AU2020230269B2 - Auris formulations for treating otic diseases and conditions - Google Patents

Abstract

A URIS FORMULATIONS FOR TREATING OTIC DISEASES AND CONDITIONS ABSTRACT Disclosed herein are compositions and methods for the treatment of otic disorders with immunomodulating agents and auris pressure modulators. In these methods, the auris compositions and formulations are administered locally to an individual afflicted with an otic disorder, through direct application of the immunomodulating and/or auris pressure modulating compositions and formulations onto the auris media and/or auris internal target areas, or via perfusion into the auris media and/or auris internal structures. AH26(25917314 1):RTK

Description

A URIS FORMULATIONS FOR TREATING OTIC DISEASES AND CONDITIONS RELATED APPLICATIONS

10001] The present application is a divisional application of Australian Patent Application No. 2018203651, which in a divisional of Australian Patent Application No. 2016228201, which is a divisional of Australian Patent Application No. 2013254940, which is a divisional of Australian Patent No. 2009239429, which is the national phase of International Application No. PCT/US2009/041320, which in turn claims the benefit of priority to U.S. Provisional Application Ser. Nos. 61/087,905, filed on August 11, 2008, 61/055,625 filed on May 23, 2008, 61/086,105 filed on August 04, 2008, 61/073,716 filed on June 18, 2008 , 61/140,033 filed on December 22, 2008, 61/127,713 filed on May 14, 2008, 61/101,112 filed on September 29, 2008, 61/094,384 filed on September 04, 2008, 61/074,583 filed on June 20, 2008, 61/060,425 filed on June 10, 2008, 61/048,878 filed on April 29, 2008, 61/046,543 filed on April 21, 2008, 61/076,567 filed on June 27, 2008, 61/076,576 filed on June 27, 2008, 61/160,233 filed on March 13, 2009, 61/086,094 filed on August 04, 2008, 61/083,830 filed on July 25, 2008, 61/083,871 filed on July 25, 2008, 61/087,951 filed on August 11, 2008, and 61/088,275 filed on August 12, 2008. The contents of each of the aforementioned applications are incorporated by cross reference in their entireties herein. BACKGROUND OF THE INVENTION

[0001a] Any reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge. 10002] Described herein are formulations for enhanced drug delivery into the external, middle and/or inner ear, including the cochlea and vestibular labyrinth; preferably with little or no systemic release of the drug. SUMMARY OF THE INVENTION 10002a] The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required. 10002b] In a first aspect, the invention relates to an intratympanic composition for the treatment or prevention of hearing loss, wherein the intratympanic composition comprises an auris sensory cell modulator and an auris acceptable gel, wherein the auris sensory cell modulator is a neurotrophin, wherein the auris acceptable gel is a thermoreversible gel comprising from 14% to 19% by weight of a poloxamer, and wherein the poloxamer is poloxamer 407. 10002c] In a second aspect, the invention relates to a method of treating or preventing hearing loss in a subject, the method comprising administering a therapeutically effective amount of an intratympanic composition to the subject, wherein the intratympanic composition comprises an auris sensory cell modulator and an auris acceptable gel, wherein the auris sensory cell modulator is a neurotrophin, wherein the auris acceptable gel is a thermoreversible gel comprising from 14% to 19% by weight of a poloxamer, and wherein the poloxamer is poloxamer 407.

10002d] In a third aspect, the invention relates to use of an intratympanic composition in the manufacture of a medicament for treating or preventing hearing loss in a subject, wherein the intratympanic composition comprises an auris sensory cell modulator and an auris acceptable gel, wherein the auris sensory cell modulator is a neurotrophin, wherein the auris acceptable gel is a thermoreversible gel comprising from 14% to 19% by weight of a poloxamer, and wherein the poloxamer is poloxamer 407.

10003] The auris formulations and therapeutic methods described herein have numerous advantages that overcome the previously-unrecognized limitations of formulations and therapeutic methods described in prior art. Sterility 10004] The environment of the inner ear is an isolated environment. The endolymph and the perilymph are static fluids and are not in contiguous contact with the circulatory system. The blood labyrinth - barrier (BLB), which includes a blood-endolymph barrier and a blood-perilymph barrier, consists of tight junctions between specialized epithelial cells in the labyrinth spaces (i.e., the vestibular and cochlear spaces). The presence of the BLB limits delivery of active agents (e.g., immunomodulators, aural pressure modulators, antimicrobials) to the isolated microenvironment of the inner ear. Auris hair cells are bathed in endolymphatic or perilymphatic fluids and cochlear recycling of potassium ions is important for hair cell function. When the inner ear is infected, there is an influx of leukocytes and/or immunoglobins (e.g. in response to a microbial infection) into the endolymph and/or the perilymph and the delicate ionic composition of inner ear fluids is upset by the influx of leukocytes and/or immunoglobins. In certain instances, a change in the ionic composition of inner ear fluids results in hearing loss, loss of balance and/or ossification of auditory structures. In certain instances, even trace amounts of pyrogens and/or microbes can trigger infections and related physiological changes in the isolated microenvironment of the inner ear.

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100051 Due to the susceptibilty of the inner ear to infections, auris formulations require a level of sterility that has not been recognized hitherto in prior art. Provided herein are auris formulations that are sterilized with stringent sterilty requirements and are suitable for administration to the middle and/or inner ear. In some embodiments, the auris compatible compositions described herein are substantially free of pyrogens and/or microbes.

Compatibilitywith Iner EarEnvironment

10006] Described herein are otic formulations with an ionic balance that is compatible with the perilymph and/or the endolymph and does not cause any change in cochlear potential. In specific embodiments, osmolarity/osmolality of the present formulations is adjusted, for example, by the use of appropriate salt concentrations (e.g.,concentration of sodium salts) or the use of tonicity agents which renders the formulations endolymph-compatible and/or perilymph-compatible (i.e. isotonic with the endolyrmph and/or perilymph), In some instances, theendolymph-compatibleand/or perilymph-compatible formulations described herein cause minimal disturbance to the environment of the inner ear and causemriimum discomfort (e.g, vertigo) to amanual (e.g., a human) upon administration.Further, the formulations comprise polxmers that are biodegradable and/or dispersable, and/or otherwise norn-toxic to the inner ear environment.In some embodiments, the formulations described hereinare free of preservatives and cause minimal disturbance (e.g., change in p or osmolarity, irritation) in auditory structures.In some enbodinments, the formulations described herein comprise antioxidants that are non-irritating and/or non-toxic tootic structures.

DosingFrequency

[00071 The current standard of care for aurisformulations requires multiple administrations of drops or injections (e.g. intratympanic injections) over several days (e.g., up to two weeks). including schedules of receiving multiple injections per day. In some embodiments, auris formulations described herein are controlled release formulations, and are administered at reduced dosing frequency compared to the current standard of care. In certain instances, when an auris formulation is administered via intratympanic injection, a reduced frequency of administration alleviates discomfort caused by multipleintratympanic injections in individuals undergoing treatment for a middle and/or inner ear disease, disorder or condition. In certain instances, areduced frequency of administration ofintratympanic injections reduces the risk of penanent damage (e.g., perforation) to the ear drum. The formulations described herein provide a constant, sustained, extended, delayed or pulsatile rate of release of an active agent into the inner ear environment and thus avoid any variability in drug exposure in treatment of otic disorders.

TherapeuticIndex

100081 Aurisformulations described herein are administered into the ear canal, or in thevestibule of the ear. Access to, for example, the vestibular and cochlear apparatus will occur through the auris media including the round window membrane, the oval window/stapes footplate, the annular ligament and through the otic capsule/temporal bone. Otic administration of the formulations described herein avoids toxicity associated with systemic administration (e.g.,hepatotoxicity, cardiotoxicity, gasrointestinal side effects, renal toxicity) of the active agents. In some instances, localized administration in the earallows 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 would otherwise have dose limithig systemic toxicity.

Prevention ofDrainageinto Eustaehian Tube 100091 In some instances, a disadvantage of liquid formulations is their propensity to drip into the eustachian tube anti cause rapid clearance of the formulation from the inner ear. Provided herein,in certain embodiments,are auris formulations comprising polymers that gel at body temperature and remain in contact with the target auditory surfaces (e.g. the round window) for extended periods of time. In some embodiments, the formulations further comprise mucoadhesives that allow the formulations to adhere to otic mucosal surfaces. In some instances, the auris formulations described herein avoid attenuation of therapeutic benefit due to drainage or leakage of active agents via the eustachian tube.

Descriptionof CerainEmbodiments 100101 Accordingly, provided herein, in some embodiments, are pharnaceutical formulations for use in the treatment ofan otic disease or condition formniulated to provide a therapeutically effective amount ofan inmunomodulating agent across the round window membrane into the cochlea, the formulation comprising: between about 0.2% to about 6% by weight of an inimunomodulating agent, or pharmaceutically acceptable prodrug or salt thereof; between about 16% to about 21% by weight of a polyoxyethylene-poivoxypropylene triblock copolymer of general formula E106 P70 E106; sterile water, q.s., buffered to provide a perilymphsuitable pH between about 6.0 and about 7.6; and substantially low degradation products of the inununomodulating agent; wherein the pharmaceutical formulation has a perilymph-suitable osrnolarity between about 250 and 320 mOsniL, less than about 50 colony forming units (cfi) of microbiological agents per grain of formulation, and less than about 5 endotoxin units (EU) per kg of body weight of asubject

100111 Provided herein,in some embodiments, are pharmaceutical formulations for use in the treatment of an otic disease or condition formulated to provide a therapeutically effective amount of an immunomodulating agent across the round window membrane into the cochlea, the fonnulation comprising: between about 0.1 mg/mi to about 70 mg/ml ofan immunomodulating agent, or phanmaceutically acceptable prodrug orsalt thereof; between about 16% to about 21% by weight of a polyoxyethylen-polyoxypropylene triblock copolymer of general formula E106 P70 E106; sterile water, qs., buffered to provide a perilymph-suitable pT between about 6.0 and about 1,6; and substantially low degradation products of the inmunomodulating agent; wherein the pharmaceutical formulation has a perilymnph-suitable osmolarity between about 250 and 320 mOsm/L, less thanabout 50 colony forming units (cfu) of microbiological agents per grain of formulation, and less than about 5 endotoxin units (EU) per kg of body weight of asubject.

100121 Insome embodiments, the immunomodulating agent is released from the formulationfor a period of at least 3 days In some embodiments, the pharmaceutical formulation is an auris acceptable thermoreversible gel. In some embodimentsthe polyoxyethylene-polyoxpropylene triblock copolymer is biodegradable, Insome embodiments. the formulations further comprise a mucoadhesive. In some embodiments, the formulations further comprise a penetration enhancer. In some embodiments,theformulations further comprisea thickening agent.Insome embodiments, the formulations further comprise a dye.

10013] In.further embodiments, provided herein are formulations further comprising a drug delivery device selectedfrom a needle andsyringe, a pump, a microinjection device, a wick, an in situ forming spongy material or combinations thereof.

100141 Insome embodimentsoftheformulations describedherein, the immuotnodulatingagent, or pharmaceutically acceptable salt thereof, has limited or nosystemic release, systemic toxicity, poor PK characteristics, or combinations thereof. In some embodiments. the inmunomodulating agent is in the form of a free base, salt, a prodrug, or a combination thereof In some embodiments, the immunomodulating agent comprises multiparticulates. In some embodiments, the immunomodulating agent is essentially in the form of micronized particles.

[00151 In some embodiments,the immunomodulatingagentisan anti-INF agent,acalcineurin inhibitor, an IKK inhibitor, an interleukin inhibitor, aTNF-a converting enzyme (TACE) inhibitor or a toll-like receptor inhibitor.

[00161 in some erbodiments, the-formulations furthercomprise animmunomodulatingagent, or phannaceutically acceptable salt thereof, as animmediate release agent.

[00171 In some embodiments, the fnnulations described herein further comprise an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a Na/K ATPase modulator, a chemotherapeutic agent, a collagen, a gamma-globulin, an interferon, an anti-microbial agent, an antibiotic, a local acting anesthetic agent, a platelet activator factor antagonist, anitric oxide synthase inhibitor, an anti-vertigo agent, a vasopressin antagonist, an anti-viral, an anti-emetic agent or combinations thereof.

[0018j In some embodiments, the pH of the composition is between about 6.0 to about 7.6 in some embodiments, the ratio of a polyoxyethylene-polyoxypropylene triblock copolymer of general formula E106 P70 E106 to a thickening agentis from about 40:1 to about 10:1. In some embodiments, the thickening agent is carboxymethyl cellulose.

[00191 In sonment embodiments. the otic disease or condition is Meniere's disease, sudden sensorineural hearing loss,noise induced hearing loss, age-related hearing loss, auto immune ear disease or tinnitus.

[0020] Also provided herein is a method of treating an otic disease or condition comprising administering to an individual in need thereof an intratympanic composition comprising between about 0.2% to about 6% by weight of an immunomodulating agent or pharmaceutically acceptable prodrug or salt thereof; between about 16% to about 21% by weight of a polyoxyethylene-polyoxypropylene triblock copolymer of general formula E106 P70 E106; sterile water, q.,buffered to provide a perilymph-suitable pH betweenabout 6.0 and about 7.6; and substantially low degradation products of the iminmunomodulating agent; wherein the pharmaceutical formulation has a perilymph-suitable osmolarity between about250and 320 mOsm/I less than about 50 colony forming units (cfa) of microbiological agents per gram of formulation, and less than about 5 endotoxin units (EU) per kg of body weight of a subject.

100211 In some embodiments of the method, the immunomodulating agent is an anti-TNF agent a calcineurin inhibitor, an IKK inhibitor, an interleukin inhibitor, aTNF-a converting enzyme (TACE) inhibitor, or a toll-like receptor inhibitor. In some embodiments of the method, the immunomodulating agent is released from the composition for a period of at least 3 days. In some embodiments of the method, the composition is administered across the round window, In some embodiments of the method, the otic disease or condition is Meniere's disease, sudden sensorineural hearing loss, age-related hearing loss, noise indhced hearing loss, auto immune ear disease or tinnitus.

[00221 Also provided hereinin sonic embodiments, are phannaceutical formulations for use in the treatment of an otic disease or condition formulated to provide a therapeutically effective amount of anaural pressure modulating agent across the round window membrane into the cochlea, the formulation comprising: between about 0.2% to about 6% by weight ofanauralpressuremodulatingagent, or

pharmaceutically acceptable prodrug or salt thereof; between about 16% to about 21% by weight of a polyoxyethylene-polyoxypropyene triblock copolymer of general fonnula E106 P70 E106; sterile water, q.s., buffered to provide a perilymph-suitable p1 between about 6.0 and about 7.6; substantially low degradation of the aural pressure modulating agent; wherein the pharmaceutical formulation has a perilymph-suitable osmolarity between about 250 and 320 mOsm/L, less than. about 50 colony forming units (cfi) ofmicrobiological agents per grain of formulation, and less than about 5 endotoxin units (EU) per kg of body weight of a subject

100231 Aso provided herein, in some embodiments, are pharmaceutical formulations for use in. the treatment of an otic disease or condition formulated to provide a therapeutically effective amount of an aural pressure modulating agent across the round window membrane into the cochlea, the formulation comprising: between about01 mg/mto about 70 mg/mL of an aural pressure modulating agent, or pharmaceutically acceptable prodrug or salt thereof; between about 16% to about21% by weight ofa polyoxyethylenepolyoxypropylene triblock copolymer of general formula E106 P70 E106; sterile water, q.s., buffered to provide a perilymph-suitable pi between about 6.0 and about 7.6; and substantially low degradation products of the aural pressure modulating agent; wherein the pharmaceutical formulation has a perlymph-suitable osmolarity between about 250and 320 mOsm/L, less than about 50 colony forming units (cfu) ofmicrobiological agents per grand of fonulation, and less thanabout 5 endotoxin units (EU) per kg of body weight ofa subject.

100241In some embodiments, the aural pressure modulating agent is released from the formulation fora period of at least 3 days. In some embodiments, the pharmaceutical formulation is an auris acceptable thermoreversible gel. In some embodimentsthe polyoxyethylene-polyoxypropylene triblock copolymer is biodegradable. in some embodiments, the formulations further comprise a round window membrane mucoadhesive. In some embodiments, the formulations further comprise a round window membrane penetration enhancer. In some embodiments, the formulations further comprise thickening agent. In some embodiments, the formulations further comprise a dye

100251 1I some embodiments of the formulations described herein, the formulations further comprise a drug delivery device selected from a needle and syringe, a pump amicroinjection device, a wick, an in situ forming spongy material or combinations thereof,

100261 In some embodiments, the aural pressuremodulating agent, or pharmaceutically acceptable salt thereof, has limited or no systemic release, systemic toxicity, poor PK characteristics, or combinations thereof. In some embodiments,the aural pressure modulating agent is administered in the form of a free base, salt, a prodrug, or a combination thereof Insome embodiments, the aural pressure modulating agent comprises multiparticulates.In some embodiments, the aural pressure modulating agent is essentially in the form of micronized particles.

[00271 In some embodiments,the aural pressure modulating agent is a modulator of aquaporin, an estrogen relatedreceptor beta modulator,a gap junction protein modulator,an NMDAreceptor modulator, an osmotic diuretic, a progesterone receptor modulator, a prostaglandin modulator, or a vasopressin receptor modulator,

[0028]1 some embodiments, the formulations described herein further comprise an aural pressure modulating agent, or pharmaceutically acceptable salt thereof, as an immediate release agent.

2} [0029] In some embodiments,the formulations described herein further comprise an additional therapeutic agent. In some embodiments, the additional therapeutic agent isINa/K ATPase modulator, a chemotherapeutic agent, a collagen, a gamma-globulin, an interferon, an anti-microbial agent, an antibiotic, alocal acting anesthetic agent, a platelet activator factor antagonist, a nitric oxide synthase inhibitor, an anti-vertigo medicine, a vasoprcssin antagonist, an anti-viral,an anti emetic agent or combinations thereof

100301 In some embodiments, the pHof the composition is between about 6.0 to about 7.6. In some embodiments,the ratio of apolyoxyethylene-polyoxypropvlene triblock copolymer of general formula El06 P70 E106 to a thickening agent is from about 40:1 to about 10:1. In some embodiments, the thickening agent is carboxymethyl cellulose.

100311 in some embodiments, the otic disease or condition is Meniere's disease, sudden sensorineural hearing loss, age-related hearing loss, noise induced hearing loss, auto immune ear disease or tinnitus.

[00321 Also provided herein is a method of treating an otic disease or condition comprising administering to an individualin need thereof anintratympanic composition comprising between about 0.2% to about 6% by weight of an aural pressure modulating agent.or pharmaceutically acceptable prodrug or salt thereof; between about 16% to about 21% by weight of a polyoxyethylene-polyoxypropylene triblock copolyiner of general formula E106 P70 E106; sterile waterq.s, bufferedtoprovideaperiymph-suitable pH between about 6,0 and about 7.6; and substantially low degradation products of the aural pressure modulating agent; wherein the pharmaceutical formulation has a perilymph-suitable osmolarity between about 250 and 320 mOsM/L, less than about 50 colony fonning units (cfu) of microbiological agents per gram of formulation, and less than about 5 endotoxin units (EU) per kg of body weight of a subject.

100331 In some embodiments, the aural pressure modulating agent is a modulator of aquaporin, an estrogen related receptor beta modulator, a gap junction protein modulator, an NMDA receptor modulator, an osmotic diuretic, a progesterone receptor modulatora prostaglandin modulator, or a vasopressin receptor modulator.

[00341 In some embodiments of the method, the aural pressure modulating agent is released from the composition for a period of at least 3 days. In some embodiments of the method, the composition is administeredacross the round window.

[00351 In some embodiments of the method, the otic disease orcondition is Meniere's disease sudden sensorineural hearing loss, age-related hearing loss, noise induced hearingloss, auto umanme

ear disease or tinnitus.

[00361 In any of the aforementioned embodiments, the term substantiallyy low degradation products" means less than 5% by weight of the active agent are degradation products of the active agent. In further embodiments, the term means less than 3% by weight of the active agent are degradation products of the active agent. In yet further embodiments, the term means less than 2% by weight of the active agent are degradation products of the active agent In further embodiments, the term means less than 1% by weight of the active agent are degradation products of the active agent.

[00371 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 thedetailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only. BRIEF DESCRIPTION OF THE FIGURES 100381 The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0039] Figure1 illustrates a comparison oftnon-sustained release formulationsand sustained release formulations.

[0040] Figure 2 illustrates the effect of concentration on viscosity of aqueous solutions of Blanos refined CMC

[0041 Figure 3 illustrates the effect of concentration on viscosity of aqueous solutions of Methocel DETAILED DESCRIPTION 100421 Systemic administration of active agents isin some instances, ineffectual in the treatment of diseases that affect inner ear structures. The cochlear canals and the cochlea, for example, are isolated from the circulatory system limiting systemic delivery of active agents to target sites in the inner ear. In sonic instances, systemic drug administration creates a potential inequality in drug concentration with higher circulating levels in the serum and lower levels in the target auris interna organstructures. In certain instances, large amounts of drug are required to overcome thisinequality in order to deliver sufficient, therapeutically effective quantities of a drug to auditory structures. In some instancessystemic drug administration also increases the likelihood ofsecondary systemic accumulation and consequent adverse side effects.

[00431 Currently available treatment for inner ear diseases also carries therisk 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 ishindered by the biological barrier presented by the blood-labyrinth-barrier (BLB), 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.

[0044} Clinical studies with steroids such as prednisolone or dexamethasone have demonstrated the benefit of having long term exposure of the steroids to the perilymph of the cochlea; this has been shown by improved clinical efficacy in improving sudden hearing loss when the steroid in question is given onmultiple occasions.

[0045} U.S. Application Publication Nos. 2006/0063802 and 2005/0214338 disclose compositions comprising arylcycloalkylamine NMDA antagonists for local administration to the inner ear. There is no disclosure of controlled release formulations, osmolarity or pH requirements, or sterility requirements for the compositions. WO 2007/038949 discloses compositions comprising arylcycloalkylamine NMDA antagonists in the treatment of inner ear disorders. No guidance is provided on pyrogenicity, sterility requirements, viscosity levels and/or controlled release characteristics of the formulation.

100461 Fernandez et al. Biomaterials,26:3311-3318 (2005) describes compositions which comprise prednisolone useful to treat inner ear disease such as Meniere's disease. Fernandez et al. do not disclose osmolarity, pyrogenicity, p1l or sterility levels of the compositions described therein.Paulson et alTheLwyngoscope, 118: 706 (2008) describe sustained release compositions which comprise dexamethasone useful in treatment of, inter alia, inner ear diseases such as Meniere's disease. Again, Paulson et al. do not disclose osmolarity, pyrogenicity, pH, orsterility requirements for the compositions described therein. 100471 C. Gang et al.,I SichuanUniv 37:456-459 (2006) describe a dexamethasonesodium phosphate (DSP) preparation. The formulation described in Gang et al comprises preservatives and adhesives and is sterilized under conditions that likely lead to breakdown of DSP. There is also no disclosure regarding osmolarity, pyrogenicity pH, or sterility requirements for the compositions described therein.

100481 Feng et al, ZhonghuaErBi Yan Hou Tou ing tiKeZa Zhi 42:443-6 (June 2007)and Feng et al, Zhonghua YiXue Za Zhi 87:2289-91 (August 2007) describe 20% and 25% poloxamer 407 solutions as non-toxic to otic structures. There is no active agent in the solutions described therein, and there is no disclosure regarding osmolaritypyrogenicity, pH. or sterility requirements for the solutions described therein 3.Daijie et a,I Cin OtorhinolarvngolHead.Neck Surg (China) 22(7) (April 2008), P. Yikun et al- J Clin. OtorhinolaryngolleadNeck Surg (China) 22(10) (May 2008), and S. Wandong et aL IClin OtrhinolaryngolleadNeckSug (China) 22(19) (October 2008) describe intratynpanic solution injections. However, Daijie et al, Yikun et al. and Wandong et al. do not disclose any otic formulations that are polymer based, or any otic formulations that are sustained release formulations. There is also no disclosure regarding osmolarity, pyrogenicity, pH, or sterility requirements for the compositions described therein,

[0049] Intratynipanic injectionof therapeutic agents isthetechniqueof injectinga therapeutic agent behind the tympanic membrane into the auris media and/or auris interna. Despite early success with this technique (Schuknecht, Laryngoscope (1956) 66, 859-870) some challenges do remain. For example, access to the round window membrane, the site of drug absorption into the auris interna, can be challenging.

