototoxcity. Q006] In some embodiments, the amount of the GSK-3 inhibitor released into the inner ear is sufficient to increase inner ear BDNF expression and wherein the toxicvity exposure limit would result in decreased inner ear BDNF expression.

[0007] In some embodiments, the auris-acceptabie gel is a thermoreversible gel. [Q008] In some embodiments, the auris-acceptable gel s capable of being injected by a narrow gauge needle or cannula through the tympanic membrane.

[0009] In some embodiments, the otic formulation has an osmolarity from about 250 to about 320 mOsm/L.

[0010] In some embodiments, the otic formulation has a gelation temperature from about 19°C to about 42° C.

[0011] In some embodiments, the otic formulation has a pH from about 7.0 to about 8.0. [0012] In some embodiments, the otic formulation comprises from about 14 wt%to about 25 wt% poloxamer 407.

[0013] In some embodiments, the otic formulation comprises from about 15 wt% to about 18 wt% poloxamer 407.

[0014] in some embodiments, the auris-aceeptable vehicle comprises triglycerides comprising medium chain fatty acids.

[0015] In some embodiments, the triglycerides are derived from glycerol and medium chain fatty acids.

[0016] In some embodiments, wherein the medium chain fatty acids are caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylenic acid (undec-10-enoic acid), lauric acid (dodecanoic acid), or any combinations thereof.

[0017] In some embodiments, the otic formulation comprises at least about 50% by weight of the triglycerides.

[0018] In some embodiments, the otic formulation comprises from about 85% to about 99.99% by weight of tire triglycerides.

[0019] In some embodiments, the otic formulation has a viscosity from about 10 cl⁵ to about 500 cP

[0020] In some embodiments, the otic formulation is free or substantially free of water, Cl- C6 alcohols or C1-C6 glycols, C1-C4 alcohols or C1-C4 glycols, or any combination thereof. [0021] In some embodiments, the GSK-3 inhibtor is multiparticulate.

[0022] In some embodiments, the GSK-3 inhibtor is dissolved in the otic formulation.

[0023] In some embodiments, the otic formulation discioed herein is used in the treatment of an otic disease or condition associated with decreased SGN density and/or branching; decreased number of Schwann cells; decreased Schwann cells or oligodendrocyte survival or proliferation; decreased SGN or auditory' nerve myelin sheaths; decrease expression of myelin-promoting genes; decreased myelin protein expression; decreased number of neural/glial precursor cells; decreased neural/glial proliferation; decreased number of hair ceil upon drug-induced ototoxcity; or combinations thereof.

[0024] in some embodiments, the otic formulation discloed herein is used in the treatment of an otic disease or condition associated with decreased inner ear BDNF expression.

[0025] In some embodiments, the otic formulation discloed herein is used in the treatment of hearing loss.

[0026] In some embodiments, the GSK-3 inhibitor in the otic formulation is not GSK-22. [0027] In some embodiments, the GSK3 inhibitor in the otic formulation is not:

[0028] In some embodiments, the GSK3 inhibitor the otic formulation is not any one of: 2~pynmidinylam oethyiamino-2-pyridinyl containing compound;

3 ~(py ridin -2-yl) - 1 H-indol-2 -ol contai n ing compoun d ;

2-pyriimdinylaminoethylamino-2-pyridyi containing compound;

N-( lH-pyrazol-4-yl)-nicotinamide containing compound

2.4-dichloropheny3-5-(lH-imidazol-2-yl)-2-pyrimidinylaminoetliylamino-2 -pyridine compound;

2.4-dichiorophenyl-5-(lH-imidazol-2-yl)-2-pyrimidinylatninoethyiammo-3-pyridine containing compound;

3-(9 fluoro-2-(piperidine-l-carbonyl)-i,2,3,4-tetrahydro-[i,4]diazepino[6,7,l- hi]indol-7-yl)-4-(imidazo[l,2-a]pyridin-3-yl)-lH-pyrrole-2,5-dione; and

1.2.3.4-tetrahydro-[l,4]diazepino[6,7, 1 -hijindolyl containing compound.

INCORPORATION BY REFERENCE Q029] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent. or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS [0030] Tiie novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0031] FIG. 1A show's perilymph, cochlear, and plasma concentration of 2.5 mg/mL GSK3 inhibitor GSK-22 in 16% P407: E1G. IB shows perilymph, cochlear, and plasma concentration of 2.5 mg/mL GSK-22. in 16% P407 that further contains 5% DMSQ; FIG. 1C shows perilymph, cochlear, and plasma concentration of 2.5 mg/mL GSK-22 in 16% P407 that further contains 5% OMSO and 87.6 mg/mL sodium valproate; FIG. ID show s perilymph, cochlear, and plasma concentration of 1.3 mg/mL CH1R99021 in 16% P407; FIG. IE shows perilymph, cochlear, and plasma concentration of 1.3 mg/mL CHTR99021 in 23.75% P407 that further contains 5% DM80 and 166.2 mg/mL sodium valproate.

[0032] FIG. 2 shows changes in inner and outer hair cells in undamaged naive cochlear explants following exposure to CHIR99021.

[Q033] FIGs. 3A and 3B show changes in the numbers of inner and outer hair cells in aminoglycoside damaged cochlear explants following exposure to CH1R99021.

[Q034] FIG. 4 shows perilymph concentrations of CHIR99021 following administration of formulations comprising various concentrations of CHIR99021 in 16% P407.

[0035] FIG. 5 show's an increase in spiral ganglion neuron (SGN) density and neural/glial precursor cells in undamaged naive cochlear explants following expposure to CHIR99021. [0036] FIG. 6 shows an increase in SGN density, Schwann cells, and myelin protein expression in undamaged naive cochlear explants following exposure to CHIR9902I.

[0037] FIG. 7 shows a dose responsive increase in SGN density in undamaged naive cochlear explants following exposure to ARA-014418.

[0038] FIG. 8 shows an increase in SGN density in undamaged naive cochlear explants following exposure to various GSK3 inhibitors.

[0039] FIG. 9 show's an increase in SGN density in undamaged naive cochlear explants following exposure to various GSK3 inhibitors. [Q040] FIG. 10 shows an increase in SGN density and Schwann cells in undamaged naive cochlear explants following exposure to various GSK3 inhibitors.

[0041] FIG. 11 shows an increase in neuron survival in dissociated spiral ganglion neuron cultures following exposure to various GSK3 inhibitors.

[0042] FIG. 12 show s preservation of HC and SGN density in cisplatin damaged cochlear explants when treated with the GSK3 inhibitor CHIR99021

[0043] FIG. 13 shows increased BDNF expression in cochlear explants when treated with the GSK3 inhibitor CHIR99021.

[0044] FIG. 14 shows synaptogenesis and/or preservation of synaptic connections in rat or mouse cochlear explant cultures when treated with the GSK3 inhibitors CHIR99021 and GSK-22.

[0045] FIG. 15 show s synaptogenesis and/or preservation of synaptic connections in rat or mouse cochlear explant cultures when treated with the GSK3 inhibitors CHIR99G21 and

GSK-22.

DETAILED DESCRIPTION

[0046] Systemic admin stration of active agents is, in some instances, ineffectual in the treatment of diseases that affect inner ear structures. The cochlear canals and the cochlea, for example, are isolated from die circulatory system limiting systemic delivery of active agents to target sites in the inner ear. In some instances, systemic drug administration creates a potential inequality in drug concentration with higher circulating levels in the serum, and lower levels in the target amis interna organ structures. In certain instances, large amounts of drag are required to overcome this inequality in order to deliver sufficient, therapeutically effective quantities of a drug to auditory structures. In some instances, systemic drag administration also increases the likelihood of secondary' systemic accumulation and consequent adverse side effects.

[0047] Currently available treatment for inner ear diseases also carries the risk of attendant side effects. For example, available methods require multiple daily doses (e.g., intratympanic injection or infusion) of drugs. In certain instances, multiple daily intratympanic injections cause patient discomfort and non-compliance. In certain instances, delivery_' of active agents to the inner ear via otic drops administered in the ear canal or via intratympanic injection is hindered by tire biological barrier presented by tire tympanic membrane the oval window membrane and/or the round window' membrane in some instances, delivery of active agents to the inner ear via otic drops or intratympanic injection causes osmotic imbalance in inner ear structures, introduces infections or other immune disorders as a result of microbial or endotoxin presence, or results in permanent structural damage (e.g. perforation of the tympanic membrane), resulting in hearing loss and the like.

[0048] Intratympanic injection of therapeutic agents is the technique of injecting a therapeutic agent behind the tympanic membrane into the auris media and/or antis interna. Some challenges remain with intratympame injections. For example, access to the round window membrane, the si te of drug absorption into the auris interna, is challenging in some instances in addition, current regimens using intratympanic injections do not address changing the osmolarity and pH of the perilymph and endolymph, and introducing pathogens and endotoxins that directly or indirectly damage inner ear.

[0049] Provided herein in one aspect are otic formulations and compositions comprising a therapeutically effective amount of an active agent, such as a GSK-3 modulator. In some embodiments, the G8K-3 modulator is a GSK-3 inhibitor. In some embodiments, the otic formulations are auris-acceptable gels. In some embodiments, the otic formulations are triglyceride based auris-acceptable formulations.

[QQ5Q] These otic pharmaceutical formulations are suitable for drug delivery into the external, middle and/or inner ear. In some instances, these otic pharmaceutical formulations and compositions are suitable for administration to humans in some instances, the otic formulations and compositions disclosed herein also meet stringent criteria for pH, osmolarity, ionic balance, sterility', endotoxin, and/or pyrogen levels. In some instances, the otic formulations and compositions are compatible with the microenvironment of the inner ear (e.g., tire perilymph).

[0051] Accordingly, provided herein, in certain embodiments, are otic formulations and compositions that are controlled release auris-acceptable formulations and compositions that locally treat auris target structures and provide extended exposure of otic active agents to the target auris structures. In certain embodiments, the otic formulations and compositions described herein are designed for stringent osmolarity and pH ranges that are compatible with auditory structures and/or the endolymph and perilymph. In some embodiments, the otic formulations and compositions described herein are controlled release formulations that provide extended release for a period of at least 3 days and meet stringent sterility' requirements. In some instances, otic formulations and compositions described herein contain lower endotoxin levels (e.g. < 0.5 EU/mL when compared to typically acceptable endotoxin levels of 0.5 EU/mL. in some instances, the otic formulations and compositions described herein contain low levels of colony forming units (e.g., <50 CPUs) per gram of the formulation or composition in some instances, the otic formulations or compositions described herein are substantially free of pyrogens and/or microbes. In some instances the otic formulations or compositions described herein are formulated to preserve the ionic balance of the endolymph and/or the perilymph.

[0052] In some instances, local administration of the otic formulations and compositions described herein avoids potential adverse side effects as a result of systemic administration of active agents in some instances, the locally applied otic fonnulations and compositions described herein are compatible with auris structures. Such compatible auris structures include those associated with the outer, middle, and/or inner ear. In some embodiments, the otic formulations and compositions are administered either directly to the desired auris structure, e.g. the cochlear region, or administered to a structure in direct communication with areas of the auris structure; in the ease of the cochlear region, for example, including but not limited to the round window membrane, the crista fenestrae cochleae or the oval window membrane.

[0053] in certain instances, the otic formulations and compositions disclosed herein controlled release formulations or compositions that provide a constant rate of release of a drug from the formulation and provide a constant prolonged source of exposure of an otic active agent to the inner ear of an individual or patient suffering from an otic disorder, reducing or eliminating any variabilities associated with other methods of treatment (such as, e.g., otic drops and/or multiple mtratympanie injections).

[Q054] In some embodiments, the otic fonnulations and compositions described herein provide extended release of the active ingredient(s) into the external ear. In some embodiments, the otic formulations and compositions described herein pro vide extended release of the active ingredient(s) into the middle and/or inner ear {auris interna), including the cochlea and vestibular labyrinth. In some embodiments, the otic fonnulations and compositions further comprise an immediate or rapid release component in combination with a controlled release component.

Certain Definitions

[0055] The term “auris-acceptable” with respect to a formulation, composition or ingredient, as used herein, includes having no persistent detrimental effect on the auris externa (or external ear or outer ear), auris media (or middle ear) and/or the auris interna (or inner ear) of the subject being treated. By “auris-pharmaceuticaliy acceptable,” as used herein, refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound in reference to the auris externa (or external ear or outer ear), auris media (or middle ear) and/or the auris interna (or inner ear), and is relatively or is reduced in toxicity to the auris externa (or external ear or outer ear), auris media (or middle ear) and the auris interna (or inner ear), i.e., the material is administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0056] As used herein, amelioration or lessening of the symptoms of a particular otic disease, disorder or condition by administration of a particular compound or pharmaceutical composition refers to any decrease of severity, delay in onset, slowing of progression, or shortening of duration, whether permanent or temporary lasting or transient that is attributed to or associated with administration of the compound or composition.

[0057] As used herein, the term ‘^"antimicrobial agent” refers to compounds that inhibit the growth, proliferation, or multiplication of microbes, or that kill microbes. Suitable “antimicrobial agents” are antibacterial agents (effective against bacteria), antiviral agents (effective against viruses), antifungal agents (effective against fungi), antiprotozoal (effective against protozoa), and/or antiparasitic to any class of microbial parasites. “Antimicrobial agents” work by any suitable mechanism against the microbes, including by being toxic or cytostatic.

[0058] “Antioxidants” are amis-pharrnaceutically acceptable antioxidants, and include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol . In certain embodiments, antioxidants enhance chemical stability where required. Antioxidants are also used to counteract the ototoxic effects of certain therapeutic agents.

[Q059] The term “auns-acceptable penetration enhancer” with respect to a formulation, composition or ingredient, as used herein, refers to the property_' of reducing barrier resistance. [006Q] “Auris externa” refers to the external (or outer) ear, and includes the pinna and the external auditory_' canal (EAC).

[0061] “Auris interna” refers to the inner ear, including the cochlea and the vestibular labyrinth, and the round window' that connects the cochlea with the middle ear.

[0062] “Auris-intema bioavailability” or ^"‘Auris media bioavailability” refers to the percentage of the administered dose of compounds disclosed herein that becomes available in the inner or middle ear, respectively, of the animal or human being studied

[0063] “Auris media” refers to the middle ear, including the tympanic cavity, auditory' ossicles and oval window, which connects the middle ear with the inner ear. [0064] “Auris-intema bioavail ability” refers to the percentage of the admin stered dose of compounds disclosed herein that becomes available in the inner ear of the animal or human being studied.

[0065] “Balance disorder” refers to a disorder, illness, or condition which causes a subject to feel unsteady, or to have a sensation of movement. Included in this definition are dizziness, vertigo, disequilibrium, and pre-syncope. Diseases which are classified as balance disorders include, but are not limited to, Ramsay Hunt's Syndrome, Meniere's Disease, mal de debarquemenf benign paroxysmal positional vertigo, labyrinthitis, and presbycusis.

[0066] ^"‘Blood plasma concentration” refers to the concentration of compounds provided herein in the plasma component of blood of a subject.

[0067] ‘ Carrier materials” are excipients that are compatible with the otic agent, the auris media, the auris interna and the release profile properties of the auris-acceptable pharmaceutical formulations. Such earner materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. “Auris-pharmaceutical!y compatible carrier materials” include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurochoiic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, alginate, carbomer, hyaluronic acid (HA), poloxamer, dextran, and the like.

[0068] As used herein, the term “cytotoxic agent” refers to compounds that are cytotoxic (i.e., toxic to a cell) effective for the treatment of otic disorders, e.g., autoimmune diseases of the ear and cancer of the ear, and are suitable for use in the formulations disclosed herein. [0069] The term “diluent” are chemical compounds that are used to dilute die otic agent prior to deli very and which are compatible with the auris media and/or auris interna.

[0070] “Drug absorption” or “absosption” refers to the process of movement of the otic agent from the localized site of administration, by way of example only, the round window membrane of the inner ear, and across a barrier (the round window membranes, as described below) into the auris interna or inner ear structures. The terms “co-administration” or the like, as used herein, are meant to encompass administration of the otic agent to a single patient, and are intended to include treatment regimens in which the otic agents are administered by the same or different route of administration or at the same or different time. [0071] The terms ‘^‘effective amount^” or “therapeutically effective amount,” as used herein, refer to a sufficient amount of the otic agent being administered that would be expected to relieve to some extent one or more of the symptoms of the disease or condition being treated. For example, the result of administration of the otic agents disclosed herein is reduction and/or alleviation of the signs, symptoms, or causes of any one of the diseases or conditions disclosed herein. For example, an “effective amount” for therapeutic uses is the amount of the otic agent, including a formulation as disclosed herein required to provide a decrease or amelioration in disease symptoms without undue adverse side effects. Tire term “therapeutically effective amount” includes, for example, a prophylactically effective amount. An “effective amount” of a otic agent composition disclosed herein is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects it is understood that “an effective amount” or “a therapeutically effective amount” varies, in some embodiments, from subject to subject, due to variation in metabolism of the compound administered, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. In some instances, it is also understood that “an effective amount” in an extended-release dosing format differs from “an effective amount” in an immediate- release dosing format based upon pharmacokinetic and pharmacodynamic considerations [0072] The terms “enhance” or “enhancing” refers to an increase or prolongation of either the potency or duration of a desired effect of the otic agent, or a diminution of any adverse symptomatology. For example, in reference to enhancing the effect of the otic agents disclosed herein, the term “enhancing” refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic agents that are used in combination with the otic agents disclosed herein. An “enhancing-effective amount,” as used herein, refers to an amount of an otic agent or other therapeutic agent that is adequate to enhance the effect of another therapeutic agent or otic agent in a desired system. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

[0073] in some embodiments, the GSK3 modulator is a GSK3 inhibitor. “GSK3 inhibitor” and “GSK-3 inhibitor” are used interchangeably herein to refer to a compound that, for example, typically exhibits an ICso (with respect to GSK3) of no more than about 100 mM and more typically not more than about 50 mM, as measured in a cell-fee assay for GSK3 inhibitory activity in some embodiments, compounds provided herein exhibit an ICso with respect to GSK3 of no more than about IQ mM, in one embodiment, no more than about 5 mM, or no more than I mM, as measured in the cell-free GSK-3 kinase assay in some embodiments the compounds provided herein exhibit an ICso with respect to GSK3 of about 1 nM or less, about 0.5 nM or less, or about 0.1 nM or less in some embodiments, the GSK3 moduiator/inhibitor modulates/inhibits G8K3ot. In some embodiments, the G8K3 modulator/inhibitor modulates/inhibits GSK3 . In some embodiments, the GSK3 modulator/inhibitor modulates/inhibits GSK3a and 08K3b. Q074] The term “inhibiting” includes preventing, slowing, or reversing the development of a condition, including any of one of the conditions described herein, or advancement of a condition in a patient necessitating treatment.

[0075] The terms “kit” and “article of manufacture” are used as synonyms.

[0076] “Local anesthetic” means a substance which causes a reversible loss of sensation and/or a loss of nociception. Often, these substances function by decreasing the rate of the depolarization and repolarization of excitable membranes (for example, neurons). By way of non-limiting example, local anesthetics include lidocame, benzocaine, prilocaine, and tetracaine.

[Q077] The term “modulate” includes the interaction with a target, for example, with the GSK-3 agents disclosed herein, the activity of GSK-3, or other direct or indirect targets that alter the activity of GSK-3, including, by way of example only, to inhibit the activity of GSK-3 or to limit the activity of tire GSK-3.

[0078] As used herein, the term “otic agent” or “otic structure modulating agent” or “otic therapeutic agent” or “otic active agent” or “active agent” or “therapeutic agent” refers to compounds that are effective for the treatment of otic disorders, e.g., otitis media, otosclerosis, autoimmune diseases of the ear and cancer of the ear, and are suitable for use in the formulations disclosed herein. An “otic agent” or “otic structure modulating agent” or “otic therapeutic agent” or “otic active agent” or “active agent” includes, but is not limited to, compounds that act as an agonist, a partial agonist, an antagonist, a partial antagonist, an inverse agonist, a competitive antagonist, a neutral antagonist, an orthosteric antagonist, an allosteric antagonist, a positive allosteric modulator of an otic structure modulating target, a negative allosteric modulator of an otic structure modulating target, or combinations thereof. [0079] The term “otic intervention” means an external insult or trauma to one or more aims structures and includes implants, otic surgery, injections, cannulations, or the like. Implants include auris-intema or auris-media medical devices, examples of which include cochlear implants, hearing sparing devices, hearing-improvement devices, short electrodes, micro- prostheses or piston-like prostheses; needles; stem cell transplants; drug delivery devices; any cell-based therapeutic; or the like. Otic surgery includes middle ear surgery, inner ear surgesyy tympanostomy, cochleostomy, labyrinthotomy, mastoidectomy, stapedectomy, stapedotomy, endolymphatic sacculotomy, or the like. Injections include intratympanic injections, intracochlear injections, injections across the round window membrane or the like. Cannuiations include intratympanic, intracochlear, endolymphatic, perilymphatic or vestibular cannuiations, or the like.

[0080] The term “penetration enhancer” refers to an agent that reduces barrier resistance (e.g., barrier resistance of the round window membrane, BLB or the like).

[0081] “Pharmacodynamics” refers to the factors which determine the biologic response observed relative to the concentration of drug at the desired site within the auris media and/or auris interna.

[0082] “Pharmacokinetics” refers to the factors which determine the attainment and maintenance of the appropriate concentration of drug at the desired site within the auris med a and/or auris interna.

[0083] In prophylactic applications, compositions containing the otic agents described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition . Such an amount is defined to be a “prophylactieally effective amount or dose.” In this use, the precise amounts also depend on the patient's state of health, weight, and the like.

[0084] As used herein, the term “subject” is used to mean an animal, preferably a mammal, including a human or non-human. The terms patient and subject are used interchangeably. [0085] “Surfactants” refers to compounds that are auris-acceptable, such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, phospholipids, lecithins, phosphatidyl cholines (c8-cl 8), phosphatidyiethaoolamines (c8-cl8), pbosphatidylglycerols (c8-cI8), sorbitan monooleate, polyoxyethylene sorbitan monooleate, pol sorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic^® (BASF), and the like. Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil: and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. in some embodiments, surfactants are included to enhance physical stability or for other purposes.

[0086] The terms “treat,” “treating” or “treatment,” as used herein, include alleviating, abating or ameliorating a disease or condition or the associated symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or controlling or stopping the symptoms of the disease or condition either prophylactically and/or therapeutically.

[0087] Pharmaceutically acceptable derivatives of a compound include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiaeetals, hemiketals, acids, bases, solvates, hydrates, or prodrugs thereof. In some embodiments, such derivatives are be readily prepared by those of skill in this art using known methods for such derivatization. Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N'~ dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxy alkylamines, ethylenediamine, N-methylglucamine, procaine, N- benzylphenethylamine, l-para-chlorobenzyl-2- pyrrolidm-i'-ylmethyibenzimidazole, diethylamine, and other alkylamines, piperazine and tris(hydroxymetliyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and inorganic salts, such as but not limited to, sodium hydrogen phosphate and disodium phosphate; and also including, but not l imited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, maiates, tartrates, citrates, ascorbates, succinates, butyrates, valerates, mesylates, and fumarates. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyi, aryl, aralkyl, and cycloalkyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sidfmic acids, and boronic acids. Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of formula C=C(QR), where R is hydrogen, alkyl, alkenyl, alkynyi, aryl, aralkyl, and cycloalkyl. Pharmaceutically acceptable enol esters include, but are not limited to, deri vatives of formula C=C(0C(0)R), where R is hydrogen, alkyl, alkenyl, alkynyi, aryl, aralkyl, and cycloalkyl. Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.

[Q088] It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configuration, or may be a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. As such, one of skill in the art will recognize that administration of a compound in its (R) torn is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (S) form.

[0089] The instant disclosure is meant to include all such possible isomers, as well as, their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)~ isomers are prepared in some instances using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0090] As used herein, alkyl, alkenyl, and alkynyl carbon chains, if not specified, contain from 1 to 20 carbons, 1 to 16 carbons or 1 to 6 carbons and are straight or branched. In certain embodiments, alkyl, alkenyl, and alkynyl carbon chains contain from 1 to 6 carbons. Alkenyl carbon chains of from 2 to 20 carbons, in certain embodiments, contain I to 8 double bonds, and the alkenyl carbon chains of 2 to 16 carbons, in certain embodiments, contain 1 to 5 double bonds. The alkenyl carbon chains of 2 to 6 carbons, in certain embodiments, contain 1 to 2 double bonds. Alkynyl carbon chains of from 2 to 20 carbons, in certain embodiments, contain 1 to 8 triple bonds, and the alkynyl carbon chains of 2 to 16 carbons, in certain embodiments, contain I to 5 triple bonds. Alkynyl carbon chains of from 2 to 6 carbons, in certain embodiments, contain 1 to 2 triple bonds. Exemplary alkyl, alkenyl and alkynyl groups herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-hutyi, sec-butyl, tert-butyl, isopentyl, neopentyl, tert- pentyl, isohexyl, vinyl, 1- propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3- butenyl, 1,3-butadienyi, ethynyl, 1-propynyl, and 2-propynyl. As used herein, lower alkyl, lower alkenyl, and lower alkynyl refer to carbon chains having from about 1 or about 2 carbons up to about 6 carbons.

[0091] The term "cycioalkyl" refers to a saturated mouo- or multicyelic ring system, in certain embodiments of 3 to 10 carbon atoms, in other embodiments of 3 to 6 carbon atoms; cycioaikenyl and cycloalkynyl refer to mono- or multicyelic ring systems that respectively include at least one double bond and at least one triple bond. Cycioaikenyl and cycloalkynyl groups may, in certain embodiments, contain 3 to 10 carbon atoms, with cycioaikenyl groups, in further embodiments, containing 4 to 7 carbon atoms and cycloalkynyl groups, in further embodiments, containing 8 to 10 carbon atoms. The ring systems of the cycioalkyl, cycioaikenyl, and cycloalkynyl groups may be composed of one ring or two or more rings which may be joined together in a fused, bridged, or spiro-connected fashion . [Q092] The term "aryl" refers to aromatic monocyclic or multi cyclic groups containing from 6 to 19 carbon atoms. Aryl groups include, but are not limited to groups such as fluorenyl, substituted fluorenyl, phenyl, substituted phenyl, naphthyl, and substituted naphthyl

[0093] The term "aralkyl" refers to an alkyl group in which one of the hydrogen atoms of the alkyl is replaced by an aryl .

[0094] Other objects, features, and advantages of the methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only.

Anatomy of the Ear

[0095] The ear serves as both the sense organ that detects sound and the organ that maintains balance and body position. The ear is generally divided into three portions: the outer ear, middle ear and the inner ear (or auris interna). The outer ear is the external portion of the organ and is composed of the pinna (auricle), the auditory canal (external auditory meatus) and the outward facing portion of the tympanic membrane, also known as the ear drum. The pinna, which is the fleshy part of the externa ear that is visible on the side of the head, collects sound waves and directs them toward the auditory canal. Thus, the function of the outer ear, in part, is to collect and direct sound waves towards the tympanic membrane and the middle ear.

[0096] The middle ear is an air-filled cavity, called the tympanic cavity, behind the tympanic membrane. The tympanic membrane, also known as the ear dram, is a thin membrane that separates the external ear from the middle ear. The middle ear lies within the temporal bone, and includes within this space the three ear bones (auditory ossicles): the malleus, the incus and the stapes. The auditory ossicles are linked together via tiny ligaments, which form a bridge across the space of the tympanic cavity. The malleus, which is atached to the tympanic membrane at one end, is linked to the incus at its anterior end, which in turn is linked to the stapes. The stapes is attached to the oval window, one of two window's located within the tympanic cavity. A fibrous tissue layer, known as the annular ligament connects the stapes to the oval window Sound waves from the outer ear first cause the tympanic membrane to vibrate. The vibration is transmitted across to the cochlea through the auditory- ossicles and oval window, which transfers the motion to the fluids in the auris interna. Tims, the auditory ossicles are arranged to pro vide a mechanical linkage between the tympanic membrane and the oval window of the fluid-filled auris interna, where sound is transformed and transduced to the auris interna for further processing. Stiffness, rigidity or loss of movement of the auditory ossicles, tympanic membrane or oval window leads to hearing loss, e.g. otosclerosis, or rigidity of the stapes bone.

[0097] The tympanic cavity also connects to the throat via the eustachian tube. The eustachian tube provides tire ability to equalize the pressure between the outside air and the middle ear cavity. The round window, a component of the auris interna but which is also accessible within the tympanic cavity, opens into the cochlea of the auris interna. The round window is covered by a membrane, which consists of three layers: an external or mucous layer, an intermediate or fibrous layer, and an internal membrane, which communicates directly with the cochlear fluid. The round window, therefore, has direct communication with the aur s interna via the internal membrane.

[0098] Movements in the oval and round window are interconnected, i.e. as the stapes bone transmits movement from the tympanic membrane to the oval window to move inward against the auris interna fluid, the round window is correspondingly pushed out and away from the cochlear fluid. This movement of the round window allows movement of fluid within the cochlea, which eventually leads in turn to movement of the cochlear inner hair ceils, allowing hearing signals to be transduced. Stiffness and rigidity in the round window leads to hearing loss because of the lack of ability of movement in the cochlear fluid. Recent studies have focused on implanting mechanical transducers onto the round window, which bypasses the normal conductive pathway through the oval windo and provides amplified input into the cochlear chamber. Q099] Auditory signal transduction takes place in the auris interna. The fluid-filled inner ear, or auris interna, consists of two major components: the cochlear and the vestibular apparatus.

[00100] The cochlea is the portion of the auris interna related to hearing. The cochlea is a tapered tube-like structure which is coiled into a shape resembling a snail. The inside of the cochlea is divided into three regions, which is further defined by the position of the vestibular membrane and the basilar membrane. The portion above the vestibular membrane is the scala vestibuli, which extends from the oval window to the apex of the cochlea and contains perilymph fluid, an aqueous liquid low in potassium and high in sodium content.

The basilar membrane defines the scala tyinpani region, which extends from the apex of the cochlea to the round window and also contains perilymph. The basilar membrane contains thousands of stiff fibers, which gradually increase in length from the round window to the apex of the cochlea. The fibers of the basement membrane vibrate when activated by sound. In between the scala vestibuli and the scala tympani is the cochlear duct, which ends as a closed sac at the apex of the cochlea. Hie cochlear duct contains endolymph fluid, which is similar to cerebrospinal fluid and is high in potassium.

[00101] The Organ of Corti, the sensory organ tor hearing, is located on the basilar membrane and extends upward into the cochlear duct. The Organ of Corti contains hair cells, which have hairlike projections that extend from their free surface, and contacts a gelatinous surface called the tectorial membrane. Although hair cells have no axons, they are surrounded by sensory nerve fibers that form the cochlear branch of the vestibulocochlear nerve (cranial nerve VIII).

[00102] As discussed, the oval window, also known as the elliptical window communicates with the stapes to relay sound waves that vibrate from the tympanic membrane. Vibrations transferred to the oval window increases pressure inside the fluid- filled cochlea via the perilymph and scala vestibuli/scala tympani, which in turn causes the membrane on the round window to expand in response. The concerted inward pressing of the oval windo /outward expansion of the round window allows for the movement of fluid within the cochlea without a change of intra-cochlear pressure. However, as vibrations travel through the perilymph in the scala vestibuli, they create corresponding oscillations in the vestibular membrane. These corresponding oscillations travel through the endolymph of the cochlear duct, and transfer to the basilar membrane. When the basilar membrane oscillates, or mo ves up and down, the Organ of Corti moves along with it. The hair cell receptors in the Organ of Corti then move against the tectorial membrane, causing a mechanical deformation in the tectorial membrane. This mechanical deformation initiates the nerve impulse which travels via the vestibulocochlear nerve to the central nervous system, mechanically transmitting the sound wave received into signals that are subsequently processed by the central nervous system.

[00103] The auris interna is located in part within the osseous or bony labyrinth, an intricate series of passages in the temporal bone of the skull. The vestibular apparatus is the organ of balance and consists of the three semi-circular canals and the vestibule. The three semi-circular canals are arranged relative to each other such that movement of the head along the three orthogonal planes in space is detected by the movement of the fluid and subsequent signal processing by the sensory organs of the semi-circular canals, called the crista ampul! axis. The crista ampu! laris contains hair cells and supporting cells, and is covered by a dome-shaped gelatinous mass called the cupula. The hairs of the hair cells are embedded in the cupula. The semi-circular canals detect dynamic equilibrium, the equilibrium of rotational or angular movements.

[00104] When the head trims rapidly, the semicircular canals move with the head, but endo!ymph fluid located in the membranous semi-circular canals tends to remain stationary. The endolymph fluid pushes against the cupula, which tilts to one side. As the cupula tilts, it bends some of the hairs on the hair cells of the crista ampuliaris, which triggers a sensory impulse. Because each semicircular canal is located in a different plane, the corresponding crista ampuliaris of each semi-circular canal responds differently to the same movement of the head. This creates a mosaic of impulses that are transmitted to the central nervous system on the vestibular branch of the vestibulocochlear nerve. The central nervous system interprets this information and initiates the appropriate responses to maintain balance. Of importance in the central nervous system is the cerebellum, which mediates the sense of balance and equilibrium.

[00105] The vestibule is the central portion of the auris interna and contains mechanoreceptors bearing hair cells that ascertain static equilibrium, or the position of the head relative to gravity. Static equilibrium plays a role when the head is motionless or moving in a straight line. The membranous labyrinth in the vestibule is divided into two sac- like structures, the utricle and the saccule. Each structure in turn contains a small structure called a macula, which is responsible for maintenance of static equilibrium. The macula consists of sensory hair cells, which are embedded in a gelatinous mass (similar to the cupula) that covers the macula. Grains of calcium carbonate, called otoliths, are embedded on the surface of the gelatinous layer.

[00106] When the head is in an upright position, the hairs are straight along the macula. When the head tilts, the gelatinous mass and otoliths tilts correspondingly, bending some of the hairs on the hair cells of the macula. This bending action initiates a signal impulse to the central nervous system, which travels via the vestibular branch of the vestibulocochlear nerve, which turn relays motor impulses to the appropriate muscles to maintain balance.

[00107] In some instances, the otic formulations described herein are placed in the outer ear. In some instances, the otic formulations described herein are placed in the middle or inner ear, including the cochlea and vestibular labyrinth: one option is to use a syringe/needle or pump and inject the formulation across the tympanic membrane (the eardrum). In some instances, for cochlear and vestibular labyrinth delivery, one option is to deliver the active ingredient across the round window membrane or even by microinjection directly into the auris interna also known as cochlear microperfusion.

GS 3 inhibition for Neural Protection and Myelination

[00108] The spiral ganglion neurons (SGNs) that innervate the hair cells of the cochlea and form the cochlear nerve to connect the organ of Corti to the brain are essential for hearing. The present disclosure recognizes that these SGNs and their synapses are sensitive to acoustic trauma ototoxins and age, all of which can contribute to their deterioration or death.

Similarly, i t is contemplated herein that the Schwann cells of the cochlea that myelinate the SGNs are sensitive to the same insults which may lead to demyeiination of the SGNs or loss or damage to the Schwann cells. Moreover, the present disclosure recognizes that Schwann ceils may have neuroprotective effects on the SGNs, which may be mediated in part by Schwann cell expression of BDNF. It is contemplated herein that damage or loss of SGNs, Schwann cells or cochlear myelination can all contribute to hearing deficits.

[00109] GSK3 is a key element ofWnt signaling, specifically the ‘canonical Wnt pathway”, whose activation results in an increase in intracellular b-catemn. When GSK3 is active, Wnt signaling and b-catenin levels are suppressed; however inhibition of GSK3 through upstream Wnt pathway components or by small molecule inhibitors, increases b- catenin. The present disclosure recognizes that this accumulation enables b-catenin to enter the nucleus of the ceil where it can affect the transcription of downstream targets involved in both proliferation and differentiation.

[00110] it is contemplated herein that GSK3 inhibition can promote myelination by increasing the number of oligodendrocytes and promoting oligodendrocyte differentiation and enhancing myelination. The present disclosure also recognizes that these effects of GSK3 inhibitors on oligodendrocytes and oligodendrocyte precursor cells occur both in vivo and ex vivo in adult and postnatal stage rodents (mice and rats), and in mouse models of demyeiination injury_' in which GSK3 inhibition promotes remyelination. It is contemplated herein that in the peripheral nervous system (PNS), similar effects of GSK3 inhibition on Schwann cell formation, myelination and survival of peripheral nerves also occur.

[00111] The present disclosure recognizes that in the cochlea, the transient loss of cochlear Schwann cells results in permanent hearing loss evidenced by reduced ABR wave amplitudes and latency it is also contemplated herein that the transient loss of cochlear Schwann ceils is associated with lack of complete myelin regeneration near the peripheral terminals of the auditory nerve (the SGN fibers). In addition, it is contemplated herein that local administration of GSK3 inhibitors abrogates cisplatin-induced ototoxicity and reduce cell death in cells within Rosenthal’s canal which houses peripheral glia and SGN cell bodies. [00112] As contemplated herein, local release of GSK3 inhibitors into the inner ear is beneficial in the treatment of various cochlear pathologies and hearing disorders. Some of these indications include: various hearing loss condition where BDNF would be indicated, as well as age-related hearing loss, sudden sensorineural hearing loss, cisplatin-induced hearing loss/otoprotection of SGNs, noise-induced hearing loss, speech-in-noise deficits/synaptopathy. It is also contemplated herein that local release of GSK3 inhibitors into the inner ear is beneficial tor hearing loss caused by the following conditions: Meniere’s disease, myelopathy or demyelinating disorders, multiple sclerosis, Schilder’s disease,

Susac's syndrome (retinocochleocerebral vascuiopathy), acute disseminated encephalomyelitis, Guillain-Barre syndrome, some or all forms of Charcot-Marie -Tooth neuropathy, neuromyelitis optica, genetic or acquired forms of auditory neuropathy, autoimmune responses or diseases, ischemia, metabolic disorders, viral infection (such as HIV, HTLV-I, measles, etc.), and ototoxic agents. Conditions that may compromise Schwann cells, spiral ganglion neurons or myelination of the auditory pathway, or that may induce an apoptotic response in these cells, also benefit from local release of GSK3 inhibitors into the inner ear.

Diseases or Conditions of the Ear

[00113] In some embodiments, the otic formulations and compositions described herein are suitable for the treatment and/or prevention of diseases or conditions associated with the outer, middle, and/or inner ear. In some embodiments, the otic formulations and compositions described herein are suitable for the treatment and/or prevention of diseases or conditions associated with the outer ear. In some embodiments, the otic formulations and compositions described herein are suitable for the treatment and/or prevention of diseases or conditions associated with the middle ear. in some embodiments, the otic formulations and compositions described herein are suitable for the treatment and/or prevention of diseases or conditions associated with the inner ear. In some embodiments, the otic formulations and compositions described herein are suitable for the treatment and/or prevention of otic diseases or conditions associated with the central nervous system (e.g. tinnitus). In some embodiments, the otic formulations and compositions described herein reduce, reverse and/or ameliorate symptoms of otic diseases or conditions, such as any one of these disclosed herein. These disorders or conditions have many causes, which include but are not limited to, infection, injury, inflammation, tumors, and adverse response to drugs or other chemical agents.

[00114] In some embodiments, the otic formulations and compositions described herein is useful for treating ear pruritus, otitis externa, otalgia, tinnitus, vertigo, ear fullness, hearing ioss, or a combination thereof in some embodiments, the otic formulations and compositions described herein are used to for the treatment and/or prevention of Meniere’s disease, sensorineural hearing loss, noise induced hearing loss, presbycusis (age related hearing loss), auto immune ear disease, tinnitus, ototoxicity, excitotoxicity, endolymphatic hydrops, labyrinthitis, Ramsay Hunt’s Syndrome, vestibular neuronitis, microvascuiar compression syndrome, hyperacusis, presbystasis, central auditory processing disorder, or auditory neuropathy. In some embodiments, the otic formulations and compositions described herein are used for the improvement of cochlea implant performance. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of Meniere’s disease. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of sensorineural hearing loss. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of noise induced hearing loss. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of presbycusis (age related hearing loss). In some embodiments, the otic formulations and compositions are used for the treatment and/or pre vention of auto immune ear disease. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of tinnitus. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of ototoxicity. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of excitotoxicity. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of endolymphatic hydrops. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of labyrinthitis. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of Ramsay Hunt’s Syndrome In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of vestibular neuronitis. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of microvascuiar compression syndrome. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of hyperacusis. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of presbystasis. In some embodiments, the otic fonnuiations and compositions are used for the treatment and/or prevention of central auditory processing disorder. In some embodiments, the otic formulations and compositions are used for the treatment and/or prevention of auditory neuropathy. In some embodiments, the otic fonnuiations and compositions are used for the improvement of cochlea implant performance.

[00115] In some embodiments, the otic formulations and compositions are used for hearing loss condition where BDNF is indicated, as well as age-related hearing loss, sudden sensorineural hearing loss, cisplatin-induced hearing loss/otoprotectioii of SGNs, noise- induced hearing loss, speech-in-noise deficits/synaptopathy. In some embodiments, the otic formulations and compositions are used for hearing loss caused by the following conditions: Meniere's disease, myelopathy or demye!inating disorders, multiple sclerosis, Schilder’s disease, Susac's syndrome (retinocoehleocerehral vasculopathy), acute disseminated encephalomyelitis, Guiliain-Barre' syndrome, all forms of Charcot-Marie-Tooth neuropathy, neuromyelitis optica, genetic or acquired forms of auditory' neuropathy, autoimmune responses or diseases, ischemia, metabolic disorders, viral infection (such as HIV, HTLV-I, measles, etc.), and ototoxic agents. In some embodiment, the otic formulations and compositions are used for condition that compromises Schwann cells, spiral ganglion neurons or myelination of the auditory pathway, or that may induce an apoptotic response in these cells.

Ear Pruritus

[00116] Ear pruritus, or itchy ear canal, is a tickling or irritating sensation that causes a desire or reflex to scratch the affected area. In some cases, redness, swelling, soreness, and flaking may develop in the affected area. Ear pruritus is caused by a variety of agents. In some embodiments, ear pruritus occurs due to either primary microbial infection within the ear or as a secondary infection from the body where it is then spread into the ear canal. In some embodiments, skin conditions, such as eczema or psoriasis, lead to skin irritations within the ear canal. Further, external irritants such as hairspray, shampoo, shower gel, or allergen, such as dust, pets, and pollen, lead to ear pruritus in some instances. In some embodiments, ear pruritus serves as an early sign for more serious complications such as otitis externa.

Otalgia

[00117] Otalgia, also known as earache or ear pain, is classified into two types, primary otalgia and referred otalgia. Primal}_' otalgia is ear pain which originates from inside of the ear. Referred otalgia is ear pain which originates from the outside of the ear. Although the etiology of referred otalgia can be complex, several well-known culprits include dental disorders, sinusitis, neck problems, tonsillitis, pharyngitis, and sensory branches from the vagus and glossopharyngeal nerves. In some cases, referred otalgia has been associated with head and neck malignancies.

Ear Fullness

[00118] Ear fullness or aural fullness is described as a feeling that the ears are clogged, stuffed, or congested. Similar to otalgia, the etiology of ear fullness is diverse with numerous underlying causes. Generally, ear fullness may also be accompanied by tinnitus, otalgia, and impaired hearing.

Hearing Loss

[00119] Hearing loss is a partial or total impairment to hearing. Hearing loss is classified into three types, conductive hearing loss, sensorineural hearing loss, and mixed hearing loss. Conductive hearing loss occurs when sound is not conducted efficiently through the external auditory canal to the tympanic membrane or eardrum. In some embodiments, conductive hearing loss involves a reduction in sound level or the ability to hear faint sounds. Treatment involves corrective medical or surgical procedures. Sensorineural hearing loss occurs when there is damage to the cochlea (inner ear), or to the nerve pathways from the cochlea to the brain. This type of hearing loss generally leads to permanent hearing loss. Mixed hearing loss is a combination of conductive hearing loss and sensorineural hearing loss in which damage occurs along both the outer and inner ear regions. [00120] The degree or severity of hearing loss is categorized into seven groups ranging from normal, slight, mild, moderate, moderately severe, severe to profound. In addition, hearing loss is stratified based on frequency in some instances. For example, a hearing loss that only affects the high tones is referred to as a high frequency hearing loss, whereas that which affects the low tones is referred to as a low frequency hearing loss. In some eases, hearing loss affects both high and low frequencies.

[00121] Hearing loss is often accompanied by additional causes and symptoms such as ceruminosis, otitis externa, otalgia, tinnitus and vertigo. In some embodiments, it has been shown that ceruminosis can decrease hearing acuity by 40-45 dB. Such impairment, especially in the geriatric population can cause difficulties in communication and even physical immobility. In some embodiments, the otic compositions and formulations disclosed herein are useful for the treatment of hearing loss.

Hair Cell Regeneration [0Q122] Hair cells in the mammalian cochlea are important for hearing. The inner and outer hair cells in the Organ of Corti sense vibrations in cochlea fluid produced by sound and transduce these into auditory nerve responses that travel to the brain for sound to be perceived. Loss of hair cells has been implicated hearing loss caused by age, exposure to loud noise, ototoxic drugs, and genetic factors in birds and amphibians, damage to hair cells triggers mechanisms that cause epithelial ceils (supporting cells) in the cochlea to transdifferentiate into new hair cells and to divide and regenerate new supporting cells and hair cells to restore hearing. This ability to regenerate hair cells has been lost in mammals. Studies in birds and amphibians have demonstrated that the Writ signaling pathway is an important component of hair cell regeneration. GSK-3 is a key element of Wnt signaling, and in particular, the so-called “canonical Wnt pathway”, whose activation results in an increase of intracellular beta-catenin. In supporting cells, beta-eatenin drives transcription of protein atonal homolog- 1 (Atoh-1), a transcription factor that is important for hair cell formation in embryonic development and drives supporting cell trans-differentiation into new hair cells. When GSK-3 is active, Wnt signaling and beta-catenin levels are suppressed; however, inhibition of GSK-3 through upstream Wnt pathw ay components or by small molecule inhibitors can increase beta-catenin translocation to nucleus where it can affect signaling of downstream pathways, and/or increase b-catenin and Atoh-1 transcription. The ratio of hair cells to supporting cells is tightly regulated through development and is likely important for fully mature Organ of Corti function. If supporting cells are not replaced after trans-differentiation into hair cells, the supporting celbhair cell ratio may not be optimal . Consequently, proliferation of supporting cells to restore an appropriate ratio during regeneration is important in some instances in some instances, Wnt pathway activation through GSK-3 inhibition influences both trans-differentiation and proliferation. In addition to the different types of supporting cells in the adult mammal ian cochlea, stem cells, or progenitor cells have been found that have the ability to form new supporting cells or hair ceils. These ceils have been characterized by expression of leucine-rich repeat-containing G- protein coupled receptor 5 (LGR5), a component of the Wnt signaling pathway. These cells represent an additional means by which new hair cells and supporting cells are generated by GSK-3 inhibition. Q0123] in some embodiments, the otic formulations or compositions described herein are useful for the regeneration of otic hair cells.

Vertigo [00124] Vertigo is described as a feeling of spinning or swaying while the body is stationary. There are two types of vertigo. Subjective vertigo is the false sensation of movement of the body. Objective vertigo is the perception that one’s surrounding are in motion. It is often accompanied by nausea, vomiting, and difficulty maintaining balance. In some embodiments, otitis externa can induce vertigo.

Meniere 's Disease

[00125] Meniere’s Disease is an idiopathic condition characterized by sudden attacks of vertigo, nausea and vomiting that lasts for 3 to 24 hours, and subside gradually.

Progressive hearing loss, tinnitus and a sensation of pressure in the ears accompanies the disease through time. The cause of Meniere’s disease is likely related to an imbalance of auris interna fluid homeostasis, including an increase in production or a decrease in resorption of auris interna fluid.

[00126] The cause of symptoms associated with Meniere’s disease is likely an imbalance of inner ear fluid homeostasis, including an increase in production or a decrease in reabsorption of inner ear fluid.

[00127] Recent studies of the vasopressin (VP)-mediated aquaporin 2 (AQP2) system in the auris interna suggest a role for VP in inducing endolymph production, thereby increasing pressure in the vestibular and cochlear structures). VP levels were found to be upregulated in endolymphatic hydrops (Meniere’s Disease) eases, and chronic administration of VP in guinea pigs was found to induce endolymphatic hydrops. Treatment with VP antagonists, including infusion of OPC-31260 (a competitive antagonist of Vi-R) into the scala tympani resulted in a marked reduction of Meniere’s disease symptoms. Other VP antagonists include WAY-140288, CL-385004, tolvaptan, conivaptan, SR 121463A and VPA 985. (Sanghi et al. Eur. Heart J. (2005) 26:538-543; Palm et al. Nephrol Dial Transplant (1999) 14:2559- 2562).

[00128] Other studies suggest a role for estrogen-related receptor b/NK3B2 (ERR/Nr3b2) in regulating endolymph production, and therefore pressure in the vestibular/cochlear apparatus. Knock-out studies in mice demonstrate the role of the protein product of the Nr3b2 gene in regulating endolymph fluid production. r3b2 expression has been localized in the endolymph -secreting stria! marginal cells and vestibular dark cells of the cochlea and vestibular apparatus, respectively. Moreover, conditional knockout of the Nr3b2 gene results in deafness and diminished endolymphatic fluid volume. In some instances, treatment with antagonists to ERR/Nr3b2 assist in reducing endolymphatic volume, and thus alter pressure in the auris interna structures.

[00129] Other treatments are aimed at deal ing with the immediate symptoms and prevention of recurrence. Low-sodium diets, avoidance of caffeine, alcohol, and tobacco have been advocated. Medications that temporarily relieve vertigo attacks include antihistamines (including meclizine (Antivert, Bonine, Dramamine, Dmmnate) and other antihistamines), and central nervous system agents, including barbiturates and/or benzodiazepines, including iorazepam or diazepam . Other examples of drugs that are useful in relieving symptoms include muscarinic antagonists, including scopolamine. Nausea and vomiting are relieved by- suppositories containing antipsychotic agents, including the phenothiazine agent prochlorperazine (Compazine, Buccastem, Stemetil and Phenotil).

[00130] Surgical procedures have also been used to relieve symptoms of Meniere s disease, including destruction of vestibular function to relieve vertigo symptoms. These procedures aim to either reduce fluid pressure in the inner ear and/or to destroy inner ear balance function. An endolymphatic shunt procedure, which relieves fluid pressure, are placed in the inner ear to relieve symptoms of vestibular dysfunction. Severing of the vestibular nerve is also employed, which controls vertigo while preserving hearing.

[00131 ] Another approach to destruction of vestibul ar function for the treatment of severe Meniere’s disease is intratympanie application of an agent that destroys sensory hair cell function in the vestibular system, thereby eradicating inner ear balance function. Various antimicrobial agents are used in the procedure, including aminoglycosides such as gentamicin and streptomycin. The agents are injected through the tympanic membrane using a small needle, a tympanostomy tube with or without a wick, or surgical catheters. V arious dosing regimens are used to administer the antimicrobial agents, including a low dose method in which less of the agents are administered over longer periods of time (e.g , one month between injections), and high dose methods in which more of the agents are administered over a shorter time frame (e.g., every week). Although the high dose method is typically⁷ more effective, it is more risky, as it results in hearing loss in some cases.

Meniere ’s Syndrome

[00132] Meniere’s Syndrome, which displays similar symptoms as Meniere’s disease, is attributed as a secondary affliction to another disease process, e.g. thyroid disease or auris interna inflammation due to syphilis infection. Meniere’s syndrome, thus, are secondary- effects to various process that interfere with normal production or resorption of endolymph, including endocrine abnormalities, electrolyte imbalance, autoimmune dysfunction, medi cations, infections (e.g. parasitic infections), or hyperlipidemia. Treatment of patients afflicted with Meniere's Syndrome is similar to Meniere's Disease.

Sensorineural Hearing Loss

[00133] Sensorineural hearing loss is a type of hearing loss which results from defects (congenital and acquired) in the vestibulocochlear nerve (also known as cranial nerve VIII), or sensory cells of tire inner ear. The majority of defects of the inner ear are defects of otic hair cells.

[00134] Aplasia of the cochlea, chromosomal defects, and congenital cholesteatoma are examples of congenital defects which result in sensorineural hearing loss. By way of non- limiting example, inflammatory diseases (e.g. suppurative labyrinthitis, meningitis, mumps, measles, viral syphilis, and autoimmune disorders), Meniere's Disease, exposure to ototoxic drugs (e.g. aminoglycosides, loop diuretics, antimetabolites, salicylates, and cisplatin), physical trauma, presbycusis, and acoustic trauma (prolonged exposure to sound in excess of 90 dB) all result in acquired sensorineural hearing loss.

[00135] If the defect resulting in sensorineural hearing loss is a defect in the auditory pathways, the sensorineural hearing loss is called central hearing loss. If the defect resulting in sensorineural hearing loss is a defect in the auditory pathways, the sensorineural hearing loss is called cortical deafness.

[00136] In some instances, sensorineural hearing loss occurs when the components of the auris interna or accompanying neural components are affected, and contain a neural, i.e. when the auditor}- nerve or auditory nerve pathways in the brain are affected, or sensory- component. Sensory hearing loss are hereditary, or it are caused by acoustic trauma (i.e. very loud noises), a viral infection, drug-induced or Meniere’s disease in some instances, neural hearing loss occurs as a result of brain tumors, infections, or various brain and nerve disorders, such as stroke. Some hereditary- diseases, such as Refsum disease (defective accumulation of branched fatty acids), also cause neural disorders affecting hearing loss. Auditory nerve pathways are damaged by demyelinating diseases, e.g. idiopathic inflammatory demyelinating disease (including multiple sclerosis), transverse myelitis, Devic’s disease, progressive multifocal leukoencephalopathy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy and anti-MAG peripheral neuropathy. [Q0137] The incidence of sudden deafness, or sensorineural hearing loss, occurs in about 1 in 5000 individuals, and are caused by viral or bacterial infections, e.g mumps, measles, influenza, chickenpox, cytomegalovirus, syphilis or infectious mononucleosis, or physical injury to the inner ear organ. In some cases, no cause is identified. In some cases, tinnitus and vertigo accompany sudden deafness, which subsides gradually. Oral corticosteroids are frequently prescribed to treat sensorineural hearing loss in some cases, surgical intervention is necessary. Other treatments include AM-101 and AM-111 compounds under development for the treatment of auris interna tinnitus and acute sensorineural hearing loss. (Auris Medical AG, Basel, Switzerland).

Noise induced hearing loss

[00138] Noise induced hearing loss (NIHL) is caused upon exposure to sounds that are too loud or loud sounds that last a long time in some instances, hearing loss occurs from prolonged exposure to loud noises, such as loud music, heavy equipment or machinery, airplanes, or gunfire. Long or repeated or impulse exposure to sounds at or above 85 decibels cause hearing loss in some cases. NIHL causes damage to the hair cells, Schwann cells, and/or the auditory nerve or the myelin sheath of the SGNs. The hair cells are small sensor - cells that convert sound energy into electrical signals that travel to the brain. In some cases, impulse sound results in immediate hearing loss that is permanent. This kind of hearing loss are accompanied by tinnitus — a ringing, buzzing, or roaring in the ears or head — which subsides over time in some cases. Hearing loss and tinnitus are experienced in one or both ears, and tinnitus continue constantly or occasionally throughout a lifetime in some instances. Permanent damage to hearing loss is often diagnosed. Continuous exposure to loud noise also damages the structure of hair cells, resulting in hearing loss and tinnitus, although the process occurs more gradually than for impulse noise.

[00139] In some embodiments, an otoprotectant prevents, reverses, reduces, or ameliorates NIHL. Examples of otoprotectants that treat or prevent NIHL include, but are not limited to, D-methionine, L-methionine, ethionine, hydroxyl methionine, methioninol, amifostine, mesna (sodium 2-suifanylethanesuifonate), a mixture of D and L methionine, normethionine, homomethionine, S-adenosyl-L-methiomne), diethyldithiocarbamate, ebselen (2-phenyl- 1, 2- benzisoselenazol-3(2H)-one), sodium thiosulfate, AM-111 (a ceil permeable INK inhibitor, (Laboratoires Auris SA8)), ieucovorin, leucovorin calcium, dexrazoxane, or combinations thereof.

Presbycusis (Age Related Hearing Loss)

[QQ140] Presbycusis (age related hearing loss (ARHL)) is the progressive bilateral loss of hearing that results from aging. Most hearing loss occurs at higher frequencies (i.e. frequencies above 15 or 16 Hz) making it difficult to hear a female voice (as opposed to male voice), and an inability to differentiate between high-pitched sounds (such as "s" and "th"). It is difficult to filter out background noise. Hie disorder is most often treated by the implantation of a hearing aid and/or the administration of pharmaceutical agents which prevent the buildup of ROS.

[00141] The disorder is caused by changes in the physiology of the inner ear, the middle ear, and/or the VIII nerve. Changes in the inner ear resulting in presbycusis include epithelial atrophy with loss of otic hair cells and/or stereocilia, atrophy of nerve cells, atrophy of the stria vascularis, and the thickening/stiffening of the basilar membrane. Additional changes which contribute to presbycusis include the accumulation of defects in the tympanic membrane and the ossicles.

[QQ142] In some instances, changes leading to presbycusis occur due to the accumulation of mutations in DNA, and mutations in mitochondrial DNA; however, the changes are exacerbated by exposure to loud noise, exposure to ototoxic agents, infections, and/or the lessening of blood flow to the ear. The latter is attributable to atherosclerosis, diabetes, hypertension, and smoking.

[00143] Presbycusis, or age-related hearing loss, occurs as a part of normal aging, and occurs as a result of degeneration of the receptor cells in the spiral Organ of Corti in the auris interna. Other causes are also attributed to a decrease in a number of nerve fibers in the vestibulocochlear nerv e, as well as a loss of flexibility of the basilar membrane in the cochlea. Most commonly, it arises from changes in the inner ear as one ages, but it also results from changes in the middle ear, or from complex changes along the nerve pathways from the ear to the brain. Certain medical conditions and medications also play a role in some instances, presbycusis results from a gradual loss of spiral ganglion neuron afferent fibers and their synapses with hair cells (ribbon synapses), causing a disconnection between the sensory cells that detect sound and the auditory nerve that transmits this information to the auditory brain. Loss of spiral ganglion neurons and hair ceils also occurs. In some cases, prior exposure to loud noise or other otic insults exacerbates this ageing process, leading to an accelerated loss of hearing. Presbycusis also involves “hidden hearing loss”, an inability to detect sound against a background noise (“speech-in-noise”) despite a lack of marked changes in hearing thresholds. These more subtle decrements in hearing have been associated with a loss of spiral ganglion neuron afferent fibers and their synaptic connections with hair cells (ribbon synapses). In some cases age may be associated with the loss of the myelin sheath around SGNs and/or the loss of Schwann cell replacement surrounding the SGNs or auditory_' nerve, which then leads to delayed auditory_' processing, speech-in-noise hearing difficulties or hearing loss.