[0050] However, intra-tympanicinjectionscreate several unrecognizedproblemsnot addressed by currently available treatment regimens, suchas changing the osmolarity and pH of the perilyph and endolymph,and introducing pathogens and endotoxins that directly orindirectly damage inner ear structures. One of the reasons the art may not have recognized these problems is that there are no approved intra-tympanic compositions:the inner ear provides sui generis formulation challenges. Thus, compositions developed for other parts of the body have little to no relevance for an intra tympanic composition.

[00514 There is no guidance in the prior art regarding requirements (e.g. level of sterilityp, osmolarity) for otic formulations that are suitable for administration to humans. There is wide anatomical disparity between the ears of animals across species. A consequenceof the inter-species differences in auditory structures is that animal models of inner ear disease are often unreliable as a tool for testing therapeutics that are being developed forclinical approval.

[00521 Provided herein are otic formulations thatmeetstringent criteria for pHiosmolarity, ionic balance, sterility, endotoxin and/or pyrogen levels. The auris compositions described herein are compatible with the microenvironment of the inner ear (e.g., the perilymph) and aresuitable for administration to humans In some embodiments, the formulations describedherein comprise dyes and aid visualization of the administered compositions obviating theneed for invasive procedures

(e.g., removal of perilymph) during preclinical and/or clinical development of intratympanic therapeutics.

[00531 Accordingly, provided herein, in certain embodiments, are controlled release auris acceltable 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 auris fonnulations described herein are polymer based formulations designed for stringent osmolarity and p1ranges that are compatible with auditory structures and/or the endolynph and perilymph. In some embodiments, the formulations described herein are controlled release formulations that provide extended release for a period of at least 3 days andmeet stringent sterility requirements. In some instancesotic 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 described herein contain low levels of colony forming units (e.g., <50 CFUs) per gram of the formulation. In some instances, the auris formulations described herein are substantially free of pyrogens and/or microbes, In some instances the auris formulations described herein are formulated to preserve the ionic balance of the endolymph and/or the perilymph. The stringent requirement for sterility and compatibility with inner ear fluids for otic formulations has not been addressed hereto.

[00541 The fornmlations describedherein representan advantage over currently available therapeutics because they are sterile controlled release otic formulations that are compatible with aurisstrucutures (eg, the perilymph) and are safe for long term administration to humans in need thereof In some instances, by providing a slow extended release of an active agent, the formulations described herein prevent an initial burst release upon administration to the inner ear; i.e., the

- II - formulations avoid causing dramatic change in the pH of the endolymph or perilymph and subsequently reduce the impact on balance and/or hearing upon administration.

[00551 In some instances, local administration of the compositions described herein avoids potential adverse side effects as a result of systemic administration of active agents. In some instances, the locally applied atris-acceptable formulations and compositions described herein are compatible with-auris structures, and 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 case of the cochlear region, for example, including but not limited to the round window membrane, the crista fenestrae cochleae or the oval window membrane,

[0056_ In certain instances, an advantage of the controlled release formulations described herein is that they 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 disorderreducing or eliminating any variabilities associated with other methods of treatment (such as, e.g, otic drops and/ormultipleintratympanic injections). 100571 The drug formulations described herein provide extended release of the active ingredient(s) into the middle and/or inner ear (auris interna), including the cochlea and vestibular labyrinth. A. further option includes an immediate or rapid release component in combination with a controlled release component. Certain Definitions 100581 The term "auris-acceptabile" with respect to a formulation, composition or ingredient, as used herein, includes having no persistent detrimental effect on the auris media (or middle ear) and the auris internal (or inner ear) of the subject being treated By "auris-pharmaceutically 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 media (or middle ear) and the auris interna (or inner ear), and isrelatively or is reduced in toxicity to the auris media (or middle ear) and the auris internal (or inner ear), i.e., thematerial is administered to an individual without causing undesirable biological effects or interacting ina deleterious manner with any of the components of the composition in which it is contained.

[0059J As used herein, amelioration or lessening of the symptoms of a particular otic disease disorderor 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. 100601 As used herein, the terms "immunomodulating agent" or "innunomodulator" or "immunomodulator agent" or "immune-mnodumlating agent" are used as synonyms.

100611 Theterm"anti-TNagent"or"antitumornecrosis factor agent" or"TNF modulator"or "TNF modulating agent"or "TNF-alpha modulator"or "anti-TNF alpha agent" are used as synonyms. The term 'anti-TNF agent" and its synonyms generally refer to agents that counteract the biological effect of TNF-a or the biological effect of pro-'NTF-a stimulus including agents which bind to and antagonize themolecular target; here, tumor necrosis factor alpha or TNF-alpha (TNF o), agents which inhibit release of TNF-a or agents which interfere withiNF-t gene expression due to pro-TNF-a stimulus. Also included are agents that indirectly antagonize the biological

activity of TNF-a b modulating targets in the general pathway ofTNF-a activation, including but not limited to targets upstream of the pathway ofTNF-alpha activation, including butnot limited to agents which increase TNF-alpha expression, activity or function

100621 As used herein, thetens"aural pressuremodulatingagent" or "aural pressure modulator" are used as synonyms and do not define the degree of efficacy. The aural pressure modulator also includes compounds that modulate theexpression or post-transcriptional processig of a fluid homeostasis protein, including vasopressin and estrogen-related receptor beta protein. Additionally, vasopressin receptor or estrogen-related receptor beta modulators include compounds that influence vasopressin receptor or estrogen-related receptor beta signalling or downstream functions wider the control of the vasopressin receptor or estrogen-related receptor beta, such as aquaporin function. Vasopressin. receptor or estrogen-related receptor beta modulating agents includes compounds that increase and/or decrease vasopressin receptor or estrogen-related receptor beta function, including antagonists, inhibitors, agonists, partial agonists and the like

[00631 "Modulator of neuron and/or hair cells of the auris"and "auris sensory cell modulating agent" are synonyms.They include agents that promote the growth and/or regeneration of neurons and/or the hair cells of the auris, and.agents that destroy neurons and/or hair cells of the auris.

[0064] 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 againstbacteria), antiviral agents (effective against viruses), antifnigal agents (effective against fungi), antiprotozoal (effective against protozoa), and/or antiparasitic to any class ofmicrobial parasites. "Antimicrobial agents" may work by any suitable mechanism against the microbes, including by being toxic or cytostatic.

[00651 The phrase "antimicrobial small molecule" refers to antimicrobial compounds that are of relatively low molecular weight, e g., less than ,000 molecular weight, that are effective forthe treatment of otic disorders, particularly otic disorders caused by pathogenic microbes. and are suitable for use in the formulations disclosed herein. Suitable "antimicrobial small molecules" include antibacterial, antiviral antifungal, antiprotozoaland antiparasitic small molecules.

100661 "Modulator of free-radicals" and "free-radical modulating agent" are synonyms. They refer to agents that modulate the production of and/or damage caused by free radicals, especially reactive oxygen species.

100671 As used herein, the terms "ion channel modulating agent" "modulator of ion channels" or "ion channel modulator" are used as synonyms and do not define the degree of efficacy. The ion channel modulator also includes compounds that modulate the expression or post-transcriptional processing of a fluid homeostasis protein, including vasopressinand estrogen-related receptor beta protein. Additionallyvasopressin receptor or estrogen-related receptor beta modulators include compounds that influence vasopressin receptor or estrogen-related receptor beta signalling or downstream functions under the controlof the vasopressin receptor or estrogen-related receptor beta, such as aquaporin function. Vasopressin receptor or estrogen-related receptor beta modulating agents includes compounds that increase and/or decrease vasopressin receptor or estrogen-related receptor beta function, including antagonists, inhibitors, agonists, partial agonists and the like. 100681 As used herein, the tern oticc agent" or oticc structure modulating agent" or "otic therapeutic agent" or "otic active agent" or "active agent" refers to compounds that are effective for the treatment of otic disorders, e.g, otitis media, otosclerosis, autoinmune 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, or a positive allosteric modulator of an otic structure modulating target, or combinations thereof. 100691 "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 tlis 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 debarquement, benign paroxysmal positional vertigo, and labyrinthitis.

[00701 "CNS modulator" and "CNS modulating agent" are synonyms. They refer to agents that decrease.diminish, partially suppress, fully suppress, ameliorate, antagonize, agonize stimulate or increase the activity of the CNS. For example, they may increase the activity of GABA by, for example, increasing the sensitivity of the GABA receptors, or they may alter the depolarization in neurons,

[00711 "Local anesthetic"means a substance which causes a reversible loss ofsensation and/ora loss of nociception, Often, these substances function by decreasing the rate of the depolarizationand repolarization of excitable membranes (for example, neurons). By way of non-limiting example, local anesthetics include lidocaine, benzocaineprilocaine, and tetracaine.

[00721 "Modulator of the GABA receptor," "modulator of the GABAreceptor""GABAA receptor modulator,"and "GABA receptor modulator," are synonyms. They refer to substances

which modulate the activity of the GAIA neurotransmitter, by; for example, increasing the sensitivity of the GABA receptor to GABA.

[00731 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.,autoinmune diseases of the ear and cancer of the ear, and are suitable foruse in the formulations disclosed herein. 100741 The phrase "cytotoxic small molecule" refers to cytotoxic compounds that are of relatively low molecular weight, e.g., less than 1,000, or less than 600-700, or between 300700 molecular weight, that are effective for the treatment of otic disorders, e.g., autoinmune diseases of the ear and cancer of the ear, and aresuitable for use in the formulations disclosed herein. Suitable "cytotoxic small molecules" include methotrexate, cyclophosphamide, and thalidomide, as well as metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of methotrexate, cyclophosphamide, and thalidomide. In certain embodimentspreferred cytotoxicsmall molecules are the pharmaceutically active metabolites of cytotoxic agents. For example, in the case of cyclophosphamide, preferred metabolites are pharmaceuticallyactive metabolites of cyclophosphamide, including but not limited to 4-hydroxycyclophosphamide, aldophosphamide. phosphoramide mustard, and combinations thereof.

[0075] "Antioxidants" are auris-pharmaceutically acceptable antioxidants, and include, for example, butylated hydroxytoluene (BIT), 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, including agents that are used in combination with tie otic agents disclosed herein.

[0076j "Auris interna" refers to the inner ear, including the cochlea and the vestibular labyrinth, and the round window that connects the cochlea with the middleear.

[0077 "Auris-intema bioavailability" or"Auris media bioavailability"refers to the percentage of theadministered dose of compounds disclosed herein that becomes available in the inner or middle ear, respectively, of the animal or human being studied. 100781 "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,

[00791 "Blood plasma concentration" refers to the concentration of compounds provided herein in the plasma component of blood of a subject.

[00801 "Auris-interna bioavailability" refers to the percentage of the administered dose of compounds disclosed herein that becomes available in the inner ear of the animal or human being studied.

J00811 Thetermauris-acceptablepenetrationenhancer" with respect to a formulation, composition or ingredient, as used herein, refers to the property of reducing barrier resistance.

[0082] "Carriermaterials" are excipients that are compatible with the otic agent, the auris media, the auris internal and the release profile properties of the auris-acceptable pharmaceutical formulations. Such carrier materials include, e.g, binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizerslubricants, wetting agents, diluents, and the like. "Auris-pharmaceutically compatible carrier materials" include, butare 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, taurocholic acidphosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan monoglyceride, diglyceride, pregelatinized starch, and the like.

[00831 The term"diluent" are chemical compounds that are used to dilute the otic agent prior to delivery and which are compatible with the auris media and/or autis interna, iS 100841 "Dispersing agents," and/or "viscosity modulating agents" and/or "thickening agents" are materials that control the diffusionand homogeneity of the otic agent through liquidmedia. Examples of diffusion facilitators/dispersing agents include but are not limited to hydrophilic polymers, electrolytes,Tween® 60 or 80, PEG, poiyvinylpyrrolidone (PVP; commercially known as Plasdone@), and the carbohydrate-based dispersing agents such as, for example, hydroxypropyl celhloses (e.g., HPC, HPC-SL, and IHPC-L), hydroxypropyl methylcelluloses (eg., HPMC K00, HPMC K4M, HPMC K15M, and HPMC Kl0M), carboxymethylcellulose. carboxymethylcellulose sodiummethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyliethylcellulose phthalate, hydroxypropylmethyileellulose acetate stearate (IIPMCAS), noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(113,3-tetramethylbutyl) phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68@, F88@ and FOS , which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g, Tetronic 908@, also known as Poloxamine 908, which is a tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, NJ.)), polyvinylpyrrolidone K12, polyvinylpyrrolidone KI7, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetate copolymer (S-630), polyethylene glycol, e.g., the polyethylene glycol has a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethyleellulose, methyleellulose, polysorbate-80, sodium alginate, gums,such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbhan monolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates, chitosans and combinations thereof. Plasticizers such as cellulose or triethyl cellulose are also be used as dispersing agents. optional dispersing agents useful in liposomal dispersions and self-emulsifying dispersions of the otic agents disclosed herein are dimyristoyl phosphatidyl choline, natural phosphatidyl choline from eggs, natural phosphatidyl glycerol froma eggs, cholesterol and isopropyl myristate.

100851 "Drug absorption" or "absorption" 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 aurs internal or inner ear structures. The terms "co-administration" or the like, as used hereinare 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. 100861 The terns "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 AIED. For example, an "effective amount" for therapeuic uses is the amount of the otic agent, including a formulation as disclosed herein required to provide a decrease or amelioration indiseasesymptoms without undueadverse side effects. The 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, thecondition being treated, the severity of the condition being treated, and the judgment of the prescribing physician It is also understood that" an effective amount" in an extended-release dosing fonnat may differ from "an effective amount" in an immediate-release dosing format based upon pharmacokinetic and pharnacodynamic considerations,

[00871 The terms "enhance" or "enhancing" refers to an increase or prolongation of either the potency or duration of a desired effect ofthe otic agent, or a diminution of any adverse symptomatology. For example, in reference to enhancing the effect ofthe otic agents disclosed herein, the term. "enhancinrefers 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.

[00881 The term "penetration enhancer" refers to an agent that reduces barrier resistance (e.g. barrier resistance of the round windowmembrane, BLB or the like).

[0089] The tem "inhibiting" includes preventing, slowing, or reversing the development of a condition, for example, AIED, or advancement of a condition in a patient necessitating treatment. 100901Theterms"kit" and"article manufacturer" are used as synonyms.

[00911 The term."modulate" includes the interaction with a target, for example, with the TNF-alpha agents disclosed herein, the activity of TNF-alpha, or other director direct targets that alter the activity ofTNF-alpha, including, by way of exarnple onlytoinhibit the activity ofTNF-alpha. or to limit the activity of the TNF-alpha,

[00921 "Phamacodynamics" 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.

[0093] "Pharmacokinetics"refers to the factors which determine the attainment and maintenance of the appropriate concentration of drug at the desired site within the aurismedia and/or auris interna

[00941 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, for example, AIED, or patients that are suffering from diseases associated with AED, including by way of example only, Ankylosing spondylitis, Systemic Lupus Erythematosus (SLE), Sjugren sSyndrome, Cogan's disease ulcerativecolitis,Wegener's granulonatosis inflammatory bowel disease, rheumatoid arthritis, scleroderma and Behet's disease. Such an amount is defined to be a "prophylactically effective amount or dose."In this use, the precise amounts also depend on the patient's state of health, weight, and the like. 00951 A "prodrug"refers to the otic agent that is converted into the parent drug in vivo. In certain embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeuticallyactive form of the compound, 'o produce a prodrug, a pharmaceutically active compound is modified such that the active compound will be regenerated upon in vivo administration. In one embodiment, the prodrug isdesigned to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, or to alter other characteristics or properties of a drug. Compounds provided herein, in some embodiments, are derivatized into suitable prodrugs. 100961 "Solubilizers" refers to auris-acceptable compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate. sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone., polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol®,propylene glycol, and dimethyl isosorbide and the like.

[00971 "Stabilizers" refers to compounds such as any antioxidation agents, buffers, acids, preservatives and the like that are compatible with the environment of the auris media and/or auis interna. Stabilizers include but arenot limited to agents that will do any of(1) improve the compatibility of excipients with a container, or a delivery system, including a syringe or a glass bottle, (2) improve the stability of a component of the composition, or (3) improve forulation 1O stability.

[00981 "Steady state,"as used herein is whenthe amount of drug administered to the auris media and/or aurisinterna is equal to the amount of drug eliminated within one dosing interval resulting in a plateau or constant levels of drug exposure within the targeted structure.

[00991 Asused herein,the term "subject"is usedtomean an animal,preferably mammal, including a human or non-human. The terms patient and subject are used interchangeably.

[001.001"Surfactants" refers to compounds that are auris-acceptable, such as sodium lauryl sulfate, sodium docusate,Tween 60 or 80, triacetin vitamin E TPGS, phospholipids, leithins, phosphatidyl cholines (c8-cl8), phosphatidylethanolanines (c8-cl8), phosphatidylglycerols (8-cl8), sorbitan mnonooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers. bile salts,glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Plronic" (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 alkylpheny ethers, eg, octoxynol 10, octoxynol 40. In some embodiments, surfactants are included to enhance physical stability or for other purposes.

[001011 The terms "treat," "treating" or "treatment," as used herein, include alleviating, abatingor ameliorating a disease or condition, for example AIED, 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.

Anatomy of the Ear

1001021The ear serves as both the enseorgan 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). As shown in the illustration abovethe 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 tympanicmembrane, 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 to collect and direct sound waves towards the tympanic membrane and the middle ear. 1001031The middle ear is an air-filled cavity, called the tympanic cavitybehind the tympanic membrane. The tympanic membrane, also known as the ear dnm, is a thinmembrane 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 nalleus, 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 rnalleus, which is attached 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 windows located winthin 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 intema. Thus, the auditory ossicles are arranged to provide amechanical linkage between the tympanic membrane and the oval window of the fluid-filled auris interna, where sound is transformed and transduced to the auris internal for further processing Stiffhess, rigidity or loss ofmovement of the auditory ossicies, tympanic membrane or oval window leads to hearing loss, eg. otoscierosis, or rigidity of the stapes bone.

[00104] The tympanic cavity also connects to the throat via the eustachian tube. The eustachian tube provides the ability to equalize the pressure between the outside air and the middle ear cavity. The round window, a component of the auris internal but which is also accessible within the tympanic

-20-~ 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 intertmediate or fibrous layer, and an internal membrane which communicates directly with the cochlear fuid. The round window, therefore, has direct communication with the auris interna via the internalmembrane.

[00105Movements 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 theround window allows movement of fluid within the cochlea, which eventually leads in turn to movement of the cochlear inner hair cells, 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 implantingmechanical transducers onto the round window, which bypasses the normal conductive pathway through the oval window and provides amplified input into the cochlear chamber.

[001061Auditory signal transduction takes place in the auris interna.The fuid-filled inner ear, or auris interna, consists of two major components: the cochlear and the vestibular apparatus. 1001071The cochlea is the portion of the auris interna related to heating 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 portionabove the vestibular membrane is the scala vestibuli, which extends from the oval window to the apex of the cochlea and contains perilviph fluid, an aqueous liquid low in potassium and high in sodium content. The basilar membrane defines the scala tympani 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 fro 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. The cochlear duct contains endolymph fluid, which dissimilar tocerebrospinalfluid and is high in potassium. 1001081 The Organ of Corti, the sensory organ for hearing, is located on the basilar membrane and extends upward into the cochlearduct. The Organ of Corti contains hair cells, whichhave 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 bysensorynerve fibers that form the cochlear branch of the vestibulocochlear nerve (cranialnerve VIIl), 1001091As discussed, the oval window, also known as the elliptical window communicates with the stapes to relay sound waves that vibrate from the typanic 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 window/outward expansion of the round window allows for the movement of fluid within the cochlea without a change ofintraochiClar pressure. However, as vibrations travel through the perilymph in the scala vestibulithey create corresponding oscillations in the vestibularmembrane. These corresponding oscillations travel through the endolymph of the cochlear duct, and transfer to the basilar membrane. When the basilar membrane oscillates, or noves 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 thatare subsequently processed by the central nervous system.

[001t10]The auris internal is located in part within the osseous or bony labyrinth, an intricateseries 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 can be detected by the movement of the fluid and subsequentsignal processing by the sensory organs of thesemi-circular canals, called the crista amupliaris. The crista ampullaris 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 detectdynamic equilibrium, the equilibrium of rotational or angular movements. (00111]When the head tuns rapidly, the semicircular canalsmove with the head, but endolymph fluid located in the membranous semi-circular canals tends to remain stationaryThe endolymph fluidpushesagainst the cupula, which tilts toone side. As the cupula tiltsit bends some of the hairs on the hair cells of the crista ampullaris, which triggers a sensory impulse. Because each semicircular canal is located in a different plane, the corresponding crista ampullaris 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 vestibuocochlear 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, 1001121 The vestibule is the central portion of the auris interna and contains mechanoreceptors bearing hair cells thatascertain 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 theinacula, Grains of calcium carbonate, called otoliths, are embedded on the surface of the gelatinous layer.

[00113] 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 in turn relays motor impulses to the appropriate muscles to maintain balance. 100114] Thedrug forinulation will first be 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). 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 theauris interna also known as cochlear iieroperfusion.

Animal Models and Human Clinical Trials

[00115]There are, at present, no intratympanic therapeutics approved for administration to humans. In some instances, a lack of suitable animal models for inner ear diseases has hindered development of intratympanic therapeutics for human use. 1001161In some instances, the use of animal models for inner ear diseases that are utilized for testing the efficacy of the formulations described herein is not accurately predictive of the efficacy of such formulations in humans. Rodent animal models for inner ear disease (e.g., innerear disease models in guinea pigs) are not amenable to allometric scaling in humans because rodents are different anatomically in the organization of the middle and inner ear.The middle ear of the guinea pig (or bulla) is a cavity that contains all of the cochlea; the cochlea is anchored to the bulla via the basal turn, its apex residing in the cavity. In contrast, the human cochlea is imbedded into the termporal bone and the only access to the human cochlea is through the round window, In some instances, from a phanmacokinetics perspective, studies in guinea pigs that overfill the bua and/or inject formulations towards the anterior quadrant of the tymnpani, or more generally away front the round window niche, will resultin high perilymph exposure because of drug diffusion through the cochlea apex. Tbis situation is not possible in humans because thehuman cochlea is imbedded into the temporal bone and as such the only access to the cochlea is on and/or through the round window or the elliptical/oval window. In addition, the ossicle chains in guinea pigs are adjacent to the round window. in some instances, the location of the ossicle chains next to the round window in guinea pig ears adversely affects the ABR threshold in experiments with guinea pigs, In contrast, the human ear is anatomicallydifferent from rodent ears; the ossicle chains and/or stapes are anatomically located away from the round window, In certain instances, an auris formulation injected intratympanically into a human ear does notmake contact with the stapes and does not adversely affect theABR threshold, Thus, in certain instances, the reliability of arnmal models of inner ear diseases as a predictor of efficacy in human clinical trials is limited by the anatomical difference between the human ear and animal ears,

[00117In some instances, a guinea pig animal modelforinner ear disease utilizes an injection via a hole drilled into the bulla. i.e., the cavity surrounding the cochlear bones. In soie instances, the bulla procedure leads to a local inflammatory reaction and a rapid accumulation of fluids within the bula cavity, a condition that lasts for several days. In some instances, an accumulation of significant volumes of fluids in the bulla (about a 1/3-1/2 of the total bulla volume) seen with the bulla injection rapidly erodes any auris formulation injected, primarily by diluting the formulation and reverting a formulation(e.ga gel formulation) to a liquid that drains away via thecustachian tube. For example, a gel formulation comprising a poloxamer will not fonn a gel at concentration below 12 14%, and at concentrations less than 15% concentration will gel at temperatures higher than 37 C. hInsome instances, a guinea pig model is of limited utility for testing the efficacy of an auris fornulation for administration to humans due the accelerated clearance of the gel fron the bulla compartment of a guinea pig. For example, in some instances, a 17% Pluronic F127 gel injection is cleared from the bulla of a guinea pig in less than 2 days.

[001181In some instances, a guinea pig animal modelforinner ear disease utilizesan injection through the tympanic membrane. In certain instances, in guinea pigs, an intratympanic infection is not associated with fluid accumulation at any of the time points evaluated (up tolO days). hi some instances,injection of an auris formulation described herein (e.g. a gel fonnulation) via the tympanic route allows for detectable amounts of the formulation (e.g.a gel) in the inner ear of a guinea pig ip to at least 5 days 1001191in some instances, animal models (e.g.,guinea pig models for inner ear diseases) utilizing intratympanic injections are limited by the volume that can be injected through the tympanic route. In the guinea pig the round window niche and membrane are located just opposite the tympanic membrane in the posterior superior quadrant. In certain instances, about 50mnL can be injected within this quadrant in a.250-350g guinea pig. In some instances, a larger volume (up to 70nL) can be injected in the posterior inferior quadrant; however most of the gel migrates towards the round window. In sone instances larger volumes (100-1 20mel) are injected in the anterior quadrant, but this action fills the bulla cavity and promotes drug transfer across the apical part of the cochlea (due to the bone structure thinness of the cochlea in rodents). In certain animal models, injection of larger volumes in any of these quadrants leads to tympanic perforation and presence of the gel in the external ear canal. In sone instances, the volume injected has an impact on the hearing threshold (measured by ABR).In the guinea pig ear for example, intratympanic injections volumes up to 50 mL do not produce any shift inhearing threshold; but volumes of 90 and 120 mL produce an ABR threshold shift within Iday. In some instances, the anatomical difference between human and animal ears and the variability in experimental outcomes lends a low predictive value to animal testing data for use insubsequent human clinical trials. Further the invasive procedures used in animal models of inner ear disease are not applicable in a clinical setting. Visualization of otic formulations 100120]Provided herein are otic fornulations that comprise a dye (e.g., a Trypan blue dye, Evans blue dye) or other tracer compound. In some instances, addition of anauris-compatible dye to an otic formulation described herein aids visualization of any administered formulation in a ear (e.g., a rodent ear and/or a human ear), In certain embodiments. anotic composition comprising a dye or other tracer compound eliminates the need for invasive procedures that are currently used in animal models to monitor the concentrations of drugs in the endolymph and/or perilymph. 100121] In some instances, intratympanic injections require the need of a specialist and the formulation needs to be delivered to aspecific site of the ear to maximize efficiency of the medication delivered. In certain instances, a visualization technique for any formulation described herein allows for visualization of a dosing site (eg.,the round window) so that the medication is applied in the proper place In some instances, a formulation comprising a dye allows visualization of the formulation during administration of the formulation to an ear (e.g., a human ear), ensures that the medication will be delivered at the intended siteand avoids any complications due to incorrect placement of a formulation.The inclusion of a dye to help enhance the visualization of the gel when applied, and the ability to visually inspect the location of the gel afteradministration without further intervention, represents an advance over currently available methods for testing intratympanic therapeutics in animal models and/or hunan trials. In someeinbodiments, dyes that are compatible with the otic compositions described herein include Evans blue (e.g, 0,5% of the total weight of an otic formulation), Methylene blue (e.g, 1% of the total weight of an otic formulation), Isosulfan blue (e.g. I% of the total weight of an otic formulation), Trypan blue (e.g., O 15% of the total weight of an otic formulation), and/or indocyanine green (e.g., 25nig/all. Other common dyes,e.g, FD&C red 40, FD&C red 3 FD&C yellow 5 FD&C yellow 6, FD&C blue 1, FD&C bhc2, FD&C green 3, fluorescence dyes (e.g, Fluorescein isotiocyanate, rhodamine, Alexa Fluors, DyLight Fluors) and/or dyes that are visualizable in conjunction with non-invasive imaging techniques such as MR, CAT scans, PET scans or the like (e.g.. Gadolinium-based MRI dyes, iodine-base dyes barium-based dyes or the like) are also contemplated for use with anyotic fonulation described herein. Other dyes that are compatible with any formulation described herein are listed in the Sigma Aldrich catalog under dyes (which is included,herein by reference for such disclosure). In some embodiments, concentration of a dye in any otic formulation described herein is less than 2%, less than 1.5%, less than 1%, less than 0.5%,less than 0.25%,less than 0.1%, or less than 100 ppm of the total weight and/or volume of any formulation described herein.

[001221In certain embodiments of such auris-compatible formulations that comprise a dye, the ability to visualize a controlled release otic formulation comprising a dye in an ear meets a long inhibitors (by way of example only CEP/KT-7515, AS601245, SPC9766 and SP600125), antioxidants, NSAIDs, neuroprotectants, glutamate modulators interleukin 1 modulators, interleukin-1 antagonists, including tumor necrosis factor-a coverting enzyme (TACE) and caspases, retinaldehydemodulator, notch modulator, gamma secretasemodulator, thalidomide latanoprost (Xalatan*) for reducing internal pressure and combinations thereof. Immunonodulating agents Anti-7VFAgents

[00266]Contemplated for use with the formulations disclosed herein are agents which reduce or ameliorate symptoms or effects as a resultof an autoimmune disease and/or inflammatory disorder,

[0 including AIED or OM. Accordingly, some embodiments incorporate the use of agents which block the effects of TNF-a, including anti-TNF agents By way of example only, anti-TNT agents include protein-based therapeutics, such as etanercept (ENBREL), infliximab (REMICADE), adalimumab (HUMIRA) and golimumab (CNTO 148), and small molecule therapeutics, suchas TACE inhibitors, IKK inhibitors or calcineurin inhibitors or combinations thereof,

[5 100267 Infliximab and adalinunab are anti-TNT monoclonal antibodies, and etanercept is a fusion protein designed to bindspecifically to the TNT protein All are currently approved for use in the treatment of rheumatoid arthritis. Golimumab, which is currently in Phase 3 clinical trials for rheumnatoid arthritis, psoriatic arthritis and ankylosing spondylitis isaflly-humanized anti-TN alpha Ig~l monoclonal antibody that targets and neutralizes both the soluble and themembrane~ bound fori of TNF-a. Other antagonists to TNF, by way of example only, includeTNF receptors (pegylated solubleTNF receptor type 1; Amgen); TNF binding factors (Onercept; Serono);TNTF antibodies (US Patent App. No. 2005/0123541; US Patent App. No. 2004/0185047), single domain antibodies against the p55 TNF receptor (US Patent App. No. 200800088713); solubleTN receptors (US Patent App.No. 2007/0249538); fusion polypeptides binding to TNF (US Patent App. No.2007/0128177); and flavone derivatives (US Patent App. No. 2006/0105967), all of which are incorporated by reference for such disclosure. The use of onercept, a solubleTNF p55 receptor, was discontinued in 2005. three phase-III clinical trials reported patients diagnosed with fatal sepsis. A risk to benefit analysis was subsequently performed, resulting in the discontinuation of the clinical trials.As discussed above, the embodiments herein specifically contemplate the use of anti-TNF agents which have been previously shown to have limited or no systemic release, systemic toxicity, poor PK characteristics of combinations thereof.

[002681Although etanercept, infiximab and adalimumab are currently approved systemic therapies for use in rheumatoid arthritis, these anti-TNT agents are not without serious adverse side effects. It is contemplated that the localized application of the anti-TNF agents to the target otic structures for treatment of autoimmune and/or inflamrmatory disorders will result in the reduction or elimination of these adverseside effects experienced with systemic treatment. Moreover, localized treatment with

- 57.

the anti-TNF agents contemplated herein will also reduce the amount of agent needed for effective treatment of the targeted disorder due, for example, to the existence of the biological bloodbarrier in the auris interna or to the lack of sufficient systemic access to the aurismedia

1002691Etanercept is a dineric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor necrosis factor receptor (TNFR) linked to the Fe portion of human IgG L The Fe component of etanercept contains the Ct2 domain, the C3 domain and. hinge region, but not the C4l domain ofIgG. Etanercept is a recombinant protein consisting of 934 amino acids, with an apparent molecular weight of approximately 150kilodaltons Etanercept binds specifically to tumor necrosis factor (TNF), and acts byinhibiting the interaction ofTNF with cell surface TNF receptors. Serious side effects with etanercept have been reported with systemic administration, including serious infections and sepsis that resulted in fatalities. Other side effects observed upon intravenous administration of etanercept include contraction of tuberculosis; onset or exacerbation of central nervous system disorders, including mental status changes, transverse myelitis, optic neuritis, multiple sclerosis and seizures resulting in permanent disability; adverse hematologic eventsincluding pancytopenia, aplastic anemia with fatal outcomes, blood dyscrasias, persistent fever, bruising, bleeding and pallor, neutropenia and cellulitis. Treatment with etanercept mayalso result in the formation of autoantibodies, which may develop into a lupus-like syndrome, as well as development ofmalignant disorders. Moreover, over one-third of patients systemically treated with etanercept experience injection site reactions including mild to moderate erythema andor itching, pain and/orswelling. Injection site bleeding and bruising has also been observed. Other side effects from the systemic administration of etanercept include headache, nausea, rhinitis, dizziness,pharyngitis, cough, asthenia, abdominal pain, rash, peripheral edema, respiratory disorder, dyspepsia, sinusitis, vomiting, mouth ulcer, alopecia and pneumonitis. Infrequent side effects includeheart failure, myocardial infarction, myocardial ischemia, hypertension, hypotension, deep vein thrombosis thrombophlebitis cholecystitis, pancreatitis, gastrointestinal hemorrhage, bursitis, polymyositis, cerebral ischemia, depression, dyspnea, pulmonary embolism, and membranous giomeruilonephropathy in rheumatoid arthritis patients. Varicella infections, gastroenteritis, depression/personality disorder, cutnaeous ulcer, esophagitis/gastritis group A streptococcal septic shock, type I diabetes mellitus, and soft tissue and post-operative wound infection was also seen in juvenile rheumatoid arthiritis patients 1002701Infliximab is a chimeric human-mouse IgGI x monoclonal antibody with an approximate molecular weight of 149 kilodaltons. Infliximab binds specifically toVTNFa with an association constant of 101M Infliximab is produced by a recombinant cell line cultured by continuous perfusion. Infliximab acts to neutralize the binding activity of TNFo by inhibiting binding of TNF to its cell surface receptors. Serious side effects as a result of systemic intravenous infusions or injections have been reported. with the use of infliximab, including fatal sepsis and serious infections. Cases of histoplasmosis, listeriosis, pneunocystosis and tuberculosis have also been observed.Hypersensitivity, including urticaria, dyspnea and hypotension have occurred upon treatment with infliximab Infusion reactions include cardiopulmonary reactions (primarily chest pain, hypotension, hypertension or dyspnea), pruritus, and combined reactions. Other hypersensitivity symptoms include fever, rash, headache, sore throat, myalgias, polyarthraligias, hand and facial edema and/or dysphagia anaphylaxis, convulsions, erytheinatous rash, laryngeal/pharyngeal edema and severe bronehospasm. Neurologic adverse events include optic neuritis, seizure and new onset or exacerbation and/or radiographic evidence of central nervous system demyelinating disorders, including multiple sclerosis. The formation of autoantibodies have also been observed, including symptoms suggestive of a lupus-like syndrome following treatment Other serious adverse events include worsening rheumatoid arthritis, rheumatoid nodules, abdominal herniaasthenia, chest pain, diaphragmatic hernia, pancytopenia, splenie infarction, splenomegaly, syncope, cerebral hypoxia, convulsions, dizziness, encephalopathy, hemiparesis, spinal stenosis, upper motorneuron lesion, cerurninosis endophthailmitis, and other infrequentoccurring side effects.

[00271]Adalimumab is a recombinant humanIgGl monoclonal antibody specific forhuman TNF. Adalimumab was created using phage display technology resulting in an antibody with human derived heavy and light chain variable regions and human IgGlK constant regions, and consists of 1330 amino acids with a molecular weight of approximately 148 kilodaltons. Adalimumab binds Zo specifically to'TNF- and blocks its interaction with both the p55 and p75 TNF cl surface receptors. Adalimuiab also lysesINF expressing cells in vitro in the presence of complement. Adalimumab does not bind or inactivate lymphotoxin (TNF-f). Serious side effects from systemic administration have been reported with the intravenous administration or injection of adalimumab, includingfatal sepsis and serious infections, including upper respiratory infections, bronchitis, urinary tract infections, pneumonia, septic arthritis, prosthetic and post-surgical infections, erysipelas cellulitis, diverticulitis, pyelonephritis, tuberculosis, and invasive opportunitistic infections caused by histoplasma, aspergillus and nocardia. Other serious adverse reactions were neurologic events, including confusion, multiple sclerosis, paresthesia, subdural hernatoma, and tremor, and the development of malignancies, including lymphoma development. The formation of autoaitibodies has also been observed, including symptoms suggestive of alupus-like syndrome following treatment. The most common adverse reaction was injection site reactions, with 20% of patients developing erythema and/or itching, hemorrhage, pain and/or swelling. Other adverse events as a result of systemic administration of adalimumab include clinical flare reaction, rash and pneumonia. Other adverse events included sinusitis, flu syndrome, nausea, abdominal pain, hypercholesterolemia, hyperlipidemia, hematuria, increased alkaline phosphatase levels, back pain, hypertension, as well as more infrequent serious adverse events, including pain, pelvic pain, thorax painarrthythnia, atrial fibrillation, cardiovascular disorder, congestive heart failure coronary artery disorder, heart arrest, hypertensive encephalopathy, myocardial infactpalpitation, pericardial effusion, pericarditis, syncope, tachycardia, vascular disorders, and other disorders. CalcineurinInhibitors 1002721 Calcineurin inhibitors are a group of stcturally diverse smallmolecules irunnoniodulators which function through the inhibition of calcineurin function. Calcineurin is a calcium-activated protein phosphatase which catalyses the dephosphorylation of cytoplasmic NFAT. Upon dephosphorylation, NFAT migrates to the nucleus and forms a regulatory complex involved in the transcription of cytokines. such asTNF-u. IL-2,IL-3 and IL-4 Inhibition of calcineurin function blocks the dephosphorylation event and subsequent cytokine transcription. An unusual aspect of calcineurin inhibition is that cyclosporine, tacrolimus and pimecrolimus are required to form a complex with an imnunophilin for the inhibitory properties to be realized (Schreiber et al, Imunol. Today (1992), 13:136-42; Liu et at, Cell (1991), 66:807~15). For cyclosporine the immunophilin is cyclophilin; tacrolimus and pimecrolimus bind to the FK506-binding protein (FKBP) H O1 ClI HO: H C1 ,H MeO MaO

0 OH 0 OH

NN H 0 H 0O &, 0 HO H H H OMe OMe

tacrolimus pimecrolimus

MeVM &C-Abu-YeGly MeLeu W 0 MeLeu I I MeLeu-DAa- A!a-MeLeu-/d

C62H 11 N 1 0 2 molwt:1202,61

cyclosporine A

[002731Cyclosporine is an I l-residue cyclic peptide produced as a metabolite of the fungus Beauveria nivea and has the chemical name cyclo[[(E)-(S,3R,4R)-3-hydroxy-4-inethyl-2 (methylanino)-6-octenoyl]-L-2-aminobutyryl-N-methylglycyl-N-methyl-L-1eucyl-L-valyl-N

It is provided in several formulations for both systemic or local administration. Sandimmune@ provides cyclosporine in three different formulations: soft gelatin capsules, an oral solution or a formulation for injection. Sandirmmune1 is indicated for prevention of organ rejection in kidney liver or heart transplants. Neoral@ and Gengraf@ provide cyclosporine in two formulations: soft gelatincapsules and an oral solution. They are indicated for prevention of organ rejection in kidney liver or heart transplants, for treatment of patients with severe active, rheumatoid arthritis, or for treatment of severe psoriasis Compared to Sandimmune, Neoral'@i and Gengrat provide increased bioavailability of cyclosporine. Restasis@ provides cyclosporine in an ophthalmic emulsion formulation. It is indicated to increase tear production in patients with reduced tear production due to ocular inflammation associated with keratoconijunctivitis sicca

[002741 Tacrolimus, also known as FK-506 or fujimycin, is a 23-membered macrolide natural product produced by Streptonyes tsukuMensis and has the chemical name [3S

[3R*[E(S*,3S*,4S*)], 4S*,5R*,8S*,9E,2R*t14R*15St16R*18S* 19St*26aR*]] 5.6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5.19-dihydroxy-342-(4-hydroxy-3 methoxycyclohexyl)-l -methylethenyl]-14,16dimethoxy~4,10,12,18-tetramethyl8(2-propenyl) 15,19-epoxy-311-pyrido[2,1-39c][1,4]oxaazacyclotricosine-;7,20,21(44,23TH)-tetrone monohydrate It is provided in formulations suitable forsystemic or topicaladministration.For systemic administration, the Progratf formulation provides an oral capsule or a sterile solution for injection. Prograf is indicated for prevention of organ rejection in liver, kidney or heart transplmts. For topical administration, the Protopic@ formulation is indicated for the treatment ofmoderate-to severe atopic dermatitis. 1002751Pimecrolimus is a seni-synthetic analog of tacrolimus and has the chemical name (IR,9S,12SJ3R.,145.17R,18EI21S,23S,24R,25S,27R)42-[(1E)-2-(1R,3R4S)-4-chloro-3 methoxycyclohexyl}-lI-methylvinyfl-l7-ethyl-1,14-dihydroxy-23,25-dimethoxy13,1921,27~ tetramethyl-11,28-dioxa-4-aza-tricyclo[22,3.1.04,9]octacos-18-ene-2,3,10,16-tetraone, i is provided in a formulation suitable for topical application and is indicated for the treatment of mild-to moderateatopie dermatitis,

[00276] Studies have shown that tacrolimus and pimecrolimus do not suppress Langerhans' cells or dermal connective tissue and therefore do not cause atrophy of the skin, unlike corticosteroids (Stuetz et al, Int. Arch. Allergy Imnunol. (2006), 141:199-212; Queille-Roussel et al, Br. J. Dermatol. (2001), 144:507-13). Because of the importance of calcineurin, systemic administration of calcineurin inhibitors leads to significant side effects. Systemic side effects are related to dose, exposure levels and duration of therapy. Prolonged elevated blood levels result in. hypertension, nephrotoxicity, psychiatric disorders, hyperlipidenia, and profound immunosuppression. Topical application of tacrolimus or pimecrolinus has shown to afford very little, if any, systemic exposure, with tacrolimus having demonstrated less than 0.5 % bioavailability after topical application.

[00277In one embodiment, the auris-acceptable controlled release immunomodulating formulation comprises a calcineurin inhibitor. In another embodiment, the auris-acceptable controlled release irmunomodulating formulation comprises cyclosporine In another embodiment, the auris acceptable controlled release inmunomodulating formiulation comprises tacrolimus. In another embodiment, the auris-acceptable controlled release immunomodulatingformulation comprises pimecrolimus. In. another embodiment, the auris-acceptable controlled release immunomodulating fornutlation comprises a calcineurin inhibitor which induces toxicity upon systernic administration.

1002781Other pharmaceutical agents that are optionally used in combination with immunomodulating-r agents for the treatment of autoinunune and/or inflammatory disorders include other agents that have been used to treat autoinnune and inflammatory disorders, including corticosteroids, local anesthetic agents, chemotherapeutic agents, including cytoxan, azathiaprine or methotrexate; treatment with collagen, ganma glulin,interferons copaxone, or combinations thereof. Accordingly, also contemplated within the scope of the embodimentsherein is the use of t0 other pharaceutical agents in combination with the immunomodulating compositions and fornalations disclosed in the treatment of autoimmune otic disorders. In addition, other pharmaceutical agents areoptionally used to treat attendant symptoms of AED or other autoimmune disorder, including vomiting, dizziness and general malaise. IKK Inhibitors

1002791The transcription ofTNF-a is dependent on the transcription factor NF-KB. In unstimulated cells, NF-K is in the cytoplasm as part of a protein complex with the protein inhibitor of NF-KB,

also known as wB. Activation of NF-KB depends on phosphorylation-induced ubiquitination of the

IB Once poly-ubiquitinated,the IB undergoes a rapid degradation through the 26S proteasome

and the free N-B migrates to the nucleus to activate pro-inflammatory gene transcription. The phosphorylation event which releases NF-kB is mediated by the 1B kinase (IKK) complex,

composed ofIKK kinases.Two IK enzymes, generally referred to as IKK- and IKK-§ (Woronicz et al. Science (1997), 278:866; Zandi et al. Cell (1997). 91:243) or IKK-I andIKK-2 (Mercurio et al Science (1997),278:860) have been discovered. Both forms of IKK can exist as homodimers and as IKK.-tIKK-f heterodimers,Another component of the I1B kinase complex is a regulatory

proteinknown as fKK-y or NEW (NF-KB-Essential Modulator) (Rothwarf et al.Nature (1998), 395:297). NEMO does not contain a catalytic domain, and thus it appears to have no direct kinase activity and it probably serves a regulatory function. Existing datasuggests that the predominant form of IKK in cells is anIKK-a/JKK-f heterodimer associated with either a dimer or a trimer of NEMO (Rothwarf et at Nature (1998) 395:297). Biochemical and molecular biology experiments have identified IKK-a and IKK- as the most likely mediators of TNF-4-induced IkB

phosphorylation and degradation, which results in NF-B activation and upregulation of families of genes involved in inflammatory processes (Woronicz et al. Science (1997); Karin, Oncogene (1999) 18:6867; Karin, .Biol. Chen.(1999) 274:27339). 100280]Many IKK-1inhibitors have been identified. SPC-839 has been extensively studied. It inhibits IKK- with an IC, of 62 nM and reduces paw edema in a rat arthritis model at 30 mgkg.