Autoimmune Inner Ear Disease [00144] Autoimmune inner ear disease (AIED) is one of the few reversible causes of sensorineural hearing loss. It is a rare disorder appearing in both adults and children that often involves a bilateral disturbance of the audio and vestibular functions of the auris interna. The origin of AIED is likely autoantibodies and/or immune cells attacking inner ear structures, but are associated with other autoimmune conditions in many cases, AIED occurs without systemic autoimmune symptoms, but up to one-third of patients also suffer from a systemic autoimmune illness, such as inflammatory' bowel disease, rheumatoid arthritis, Ankylosing spondylitis, Systemic Lupus Erythematosus (8LE), Sjogren's Syndrome, Cogan’s disease, ulcerative colitis, Wegener's granulomatosis and scleroderma. Behpet’s disease, a multisystem disease, also commonly has audio vestibular problems. There is some evidence for food-related allergies as a cause for cochlear and vestibular autoimmunity, but there is presently no agreement as to its importance in the aetiology of the disease. A classification scheme for AIED has been developed (Harris and Keithley, (2002) Autoimmune inner ear disease, in Otorhinolaryngology Head and Neck Surgery 91, 18-32). [00145] Tire immune system normally performs a crucial role in protecting the auris interna from invasive pathogens such as bacteria and viruses. However, in AIED the immune system itself begins to damage the delicate auris interna tissues. It is well established that the auris interna is fully capable of mounting a localized immune response to foreign antigens. When a foreign antigen enters the auris interna, it is first processed by immunocompetent cells which reside in and around the endolymphatic sac. Once the foreign antigen has been processed by these immunocompetent cells, these cells secrete various cytokines which modulate the immune response of the auris interna. One result of this cytokine release is to facilitate the influx of inflammatory cells which are recruited from the systemic circulation. These systemic inflammatory cells enter the cochlea via diapedesis through the spiral modiolar vein and its tributaries and begin to participate in antigen uptake and deregulation just as it occurs in other parts of the body. Interleukin 1 (IL-I) plays an important role in modulating the innate (nonspecific) immune response and is a known acti vator of resting T helper cells and B-eells. T helper cells, once activated by IL-1, produce IL-2. IL-2 secretion results in differentiation of pluripotent T-cells into helper, cytotoxic and suppressor T-cell subtypes. IL-2 also assists T helper cells in the activation of B lymphocytes and probably plays a pi votal role in the immunoregulation of the immune response of the auris interna. IL- 2 has been identified within the perilymph of the auris interna as early as 6 h after antigen challenge with peak levels at 18 h after antigen challenge. The perilymphatic levels of IL-2 then dissipate, and it is no longer present within the perilymph at 120 hours post antigen challenge (Gloddek, Acta Otolaryngol. (1989) 108, 68-75). Q0146] Both IL-Ib and tumor necrosis factor-cx (TNF-a) play a key role in the initiation and amplification of the immune response. IL-Ib is expressed by the fibrocytes of the spiral ligament in the presence of trauma such as surgical trauma or acoustic trauma in a nonspecific response. THF-a is expressed either by infiltrating systemic cells or by resident cells contained within the endolymphatic sac in the presence of antigen. THF-a is released as part of the adaptive (specific) immune response in animal models. When antigen is injected into the auris internas of mice, IL-Ib and TNF-a are both expressed and a vigorous immune response occurs. However, when antigen is introduced to the auris in terna via the cerebral spinal fluid without trauma to the auris interna, only TNF-a is expressed and the immune response in minimal (Satoh, J Assoc. Res. Otolaryngol (2003), 4, 139-147). Importantly, cochlear trauma in isolation also results in a minimal immune response. These results suggest that both the nonspecific and specific components of the immune response act in concert in the auris interna to achieve a maximal response. Q0147] Accordingly, if the cochlea is traumatized and an antigen is injected (or in the case of autoimmune disease, the patient has immune cells directed against auris interna antigens), both the nonspecific and the specific immune responses are activated simultaneously. This results in the concurrent production of IL-Ib as well as THF-a which causes a greatly amplified level of inflammation leading to substantial damage to the auris interna.

Subsequent experi ments in animal models confirm that an important step in i mmune- mediated damage requires that the auris interna be conditioned by the non-specific innate immune response before the specific adaptive immune response leads to enough inflammation to result in damage. As a result, in some instances, agents which downregulate or block the specific immune responses prevent the excessive immune response seen when both the specific and nonspecific immune responses are simultaneously activated.

Tinnitus Q0148] Tinnitus is defined as the perception of sound the absence of any external stimuli. In some eases, it occurs in one or both ears, continuously or sporadically, and is most often described as a ringing sound. It is most often used as a diagnostic symptom for other diseases. There are two types of tinnitus: objective and subjective. The former is a sound created in the body which is audible to anyone. The latter is audible only to the affected individual. Studies estimate that over 50 million Americans experience some form of tinnitus. Of those 50 million, about 12 million experience severe tinnitus.

[00149] In certain instances, tinnitus results from damage to otic structures (e.g. stereocilia), the dysfunction of one or more molecular receptors, and/or the dysfunction of one or more neural pathways. In certain instances, tinnitus results from excitotoxicity caused by abnormal activity of an NMDA receptor. In certain instances, tinnitus results from by dysfunction of an <x9 and/or alt⁾ acetylcholine receptor. In certain instances, tinnitus results from damage to the vestibulocochlear nerve. In certain embodiments, a reduction in n euro transmiter reuptake (e.g. the increase in extracellular neurotransmitters) treats, and/or ameliorates the symptoms of tinnitus. In certain embodiments, antagonism of an NK1 receptor treats, and/or ameliorates the symptoms of tinnitus. In certain embodiments, a reduction in neurotransmitter reuptake and antagonism of an NK1 receptor treats, and/or ameliorates the symptoms of tinnitus.

[00150] There are several treatments for tinnitus. Lidocaine, administered by IV, reduces or eliminates the noise associated with tinnitus in about 60-80% of sufferers. Selective neuro transmitter reuptake inhibitors, such as nortriptyline, sertraline, and paroxetine, have also demonstrated efficacy against tinnitus. Benzodiazepines are also prescribed to treat tinnitus.

Ototoxicity

[00151] Ototoxicity refers to hearing loss caused by a toxin. The hearing loss are due to trauma to otic hair cells, the cochlea, and/or the cranial nerve VIII. Multiple drugs are known to be ototoxic. Often ototoxicity is dose-dependent. It is permanent or reversible upon withdrawal of the drug.

[00152] Known ototoxic drags include, but are not limited to, the aminoglycoside class of antibiotics (e.g., gentamicin, and amikacin), some members of the macrolide class of antibiotics (e.g., erythromycin), some members of the glycopeptide class of antibiotics (e.g., vancomycin), salicylic acid, nicotine, some chemotherapeutic agents (e.g., actinomycin, bleomycin, cisplatin, carboplatin and vincristine), and some members of the loop diuretic family of drags (e.g., furosemide) .

[00153] Cisplatin and the aminoglycoside class of antibiotics induce the production of reactive oxygen species (“ROS”). ROS damages cells directly by damaging DNA, polypeptides, and/or lipids. Antioxidants pre vent damage of ROS by preventing their formation or scavenging free radicals before they damage the cell. Both cisplatin and the aminoglycoside class of antibiotics are also thought to damage the ear by binding melanin in the stria vascularis of the inner ear.

[00154] Salicylic acid is classified as ototoxic as it inhibits the function of the polypeptide prestin. Prestin mediates outer otic hair cell motility by controlling the exchange of chloride and carbonate across the plasma membrane of outer otic hair cells it is only found in the outer otic hair ceils, not the inner otic hair ceils. Accordingly, in some embodiments, the use of the controlled release auris-compositions described herein, ameliorates or lessens ototoxic effects of chemotherapy, including but not limited to cisplatin treatment, aminoglycoside or salicylic acid administration, or other ototoxic agents.

Excitotoxicity

[00155] Exc totoxicity refers to the death or damaging of neurons and/or otic hair cells by glutamate and/or similar substances.

[001S6] Glutamate is the most abundant excitatory neurotransmitter the central nervous system. Pre-synaptic neurons release glutamate upon stimulation. It flows across the synapse, binds to receptors located on post-synaptie neurons, and activates these neurons.

The glutamate receptors include the NMD A, AMPA, and kainate receptors. Glutamate transporters are tasked with removing extracellular glutamate from the synapse. Certain events (e.g ischemia or stroke) damage the transporters. This results in excess glutamate accumulating in the synapse. Excess glutamate in synapses results in the over-activation of the glutamate receptors.

[00157] The AMPA receptor is activated by the binding of both glutamate and AMPA. Activation of certain isoforms of the AMPA receptor results in the opening of ion channels located in the plasma membrane of the neuron. When the channels open, Na^÷ and Ca² ions flow into the neuron and K ions flo out of the neuron.

[00158] The NMD A receptor is activated by the binding of both glutamate or NMDA together with a co-agonist glycine or D-serine. Activation of the NMDA receptor, results in the opening of ion channels located in the plasma membrane of die neuron. However, these channels are blocked by Mg²⁺ ions. Activation of the AMPA receptor results in the expulsion of Mg²⁺ ions from the ion channels into the synapse. When the ion channels open, and the Mg^2÷ ions evacuate the ion channels, Na⁺ and Ca²⁺ ions flow' into the neuron, and K⁺ ions flow out of the neuron.

[00159] Excitotoxicity occurs when the NMDA receptor and AMPA receptors are over- activated by the binding of excessive amounts of ligands, for example, abnormal amounts of glutamate. The over-activation of these receptors causes excessive opening of the ion channels under their control. This allows abnormally high levels of Ca²⁺ and Na^ to enter the neuron. Hie influx of these levels of Ca²⁺ and Na⁺ into the neuron causes the neuron to fire more often, resulting in a rapid buildup of free radicals and inflammatory compounds within the cell. Tire free radicals eventually damage the mitochondria, depleting the cell’s energy stores. Furthermore, excess levels of Ca²⁺ and Na⁺ ions activate excess levels of enzymes including, but not limited to, phospholipases, endonucleases, and proteases. The over- activation of these enzymes results in damage to the cytoskeleton, plasma membrane, mitochondria, and DNA of the sensory neuron.

Endolymphatic Hydrops

[Q0160] Endolymphatic hydrops refers to an increase in the hydraulic pressure within tire endolymphatic system of the inner ear. The endolymph and perilymph are separated by thin membranes which contain multiple nerves. Fluctuation in the pressure stresses the membranes and the nerves they house if the pressure is great enough, disruptions form in the membranes. This results in a mixing of the fluids which leads to a depolarization blockade and transient loss of function. Changes in the rate of vestibular nerve firing often lead to vertigo. Further, the Organ of Corti are affected in some cases. Distortions of the basilar membrane and the inner and outer hair cells leads to hearing loss and/or tinnitus. [00161] Causes include metabolic disturbances, hormonal imbalances, autoimmune disease, and viral, bacterial, or fungal infections. Symptoms include hearing loss, vertigo, tinnitus, and aural fullness. Nystagmus is also present in some instances. Treatment includes sy stemic administration of benzodiazepine, diuretics (to decrease the fluid pressure), corticosteroids, and/or anti-bacterial, anti-viral, or anti-fungal agents.

Labyrinthitis

[00162] Labyrinthitis is an inflammation of the labyrinths of the ear which contain the vestibular system of the inner ear. Causes include bacterial, viral, and fungal infections. It is also caused by a head injur ' or allergies in some instances. Symptoms of labyrinthitis include difficulty maintaining balance, dizziness, vertigo, tinnitus, and hearing loss. In some cases, recovery takes one to six weeks; however, chronic symptoms are present for years. [Q0163] There are several treatments for labyrinthitis. Prochlorperazine is often prescribed as an antiemetic. Serotonin-reuptake inhibitors have been shown to stimulate new neural growth within the inner ear. Additionally, treatment with antibiotics is prescribed if the cause is abacterial infection, and treatment with corticosteroids and antivirals is recommended if the condition is caused by a viral infection.

Ramsay Hunt 's Syndrome (Herpes Zoster Infection) [00164] Ramsay Hunt's syndrome is caused by a herpes zoster infection of the auditor - nerve. Tiie infection causes severe ear pain, hearing loss, vertigo, as well as blisters on the outer ear, in the ear canal, as well as on the skin of the face or neck supplied by the nerves.

In some cases, facial muscles also become paralyzed if the facial nerves are compressed by the swelling. Hearing loss are temporary or permanent, with vertigo symptoms usually lasting from several days to weeks.

[00165] Treatment of Ramsay Hunt's syndrome includes administration of antiviral agents, including acyclovir. Other antiviral agents include famciclovir and va!acyc!ovir.

Combination of antiviral and corticosteroid therapy are also employed to ameliorate herpes zoster infection. Analgesics or narcotics are also administered to relieve the pain, and diazempam or other central nervous system agents to suppress vertigo. Capsaicin, lidocaine patches and nerve blocks are also used. Surgery is also performed on compressed facial nerves to relieve facial paralysis.

Vestibular Neuronitis

[00166] Vestibular neuronitis, or vestibular neuropathy, is an acute, sustained dysfunction of the peripheral vestibular system. It is theorized that vestibular neuronitis is caused by a disruption of afferent neuronal input from one or both of the vestibular apparatuses. Sources of this disruption include viral infection, and acute localized ischemia of the vestibular nerve and/or labyrinth. Vestibular neuronitis is characterized by sudden vertigo attacks, which manifests as a single attack of vertigo, a series of attacks, or a persistent condition w hich diminishes over a matter of weeks. Symptoms typically include nausea, vomiting, and previous upper respiratory_' tract infections, although there are generally no auditory_' symptoms. Tire first attack of vertigo is usually severe, leading to nystagmus, a condition characterized by flickering of the eyes involuntarily toward the affected side. Hearing loss does not usually occur

[00167] In some instances, vestibular neuronitis is caused by inflammation of the vestibular nerve, the nerve that connects the inner ear to the brain, and is likely caused by viral infection. Diagnosis of vestibular neuronitis usually involves tests for nystagmus using eleetronystagmography, a method of electronically recording eye movements. Magnetic resonance imaging is also performed to determine if other causes play a role in the vertigo symptoms.

[00168] Treatment of vestibular neuronitis typically involves alle viating the symptoms of the condition, primarily vertigo, until the condition clears on its own. Treatment of vertigo is often identical to Meniere's disease, and optionally includes meclizine, lorazepam, prochiorperazine, or scopolamine. Fluids and electrolytes are intravenously administered if the vomiting is severe. Corticosteroids, such as prednisolone, are also given if the condition is detected early enough.

[00169] In some embodiments, the compositions disclosed herein are administered tor the treatment of vestibular neuronitis. Further, in some embodiments, the compositions are administered with other agents that are typically used to treat symptoms of the condition, including anticholinergics, antihistamines, benzodiazepines, or steroids. Treatment of vertigo is identical to Meniere’s disease, and include meclizine, lorazepam, prochlorperazine or scopolamine. Fluids and electrolytes are also intravenously administered if the vomiting is severe.

[00170] The most significant finding when diagnosing vestibular neuronitis is spontaneous, unidirectional, horizontal nystagmus it is often accompanied by nausea, vomiting, and vertigo. It is, generally, not accompanied by hearing loss or other auditory symptoms.

[00171] There are several treatments for vestibular neuronitis. HI -receptor antagonists, such as dimenhydrinate, diphenhydramine, meclizine, and promethazine, diminish vestibular stimulation and depress labyrinthine function through anticholinergic effects. Benzodiazepines, such as diazepam and lorazepam, are also used to inhibit vestibular responses due to their effects on the GABAA receptor. Anticholinergics, for example scopolamine, are also prescribed. They function by suppressing conduction in the vestibular cerebellar pathways. Finally, corticosteroids (i.e. prednisone) are prescribed to ameliorate the inflammation of the vestibular nerve and associated apparatus.

Microvascular compression syndrome

[00172] Microvascular compression syndrome (MCS), also called "vascular compression" or "neurovascular compression”, is a disorder characterized by vertigo and tinnitus. It is caused by the irritation of Cranial Nerve VIII by a blood vessel. Other symptoms found in subjects with MCS include, but are not limited to, severe motion intolerance, and neuralgic like "quick spins". MCS is treated with earbamazepine, TRILEPTAL®, and baclofen it is also surgically treated for some cases.

Auditory Nerve Tumors Q0173] Auditory nerve tumors, including acoustic neuroma, acoustic neurinoma, vestibular schwannoma and eighth nerve tumor) are tumors that originate in Schwann ceils, cells that wrap around a nerve. Auditory nerve tumors account for approximately 7-8% of ail tumors originating in the skull, and are often associated with the diagnosis of neurofibromatosis in a patient. Depending upon the location of the tumor, some symptoms include hearing loss. tinnitus, dizziness and loss of balance. In some cases, other more serious symptoms develop as the tumor becomes larger, which compresses against the facial or trigeminal nerve, which affect connections between the brain and the mouth, eye or jaw. Smaller tumors are removed by microsurgery', or stereotactic radiosurgical techniques, including fractionated stereotactic radiotherapy. Malignant Schwannomas are treated with chemotherapeutic agents, including vincristine, adriamycin, cyclophosphamide and imidazole carboxamide.

A uditory Neuropathy

[00174] Auditory neuropathy (AN) is also known as auditory_' neuropathy/auditory dys- synchrony (AN/AD) or auditory neuropathy spectrum disorder (ANSD). Auditory neuropathy describes hearing loss in which the outer hair cells within the cochlea are present and functional, but auditory information is not properly transmited to the auditory nerve and brain.

Benign Paroxysmal Positional Vertigo

[0017S] Benign paroxysmal positional vertigo is caused by the movement of free floating calcium carbonate crystals (otoliths) from the utricle to one of the semicircular canals, most often the posterior semicircular canal. Movement of the head results in the movement of the otoliths causing abnormal endolymph displacement and a resultant sensation of vertigo. The episodes of vertigo usually last for about a minute and are rarely accompanied by other auditory symptoms.

Cancer of the Ear

[00176] Although the cause is unknown, cancer of the ear is often associated with long-term and untreated otitis, suggesting a link between chronic inflammation and development of the cancer, at least in some cases. In some instances, tumors in the ear are benign or malignant, and they exist in the external, middle, or inner ear. Symptoms of ear carreer include otorrhea, otalgia, hearing loss, facial palsy, tinnitus, and vertigo. Treatment options arc limited, and include surgery-, radiotherapy, chemotherapy, and combinations thereof. Also, additional pharmaceutical agents are used to treat symptoms or conditions associated with the cancer, including corticosteroids in the case of facial palsy, and an timicrobial agents when otitis is present.

[00177] Systemic administration of conventional cytotoxic agents have been used to treat cancer of the ear, including systemic administration of cyclophosphamide (in CHOP chemotherapy) in combination with radiotherapy and methotrexate, and perfusion of methotrexate through the external carotid artery. However, such treatments requiring systemic administration of the active agents suffer from the same drawbacks discussed herein. Namely, relatively high doses of the agents are required to achieve the necessary therapeutic doses in the ear, which result in an increase of undesired, adverse side effects. Accordingly, in some embodiments, the local administration of the active agents in the compositions and formulations disclosed herein results in treatment of cancer of the ear with lower effective doses, and with a decrease in the incidence and/or severity of side effects. Typical side effects of systemic administration of cytotoxic agents, e.g., methotrexate, cyclophosphamide, and thalidomide, for the treatment of cancer of the ear include anemia, neutropenia, bruising, nausea, dennatitis, hepatitis, pulmonary fibrosis, teratogenicity, peripheral neuropathy, fatigue, constipation, deep vein thrombosis, pulmonary edema, atelectasis, aspiration pneumonia, hypotension, bone marrow suppression, diarrhea, darkening of skin and nails, alopecia, changes in hair color and texture, lethargy, hemorrhagic cystitis, carcinoma, mouth sores, and decreased immunity'.

[00178] In some embodiments, the compounds have anti-inflammatory_' properties and are used in the formulations and compositions disclosed herein for the treatment of inflammatory' disorders of the ear, including AIED.

[00179] Although systemic administration of methotrexate, cyclophosphamide, and thalidomide is currently used to treat or is being investigated for the treatment of otic disorders, such as inflammatory otic disorders, including AIED, Meniere’s disease, and Behcet’s disease, as well as cancer of the ear, the cytotoxic agents are not without the potential for serious adverse side effects. Moreo ver, cytotoxic agents, which demonstrate efficacy but are otherwise not approvable because of safety considerations, are also contemplated within the embodiments disclosed herein. In some embodiments, the localized application of the active agents to the target otic structures for treatment of autoimmune and/or inflammatory disorders, as well as cancer of the ear, results in the reduction or elimination of adverse side effects experienced with systemic treatment. In some embodiments, the localized treatment with the active agents contemplated herein reduce the amount of agent needed for effective treatment of the targeted disorder due, for example, to increased retention of the active agents in the auris interna and/or media, to the existence of the biological blood barrier in the auris interna, or to the lack of sufficient systemic access to the auris media.

[00180] in some embodiments, acti ve agents used in the compositions, formulations, and methods disclosed herein are metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of active agents. In some embodiments, the active agents are metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of active agents that retain at least partially the cytotoxicity and anti-inflammatory properties of the parent compounds.

Central Auditory Processing Disorder

[00181] Central auditory processing disorder (CAPD), also referred as auditory processing disorder (APD), is a general term for describing a variety of disorders that affect the way the brain processes auditory_'· information. Individuals with CAPD usually have normal structure and function of the outer, middle and inner ear (peripheral hearing). However, these individuals are unable to process the auditory information, which leads to difficulties in recognizing and interpreting sounds, especially the sounds from speech. Central auditory processing disorder is either developmental or acquired and is believed to arise from dysfunction in the central nervous system.

Cholesteatoma

[00182] A cholesteatoma is a hyperproliferative cyst often found in the middle ear. Cholesteatoma are classified as congenital or acquired. Acquired cholesteatomas result from retraction of the ear drum (primary) and/or a tear in the ear dram (secondary).

[Q0183] Tire most common primary_' cholesteatoma results from the pars fiaccida retracting into the epitympanum. As the pars fiaccida continues to retract, the lateral wall of tire epitympanum slowly erodes. This produces a defect in the lateral wall of the epitympanum that slowly expands. A less common type of primary' acquired cholesteatoma results from the retraction of the posterior quadrant of the tympanic membrane retracts into the posterior middle ear. As the tympanic membrane retracts, squamous epithelium envelops the stapes and retracts into the sinus tympani. Secondary cholesteatomas result from injury to the tympanic membrane (e.g. a perforation resulting from otitis media; trauma; or a surgically- induced injury).

[Q0184] Complications associated with a growing cholesteatoma include injury to the osteoclasts and, in some cases, deterioration of the thin bone layer separating the top of the ear from the brain. Damage to the osteoclasts results from the persistent application of pressure to the bones resulting from the expansion of the cholesteatoma. Additionally, the presence of multiple cytokines (e.g TNF-a, TGF-bI, TOR-b2, II- 1 , and IL-6) in the epithelium of the cholesteatoma results in further degradation of the surrounding bones. [00185] Patients with a cholesteatoma often present with earache, hearing loss, mucopurulent discharge, and/or dizziness. Physical examination confirms the presence of a cholesteatoma. Symptoms that are identified upon physical examination include damage to the ossicles, and a canal filled with mucopus and granulation tissue. [00186] There is currently no effective medical therapy for cholesteatomas. As a cholesteatoma has no blood supply, it cannot be treated with systemic antibiotics. Topical administration of antibiotics often fails to treat a cholesteatoma.

Drug-Induced Inner Ear Damage

[00187] Damage from the administration of drugs, including certain antibiotics, diuretics (e.g. ethacrynic acid and furosemide), aspirin, aspirin-like substances (e.g. salicylates) and quinine. Deterioration of the aims interna organ are hastened by impaired kidney function, which results in decreased clearance of the affecting drugs and their metabolites. In some instances, these drugs affect both hearing and equilibrium, but likely affects hearing to a greater extent.

[00188] For example, neomycin, kanamyein, amikacin have a greater effect on hearing than on balance. The antibiotics viomycin, gentamicin and tobramycin affect both hearing and equilibrium. Streptomycin, another commonly administered antibiotic, induces vertigo more than loss of hearing, and leads to Dandy's syndrome, where walking in the dark becomes difficult and induces a sensation of the environment moving with each step. Aspirin, when taken in very high doses, also leads to temporary hearing loss and tinnitus, a condition where sound is perceived in the absence of external sound. Similarly, quinine, ethacryinic acid and furosemide result in temporary or permanent hearing loss in some instances.

Hereditary Disorders

[00189] Hereditary_' disorders, including Scheibe, Mondini-Michelle, Waardenburg, Michel, Alexander’s ear deformity, hypertelorism, Jervell-Lange Nielson, Refsum and Usher syndromes, are found in approximately 20% of patients with sensorineural hearing loss in some instances, congenital ear malformations result from defects in the development of the membranous labyrinthine, the osseous labyrinthine, or both. Along with profound hearing loss and vestibular function abnormalities, hereditary deformities are associated with other dysfunctions, including development of recurring meningitis, cerebral spinal fluid (CSF) leaks, as well as perilymphatic fistulas. Treatment of chronic infections is necessitated in hereditary disorder patients.

Hyperacusis

[00190] Hyperacusis is a condition characterized by an increased sensitivity to certain frequency and volume ranges of sound (a collapsed tolerance to usual environmental sound). In some eases, hyperacusis occur gradually and in other cases, hyperacusis appears suddenly A person with severe hyperacusis has difficulty tolerating everyday sounds, wherein some of these sounds seem unpleasantly or painfully loud to the afflicted person but not to others. Otic Disorders caused by Free Radicals

[00191] Free radicals are highly reactive atoms, molecules, or ions the reactivity of which results from the presence of unpaired electrons. Reactive oxygen species (“ROS”) form as a result of sequential reduction of molecular oxygen . Examples of reactive oxygen species of interest (“ROS”) include, but are not limited to, superoxide, hydrogen peroxide, and hydroxyl radicals. ROS are naturally produced as a by-product of the production of ATP. in some cases, ROS also results from the use of cisplatin, and aminoglycosides. Further, stress to stereocilia caused by acoustic trauma results in otic hair cells producing ROS.

[00192] In some instances, ROS damages ceils directly by damaging nuclear DNA and mitochondrial DNA . Damage to the former leads to mutations which impair the functioning of auditory_' cells and/or apoptosis. Damage to the latter often results in decreased energy production and increased ROS production both of which leads to impaired cellular functioning or apoptosis. Further, ROS also damages or kills cells by oxidizing the polydesaturated fatty acids which comprise lipids, oxidizing the amino acids which comprise proteins, and oxidizing co-factors necessary- for the activity of enzymes. Antioxidants ameliorate damage by caused by ROS by preventing their formation, or scavenging the ROS before they damage the ceil.

[00193] Damage to mitochondria by ROS is often seen in hearing loss, especially hearing loss due to aging. The loss of ATP correlates to a loss in neural functioning in the inner ear. it also leads to physiological changes in the inner ear. Further, damage to mitochondria often results in an increased rate of cellular degradation and apoptosis of inner ear cells. The cells of the stria vascularis are the most metabolically active due to the vast energy requirements needed to maintain the ionic balance of fluids in the inner ear. Thus, the cells of the stria vaseuians are most often damaged or killed due to damage of the mitochondria.

Presbystasis

[00194] Presbystasis, also known as disequilibrium of aging, is a disorder wherein affected individuals have generalized imbalance, but without spinning vertigo. The generalized imbalance is typically noticed during walking.

Postural Vertigo

[00195] Postural vertigo, otherwise known as positional vertigo, is characterized by sudden violent vertigo that is triggered by certain head positions. This condition is caused by- damaged semicircular canals caused by physical injury to the amis interna, otitis media, ear surgery, or blockage of the artery to the auris interna. [00196] Vertigo onset in patients with postural vertigo usually develops when a person lies on one ear or tilts the head back to look up. Vertigo is accompanied by nystagmus. In severe cases of postural vertigo, the vestibular nerve is severed to the affected semicircular canal. Treatment of vertigo is often identical to Meniere’s disease, and includes meclizine, lorazepam, prochlorperazine or scopolamine. Fluids and electrolytes are intravenously administered if the vomiting is severe.

Recurrent Vestibulopathy

[00197] Recurrent vestibulopathy is a condition wherein the subject experiences multiple episodes of severe vertigo. The episodes of vertigo last for minutes or hours. Unlike Meniere’s Disease, it is not accompanied by hearing loss. In some cases it develops into Meniere’s Disease or Benign Paroxysmal Positional Vertigo. Treatment is similar to that of Meniere’s Disease.

Vertigo

[00198] Vertigo is described as a feeling of spinning or swaying while the body is stationary. There are two types of vertigo. Subjective vertigo is the false sensation of movement of the body. Objective vertigo is the perception that one’s surrounding are in motion. It is often accompanied by nausea, vomiting, and difficulty maintaining balance.