Carboline PS-1.145 inhibits the IKK complex with an IC of 150 nM and reduces the production of TNF-ct in LPS-challenged mice. BMS-345541,an allosteric inhibitor, inhibits IKK- with an IC3 of 0.3 M, In the mouse collagen-induced arthritis model it significantly reduced the severity of disease at a 30mg/kg dose..A scientific review ofIK inhibitors has been published (Karin et al,Nature Reviews Drug Discovery (2004), 3, 17-26), incorporated herein by reference for such disclosure. 0

N Me H N N

/ N MC N.N NN H

SPC-839 PS-1145 BMS-345541

[00281]In one embodiment, the auris-acceptable controlled release imunomodulating formulation comprises an IKK inhibitor. In a further embodiment, the auris-acceptable controlled release imnunomodulating formulation comprises a IKK4 inhibitor. In another embodiment, the auris acceptable controlled release immnomodulating formulation comprisesa IKK inhibitor which induces toxicity upon systemic administration. In an additional embodiment, the auris-acceptable controlled release immunomodulating formulation comprises a IKK inhibitor which is not orally absorbed. In an additional embodiment, the aris-acceptable controlled release immunomodulating formulation comprises an IKK inhibitor selected from SPC-839,PS-I145,BMS-345541,or SC-514 In an additional embodiment, the auris-acceptable controlled release immunomodulating formulation comprises an IKK inhibitor selected from compounds disclosed in the following group of patent publications: WOl 99901441, W02001068648, W02002060386, W02002030353, W020030 2 9242, WO2003010163, W02001058890, W02002044 153, W02002024679, W02002046171, W02003076447, W02001030774 W02001000610, W02003024936, W02003024935, W02002041843, W0200230423, W02002094265. W02002094322, W02005113544 and W02006076318, all of which are incorporated by reference herein for such disclosure. Interlenkin inhibitors

[00282] Intereukins are a class of cytokines. In certain instances, they are signaling molecules secreted by leukocytes having encountered a pathogen. In certain instances, the secretion of interleukins activates and recruits additional leukocytes to the site ofinfection. In certain instances, the recruitment of additional leukocytes to thesite of infection results in inflaunation (due to the increase in leukocyte containing lymph). IL-1a, IL-I$, IL-2, and IL-8 arefound in middle ear effusions. In certain instances, IL-a and IL- 3are also found in the epithelium of cholesteatomas 1002831il-1 is a class of interleukins comprised of IL-la, and IL-1 IL-I is made by macrophages, B cells, monocytes, and dendritic cells (DC). It binds to receptors ILR/CDI121a and

ILlR2/CD121b. The binding of IL- Ito its receptors results in an increase in cell-surface adhesion factors, This enables themigration of leukocytes to the site of infection 100284IL-2 is made by TM-I1 cells and binds to the receptors CD25/IL2Ra, CD2212Rb, and CDI132/IL2Rg. 11-2 secretion is stimulated by the binding of an antigen to a T-1 cell. The binding of IL-2 to a receptor stimulates the growth, and differentiation of memory T cells 10028511L-8 is made by macrophages. lymphocytes, epithelial cells, and endothelial cells. It binds to CXCRI/ILSRamd CXCR2/ILSRa/CD28. Secretion of ILS initiates neutrophilchemotaxis to the site ofinfection. 1002861In some embodiments, a subject in need thereof is administered an inhibitor of a pro inflannatoryinterleukin. Insome embodiments, thepro-inflammatory interleukinis IL.-1a, IL-, IL-2, orI-. In some embodimentsthe inhibitor of a pro-inflammatory interleukin is a WS-4 (an antibody against 1-8); [Ser iL-8]; or [Ala IL-S] (See U.S. Patent No, 5,451,399 which is hereby incorporated by reference for disclosures relating to these peptides);IL-1RA; SB 265610 (N-(2 Bromophenyl)-N'-(7-cyano-1H-benzotriazol-4-y)urea); SB 225002(N-(2-Bromophenyl)-N'(2

[5 hydroxy-4-nitrophenyl)urea); SB203580 (4-(4-Fluorophenyl)-2-(4-methylsulfivl phenyl)-5-(4 pyridyl) 1H-imidazole); SB272844 (GlaxoSnithKline); SB517785 (GlaxoSmithKline); SB656933 (GlaxoSmithKline); Sch527123 (2hydroxy-NN-dimethyl-3-{2~[[(R)-1-(5-methyl-furan-2-yi) propyllamino]-3.4-dioxo-cyclobut-I-enylamino}-benzamide); PD98059(2-(2-amino-3 methoxyphenyl)-411-1-Benzopyran-4-one); reparixin; N[4-chloro-2hydroxy-3-(piperazine-l !0 sulfonyl)phenyl]-N-(2-chloro-3-fluorophenyl)urea ptoluenesulfonate (See WO/2007/150016 which is hereby incorporated by reference for disclosures relating to this compound); sivelestat; bG3iP (CXCLS((3-74))K11R/G3IP); basiliximab; cyclosporin A; SDZ RAD (40-O-(2-hydroxyethyl) rapamycin); FR235222 (Astellas Pharma); daclizumab; anakinra; AFI2198 (Ac-Phe-Glu-Trp-Thr Pro-Gly-Trp-Tyr-G-i n ToH or combinations thereof PlateletActivatingFactorAntagomnists 002871Platelet activating factor antagonists are contemplated for use in combination with the immunomodulating formulations disclosed herein. Platelet activating factorantagonists include, by way of example only, kadsurenone, phomactin G, ginsenosides, apafant (4-(2- chlorophenyl)-9 methyl.-2[3(4-morpholinyl)-3-propanol-1- yl[611- thieno[3:2.f{[1 .2.4]triazolo]4,3-1]]1.4]diazepine), A-85783, BN-52063, BN-52021, BN-50730 (tetrahedra-47,8,10 methyl-I (chloro-l phenyl)-6 (methoxy-4 phenyl-carbamoyl)-9 pyrido [4',3'-4,5] thieno [3,2f] triazolo-1,2,4 [4,3-a] diazepine 1,4), BN 50739, SM-12502, RP-55778, Ro 24-4736 SR27417A, CV-6209, WEB 2086,VEB 2170, 14-deoxyandrographolide, CL 184005, CV-3988, TC-309, PMS-601,TCV-309 and combinations thereof TAT-a Converting Enzyme (TACE) Inhibitors

[00288] TNF-a is initially expressed on the cell surface as a 26 kDa, 233-amino acid,membrane bound precursor protein, Proteolytic cleavage of the membrane-bound TNT-aby thematrix metalloproteinase TNF-c converting enzyme occurs between Ala-76 and Val77 and results in a 17 kDa matureTNF-a which exists as asoluble trimer. Inhibition of the proteolyti cleavage could provide an alternative to the use of protein-based therapeutics in anti-inflammatory therapy. One potential complication, however, is that TACE is thought to be involved in the processing of other proteins in addition to TNF-a. For example, in a phase II clinical trial,indications of toxic effects in the liver occurred as a result oflTACE inhibition, (Car et al, Society of Toxicology, 46z Annual Meeting, Charlotte, North Carolina, March 25-29,2007). The hypothesis for thismechanism-based toxicity is that TACE also acts on other membrane bound proteins, such as TNFRI and TNFRI.

[002891While toxicities following oral administration are problematic for a dug administered systemically, local delivery to the site of action overcomes this problem Inhibitor GW3333 has a TACE IC of 40 nIM and an ICf of 0,97 pM for inhibiting TNF-a production in the LPS-induced human PBMC cells (Conway et al. IPharmacol. Exp.'her.(2001), 298:900). Nitroarginine analog . A has an IC ,TACE ICo of 4 M and an IC of 0.034 pM for inhibitingTNF-u production in the LPS-induced MonoMac-6 cells (Musso etal, Bioorg. Med. Chem. Letta (2001) 112147), but lacks oral activity, A scientific review of'NF-a converting enzymne inhibitors has been published (Skotnicki et at Annual Reports in Medicinal Chemistry (2003), 38. 153-162),incorporated by reference herein for such disclosure

N

Hq

9Hy' H QH ,NO

[."( H 4kN 'H OrK,0 0r a H No

N NH 2 GW3333 nitroaginne analog A H 1002901Accordingly, in one embodiment, the auris-acceptable controlled release anti-TNF formulation comprises a TACE inhibitor. In another embodiment, the auris-acceptable controlled release anti-TNF formulation comprises a T'ACE inhibitor which induces toxicity upon systemic administration. In additional embodiments, theauris-acceptable controlled releaseanti-TNF formulation comprise a TACE inhibitor which is not orally absorbed. In another embodiment, the auris-acceptable controlled release anti-TNF formulation comprises aTACE inhibitor selected from Nitroarginine analog A, GW3333,ITMI-1, BMS-561392, DPC-3333, TMI-2, BMS-566394, TMI 005, apratastat, GW4459, W-3646, IK-682,G-5402 01-245402 BB-2983, DPC-A38088,DPH~ 067517, R-618,or CI-138,

Toll-likeReceptorinhibitors 1002911Toll-like receptors (TLR) are a family of at least 12 pattern recognition cell-surface and intracellular receptors, The family is defined by the presence of two domains: a ligand-binding domain with multiple leucine-rich repeatsand a short Toll/U-I receptor domain; the latter controllingthe initiation of downstreai-signaling cascades In certain instances, the receptors are activated by the binding of structurally conserved molecules (i.e. the "patterns") found on pathogens. Each receptor recognizes and binds to specific conserved molecules found on pathogens (e.g. TLR2- lipopeptides; TLR3- viral dRNA; TLR4 LPS; TLR5 - flagellin; TLR9 - CpG DNA) In certain instances, the binding of a TLR to a pathogen, initiates theTR signaling cascade which ultimately leads to the activation of various cytokines, chemokines, and antigen-specific and non-specific immune responses. In certain instances, the expression of TLR2 and/or TLR4 is up regulated upon exposure to nontypeable Hemophilus influenza (NTHi). Infection byNT!!i is a common cause of otitis media.

[002921Toll-like receptors belong to a class of singlemembrane-spanning non-catalyticreceptors that recognize structurally conserved molecules derived from breached microbes are believed to play a key role in the innate immune system. Toll-like receptors thus recognize molecules that are broadly shared by pathogens, but are distinguishablefrom the host molecules. These receptors form a superfamily with Interleukin-1 receptors, and have in common a Toll-like receptor domain Toll like receptor agonists, such as CQ-07001,can stimulate Toll-like receptor 3 function, triggering nti inflammatory and tissue regeneration activity. Toll-like receptor modulators, thus, have implication for use in bothauris interna disorders, including AIED, and auris media diseases, including otitis media. In some embodiments, toll-like receptor modulators include toll-like receptor antagonist, partial agonist, inverse agonist, neutral or competitive antagonist, allosteric antagonist, and/or orthosteric antagonist. Other toll-like receptor modulators include but are not limited to polyinosinic-polycytidylic acid [poly(:C)], polyAU, other nucleic acid molecules, including dsRNA agonists (such as AMPLIGEN@ Hemispherx ,Inc,Rockville MD; and POLYADENUR@, Ipsen), and are also contemplated within the scope of the embodiments disclosedherein.

002931In some embodiments, the TLR inhibitor is an ST2 antibody; sST2-Fc (functional marine soluble:ST2-human IgG1 Fc fusion protein; see Biochemical and Biophysical Research Communications, 29 December 2006, voL351, no. 4, 940-946 which is herein incorporated by reference for disclosures related to sST2-Fe); CRX-526 (Corixa); lipid IVA; RSLA (Rhodobacter sphaeroideslipid A); E5531 ((6-0-{2-deoxy-6-O-methyl-4-O-phosphono-3-0-[(R)-3-Z-dode-5 endoyloxydec1]-2-[3-oxo-tetradecanovlaminol-3-O-phosphono-+-D-glucopyranose tetrasodium salt); E5564 (a-D-Glucopyranose3-0-decii-2-deoxy-6-0-[2-deoxy-3-0-[(3R)-3-methoxydecyl-6 O-methyl-2-[[(l1Z)--oxo-11-octadecenyl]amino-4-O-phosphonot-D-gucopyranosyl]-2-(1,3 dioxotetradecyl)amino]-l -(dihydrogen phosphate), tetrasodium salt); compound 4a

(hydroeinnamoyl-L-vaiyl pyrroidine;see PNAS, June 24, 2003, vol. 100, no. 13, 7971-7976 which is herein incorporated by reference for disclosures related to compound 4a); CPG 52364 (Coley Pharmaceutical Group); LY294002 (2(4-Morpholinyl)-8-phenyl-4-I -benzopyran-4-one); PD98059 (2-(2-amino-3-methvxyphenyl)-41-1-Benzopyran-4-one); chloroquine; and an immune S regulatory oligonucleotide (for disclosures relating to IROs seeU.S Patent ApplicationPublication No. 2008/0089883) Auto-hmmune Agents 100294]Also contemplated for use with the formulations disclosed hereinare agents which reduce or ameliorate symptoms or effects as a result of autoimmune disease, including autoimmune inner ear disease (AIlD).Accordingly, some embodiments may incorporate the use of agents which block the effects of TNF-, including but not limited to anti-TNF agents. By way of example only, some anti TNF agents include etanercept (ENBREL), infliximab (REMICNDET) and adalimumab (IUMIRA@), or combinations thereof Other pharmaceutical agents to treat autoirmune disorders include chemotherapeutic agents, including cytoxan, azathiaprine or methotrexate; treatment with

[5 collagen, gamnma globulin, interferons. copaxone, or combinations thereof. IL-I Modulators

[002951 Interleukin-I (IL-1) is a plciotropic cytokine that plays a role in the modulation of local as well as systemic inflammation, immune regulation and hemopoiesis. IL-i§, a member of the IL-1 family, has been implicated in angiogenesis processes, including tumor angiogenesis, In addition, IL-1 has beenshown to stimulate the synthesis of inflammatory eicosanoids in macrophages, fibroblasts, synovial cells and chondrocytes, and is believed to contribute to leukocyteactivation and tissue destruction in arthritic modelsinterfering with I1 activity, therefore5is anapproach for developing a disease modifying therapy for chronic inflamrmnatory disesaes, such as AIED and otitis media. In some embodiments,fL-1 modulators include an IL- antagonist, partial agonist, inverse agonist, neutral or competitive antagonist, allosteric antagonist, and/or orthosteric antagonist. In some embodiments, IL-I modulators include but are not limited to antibodies that specifically recognize IL-] subunits or its receptors, proteins, peptides, nucleic acids, and small molecule therapeutics. In some embodiments, ILL-1 modulators areIL- antagonists, including, for example, AF12198, IL-1 natural antagonists, inactive receptor fragments that bind to IL-1 molecule, and antisense molecules or factors that block expression of IL- cytokine proteins. In some embodiments IL-1 antagonists are IL- antibodies including, by way of example, anakinra (Kinaret@) and ACZ885 (Canakinumab@). In some embdodiments, modulators of IL-1 are antibodies that modulate cytokines and/or growth factors that affect the release and/or expression of IL-i, including, by way of example, ranibizumnab, tefibazurab,and bevacizumab. In some embodiments, IL-1 modulators are IL-I traps that attach to IL-1 and neutralize IL-l before it can bind to cell surface receptors and include, but are not limited to, rilonocept (ArcalystR).

RN~i

[002961In some embodiments, where inhibition or down-regulation of a target is desired (e.g.genes encoding one or more calcineurins, IKKs, TACEs, TLRs, or cytokines), RNA interferenceare utilized.. In some embodiments, the agent that inhibits or down-regulates the target is an siRNA molecule. In certain instances, the siRNA molecule inhibits the transcription of a target by RNA interference (RNAi). In some embodiments, a double stranded RNA (dsRNA) molecule with sequences complementary to a target is generated (e.g. by PCR). In some embodiments, a 20-25 bp siRNA molecule with sequences complementary to a target is generated. In some embodiments, the 20-25 bp siRNA molecule has 2-5 bp overhangs on the 3' end of each strand, and a 5' phosphate terminusand a 3' hydroxyl terminus.In some embodiments, the 20-25 bp siRNA molecule has blunt ends. For techniques for generating RNA sequences see Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), jointly referred toherein as "Sambrook"); Current Protocols in Molecular Biology (F. M. Ausubet et al., eds., 1987, including supplements through 2001); Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc. New York, 2000) which are hereby incorporated by reference for such disclosure.

[002971 i some embodiments,the dsRNA or siRNA molecule is incorporated intoa controlled release auris-acceptable microsphere or microparticle, hydrogel, liposome, actinic radiation curable gel, solvent-release gel, xerogel, paint,foam, in situ forming spongy material, or thermoreversible gel. In some embodiments, the auris-acceptable microsphere, hydrogel, liposome, paint, foam, in situ forming spongy material, nanocapsule or nanosphere or thermoreversible gel isinjected intothe imer ear. In some embodiments, the auris-acceptable microsphere ormicroparticle, actinic radiation curable gel, solvent-release gel, hydrogel, liposome, or thermoreversible gel is injected through the round window membrane, In some embodiments the auris-acceptable microsphere, hydrogel, liposome, paint, foam, in situ forming spongy material, actinic radiation curable get solvent-release gel, nanocapsule or nanosphere or thennoreversible gel is injected into the cochlea, the Organ of Cortithe vestibular labyrinth, or a combination thereof

[002981Incertain instances, after administration of the dsRNA or siRNA molecule, cells at the site of administration (e.g. the cells of cochlea, Organ ofCortandorthevestibularlabyrinth)are transformed with the dsRNA or siRNA molecule. In certain instances following transformation, the dsRNA moleculeis cleaved into multiple fragments of about 20-25 bp to yield siRNA molecules. In certain instances, the fragments have about 2bp overhangs on the 3 end of each strand. 1002991In certain instances, an siRNA molecule is divided into two strands (the guide strand and the anti-guide strand) by an RNA-induced Silencing Complex (RISC).In certain instances, the guide strand is incorporated into the catalytic component of the RISC (i.e. argonaute). In certain instances, the guide strand binds to a complementary target mRNA sequence. In certain instances, the RISC cleaves the target mRNA. In certain instances, the expression of the target gene is down-regulated.

1003001In some embodiments, a sequence complementary to a targetis ligated into a vector. In someembodiments, the sequence is placed between two promoters. In some embodiments, the promoters are orientatedin opposite directions. in some embodiments, the vector is contacted with a cell. In certain instances, a cell is transformed with the vector. In certain instances following transformation, sense and anti-sense strands of the sequence are generated. In certain instances, the sense and anti-sense strands hybridize to form a dsRNA molecule which is cleaved into siRNA molecules. In certain instances, the strands hybridize to form an siRNA molecule. Insome o embodiments, the vector is a plasmid (e.g pSUPR; pSUPERneo; pSUPERneo+gfp). 100301]Insome embodiments, the vector is incorporated into a controlled-release auris-acceptable uicrosphere or nicroparticle, hydrogen, liposome, or thermoreversible gel In some embodiments, the auris-acceptable microsphere, hydrogel, liposome, paint, am, insitufonning spongymaterial., nanocapsule or nanosphere or thernoreversible gel is injected into the inner ear, In some :5 embodiments, the auris-acceptable microsphere or microparticle, hydrogel, liposome, or thermoreversible gel. In some embodiments, the auris-acceptable microsphere, hydrogel, liposome, paint, foam, in situ forming spongy material, nanocapsule or nanosphere or thermoreversible gel is injected into the cochlea, the Organ of Corti, the vestibular labyrinth, or a combination thereof. Aural Pressure Modulators Aquaporin 100302]Contemplated for use with the formulations disclosed herein are agents that treat disorders of theauris, and/or modulate the cells and structures of the auris. In certain instances, an aquaporin is involved in fluid homeostasis. In certain instances, AQP2 iRNA is elevated in rats treated with vasopressin above the levels observed in control animals..In certain instances, Aquaporin- is expressed in the cochlea and endolynphatic sac. In certain instances, Aquaporin-1 is expressed in the spiral ligament, the Organ of Corti, the scala tympani, and the endolymphatic sac, Aquaporin-3 is expressed in the stria vasculatris, the spiral ligament, the Organ ofCorti, the spiral ganglion and the endolymphatic sac. In certain instances, aquaporin 2 (AQP2) mRNA is elevated above normal levels in individuals with endolymphatic hydrops. 100303]Accordingly, some embodiments incorporate the use of agents that modulate an aquaporin. In some embodiments, the aquaporin is aquaporin 1, aquaporin 2 and/or aquaporin 3.In some embodiments, the agent that modulates an aquaporin (e.g, aquaporin 1, aquaporin 2 or aquaporin 3) is an aquaporin antagonist, partial agonist, inverse agonist, neutral or competitive antagonist, allosteric antagonist, and/or orthosteric antagonist. In some embodiments, the aquaporin antagonist, partial agonist, inverse agonist, neutral or competitive antagonist, allosteric antagonist, and/or orthosteric antagonist includes, but is not limited to, substance P RU-486; tetraethylamnionium

(TEA); an anti-aquaporin antibody; a vasopressin and/or a vasopressin receptor antagonist, partial agonist inverse agonist, neutral or competitive antagonist, allosteric antagonist, and/or orthosteric antagonist; or combinations thereof. Estrogen-RelatedReceptor Beta Modulators

[00304] Estrogen-related receptor beta (ERR-beta also known as Nr3b2), an orphan nuclear receptor, is specifically expressed in and controls the development of the ndolymph-producingcells of the inter ear: the strialmarginal cells in the cochlea and the vestibular dark cells in the ampulla and utricle. (Chen et al. Dev. Cell. (2007) 13:325-337), Nr3b2 expression has been localized in the endolymph-secreting strial marginal cells and vestibular dark cells ofthe cochlea and vestibular apparatus, respectively. Studies in knockout mice have shown that strial marginal cells in these animals fail to express multiple ion channel and transporter genes, suggesting a role in the development andor function of endolymph producing epithelia. Moreover, conditional knockout of the Nr3b2 gene results in deafness and diminished endolynmphatic fluid volume.

[0305IOtherstudies suggest a role for estrogenrelated receptor /NR3B2 (ERRINr3b2) in regulating endolymph production, and therefore pressure in the vestibularcochlear apparatus. Treatinent with antagonists to ERR/Nr3b2 may assist in reducing endolymphatic volumeand thus alter pressure in the auris interna structures. Accordingly, agents which antagonize ERR/Nr3b2 expression, protein production or proteintfunction are contemplated as useful with the formulations disclosed herein. 20 GAP Junction Proteins

[003061 Contemplated for use with the formulations disclosed herein are agents that treat disorders of the auris, and/or modulate the cells and structures of the auris. Gap junctions are intracellular connections. In certain instances, a gap junction connects the cytoplasm of two cells. In certain instances, a gap junction facilitates the passage of small molecules (e.g. IU) and ions between the cells, In certain instances, gap junctions are formed of connexins (e.g. six connexins forn a connexon and two connexons forn a gap junction), There are multiple connexins (e.g. Cx23, Cx25, Cx26, Cx29, Cx30, Cx30.2, Cx30.3, Cx3l, Cx3l.1, Cx31.9, Cx32, Cx33, Cx36, Cx37, Cx39,Cx40, Cx40.1, Cx43, Cx45, Cx46, Cx47, Cx50, Cx59, and Cx62), In certain instances, of Cx26 and Cx43 are expressed in a spiral limbus, a spiral ligament, a stria vascularis, cells of the Organ of Corti. In certain instances, non-syndromic deafness is associated with mutations in genes (e.g, GJB2) encoding connexins (e.g. Cx26)In certain instances, sensorineural hearing loss is associated with mutations in genes encoding connexins (e.g. Cx26). In certain instances, the expression of Cx26 and Cx43 is upregulated in a cholesteatoma. In certain instances, the expression of Cx26 is upregulated following acoustic trauma. In certain instances, gap junctions facilitate the movement of K ,ions in endolymph.

1003071Accordingly, some embodiments disclosed herein incorporate the use of agents that modulate gap junction proteins, In some embodiments,; the gap junction protein is a connexin. In some embodiments, the agent that modulates a connexin is a connexin agonist, partial agonist, and/or positive allosteric modulator of a connexin. In some embodiments, the connexin agonist, partial agonist, and/or positive allosteric modulator includes, but is not limited to, astaxanthin; rotigaptide; adenosine; corticotropin-releasing hormone; or combinations thereof Vasopresinand the Vasopressin Receptor

100308]Vasopressin (VP) is a hormone that plays an important part incirculatory andwater homoeostasis. This hormone is synthesised by neurosecretory cells located predominantly in two specific hypothalamic nuclei-the supraoptic nucleus and the paraventricular nucleus. These neurons have axons that terminate in the neural lobe of the posterior pituitary gland (neurohypophysis) in which they release vasopressin. The three vasopressin receptor subtypes (VPIa, VPIb and VP2) all belong to the G-protein coupled receptor family and have differing tissue distributions. The VPla receptor is predominantly located in the vascular smooth muscle, hepatocytes and blood platelets. The VPlb receptors are found in the anterior pituitary. The VP2 receptors are localized in the collecting duct of the kidney and regulate the presentation of aquaporin-2 channels at the apical cell surface. The effect of modulation of the VP2 subtype provides readily observed changes in urine volume and electrolyte levels to determine the pharmacological effects ofanti-diuresis.

[003091Vasopressin regulates systemic osmolality by controlling urinary volume and composition. Vasopressin is secreted in response to increases in plasma tonicity (very sensitive stimulus) or to decreases in plasma volume (less sensitive stimulus). Vasopressin mainly regulates urinary volume by binding to the VP receptor in the collecting duct of the kidney. The VP receptor also exists in the inner ear of rodents, and aquaporin-2 (AQP2) a VP mediated water channel protein, is also expressed (Kitano etal Neuroreport (1997),8:2289-92), Water homeostasis of the inner ear fluid was confirmed to be regulated using the VP-AQP2 system (Takeda et al. Hear Res (2000), 140:--6; Takeda et al. Hear Res. (2003), 182:9-18). A recent study looked at tissue expression of VP2 and AQP2 in human endolymphatic sac by immunohistochemistry and noted that VP2 and AQP2 were located in the epithelial layer of the endolymphatic sac but not in surrounding connective tissue (Taguchi etal, Laryngoscope (2007), 117:695-698). Studies on the systemic administration of vasopressio in the guinea pig showed the development of endolymphatic hydrops (Takeda et al Hear Res (2000), 140:1-6). Additionally, the aquaporin-4 knockout mouse, while otherwise healthy, is deaf (Beitz et at., Cellular and Molecular Neurobiology (2003) 23(3):315-29), This suggests that transport of water and solutes in a manner similar to that of the kidney may play a role in fluid homeostasis of the endolymphatic sac, A mutant human VP2 receptor protein (D136A) has been identified and characterized as constitutively active (Morin et al., FEBS Letters (1998) 441(3):470

5). This hormone-independent activation of the VP2 receptor could play a role in the etiology of conditions such as Meniere's disease.

[003101Contemplated for use with the formulations disclosed herein are agents that treat disorders of the auris, andior modulate the cells (e.gauris sensory cells) and structures of the auris. In certain instances, VP is involved in fluid honeostasis. In certain instances, VP is involved in endolymph and/or perilymph-homeostasis. In certain instances, an increase inendolymph volume increases pressure in the vestibular and cochlear structures. In certain instances, plasma levels of VP are elevated above normal levels in endolymphatic hydrops and/or Meniere's Disease. VasopressinReceptor Modulators L0 1003111Vasopressin receptor modulators can be differentiated based upon their efficacy relative to the vasopressin peptide hormone. A vasopressin receptor full agonist is a mimic of the native peptide. A vasopressin receptor antagonist blocks the effect of the native peptide. A partial agonist can serve as a mimic of the native peptide and induce a partial response, or in the presence of elevated levels of the native peptide; a partial agonist competes with the native peptide for receptor

[5 occupancy and provides a reduction in efficacy, relative to the native peptide alone. For a vasopressin receptor with constitutive activity,an inverse agonist serves to reverse the activity ofthe

receptor.

[00312JAccordingly, some embodiments incorporate the use of agents that modulate vaspressin and/or a vasopressin receptor. In some embodiments, the agent that modulates vasopressin and/or a vasopressin receptor is avasopressin and/or a vasopressin receptor antagonistpartialagonist, inverse agonist, neutral or competitive antagonist, allosteric antagonist, and/or orthosteric antagonist. In some embodiments, the vasopressin and/or a vasopressin receptor antagonist, partial agonistinverse agonist, neutral or competitive antagonist, allosteric antagonist, and/or orthosteric antagonist includes, but is not limited to, an anti-vasopressin antibody; an anti-vasopressin receptor antibody; lithium: OPC-31260 (()-5-dimethylamino-1-(4- [2-methyibenzoylaminobenzoyl) 2,3,4,5-tetrahydro-I 1-benzazepin hydrochloride); WAY-140288 (N44-[3 (Dimethylaninomethyl)-10,11-dihydro-5H-pyrrolo[2,I-c][1,4jbenzodiazepin-10-ycarbonyl]-2 methoxyphenyl]biphenyl-2-carboxainide); CL-385004(5-Fluoro-2-methyl-N-[5-(5H-pyrrolo2,1 c][1 ]benzodiazepine-l0(11i)-yl carbonyl)-2-pyridinylbenzamide); relcovaptan,lixivaptan (VPA 985); tolvaptan; conivaptan; SR 121463A (1-(4-(N-tert-butylcarbamoyl)-2 methoxybenzenesulfbnv)-5-ethoxy-3-spiro-(4-(2-morpholinoethoxy)csyclohexane)indol-2-one fumarate); SR-49059 ((2S)-1-[[(2R,3S)-5-Chloro-3~(2-chlorophenyi)-1-[(3,4-di methoxyphenyl)sulfonyl)~2,3dihydro-3hydroxy11-indol- 2-ylcarbonyl-2 pyrroidinecarboxamide), Lixivaptan (VPA 985); AC-94544 (ACADIA Pharmaceuticals Inc.); AC 88324 (ACADIA Pharmaceuticals Inc.);AC-110484 (ACADIA Pharmaceuticals Inc.); or combinations thereof.

1003131 Recent studies have suggested a role for vasopressin in regulating auris intema pressure by regulating endolymph production, therepy mediating the pressure present in vestibular and cochlear structures (Takeda et al. HearingRes. (2006) 218:89-97).Treatment with vasopressin antagonists, including OPC-31260, resulted in the marked reduction of Menieres disease symptoms. Accordingly, vasopressin antagonists are contemplated as useful with the formulations disclosed herein. Examples of vasopressin antagonists include, but are not limited to OPC-31260 WAY 140288, CL 385004, tolvaptan, conivaptan, SR 121463A, VPA 985 valium (diazepam) benzodiazepines and combinations thereof. Testing of vasopressin antagonistsmay include testing and calculating hydrops reduction with treatment in a guinea pig animal model See, e.g., Chi etal. "The quantification of endolymphatic hydrops in an experimental animal model with guinea pigs", J. Oto-Rhino-LatynoL (2004) 66:56-61.

1003141Agonists of the VP2 receptor are known including OPC-51803 and related analogs (Kondo et al, J. Med, Chem. (2000) 43:4388; Nakamura et al., Br. J. Pharmacol (2000) 129(8)1700; Nakamure et al., J.Pharmacol. ExpTher.(2000)295(3):1005) and WAY-VNA-932 (Caggiano S Drugs Gut (2002) 27(3):24 8). Antagonists of the VP2 receptor includelixivaptan, tolvaptan, conivaptan, SR-121463 and OPC-31260 (Martin et aL, J, Am. Soc. Nephrol. (1999) 10(10):2165; Gross et aL, Exp. Physiol. (2000) 85: Spec No 253S; Wong et al.,Gastroent April 2000, vol 118, 4 Suppl. 2. Part 1); Norman et aDrugs Fut (2000)25(11):1121; Inoue et al, Cin. Pham. Therap. (1998) 63(5):561). In testing against the constitutively activated D136A mutant VP2 receptor, SR 1211463 and OPC-31260 behaved as inverse agonist (Morin et al., FEBS Letters (1998) 441(3):470 75).

NN NN . IN, N

modulat receptor NMDA

AIDA ReceptorAModuiators 1003151 Contemplated for use with the formulations disclosed herein are agents that modulate the 5deceneration of neurons and/orhair cells of the a.-is, and agents,,for treatingor ameliorating hearing disorders such as tinnitus. Accordingly, some embodiments incorporate the use of agents which muodulate NMDA receptors. 1003161In certain instances,the over-activation of the NMDA glutamate receptors by the binding of excessive amounts of glutamate, results in the excessive opening of the ion channels under their control. In certain instances, this results in abnormally high. levels of Ca and Na entering the neuron. In certain instances, the influx of Ca and Na into the neuron activates multiple enzymes including, but not limited to, phospholipases, endonucleases, and proteases. In certaintinstances, the over-activation of these enzymes results in tinnitus, and/or damage to the cytoskeleton, plasma membrane, mitochondria, and DNA of the neuron, In certain instances, the NMDA receptor modulator neramexane treats, and/or ameliorates the symptoms of timitus. 1003171In some embodiments, the agent that modulates the NMDA receptor is an NMDA receptor antagonist, In some embodiments, the agent that modulates an NMDA receptor is an NMDA receptor antagonist, partial agonist,inverse agonist, neutral or competitiveantagonist, allosteric antagonist and/or orthosteric antagonist. In some embodiments, the agent which antagonizes the NMDA receptor includes, but is not limited to, I -aminoadamantane.dextronethorphan, dextrorphan, ibogaine, ketanine, nitrous oxide, phencyclidine, riluzole, tiletamine, memantine, neramexane. dizocilpine, aptiganel, remacimide, 7-chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic acid, I-aminocyclopropanecarboxylic acid (ACPC),AP7 (2-amino-7 phosphonoheptanoic acid), APV (R2-amino-5-phosphonopentanoate), CPPene (3-{(R)2 carboxypiperazin-4-ylj-prop-2-enyl- -phosphonic acid); (+)-(1S.2S)-i-(4-hydroxy-phenyl)-2-(4 hydroxy-4-phenylpiperidino)-I -pro-panol; (1S 2)--(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy 4-phenylpiperi-dino)-1-propanol;(3R, 4S)3-(4(44-fuorophenyl)-4-hydroxypiperidin-1-y1 ) chroman-4;7-diol; (IR*,?R*)-1-(4-hydroxv-3-methylphenyl)-2-(4-(4-fluorophenyl)-4 hydroxypiperidin-1-yl)-propan-l-ol-mesylate; and/or combinations thereof. ENaC Receplor Modulators

10031.81The epithelial sodium channel (ENaC, sodium channel non-neuronal I (SCNNI) or amiloride sensitive sodium channel (ASSC)) is a membrane-bound ion-channel that is pemeable for Li-ions, protons and Na-ions. The ENaC is located in the apical membrane of polarized epithelial cells and is involved in transepithelial Nation transport. Na4K+-ATPase is also involved in Na' transport and ionhoneostasis.

1003191ENaC plays a role in the Na-- and K+-ion homeostasis of blood, epithelia and extraepithelial fluids by resorption of Na+-ions. Modulators of theactivity of ENaC modulate aural pressure and include, by way of example, themineralcorticoid aldosterone, triamterene and amiloride.

Osmotic Diuretics t003201Contemplated for use with the compositions disclosed herein are agents which regulate aural pressure. Accordingly, some embodiments comprise osmotic diuretics. An osmotic diuretic is a substance that produces an osmotic gradient between two spaces. In certain instances, an osmotic diuretic produces an osmotic gradient between the endolynphatic and perilymphatic spaces. In certain instances, an osmotic gradient between the endolynphatic and perilymphatic spaces exerts a dehydrating effect on the endolyrmphatic space. in certain instances, dehydrating the endolymphatic space decreases aural pressure.

[00321] Accordingly, in some embodiments of the compositions and formulations disclosed herein, the aural pressure modulator is an osmotic diuretic. in some embodiments, the osmotic diuretic is erythritol, mannitol, glucose, isosorbide, glycerol; urea; or combinations thereof 100322]In some instances, contemplated for use in combination with the aural pressure modulating formulations disclosed herein are diuretic agents, A diuretic agent isa drug that elevates the rate of urination. Such diuretics include triamterene amiloride, bendroflumethiazide, hydrochlorothiazide, furosemide, torsemide, bumetanide.acetazolamide, dorzolamide and combinations thereof.

ProgesteroneReceptors

[00323jContemplated for use with the formulations disclosed herein are otic therapeutic agents that treat disorders (e.g., inflammation) of the auris, and/or modulate the cells and structures of the auris. Progesterone is a steroidal hormone. In certain instances, progesterone is a ligand for a progesterone receptor. In certain instances, progesterone is found in the brain. In certain instances, progesterone affects synaptic functioning. In certain instances, progesterone is associated with partial or complete loss of hearing. In certain instances, females taking progesterone and estrogen experienced greater hearing loss than females taking estrogen alone (e.g. about 10% to about 30%).

[003241Accordingly, some embodiments incorporate the use of agents that modulate progesterone and/ora progesterone receptor.In some embodiments, the agent that modulates progesterone and/or a progesterone receptor is a progesterone and/or progesterone receptor antagonist, a partial agonist, an inverse agonist, a neutral or competitive antagonist, an allosteric antagonist, and/or an orthosteric antagonist. In other embodiments, the agent that modulates progesterone and/or aprogesterone receptor includes, but is not limited to, RU-486 ((11.17 b)-f1-[4-(Dimethylamino)phenyl]-1I7 hydroxy-I7-(I-propyn yI)-estra-49-dien-3-one); CDB2914 (17a-acetoxy-11-j-[4-N,N dimethylaminophenylj-19-norpregna-4,9-iene-3.20-dione); CDB-4124 (17a-acetoxy-21-methoxy 11p-[4-N,N-dimethyaminophenyl]-19-norpregna-4,9- diene-3,20-dione); CDB-4453 (17a-acetoxy 21-methoxy-11 p-[4--N-methylaminophenyl]-19-norpregna-4,9-diene-3,20-dione); RTI 3021-022 (Research Triangle Institute); ZK 230211 (11-(4-acetylphenyl)-17-hydroxy-7-(1,1,2,2,2 pentafluoroethyl)estra-4,9-dien-3 -one); ORG 31710 (11-(4-dimethylaminophenyl)6-methyl-45 dihydro(estra-4,9-diene-17,2-(3H)-furan)-3-one); ORG 33628 (Organon); onaprisione (ZK 98299); asoprisnil; ulipristal; a anti-progesterone antibody; ananti-progesterone receptor antibody; or combinations thereof. Prostaglandins

[003251Prostaglandins are members of a group of fatty-acid derived compounds and depending upon the subtype, participate ina variety of functions including control of constriction or dilation in vascular smooth muscle cells, aggregation or disaggregation of platelets, sensitization of spinal neurons to pain, increase or decrease in intraocular pressure, regulation of inflammatorymediation, regulation of calcium movement, control of hormone regulation and control of honnonal regulation. Prostaglandins have both paracrine and autocrine functions, and are a subclass of eicosanoid compounds.

1003261Prostaglandin analogues, such as latanoprost, travoprost, unoprostonc, minprostin F2 alpha and biitoprost, have been shown in reduce intra-ocular pressure in glaucoma patients by enhancing the uveoseleral outflow, possibly through vasodilation mechanisms, in addition to effects on the trabecular meshwork. Insensorincural hearing loss animal models, noise exposure induces 8

isoprostaglandin F2c production in the cochlea, concomitant with an increase in vasoconstriction and reduced blood flow. Treatment with SQ29548, a specific antagonist of 8-isoprostaglandin F2t, prevents these noise-induced changes in cochlear blood flow and vascular condictance. Further. the prostaglandin analogue JB004/A improves hearing, and treats,and/or the symptoms of tinnitus and vertigo in patients suffering from Minire's disease. Inhibition of prostaglandin F2 function also reduces tinntius in patients suffering fom Mcniere's disease, as well as improvements in hearing and vertigo. Finally, prostaglandins have beenimplicated in chronic inflammation associated with otitis media.

[00327JAccordingly, one embodiment disclosed herein is the use of prostaglandin modulators, including latanoprost, travoprost unoprostone, minprostin F2-alpha, bimtoprostand SQ29548, and JB004/A. (Synphora AB) to ameliorate or decrease inner ear and middle ear disorders, including Meniere's disease, tinnitus,vertigo, hearing loss and otitis media. RNAi

[00328]In some embodiments, where inhibition or down-regulation of a target is desired (e.g. genes ERR, and Nr3b2), RNA interferenceare utilized. In some embodiments, the agent that inhibits or down-regulates the target is an siRNA molecule. In certain instances, the siRNA molecule is as described herein. Cytotoxic Agents

[003291 In some instances, immunomodulators and/or aural pressure modulators are useful in treatment of inflammatory otic disorders.

[003301Any cytotoxic agent useful for the treatment of otic disorders, e.g., inflanunatory diseases of the ear or cancer of the ear, is suitable for use in the formulations and methods disclosed herein. In certain embodiments, the cytotoxic agent is an antimetabolite, an antifolate, an alkylating agent, a DNA intercalator, an anti-TNF agent, an anti-angiogenic agent, an anti-inflammatory agent, and/or an immunomodulatory agent. in some embodiments, the cytotoxic agent is a protein, a peptide, an antibody, DNAa carbohydrate, aninorganic molecule, or an organic molecule. In certain embodiments, the cytotoxic agents are ytotoxic small molecules. Typically, cytotoxic small molecules are of relatively low molecular weight e.g., less than 1,000, or less than 600-700, or between 300-700 molecular weight.in some embodiments, the cytotoxic small molecules will also have anti-inflammatory properties. 1003311In certain embodimentsthe cytotoxic agents are methotrexate (RIEUMATREX, Amethopterin) cyclophosphamide (CYTOXAN@), and thalidomide (THALIDOMID). All of the compounds can be used to treat cancer, including cancer of the ear. Further, all of the compounds have anti-inflammatory properties and can be used in the formulations and compositions disclosed herein for the treatment of inflanunatory disorders of the ear,including ATED. 1003321Although systemic administration of methotrexate, cyclophosphamide and thalidoide is currently used to treat or is being investigated for the treatment of otic disorders, such as inflammatory otic disorders, including.AIED, Menieres disease, and.Behgetfs disease, as well as cancer of the ear, the cytotoxic agents are not without the potential for serious adverse side effects. Moreover, cytotoxic agents which demonstrate efficacy but are otherwise not approvable because of safety considerations is also contemplated within the embodiments disclosed hereinIt is contemplated that localized application of the cytotoxic agents to the target otic structures for treatment of autoinmune and/or inflammatory disorders, as well as cancer of the ear, will resultin the reduction or elimination of adverse side effects experienced with systemic treatment. Moreover, localized treatment with the cytotoxic agents contemplated herein will also reduce the amount of agent needed for effective treatment of the targeted disorder due, for exampleto increased retention

[0 of the active agents in the auris internal and/or media, to the existence ofthe biological blood barrier in the auris internal, or to the lack of sufficient systemic access to the auris media.

[003331In some embodiments, cytotoxic agents used in the compositions, formulations, and methods disclosed herein are metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of cytotoxic agents, includingmethotrexate, cyclophosphamide, and thalidomide. Particularly preferred

[5 are metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of cytotoxic. agents, e.g., methotrexate, cyclophosphamide.and thalidomide, that retain at least partially the cytotoxicity and anti-inflammatory properties of the parent compounds. in certain embodiments, analogues of thalidomide used in the formulations and compositions disclosed herein arelenalidomide (REVLIMID@) and CC-4047 (ACTIMID@). 1003341Cyclophosphamide is a prodrug that undergoes in vivo metabolism when administered systemically. The oxidized metabolite 4-hydroxycyclophosphamide exists in equilibrium with aldophosphamide, and the two compounds serve as the transport forms of the active agent phosphoramide mustard and the degradation byproduct aerolein.'Thus, in someembodiments, preferred cyclophosphamide metabolites for incorporation into the formulations and compositions disclosed herein are 4-hydroxycyclophosphamide aldophosphamide, phosphoramide mustard, and combinations thereof

[00335]Other cytotoxic agents used in the compositionsformulations, and methods disclosed herein, particularly for the treatment of cancer ofthe ear, are any conventional chemotherpetic agents, including acridine carboxamideactinomycin 17-N-allyiamino-17-demethoxygeldanamycin, aminopterin, amsacrine, anthracycline, antineoplastic, antineoplaston, 5-azacytidine, azathioprine B1L22, bendamustine, biricodar, bleomycin, bortezomib, bryostatin, busulfan, calyculin, camptothecin, capecitabine, carboplatin, chlorambucil, cisplatin, cladribine, clofarabine, cytarabine, dacarbazine, dasatinib, daunonibicin. decitabine, dichloroacetic acid, discodermolide, docetaxel, doxorubicin, epirubicin, epothilone, cribulin, estramustine, etoposide, exatecan, exisulind, ferruginol floxuridine, fludarabine, fluorouracil, fosfestrol, fotemustine, gemcitabine, hydroxyurea, IT-0 lidarubicin, ifosfamide, imiquimod, irinotecan, iroftlven, ixabepilone, laniquidar, lapatinib,

- 78'N,- lenalidomide, loniustine, lurtotecan, mafosfamide, iasoprocolrmechlorethamine, melphalan, iercaptopurine, iitomycin, mitotane, initoxantrone, nelarabine, nilotinib, oblimersen, oxaliplatin, PAC-1, paclitaxelpenetrexed, pentostatin, pipobroman, pixantrone, plicamycin, procarbazine, proteasome inhibitors (e.g, bortezonib), raltitrexed rebeccamycin, rubitecan, SN38, salinosporanide A, satraplatin, streptozotocin, swainsonine, tariquidar, taxane, tegafur-uracil, temozolomide, testolactone, thioTEPAztioguanine, topotecan, trabectedin, tretinoin, triplatin tetranitrate, tris(2-chloroethvljamine, troxacitabine, uracil mustard, valirubicin, vinblastine, vincristine, vinorelbine vorinostat. and zosuquidar Auris Sensory Cell Modulators

[00336]In some instances, immunomodulators and/or aural pressure modulators niodulate the function of neurons and/or auris sensory cells. Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration of neurons and/or hair cells of the auris, promote the growth of neurons and/or hair cells of theauris, and agents for treating or ameliorating heaing loss or reduction resulting from destroyed, stunted, malfunctioning, damaged, fragile or missing hairs in the inner ear, Accordingly, some embodiments incorporate the use of agents which promote the survival of neurons and otic hair cells, and/or the growth of neurons and otic hair cells. In some embodiments, the agent which promotes the survival of otic hair cells is a growth factor, In some embodiments, the growth factor modulator is a growth factormodulator antagonist, partial agonist, inverse agonist, neutral or competitive antagonist, allosteric antagonist, and/or orthosteric antagonist.

Amiostine

[003371Contemplated for use with the fornmlations disclosed herein are agents that modulate the degeneration of neurons and/or hair cels of the auris, and agents for treating or ameliorating hearing loss or reduction resulting from destroyed, stuntedmalfunctioning, damaged, fragile or missing hairs in the inner ear. Accordingly, some embodiments incorporate the use of agents which rescue neurons and otic hair cells from cisplatin-induced ototoxicity.

100338]Amifostine (also known as WR-2721, or ETIYOL®) is a cytoprotective agent. In certain instances, it prevents or ameliorates the damage to neuron and otic hair cells caused by cisplatin. In certain instances, doses at or above 40 mg/kgare needed to protect against orameliorate the ototoxic effects of cisplatin,

Anti-intercellular adhesion molecule -iantibodyy

[003391Contemplated for use with the formulations disclosed herein are antibodies to anti intercellular adhesion molecule (ICAM), In some instances, ICAM blocks the cascade ofreactive oxygen species associated with exposure to noise. In sonic instances modulation of the cascade of reactive oxygen species associated with exposure to noise ameliorates or reduces the degeneration of neurons and/or hair cells of the auris. Accordingly, some embodiments incorporate the use of agents that are antibodies to ICAMs (e.g, anti-ICAM- Ab, anti-ICAM-2.Ab or the like). Modulation of/Atoh/Math! 1003401Contemplatedfor use with thefornnulations disclosed herein are agents that promote the growth and/or regeneration of neurons and/or oic hair cells. Atohl is a transcription factor which binds to an E-box, In certain instances, it is expressed during the development of the hair cells of the vestibular and auditory systems. In certain instances, mice with Atohl knocked-out did not develop otic hair cells, In certain instances, adenoviruses expressing AtohI stimulate the growth and/or regeneration of otic hair cells in guinea pigs treated with ototoxic antibiotics Accordingly, some !0 embodiments incorporate modulation of the Atohi gene.

[003411In some embodiments, a subject is administered a vector engineered to carry the human Atohl gene (the "Atohl vector"). For disclosures of techniques for creating the Atohl vector see US. Pub. No. 2004/02475750, which ishereby incorporated by reference for those disclosures. in some embodiments, theAtoh Ivector is a retrovirus. In some embodiments, the Atohl vector is not .5 a retrovirus (e.g. it is an adenovirus a lentivirus; or a polymeric delivery system suchas METAFECTENE, SUPERFECT, EFFECTENE@, or MIRUS TRANSIT).

[00342n hlsome embodiments, the Atohl vector is incorporated into a controlled-release aris acceptable microsphere or microparticle, hydrogel, liposome, or thermoreversible gel.In sone embodiments, the auris-acceptable inicrosphere, hydrogelliposome, paint, foam, in situ forming spongy material, nanocapsule or nanosphere or thermoreversible gel is injected into the inner ear. In some embodiments, the auris-acceptable microsphere or microparticle, hydrogel, liposome,or thermoreversible gel In some embodiments, the auris-acceptable microsphere, hydrogel, liposome, paint, foam, in situ formingspongy material, nanocapsule ornanosphere or thermoreversible gel is injected into the cochlea, the Organ of Corti, the vestibular labyrinth, or a combination thereof.

[003431In certain instances, after administration of the Atohi vector, the Atohl vector infects the cells at the site of administration (e.g. the cells of cochlea, Organ of Corti, and/or the vestibular labyrinth). In certain instances the AtohI sequence is incorporated into the subject's genome (e.g. when the Atohl vector is a retrovirus), h certain instances the therapy will need to be periodically re-administered (e.g. when the Atohl vector is not a retrovirus).in some embodiments, the therapy is re-administered annually, In some embodiments, the therapy is re-administered semi-annually. In some embodiments, the therapy is re-administered when the subject's hearing loss is moderate (i.e. the subject cannot consistently hear frequencies less than 41 db to 55 dB) to profound (i.e. the subject cannot consistently hear frequencies less than 90 dB).

[0034411n some embodiments, subject is administered the Atohl polypeptide. In some embodiments, the Atol polypeptide is incorporated into controlled-release auris-acceptable mnicrosphere or microparticle, hydrogen, liposome, or thermoreversible gel. In some embodiments, the auris-aeceptable nicrosphere, hydrogen, liposome, paint, foam, in situ foringspongymaterial, nanocapsule or nanosphere or thermoreversible gel. In some embodiments, the auris-acceptable microsphere or microparticle, hydrogel, liposome, or thermoreversible gel. In some embodiments, the auris-acceptable microsphere, hydrogel, liposome, paint, foam, in situ forming spongy material, nanocapsule or nanosphere or therrnoreversiblegel is injected into the inner ear, In some embodiments, the auris-acceptable microsphere or microparticle, hydrogen, liposome, or thermoreversible gel. In some embodiments. the auris-acceptable microsphere, hydrogel, liposome, paint, foam, in situ forming spongy material, nanocapsule or nanosphere or thermoreversible gel is injected into the cochlea, the Organ of Corti, the vestibular labyrinth, or a combination thereof In tO some embodiments, the auris-acceptable microsphere or microparticle, hydrogel, liposome, or thermoreversible gel. In some embodiments, the auris-acceptable microsphere, hydrogel, liposome, paintfoam,insituformingspongymaterial, nanocapsule or nanosphere or thermoreversible gel is placed in contact with the round window membrane.