[00199] While not wishing to be bound by any one theory, it is hypothesized that vertigo is caused by an over-accumulation of fluid in the endolymph. This fluid imbalance results increased pressure on the cells of the inner ear which leads to the sensation of movement.

Hie most common cause of vertigo is benign paroxysmal positional vertigo, or BPPV. In some cases, it is brought on by a head injury, or a sudden change of blood pressure. It is a diagnostic symptom of several diseases including superior canal dehiscence syndrome.

Local otic administration

[00200] Also provided herein are methods, formulations, and compositions for local delivery of therapeutic agents (otic agents) to auris externa, aims media, and/or auris interna structures. In some embodiments, local delivery' of the therapeutic agent (otic agent) overcomes the toxic and attendant side effects of systemic delivery. In some embodiments, access to the vestibular and cochlear apparatus is through the auris media and includes the round window membrane, the oval window/stapes footplate, the annular ligament and through the otic capsule/temporal bone.

[00201] Provided herein, in certain embodiments, are otic formulations and compositions that remain in contact with the target auditory surfaces (e.g., the round window) for extended periods of time. In some embodiments, the otic formulations and compositions further comprise mucoadhesives that allow the otic form illations to adhere to otic mucosal surfaces in some instances, the formulations and compositions described herein avoid attenuation of therapeutic benefit due to drainage or leakage of active agents via the eustaeliian tube.

[00202] In some embodiments, the localized treatment of the auris externa, auris media and/or auris interna affords the use of previously undesired therapeutic agents, including agents with poor PK profiles, poor uptake, low systemic release and/or toxicity issues. In some embodiments, localized targeting of the otic agent formulations and compositions reduces the risk of adverse effects with previously characterized toxic or ineffective therapeutic agents (otic active agents). Accordingly, also contemplated within the scope of the embodiments described herein is the use of active agents and/or agents that have been previously rejected by practitioners because of adverse effects or ineffectiveness of the therapeutic agent (otic agent).

100203] In some embodiments, specifically targeting the auris externa, auris media and/or auris interna structures avoids the adverse side effects usually associated with systemic treatment. In some embodiments, the otic formulations and compositions described herein are controlled release therapeutic agent formulations and compositions that treat otic disorders by providing a constant, variable and/or extended source of a therapeutic agent (otic agent) to the individual or patient suffering from an otic disorder, thereby reducing or eliminating the variability of treatment. Accordingly, one embodiment disclosed herein is to provide a formulation or composition that enables at least one therapeutic agent (otic agent) to be released in therapeutically effective doses either at variable or constant rates such as to ensure a continuous release of the at least one therapeutic agent (otic agent). In some embodiments, the therapeutic agents (otic agents) disclosed herein are administered as an immediate release formulation or composition. In other embodiments, the therapeutic agents (otic agents) are administered as a controlled release formulation, released either continuously or in a pulsatile manner, or variants of both . In still other embodiments, the therapeutic agent (otic agent) formulation or composition is administered as both an immediate release and controlled release formulation or composition, released either continuously or in a pulsatile manner, or variants of both. The release is optionally dependent on environmental or physiological conditions, for example, the external ionic environment (see, e.g. Oros^® release system, Johnson & Johnson).

[00204] In addition, the otic compositions or formulations included herein also optionally include carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. Such carriers, adjuvants, and other excipients are compatible with the environment in the auris externa, auris media and/or auris interna. Accordingly, specifically contemplated are carriers, adjuvants and excipients that lack ototoxicity or are minimally ototoxic in order to allow effective treatment of the otic disorders contemplated herein with minimal side effects in the targeted regions or areas. To prevent ototoxicity, otic compositions or formulations disclosed herein are optionally targeted to distinct regions of the auris externa, auris media and/or auris interna, including but not limited to the tympanic cavity, vestibular bony and membranous labyrinths, cochlear bony and membranous labyrinths and other anatomical or physiological structures located within the auris interna.

Treatment

[00205] Provided herein are otic formulations and compositions suitable for the treatment of any otic condition, disease or disorder (e.g., outer, middle and/or inner ear disorders) described herein, comprising administration of a therapeutic agent (otic agent) described herein to an individual or patient m need thereof. The formulations and compositions described herein are suitable for the treatment of any disease described herein. In some instances, the treatment is long-term treatment for chronic recurring disease in some instances, the treatment is prophylactic administration of an otic formulation described herein for the treatment of any otic disease or disorder described herein. In some instances, prophylactic administration avoids occurrence of disease in individuals suspected of having a disease or in individuals genetically predisposed to an otic disease or disorder. In some instances the treatment is preventive maintenance therapy. In some instances, preventive maintenance therapy avoids recurrence of a disease.

[00206] In some instances, because of their otic compatibility and improved sterility, the otic formulations and compositions described herein are safe for long-term administration. In some embodiments, the otic formulations and compositions described herein have very low ototoxicity.

[00207] In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least one day, three days, five days, one week, two weeks, three weeks, a month, two months, three months, four months, five months, six months, or a year. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least three days in some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least five days. In some embodiments, the otic formulations and composi lions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least one week. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least two weeks. In some embodiments, die otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least three weeks. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least a month . In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least two months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) tor a period of at least three months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least four months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least five months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least six months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of at least a year.

[00208] In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about a day, three days, five days, one week, two weeks, three weeks, a month, two months, three months, four months, five months, six months, or a year. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about three days. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about five days. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about one week in some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent for a period of about two weeks. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about three weeks. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about a month in some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about two months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about three months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about four months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about five months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about six months. In some embodiments, the otic formulations and compositions described herein provide a sustained release of a therapeutic agent (otic agent) for a period of about a year.

[00209] In one aspect, provided herein are controlled release compositions and formulations to treat and/or prevent diseases or conditions associated with the ear. In some instances, these diseases or conditions associated with the ear include the outer, the middle ear and/or inner ear.

[00210] In some embodiments, disease or condition is ear pruritus, otitis externa, otalgia, tinnitus, vertigo, ear fullness, hearing loss, or a combination thereof.

[00211] Other otic diseases or conditions include autoimmune inner ear disorder (AIED), Meniere’s disease, endolymphatic hydrops, noise induced hearing loss (NIHL), sensorineural hearing loss (SNL), tinnitus, otosclerosis, balance disorders, vertigo and the like. In some embodiments, the disease or condition associated with the ear is Meniere’s disease, sensorineural hearing loss, noise induced hearing loss, presbycusis (age related hearing loss), auto immune ear disease, tinnitus, ototoxicity, excitotoxicity, endolymphatic hydrops, labyrinthitis, Ramsay Hunt’s Syndrome, vestibular neuronitis, microvascular compression syndrome, hyperacusis, presbystasis, central auditory processing disorder, auditory neuropathy, or improvement of cochlea implant performance.

[Q0212] The etiology of several ear diseases or disorders consists of a syndrome of progressive hearing loss, including noise induced hearing loss and age-related hearing loss, dizziness, nausea, nystagmus, vertigo, tinnitus, inflammation, swelling, infection and/or congestion. These disorders have many causes, such as infection, exposure to noise, injury', inflammation, tumors, and/or adverse response to drugs or other chemical agents. Several causes of hearing and/or equilibrium impairment are attributed to inflammation and/or an autoimmune disorder and/or a cytokine -mediated inflammatory response.

Therapeutic Agents

[00213] In some embodiments, the otic formulations and compositions described herein have pH and osmolarity that are auris-acceptable. In some embodiments, the otic formulations and compositions described herein meet the stringent sterility requirements described herein and are compatible with the endolympb and/or the perilymph. Pharmaceutical agents that are used in conjunction with the formulations and compositions disclosed herein include agents that ameliorate or lessen otic disorders, including auris interna disorders, and their attendant symptoms, which include but are not limited to hearing loss, nystagmus, vertigo, tinnitus, inflammation, swelling, infection and congestion. Otic disorders have many causes and include infection, injury', inflammation, tumors and adverse response to drugs or other chemical agents that are responsive to the pharmaceutical agents disclosed herein in some embodiments, pharmaceutically active metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of the otic agents disclosed herein are used in the formulations.

[00214] For some embodiments, wherein the formulation or composition is designed such that the active ingredient has limited or no systemic release, therapeutic agents that produce systemic toxicities (e.g., liver toxicity) or have poor PK characteristics (e.g. short half-life) are also optionally used. Thus, in some embodiments, therapeutic agents that have been previously shown to be toxic, harmful or non-effective during systemic application, for example through toxic metabolites formed after hepatic processing, toxicity of the drag in particular organs, tissues or systems, through high levels needed to achieve efficacy, through the inabilit to be released through systemic pathways or through poor PK characteristics, are useful. The formulations and compositions disclosed herein are contemplated to be targeted directly to otic structures where treatment is needed; for example, one embodiment contemplated is the direct application of the otic formulations disclosed herein onto the round window membrane or the crista fenestrae cochlea of the auris interna, allowing direct access and treatment of the auris interna, or inner ear components. In other embodiments, the otic formulations and compositions disclosed herein are applied directly to the oval window. In yet other embodiments, direct access is obtained through microinjection directly into the auris interna, for example, through cochlear microperfusion. Such embodiments also optionally comprise a drug delivery device, wherein the drug delivery device delivers the otic formulations through use of a needle and syringe, a pump, a microinjection device, a spongy material or any combination thereof.

[00215] In still other embodiments, application of any otic formulation or composition described herein is targeted to the auris media through piercing of the intratympanic membrane and applying the otic agent formulation directly to the auris media structures affected, including the walls of the tympanic cavity or auditory ossicles. In some embodiments, the auris active agent formulations and compositions disclosed herein are confined to the targeted auris media structure, and will not be lost, for example, through diffusion or leakage through tire eustachian tube or pierced tympanic membrane in some embodiments, the otic formulations and compositions disclosed herein are delivered to the auris externa in any suitable manner, including by cotton swab, injection or ear drops. Also, in other embodiments, the otic formulations and compositions described herein are targeted to specific regions of the auris externa by application with a needle and syringe, a pump, a microinjection device, a spongy material, or any combination thereof. For example, in the case of treatment of otitis externa, antimicrobial agent formulations disclosed herein are delivered directly to the ear canal, where they are retained, thereby reducing loss of the active agents from the target ear structure by drainage or leakage.

WNT Modulators

[00216] In some embodiments, active agents compatible with the formulations described herein include agents that modulate re-growth of damaged auris sensory hair cells. In some instances, modulation of the WNT pathway promotes morphogenesis and/or re-growth of damaged auris sensory hair cells. WNT signaling proteins include protein products encoded by genes such as WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT j 0B. WNT1 1, or WNTI6. In some embodiments, the therapeutic agent is a modulator of WNT. Modulators of the WNT pathway include, and are not limited to, 2-amino-4-[3,4- (methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl}pyrimidine, the signaling molecule Cerberus, or the like. In some embodiments, the therapeutic agent is 2 amino-4~[3,4~ (methylenedioxy)benzyl-amino]-6-(3-metlioxyphenyl)pyrimidine, the signaling molecule Cerberus, or the like.

GSK-3 Modulators

[00217] In some embodiments, the acti ve agents compatible with the formulation described herein are GSK-3 modulators. In some embodiments, the WNT modulator is a GSK-3 modulator. Glycogen synthase kinase-3 (GSK-3) is a serine/direonine protein kinase that is encoded by two known genes, GSK-3 alpha (GSK-3 A) and GSK-3 beta (GSK-3B). In some embodiments, the GSK-3 modulator is a GSK-3 inhibitor. Examples of GSK-3 inhibitors include, but are not limited, to the compounds disclosed in W02007/I06537,

W02009/035634, W02009/035684, and W02010/104205, winch are incorporated by- reference for the disclosure of such compounds.

[00218] Additional examples of GSK-3 inhibitors include, but are not limited to, the compounds disclosed in Phukan et al., British Journal of Pharmacology (2010), 160, 1-19; and Palomo et al., Expert Opinion On Therapeutic Patents (2017), Vol. 27, No. 6, 657-666, which are incorporated by reference for the disclosure of such compounds.

[00219] In some embodiments, the GSK-3 inhibitor is lithium. In some embodiments, the GSK-3 inhibitor is an azaindoie, a benzimidazole, a benzoxazole, a benzthiazole, an imidazole, an indole, an oxazole, a piperidine, a purine, a pyrazole, a pyrrole, a pyrazine, a pyridine, a pyrimidine, a quinazoline, a thiazole, a thiophene, or a derivative thereof.

[Q0220] In some embodiments, the GSK-3 inhibitor is pyrimido[l,2-a]pyrimidin-4-one, pyrrole-2, 5-dione, pyrrole-2, 5-dione, thiazo-l,2-one, pyrrolo[2,3-c]azepin-8-one, 2-phenyl- benzoimidazole, or pyridazin-3-one. in some embodiments, the GSK-3 inhibitor is any one of the following compounds:

[00221] Examples of other GSK-3 inhibitors include, but are not limited to, CHIR-98023, CHIR-73911, CHIR-98014, LY-317615 (Enzastaurm), NP-12 (NP-031112), Neu-120, CP- 70949, SAR-502250, VX-608, TDZD-8, and KUSTU-144 (cazpaullone).

[00222] In some embodiments, the GSK-3 inhibitor is any one of the following compounds:

[00223] In some embodiments, the GSK-3 inhibitor is any one of the following compounds:

[00224] In some embodiments, the GSK-3 inhibitor is AZD1080, tideglusib, and LY2090314. In some embodiments, the GSK-3 inhibitor is any one of the following compounds:

[00225] In some embodiments, the GSK-3 inhibitor is valproic acid, indirubin, BK), SB415286, SB216763, CH1R99021, AR-A014418, L803mts (Myr-GKEAPPAPPQpSP), macrocyclc GM, kenpaullone, or derivatives thereof. In some embodiments, the GSK-3 inhibitor is an oxime or acetoxime derivative of BIO. in some embodiments, the GSK-3 inhibitor is any one of the following compounds:

[00226] In some embodiments, die GSK-3 inhibitor is L803F (KEAPPAPPQS(p)PF), L806- mts (Myr-GKEAPPAPPPS(p)P), VP0.7, 7, luteolin, or derivatives thereof. In some embodiments, the GSK-3 inhibitor is any one of the following compounds:

Additional GSK-3 Inhibitors Q0227] Other examples of GSK-3 inhibitors include, but are not limited to, the compounds disclosed in Kramer et ai., Ini J Alzheimers Dis. 2012; 2012: 381029, which is incorporated by reference tor the disclosure of such compounds.

100228] In some embodiments, the GSK-3 inhibitor is lithium chloride. In some embodiments, the GSK-3 inhibitor is a lithium salt. In some embodiments, the GSK-3 inhibitor is lithium carbonate. In some embodiments, the GSK-3 inhibitor is a covalent or irreversible inhibitor, such as TDZD NP-12. in some embodiments, the GSK-3 inhibitor is a peptide derived from FRATl, such as FRATtide.

Maleimides

[00229] In some embodiments, the GSK-3 inhibitor is a maleimide or a derivative thereof in some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table:

Table 1.

[00230] In some embodiments, the GSK-3 inhibitor is a maleimide, such as an indo!yl- maleirnide. In some embodiments, the indolyl-maleimide is SB-216763. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table: Table 2.

[00231] In some embodiments, the GSK-3 inhibitor is a maleimide, such as a bisindoiyi- maleimide. In some embodiments, the GSK-3 inhibitor is a maleimide, such as a benzofuranyl-indolyl-maleimide. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table:

Table 3.

QQ232] In some embodiments, the GSK-3 inhibitor is a maieimide, such as a benzoje]isoindole-I,3-dione. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table:

Table 4.

Staurosporine and organometallic compounds

[00233] In some embodiments, the GSK-3 inhibitor is Staurosporine. In some embodiments, the GSK-3 inhibitor is an organometallic inhibitor. In some embodiments, the GSK-3 inhibitor is an organometallic inhibitor, which is also amaleimide. In some embodiments, the GSK-3 inhibitor is A-OSl. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following tables:

Table 5a.

Table 5b.

Indoles

100234] In some embodiments the GSK-3 inhibitor is an indole derivative. In some embodiments, the GSK-3 inhibitor is an indole derivative, which is an indirubine. In some embodiments, the indirubine is indirubin. In some embodiments, the indirubine is indirubin- 3’ -monoxime. In some embodiments, the indirubine is BIO. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown m the following table:

Table 6.

Paullones Q0235] In some embodiments, the GSK-3 inhibitor is a pauiione derivative. In some embodiments, the pauiione is alsterpaullone. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table:

Table 7.

Pyrazolamid.es Q0236] In some embodiments, the GSK-3 inhibitor is a pyrazo!amide derivative. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table: Table 8.

Q0237] in some embodiments, the G8K-3 inhibitor is a pyrazoiamide inhibitor, which has the structure of the following compound, Compound 78 (GSK-22 or FX-22).

78

Pyrimidines Q0238] in some embodiments, the GSK-3 inhibitor is a pyrimidine derivative. In some embodiments, the GSK-3 inhibitor is a pyrimidine inhibitor, such as CHIR 99021. In some embodiments, the GSK-3 inhibitor is a pyrimidine inhibitor, such as CHIR 98014. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table:

Table 9.

Furopynrmdmes

[00239] In some embodiments, the GSK-3 inhibitor is a furopyrimidine derivative. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table: Table 10.

Oxadiazoles Q0240] In some embodiments, the GSK-3 inhibitor is an oxadizao!e derivative. In some embodiments, tire GSK-3 inhibitor is a 1,2,5-oxadizaoIe derivative. In some embodiments, the GSK-3 inhibitor is a 1,3,4-oxadizaoIe derivative. In some embodiments, the GSK-3 inhibitor is a 1 ,2,4-oxadizaole derivative. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following tables:

Table 11.

Table 12.

Table 13,

Thiazoles

[00241] In some embodiments, the GSK-3 inhibitor is atlnazole derivative in some embodiments, the GSK-3 inhibitor is a th iazoiylurea derivative. In some embodiments, the thiazolylurea derivative is AR-A0144I8. In some embodiments, the GSK-3 inhibitor is a benzothiazole derivative. In some embodiments, tire GSK-3 inhibitor is any one of the compounds shown in the following tables:

Table 14a.

Table 14b.

Miscellaneous heterocycles A. Benzimidazoles

[00242] In some embodiments, the GSK-3 inhibitor is a benzimidazole derivative in some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table: Table 15a.

B. 1 -Aza-9-oxafluorenes

[00243] In some embodiments, the GSK-3 inhibitor is a l-aza-9-oxaiIuorene derivative. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table:

Table 15b.

C. Xanthines

[Q0244] In some embodiments, the GSK-3 inhibitor is a xanthine derivative. In some embodiments, the xanthine derivative is propentofyliine (PPF, compound 138).

138

D Pyrazolones

[00245] In some embodiments, the GSK-3 inhibitor is a pyrazolone derivative. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table: Table 15c.

[00246] In some embodiments, the GSK-3 inhibitor is a pyrazolone derivati ve with the structure of compound 144.

144 E. Hydantoins

[00247] In some embodiments, the GSK-3 inhibitor is an imidazolidine-2,4-dione, or hydantoin, derivative. In some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table:

Table 15d.

F. Pyridones

[00248] in some embodiments, tire GSK-3 inhibitor is a pyridone derivative in some embodiments, the GSK-3 inhibitor is any one of the compounds shown in the following table: Table 15e.

G. Alkaloid Natural Products QQ249] In some embodiments, the GSK-3 inhibitor is an alkaloid derivative. In some embodiments, the GSK-3 inhibitor is an alkaloid derivative derived from marine organisms. In some embodiments, the GSK-3 inhibitor a manzamine, a meridianin, hymenialdisme, or dibromocantbarelline. In some embodiments, the GSK-3 inhibitor is a compound selected from the following structures:

Manzamine A Meridianin D Hymenia!disine Difaromocanthare!iine 151 152 153 154

100250] In some embodiments, the GSK-3 inhibitor is lithium chloride, lithium carbonate, NP-12, NP-103, CG-301338, XD-4241, SB-415286, SAR-502250, or CEP-16805.

Amount of Therapeutic Agent QQ251] In some embodiments, the otic formulation or composition comprises between about 0.001% to about 99.99%._> by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation os- composition comprises between about 0.001% to about 99.9% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic fonnulation or composition comprises between about 0.001% to about 99% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.001% to about 90% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.001% to about 80% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.001% to about 70% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.001% to about 60% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.001% to about 50% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, tire otic formulation or composition comprises between about 0.001% to about 40% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.001% to about 30% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.001% to about 2.0% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof In some embodiments, the otic formulation or composition comprises between about 0.001% to about 15% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.001% to about 10% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.001% to about 7% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof In some embodiments, the otic formulation or composition comprises between about 0.001% to about 5% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof In some embodiments, the otic formulation or composition comprises between about 0.001% to about 3% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof In some embodiments, the otic formulation or composition comprises between about 0.001% to about 2% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.001% to about 1% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. Q0252] in some embodiments, the otic formulation or composition comprises between about 0.01% to about 99.99% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 99.9% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.01 % to about 99% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 90% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.01% to about 80% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 70% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.01% to about 60% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.01% to about 50% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 40% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.01% to about 30% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.01% to about 20% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 15% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.01% to about 10% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof In some embodiments, the otic formulation or composition comprises between about 0.01% to about 7% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 5% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.01% to about 3% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof In some embodiments, the otic formulation or composition comprises between about 0.01% to about 2% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.01% to about !% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof.

[00253] In some embodiments, the otic formulation or composition comprises between about 0.1 % to about 40% by weight of the therapeutic agen t, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 0.1% to about 30% by weight of tire therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.10% to about 20% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.10% to about 15% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.10% to about 10% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.10% to about 7% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.10% to about 5% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.10% to about 3% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0 10% to about 2% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 0.10% to about 1% by weight of the therapeutic agent, or pharmaceutically acceptable prodnig or salt thereof.

[00254] In some embodiments, the otic formulation or composition comprises between about 1% to about 40% by weight of the therapeutic agent, or pharmaceutically⁷ acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises between about 1% to about 30% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about 1% to about 20% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises between about 1% to about 15% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises between about !%to about 10% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 1% to about 7% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 1% to about 5% by weight of die therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof In some embodiments, the otic formulation or composition comprises between about l%to about 3% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises between about 1 % to about 2% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or sal t thereof [00255] In some embodiments, the otic formulation or composition comprises about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2% about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, or about 40% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereo In some embodiments, the otic formulation or composition comprises about 0.01% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.02% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises about 0.03% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or sal t thereof In some embodiments, the otic formulation or composition comprises about 0.04% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic fonnuiation or composition comprises about 0.05% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises about 0.06% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.07% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.08% by weight of the therapeutic agent, or pharmaceutical iy acceptable prodrug or sait thereof in some embodiments, the otic formulation or composition comprises about 0.09% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.1 % by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic forsmilation or composition comprises about 0.2% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.3% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.4% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.5% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.6% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or sait thereof. In some embodiments, the otic formulation or composition comprises about 0.7% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.8% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises about 0.9% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 1% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or sait thereof. In some embodiments, the otic formulation or composition comprises about 2% by weight of th e therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 3% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 4% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or sait thereof. In some embodiments, the otic formulation or composition comprises about 5% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises about 6% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some 9 embodiments, the otic formulation or composition comprises about 7% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises about 8% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or sal t thereof. In some embodiments, the otic formulation or composition comprises about 9% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 10% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 11% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises about 12% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises about 13% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 14% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof in some embodiments, the otic formulation or composition comprises about 15% by weight of the therapeutic agent, or pharmaceutically acceptable prodrag or salt thereof. In some embodiments, the otic formulation or composition comprises about 16% by weight of the therapeutic agent, or pharmaceutically acceptable prodnig or salt thereof. In some embodiments, the otic formulation or composition comprises about 17% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, foe otic formulation or composition comprises about 18% by weight of foe therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 19% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof. In some embodiments, the otic formulation or composition comprises about 20% by weight of the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof.

Devices

[00256] Also contemplated herein are the use of devices for the delivery of foe pharmaceutical formulations and compositions disclosed herein, or alternatively for the measurement or surveillance of the function of the auris formulations disclosed herein. For example, in one embodiment pumps, osmotic devices or o ther means of mechanically delivering pharmaceutical formulations and compositions are used for the delivery of the pharmaceutical formulations disclosed herein. Reservoir devices are optionally used with the pharmaceutical drag delivery units, and reside either internally along with the drug delivery unit, or externally of the auris structures.

[00257] Other embodiments contemplate the use of mechanical or imaging devices to monitor or survey the hearing, balance or other auris disorder. For example, magnetic resonance imaging (MRJ) devices are specifically contemplated within the scope of the embodiments, wherein the MR! devices (for example, 3 Tesla MRI devices) are capable of evaluating Meniere Disease progression and subsequent treatment with the pharmaceutical formulations disclosed herein. See, Carfrae et al. Laryngoscope 118:501-505 (March 2008). Whole body scanners, or alternatively cranial scanners, are contemplated, as well as higher resolution (7 Tesla, 8 Tesla, 9.5 Tesla or 11 Tesla for humans) are optionally used in MRI scanning.

Auris- Acceptable Gel Forinulations/Compositions

[00258] In some embodiments, the auris-acceptable formulations or compositions described herein are gel formulations or gel compositions.

[00259] In some embodiments, the otic gei formulations or compositions that include at least therapeutic agent and a pharmaceutically acceptable difuent(s), excipient(s), or carrier(s). In some embodiments, the otic gel formulations or compositions include other medicinal or pharmaceutical agents; carriers; adjuvants; preserving, stabilizing, wetting or emulsifying agents; solution promoters; salts for regulating the osmotic pressure; and/or buffers in some embodiments, the otic gel formulations or compositions comprises (i) a therapeutic agent, (ii) a gelling and viscosity enhancing agent, (iii) a pH adjusting agent, and (iv) sterile water. [00260] Gels, sometimes referred to as jellies, have been defined in various ways. For example, the United States Pharmacopoeia defines gels as semisolid systems consisting of either suspensions made up of small inorganic particles or large organic molecules interpenetrated by a liquid. Gels include a single-phase or a two-phase system. A single phase gel consists of organic macromolecules distributed uniformly throughout a liquid in such a manner that no apparent boundaries exist between the dispersed macromolecules and the liquid. Some single-phase gels are prepared from synthetic macromolecules (e.g., carbomer) or from natural gums (e.g., tragacanth). In some embodiments, single-phase gels are generally aqueous but will also be made using alcohols and oils. Two-phase gels consist of a network of small discrete particles.

-Bi- [00261] Gels can also be classified as being hydrophobic or hydrophilic. In certain embodiments, the base of a hydrophobic gel consists of a liquid paraffin with polyethylene or fatty oils gelled with colloidal silica or aluminum or zinc soaps. In contrast, the base of hydrophilic gels usually consists of water, glycerol, or propylene glycol gelled with a suitable gelling agent (e.g., tragacanth, starch, cellulose derivatives, carboxyvinylpolymers, and magnesium-aluminum silicates) in certain embodiments, the rheology of the formulations or devices disclosed herein is pseudo plastic, plastic, thixotropic, or dilatant.

[Q0262] In one embodiment the enhanced viscosity auris-acceptable formulation described herein is not a liquid at room temperature. In certain embodiments, the enhanced viscosity formulation is characterized by a phase transition between room temperature and body temperature (including an individual with a serious fever, e.g., up to about 42 °C). In some embodiments, the phase transition occurs at about 1 °C below' body temperature, at about 2 °C below body temperature, at about 3 °C belo body temperature, at about 4 °C belo body temperature, at about 6 °C below body temperature, at about 8 °C below_' body temperature, or at about 10 °C below body temperature. In some embodiments, the phase transition occurs at about 15 °C below' body temperature, at about 20 °C below' body temperature, or at about 25 °C below body temperature. In specific embodiments, the gelation temperature (Tgei) of a formulation described herein is about 20 °C, about 25 °C, or about 30 °C In certain embodiments, the gelation temperature (Tgel) of a formulation described herein is about 35 °C or about 40 °C. In one embodiment, administration of any formulation described herein at about body temperature reduces or inhibits vertigo associated with intratympanic adminis tration of otic formulations. Included within the definition of body temperature is the body temperature of a healthy individual or an unhealthy individual, including an individual with a fever (up to ~42 °C). In some embodiments, the pharmaceutical formulations or devices described herein are liquids at about room temperature and are administered at or about room temperature, reducing or ameliorating side effects such as, for example, vertigo. [00263] Polymers composed of polyoxypropylene and polyoxyethylene form thermoreversible gels when incorporated into aqueous solutions. These polymers have the ability to change from the liquid state to the gel state at temperatures close to body temperature, therefore allowing useful formulations that are applied to the targeted auris stmcture(s). The liquid state-to-gei state phase transition is dependent on the polymer concentration and the ingredients in the solution.

[00264] Poioxamer 407 (PF-I27) is a nonionic surfactant composed of polyoxyethylene- polyoxypropylene copolymers. Other poloxamers include 188 (F-68 grade), 237 (F-87 grade), and 338 (F-108 grade). Aqueous solutions of poloxamers are stable in the presence of acids, alkalis, and metal ions. PF-127 is a commercially available polyoxyethylene- polyoxypropylene triblock copolymer of general formula E106 P70 El 06, with an average molar mass of 13,000. Tie polymer can be further purified by suitable methods that w ill enhance gelation properties of the polymer. It contains approximately 70% ethylene oxide, which accounts for its hydrophilicity . it is one of the series of poloxamer ABA block copolymers, whose members share the chemical formula shown below. hydrophilic hydrophilic

hydrophobic

[00265] PF-127 is of particular interest since concentrated solutions (>20% w/w) of the copolymer are transformed from low viscosity transparent solutions to solid gels on heating to body temperature. This phenomenon, therefore, suggests that when placed in contact with the body, the gel preparation will form a semi-solid structure and a sustained release depot. Furthermore, PF-127 has good solubilizing capacity, low toxicity and is, therefore, considered a good medium for drag delivery systems.