[003451In some embodiments, a subject is administered a pharmaceutically acceptable agent which modulates the expression of the Atohl gene or activity of the Atohl polypeptide In some embodinents, the expression of the Atohl gene or activity of the Atohl polypeptide is upregulated. In some embodiments, the expression of the Atohl gene or activity of the Atohl polypeptide is down-regulated. j00346]In certain instances, a compound which agonizes or antagonizes.Atohl is identified (e.g. by )0 use of a high throughput screen) In some embodiments, a construct is designed such that a reporter gene is placed downstream of an F-box sequence. In some embodiments, thereporter gene is luciferase, CAT, GFP, P-lactamase orf-galactosidase. In certain instances, the Atohl polypeptide binds to the F-box sequence and initiates transcription and expression of the reporter gene. In certain instances, an agonist of Atohi aids or facilitates the binding of Atohl to the E-box sequence, thus increasing transcription and expression of the reporter gene relative to a pre-determined baseline expression level. I certain instances, an antagonist of Atol .blocks the binding of Atohl to the E box, thus decreasing transcriptionand expression of the reporter gene relative to a pre-determined baseline expression level. BRA-3Aodulators

100347] Contemplated for use with the formulations disclosed herein are agents that promote the growth and/or regeneration of neurons and/or otic hair cells. BRN-3 is a group of transcription factors that include, but are not limited to, BRN-3a, BRN-3b, and BRN-3c. In certain instances, they are expressed in postmitotic hair cells.In certain instances, the hair cells of mice with BRN-3c knocked-out did not develop stereocilia and/or underwent apoptosis. In certain instances, BRN3 genes regulate the differentiation of inner ear supporting cells into inner ear sensory cells, Accordingly, some embodiments incorporate modulation of the BRN3 genes, andlor polypeptides.

100348]In some embodiments, a subject is administered a vector engineered to caya human BRN 3 gene (the "BRN3 vector") i some embodiments, the BRN3 vector is a retrovirus. In some embodiments the BRN3 vector is not a retrovirus (e.g. it is an adenovirus; a lentivirus; ora polymeric delivery system such as METAFECTENE@, SUPERFECT@, EFFECTENE@, or MlRUS'TRANSIT@).

[003491In some embodiments, the subject is administered the BRN3 vector before, during, or after exposure to an ototoxic agent (e.g an aminoglycoside or cisplatin), ora sound of sufficient loudness to induce acoustic trauma.

003501In some embodiments, the BRN3 vector is incorporated into a controlled-release auris acceptable inicrosphere or microparticle, hydrogel, liposome, or thermoreversible gel. In some embodiments, the auris-acceptable microsphere, hydrogel, liposome paint, foam, in situ forming spongy material,nanocapsule ornanosphere orthemnoreversible gel is injected into the inner ear In some embodiments, the auris-acceptable microsphere or microparticle, hydrogelliposome, or thermoreversible gel. In somic embodiments, the auris-acceptable microsphere hydrogel, liposome paint, foam, in situ forming spongy material, nanocapsule or nanosphere or thermoreversible gel is injected into the cochlea, the Organ of Corti, the vestibular labyrinth, or a combination thereof.

1003511In certain instances, after administration of the BRN3 vector, the BRN3 vector infects the cells at the site of administration (e.g. the cells of cochlea, Organ of Corti, and/or the vestibular labyrinth). In certain instances the BRN3 sequence is incorporated into the subject's genoine (e.g. when the BRN3 vector is a retrovirus)I h certain instances the therapy will need to be periodically re-administered (e.g. when the BRN3 vector is not a retrovirs).

1003521In some embodiments, a subject is administered a BRN3 polypeptidein some embodiments, the BRN3 polypeptideis incorporated into contoled-releasearis-acceptable inicrosphere or microparticle, hydrogel, liposome, or thermoreversible gel. In. some embodiments, the auris-acceptable microsphere, hydrogel, liposome, paint, foam, in situ forming spongy material, nanocapsule or nanosphere or thermoreversible ge .In some embodiments, the auris-acceptable microsphere ormicropaticle, hydrogel, liposome, or thermoreversible gel. In some embodinents, the atis-acceptable microsphere, hydrogelliposome, paint, foam, in situ forningspongy maternal, nanocapsule or nanosphere or thermoreversible gel is injected into the inner ear. In some embodiments, the auris-acceptable microsphere or microparticle, hydrogel, liposome, or thermoreversible gel In some embodiments, the auris-acceptable microsphere, hydrogel, liposome, paint, foam, in situ forming spongy material,nanocapsule or nanosphere or thermoreversible gel is injected into the cochlea, the Organ of Corti, the vestibular labyrinth, or a combination thereof. In some embodiments, the auris-acceptable nicrosphere or microparticle, hydrogel, liposome, or thermoreversible gel. In some embodiments, the auis-acceptable microsphere, hydrogel, liposome paint, foam, in situ forming spongy material nanocapsule or nanosphere or thermoreversible gel is placed in contact with the round window ieibrane,

[00353]In some embodiments,a subject is administered a pharmaceutically acceptable agent which modulates the expression of the BRN3 gene or activity of the BRN3 polypeptide. In some embodiments, the expression of the BRN3 gene or activity of the BRN3 polypeptide is up-regulated. in some embodiments, the expression of the BRN3 gene or activity of the BRN3 polypeptide is down-regulated.

t00354]in some embodimentsa compound which agonizes or antagonizes BRN3 is identified (e.g by use of a high throughput screen), In someembodimentsa construct is designed such that a .0 reporter gene is placed downstream of a BRN3 binding site. in some embodiments, the BRN3 bindingsite has the sequence ATGAATTAAT (SBNR3). In some embodiments, the reporter gene is luciferase, CAT, GFP, i-lactamase or 3-galactosidase, In certain instances, the BRN3 polypeptide binds to the SBNR3 sequence and initiates transcription and expression of the reporter gene. In certain instances,an agonist of BRN3 aids or facilitates the binding of BRN3 to the SBNR3 .5 sequence thus increasing transcription and expression of the reporter gene relative to a pre determined baseline expression level. In certain instances, an antagonist of BRN3 blocks the binding of BRN3 to the SBNR3, thus decreasing transcription and expression of thereporter gene relative to a pre-determined baseline expression level. Carbamates

?0 [003551Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration of neurons and/or hair cells of the auris, and agents for treating or ameliorating hearing loss or reduction resulting from destroyed, stunted, malfunctioning damaged, fragile or missing hairs in the inner ear. In certain instances, carbanate compounds protect neurons and otie hair cells from glutamate-induced excitotoxicity. Accordingly, some embodiments incorporate the use of carbamate compounds. In some embodiments, the carbamate compounds are 2-phenyl-1,2 ethanediol monocarbomates and dicarbamates, derivatives thereof, andor combinations thereof Estrogen Receptors 1003561In some enbodiments, the agent that promotes the survival of otic hair cells is an Estrogen Receptor agonist. in some embodiments, the estrogen receptor agonist is a partial agonist or inverse agonist,

[003571In certain instances, Estrogen Receptor p (ER§) is expressed in an outer hair cell, an inner hair cell, a spiral ganglion neuron, or combinations thereof. In certain enbodiinents, agonism of ERa and/or ER p3ameliorates hearing loss resulting from acoustic trauma. In certain embodiments, agonism of ERu and/or ERp increasesand/or up-regulates the expression of aneurotroph gene and/or the activity of a neurotroph polypeptide (e.g. BDNF), In certain ebodiments, antagonism of ERa and/or ERfincreases hearing loss resulting from acoustic trauma. In certain embodiments, antagonism of ERa andor ERp down-regulates the expression of a neurotroph gene andor the activity of a neurotroph polypeptide (e.g BDNF),

[00358]1n some embodiments, theERa agonist is PPT (4,44"-(4Propy[11]-pyrazole1,3,5 triyl)trisphenol): SKF-82958 (6chloro-7,8-dihydroxy-3-aliyl-I-phenvl-2,3,4,5-tetrahydro-11H-3 benzazepine); estrogen; estradiol; estradiol derivatives, including but not limited to 17§ estradiol, estrone, estriol, synthetic estrogen compositions or combinations thereof. In some embodiments,the ERfi agonist is ERp-1.31, phytoestrogen, MK 101 (bioNovo); VG-1010 (bioNovo); DPN (diarylpropiolitrile); ERB-041 ; WAY-202196; WAY-214156; genistein; estrogen; estradiol; estradiol derivatives, including but not limited to 17-4 estradiol, estrone, estriol, synthetic estrogen compositions or combinations thereof Other ERJ agonists include select benzopyrans and triazolo tetrahydrofluorenones, disclosed in LUS. Patent No. 7,279,499, and Parker et al., Bioorg. & Med. Chem. Ltrs, 16: 4652-4656 (2006) each of which is incorporated herein by reference for such disclosure. In some embodiments, a neurotroph is administered before, after, or simultaneously with an Estrogen Receptor [ (ERP) agonist. In some embodiments, the neurotroph is BDNF, CNTF, Gi)NF, neurotrophin-3, neurotrojhin-4, and/or combinations thereof. Fatty Acids

[00359 Contemplated for use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris. Accordingly, sonic embodiments incorporate the use offatty acids In certain instances, themembranesurrounding auditory neurons and the vestibulocochlear nerve comprise fatty acids. In certain instances, a deficiency in omega-3 fatty acid results in a decreased response to auditory stimuli. In certain instances, maternal deficiency of alpha-linolenic acid (ALA) leads to offspring with hearing deficiency. In some embodiments, the fatty acid includes but isnot limited to an omega-3 fatty acid an omega-6 fatty acid, or combinations thereofIn some embodiments, the omega-3 fatty acid is Q Linolenic acid, Stearidonic acid, Eicosatrienoic acid, Eicosatetraenoic acid, Eicosapentaenoic acid, Docosapentaenoic acid, Clupanodonic acid, Docosahexaenoic acid, Tetracosapentaenoic acid, Tetracosahexaenoic acid (Nisinic acid), or combinations thereof In some embodiments, the omega 3 fatty acid is a-Linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, or combinations thereof;in some embodiments, the omega--6 fatty acid is Linoleic acid, Gainma-linolenic acid, Eicosadienoic acid, Diihomo-gamma-linolenic acid, Arachidonic acid, Docosadienoic acidAdrenic acid, Docosapentaenoic acid, Calendic acid, or combinations thereof. Gamma-SecretaseInhibitors

[003601 Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration of neurons and/or hair cells of the auris, and agents for treating or amelioratinghearing loss orreduction resulting from destroyed, stunted, malfunctioning, damaged, fragile or missing hairs in the inner ear, Accordingly, sonic embodiments incorporate the use ofagents which inhibit

Notchi signaling, Notchl is a transmembrane polypeptide which participates in cell development. In some embodiments, the agents which inhibit Notchl signaling arey-secretase inhibitors, In certain instances, the inhibition of Notchl byay-secretase inhibitor-following treatment with an ototoxic agent, results in the production of otic hair cells In some embodiments, the -secretase inhibitor is LY450139 (hydroxylvaleryl monobenzocaprolactam), L685458 (1S-benzyl-4R[I-[I -S-carbamoy-2 phenethvlcarbamovl)-1.S-3-methylbutylcarbamoyll-2R-hydroxy-5-phenylpentyllcarbamic acid ter

butyl ester); LY411575 (N[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl-N[(7S)5-methyl-6 oxo-6,7-dihydro-51-Idibenzo[bid]azepin-7yl]-L-alaninamide), MK-0752 (Merck), tarenflurbil, and/or BMS-299897 (2[(R)+[(4-chloropheny)sulfony(2,5-difluorophenyl)amino]ethyl]-5 fluorobenzenepropanoic acid). Glutanate-ReceptorModulators

[003611Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration of neurons and/or hair cells of the auris, and agents for treating or ameliorating hearing loss or reduction resulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodiments incorporate the use of agents which modulate glutamate receptors. In some embodiments. the glutamate receptor is the AMPA receptor, the NMDA receptor, and/or a group II or III mGlu receptor.

f003621In some embodiments, theagent that modulates the AMPA receptor is anAMPA receptor antagonist. n some embodiments, the agent which antagonizes the AMPA receptors is CNQX (6 cyano-7-nitroquinoxaline-2,3-dione); NBQX (2,3-dihydroxy-6-nitro-7-ulfamoyl benzo[f quinoxaline-2 3-dione); DNQX (6,7-dinitroquinoxaline-2,3-dione); kynurenic acid; 2,3 dihydroxy-6-nitro-7-sulfamoylbenzo[fquinoxaline; or combinations thereof

[00363]In some embodiments, the agent that modulates the NMDA receptor is an NMDA receptor antagonist. In some embodiments, .theagent which antagonizes the NMDA receptor is I aminoadamantane. dextromethorphandextrorphan, ibogaine, ketamine, nitrous oxide, phencyclidine, riluzole, tiletaiine, memantine, dizocilpine, aptiganel, remacimide, 7 chiorokynurenate, DCKA (5;7-dichlorokynurenic acid), kynurenic acid, 1 aminocyclopropanecarboxylic acid (ACPC), AP7 (2-aniino-7-phosphonoheptanoic acid), A V (R-2 amino-5-phosphonopentanoate), CPPene (3[(R)-2-carboxypiperazin-4-yl]-prop-2-enyvl-I phosphonic acid); (+)-(IS, 2)-1-(4-hydroxy-phenyl)-2-(4-hydroxy-4-phenylpiperidino)-I-pro panol; (1, 2S)-I-(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy-4-phenylpiperi-dino)-I-propanol; (3R, 4S)-3-(4-(4-fluorophenyl)-4-hydroxypiperidin-I-y-)-chroman-4,7-diol; (1R*, 2R*)-1-(4 hydroxy-3-methylphenyl)-2-(4-(4-fluoro-phenyl)-4-hydroxypiperidin-I-yl)-propan-l-ol-mesylate; and/or combinations thereof, 100364 In certain instances, the over-activation ofthe AMPA and NMDA glutamate receptors by the binding of excessive amounts of glutamateresults in the excessive opening of the ion channels under their control. In certain instances, this results in abnormally high levels of C and Na' entering the neuron. In certain instances, the influx of Ca and Na into the neuron activates multiple enzymes including, but not limited to, phospholipases, endonucleases, and proteases. In certain instances, the over-activation of these enzymes results in damage to the cytoskeleton, plasma membrane, mitochondria, and DNA of the neuron. Further, in certain instances, the transcription of multiple pro-apoptotic genes and anti-apoptotic genes are controlled by Ca' levels.

[00365] The mGlu receptors, unlike the AMPAand NMDA receptors, do not directly control an ion channel. However, in certain instances, they indirectly control the opening of ion channels by the activation of biochemical cascades. The mGlu receptors are divided into three groups. in certain instances, the members of groups 11 and. III reduce or inhibit post-synaptic potentials by preventing or decreasing the formation of cAMP. In certain instances, this causes a reduction in the release of neurotransmitters, especially glutamate. GRM7 is the gene which encodes the mGlu7 receptor, a group III receptor. In certain instances, the agonism of mGIu7 results in a decrease in synaptic concentrations of glutamate. This ameliorates glutamate excitotoxicity.

[00366in some embodiments, the glutamate receptor is a group II mGlu receptor. In some embodiments, theagent which modulates the group IImGlu receptor is a group II mGlu receptor agonist. In some embodiments, the group 11miGlu receptor agonist is LY389795 ((-)-2-thia-4 aminobicyclo-hexane-4,6-dicarboxylate); LY379268 ((-)-2-oxa-4-aminobicyclo-hexane-4,6 dicarboxylate); LY354740 ((+)-2-aminobicyclo-hexane-2,6dicarboxylate);DCG-IV ((2S,2R3'R)-2 (23-dicarboxvcyclopropyl)glyine); 2R,4R-APDC (2R,4R-4-aminopyrrolidine-2,4-dicarboxyate), (S)-3C4HPG ((S)-3-carboxy-4-hydroxyphenylglycine); (S)-4C3HPG ((S)-4-carboxy-3 hydroxyphcnylglycine);..-CCG-I ((2S,1'S,2'S)-2-(carboxycyclopropyl)glycine); and/or combinations thereof.

[003671In some embodiments, the mGlu receptor is a group IIImGlu receptor. In some embodiments, the group I m~lu receptor is mGlu7. In some embodiments, the agent which modulates the group III mGlu receptor is a group III mGlu receptoragonist. In some embodiments, the group I mGlu receptor agonist is ACPT- ((S,3R,4S)--aminocyclopentane-1,3,4-tricarboxvlic acid); L-AP4 (L-(4)-2-Amino-4-phosphonobutyric acid); (S)-3,4-DCPG ((S)-3,4 dicarboxyphenylglycine); (RS)-3,4-DCPG ((RS)-3j4-dicarboxyphenylglycine); (RS)-4 phosphonophenylglycine ((RS)PPG); AMN082(,Nbis(diphenylmethyl)-I,2-ethancdiamine dihydrochloride); DCG-1V ((2S,2'R3'R)-2-(2,3'-di.carboxycyclopropyl)glycine); and/or combinations thereof. In some embodiments, the mGlu receptor is naGlu7. In some embodiments, the agonist of mGlu7 is AMN082.In some embodiments, the miGlu receptor modulator is35 Dimethyl pyrrole-2.4-dicarboxylicacid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester (3,5-dimethyl PPP); 3,3'-difluorobenzaldazine ()F13) 3,3-dimlethoxybenzaldazine (DMeOB), 3,3' dichlorobenzaldazine (DCB) and other allosteric modulators of mGluRj disclosed in Mol.

Pharmacol. 2003, 64, 731-740; (E)-6-methyl-2-(phenyldiazenyi)pyridin-3-ol (SIB 1757);(E)-2 methyl-6-styrylpyridine (S11 1893); 2-methyli-6-(phenylethynyl)pyridine (MPEP)2-methyl4-((6 tnethylpyridin-2-yl)ethynyl)thiazole (MTEP) 7-( Hydroxyimino)cyclopropa[b]chromen-I I carboxylate ethyl ester (PCCOEt), N-cyclohexyl-3-methylbenzo[d]thiazolo[3,2-a]imidazole2 carboxamide (YM-298198), tricyclo[3.3,3.1]nonanyl quinoxaline-2-carboxamide (NPS 2390); 6 methoxy-N-(4-methoxyphenyl)quinazolin4-amine (LY 456239); mGluR1 antagonists disclosed in W02004/058754 and W2005/009987;2-(4-(2,3-dihydro-IH-inden-2-ylamino)-5,6.7,8 tetrahydroquinazolin-2-ylthio)ethanol; 3-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile, 2-(2 methoxy-4-(4-(pyridin-2-yl)oxazol-2-yl)phenyl)acetoitriie; 2(4-(benzo[d]oxazol-2-yl)-2 to methoxyphenyl)acetoniti-le; 6-(3-methoxy-4-(pyridin-2-yl)phenyl)inidazo[2,1-bthiazole; (S)-(4 flu orophenyl)(3-(3-(4-fluorophenyl)- 12,4-oxadiazol-5-yl)piperidin-1-y~methanone (ADX47273) and/or combinations thereof

[003681in some embodiments, a glutamate receptor modulator is a nootropic agent. Contemplated for use with the formulations disclosed herein are nootropic agents that modulate neuronal signalling

[5 by activating glutamate receptors. Insome instances, nootropic agents treat or ameliorate hearing loss (e.g, NIL) or tinnitus. Accordingly, some embodiments incorporate the use ofnootropic agents including, and not limited to, piracetamOxiracetam, Aniracetam Praniracetam, Phenylpiracetam (Carphedon) Etiracetarn, LevetiracetanNefiracetam, NicoracetamRolziracetam, Nebracetam, Fasoracetan, Coluracetam, Dimiracetam, Brivaracetam. Seletracetam, and/or Rolipram M0 for the treatment of N-IL or tinnitus. GrowthFactors

[00369]Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration of neurons and/or hair cells of the auris, promote the survival and/or growth ofneurons and/or hair cellsof the ais, and agents for treatingor ameliorating hearing loss or reduction resulting from destroyed, stunted, malfunctioning, damaged, fragile or missing hairs in the inner ear. Accordingly, some embodiments incorporate the use of agents which promote the survival of neurons and otichair cells, and/or the growth of neurons and otic hair cells. In some embodiments, the agent which promotes the survival of otic hair cells is a growth factor. In some embodiments, the growth factor is a neurotroph. In certain instances, neurotrophs are growth factors which prevent cells from initiating apoptosis repair damaged neurons and otichair cells, and/or induce differentiation in progenitor cells. In some embodiments, the neurotroph is brain-derived neurotrophic factor (.BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor (GDNF), neurotrophin-3, neurotrophin-4, and/or combinations thereof In some embodiments, the growth factor is a fibroblast growth factor (FGF), an insulin-like growth factor (IGF), an epidenral growth factor (EGF), a platlet-derived growth factor (PGF) and/or agonists thereof In some enibodiments, the growth factor is an agonist of the fibroblast growth factor (FGF) receptor, standing need for suitable testing methods that are applicable to the development of intratympanic otic compositions suitable for human use. In certain embodiments of such auris-compatible formulations that comprise a dye, the ability to visualize a controlled release otic formulation comprising a dye allows for testing of any otic formulation described herein in human clinical trials. Diseases of the Ear 100123]1The formulations described herein are suitable for the treatment and/or prevention of diseases or conditions associated with the middle and inner ear, including the cochlea, including vertigo, tinnitus, hearing loss, otoselerosis, balance disorders, andM niere's disease (endolymphatic hydrops).

[00124] The formulations described herein reduce, reverse and/or amelioratesymptoms of otic disorders (e.g.,auris interna disorders) which include but are not limited to hearing loss, nystagmus, vertigo, tinnitus, inflammation, swelling, infection and congestion. These disorders may have many causes, such as infection, injury, inflammation, tumors and adverse response to drugs or other chernical agents. Meniere 's Disease

[001251Meniere's Disease is an idiopathic condition characterized by sudden attacks of vertigo, nausea and vomiting that may last for 3 to 24 hours, andmay 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 internal fluid homeostasis, including an increase in production or a decrease in resorption of auris internal fluid. 1001261The 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. 100127]Although the cause of Meniere's disease is unknown certain evidence suggests a viral etiology for the disease. Specifically, histopathologic analysis of temporal bones in patients with Meniere's disease revealed viral ganglionitis, Also, viral DNA has been observed in the ganglia of patients with Meniere's disease at a higher rate than in healthy patients. Oliveira et al. ORL (2008) 70: 42-51, Based on these studies, a pilot study of intratympanic injection of the antiviral agent ganciclovir was conducted, resulting in an improvement of patients suffering from Meniere's disease. Guyot et al. ORL (2008) 7021-27 Accordingly, controlled release fonulations disclosed herein comprising antiviral agents, e.g., ganciclvir, acyclovir, famovir, and valgancyclovir, is administered to the ear for localized treatment of Meniere's disease.

[001281 Recent studies of the vasopressin (VP)-mediated aquaporin 2 (AQP2) system in the auris intema suggest a role for VP in inducing endolymph production, thereby increasing pressure in the vestibular and cochlear structures. (Takeda et al.NearingRes, (2006) 218:89-97). VP levels were found to be upregulated in endolymphatic hydrops (Meniere's Disease) cases, and chronic.

administration of VP in guinea pigs was found to induce endolymphatic hydropsTreatment with VP antagonists, including infusion of OPC-31260 (a competitive antagonist of V-R) into thescala tympani resulted in a marked reduction of Meniere's disease symptoms. (Takeda et al HearingRes (2003) 182:9-18). Other VPantagonists include WAY-140288, CL-385004, toivaptan, conivaptan, SR 121463A and VPA 985. (Sanghi et al. Eur. HeartJ (2005) 26:538-543; Palm et al. NephroL Dial Transplant(1999) 14:2559-2562).

1001291Other studies suggesta role for estrogenrelated receptor /NR3B2 (ERR/Nr3b2) in regulating endolymph production, and therefore pressure in the vestibular/cochlear apparatus. (Chen et al. Dev, Cell (2007) 13.325-337). Knock-out studies in mice demonstrate the role of the protein product of the Nr3b2 gene in regulating endolyiphfluid production. Nr3b2 expression has been localized in the endolymph-secreting strial 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. Treatment with antagonists to ERR.Nr3b2 may assist in reducing endolymphatic volume, and thus alter pressure in the auris internal structures,

[001301Other treatments are aimed at dealing with the immediate symriptoms and prevention of recurrence. Low-sodium diets, avoidance of caffeine, alcohol, and tobacco have been advocated. Medications that may temporarily relieve vertigo attacks include antihistamines (includingmeclizine (Antivert, Bonine, Dramamine, Driminate) and other antihistamines), and central nervous system agents, including barbiturates and/or benzodiazepines, including lorazepam 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 anipsychotic agents, including the phenothiazine agent prochlorperazine (Compazine, Buccastem, Stenetil andPhenotil)

[00131] Surgical procedures havealso 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 andor 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 may also be employed, which may control vertigo while preserving hearing.

[001321Another approach to destruction of vestibular function for the treatment of severe Menieres disease is intratympanic 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 gentanicin and streptomycin. The agents are injected through the tympanic membrane using a small needle, a tympanostomny tube with or without a wick, orsurgical catheters. Various 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 typically more effective, it is more risky, as it may result in hearing loss. 1001331Accordingly, formulations disclosed herein are also useful for administration of antimicrobial agents, e.g., gentanicin and streptomycin, for disabling the vestibular apparatus to treat Meniere's disease. The formulations disclosed herein are used to maintain steady release of the active agents inside the tympanic membrane, thereby avoiding theneed for multiple injections or the insertion of a tympanostomy tube. Furtherby keeping the active agents localized in the vestibularsystem, the formulations disclosedlherein can also be used to administer higher doses of the antimicrobial agents with a decreased risk of hearing loss. Meniere'sSyndrome 1001341Meniere'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 syphillis infection. Meniere's syndrome, thusare secondary effects to various process that interfere with normal production or resportption of endolymph, including endocrine abnormalities, electrolyte imbalance, autoimmune dysfuntion, medications, infections (eg parasitic infections) or hyperlipidemia. Treatment of patients afflicted with Meniere's Syndrome is similar to Meniere's Disease. SensorineuraiHearingLoss

[00135] 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 the inner ear. The-majority of defects of the inner ear are defects of otic hair cells,

[001361Aplasia of the cochlea, chromosomal defects, and congenital cholesteatorna are examples of congenital defects which can 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. aninoglycosides loop diuretics, antimetabolites, salicylates, and cisplatin), physical trauma, presbyacusis, and acoustic trauma (prolonged exposure to sound in excess of 90 dB) canall result in acquired sensorineural hearing loss. 1001371If the defect resulting in sensorineural hearing loss is a defectin 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.

[001381Insome instances, sensorineural hearing loss occurs when the components of the auris intera or accompanyingneural components are affected, and may contain a neural, i,e. when the auditory nerveor 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.Neural hearing loss may occur as a result of brain tumors, infections, or various brain and nerve disorders such as stroke, Some hereditary diseases, such as Refsum's disease (defective accumulation of branched fatty acids), may also cause neural disorders affecting hearing loss.Auditory nerve pathways are damaged by demyclinating diseases, e.g. idiopathic inflammatory demyelinating disease (including multiple sclerosis), transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, Guillain-Barresyndrome, chronic inflammatory demyelinating polyneuropathy and anti-MAG peripheral neuropathy

[00139j The incidence of sudden deafess, or sensorineural hearing loss, occurs in about I in 5000 individuals, and are caused by viral or bacterial infections, e.g.mumps, measles, influenza, chickenpox, cytomegalovirus, syphillis or infectious mononucleosis, or physical injury to the inner ear organ. In some cases, no cause can be identified. Tinnitus and vertigo may accompany sudden deafness, which subsides gradually. Oral corticosteroids are frequently prescribed to treat sensorineural hearing loss In some cases, surgical intervention are necessary. Other treatments include AM-101 andAM-111, compounds under development for thetreatment of auris interna tinnitus andacutesensorineural hearing loss, (Auris Medical AGBasel, Switzerland). Noise induced hearingloss 001401Noise induced hearing loss (NIHL) is caused upon exposure to sounds that are too loud or loud sounds that last a long time. Hearing loss may occur 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 can cause hearing loss. NIHL causes damage to the hair cells andlor the auditory nerve The hair cells are small sensory cells that convert sound energy into electrical signals that travel to the brain. Impulse sound can result in immediate hearing loss that are permanent. Thiskind of hearing loss are accompanied by tinnitus-a ringing, buzzing, or roaring in the ears or head -which may subside over time. Hearing loss and tinnitus are experienced in one or both ears, and tinnitus may continue constantly or occasionally throughout a lifetime. Permanent damage to hearing loss is often diagnosed. Continuous exposure to loud noise also damages the structure ofhair cells, resulting in hearing loss and tinnitus, although the process occurs more gradually than for impulse noise.

[001411in some embodiments, an otoprotectant can reverse. reduce or ameliorate NIHL. Examples of otoprotectants that treat or prevent NI-iL include, but are not limited to, D-methionine, L methionine, ethionine, hydroxyl methionine, methioninol, anifostine, mesna (sodium 2 sulfanylethanesufonate), a mixture of D and L methionine, normethionine, homomethionine, S adenosyl-L-methionine), diethyldithiocarbamate, ebselen (2-phenyl-1,2-benzisoselenazol-3(2H1-) one), sodium thiosulfate, AM-I11 (a cell permeable JNK inhibitor, (Laboratoires Auris SAS)), leucovorin, leucovorin calcium, dexrazoxane, or combinations thereof

1001421Although there is currently no treatment for noise-induced hearingloss, several treatment regimens have been experimentally developed, including treatment with insulin-like growth factor 1 (IGF-l) and antioxidant therapy, including treatment with alpha lipoic acid. (Lee et al. Otol. NeWurotol. (2007) 28976-981).

Tinnitus 100143]Tinnitus is defined as the perception of sound in the absence of any external stimuli. It may occur in one or both ears, continuously or sporadically, and is most often described as a ringing sound. It is most often usedas 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 theaffected individual. Studies estimate that over 50 million Americans experience some form of tinnitus. Of those 50 million, about 12million experience severe tinnitus.

[001441In certain instances, tinnitus results from damage to otic structures (eg. stereocillia), 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 fom by dysfunction of ann9 and/or a10 acetylcholine receptor. In certain instances, tinnitus results from damage to the vestibulocochlear nerve.In certain embodiments, a reduction in neurotransmitter reuptake (e.g. the increase in extracellular neurtotransmitters) treats, and/or ameliorates the symptoms of tinnitus. In certain embodiments, antagonism of anNK1 receptor treats, and/or ameliorates the symptoms of tinnitus. In certain embodiments, a reduction in neurotransmitter reuptake and antagonism of an NK Ireceptor treats, and/or ameliorates the symptoms of tinnitus. 1001451There are several treatments for tinnitus, Lidocaine, administered by IV, reduces or eliminates the noise associated with tinnitus in about 60-80% of sufferers. Selective neurotransmitter reuptake inhibitors, such as nortriptyline, sertralines and paroxetine, have also demonstrated efficacy against tinnitus. Benzodiazepines are also prescribed to treat tinnitus, Autoimmune InnerEar Disease

[00146]Autoinnmune inner ear disease (AIED) is one of the few reversible causes of sensorineural hearing loss, It is a rare disorderappearing 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/orimmune cells attacking imer ear structures, but areassociated with other autoimmune conditions. In many cases, AIED occurs without systemic autoimmunesymptomsbut up to one-third of patients also suffer from systemic autoimmune illness, such as inflammatory bowel disease, rheumatoid arthritis, Ankylosingspondylitis, Systemic Lupus Erytheiatosus (SLE), SjCgren's Syndrome, Cogan's disease, ulcerative colitis, Wegener's granulomatosis and scleroderma, Behget's disease, a multisystem disease,also commonly has audiovestibular problems,

There is some evidence for food-related allergies as a cause for cochlear and vestibular autoimnunity but there is presently no agreement as to its importance in the actiology of the disease. A classification scheme forMAED has been developed (Harris and Keithley, (2002) Autoimmune inner ear disease, in tarhinolaryngologyHead and NeckSurgery. 91, 18-32) 100147]The immune system normally performs a cruical role in protecting the auris interna from invasive pathogens such as bacteria and viruses. However, inAIED the immune system itself begins to damage the delicate auris internal tissues. It is well established that the auris intema is fully capable ofmounting a localized immune response to foreign antigens, (Harris, OolaryngoL Head Neck Surg (1983) 91 18-32). When a foreign antigen enters the auris internal, it is first processed by immunocompetent cellswhich reside in andiaround the endolymphatic sac. Once the foreignantigen has been processed by these immunocompetent cells, these cellssecrete various cytokines which modulate the immune response of the auris intena. One result of this cytokine release is to facilitate the influx of inflammatory eels which are recruited from the systemic circulation. These systemic inflanmatory 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 (Harris,AcOtaOolaryngl. (1990) 110, 357-365). Interleukin I (IL-1) plays an important role in modulating the innate (nonspecific) immune response and is a known activator of resting T helper cells and B-cells. helper cells, once activated by IL-1, produce IL-2 IL-2 secretion results in differentiation of pluripotet T-cells into helper, cytotoxic and suppressor T-cell subtypes. IL-2 also assists Thelper cells in the activation of B lymphocytes and probably plays a pivotal 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 hafter antigen challenge. The perilymphatic levels of IL-2 then dissipate, and it is no longer present within the perilynph at 120 hours post antigen challange (Gloddek,Acta Otolarngol (1989) 108, 6875).

[00148]Both IL-1 andtumor necrosis factor-(TNF-) may play akeyrole in.the initiation and amplification of the immune response, IL-1 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 is expressed eitherby infiltratingsystemic cells or byresident cells contained within the endolymphatic sac in the presence of antigen. THF-uis released as part of the adaptive (specific)

immune response in animal models. When antigen is injected into the auris intemas of mice. IL-l and TNF-u areboth expressed and a vigorous immune response occurs.However, when antigen is introduced to the auris interna via the cerebral spinal fluid without trauma to the auris internal, only TNF-u is expressed and the immune response inmiimal (Satoh,J Assoc. Re. Otolarvngol (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 oftheimmune

response may act in concert in theauris internal to achieve amaximal response.

1001491Accordingly, 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 thespecific immune responses can be activated simultaneously.This results in the concurrent production ofIL-i [as well as TIF-u which causes a greatly amplified level of inflammation leading to substantial damage to the auris intea. Subsequent experiments in animal models confirm that an important step in immune-mediated damage requires that the auris interna be conditioned by thenon-specific innate immune response before the specific adaptive immune response can lead to enough inflammation to result in damage (Hashimoto, AudiolNeurootol. (2005), 10, 35-43). As aresult, agents which downregulate or block the specific immune response,

and in particular the effectof TNF-, might be able to prevent the excessive immune response seen when both thespecific and nonspecific immune responses are simultaneously activated (Satoh, Laryngoscope (2002) 112,1627-1634). 1001501Treatment of autoinunune eardisease, thus, may consist of anti-iNF agents. Trials using etanercept (ENBREL*).an anti-TNF drug, is emerging as a promising agent for treatment of autoimmune inner ear disease. (Rahmen et al., Oto NeuroL (2001) 22:619-624; Wang et al., Otology& Neuroology (2003) 24:52-57). Additionally, the anti-TNF agentsintflixi-mab (REMICADE) and adalimumab (IIUMIRA) may also be useful in treatment of autoimmune auris internal disorders. Trial protocols include injections of anti-TNF agents as an injection on a twice weekly basis. 1001511In addition, steroids have been used, e.g, prednisone or decadron, have also been tried with some success, Chemotherapeutic agents, e.g. cytoxan, azathiaprine or methotrexate are used on a long-term basis to treat autoimmune inner ear disorders. (Sismanis et al Langoscope (1994) 104:932-934; Sismanis et al., Otolaryngol(1997) 116:146152; Harris et al.AMIA (2003) 290:1875 1883). Plasmapheresis procedures have also been tried with some success. (Luetie et al. Am.J. Otol. (1997) 18:572-576). Treatment with oral collagen (Kim et al. Ann. Otol Rhinof Larynogol. (2001) 110: 646-654), gamma globulin infusions or other immunemodulating drugs (e.g. beta-interferon, alphainterferon or copaxone) may also be used to treat autoiniune inner ear disorders. 1001521Certain evidence suggests that viral infection is a factor in the initiation of the inflammatory response that results in AIED. Various autoimmune conditions are induced or enhanced by a variety of DNA and RNA virus infections. Acute or persistent viral infections induce or enhance autoimmune diseases in animal models as well. Similar antigenic determinants have also been observed on viruses and host components. OldstoneM.B.A. J. Autoimmun. (1989) 2(suppl): 187 194. Further, serological tests have identified viral infectionin at least one patient diagnosed with a systemic autoinmnune disorder that is often associated with AiED (Coga's syndrome). Garcia Berrocal, et al O.RL. (2008) 70: 16-20.

[00153] Accordingly, in some embodiments, controlled release antimicrobial agent compositions and formulations disclosed herein are administered for the treatment of AIED. Particularly, in certain embodiments, fonnulations disclosed herein comprising antiviral agents are administered for treatment of AIED. hi other embodiments, the antimicrobial agentfonnulations disclosed herein are administered for the treatment of MED in conjunction with other pharmaceutical agents useful for treating the same conditions or symptoms of the same conditions, including steroids, cytotoxic agents, collagen, gamma globulin infusion, orother immune modulating drugs. Steroids include. e,g., prednisone or decadron. Cytotoxic agents for the treatment of AIED include, e,g.,methotrexate, cyclophosphamide, and thalidomide. Plasmapheresis procedures are optionally used. Treatment with oral collagen, gamma globulin infusions, or other immune modulating drugs (e.g. beta-interferon, alpha-interferon or copaxone) is also optionally used in combination with the antimicrobial agent formulations disclosed herein. The additional pharmaceutical agents are optionally administered together with the controlled release formulations disclosed herein, or through othermodes of administration, e.g, orally, by injection, topically, nasally or through any other suitable means. The additional pharmaceutical agents are optionally co-administeredor administered at different time periods. Auditory Nerve Tunors 1001541Auditory nerve tumors, including acoustic neuroma, acoustic neurinoma. vestibular schwannoma and eighth nerve tumor) are tumors that originatein Schwann cells, cells that wrap around a nerve. Auditory nerve tumors account for approximately 7-8% of all 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, Other more serious symptoms may develop as the tumor becomes larger, which may compress against the facial or trigemminal nerve, which may affect connections between the brain and the mouth, eye or jaw Smaller tumors are removed by microsurgery, or sterotactic radiosurgical techniques, including fractionated sterotactic radiotherapy. Malignant Schwanuomas are treated with chemotherapeutic agents, including vineristine, adriamycin, cyclophosphamide and imidazole carboxamide, Benign ParysmnalPositionalVertigo

[00155]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 auditorysymptoms. Cancer frthe Ear

[001561Although the cause is unknown, cancer of the ear is often associated with long-term and untreated otitis, suggesting a link between chronic inflannnation and development of the cancer, at least in some cases. Tumors in the ear can be benign or malignantsandtheycan exist intheexternal, middle, or inner ear. Symptoms of ear cancer include otorrhea, otalgia, hearing loss, facial palsy, tinnitus, and vertigo. Treatment options are 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 antimicrobial agents whenotitisis present.

[001571Systemic administration of conventional cytoxic agents have been used to treat cancer of the ear, including systemic administration of cyclophosphamide (in CHOP chemotherapy) in combination with radiotherapy and methotrexate, Merkus, P., et al. J Oorhinlaryngo. Relat. Spec. (2000) 62:274-7, and perfusion of methotrexate through the external carotid artery Mahindrakar, N. H. J Laryngol. Oo (1965) 79:921 -5However, treatments requiring systemic administration of the active agents suffer from the same drawbacks discussed above. Namely, relatively high doses of the agents are required to achieve the necessary therapeutic doses in theear, whichresult in an increase of undesired, adverse side effects.Accordingly, local administration of the cytotoxic agents in the compositions and forumlations disclosed herein results in treatment of cancer of the ear with lower effective doses, and with a decrease in the incidence and/orseverity 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, dermatitis, 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.

[00158In certain embodiments, the cytotoxic agents are methotrexate (RHEUMATREX@, Armethopterin) cyclophosphamide (CYTOXAN@,) and thalidomide (THALIDOMID@). All of the compounds canbe used to treat cancer, including cancer ofthe ear. Further, all of the compounds have anti-inflammatory properties and. can be used in the formulations and compositions disclosed herein for the treatment of inflammatory disorders of the ear, including AIED. tOO591 Although systemic administration of methotrexate, cyclophosphanide, and thalidomide is currently used to treat or is being investigated for the treatment of otic disorders, such as inflammatory otic disorders, including AlED, Meniere's disease, and Beget's disease, as well as cancer of the ear, the cytotoxicagents are not without the potential for serious adverse side effects. Moreover, cytotoxic agents which demonstrate efficacy butare otherwise not approvable becauseof safety considerations is also contemplated within the embodiments disclosed herein. It is contemplated that localized application of the cytotoxic agents to the target otic structures for treatment of autoimmune and/or inflammatory disorders, as well as cancer of the ear, will result in the reduction or elimination of adverse side effects experienced with systemic treatment Moreover, localized treatment with the cytotoxic agents contemplated herein will also reduce the amount of agent needed for effective treatment of the targeted disorder due, for exampleto increased retention of the active agents in the auris interna and/or mediato the existence of the biological blood barrier in the auris internal, or to the lack of sufficient systemic access to the auris media. 100160 In some embodiments, cytotoxic agents used in the compositions, formulations, and methods disclosed herein are metabolites, salts, polynorphs, prodnigs, analogues, and derivatives of cytotoxic agents, including methotrexate, cyclophosphamide, and thalidomide. Particularly preferred are metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of cytotoxic agents, e.g., methotrexate, cyclophosphamide, andthalidomide, that retain at least partially the cytotoxicify and anti-inflamnatory properties of the parent compounds. In certain embodiments, analogues of thalidomide used in theformulations and compositions disclosed herein arelenalidomide (REVLIMID) and CC-4047 (ACTIMID@). 1001611Cyclophosphamide is a prodrug that undergoes in vivo metabolism when administered systemically. The oxidized metabolite 4-hydroxycyclophosphamide exists in equilibrium with aldophosphamide, and the two compounds serve as the transport forms of the active agent phosphorarnide mustard and the degradation byproduct acrolein. Thus, in some embodiments, preferred cyclophosphamide metabolites for incorporation into the formulations and compositions disclosed herein are 4-hydroxycyclophosphamide. aldophosphamide, phosphoramide mustard, and combinations thereof, 100162 Other cytotoxic agents used in the compositions, formulations, and methods disclosed herein, particularly for the treatment of cancer of thecar, areany conventional chemotherpeutic agents, including acridine carboxamide actinomycin, 17-N-allylamino-17-demethoxygeldanamycin, aminopterin, amsacrine, anthracycline, antineoplastic, antineoplaston, 5-azacytidine, azathioprine, BL22, bendamustine, biricodar, bleomycin, bortezonib, bryostatin, busulfan calyculin, camptothecin, capecitabine, carboplatin, chlorambucilcisplatin, cladribine cofarabine, cytarabine, dacarbazine, dasatinib, daunorubicin, decitabine, dichloroaceticacid, discodermolide, docetaxel, doxorubicin, epirubici, epothilone, eribulin, estramustine., etoposide, exatecan, exisulind, ferruginol, floxuridine, fiudarabine, fluorouracil, fosfestrol, fotenustine, gemcitabinehydroxyurea, I'-101, idarubicin, ifosfamide, imiquimodirinotecan, irofulven, ixabepilone, laniquidar, lapatinib, lenalidomide, lomustine, lurtotecan, mafosfamide, inasoprocolmechlorethanine, melphalan, mercaptopurine, ritomycin, mitotane, mitoxantrone, nelarabine, nilotinib, oblimersen, oxaipatin, PAC-1, paclitaxel., pemetrexed, pentostatin, pipobroman, pixantrone, plicamycin, procarbazine proteasome inhibitors (e.g., bortezomib), raltitrexed, rebeccamycin, rubitecan, SN38, salinosporamide A, satraplatin, streptozotocii. swainsonine,tariquidar, taxane, tegafur-uracil, temozolomide, testolactone, thioTEPA, tioguanine, topotecan, trabectedin, tretinoin, triplatin tetranitrate, tris(2-chloroethyl)anine, troxacitabine, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat. and zosuquidar. Cholesteatome 1001631A cholesteatoma is a hyperproliferative cyst often found in the middleear. Cholesteatona are classified as congenital or acquired. Acquired cholesteatomas result from retraction of the ear drum (primary) and/or a tear in the ear drum (secondary).

[00164JThe most common primary cholesteatoma results from the pars flaccida retracting into the epitympanum. As the pars flaccida continues to retract, the lateral wall of the 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 ofthe tympanic membrane retracts into the posterior middle ear. As the tynpanic 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). 1001651Complications associated with a growing cholesteatona 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-1, TGF-132, 11-1, and IL-6) in the epithelium of the cholesteatoma can result in further degradation of the surrounding bones. 1001661Patients with a cholesteatoma often present with earache, hearingloss,mucopurulent discharge, and/or dizziness. Physical examination can confirm the presence of acholesteatona Symptoms which can be identified upon physical examination include damage to the ossicies, and a canal filled with mucopus and granulation tissue. 1001671There is currently no effective medical therapy for cholesteatomas. As a cholesteatomia has no blood supply, it cannot be treated with systemic antibiotics. Topical administration of antibiotics often fails to treat a cholesteatoma, Drug-InducedInnerEar.Damage 1001681TDamage from the administration of drugs, including certain antibiotics, diuretics (e.g. ethacrynic acid and fuirosemide), aspirin, aspirin-likesubstances (e.g. salicylates) and quinine. Deterioration of the auris interna organ are hastened by impaired kidney function, which results in decreased clearance of the affecting drugs and their metabolites, The drugs may affect both hearing and equilibrium, but likely affects hearing to a greater extent.

[00169]For example, neomycin, kanamycin, anikacin have a greater effect on hearing than on balance. The antibiotics viomycin, gentamicin and tobramycin affect both hearing and equilibirun. Streptomycin, another commonly administered antibiotic induces vertigo more than loss of hearing, and can lead. 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, may also lead to temporary hearing loss and tinnitus, a condition where sound is perceived in the absence of external sound. Similarly, quinine, ethacryinic acid and furosemide can result in temporary or permanent hearing loss. ExcitotoxicitV 1001701Excitotoxicity refers to the death or damaging of neurons and/or otic hair cells byglutamate and/or similar substances.

[00171]Glutamate is the most abundantexcitatory neurotransmitter in the central nervous system. Pre-synaptic neurons release glutamate upon stimulation. It flows across the synapse, binds to receptors located on post-synaptic neurons, and activates these neurons. The glutamate receptors include theNIDA, AMPA, and kainate receptors. Glutamate transporters are tasked with removing extracellular glutamate from the synapse. Certain events (e.g. ischemia or stroke) can 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

[001721The AMPA receptor is activated by the binding of both glutamate and AMPA. Activation of certain isoforms of the AMPA receptor results in the opening ofion channels locatedin the plasma membrane of the neuron. When the channels open, Na' and Ca ions flow into the neuron and K' ions flow out of theneuron. 1001731The NMDA receptor is activated by the binding of both glutamate and NMDA. Activation of the NMDA receptor, results in the opening of ion channels located in the plasma membrane of the neuron. However, these channels are blocked by Mg ions. Activation of the AMPA receptor results in the expulsion of Mg" ionsfromn the ion channels into the synapse. When the ion channels open, and the Mg ions evacuate the ion channels, Na4 and Ca ions flow into the neuron, and K' ions flow out of theneuron. {OO174 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 Ca iand Na'to enter the neuron. The influx of these levels of Ca 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. The free radicals eventually damage the initochondria, depleting the cell's energy stores. Furthermore, excess levels of Ca 2 'and Na ions activate excess levels of enzymes including, but notlimited 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. E'ndolymphaicHydrops

[00175]Endolymphatic hydrops refers to an increase in the hydraulic pressure within the endolymphatic system of the inner ear. The endolymph and perilymph are separated by thin membranes which contain multiple nerves. Fluctuationin the pressure stresses the membranes and the nerves they house. If the pressure is great enough, disruptions may form in the membranes. This results in a mixing of the fluids which can lead 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 may also be affected. Distortions of the basilar membrane and the inner and outer hair cells can lead to hearing loss and/or tinnitus.