[00266] In an alternative embodiment, the thermogel is a PEG-PLGA-PEG triblock copolymer (Jeong et al, Nature (1997), 388:860-2; Jeong et al, I. Control. Release (2000),

63: 155-63; Jeong et al, Adv. Drag Delivery Rev. (2002), 54:37-51). Tie polymer exhibits sol-gel behavior over a concentration of about 5% w/w to about 40% w/w. Depending on the properties desired, the lactide/glycolide molar ratio in the PLGA copolymer ranges from about 1 : 1 to about 20: 1. Tie resulting coploymers are soluble in water and form a free- flowing liquid at room temperature but form a hydrogel at body temperature. A commercially available PEG-PLGA-PEG triblock copolymer is RESOMER RGP t50106 manufactured by Boehringer Ingelheim. This material is composed of a PGLA copolymer of 50:50 poly(DL-lactide-co-glyeolide), is 1014 w/w of PEG, and has a molecular weight of about 6000.

[00267] EcGel is a tradename of MacroMed Incorporated for a class of low molecular weight, biodegradable block copolymers having reverse thermal gelation properties as described in U.S. Pat. Nos. 6,004,573, 6,117949, 6,201,072, and 6,287,588. It also includes biodegradable polymeric drag carriers disclosed in pending U.S. patent application Ser Nos. 09/906,041, 09/559,799 and 10/919,603. The biodegradable drug carrier comprises ABA- type or BAB-type triblock copolymers, or mixtures thereof, wherein the A-blocks are relatively hydrophobic and comprise biodegradable polyesters or poIy(orthoester)s, and the B-biocks are relatively hydrophilic and comprise polyethylene glycol (PEG), said copolymers having a hydrophobic content of between 50.1 to 83% by weight and a hydrophilic content of between 17 to 49.9% by weight, and an overall block copolymer molecular weight of between 2000 and 8000 Daltons. The drug carriers exhibit water solubility at temperatures below normal mammalian body temperatures and undergo reversible thermal gelation to then exist as a gel at temperatures equal to physiological mammalian body temperatures. The biodegradable, hydrophobic A polymer block comprises a polyester or pofyCortho ester), in which the polyester is synthesized from monomers selected from the group consisting of D,L- lactide, D-lactide, L-laetide, D,L-laetic acid, D-lactic acid, L-lactic acid, glyeolide, glycolic acid, e-caprolactone, e-hydroxyhexanoic acid, y-butyroiactone, g-hydroxybutyric acid, d- va!eroiactone, d-hydroxy valeric acid, hydroxybutyric acids, malic acid, and copolymers thereof and having an average molecular weight of between about 600 and 3000 Daltons.

The hydrophilic B-block segment is preferably polyethylene glycol (PEG) having an average molecular weight of between about 500 and 2200 Daltons.

[00268] Additional biodegradable thermoplastic polyesters include AtriGeP (provided by Atrix Laboratories, Inc.) and/or those disclosed, e.g., in U.S. Patent Nos. 5,324,519; 4,938,763; 5,702,716; 5,744,153; and 5,990,194; wherein tire suitable biodegradable thermoplastic polyester is disclosed as a thermoplastic polymer. Examples of suitable biodegradable thermoplastic polyesters include polylactides, polyglycol ides, polycaprolactones, copolymers thereof, terpolymers thereof, and any combinations thereof.

In some such embodiments, the suitable biodegradable thermoplastic polyester is a polylactide, a poiyglyeolide, a copolymer thereof, a terpolymer thereof, or any combination thereof in one embodiment, the biodegradable thermoplastic polyester is 50/50 polyiDL- iaetide-eo-glycolide) having a carboxy terminal group; is present in about 30 wt. % to about 40 wt. % of the formulation; and has an average molecular weight of about 2.3,000 to about 45,000. Alternatively, in another embodiment, the biodegradable thermoplastic polyester is 75/25 poly (DL-lactide-co-glycolide) without a carboxy terminal group; is present in about 40 wt. % to about 50 wt. % of the formulation; and has an average molecular weight of about 15,000 to about 24,000. In further or alternative embodiments, the terminal groups of the poly(DL~lactide-co-g!ycolide) are either hydroxyl, carboxyl, or ester depending upon the method of polymerization. Polycondensation of lactic or glycolic acid provides a polymer with terminal hydroxyl and carboxyl groups. Ring-opening polymerization of the cyclic iactide or glycolide monomers with water, lactic acid, or glycolic acid provides polymers with the same terminal groups. However, ring-opening of the cyclic monomers with a monofunctional alcohol such as methanol, ethanol, or !-dodecanol provides a polymer with one hydroxyl group and one ester terminal groups. Ring-opening polymerization of the cyclic monomers with a dioi such as 1,6-hexanediol or polyethylene glycol provides a polymer with only hydroxyl terminal groups.

[Q0269] In some embodiments, any active composition described herein comprises purified thermosensitive polymer in some embodiments, any active composition described herein comprises fractionated a purified thermosensitive polymer composed of polyoxyethylene-polyoxypropy!ene copolymers. In some of such embodiments, the thermosensitive polymer is a poloxamer.

[00270] lire purification of poioxamers is based on the removal of low molecular weight components (e.g., oligomers, unreacted material and/or other unwanted impurities that are produced during manufacturing or storage) and/or large molecular weight components (components from unwanted polymer-polymer reactions). The resulting purified product has a narrower PD1 with approximately the same molecular weight as tire original material in some embodiments, a purified poloxamer has better gelling characteristics (e.g., a lower Tgei tor the same % poloxamer concentration while providing a higher viscosity in the gel state). [00271] As used herein, a purified thermosensiti ve polymer has low* polydispersity (i.e., a narrow distribution of molecular weights amongst the individual polymer chains therein). For example, commercially available poioxamers contain certain impurities such as poly(oxyethylene) homopolymer and poly(oxyethyiene)/poly(oxypropyiene) diblock polymers due to the nature of tire manner in which they are produced lire relative amounts of these byproducts increase as the molecular weights of the component blocks increase. In some instances, in commercially available poloxamer 407, byproducts may constitute from about 15 to about 50% by weight of the polymer depending upon the manufacturer, thereby resulting in high polydispersity. Example 15 illustrates a procedure for fractionation of P407 that reduces polydispersity in commercially available P407.

[QQ272] In some embodiments, super critical fluid extraction technique is used to fractionate polyoxyalkylene block copolymers. See, U.S. Pat. No. 5,567,859, the disclosure for fractionation of polymers described therein is incorported herein by reference. In this technique, lower molecular weight fractions in commercially purchased polymer are removed in a stream of CO? maintained at a pressure of 2200 pounds per square inch (psi) and a temperature of 40 °C, thereby providing purified polymer having low poiydispersity.

[00273] . In some embodiments, gei permeation ehromoatography allows for isolation of fractions of polymers. See, European Patent Application WO 92/16484; the use of gei permeation chromatography to isolate a fraction of poloxamer hav ing low poiydispersity and saturation described therein is incorporated herein by reference.

[00274] In some embodiments, one or more of the blocks is purified prior to manufacture of the copolymer. By way of example, purifying ei ther the polyoxypropylene center block during synthesis of the copolymer, or the copolymer product itself (See, U.S.

Pat. Nos. 5,523,492, and 5,696,298, incorporated herein by reference for such disclosure) allows for manufacture of purified poioxamers.

[00275] in some embodiments, fractionation of polyoxyalkylene block copolymers is acheived by batchwise removal of low molecular weight species using a salt extraction and liquid phase separation technique (See, U.S. Pat. No. 5,800,711, which process of purification of polymers described therein is incorporated herein by reference). Such fractionation produces polyoxyalkylene block copolymers (e.g., poloxamer 407, poloxamaer 188 or the like) having improved phy sical characteristics including increased gel strength, decreased poiydispersity, higher average molecular weight, decreased gelling concentration and/or extended gel dissolution profiles compared to commercially available poioxamers (e.g., P407 NF grade from BASF). Other processes for purification and/or fractionation of polymers are described in, for example, US 6,977,045 and US 6,761,824 which processes are ineosporated herein by reference.

[00276] In some instances, low' molecular weight contaminants of polymers (e.g., poioxamers) cause deleterious side effects in vivo; the use of purified poioxamers in pharmaceutical formulations described herein reduces such in vivo side effects.

[00277] Accordingly, also contemplated within the scope of embodiments presented herein are formulations comprising purified poly(oxyethylene)/poly(oxypropylene) triblock polymers that are substantially free of the poly(oxyethylene) liomopolymers and/or poly(oxypropylene)/poly(oxyethylene) diblock byproducts, thereby narrowing the molecular weight distribution of block copolymers, (i.e., providing low poiydispersity). In some embodiments, such purified poly(oxyethylene)/poly(oxypropylene) triblock polymers (e.g., fractionated poioxamers) allow for formulation of active compositions that comprise lower concentrations of the poly(oxyethylene)/poIy(oxypropylene) tribiock polymers compared to active compositions that comprise non-fractionated poly(oxyethylene)/poly(oxypropylene) triblock polymers.

[00278] Advantageously, such compositions comprising lower concentrations of fractionated poly(oxyethylene)/poiy(oxypropylene) triblock polymers (e.g., poloxamers) retain gelation properties (e.g., gelation between abortt 15 °C and about 42 °C) and sustained release characteristics (e.g., sustained release of dexamethasone over at least 3 days, 5 days or 7 days) despite having a lower concentration of the poly(oxyethylene)/poly(oxypropylene) triblock polymer (e.g., poioxanier).

[QQ279] Accordingly, by way of example, a formulation comprising rnicronized dexamethasone and lower concentrations of fractionated P407 (e.g., between about 5% to about 14% P407) has gelation properties and/or sustained release characteristics that are substantially the same or better than the gelation properties and/or sustained release characteristics of a formulation comprising rnicronized dexamethasone and non-fractionated P407 (e.g., between about 14.5% to about 25% of P407 NF from BASF).

[Q0280] In some embodiments, pharmaceutical formulations described herein comprise gelation temperature modifying agents. A “gelation temperature modifying agent ’ or a “gel temperature modifying agent” is an additive added to any formulation described herein, and changes the gelation temperature of the formulation such that the gel temperature of the formulation is maintained between about 14 °C and about 42 °C. A gel temperature modifying agent increases or decreases the gelation temperature of any formulation described herein such that the formulation maintains a gelation temperature of between about 14 °C and about 42 °C,

[00281] In some embodiments, a gel temperature modifying agent is a gel temperature increasing agent. For example, where a formulation comprising a thermosensitive polymer has a gelation temperature below 14 °C, addition of a gel temperature increasing agent (e.g., P188, P388, cyclodextrin, carboxymethyi cellulose, hyaluronic acid, CARBOPOL®, Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, Tween 85, n methyl pyrrolidone, short chain fatty acid salts (e.g., sodium oleate, sodium caprate, sodium caprylate or the like) increases the gelation temperature of the formulation to above 14 °C, to between about 14 °C and about 42 °C.

[Q0282] In some embodiments, a gel temperature modifying agent is a gel temperature decreasing agent. For example, where a formulation comprising a thermosensitive polymer has a gelation temperature above 42 °C, addition of a gel temperature decreasing agent (e.g., P188, P388, cyclodextrin, carboxymethyi cellulose, hyaluronic acid, CARBOPOL®, Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, Tween 85, n methyl pyrrolidone, faty acid salts (e.g., sodium oleate, sodium caprate, sodium caprylate or the like) decreases the gelation temperature of the formulation to below 42. °C, to between about 14 °C and about 42 °C.

[00283] in some embodiments, a gel temperature modifying agent is a pH sensitive polymer (e.g., chitosan). in some embodiments, a gel temperature modifiying agent is a thermosensitive polymer. In some embodiments, a gel temperature modifying agent is an ion-sensitive polymer (e.g., alginates gel in the presence of calcium ions). In some embodiments, a gel temperature modifying agent is an acrylic acid-based polymer (e.g., CARBOPOL®). In some embodiments, a gel temperature modifiying agent is a cellulose based polymer (e.g., hydroxypropylmethy! cellulose, carboxymethyl cellulose, or the like).

In some embodiments, a gel temperature modifying agent is an alkyl aryl polyether alcohol- based polymer (e.g., TYLOXAPOL®).

[00284] In some embodiments, a gel temperature modifiying agent is a poloxamer.

By way of example, addition of not more than about 5% poloxamer 188 to a formulation comprising about 16% P407 increases the gelation temperature of a 16% P407 formulation by about 5 °C.

[00285] In one embodiment, a pharmaceutical formulation described herein is a liquid at about room temperature in certain embodiments, the pharmaceutical formulation is characterized by a phase transition between about room temperature and about body temperature (including an individual with a serious fever, e.g., up to about 42 °C) In some embodiments, the phase transition occurs between at least about 1 °C below body temperature and body temperature, between at least about 2 °C below body temperature and body temperature, between at least about 3 °C below body temperture and body temperature, between at least about 4 °C below body temperature and body temperature, between at least about 6 °C below body temperature and body temperature, between at least about 8 °C below body temperature and body temperature, between at least about 10 °C below body temperature and body temperature, between at least about 15 °C below body temperature and body temperature, or between at least about 20 °C below body temperature and body temperature.

[Q0286] In some embodiments, a formulation described herein has a gelation temperature of between about 5 °C, 10 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, or 20 °C, and about 25 °C, 28 °C, 30 °C , 33 °C, 35 °C , 37 °C , 40 °C or 42 °C. In some embodiments, a formulation described herein has a gelation temperature of betw een about 5 °C and about 42 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 10 °C and about 42 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 14 °C and about 42. °C. In some embodiments, a formulation described herein has a gelation temperature of between about 14 °C and about 40 °C. in some embodiments, a formulation described herein has a gelation temperature of between about 14 °C and about 37 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 14 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 16 °C and about 35 °C. in some embodiments, a formulation described herein has a gelation temperature of between about 18 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 42. °C. In some embodiments, a formulation described herein has a gelation temperature of betw een about 20 °C and about 37 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 30 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 28 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 25 °C.

[00287] Since the polymer systems of thermoreversihle gels dissolve more completely at reduced temperatures, methods of solubilization include adding the required amount of polymer to the amount of water to be used at. reduced temperatures. Generally after wetting the polymer by shaking, the mixture is capped and placed in a cold chamber or in a thermostatic container at about 0-10 °C in order to dissolve the polymer. The mixture is stirred or shaken to bring about a more rapid dissolution of the thermoreversihle gei polymer. The active agent and various additives such as buffers, salts, and preservatives are subsequently added and dissolved. In some instances the active agent and/or other pharmaceutically active agent is suspended if it is insoluble in water. The pH is modulated by the addition of appropriate buffering agents. Round window membrane mucoadhesive characteristics are optionally imparted to a thermoreversihle gel by incorporation of round window membrane mucoadhesive earbomers, such as Carbopol® 934P, to the formulation (Majithiya et ai, AAPS PhaimSeiTech (2006), 7(3), p. El; EP0551626, both of which is incorporated herein by reference for such disclosure).

[00288] In one embodiment are auris-acceptable pharmaceutical gel formulations which do not require the use of an added viscosity enhancing agent or viscosity modulating agent. Such gel formulations incorporate at least one pharmaceutically acceptable buffer. In one aspect is a gel formulation and a pharmaceutically acceptable buffer in another embodiment, the pharmaceutically acceptable excipient or carrier is a gelling agent.

[00289] In other embodiments, useful auris-acceptable pharmaceutical formulations also include one or more pH adjusting agents or buffering agents to provide an endolymph or perilymph suitable pH. Suitable pH adjusting agents or buffers include, but are not limited to acetate, bicarbonate, ammonium chloride, citrate, phosphate, pharmaceutically acceptable salts thereof, and combinations or mixtures thereof Such pH adjusting agents and buffers are included in an amount required to maintain pH of the formulation from a pH of about 5 to about 9, in one embodiment a pH from about 6.5 to about 7.5, and in yet another embodiment at a pH of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In one embodiment, when one or more buffers are utilized in the formulations of the present disclosure, they are combined, e.g., with a pharmaceutically acceptable vehicle and are present in the final formulation, e.g., in an amount ranging from about 0.1% to about 20%, from about 0.5% to about 10%. in certain embodiments of the present disclosure, the amount of buffer included in the gel formulations are an amount such that the pH of the gel formulation does not interfere with the auns media or auris interna’ s natural buffering system, or does not interfere with the natural pH of the endolymph or perilymph, depending on where in the cochlea the otic formulation is targeted. In some embodiments, from about 10 niM to about 200 niM concentration of a buffer is present in the gel formulation. In certain embodiments, from about a 5 mM to about a 200 mM concentration of a buffer is present. In certain embodiments, from about a 20 mM to about a 100 mM concentration of a buffer is present in one embodiment is a buffer such as acetate or citrate at slightly acidic pH. In one embodiment the buffer is a sodium acetate buffer having a pH of about 4.5 to about 6.5. In one embodiment the buffer is a sodium citrate buffer having a pH of about 5.0 to about 8.0, or about 5.5 to about 7.0.

[00290] In an alternative embodiment, the buffer used is tris(hydroxymethyl)aminomethane, bicarbonate, carbonate, or phosphate at slightly basic pH. In one embodiment, the buffer is a sodium bicarbonate buffer having a pH of about 6.5 to about 8.5, or about 7.0 to about 8.0.

In another embodiment the buffer is a sodium phosphate dibasic buffer having a pH of about 6.0 to about 9.0.

[00291] Also described herein are control! ed-release formulations or devices a viscosity enhancing agent or viscosity modulating agent. Suitable viscosity-enhancing agents or viscosity modulating agents include by way of example only, gelling agents and suspending agents. In one embodiment, the enhanced viscosity formulation does not include a buffer. In other embodiments, the enhanced viscosity formulation includes a pharmaceutically acceptable buffer. Sodium chloride or other tonicity agents are optionally used to adjust tonicity, if necessary.

[00292] In some embodiments is an enhanced viscosity formulation, comprising from about 0.1 inM and about 100 niM of an acti ve agent, a pharmaceutically acceptable viscosity enhancer or viscosity modulating agent, and water for injection, the concentration of the viscosity enhancer or viscosity modulating agent in the water being sufficient to provide an enhanced viscosity formulation with a final viscosity from about 100 to about 100,000 cP. in certain embodiments, the viscosity of the gel is in the range from about 100 to about 50,000 cP, about 100 cP to about 1,000 cP, about 500 cP to about 1500 cP, about 1000 cP to about 3000 cP, about 2000 cP to about 8,000 cP, about 4,000 cP to about 50,000 cP, about 10,000 cP to about 500,000 eP, about 15,000 cP to about 1,000,000 cP. in certain embodiments, the viscosity of the gel is in the range from about 100 to about 50,000 cP, about 100 cP to about 1,000 cP, about 500 cP to about 1500 cP, about 1000 cP to about 3000 cP, about 2000 eP to about 8,000 cP, about 4,000 cP to about 50,000 cP, about 10,000 cP to about 500,000 cP, about 15,000 cP to about 3,000,000 cP. In other embodiments, when an even more viscous medium is desired, the biocompatible gel comprises at least about 35%, at least about 45%, at least about 55%, at least about 65%, at least about 70%, at least about 75%, or even at least about 80% or so by weight of the active agent. In highly concentrated samples, the biocompatible enhanced viscosity formulation comprises at least about 25%, at least about 35%, at least about 45%, at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, or more by weight of the active agent. [Q0293] in further or alternative embodiments, the otic gel formulations are capable of being administered on or near the round window membrane via mtratympanie injection In other embodiments, the otic gel formulations are administered on or near the round window or the crista fenestrae cochleae through entry via a post-auricular incision and surgical manipulation into or near the round window or the crista fenestrae cochleae area. Alternatively, the otic gel formulation is applied via syringe and needle, wherein the needle is inserted through the tympanic membrane and guided to the area of the round window or crista fenestrae cochleae. The otic gel formulations are then deposited on or near the round window or crista fenestrae cochleae for localized treatment of autoimmune otic disorders. In other embodiments, the otic gel formulations are applied via microcathethers implanted into the patient, and in yet further embodiments the formulations are administered via a pump device onto or near the round window membrane in still further embodiments, the otic gel formulations are applied at or near the round window membrane via a microinjection device. In yet other embodiments the otic gel formulations are applied in the tympanic cavity in some embodiments, the otic gel formulations are applied on the tympanic membrane in still other embodiments, the otic gel formulations are applied onto or in the auditory canal.

Triglyceride Based Otic Formulations and Compositions

[00294] Provided herein in one embodiment are otic formulations and compositions comprising triglycerides. Triglycerides are esters derived from glycerol and three fatty acids in some instances, these fatty acids are saturated fatty acids, unsaturated fatty acids, or a combination thereof. Provided herein in one aspect, is an otic formulation or a composition comprising a therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof; and triglycerides comprising medium chain fatty acids; wherein the triglycerides are present in an amount that is sufficient to stabilize the therapeutic agent for injection into the ear, and wherein the otic pharmaceutical formulation or composition comprises at least about 50% by weight of the triglycerides.

[00295] In some instances, these triglycerides are medium chain triglycerides (MCTs). In some embodiments, these triglycerides comprise medium chain faty acids.

[00296] In some embodiments, the triglycerides are derived from glycerol and medium chain fatty acids. In some embodiments, each medium chain fatty acid independently comprises 6 to 12 carbon atoms in the carbon chain. In some embodiments, each medium chain fatty acid independently comprises 8 to 12 carbon atoms in the carbon chain in some embodiments, each medium chain fatty acid independently comprises 6, 7, 8, 9, 10, 11, or 12 carbon atoms in the carbon chain. In some embodiments, each medium chain fatty acid independently comprises 8 or 10 carbon atoms in the carbon chain. In some embodiments, the medium chain fatty acids are caproic acid (hexasioic acid), enanthie acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecyienic acid (undec-lG-enoic acid), lauric acid (dodecanoic acid), or a combination thereof. In some embodiments, the medium chain faty acids are caprylic acid (octanoic acid), capric acid (decanoic acid), or a combination thereof.

[00297] In some embodiments, the triglycerides comprising medium chain fatty acids are balassee oil, coconut oil, cohune oil, palm kernel oil, tucum oil, or combinations thereof. In some embodiments, triglycerides comprising medium chain fatty acids are coconut oil, cohune oil, palm kernel oil, tucum oil, or any combinations thereof. In some embodiments, the triglycerides comprising medium chain fatty acids are balassee oil. in some embodiments, the triglycerides comprising medium chain fatty acids are coconut oil. In some embodiments, the triglycerides comprising medium chain fatty acids are cohune oil. In some embodiments, the triglycerides comprising medium chain faty acids are palm kernel oil. In some embodiments, the triglycerides comprising medium chain fatty acids are tucum oil. [00298] .Provided herein in one aspect, is an otic formulation or a composition comprising a therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof; and triglycerides comprising long-chain fatty acids; wherein the triglycerides are present in an amount that is sufficient to stabilize the therapeutic agent for injection into the ear, and wherein the otic pharmaceutical formulation or composition comprises at least about 50% by weight of the triglycerides.

[00299] In some instances, these triglycerides are long-chain triglycerides (LCTs). In some embodiments, these triglycerides comprise long-chain fatty acids in some embodiments, the triglycerides are derived from glycerol and at least two long-chain fatty acids. In some embodiments, the triglycerides are derived from glycerol and three long-chain fatty acids. In some embodiments, the triglycerides are derived from glycerol, two long-chain fatty acids, and one medium-chain fatty acid.

[00300] In some embodiments, the triglycerides are derived from glycerol and at least two long-chain fatty acids. In some embodiments, each long-chain faty- acid independently comprises greater than 12 carbon atoms in a carbon chain. In some embodiments, each long- chain fatty acid independently comprises 13 to 38 carbon atoms in a carbon chain. In some embodiments, the long-chain fatty acids are saturated long-chain fatty acids, unsaturated long-chain fatty acids, or a combination thereof. In some embodiments, each long-chain fatty acid independently comprises 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 carbon atoms in a carbon chain. In some embodiments, each long -chain fatty acid independently comprises 13 to 24 carbon atoms in a carbon chain. In some embodiments, the long-chain fatty acids are saturated long-chain fatty acids, unsaturated long-chain fatty acids, or a combination thereof. In some embodiments, each long-chain faty- acid independently comprises 13, 14, 15, 16, 17, 18, 19, 20, 2.1, 22, 23, or 24 carbon atoms in a carbon chain. In some embodiments, each long-chain fatty acid independently comprises 13 to 22 carbon atoms in a carbon chain. In some embodiments, the long-chain fatty acids are saturated long-chain fatty acids, unsaturated long -chain faty acids, or a combination thereof. In some embodiments, each long-chain faty acid independently comprises 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms in a carbon chain. [00301] In some embodiments, the long-chain fatty acids are tridecylic acid (tridecanoic acid), myristic acid (tetradecanoic acid), pentadecylic acid (pentadecanoic acid), palmitic acid (hexadecanoic acid) , margaric acid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecylic acid (nonadecanoic acid), arachidic acid (eicosanoic acid), heneicosylic acid (heneicosanoic acid), behenic acid (docosanoic acid), tricosylic acid (tricosanoic acid), lignoceric acid (tetracosanoic acid), pentacosybc acid (pentacosanoic acid), cerotic acid (hexacosanoie acid), heptacosylic acid (heptacosanoic acid), montame acid (octacosanoic acid), nonacosylic acid (nonacosanoic acid), melissic acid (triacontanoic acid), henatriacontylic acid (henatriacontanoic acid), lacceroic acid (dotriacontanoic acid), psyilic acid (tritriacontanoic acid), geddic acid (tetratriacontanoic acid), ceropiastic acid (pentatriacontanoic acid), bexatriacontylic acid (hexatriacontanoic acid), lieptatriacontanoic acid (heptatriacontanoic acid), octatriacontanoic acid or a combination thereof.

[00302] In some embodiments, the long-chain fatty acids are a-lmoienic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, g-linolemc acid, dihomo-g- iinolenic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, paullinic acid, oleic acid, elaidic acid, gondoic acid, erucic acid, nervonic acid, mead acid, or a combination thereof.

[00303] In some embodiments, the triglycerides comprising long-chain fatty acids are co oil, peanut oil, safflower oil, soybean oil, sunflower seed oil sesame oil, olive oil, castor oil, cotton seed oil, and fish oil, or combinations thereof.

[00304] In some embodiments, the triglycerides are derived from glycerol, two long-chain fatty acids, and one medium-chain fatty acid. In some embodiments, each long-chain fatty acid is independently any one of the long-chain fatty acids described herein. In some embodiments, the medium-chain fatty acid comprises 6 to 12 carbon atoms in a carbon chain. In some embodiments, the medium-chain fatty acid comprises 8 to 12 carbon atoms in a carbon chain. In some embodiments, the medium-chain fatty acid comprises 6, 7, 8, 9, 10, 11, or 12 carbon atoms in a carbon chain. In some embodiments, the medium-chain fatty acid comprises 8 or 10 carbon atoms in a carbon chain. In some embodiments, the medium-chain fatty acid is caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonie acid (nonanoic acid), caprie acid (decanoic acid), undecylenic acid (undec-10-cnoic acid), and lauric acid (dodecanoie acid). In some embodiments, the medium- chain fatty_' acid is caprylic acid (octanoic acid), eapric acid (decanoic acid).

[00305] In some embodiments, the otic pharmaceutical formulation has triglycerides an amount that is sufficient to stabilize the therapeutic agent for injection into the ear. In some embodiments, the otic pharmaceutical formulation has triglycerides in an amount that is sufficient to provide sufficient retention time in the ear. In some embodiments, the ear is the outer ear, middle ear, or inner ear. In some embodiments, the otic pharmaceutical formulation has triglycerides in an amount that is sufficient to provide sustained release of the therapeutic agent. In some embodiments, the otic formulation has triglycerides in an amount that is sufficient to allow delivery of the formulation via a narrow gauge needle.

[00306] In some embodiments, the otic pharmaceutical formulation comprises between about 50% to about 99.9% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 55% to about 99.9% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 60% to about 99.9% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 65% to about 99.9% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 70% to about 99.9% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 75% to about 99.9% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 80% to about 99.9% by the weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 85% to about 99.9% by the weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 90% to about 99.9% by the weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 95% to about 99.9% by the weight of the triglycerides.

[00307] In some embodiments, the otic pharmaceutical formulation comprises between about 50% to about 99.99% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 55% to about 99.99% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 60% to about 99.99% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 65% to about 99.99% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 70% to about 99.99% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 75% to about 99.99% by weight of the triglycerides . In some embodiments, the otic pharmaceutical formulation comprises between about 80% to about 99.99% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 85% to about 99.99% by weight of the triglycerides. in some embodiments, the otic pharmaceutical formulation comprises between about 90% to about 99.99% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 95% to about 99.99% by weight of the triglycerides.

[00308] In some embodiments, the otic pharmaceutical formulation comprises between about 50% to about 95% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 55% to about 95% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 60% to about 95% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 65% to about 95% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 70% to about 95% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 75% to about 95% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 80% to about 95% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 85% to about 95% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 90% to about 95% by weight of the triglycerides.

[00309] In some embodiments, the otic pharmaceutical formulation comprises between about 50% to about 55% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 55% to about 60% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 60% to about 65% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 65% to about 70% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 70% to about 75% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 75% to about 80% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 80% to about 85% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 85% to about 90% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 90% to about 95% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 95% to about 99% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 95% to about 99.9% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 95% to about 99.99% by weight of the triglycerides.

[00310] In some embodiments, die otic pharmaceutical formulation comprises between about 50% to about 60% by weight of the triglycerides in some embodiments, the otic pharmaceutical formulation comprises between about 60% to about 70% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 70% to about 80% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 80% to about 90% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 90% to about 99% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 90% to about 99.9% by weight of the triglycerides. In some embodiments, the otic pharmaceutical formulation comprises between about 90% to about 99.99% by weight of the triglycerides.

[00311] In some embodiments, the triglycerides in any one of the otic fonnulations and compositions described herein are replaced with at least one of the following components in the corresponding amounts of triglyceride in the formulation or composition disclosed herein: mineral oil or any corresponding higher alkanes; Vaseline (petroleum jelly); silicone oil (polydimethylsiloxane) in different molecular weights; beeswax dissolved in any of the oils disclosed herein.

[00312] In some embodiments, the otic formulation or composition further comprises at least one viscosity modulating agent. In some embodiments, the at least one viscosity modulating agent is silicon dioxide, povidone, carbomer, poloxamer, or a combination thereof. In some embodiments, the viscosity modulating agent is silicon dioxide. In some embodiments, the viscosity_' modulating agent is povidone. In some embodiments, the viscosity modulating agent is carbomer. In some embodiments, the viscosity modulating agent is poloxamer. In some embodiments, the viscosity modulating agents are silicon dioxide and povidone. In some embodiments, the viscosity_' modulating agents are silicon dioxide and carbomer. In some embodiments, the viscosity modulating agents are silicon dioxide and poloxamer. in some embodiments, the poloxamer is P407.

[00313] in some embodiments, the otic formulation or composition comprises between about 0.01 % to about 40% by weight of the povido e. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 35% by weight of the povidone. In some embodiments, tire otic formulation or composition comprises between about 0.01% to about 30% by weight of the povidone. In some embodiments, the otic formulation or composition comprises between about 0.01 % to about 25% by weight of the povidone. In some embodiments, the otic formulation or composition comprises between about 0.01 % to about 20% by weight of the povidone. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 15% by weight of the povidone. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 10% by weight of the povidone. In some embodiments, the otic formulation or composition comprises about 0.01% to about 7% by weight of the povidone. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 5% by weight of the povidone. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 3% by weight of the povidone. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 2% by weight of the povidone. In some embodiments, the otic formulation or composition comprises about 0.01% to about 1% by weight of the povidone. Q0314] In some embodiments, the otic formulation or composition comprises between about 0.01%₎ to about 40%. by weight of the carbomer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 35% by weight of the carbomer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 30% by weight of the carbomer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 25% by weight of the carbomer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 20%- by weight of the carbomer. In some embodiments, the otic formulation or composition comprises between about 0.01 % to about 15% by weight of the carbomer. In some embodiments, the otic formulation or composition comprises between about 0.01 % to about 10% by weight of the carbomer. In some embodiments, the otic formulation or composition comprises about 0.01 % to about 7% by weight of the carbomer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 5% by weight of the carbomer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 3%_> by weight of the carbomer. in some embodiments, the otic formulation or composition comprises between about 0.01%₎ to about 2% by weight of the carbomer. In some embodiments, the otic formulation or composition comprises about 0.01% to about 1% by weight of the carbomer.

[00315] In some embodiments, the otic formulation or composition comprises between about 0.01% to about 40% by weight of the poloxamer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 35% by weight of the poloxamer. in some embodiments, the otic formulation or composition comprises between about 0.01 % to about 30% by weight of the poloxamer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 25% by weight of the poloxamer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 20% by weight of the poloxamer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 15% by weight of the poloxamer. in some embodiments, the otic formulation or composition comprises between about 0.01% to about 10% by weight of the poloxamer. in some embodiments, the otic formulation or composition comprises about 0.01% to about 7% by weight of the poloxamer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 5% by weight of the poloxamer. in some embodiments, the otic formulation or composition comprises between about 0.01% to about 3% by weight of the poloxamer. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 2% by weight of the poloxamer. In some embodiments, the otic formulation or composition comprises about 0.01% to about 1% by weight of the poloxamer.

[Q0316] in some embodiments, the otic formulation or composition comprises between about 0.01% to about 20% by weight of the silicon dioxide. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 15% by weight of the silicon dioxide. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 10% by weight of the silicon dioxide. In some embodiments, the otic formulation or composition comprises about 0.01% to about 7% by weight of the silicon dioxide in some embodiments, the otic formulation or composition comprises between about 0.01% to about 5% by weight of the silicon dioxide. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 3% by weight of the silicon dioxide. In some embodiments, the otic formulation or composition comprises between about 0.01% to about 2% by weight of the silicon dioxide. In some embodiments, the otic formulation or composition comprises about 0.01% to about 1% by weight of the silicon dioxide.

[00317] In some embodiments, the otic triglyceride based pharmaceutical formulations have triglycerides in an amount that is sufficient to stabilize the therapeutic agent for injection into the ear. In some embodiments, the injection is into the outer ear. In some embodiments, the injection is into the middle ear. In some embodiments, the injection is intratympanic. In some embodiments, the injection is into the inner ear. In some embodiments, the otic triglyceride based pharmaceutical formulations have triglycerides in an amount that is sufficient to provide sufficient retention time in the ear. In some embodiments, the sufficient retention time in the ear is for the middle ear. In some embodiments, the sufficient retention time in the ear is for the inner ear. in some embodiments, the sufficient retention time in the ear is for the outer ear. in some embodiments, the outer ear is the external auditory canal, the outer surface of tire tympanic membrane, or a combination thereof. In some embodiments, the outer ear is the external auditory canal. In some embodiments, the otic triglyceride based pharmaceutical formulations have triglycerides in an amount that is sufficient to provide sustained release of the therapeutic agent. In some embodiments, the sustained release of the therapeutic agent is m the outer ear. In some embodiments, the sustained release of the therapeutic agent is in the middle ear. In some embodiments, the sustained release of the therapeutic agent is in the inner ear. Q0318] In some embodiments, the otic composition or formulation is free or substantially free of water. In some embodiments, the otic composition or formulation comprises less than 10% by weight of w ater in some embodiments, the otic composition or formulation comprises less than 9% by weight of water. In some embodiments, the otic composition or formulation comprises less than 8% by weight of water. In some embodiments, the otic composition or formulation comprises less than 7% by weight of water. In some embodiments, the otic composition or formulation comprises less than 6% by weight of water in some embodiments, the otic composition or formulation comprises less than 5% by weight of water. In some embodiments, the otic composition or formulation comprises less than 4% by weight of water in some embodiments, the otic composition or formulation comprises less than 3% by weight of water. In some embodiments, the otic composition or formulation comprises less than 2% by weight of water. In some embodiments, the otic composition or formulation comprises less than 1% by weight of water. In some embodiments, the otic composition or formulation comprises less than 0.5% by weight of water in some embodiments, the otic composition or formulation comprises less than 0.1% by weight of water. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 50 ppm of water. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 25 ppm of water in some embodiments, an otic composition or formulation disclosed herein comprises less than about 20 ppm of water. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 10 ppm of water. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 5 pprn of w ater in some embodiments, an otic composition or formulation disclosed herein comprises less than about 1 ppm of water.

[00319] In some embodiments, the otic composition or formulation is free or substantially free of poloxamer. In some embodiments, the otic composition or formulation is free or substantially free of poloxamer 407

[Q0320] In some embodiments, the otic composition or formulation is free or substantially free of C1-C6 alcohols or C1-C6 glycols. In some embodiments, the otic composition or formulation is free or substantially free of C1-C6 alcohols. In some embodiments, the otic composition or formulation is free or substantially free of C1-C6 glycols. In some embodiments, the otic composition or formulation comprises less than 10% by weight of Cl- C6 alcohols or C1-C6 glycols. In some embodiments, the otic composition or formulation comprises less than 9% by weight of C1-C6 alcohols or C1-C6 glycols. In some embodiments, the otic composition or fonnuiation comprises less than 8% by weight of Cl- C6 alcohols or C1-C6 glycols. In some embodiments, the otic composition or formulation comprises less than 7% by weight of C1-C6 alcohols or C1-C6 glycols in some embodiments, the otic composition or formulation comprises less than 6% by weight of Cl - C6 alcohols or C1-C6 glycols. In some embodiments, the otic composition or formulation comprises less than 5% by weight of C1-C6 alcohols or C1-C6 glycols in some embodiments, the otic composition or formulation comprises less than 4% by weight of Cl~ C6 alcohols or C1-C6 glycols. In some embodiments, the otic composition or formulation comprises less than 3% by weight of C1-C6 alcohols or C1-C6 glycols in some embodiments, the otic composition or formulation comprises less than 2% by weight of Cl- C6 alcohols or C1-C6 glycols. In some embodiments, the otic composition or fonnuiation comprises less than 1% by weight of C1-C6 alcohols or C1-C6 glycols. In some embodiments, the otic composition or formulation comprises less than 0.5% by weight of Cl- C6 alcohols or C1-C6 glycols. In some embodiments, the otic composition or fonnuiation comprises less than 0.1% by weight of C1-C6 alcohols or C1-C6 glycols. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 50 ppm of each of C1-C6 alcohols or C 1-C6 glycols. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 25 ppm of each of Cl~ C6 alcohols or C1-C6 glycols. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 20 pprn of each of C1-C6 alcohols or C1-C6 glycols. in some embodiments, an otic composition or formulation disclosed herein comprises less than about 10 ppm of each of C1-C6 alcohols or C1-C6 glycols. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 5 ppm of each of C1-C6 alcohols or C1-C6 glycols. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 1 ppm of each of Cl- C6 alcohols or C1-C6 glycols.

[00321] In some embodiments, the otic composition or formulation is free or substantially free of CI-C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation is free or substantially free of C1-C4 alcohols. In some embodiments, the otic composition or formulation is free or substantially free of C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 10% by weight of Cl - C4 alcohols or C1-C4 glycols in some embodiments, the otic composition or formulation comprises less than 9% by weight of C1-C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 8% by weight of Cl- C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 7% by weight of C1-C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 6% by weight of Cl- C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 5% by weight of C1-C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 4% by weight of Cl- C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 3% by weight of C1-C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 2% by weight of Cl- C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 1% by weight of C1-C4 alcohols or C 1 -C4 glycols. In some embodiments, the otic composition or formulation comprises less than 0.5% by weight of Cl- C4 alcohols or C1-C4 glycols. In some embodiments, the otic composition or formulation comprises less than 0.1% by weight of C1-C4 alcohols or Cl -04 glycols. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 50 ppm of each of C1-C4 alcohols or C1-C4 glycols in some embodiments, an otic composition or formulation disclosed herein comprises less than about 25 ppm of each of Cl- C4 alcohols or CI-C4 glycols in some embodiments, an otic composition or formulation disclosed herein comprises less than about 20 ppm of each of C1-C4 alcohols or C1-C4 glycols. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 10 ppm of each of C1-C4 alcohols or C1-C4 glycols. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 5 ppm of each of C1-C4 alcohols or C1-C4 glycols. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 1 ppm of each of Cl~ C4 alcohols or C1-C4 glycols.

[00322] By way of non-limiting example, the use of the following commonly used solvents should be limited, reduced or eliminated when formulating agents for administration to the ear: alcohols, propylene glycol, and cyclohexane. Thus, in some embodiments, an otic composition or formulation disclosed herein is free or substantially free of alcohols, propylene glycol, and cyclohexane. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 50 ppm of each of alcohols, propylene glycol, and cyclohexane. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 25 ppm of each of alcohols, propylene glycol, and cyclohexane. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 20 ppm of each of alcohols, propylene glycol, and cyclohexane. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 10 ppm of each of alcohols, propylene glycol, and cyclohexane in some embodiments, an otic composition or formulation disclosed herein comprises less than about 5 ppm of each of alcohols, propylene glycol, and cyclohexane. In some embodiments, an otic composition or formulation disclosed herein comprises less than about 1 ppm of each of alcohols, propylene glycol, and cyclohexane. Q0323] In some embodiments, therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof, is multiparticulate. In some embodiments, the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof, is essentially in the form of micronized particles. In some embodiments, the therapeutic agent, or pharmaceutically acceptable prodrug or salt thereof, is essentially dissolved in the otic pharmaceutical formulation or composition.

Carriers Q0324] In other embodiments, the otic pharmaceutical formulations described herein is an auris-aeceptabie hydrogel; in yet other embodiments, the otic pharmaceutical formulations is an auris-acceptable oil. In some embodiment, the active agents is provided as microsphere, microparticle, or nanoparticles; in still other embodiments, the active agent is provided as polymer-containing particles. In some embodiments, theactive agent is provided as unencapsulated or uncoated particles, such as unencapsulated or uncoated multiparticulates or micron-sized particles. In some embodiments, the otic pharmaceutical formulations provide cm auris-acceptable paint; in still further embodiments, otic pharmaceutical formulations provide an auris-acceptable in situ forming spongy material. In some embodiments, the otic pharmaceutical formulations provide an auris-acceptable solvent release gel. In some embodiments, the otic pharmaceutical formulations provide an actinic radiation curable gel. Further embodiments include a thermore versible gel in the otic pharmaceutical formulation, such that upon preparation of the gel at room temperature or below, the formulation is a fluid, but upon application of the gel into or near the auris interna and/or aur s media target site, including the tympanic cavity, round window membrane, or the crista fenestrae cochleae, the otic-pharmaceutical formulation stiffens or hardens into a gel-like substance.

[00325] Suitable carriers for use in a formulation or composition described herein include, but are not limited to, any pharmaceutically acceptable solvent. For example, suitable solvents include polyalkyiene glycols such as, but not limited to, polyethylene glycol (PEG) and any combinations or mixtures thereof. In other embodiments, the base is a combination of a pharmaceut cally acceptable surfactant and solven t.

[00326] In some embodiments, other excipients include, sodium stearyl fumarate, diethanolamine cetyl sulfate, isostearate, poly ethoxy late d castor oil, benzalkonium chloride, nonoxyl 10, octoxynol 9, sodium lauryl sulfate, sorbitan esters (sorbitan mono!aurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioieate, sorbitan trioleate, sorbitan tristearate, sorbitan iaurate, sorbitan oleate, sorbitan painutate, sorbitan stearate, sorbitan di oleate, sorbitan sesqui -isostearate, sorbitan sesquistearate, sorbitan tri-isostearate), lecithins, phospholipids, phosphatidyl cholines (c8-cl8), phosphatidylethanolamines (c8-cl8), phosphatidylglycerols (c8-cl8), pharmaceutical acceptable salts thereof and combinations or mixtures thereof.

[00327] In further embodiments, the carrier is polyethylene glycol. Polyethylene glycol is available many different grades having varying molecular weights. For example, polyethylene glycol is available as PEG 200: PEG 300; PEG 400; PEG 540 (blend); PEG 600; PEG 900; PEG 1000; PEG 1450; PEG 1540; PEG 2000; PEG 3000; PEG 3350; PEG 4000; PEG 4600, and PEG 8000. For purposes of the present disclosure, all grades of polyethylene glycol are contemplated for use in preparation of a formulation described herein in some embodiments the polyethylene glycol used to prepare a formulation described herein is PEG 300.

[00328] In other embodiments, the carrier is a polysorbate. Polysorbates are nonionic surfactants of sorbitan esters. Polysorbates useful in the present disclosure include, but are not limited to polysorhate 20, polysorbate 40, polysorbate 60, poiysorbate 80 (Tween 80) and any combinations or mixtures thereof in further embodiments, poiysorbate 80 is utilized as the pharmaceutically acceptable carrier.

[00329] In one embodiment, water-soluble glycerin-based auris-acceptable enhanced viscosity formulations utilized in the preparation of pharmaceutical deliver) vehicles comprise at least one active agent containing at least about 0.1% of the water-soluble glycerin compound or snore. In some embodiments, the percentage of active agent is varied between about 1% and about 95%, between about 5% and about 80%, between about 10% and about 60% or more of the weight or volume of the total pharmaceutical formulation. In some embodiments, the amount of the compound(s) in each therapeutically useful formulation is prepared in such a way that a ssii table dosage will he obtained in any g ven un t dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations are contemplated herein.

[00330] If desired, the auris-acceptable pharmaceutical gels also contain co-solvents, preservatives, cosolvents, ionic strength and osmolality adjustors and other excipients in addition to buffering agents. Suitable auris-acceptable water soluble buffering agents are alkali or alkaline earth metal carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate, carbonate, and trometliamme (TKIS). These agents are present in amounts sufficient to maintain the pH of the system at 7.4±0.2 and preferably, 7.4. As such, the buffering agent is as much as 5% on a weight basis of the total formulation.

[00331] Cosolvents are used to enhance the active agent solubility, however, some active agents are insoluble. These are often suspended in the polymer vehicle with the aid of suitable suspending or viscosity enhancing agents.

[00332] Moreover, some pharmaceutical excipients, diluents or carriers are potentially ototoxic. For example, benzaikonmm chloride, a common preservative, is ototoxic and therefore potentially harmful if introduced into the vestibular or cochlear structures. In formulating a contrail ed-release formulation, it is advised to avoid or combine the appropriate excipients, diluents or carriers to lessen or eliminate potential ototoxic components from the formulation, or to decrease the amount of such excipients, diluents, or carriers. Optionally, a controlled-release formulation includes otoprotective agents, such as antioxidants, alpha lipoic acid, calcium, fosfomycin or iron chelators, to counteract potential ototoxic effects that may arise from the use of specific therapeutic agents or excipients, diluents, or carriers.

[00333] In some embodiments, therapeutically acceptable otic formulations are:

[00334] The formulations disclosed herein alternati vely encompass an otoprotectant agent in addition to the at feast one active agent and/or excipients, including but not limited to such agents as antioxidants, alpha hpoic acid, calcium, fosfomycin or iron chelators, to counteract potential ototoxic effects that may arise from the use of specific therapeutic agents or excipients, diluents or carriers.

[00335] In some embodiments, the percentage of active pharmaceutical ingredient is varied between about 0.01% and about 20%, between about 0.01% and about 10%, between about 0.01% and about 5% or more of the weight or volume of die total pharmaceutical formulation or composition. In some embodiments, the amount of the compound(s) in each therapeutically useful formulation or composition is prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations are contemplated herein and the preparation of such pharmaceutical formulations or compositions is presented herein.

Modes of Otic Administration Q0336] In some embodiments, the auris formulations or compositions described herein are administered into the ear canal, or in the vestibule of the ear. Access to, for example, the vestibular and cochlear apparatus occurs through the auris media including the round wnndow membrane, the oval window/stapes footplate, the annular ligament and through the otic capside/temporal bone. In some embodiments, otic administration of the formulations or compositions described herein avoids toxicity associated with systemic administration (e.g., hepatotoxicity, cardiotoxicity, gastrointestinal side effects, and renal toxicity) of the active agents. In some instances, localized administration in the ear allows an active agent to reach a target organ (e.g., inner ear) in the absence of systemic accumulation of the active agent. In some instances, local administration to the ear provides a higher therapeutic index for an active agent that otherwise have dose-limiting systemic toxicity.

[00337] Pro v ided herein are modes of treatment for otic formulations or compositions that ameliorate or lessen otic disorders described herein. Drags delivered to the inner ear have been administered systemicaliy via oral, intravenous or intramuscular routes. However, systemic administration for pathologies local to the inner ear increases the likelihood of systemic toxicities and adverse side effects and creates a non-productive distribution of drag in which high levels of drag are found in the serum and correspondingly lower levels are found at the inner ear. [00338] Provided herein are methods comprising the administration of said auris formulations or compositions on or near the round window membrane via intratympanie injection. In some embodiments a composition disclosed herein is administered on or near the round window' or the crista fenestrae cochleae through entry via a post-auricular incision and surgical manipulation into or near the round window or the crista fenestrae cochleae area. Alternatively, a formulation or composition disclosed herein is applied via syringe and needle, wherein the needle is inserted through the tympanic membrane and guided to the area of the round window or crista fenestrae cochleae. In some embodiments, a formulation or composition disclosed herein is then deposited on or near the round window' or crista fenestrae cochleae for localized treatment. In other embodiments, a formulation or composition disclosed herein is applied via microcathethers implanted into the patient, and in yet further embodiments a composition disclosed herein is administered via a pump device onto or near the round window' membrane in still further embodiments, a formulation or composition disclosed herein is applied at or near the round window membrane via a microinjection device in yet other embodiments, a formulation or composition disclosed herein is applied in the tympanic cavity. In some embodiments, a formulation or composition disclosed herein is applied on the tympanic membrane. In still other embodiments, a formulation or composition disclosed herein is applied onto or in the auditory_' canal. The formulations or compositions described herein, and modes of administration thereof, are also applicable to methods of direct instillation or perfusion of the inner ear compartments. Thus, the formulations or compositions described herein are useful in surgical procedures including, by way of non-limiting examples, cochleostomy, labyriiithotomy, mastoidectomy, stapedectomy, endolymphatic sacculotomy or the like.

Intratympanie Injections

[00339] In some embodiments, a surgical microscope is used to visualize the tympanic membrane. In some embodiments, the tympanic membrane is anesthetized by any suitable method (e.g., use of phenol, lidocaine, and xyiocaine). In some embodiments, the anterior- superior and posterior-inferior quadrants of the tympanic membrane are anesthetized.

[00340] In some embodiments, a puncture is made in the tympanic membrane to vent any gases behind the tympanic membrane. In some embodiments, a puncture is made in the anterior-superior quadrant of the tympanic membrane to vent any gases behind the ty mpanic membrane. In some embodiments, the puncture is made with a needle (e.g., a 25 gauge needle). In some embodiments, the puncture is made with a laser (e.g., a COi laser). In one embodiment the delivery system is a syringe and needle apparatus that is capable of piercing the tympanic membrane and directly accessing the round window membrane or cr sta fenes trae cochleae of the auris in terna.

[00341] In one embodiment, the needle is a hypodermic needle used for instant delivery of the formulation. The hypodermic needle is a single use needle or a disposable needle. In some embodiments, a syringe is used for delivery of the pharmaceutically acceptable otic agent-containing compositions as disclosed herein wherein the syringe has a press-fit (Luer) or twist-on (Luer-lock) fitting. In one embodiment, the syringe is a hypodermic syringe. In another embodiment, the syringe is made of plastic or glass. In yet another embodiment, the hypodermic syringe is a single use syringe. In a further embodiment, the glass syringe is capable of being sterilized. In yet a further embodiment, the sterilization occurs through an autoclave. In another embodiment, the syringe comprises a cylindrical syringe body wherein the formulation is stored before use. In other embodiments, the syringe comprises a cylindrical syringe body wherein the pharmaceutically acceptable otic formulations or compositions as disclosed herein is stored before use which conveniently allows for mixing with a suitable pharmaceutically acceptable buffer. In other embodiments, the syringe contains oilier excipients, stabilizers, suspending agents, diluents, or a combination thereof to stabilize or otherwise stably store the otic agent or other pharmaceutical compounds contained therein.

[00342] In some embodiments, the syringe comprises a cylindrical syringe body wherein the body is compartmentalized in that each compartment is able to store at least one component of the auris-aeceptable otic formulation. In a further embodiment, the syringe having a compartmentalized body allows for mixing of the components prior to injection into the auris media or auris interna. In other embodiments, the delivery system comprises multiple syringes, each syringe of the multiple syringes contains at least one component of the formulation such that each component is pre-mixed prior to injection or is mixed subsequent to injection. In a further embodiment, the syringes disclosed herein comprise at least one reservoir wherein the at least one reservoir comprises an otic agent, or a pharmaceutically acceptable buffer, or a viscosity enhancing agent, or a combination thereof. Commercially available injection devices are optionally employed in their simplest form as ready-to-use plastic syringes with a syringe barrel, needle assembly with a needle, plunger with a plunger rod, and holding flange, to perform an intratympanic injection.

[00343] In some embodiments, a needle is used to deliver the formulations or compositions described herein. In some embodiments, a needle punctures the posterior-inferior quadrant of tiie tympanic membrane. In some embodiments, the needle is a standard gauge needle. In sortie embodiments, the needle is a narrow gauge needle in some embodiments, the needle is wider than an 18 gauge needle. In another embodiment, the needle gauge is from about 18 gauge to about 30 gauge. In some embodiments, the needle gauge is from about 2.0 gauge to about 30 gauge. In some embodiments, the needle gauge is from about 25 gauge to about 30 gauge. In some embodiments, the needle gauge is about 18 gauge, about 19 gauge, about 20 gauge, about 21 gauge, about 22 gauge, about 23 gauge, about 24 gauge, about 25 gauge, about 26 gauge, about 2.7 gauge, about 28 gauge, about 29 gauge, or about 30 gauge. In a further embodiment, the needle is a 25 gauge needle. Depending upon the thickness or viscosity of a formulation or composition disclosed herein, the gauge level of the syringe or hypodermic needle is varied accordingly. In some embodiments, the formulations or compositions described herein are liquids and are administered via narrow gauge needles or cannulas (e.g., 22 gauge needle, 25 gauge needle, or cannula), minimizing damage to the tympanic membrane upon administration. The formulations or compositions described herein are administered with minimal discomfort to a patient.

[Q0344] In some embodiments, an otoendoscope (e.g., about 1.7 mm m diameter) is used to visualize the round window membrane. In some embodiments, any obstructions to the round window membrane (e.g., a false round window membrane, a fat plug, fibrous tissue) are removed.

[00345] In some embodiments, a formulation or composition disclosed herein is injected onto the round window membrane. In some embodiments, 0.1 to 0.5 cc of a formulation or composition disclosed herein is injected onto the round window membrane.

[Q0346] In some embodiments, the tympanic membrane puncture is left to heal spontaneously in some embodiments, a paper patch myringoplasty is performed by a trained physician. In some embodiments, a tympanoplasty is performed by a trained physician. In some embodiments, an individual is advised to avoid water. In some embodiments, a cotton ball soaked in petroleum-jelly is utilized as a barrier to water and other environmental agents. Other Delivery Routes

[00347] In some embodiments, a formulation or composition disclosed herein is administered locally to the outer ear, such as the external auditory canal, the outer surface of the tympanic membrane, or a combination thereof in some embodiments, the formulations or compositions described herein are not administered through the tympanic membrane. [00348] In some embodiments, a formulation or composition disclosed herein is administered to the inner ear. In some embodiments, a formulation or composition disclosed herein is administered to the inner ear via an incision in the stapes footplate. In some embodiments, a formulation or composition disclosed herein is administered to the cochlea via a cochleostomy. in some embodiments, a formulation or composition disclosed herein is administered to the vestibular apparatus (e.g.. semicircular canals or vestibule)

[00349] In some embodiments, a formulation or composition disclosed herein is applied via syringe and needle. In other embodiments, a formulation or composition disclosed herein is applied via microcatheters implanted into the patient. In some embodiments, a formulation or composition disclosed herein is administered via a pump device. In still further embodiments, a formulation or composition disclosed herein is applied via a microinjection device. In some embodiments, a formulation or composition disclosed herein is administered via a prosthesis, a cochlear implant, a constant infusion primp, or a wick.

[00350] In some embodiments, the deliver}- device is an apparatus designed tor administration of therapeutic agents to the middle and/or inner ear. By way of example only: GYRUS Medical GmbH offers micro-otoscopes for visualization of and drag delivery to the round window niche; Arenberg has described a medical treatment device to deliver fluids to inner ear structures in U.S. Patent Nos. 5,421,818; 5,474,529; and 5,476,446, each of which is incorporated by reference herein for such disclosure. U.S. Patent Application No.

08/874,208, which is incorporated herein by reference for such disclosure, describes a surgical method for implanting a fluid transfer conduit to deliver therapeutic agents to the inner ear. U.S. Patent Application Publication 2007/0167918, which is incorporated herein by reference for such disclosure, further describes a combined otic aspirator and medication dispenser for intratympanie fluid sampling and medicament application.

Certain Non-Limiting Embodiments

[00351] Provided below are certain non-limiting embodiment of the present disclosure.

Embodiment 1 An otic formulation comprising a therapeutically effective amount of a GSK-3 modulator and an auris-acceptable vehicle, wherein the amount of the GSK-3 modulator released into the inner ear is sufficient to: increase SGN density and/or branching; increase the number of Schwann cells; increase Schwann cells or oligodendrocyte survival or proliferation; preserve of restore SGN or auditory nerve myelin sheaths; increase expression of myelin-promoting genes; increase myelin protein expression; increase the number of neurai/glial precursor cells; increaseneural/glial proliferation; preserve hair cells upon drug -induced ototoxcity; or combinations thereof.

Embodiment 2. The otic formulation of embodiment 1 , wherein GSK-3 modulator is a GSK-3 inhibitor.

Embodiment 3. The otic formulation of embodiments 1 or 2, wherein the auris- acceptable vehicle is an auris-aeceptable gel.

Embodiment 4. The otic formulation of embodiment 3, wherein the auris-aeceptable gel is a thermoreversible gel.

Embodiment 5 The otic formulation of embodiments 3 or 4, wherein the auris- aeceptable gel has a gelation viscosity from about 15,000 cP and about 3,000,000 eP. Embodiment 6. The otic formulation of embodiment 5, wherein the auris-aeceptable gel has a gelation viscosity from about 100,000 cP to about 500,000 cP.

Embodiment 7. The otic formulation of embodiment 5, wherein the auris-aeceptable gel has a gelation viscosity from about 250,000 cP to about 500,000 cP.

Embodiment 8. The otic formulation of any one of embodiments 3-7, wherein the auris-aeceptable gel is capable of being injected by a narrow gauge needle or cannula through the tympanic membrane.

Embodiment 9. The otic formulation of any one of embodiments 3-8, wherein the otic formulation has an osmolanty from about 100 mOsm/'L to about 1000 mOsm/L.

Embodiment 10. The otic formulation of embodiment 9, wherein the otic formulation has an osmolarity from about 150 to about 500 mOsm/L.

Embodiment 11. The otic formulation of embodiment 9, wherein the otic formulation has an osmolarity from about 200 to about 400 mOsm/L.

Embodiment 12. The otic formulation of embodiment 9, wherein the otic formulation has an osmolarity from about 250 to about 320 mOsm/L.

Embodiment 13. The otic formulation of any one of embodiments 3-12, wherein the otic formulation has a gelation temperature from about 19°C to about 42°C.

Embodiment 14. Tire otic formulation of any one of embodiments 3-13, wherein the otic formulation has a pH from about 7.0 to about 8.0.

Embodiment 15. The otic formulation of any one of embodiments 3-14, wherein the auris-aeceptable gel comprises a copolymer of polyoxyethylene and polyoxypropylene. Embodiment 16. The otic formulation of embodiment 15, wherein the copolymer of polyoxyethylene and polyoxypropylene is poloxamer 407. Embodiment 17. Hie otic formulation of embodiment 16, wherein the otic formulation comprises from about 14 wt% to about 18 \vt% poloxamer 407.

Embodiment 18. The otic formulation of embodiment 16, wherein the otic formulation comprises from about 15 wt%to about 17 wt% poloxamer 407.

Embodiment 19. The otic formulation of embodiment 16, wherein the otic formulation comprises about 16 wt% poloxamer 407.

Embodiment 20. The otic formulation of embodiments 1 or 2, wherein the auris- accep table vehicle comprises triglycerides comprising medium chain fatty acids. Embodiment 21. Hie otic formulation of embodiment 20, wherein the triglycerides are derived from glycerol and medium chain fatty acids.

Embodiment 22 The otic formulation of embodiments 20 or 21, wherein each medium chain fatty acid independently comprises 6 to 12 carbon atoms in the carbon chain. Embodiment 23. The otic formulation of embodiments 20 or 21, wherein each medium chain faty acid independently comprises 8 to 12 carbon atoms in the carbon chain. Embodiment 24. Hie otic formulation of any one of embodiments 20-23, wherein the medium chain faty acids are saturated medium chain faty acids, unsaturated medium chain fatty acids, or any combinations thereof.

Embodiment 25. The otic formulation of any one of embodiments 20-24, wherein the medium chain faty acids are caproie acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylenic acid (undec-10-enoic acid), lauric acid (dodecanoic acid), or any combinations thereof.

Embodiment 26. Hie otic formulation of embodiment 20, wherein the triglycerides comprising medium chain faty acids are baiassee oil, coconut oil, cohune oil, palm kernel oil, tucum oil, or any combinations thereof.

Embodiment 27. The otic formulation of any one of embodiments 20-26, wherein the otic formulation comprises at least about 50% by weight of the triglycerides.

Embodiment 28. The otic formulation of any one of embodiments 20-2.6, wherein the otic formulation comprises from about 50% to about 99.99% by weight of the triglycerides, about 55% to about 99.99% by weight of the triglycerides, about 60% to about 99.99% by weight of the triglycerides, about 65% to about 99.99% by weight of the triglycerides, about 70% to about 99.99% by weight of the triglycerides, about 75% to about 99.99% by weight of the triglycerides, about 80% to about 99.99% by weight of the triglycerides, about 85% to about 99.99% by weight of the triglycerides, about 90% to about 99.99% by weight of the triglycerides, or about 95% to about 99.99% by weight of the triglycerides.

Embodiment 29 The otic formulation of embodiments 27 or 28, wherein the otic formulation has triglycerides in an amount that is sufficient to allow delivery of the formulation via a narrow gauge needle.

Embodiment 30. The otic formulation of any one of embodiments 20-29, wherein the otic formulation further comprises at least one viscosity modulating agent.

Embodiment 31 . Embodiment 1. The otic formulation of embodiment 30, wherein the at least one viscosity modulating agent is silicon dioxide, povidone, carbomer, poloxamer, or a combination thereof.

Embodiment 32 The otic formulation of embodiment 31 , wherein the viscosity modulating agent is silicon dioxide.

Embodiment 33. The otic formulation of embodiment 31, wherein the viscosity modulating agents are silicon dioxide and povidone.

Embodiment 34. Hie otic formulation of embodiment 33, wherein the otic formulation comprises between about 0.01% to about 20% by weight of the povidone, about 0.01% to about 15% by weight of the povidone, about 0.01% to about 10% by weight of the povidone, about 0.01% to about 7% by weight of the povidone, about 0.01% to about 5% by weight of the povidone, about 0.01% to about 3% by weight of the povidone, about 0.01% to about 2% by weight of the povidone, or about 0.01% to about 1% by weight of the povidone. Embodiment 35. The otic formulation of embodiment 31, wherein the viscosity modulating agents are silicon dioxide and carbomer.

Embodiment 36. Hie otic formulation of embodiment 35, wherein the otic formulation comprises between about 0.01% to about 20% by weight of the carbomer, about 0.01% to about 15% by weight of the carbomer, about 0.01 % to about 10% by weight of the carbomer, about 0.01 % to about 7% by weight of the carbomer, about 0.01% to about 5% by weight of the carbomer, about 0.01%to about 3% by weight of the carbomer, about 0.01% to about 2% by weight of the carbomer, or about 0.01% to about 1% by weight of the carbomer. Embodiment 37. Hie otic formulation of embodiment 31, wherein the viscosity modulating agents are silicon dioxide and poloxamer.

Embodiment 38. The otic formulation of embodiment 37, wherein the otic formulation comprises between about 0.01% to about 20% by weight of the poloxamer, about 0.01% to about 15% by weight of the poloxamer, about 0.01 % to about 10% by weight of the poloxamer, about 0.01% to about 7% by weight of the poloxamer, about 0.01% to about 5% by weight of the poloxamer, about 0.01% to about 3% by weight of the poloxamer, about 0.01% to about 2% by weight of the poloxamer, or about 0.01% to about 1% by weight of the poloxamer.

Embodiment 39. The otic formulation of any one of embodiments 31-38, wherein the otic formulation comprises between about 0.01%₎ to about 10%₎ by weight of the silicon dioxide, about 0.01% to about 7% by weight of the silicon dioxide, about 0.01% to about 5% by weight of the silicon dioxide, about 0.01% to about 3% by weight of the silicon dioxide, about 0.01% to about 2% by weight of the silicon dioxide, or about 0.01% to about 1% by weight of the silicon dioxide.

Embodiment 40. The otic formulation of any one of embodiments 2.0-39, wherein the otic formulation has a viscosity between about 10 cP to about 10,000 cP, about 10 cP to about 5,000 cP, about 10 cP to about 1,000 cP, about 10 cP to about 500 cP, about 10 cP to about 250 cP, about 10 cP to about 100 cP, or about 10 cP to about 50 cP.

Embodiment 41. The otic formulation of any one of embodiments 20-40, wherein the otic formulation comprises between about 0.01% to about 20% by weight of the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, about 0.01%₎ to about 15%_> by weight of the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, about 0.01 % to about 10% by weight of the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, about 0.01% to about 7% by weight of the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, about 0.01% to about 5% by weight of the GSK-3 modulator, or pharmaceutically acceptable prodrag or salt thereof, about 0.01% to about 3% by weight of the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, about 0.01% to about 2% by weight of the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, or about 0.01% to about 1% by weight of the GSK-3 modulator, or pharmaceutically acceptable prodrag or salt thereof.

Embodiment 42. The otic formulation of any one of embodiments 20-41, wherein the otic formulation is free or substantially free of water, C1-C6 alcohols or C1-C6 glycols, Cl- C4 alcohols or C1-C4 glycols, or any combination thereof.

Embodiment 43. The otic formulation of any one of embodiments 1-42, wherein the GSK-3 modulator has a mean dissolution time of about 30 hours.

Embodiment 44. The otic formulation of any one of embodiments 1-43, wherein the GSK-3 modulator is released from the formulation over a period of at least 3 days. Embodiment 45. The otic formulation of any one of embodiments 1-44, wherein the GSK-3 modulator is released from the formulation over a period of at least 4 days. Embodiment 46. Hie otic formulation of any one of embodiments 1-45, wherein the GSK-3 modulator is released from the formulation over a period of at least 5 days. Embodiment 47. The otic formulation of any one of embodiments 1-46, wherein the GSK-3 modulator is released from the formulation over a period of at least 7 days. Embodiment 48. The otic formulation of any one of embodiments 1-47, wherein the GSK-3 modulator is released from the formulation over a period of at least 14 days. Embodiment 49. The otic formulation of any one of embodiments 1-48, wherein the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, is multiparticulate. Embodiment 50. Hie otic formulation of any one of embodiments 1-49, wherein the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, is essentially in the form of micronized particles.

Embodiment 51. The otic formulation of any one of embodiments 1 -49, wherein the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, is essentially in the form of nanosized particles.

Embodiment 52. Hie otic formulation of any one of embodiments 1-48, wherein the GSK-3 modulator, or pharmaceutically acceptable prodrug or salt thereof, is essentially dissolved in the otic formulation.

Embodiment 53. The otic formulation of any one of embodiments 1-52, further comprising a drug delivery device selected from a needle and syringe, a pump, a microinjection device, a wick, a spongy material, and combinations thereof.

Embodiment 54. The otic formulation of any of embodiments 1-53, further comprising an antioxidant.

Embodiment 55. Hie otic formulation of any of embodiments 1-54, further comprising a mucoadhesive.

Embodiment 56. The otic formulation of any of embodiments 1-55, further comprising a penetration enhancer.

Embodiment 57. The otic formulation of any of embodiments 1-56, further comprising a preservative.

Embodiment 58. Hie otic formulation of any of embodiments 1-57, further comprising a thickening agent or viscosity modulator agent.

Embodiment 59. The otic formulation of any of embodiments 1-58, further comprising a chelator.

Embodiment 60. The otic formulation of any of embodiments 1-59, further comprising an excipient that increases the release rate of the therapeutic agent. Embodiment 61 . Hie otic formulation of any of embodiments 1-60, further comprising an excipient that decreases the release rate of the therapeutic agent.

Embodiment 62. The otic formulation of any of embodiments 1-61, for use in the treatment of an otic disease or condition associated with decreased SGN densi ty and/or branching; decreased number of Schwann cells; decreased Schwann cells or oligodendrocyte survival or proliferation; decreased SGN or auditory nerve myelin sheaths; decrease expression of myelin-promoting genes; decreased myelin protein expression; decreased number of neural/glial precursor cells; decreased neural/glial proliferation; decreased number of hair cell upon drug-induced ototoxcity; or combinations thereof.

Embodiment 63 The otic formulation of embodiment 62, wherein the otic disease or condition is hearing loss.

Embodiment 64. The otic formulation of any one of embodiments 1-63, wherein the otic formulation regenerates otic hair cells.

Embodiment 65. The otic formulation of any one of the embodiment 1-64, wherein the amount of the GSK-3 modulator released into the inner ear is below a toxicity exposure limit. Embodiment 66. Hie otic formulation of embodiment 65, wherein release of the GSK-3 modulator above the toxicity exposure limit would result in at least one of: decreased SGN density and/or branching; decreased number of Schwann cells; decreased myelin protein expression; decreased number of neural/glial precursor cells; decreased neuron survival upon SGN dissociation;

Embodiment 67. Hie otic formulation of embodiments 1-66, wherein the GSK3 inhibitor is not:

Formula I

Embodiment 68. The otic formulation of embodiments 1-66, wherein the GSK3 inhibitor is not any one of:

2-pyriinidinylaminoethylainino-2~pyndinyl containing compound;

3-(pyridin-2-yl)-lH-indo3-2~ol containing compound;

2-pyrimidinylaminoethylamino-2-pyridyi containing compound;

N-( lH-pyrazoi-4-yl)-nicotinamide containing compound

2.4-dichiorophenyi-5-(lH-imidazol-2~yl)-2~pyrmudinyiaminoe†hylamino~2-pyridine compound;

2.4-dichiorophenyl-5-(lH-imidazo3-2-yl)-2-pyrimidinylaminoethyiamino-3-pyridine containing compound;

3-(9 fluoro-2-(piperidme-l~carbonyl)~l,2,3,4-tetrahydro~[l,4]diazepino[6,7,l- hi]indol-7-yl)-4-(imidazo[l,2-a]pyridin-3-yl)-lH-pyrrole-2 5-dione; and

1.2.3.4-tetrahydro-j l,4]diazepino[6,7, i-hi]indoiyi containing compound. Embodiment 69. The otic formulation of embodiment 1-66, wherein the G8K3 inhibitor is not G8K-22.

EXAMPLES

Example A l - Pharmacokinetics of GSK-3 Modulator Formulations GSK-3 Modulator Solution in P407 Q0352] A stock solution of poloxamer 407, such as a 16% w/w stock solution, is prepared by slowly adding it to a cold buffer solution to provide an appropriate final concentration (such as a final concentration in stock solution of about 50 niM tromethamine, about 77 mM sodium chloride, pH 7.7). This solution is sterilized by filtration. Then any one of the GSK- 3 modulators described herein is suspended with an appropriate amount of poloxamer 407 solution to reach appropriate concentrations, such as 0.6%, 1.5%, 3.0%, and 6.0% w/w. Pharmacokinetics

[00353] Female rats (Charles River) weighing 200-300 g of approximately 12.-16 weeks of age serve as subjects (N = 4 per group). Prior to any procedures, animals are anesthetized using a combination of xylazine (10 mg kg) and ketamine (90 mg/kg) for up to an hour via the intraperitoneal route if needed, an intraoperative booster is administered intraperitoneal representing a one-tenth of the original dose.

[00354] lntratympamc injection - Each animal is positioned so that the head is tilted at an angle to favor injection towards the round window niche. Briefly, under visualization with an operating microscope, 20 pL of the formulation is injected using a 25G (Gauge) 11/2 needle through the tympanic membrane into the superior posterior quadrant. Formulations are del ivered using a perfusion pump at the rate of 2 pL/sec. Contact with the round window membrane is maintained for 30 minutes by placing the animal in a recumbent position.

During the procedure and until recovery, animals are placed on a temperature controlled (40 °C) heating pad until consciousness is regained at which time they are returned to the vivarium.

[00355] Perilymph sampling procedure - The skin behind the ear of anesthetized rats is shaved and disinfected with povidone-iodine. An incision is then made behind the ear, and muscles are carefully retracted from over the bulla. A hole is drilled through the bulla using a dental burr so that the middle ear is exposed and accessed. The cochlea and the round window membrane are visuali zed under a stereo surgical microscope. The basal turn of bulla is cleaned by using small cotton ball. A unique microhole is hand drilled through the bony shell of the cochlea (cochlear capsule) adjacent to tire round window. Perilymph (about 2 mΐ,) is then collected using a microcapillary_' inserted into the cochlear scalatympani. Perilymph samples are added to a vial containing 18 pL of acetonitrile and stored at -80 °C until analysis.

Analytical method

[00356] Concentrations of the GSK-3 modulator are determined using LC-MS.

[00357] Samples are extracted by protein precipitation, then vortexed and centrifuged. The supernatant is collected and diluted with two folds of water. Samples are analyzed by re versed phase HPLC.

[00358] Carbutamide is used as the internal standard (I.S.). Mass spectrometry is carried out using an AB Sciex API 5500 Q-Trap MS equipped with a Turbo lonSpray source. ESI mass spectra are acquired in positive MRM mode. Peak areas of the GSK-3 modulator are determined using Analyst 1.6 (Applied Biosystems) The calibration curves are obtained by fitting the peak area ratios of analyte/intemal standard (IS) and the standard concentrations using a quadratic regression analysis (1 /concentration²). Sample GSK-3 modulator concentrations are then interpolated using the equations derived from the calibration curves, using the peak area ratios derived from the software.

Example A2 - Effect of GSK-3 Modulators on Hair Cell Survival aud Morphological Preservation in Rat Cochlear Explant Cultures

Cochlear explants [00359] Postnatal (P2-P3) Sprague Dawiey rat pups (Charles River) of both sexes are anesthetized by isof!urane inhalation tor 2 minutes then decapitated. Temporal bones are removed and transferred to a cell culture dish with ice-cold Ca²VMg²⁺~contaming phosphate- buffered saline (PBS; Invitrogen). Under microscopic visualization, the cochlear capsule is carefully removed from the temporal bone using forceps and is transferred to a new cell culture dish containing ice-cold PBS. The cochlea is then dissected from the cochlear capsule using fine forceps. The stria vascularis is removed from the cochlear tissue and discarded. Dissected cochlear epithelia are transferred to permeable membrane inserts (Millicell Organotypic Cell Culture Inserts, Millipore) with up to 5 cochleae per membrane, inserts are then placed into 1 mL of a suitable cell culture media (such as, Dulbecco’s modified Eagle's medium [high glucose, giutamax, 25 mM HEPES] with 10% fetal bovine serum, 1% N2 supplement and 10 units/mL penicillin) in 35 mm sterile wells such that the explants could be bathed in media. The wells are then covered and explants are incubated for at least 18 hours in a humidified chamber at 37°C with 5% COi prior to treatment.

[00360] For evaluation of hair cell survival and morphological preservation, after the initial 18 hour culture period, explants adhered to the membrane inserts are rinsed twice in ceil culture media without penicillin, then are placed into new media without penicillin but including 50 pM of gentamicin to induce damage or in media without gentamicin for naive controls. Explants are maintained in control media or gentamicin-containing media for 16 hours to induce moderate hair cell damage, or for 24 hours to induce severe hair cell damage. After the prescribed amount of time, the explants are washed twice in penicillin-free media then are placed into new penicillin-free media containing an appropriate amount of any one of the GSK-3 modulators described herein. GSK-3 modulators stocks are made by solubilizing the compounds in DMSQ. Control explants are maintained in media containing an equivalent amount of DMSQ only. The cultures are incubated at 37°C for 24-72 hours before being fixed and processed for analysis.

Immunohistochemistry

[00361] Cochlear samples are immuno-stained for the protein Myosin Vila, a known marker of cochlear hair cells, as well as labeled with DAPI to identify cell nuclei. Cochlear samples are fixed in 4% paraformaldehyde after treatment then permeabilized in 0.5% Triton in PBS (PBST) for 1 hour, followed by overnight incubation at 4°C in Myosin Vila primary antibody (rabbit polyclonal antibody, 1 : 1000, Proteus Biosciences) in 10% goat serum (Sigma). Samples are then rinsed in PBST 2 times for 10 minutes each then incubated in alexa-488 conjugated goat anti-rabbit secondary' antibody (1:1000) for 2 hours at room temperature. Samples are rinsed 2 times in PBS then incubated in DAPI (1 :3Q00) in PBS tor 5 minutes prior to mounting on slides.

Analytical Method

[00362] The hair cell survival and morphological preservation are determined by evaluate tiie number and morphology of inner and outer hair cells at three different regions (basal, middle and apical) in each cochlear explant. For quantification, samples are imaged with an LSM880 laser-scanning eonfoeal microscope (Zeiss). The entire explant is simultaneously imaged for Myosin Vila and DAPI labeling (with a 488 and 350 wavelength laser, respectively) using a 20X objective in a 3-dimensional X-Y -Z plane in which the Z-plane consisted of a stack 25-50 pin in depth imaged at 2.5 pm intervals. The total length of the organ of Corti in each explant is determined and is divided into 4 equal length regions. Basal hair cells are evaluated at the region approximately 25% of the total cochlear length from the base: middle hair cells are evaluated at the region 50% of the length from tire base, and apical hair cells are evaluated at the 75% length region. Hair cell counts are obtained by placing a 200 pm length scale bar parallel to the rows of hair cells at each region and manually counting the total number of outer hair cells (OHCs) and inner hair cells (IHCs) within the 200 pm region. DAPI staining is analyzed as confirmation of the presence/absence of hair cell nuclei. Image analysis and quantification is performed using Zeiss Zen Blue software. Hie average number of inner and outer hair cells from the 3 regions is then determined for each cochlea and the group averages are calculated per condition.

Example B1 - Preparation of a Therinoreversible Gel Formulation Table A. Thermoreversible Gel GSK-3 Modulator Otic Formulation

[00363] An exemplary hatch of gel formulation containing, for example, 1.5% of a GSK-3 modulator described herein is prepared by dissolving Poloxamer 407 (BASF Corp.) in 50 mM Tris buffer and 77 niM Nad solution with a pH between 5.5-8.0 The appropriate amount of the GSK-3 modulator powder is added and the formulation is mixed until a homogenous suspension is produced. The mixture is maintained below room temperature until use.

Example B2 - In Vitro Comparison of Gelation Temperature

100364] The effect of Poloxarner 188 and any one of the GSK-3 modulators described herein on the gelation temperature and viscosity of Poloxarner 407 formulations is evaluated with the purpose of manipulating the gelation temperature

[Q0365] A 25% Poloxarner 407 stock solution in PBS buffer and Poloxarner 188NF from BASF are used. An appropriate amount of the GSK-3 modulator is added to the solutions described in Table B to provide a 2% formulation of the GSK-3 modulator

[00366] A PBS buffer (pH 7.3) is prepared by dissolving 805.5 mg of sodium chloride (Fisher Scientific), 606 mg of sodium phosphate dibasic anhydrous (Fisher Scientific), 247 mg of sodium phosphate monobasic anhydrous (Fisher Scientific), then QS to 200g with sterile filtered DI water.

Table B. Preparation of Samples Containing Poloxarner 407/Poloxamer 188

[00367] Gelation temperature of the above formulations are measured using procedures described herein.

[Q0368] An equation is fitted to the data obtained and is utilized to estimate the gelation temperature of F127/F68 mixtures (for 17-20% FI 27 and 0-10% F68).

Tg_6i= -1.8 (%F!27) + 1.3 (%F68) +53

[00369] An equation is fitted to the data obtained and can be utilized to estimate the Mean Dissolution Time (hr) based on the gelation temperature of F127/F68 mixtures (for 17-25% F127 and 0-10% F68), using results obtained in examples above: MDT — 0.2 Tgd) + 8 .

Example B3 - Preparation of Medium Chain Triglyceride Formulations [00370] Formulations 1 , 2, and 3 are prepared with the appropriate amounts of a GSK-3 modulator and medium chain triglycerides (CRODAMOL, GTCC-LQ-(MV), PhEur) as shown in the below' table (Table C).

[00371] The formulations are prepared by adding the target weight percentage of any one of the GSK-3 modulators described herein to the appropriate amount of medium chain triglyceride for a total volume of about 100 mb. The formulations are mixed until complete dissolution. The formulations are then sterilized by passing the formulations through 0.22 pm sterilizing grade filters under aseptic conditions. The sterilized solutions are then filled into vials or pre-filled syringes, which were then used to test the formulations.

Example B4 - Additional Preparation of Medium Chain Triglyceride Formulations - GSK-3 Modulator

[00372] Formulations 4, 5, and 6 are prepared with the appropriate amounts of the GSK-3 modulator and medium chain triglyceride (CRODAMOL, GTCC-LQ-(MV), PhEur) as shown in the below_' table (Table D).

[00373] The formulations are prepared by adding the target weight percentage of the GSK-3 modulator to the appropriate amount of medium chain triglyceride. The formulations are mixed until complete dissolution. The formulations are then sterilized by passing the formulations through 0.22 pm sterilizing grade filters under aseptic conditions. The sterilized solutions are then filled into vials or pre-filled syringes, which are then used to test the formulations.

Example B5 -Preparation of Medium Chain Triglyceride Formulations [00374] Medium chain triglyceride formulations are prepared with the appropriate amount of GSK-3 modulator, medium chain triglyceride, and viscosity modulating agents as shown in the below tables (Tables E~H).

GSK-3 Modulator Solution in MCT

[00375] The formulation is prepared by dissolving the appropriate amount of a GSK-3 modulator and one or more than one of the viscosity modulating agents, such as PVP, earhomer, and P407, in water for injection and sterile filtering the solution . The sterilized solution is lyophilized to form the dry cake. The appropriate amount of the dry cake is aseptically added to the appropriate amount of sterile filtered medium chain triglyceride. The formulation is mixed until a uniform suspension is achieved. If needed, the suspension is homogenized to reduce the particle size to below 10 microns (D50). Then the appropriate amoun t of sterilized silicon dioxide is added to the suspension, if needed. The final formulation is mixed until a uniform suspension is achieved and then is filled into vials. GSK-3 Modulator Suspension in MCT and Si02

[Q0376] Tire formulation is prepared by adding the target weight percentage of a GSK-3 modulator that has been micronized and gamma irradiated to the appropriate amount of medium chain triglyceride that has been sterilized via filtration. The formulation is mixed until a uniform suspension is formed. The appropriate amount of Si02 is then added and is mixed until uniform. The resulting uniform suspension is then filled into vials.

GSK-3 Modulator Nano-Suspension in MCT and Si02

[00377] The formulation is prepared by adding the target weight percentage of a GSK-3 modulator that has been micronized and gamma irradiated to the appropriate amount of medium chain triglyceride that has been sterilized via filtration. The formulation is mixed until a uniform suspension is formed. Ball milling equipment is then used to reduce the particle size to below 0.2 p . The appropriate amount of Si02. is then added and is mixed until uniform. The resulting uniform suspension is then filled into vials.

Table E

Table F

Table G

Table H

Example (T — Effects of GSK-3 inhibitors on Hair Cell Formation in Rat Cochlear Explant Cultures

Cochlear explants

[00378] Postnatal (P2-P3) Sprague Dawley rats pups (Charles River) of both sexes were anesthetized by isoflurane inhalation for 2 minutes then decapitated. Temporal bones were removed and transferred to a cell culture dish with ice-cold Ca^2÷/Mg² -containing phosphate- buffered saline (PBS; Invitrogen). Under microscopic visualization, the cochlear capsule was carefully removed from the temporal bone using forceps and transferred to a new cell culture dish containing ice-cold PBS. The cochlea was then dissected from the cochlear capsule using fine forceps lire stria vascularis was removed from the cochlear tissue and discarded. Dissected cochlear epitheha were transferred to permeable membrane inserts (Milhcell Organotypic Cell Culture inserts, Millipore) with up to 5 cochleae per membrane, inserts were then placed into 1 inL of cell culture media (Dulbeceo s modified Eagle’s medium [high glucose, glutamax, 25 mM HEPES] with 10% fetal bovine serum, 1% N2 supplement and 10 units/mL penicillin) in 35 mm sterile wells such that the explants could be bathed in media. The wells were then covered and explants were incubated tor at least 18 hours in a humidified chamber at 37°C with 5% CO? prior to treatment.

[00379] For evaluation of hair cell formation, after the initial 18 -hour culture period, explants adhered to the membrane inserts were rinsed twice in cell culture media without penicillin, then were placed into ne media without penicillin but including 50 mM of gentamicin to induce damage or in media without gentamicin for naive controls. Explants were maintained in control media or gentamicin-containing media for 16 hours to induce hair cell damage, after which the explains were washed twice in penicillin-free media then were placed into new' penicillin-free media containing 3 or 10 mM of CHIR99Q21. CHIR99021 stock solution was made by solubilizing the compound in DMSO. Control explants were maintained in media containing an equivalent amount of DMSO only. The cultures were incubated at 37°C for 24-72 hours before being fixed and processed for analysis. Immunohistochemistry

[00380] Cochlear samples were immuno-stained for the protein Myosin Vila, a known marker of cochlear hair cells, as well as labeled with DAPI to identify cell nuclei. Cochlear samples w ere fixed in 4% paraformaldehyde after treatment then permeahilized in 0.5% Triton in PBS (PBS^'T) for 1 hour, followed by overnight incubation at 4°C in Myosin Vila primary antibody (rabbit polyclonal antibody, 1 :1000, Proteus Biosciences) in 10% goat serum (Sigma). Samples wure then rinsed in PBST 2 times for 10 minutes each then incubated m alexa-488 conjugated goat anti-rabbit secondary antibody (1: 1000) for 2 hours at room temperature. Samples were rinsed 2 times in PBS then incubated in DAPI (1:3000) in PBS for 5 minutes prior to mounting on slides.

A nalytical Method Q0381] The number of hair cells was determined by counting the number of inner and outer hair cells at three different regions (basal, middle and apical) in each cochlear explant. For quantification, samples were imaged with an LSM880 laser-scanning confocal microscope (Zeiss). The entire explant was simultaneously imaged for Myosin Vila and DAPI labeling (with a 488 and 350 wavelength laser, respectively) using a 20X objective in a 3 -dimensional X-Y-Z plane in which the Z-plane consisted of a stack 25-50 pm in depth imaged at 2.5 pm intervals. The total length of the organ of Corti in each explant was determined and was divided into 4 equal length regions. Basal hair cells were counted at the region approximately 25% of the total cochlear length from the base; middle hair cells were counted at the region 50% of the length from the base, and apical hair cells wure counted at the 75% length region. Hair cell counts were obtained by placing a 200 pm length scale bar parallel to the rows of hair cells at each region and manually counting the total number of outer hair cells (OHCs) and inner hair cells (IHCs) within the 200 pm region. DAPI staining was analyzed as confirmation of the presence/absence of hair cell nuclei. Image analysis and quantification was performed using Zeiss Zen Blue software. The average number of inner and outer hair cells from the 3 regions was then determined for each cochlea and the group averages were calculated per condition.

Results [Q0382] Exposure of rat cochlear explants to CHIR9902I GSK-3 inhibitor resulted in an increase in the number of inner and outer hair cells present throughout the cochleae under ail tested conditions (naive undamaged and aminoglycoside damaged) when compared to untreated controls or compared to gentamicin damage alone. Tire effects of CHIR99021 were evaluated at 3 and 10 mM.

[00383] Undamaged explants : FIG. 2 shows an increase in inner and outer hair cells in undamaged naive cochlear explants following exposure to CHIR9902.1. The image at right shows the increase in hair cells after CHIR99021 treatment in an «undamaged cochlear explant compared to a naive untreated explant (left image). Hair cells are stained with Myosin Vila in red.

[00384] Aminoglycoside damaged explants. FIGs. 3A and 3B show an increased number of inner and outer hair cells in aminoglycoside damaged cochlear explants following exposure to CHIR99Q21. FIG. 3A shows representative images showing thhe increase m hair cells after CHIR99021 treatment in a ggentamicin damaged cochlear explant (right) compared to an explant exposed only to gentamicin (middle). Hair cells are stained with Myosin Vila in green. FIG. 3B show's quantification of the increased nummberr of hair cells in gentamicin (‘Vent ’) damaged explants exposed to 3 mM CHIR99021 commpared to undamaged naive or untreated gentamicin-damaged explants. Inner hair cells (IHCs) are shown in the upper panel and outer hair cells (OHCs) are shown in the lower panel. For each explant, the number of hair cells in the basal and middle regions was averaged and is plotted as the number of hair cells per 200 pM length of the cochlea. Error bars represent standard error of the mean. Example C2 — GSK3 Inhibitors Formulation Pharmacokinetics GSK3 inhibitors in P407

[Q0385] A CHIR99021 formulation was prepared using the following procedure: A 16% w/w stock solution of pofoxamer 407 (P407) was prepared by slowly adding it to a cold buffer solution (final concentration in stock solution of 50 mM tromethamine, 77 mM sodium chloride, pH 7.7). Tins solution was sterilized by filtration. Then CHIR99021 was suspended with an appropriate amount of poloxamer 407 solution to reach concentrations of 0.15%, 0.5%, 1.5%, and 3.0% w/w.

Pharmacokinetics

[00386] Female rats (Charles River) weighing 200-300 g of approximately 12-16 weeks of age served as subjects (N = 4 per group). Prior to any procedures, animals were anesthetized using a combination of xylazine (10 mg/kg) and ketamine (90 mg/kg) for up to an hour via the intraperitoneal route. If needed, an intraoperative booster was administered intraperitoneal representing a one-tenth of the original dose.

[00387] Jntratympanic injection : Each animal was positioned so that the head was tilted at an angle to favor injection towards the round window niche. Briefly, under visualization with an operating microscope, 20 pL of the formulation was injected using a 25G (Gauge) 11/2 needle through the tympanic membrane into the superior posterior quadrant. Formulations were del ivered using a perfusion pump at the rate of 2. pL/sec. Contact with the round window membrane was maintained for 30 minutes by placing the animal in a recumbent position. During the procedure and until recovery, animals were placed on a temperature controlled (40 °C) heating pad until consciousness was regained at which time they were returned to the vivarium.

[00388] Perilymph sampling procedure: The skin behind the ear of anesthetized rats was shaved and disinfected with povidone-iodine. An incision was then made behind the ear, and muscles were carefully retracted from over the bulla. A hole was drilled through the bulla using a dental burr so that the middle ear was exposed and accessed. The cochlea and the round window membrane were visualized under a stereo surgical microscope. The basal turn of bulla was cleaned by using small coton ball. A unique microhole was hand drilled through the bony shell of the cochlea (cochlear capsule) adjacent to the round window. Perilymph (about 2 pL) was then collected using a microcapiilary inserted into the cochlear seala tympani. Perilymph samples were added to a vial containing 18 pL of acetonitrile, stored at -80 °C until analysis.

A nafytical method

[00389] Concentrations of CHIR99021 w'ere determined using LC-MS.

[00390] FIG. 4 show's perilymph concentrations of CH1R99021 following administration of formulations comprising various concentrations of CHIR99021 in 16% P407. Data are presented as mean ± 8EM of arbitrary units.

Example C3 — Effects of GSK3 inhibitors on neural protection, SGN density, 8GM branching, myeiinatiion, and Schwann cell formation/survival in rate cochlear explant cultures

Coclear Explants

[00391] Postnatal (P0-P5) Sprague Dawley rat pups (Charles River) of both sexes were anesthetized by isofiurane inhalation for 2 minutes then decapitated. Temporal bones were removed and transferred to a cell culture dish with ice-cold Ca² vMg²⁺~containing phosphate- buffered saline (PBS; Invitrogen). Under microscopic visualization, the cochlear capsule w'as carefully removed from the temporal bone using forceps and transferred to a new cell culture dish containing ice-cold PBS. The cochlea was then dissected from the cochlear capsule using fine forceps. The stria vascularis was removed from the cochlear tissue and discarded. Dissected cochlear epithelia were transferred to permeable membrane inserts (Millicell Organotypic Cell Culture inserts, Millipore) with up to 5 cochleae per membrane, inserts were then placed into 1 mL of cell culture media (Duibecco’s modified Eagle’s medium [high glucose, glutamax, 25 mM HEPES] with 2-10% fetal bovine serum,

1 % N2 supplement and 10 units/mL penicillin) in 35 m sterile wells such that the explants could be bathed in media. The wells were then covered and explants were incubated for at least 12 hours in a humidified chamber at 37°C with 5% CO2 prior to treatment.

[00392] For evaluation of drag effects on spiral ganglion neurons, neural or glial precursor cells, Schwann cells, or myelin formation, after the initial 18-hour culture period, explants adhered to the membrane inserts were rinsed twice in cell culture media without penicillin, then were placed into new media without penicillin but including 4-50 mM of cisplatin to induce cell or myelin damage or in control media without cisplatin for 24 hours. After 24 hours, explants were placed into fresh media with or without cisplatin and containing 3 mM to 25 mM of a GSK3 inhibitor (such as CMR99021, ARA-014418, or LiCi). CHIR99021 and ARA-014418 stock solution was made by solubilizing the compounds in DMSG; LiCl stock solutions were made by solubilizing compound in PBS. Control explants were maintained in media containing an equivalent amount of DMSO or PBS, respectively, with or without cisplatin. The cultures were incubated at 37°C for 24-72 hours before being fixed and processed for analysis.

Spiral Ganglion Dissection and Culture

[Q0393] Postnatal Sprague Dawley rats (P2-8) of both sexes were anesthetized with isoflurane and decapitated. Temporal bones were removed and transferred to a cell culture dish with ice-cold Ca²⁺/Mg²⁺-containing phosphate-buffered saline (PBS; Invitrogen). Under microscopic visualization, the cochlear capsule was carefully removed from the temporal bone using forceps and transferred to anew cell culture dish containing ice-cold PBS. The cochlea was then dissected from the cochlear capsule using fine forceps. The stria vascularis and the organ of Corti were removed from the cochlear tissue, and the spiral ganglion neurons were subsequently detached from the modiolus. Hits strand, containing spiral ganglion neurons, was transferred to a 1.5 ml. mserocentrifuge tube containing 0.5 mL ice- cold Ca²⁺/Mg²⁺-free Hank’s balanced salt solution (HBSS; invitrogen). Once ~12 of these strands (representing 6 animals) were collected in cold HBSS, enzymatic and mechanical dissociation proceeded as described below. Alternatively, some dissected SGN strands were plated without dissociation into culture wells containing the same culture media and incubated and were treated with GSK3 inhibitors as whole explants as described below. [00394] For SGN dissociation, 0.5 mL of warm (37°C) FIBSS mixed with 1 mg/niL Thermolysin (Promega) was added to the spiral ganglion collection (for a final concentration of 0.5 mg/mL Thermolysin in a volume of ~1 mL) and incubated at 37°C for 30-35 minutes. The cells were then briefly centrifuged, the supernatant discarded and the cells were washed twice with culture medium (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum; see below). The cells were resuspended in 1 ml of culture medium and mechanically dissociated with a 1000 mΐ pipette for 4 triturations. After 4 triturations, the cells were briefly centrifuged and the supernatant applied to a 40 pm cell strainer (Millipore). This was repeated until the tissue w'as fully dissociated with no visible cell clusters remaining.

Survi ving ceils were counted using the Countess P (Thermo Fisher) using trypan blue, and then seeded into a 96-well plate (pre-coated with poly-L-omithine and laminin (Coming) and then incubated for 3-4 hours with 10 ug/mL poly-L-lysine) at a density of 1 .4 x 10⁴ cells per w'ell.

[Q0395] Treatments with GSK3 inhibitors were conducted for 4 days at 37°C. Immediately after seeding, test agents were added to the culture medium prepared at iOx concentration and the volume was then diluted 10-fold when added to the seeded cells. The next day, the adhered ceils were washed once with serum-free culture medium and then fresh serum-free medium was applied IOx concentrated treatments were again added to the cells with a 10- fold dilution. The cultures were kept in an incubator for an additional 3 days before being fixed, stained, and imaged.

Imrnunohistochemistrv

[00396] Cochlear samples were immuno-stained for various combinations of the following proteins: Myosin Vila (a known marker of cochlear hair cells); b-tubulm III or neurofilament (known markers of spiral ganglion neurons); Sox 10 (known marker of glia and Schwann ceils); MBP, MPZ, Cellmask or anti-myelin 2.B5 (myelin markers); and DAPI to identify ceil nuclei. Cochlear samples or dissociated cells were fixed in 4% paraformaldehyde after treatment then permeabihzed in 0.5% Triton in PBS (PBST) for 1 hour, followed by incubation in primary antibodies (overnight at 4°C or for 3 hours at room temperature). Explants were then rinsed in PBST two times for 10 minutes each then incubated in Alexa f!uorescently conjugated species-specific secondary antibodies (Invitrogen) tor 2 hours at room temperature or overnight at 4°C. Samples were rinsed 2 times in PBS then incubated in DAPI (1 :3000) diluted in PBS for 5 minutes prior to mounting on slides using Fluoromount- G mounting medium; alternatively, samples could be mounted in DAPI Fluoromount-G. aPCR

[00397] After GSK3 treatment, cell culture media is removed from SGN explants or dissociated SGNs and the remaining cells are lysed, mRNA was extracted, and q-PCR is then performed for the myelin proteins MPZ or PMP22 using rat-specific primers.

BDNF ELISA Q0398] Dissociated SGNs are seeded at a density of 2e4 celis/well in a 96 well plate and maintained in 100-2Q0pLs of cell culture media with or without GSK3 inhibitors tor 96 hours. The culture media is then pipetted off without disturbing the cells and the media was subjected to ELISA using anti -BDNF antibodies.

Analytical Method

[00399] The effects on SGNs, Schwann cells or myelination was determined by qualitatively evaluating each irnmunostained cochlear explant using a standard fluorescent microscope or by analyzing a z series of images obtained with a confocal laser-scanning microscope.

[00400] For quantitative analysis, the number of SGN dendritic fibers, SGN soma, glial or neural precursor cells or Selrwann cells in the different regions (basal, middle and apical) of each cochlear expiant or SGN preparation w¾s determined. For quantification of dissociated SGNs, the number of SGNs (identified by neurofilament staining) surviving in each well was counted. For quantification in whole cochlear explants, samples were imaged with an LSM880 laser-scanning confocal microscope (Zeiss) as described below'. The entire explant was simultaneously imaged for the various cell type-specific markers a 20X, 40X, or 63X objective in a 3 -dimensio al X-Y-Z plane in which the Z-plane consisted of a stack 15-60 mih in depth imaged at 0.5-5.0 pm intervals. The total length of the organ of Corti in each explant was determined and was divided into 4 equal length regions. Basal cells were counted at the region approximately 25% of the total cochlear length from the base; middle cells were counted at the region 50% of the length from the base, and apical ceils were counted at the 75% length region. Cell counts were obtained by digitally placing an appropriately sized box onto the image and manually counting the total number of cells within the 200 pm region. Image analysis and quantification was performed using Zeiss Zen Blue software. The average number of cells from the 3 regions w'as then determined for each cochlea and the group averages were calculated per condition.

Results [0Q401] Exposure of rat cochlear explants to G8K3 inhibitors (including CHIR-99021, ARA-014418, 8B216763, CHIR-98014 and TWS-119) resulted in an increase in SGN density, neurite branching, neural/glial precursor cells, Schwann cells, and ravelin protein expression throughout the cochlea in naive undamaged explants when compared to untreated controls. G8K3 inhibitors also increased neuron survival in dissociated SGN explants. in addition, pre-treatment of cochlear explants with CH1R99021 followed by co-incubation in cisplatin resulted in the protection of hair cells and spiral ganglion neurons from cisplatin- induced damage compared to cisplatin damage alone. The effects of G8K3 inhibitors were evaluated at doses between 1.0 and 50 mM of undamaged rat cochlear explants to GSK3 inhibitors (including CHTR99021 and ARA-014418) resulted in an increase in SGN density, neurite branching, neural/ghal precursor cells, Schwann cells, and myelin protein expression throughout the cochlea compared to untreated controls in addition, pre-treatment with CH1R99021 followed by co-incubation in cisplatin resulted in the protection of hair cells and spiral ganglion neuron density from cisplatin-snduced damage compared to cisplatin damage alone. The effects of CHIR99021 were evaluated at doses between 3 and 50 mM. ARA- 014418 was evaluated at 100 mM.

[Q04Q2] FIG. 5 shows an increase in SGN density and neurai/glial precursor cells m undamaged naive cochlear explants following exposure to CHTR99021. The images show tiie increase in neuronal density and ectopic neural/glial precursor cells after CHIR99Q21 (“CHIR”) treatment alone or with VPA in undamaged cochlear explants (middle and right, respectively) compare to naive untreated explants (left). Cells were stained with neurofilament. Asterixes indicate ectopic precursor cells.

[00403] FIG. 6 shows an increase in SGN density, Schwann ceils and myelin protein expression in undamaged naive cochlear explants following exposure to CHIR99Q21. The images show the increase in neuronal density (immunostained for neuroftlament, red), Schw'ann cells (immunostained tor SoxiO, green) and myelin protein (nnmunostained for MBP, blue) after 10 or 50 mM CHIR99021 (/‘CHIR ’) treatment for 72. hrs in undamaged cochlear explants (middle and right columns, respectively) compared to naive untreated explants (left column). The top row shows all antibody staining in color, the bottom rows show' individual channels in white as labeled.

[Q04Q4] FIG. 7 shows dose responsive increase in SGN density in undamaged naive cochlear explants following exposure to ARA-014418. Representative images showing the increase in neuronal density (immunostained for neurofi lament, white) after 1.0, 10 or 25 mM ARA-014418 treatment for 96 hrs in undamaged cochlear explants compared to naive untreated explants (left column).

[00405] FIG. 8 shows increase in SGN density in undamaged naive cochlear explants following exposure to various GSK3 inhibitors. Representative images showing the increase in neuronal density (immunostained for neurofilament, white) after treatment with CHIR- 99021 (10 mM), TWS-119 (25 mM) or SB216763 (1.0 mM) for 96 hrs in undamaged cochlear explants compared to naive untreated explants (left column) .

[00406] FIG. 9 sho 's increase in SGN density in undamaged naive cochlear explants following exposure to various GSK3 inhibitors. Representative images showing the increase in neuronal density (immunostained for neurofiiament white) after treatment with CH1R- 99021 (10 mM), TWS-119 (25 mM) or SB216763 (1.0 mM) for 96 hrs in undamaged cochlear explants compared to naive untreated explants (left column).

[00407] FIG. 10 shows the increase in SGN density and Schwann cells in undamaged naive cochlear explants following exposure to various GSK3 inhibitors. (÷+) or (+) denotes the relative degree of increase in SGN density or Schwann cell density observed.

[00408] FIG. 11 show's increase in neuron survival in dissociated spiral ganglion neuron cultures following exposure to various GSK3 inhibitors. (A) Representative images showing the increase in neuron survival (immunostained for neurofiiament, white) in dissociated SGN cultures after treatment with a GSK3 inhibitor (ARA-014418, 1.0 mM) compared to an untreated control (naive). (B) Graph showing the quantified increase in survival of dissociated SGNs (quantified as the number of survi ving neurit.es per well) after treatment with ARA-014418 (ARA, 1.0 mM) or SB2I6763 (SB, 25 pM) compared to untreated SGNs (naive, 0.0 mM). Error bars represent S.E.M.

[004Q9] FIG. 12 shows preservation of HC and SGN density cisplatin damaged cochlear explants when treated with GSK3 inhibitor CHIR9902I. Representative images showing the preservation of neuronal density and protection of hair cells after pre-treatment with CH1R99021 followed by co-treatment with 10 mM cisplatin (Cis) in cochlear explants for 72 hrs. Naive explant without cisplatin treatment (left), cisplatin only (middle left) compared to treatment with the GSK3 inhibitor CHIR99021 at 10 pM (middle right) or 50 mM (right). Hair cells are immunolabeled with myosin7a (Myo7a, green) and spiral ganglion neurons are immunoiabeled with neurofiiament (red).

Example C4 - Effects of GSK3 inhibition on BDNF expression in cochlear explants [00410] Formulations comprising selected G8K3 inhibitors (CHIR99021, Li Cl, ARA- 014418, and SB216763) are prepared. Various doses of the formulations are applied to dissociated SGNs in vitro.

[00411] Methods and Materials

[00412] Cochlea from p2 rat pups were harvested and transferred to a cell tak coated mesh insert. For the first day of culture, cochlea were treated with DMEM media supplemented with 10% FBS, 1%N2, 25ug/mL Amphotericin B, and 50.000U/L Penicillin. Following the acclimation day explains were transferred to treatment DMEM media supplemented with 10% FBS and 1% N2. Treatment media also contained a range of different concentrations of the GSK3 inhibitor CHTR 99021 or DMSO as a vehicle control. Cochlear explants were treated continuously for 48-96 hours after which the media and tissue were collected separately in pre-weighed tubes.

[00413] For determining BDNF levels in rat cochlear explants, tissue specimens were removed from ~80°C storage on the day of analysis and homogenized in a solution of freshly prepared extraction buffer with the addition of protease inhibitors. Homogenates were centrifuged to remove bone and cellular debris and the resulting supernatant analyzed by ELISA. Cell culture media samples were also analyzed by ELISA using a cell culture media specific standard curve.

[00414] Result

[00415] As shown in FIG. 13, treatment of cochlear explants with GSK3 inhibitors, such as CHIR 99021, increased BDNF expression, as observed upon quantification using ELIIIS with anti -BDNF antibodies.

Example C5 - Effects of GSK3 inhibitors on SGN myelin at ion or myelin markers in P8~ P10 SGN explants

[00416] Formulations comprising selected GSK3 inhibitors (CHHIR99021, LiCl, ARA- 014418, and SB216763) are prepared. Various doses of the formulations are administered to SGN explants in vitro. Increased staining for myelin is observed by immunohistochemistry using antibodies against myelin proteins. Increased expression of peripheral myelin markers such as MPZ and PMP22 is evaluated by qPCR or Western blot.

Example C6 - Evaluation of ectopic neura!/glial precursor cel! proliferation in cochlear explants

[00417] Formulations comprising selected GSK3 inhibitors (CHHIR99021, LiCl, ARA- 014418, and SB216763) are prepared. Various doses of the formulations are administered to cochlear expiants or SGN explants. Ectopic precursor cells are positive tor proliferation markers such as BrdU/EdU or Ki67.

Example C7- Effect of GSK3 modulators on supporting ceil or Schwann cel! proliferation in rat cochlear explant cultures

[00418] Formulations comprising selected GSK3 inhibitors (CHHIR99G21, LiCl, ARA- 014418, and 8B216763) are prepared. Various doses of the formulations are applied to cochlear explant cultures in vitro. Proliferative supporting cells or Schwann cells are positive for proliferation markers such as BrdU/EdU or Ki67.

Example €8 - Effect of G8K-3 Modulators on Supporting Cell Formation in Rat Cochlear Explant Cultures

[00419] Formulations comprising selected GSK3 inhibitors (CHHIR99Q21, LiC!, ARA- 014418, and SB216763) are prepared. Various doses of the formulations are applied to cochlear explant cultures in vitro. De novo formation of supporting cells is confirmed by quantifying the number of supporting cells in cochlear explants or by qpCR for expression of supporting cell genes. Supporting cells are identified by the expression of supporting cell markers such as Sox2, FGFR3, Lgr5, S100A, p75, among others.

Example C9 - Effect of GS&-3 Modulators on Supporting Cell Transdifferentiation in Rat or Mouse Cochlear Explaut Cultures

[00420] Formulations comprising selected GSK3 inhibitors (CHHIR99G21, LiCl, ARA- 014418, and 8B216763) are prepared. Various doses of the formulations are applied to cochlear expfant cultures in vitro. Transdifferentiation of supporting cells into hair cells is determined based on qualitatively or quantitatively evaluating the residual Sox2 expression in hair cells. Alternatively, transdifferentiation can be evaluated using cochlear explants derived from reporter mice with specific genetic recombinations such that only support cell lineages will express either tdTomato or GFP, or some other endogenous label. Exp3 anted cochleae from these mice can be used to lineage trace supporting cells such that any expression of GFP or tdTomato in a hair ceil is indicative of that cell having transdifferentiated from a supporting cell.

Example CIO - Effect of GSK-3 Modulators on synaptogenesis and/or preservation of synaptic connections in rat or mouse cochlear exp!ant cultures [Q0421] Formulations comprising selected GSK3 inhibitors (CHH1R99021, LiCl, ARA- 014418, and SB216763) are prepared. Various doses of the formulations are administered to either naive cochlear explants or explants treated with excitotoxic agents to damage the normal synapses. Increased synaptogenesis or synaptic preservation following treatment or following excitotoxic damage then treatment is determined by quantifying the number of ribbon synapses per inner hair cell using presynaptic ribbon markers such as CtBP2 and post- synaptic markers such as PSD-95, or GluR2, among others

[00422] Two G8K3 inhibitors were tested for their ability to affect the connections between afferent fibers of type 1 spiral ganglion neurons (SGNs) and inner hair cells in rat cochlear explants. Compared with untreated controls, both CHIR9902I (3 uM) and GSK-22 (100 nM) increased th e number of type 1 SGN afferent fibers synapsing with inner hair ceils in neonatal cochlear explants. QQ423] Methods and Materials

[00424] Preparation of cochlear ex plants: temporal bones were obtained from postnatal day 3 Sprague-Dawley rat pups and further dissected in HBSS The cochlear capsule was cut open, the modiolus was excised, and the remaining tissue was placed on a porous cell culture insert (Miilipore, catalog # P1CMORG50) in lmL of media. The media consisted of DMEM (Gibco, catalog #10564), 10% FBS, N2 supplement, penicillin, amphotericin B (Gibco, catalog #15290018). CHIR99021 (3 uM) or GSK-22 (100 nM) was added to the culture medium and explants were then cultured for 72h prior to fixation (4% PFA) and immunostaining for Myosin7a (Proteus, catalog # 25-6790), CtBP2 (BD Biosciences, catalog# 612044), PSD-95 (Miilipore, catalog # MABN68) and neurofilament (Abeam, catalog# ab 134459).

[00425] Immunostaining and image analysis: Explants were immunostained with Myo7A (blue) to identify hair cells and neurofilament (yellow) to label SGNs. The number of type I SGNs synapsing with inner hair cells w as calculated by counting all of the fibers approaching the hair cells and then subtracting those that continue to the outer hair cell layer. The pre synaptic puncta were identified by an antibody that labels CtBP2 (green). The postsynaptic puncta were identified by an antibody that labels PSD-95 (red).

[00426] Result

[00427] As shown in FIGs. 14 and 15, GSK23 inhibitors, such as CHIR99021 and GSK-22, increased synaptogenesis or synaptic preservation following treatment or following excitotoxic damage then treatment, as determined by quantifying the number of ribbon synapses per inner hair cell using presynaptic ribbon markers such as QBP2 and post synaptic markers such as PSD-95, or GluR2, among others.

[00428] While preferred embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Various alternatives to the embodiments described herein are optionally employed. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WHAT IS CLAIMED IS:

1. An otic formulation comprising a therapeutically effective amount of a GSK-3 inhibitor and an auris-acceptahle vehicle, wherein the amount of the GSK-3 inhibitor released into the inner ear is sufficient to: increase hair cell numbers; increase SGN density and/or branching; increase the number of Schwann cells; increase myelin protein expression; preserve hair cells upon drag-induced ototoxcity; or combinations thereof, and wherein the amount of the GSK-3 inhibitor released into the inner ear is below a toxicity exposure limit.

2. lire otic formulation of claim 1, release of the GSK-3 inhibitor above the toxicity exposure limit would result in at least one of: decreased SGN density and/or branching; decreased number of Schwann cells; decreased myelin protein expression; and decreased hair cell preservation upon drag-induced ototoxcity_'.

3. The otic formulation of claim 1, wherein the amount of the GSK-3 inhibitor released into the inner ear is sufficient to increase inner ear BDNF expression and wherein the toxicvity exposure limit would result in decreased inner ear BDNF expression.

4. The otic formulation of claim 1, w herein the auris-acceptahle vehicle is a thermoreversible gel .

5. The otic formulation of claim 4, wherein the auris-acceptable gel is capable of being injected by a narrow gauge needle or cannula through the tympanic membrane.

6. The otic formulation of claim 5, wherein the otic formulation has an osmolarity from about 250 to about 320 mOsm/L.

7. The otic formulation of claim 6, wherein the otic formulation has a gelation temperature from about 19°C to about 42°C.

8. The otic formulation of claim 7, wherein the otic formulation has a pH from about 7.0 to about 8 0

9. The otic formulation of claim 4, wherein the otic formulation comprises from about 14 wt% to about 25 wt% poloxamer 407. 10 The otic formulation of claim 4, wherein the otic formulation comprises from about 15 wt% to about 18 wt% poloxamer 407.

11 The otic formulation of claim 1 , wherein the auris-acceptable vehicle comprises triglycerides comprising medium chain fatty acids.

12 The otic formulation of claim 11, wherein the triglycerides are derived from glycerol and medium chain fatty acids.

13. The otic formulation of any one of claims 12, wherein the medium chain fatty acids are caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylenic acid (undec-10-enoic acid), lauric acid (dodecanoic acid), or any combinations thereof.

14. The otic formulation of any one of claims 20-26, w herein the otic formulation comprises at least about 50% by weight of the triglycerides.

15. The otic formulation of any one of claims 20-26, wherein the otic formulation comprises from about 85% to about 99.99% by weight of the triglycerides.

16. The otic formulation of any one of claims 20-39, wherein the otic formulation has a viscosity from about 10 cP to about 500 cl⁵.

17 The otic formulation claim 16. wherein the otic formulation is free or substantially free of water, C1-C6 alcohols or C1-C6 glycols, C1-C4 alcohols or C1-C4 glycols, or any combination thereof.

18 The otic formulation of claim 1, wherein the GSK-3 inhibtor is multiparticulate.

19 The otic formulation of claim I, wherein the GSK-3 inhibtor is dissolved in the otic formulation. 0 The otic formulation of claim 1, wherein the GSK-3 inhibitor is not GSK-22.

21 The otic formul ation of claim 20, wherein the GSK3 inhibitor is not:

Formula I

22. The otic formulation of claim 21, wherein the GSK3 inhibitor is not any one of:

2-pyrimidinylaminoethylamino-2-pyridinyl containing compound;

3-{pyridin-2.-yl)~ 1 H-indol -2-ol containing compound;

2-pyrimidinylaminoetliylamino-2-pyridyl containing compound;

N-( lH-pyrazol-4-yl)-nicotinamide containing compound

2, 4-dichlorophenyl-5-(lH-imidazol-2-yl)-2-pyrimidinylaminoethylammo-2 -pyridine compound;

2.4-dichiorophenyl-5-(lH-imidazol-2-yl)-2-pyrimidinylaminoethyiamino-3-pyridine containing compound;

3-{9 fluoro-2-(piperidine-l-carboiiyl)-l ,2,3.4-tetrahydro~[I ,4]diazepino[6,7, 1- hi]indol-7-y3)-4-(imidazo[l,2-a]pyridin-3-yl)-lH-pyrrole-2,5-dione; and

1.2.3.4-tetrahydro-j l,4]diazepino 6,7, 1 -hijindolyl containing compound.

23. Hie otic formulation of claim 1, for use in the treatment of an otic disease or condition associated with decreased SGN density and/or branching; decreased number of Schwann cells; decreased Schwann cells or oligodendrocyte survival or proliferation; decreased SGN or auditor_}' nerve myelin sheaths; decrease expression of myelin-promoting genes; decreased myelin protein expression; decreased number of neural/glial precursor cells; decreased neural/glial proliferation; decreased number of hair cell upon drug-induced ototoxcity; or combinations thereof.

24. The otic formulation of claim 1, for use in the treatment of an otic disease or condition associated with decreased inner ear BDNF expression

25. The otic formulation of claim 1, for use in the treatment of hearing loss.