100176]Causes include metabolic disturbances. hormonal imbalances, autoimmune disease, and viral, bacterial, or fungal infections. Symptoms include hearing loss, vertigo, tinitus, and aural fullness. Nystagmus may also be present. Treatment includes systemic administration of benzodiazepine, diuretics (to decrease thefluid pressure), corticosteroids, and/or anti-bacterial, anti viral, or anti-fungal agents. HereditaryDisorders 001771Hereditary disorders, including Scheibe, Mondini-Michele, Waardenburg's, Michel, Alexander's ear deformity hypertelorism.Jervell-Lange Nielson, Refsum's and Usher's sydromes, are found in approximately 20% of patients with sensorineural hearing loss. Congenital ear malformations may 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 may also be associated with other dysfunctions, including development of recurring menigitis, cerebral spinal fluid (CSF) leaks, as well as perilymphatic fistulas. Treatment of chronic infections are necessitated in hereditary disorder patients. nflaninmatoryDisorders othe Auris Media

[001781 Otitis media (OM), which includes acute otitis media (AOM), otitis media with effusion (OME) and chronic ofitis media as examples, is a condition affecting both adults and children. OM susceptibility is multifactorial and complexincluding environmental, microbial and hostfactors. Bacterial infection accounts for a large percentage of OMcases, with more than 40% of cases attributed to Sirepiacoccuspneumoniaeinfection, However, viral causes, as well as other microbial agents, may also account for OM conditions, 1001791Regardless of the causative agent, increases in cytokine production, including interleukins and"TNF, have been observed in the effluent media of individuals afflicted with OM. I.-13, iL-6 and TNF-uare acute-phase cytokines that promote acute inflammatory response after infection with viruses and bacteria. Genetic studies supports this link between cytokines and OM by demonstrating a correlation in the occurrence of TNF- SNP (single-nucleotide polymorphisms) and an increased susceptibility for OM in pediatric patients suffering from AOM and with a subsequent need for placement of tympanostomy tubes. (Patel et al. Pediatrics(2006) 118:2273-2279) In animal models of ON! induced with pneumococci innoculations, TNF-c and interleukins levels were found to increase in early developmental phase of OM, with TNT-a levels steadily increasing 72 hours after innoculation. Moreover, higher TNF-a levels have been associated with a history of multiple tympanostomy tube placements, indicating a role for TNF-u in chronic OM cases, Finally, direct injection of TNF-a and interleukins has been shown to induce middle ear inflammation in a guinea pig model. These studies support the role that cytokines may play in the origin and maintenance of OM in the auris media.

[100180JBecause OM can be caused by a virus, bacteria or both, it is often difficult to identify the exact cause and thus the most appropriate treatment. Treatment options of OM in the auris media to include treatment with antibiotics, such as aoxicillin, clavulanate acid, trimethoprim sulfamethoxazole, cefuroxime, clarithromycin and azithromycin and other cephalosporins, macrolides, penicillins orsulfonamides. Surgical intervention is also available, including a myringotomy, an operation to insert a tympanostomy tube through the ympanic membrane and into the patient's middle ear to drain the fluid and balance the pressure between the outer and middle ear, Antipyretics and analgesics, including benzocaine, ibuprofen and acetaniinophen, may also be prescribed to treat accompanying fever or paineffects Pre-treatment withTNF inhibitors in experimental lipopolysaccharide (LPS)-induced OM animal models has been shown to suppress development of OM, suggesting a role in the treatment of OM or OME.In addition, treatment of such conditions include use of TNF- inhibitors in combination with other inflammatory response mediators, includingplatelet activating factorantagonists, nitric oxide synthase inhibitors and histamine antagonists,

[00181]As discussed above, methotrexate, eyclophosphamide,and thalidomide are all cytotoxic small molecule agents that are systemically administered to treat AIED. Thus, the compounds are useful in the compositions and formulations disclosed herein for the treatment of inflammatory disorders of the auris media, including OM, by having a direct anti-inflammatory effect, particularly by interfering with TNF activity. In other embodiments, metabolites, salts, polymorphs; prodrugs, analogues, and derivatives of methotrexate, cyclophosphamide, and thalidomide that retain the ability of the parent cytotoxic agents to treat inflammatory disorders of the auris media, Including OM, are useful in the formulations disclosed hereinfor the treatment of inflammatorydisorders of

the auris media, including OM. In certain embodiments, preferred metabolites of cyclophosphanmide for incorporation into the compositions and fonnulations disclosed herein include 4 hydroxycyclophosphamide, aldophosphanmide, phosphorarnide mustard, or combinations thereof.

[001821In additionother otic disordershave inflammatory response aspects or are tangentially related to autoimmune conditions. including Meniere's disease and non-sudden hearing loss or noise induced hearing loss.. These disorders are also explicitly contemplated as benefiting from the cytotoxic agent formulations disclosed herein, and therefore are within the scope of the embodiments disclosed. Ingiammatory Disordersofthe Auris externa

[00183]Otitis extena (OE), also referred to asswimmer's ear, is an inflammation and/or infection of the external ear OE is often caused by bacteria in the outer ear, which establish infection following damage to the skin of the ear canal. Primary bacterial pathogens that cause OE are PseudononsaerugnosaandStaphylococcus aureus, but the condition is associated with the

presence of many other strains of gram positive and negative bacteria. OE is also sometimes caused by fungal infection in the outer ear, including Candida alluicans and Aspergilus. Symptoms ofOE include otalgia, swelling, and otorrhea. If the condition progressessignificantly, OE may cause temporary conductive hearing loss as a result of the swelling and discharge. 100184] Treatment of OE involves eliminating the aggravating pathogen from the ear canal and reducing inflanimation, which is usually accomplished by administering combinations of antimicrobial agents, e.g, antibacterial and antifungal agents, with anti-inflammatory agents, e.g., steroids. Typical antibacterial agents for the treatment of OE include aminoglycosides (e.g. neomycin, gentamycin, and tobramycin), polymyxins (e.g., polymyxin B), fluoroquinolone (e.g., ofloxacin and ciprofloxacin), cephalosporins (e.g., cefuroxime, ceflacor, cefprozil, loracarbef, cefindir, cefixime, cefpodoxime proxetil, cefibuten, and ceftriaxone), penicillins (e.gamoxicillin, amoxicillin-clavulanate, and penicillinase-resistant penicillins) and combiiations thereof Typical antifungal agents for the treatment ofGE includeclotrimazole, thimirasol, M-cresyl acetate, tolnaftate, itraconazole, and combinations thereof. Acetic acid is also administered to the ear, alone and in combination with other agents, to treat bacterial and fungal infections. Ear drops are often used as the vehiclefor administration of the active agents. In the case that ear swelling has progressed substantially and ear drops do not penetratesignificantly into the ear canal, a wick can be inserted into the ear canal to facilitate penetration of the treatment solutions. Oral antibiotics are also administered in the case of extensive soft tissue swelling that extends to the face and neck. When the pain of OE is extremely severe such that it interferes with normal activity, e.g, sleeping, pain relievers such as topical analgesics or oral narcotics are given until the underlyinginflammation and infection are alleviated.

[001851Notablysome types of topical ear drops, such as ear drops containing neomycinare safe and effective for use in the ear canal, but can be irritating and even ototoxic to the aurismedia, prompting concern that such topical preparations should not be used unless the tympanic membrane is known to be intact. Utilization of the fonrulations disclosed herein for the treatment of OE allows for use of active agents that arc potentially damaging to the auris media, even when the tympanic membrane is not intact. Specifically, the controlled release formulations disclosed herein can be applied locally in the external earvith improved retention time, thus eliminating concern that the active agents will leak out of theear canal into the auris media. Furthermore, otoprotectants can be added when ototoxic agents, such as neomycin, are used.

[001861Treatment of severe OEwith the compositions disclosed herein, particularly highly viscous and/or mucoadhesive formulations, also obviates the need for extended use of an ear wick. Specifically, the compositions disclosed herein have increased retention. time in the ear canal as a result of the forndation technology, thus eliminating the need for a device to maintain their presence in the outer ear. The formulations can be applied in the outer earwith a needle or an car dropper, and the active agents can be maintained at the site of inflammation without the aid of an ear wick.

[00187]In some embodiments, the treatment of OE with antimicrobial formulations disclosed herein encompasses the treatment of granular myringitis, a specific form of OE characterized by chronic inflammation of the pars tensa of the tympanic membrane. The outer epithelial and underlying fibrous layers of the tympanic membrane are replaced by a proliferating granulation tissue. The predominantsymptom is foul-smelling otorrhea, A variety of bacteria and fungi cause the condition, including Proteus and Psuedomonasspecies. Accordingly, antimicrobial agent formulations disclosed herein comprising antibacterial or antifungal agents are useful for the treatment of granular myringitis.

[001881In some embodiments, the treatment of OE with antimicrobial formulations disclosed herein encompasses the treatment of chronic stenosing otitis externa, Chronic stenosing otitis externa is characterized by repeated infections, typically caused by bacteria or fungi. The primary symptoms are pruritus in theear canal, otorrhea, and chronic swelling. Antimicrobial agent formulations disclosed herein comprising antibacterial or antifungal agents are useful for the treatment of chronic stenosing otitis externa.

[001891In some embodiments, the treatment of OE with antimicrobial forulations disclosed herein encompasses the treatment of malignant or necrotizing external otitis, an infection involving the temporal and adjacent bones. Malignant external otitis is typically a complication of external otitis. It occurs primarily in persons with compromised immunity, especially in older persons with diabetes mellitus. Malignant external otitis is often caused by the bacteria Pseudomonasaeruginosa Treatment typically involves correction of immunosuppression when possible, in conjunction with antibacterial therapyand pain relievers. According, antimicrobial agent formulations disclosed herein are useful for the treament of malignant or nerotizing external otitis,

[001901Otitis inedia (OM), which includes acute otitis media (AOM), chronic otitis media, otitis media with effusion (OME), secretory otitismedia, and chronic secretory otitismedia as examples, is a condition affecting both adults and children. OM susceptibility is multifactorial and complex, including environmental, microbial and host factors. Bacterialinfectionaccounts for a large percentage of OM cases, with more than 40% of cases attributed to Streptococusmeumoniae infection, However, viruses, as well as other microbes, may also account for OM conditions,

100191]Because OM can be caused by a virus, bacteria or both, it is often difficult to identify the exact cause and thus the most appropriate treatment, Treatment options for OM include antibiotics, such as penicillins (e.g., amoxicillin and amoxicillin-clavulanate), clavulanate acid, trimethoprim sulfamethoxazole, cephalosporins (e.g. cefuroxine, cefacor, cefprozil, loracarbef cefindir, cefixime, cefpodoxime proxetil, cefibuten, and cefitriaxone), macrolides and azalides (e.g., erythromycin, clarithronycin, and azithromycin), sulfonamides, and combinations thereof Surgical intervention is also availableincluding myringotomy, an operation to insert a tympanostomy tube through the tympanic membrane and into the patients middle ear to drain the fluid and balance the pressure between the outer and middle ear. Antipyretics and analgesics, including benzocaine, ibuprofen and acetaminophen, may also be prescribed to treat accompanying fever or pain effects.

[00192]Regardless of the causative agent, increasesin cytokine production, including interleukins and TNT, have been observed in the effluentmedia of individuals afflicted with OM I-i3 , IL-6

and TNF.aare acute-phase cytokines that promote acute inflammatory response after infection with viruses and bacteria. Moreover, higher TNF-o- levels have been associated witha history of multiple

tympanostomy tube placementsindicating a role for TNT-a in chronic OM cases. Finally, direct injection ofTNF-c and interleukins has been shown to inducemiddle ear inflammation in a guinea pig model These studies support the role that cytokines may play in the origin and maintenance of OM in theauris media. Thus, treatment of OM includes the use of antimicrobial agents in

conjunction with anti-inflammatory agents to eliminate the pathogen and treat the symptoms of inflammation. Such treatments include use of steroids,'TNF-a inhibitors, platelet activating factor antagonists, nitric oxide synthase inhibitors, histamine antagonists, and combinations thereofin conjunction with theantimicrobial formulations disclosed herein.

[001931Mastoiditis is an infection of the mastoid process, which is the portion of the temporal bone behind the ear. It is typically caused by untreated acute otitis media. Madtoiditis are acute or chronic. Symptoms include pain, swelling, and tenderness in the mastoid region, as well as otalgia, erythematousand otorrhea. Mastoiditis typically occurs as bacteria spread from the middle ear to the mastoid air cells, where the inflammation causes damage to the bony structures. The most common bacterial pathogens are Streptococcus pneumoniae,Streptococcuspyogenes,

Staphylococcus aures, and gram-negative bacilli, Accordingly, antimicrobial agent formulations disclosed herein comprising antibacterial agents effective against the bacteria are useful for the treatment of mastoiditis, including acute mastoiditis and chronic mastoiditis. 1001941Bullous myringitis is an infection of the tympanic membrane, caused by a variety of bacteria and viruses, includingMvoplasmabacteria. The infection leads to inflammation of the tympanic membrane and nearby canal, and causes the formation of blisters on the ear drum. The primary symptomof Bullous myringitis is pain, which are relieved through the administration of analgesics. Antimicrobial formulations disclosed herein comprising antibacterial and antiviral agents are useful for the treatment of Bullous myringitis.

[001951Eustachian tubal catarrhor Eustachiansalpingitisis caused from inflammation and swelling of the Eustachian tubes, resulting in a build-up of catarrh. Accordingly, antimicrobial formulations disclosed herein are useful for the treatment of Eustachian salpingitis

[001961Labyrinthitis, e.g., serous labyrinthitis, is an inflammation of the inner ear that involves one or more labyrinths housing the vestibular system. The primary symptom is vertigo, but the condition is also characterized by hearing loss, tinnitus, and nystagmus. Labrynthitis maybe acute, lasting for one to six weeks and being accompanied by severe vertigo and vomiting, or chronic, with symptoms lasting formonths or even years. Labyrinthitis is typically caused by viral or bacterial infection. Accordinglyantimicrobial formulations disclosed herein comprising antibacterial and antiviral agents are useful for the treatment of labyrinthitis.

[001971Facial nerve neuritis is a form of neuritis, an inflammation of the peripheral nervous system, afflicting the facial nerve.The primary symptoms of the condition are a tingling and burning sensation, and stabbing pains in the affected nerves. In severe cases, there are numbness, loss of sensation, and paralysis of the nearby muscles, The condition is typically caused by herpes zoster or herpes simplex viral infection, but has also been associated with bacterial infection, e.g, leprosy. Accordingly, antimicrobial formulations disclosed herein comprising antibacterial and antiviral agents are useful for the treatment of facial nerve neuritis. 1001981In some embodiments, antimicrobial formulations disclosed herein are also useful for the treatment of temporal bone osteoradionecrosis. Kinetosis 1001991Kinetosis, also known as motion sickness,is a condition in which there is a disconnection between visually perceived movement and the vestibular system's sense of movement. Dizziness, fatigue, and nausea are the most common symptoms of kinetosis. Dimenhydrinate, cinnarizine, and meclizine are all systemic treatments for kinetosis. Additionally, benzodiazepines and antihistamines have demonstrated efficacy in treating kinetosis.

Labyrinthitis

[002001Labyrinthitis is an inflammation of the labyrinths of the ear which contain the vestibular system of the inner ear. Causes include bacterial viral, andfungal infections. It may also be caused by a head injuryor allergies. Symptoms of labyrinthitis include difficulty maintaining balance, dizziness, vertigo, tinnitus, and hearing loss. Recovery may take one to six weeks; however, chronic symptoms are present for years.

t002011There 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 a bacterial infection, and treatment with corticosteroids and antivirals is recommended if the condition is caused by a viral infection.

Mal de Debarquement

[00202]Mal de debarquement is a condition which usually occurs subsequent to a sustained motion event, for example, a cruise, car trip, or airplane ride. It is characterized by a persistent sense of motion, difficulty maintaining balancefatigue, and cognitive impairment. Symptoms may also include dizziness, headaches, hyperacusis, and"or tinnitus. Symptoms often last in excess of a month. Treatment includes benzodiazepines, diuretics, sodium channel blockers, and tricyclic antidepressants.

Microvascularcompression syndrome

1002031Microvascular 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 VII by a blood vessel Other symptoms found in subjects with MCS include, but are not limited to, severe motion intolerance, and neuralgiclike "quickspins". MCS is treated with carbamazepine. TRILEPTAL@, and baclofen. It can also be surgically treated.

OtherMicrobialIhfections Causing CochleovestibularDisorders

[002041Other microbial infections are known to cause cochleovestibular disorders, including hearing loss. Such infections include rubella, cytomegalovirus, mononucleosis, varicella zoster (chicken pox), pneumonia, Borrelia species of bacteria (Lyme disease), and certain. fungal infections. Accordingly, controlled release antimicrobial agent formulations disclosed herein are also used for localized treatment of these infections in the ear. Otic Disorderscaused by FreeRadicals 1002051Free radicals are highly reactive atoms, molecules, or ions the reactivity of which results from the presence of unpaired electrons. Reactive oxygen species ("ROS") formas a result of sequential reduction of molecular oxygen. Examples of reactive oxygen species of interest ("ROS") include, but are not limited to, superoxide, hydrogen peroxideand hydroxyl radicals. ROS are naturally produced as a by-product ofthe production of ATP. ROS can also result front the use of cisplatin, and aminoglycosides. Further, stress to stereocila caused by acoustic trauma results in otic hair cells producing ROS. 1002061ROS can damage cells directly by damaging nuclear DNA and mitochondrial DNA Damage to the former can lead tomutations 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 can lead to impaired cellular functioning or apoptosis. Further, ROS can also damage or kill 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. Arntioxidants can ameliorate damagelby caused by ROS by preventing their formation, or scavenging the ROS before they can damage the cell. {00207Damage to mitochondria by ROS is often seen in hearing loss, especially hearing loss due to S agingThe loss of ATP correlates to a loss in neural functioning in the inner ear. It can also lead 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 vastenergy requirements needed to maintain the ionic balance of fluids in the inner ear. Thus, the cells of the stria vascularis are most often damaged or killed due to damage of the mitochondria. Otosdelrosis

[00208]Otosclerosis is an abnormal growth of bone in themiddle ear, which prevents structures within the ear from transducing vibration, which causes hearing loss. Otoscelorosis usually effects the ossicles, in particular te stapes, which rests in the entrance to the cochlea in the oval window. The abnormal bone growth fixates the stapes onto the oval window preventing sound passing waves from traveling to the cochlea. Otoscelorosis may cause a sensorineural hearing loss, i.e. damaged nerve fibers or-hearing hair cells, or conductive hearing loss.

[002091'reatment of otoscelrosis may include surgery to remove the fixated stapes bone, called a stapedectomy. Fluoride treatment may also be used, which will not reverse the hearing loss but may slowthe development of otoscelorosis.

Ototoxicity

[002101Ototoxicity 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 VIL Multiple drugs are known to be ototoxic. Often ototoxicity is dose-dependent, It are permanent or reversible upon withdrawal of the drug. 1002111Known ototoxic drugs 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 (eg vancomycin), salicylic acid, nicotine, some chemotherapeutic agents (eg. actinonycin,beomycin, cisplatin, carboplatin and vincristine)hand some members of the loop diuretic family of drugs (e.g. furosemide).

[002121Cisplatin and theaminoglycoside class of antibiotics induce the production ofreactive oxygen species ("ROS"). ROS can damage cells directly by damaging DNA, polypeptides, and/or lipids. Antioxidants prevent damage of ROS by preventingtheir formation or scavengingfree radicals before they can damage the cell. Both cisplatin and the aminoglycoside class of antibiotics are also thought to damage the ear by binding melaninin the stria vascularis of the inner ear.

[00213]Salicylicacid 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 cells, not the inner otic hair cells. Accordingly, disclosed herein is the use of controlled release auris compositions comprising antioxidants to prevent, ameliorate or lessen ototoxic effects of chemotherapy, including but not limited to isplatin treatment, aminoglycoside or salicylic acid administration, or other ototoxic agents. Postural Vertigo

[002141 Postural vertigo, otherwise koi as positional vertigo, is characterized bysudden violent vertigo that is triggered by certain head positions. This condition are caused by damaged semicircular canals caused by physical jury to the auris internal, otitis media, ear surgery or blockage of the artery to the auris internal. t002151Vertigo onset in patients with postural vertigo usually develops when a person lies on one ear or tilts the head back tolookup. 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 may include meclizine, lorazepan prochlorperazine or scopolamine. Fluids and electrolytes may also be intravenously administered if the vomiting is severe Presh'cusis(Age Related HearingLoss)

[002161Presbycusis (or presbyacusis orage 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 z) 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"). i is difficult to filter out background noise. The disorder is most often treated by the implantation of a hearing aid and/or the administration of pharmaceutical agents which prevent the build up of ROS.

[00217]The disorder is caused by changes in the physiology of the inner ear, the middle ear, and/or the VII nerve. Changes in the inner ear resulting in presbycusis include epithelial atrophy with loss of otic hair cells and/or stereocilia, atrophy ofnerve cells, atrophy of thestria vascularisand the thickening/stiffening of the basilar membrane. Additional changes which can contribute to presbycusis include the accumulation of defects in the tympanic membrane and the ossicles.

[00218] Changes leading to presbyusis can occur due to the accumulation of mutations in DNA, and rautations in mitochondrial DNA; however, the changes are exacerbated by exposure to loud noise, exposure to ototoxic agents, infectionsand/or the lessening of blood flow to the ear The latter is attributable to atherosclerosis, diabetes, hypertension, andsmoking.

[00219]Presbycusis, or age-related hearing loss, occurs as a part of normal aging, and occurs as a result of degeneration of thereceptor cells in the spiral Organ of Corti in the auris interna. Other causes may also be attributed to a decrease in a number of nerve fibers in the vestibulocochlear nerve, as well as a loss offlexibility of the basilarmembranein the cochlea, There is currently no known cure for permanent hearing damageas a result of presbycusis or excessive noise, although treatment regimens have been proposedincluding treatment with antioxidants such as alpha lipoic acid. (Seidman etat Am.J. Otol. (2000) 21:161-167). RasayRunt's Syndrome (HerpesZosterinfection)

[002201Ramsay Hunt's syndrome is caused by a herpes zoster infection of the auditory nerveThe infection may cause 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 orneck supplied by the nerves. Facial muscles may also become paralyzed if the facial nerves are compressed by the swellingHearing loss are temporary or

permanent, with vertigo symptoms usually lasting from several days to weeks

[002211Treatment of Ramsay Hunt's syndrome includes administration of antiviral agents, including acyclovir, Other antiviral agents include fameiclovir and valacyclovir. Combination of antiviral and corticosteroid therapy may also be employed to ameliorate herpes zoster infection. Analgesics or narcotics may also be administered to relieve the pain, and diazempam or other central nervous system agents to suppress vertigo. Capsaicin, lidocaine patches and nerve blocks may also be used. Surgery may also be performed on compressed facial nerves to relieve facial paralysis. Recurrent Vestibulopathy 1002221Recurrent vestibulopathyis acondition whereinthesubject experiences multiple episodes of severe vertigo The episodes of vertigo may last forminutes or hours. Unlike Meniere's Disease, it is not accompanied by hearing loss. In some cases it may develop into Meniere's Disease or Benign Paroxysmal Positional Vertigo. Treatment is similar to that of Meniere's Disease.

Syphilis Infection 1002231Syphillis infection may also lead to congenital prenatal hearing loss, affecting approximately 11 .2 per 100,000 live births in the United States, as well as sudden hearing loss in adults, Syphilis is a venereal disease, caused by the spirochete Treponema pallidum, which in its secondary and tertiary stages may result in auditory and vestibular disorders due tomembranous labyrinths, and secondarily include meningitis. 100224]Both acquired and congenital syphilis can cause otic disorders. Symptoms of cochleovestibular disorders resulting from syphilis are oftensimilar to those of other otic disorders, such as AIED and Meniere's disease, and include finnitus, deafness, vertigo, malaise, sore throat, headaches, and skin rashes. Syphilis infection may lead to congenital prenatal hearing loss, affecting approximately 11.2 per 100,000 live births in the United States, as well as sudden hearing loss In adults.

[00225] Treatment with steroids and antibiotics, including penicillins (e.g. benzathine penicillin G (BICILLIN LA), are effective in eradicating the spirochete organism, However, Treponemas may remain inthe cochlear and vestibular endolymph even after eradication from other sites in the body.

Accordingly long term treatment with penicillins are warranted to achieve complete eradication of the spirochete organism from the endolymph fluid. 1002261Treatment of otosyphilis (syphilis presenting otic symptoms) typically includes a combination of steroids (e.g., prednisilone) and antibacterial agents (esgbenzathine penicillin G (BICILLIN LA). penicillin G procaine, doxycycline, tetracycline, ceftriaxone. azithromycin). Such treatments are effective in eradicating the spirochete organism. However, Treponemas mayremain in the cochlear and vestibular endolymph even after eradication from other sites in the body. Accordingly, long te treatment with penicillin are required to achieve complete eradication of the spirochete organism from the endolyrph fluid. Also,in the case of a severe or advanced case of syphilisauricosuric drug, suchas probenecid, are administered in conjunction with the antibacterial agent to increase its efficacy. Temporal Bone Fractures

[00227] The temporal bone,which contains part of the ear canal, the middle ear and the auris interma, is subject to fractures from blows to the skull or other injuries. Bleeding from the ear or patchy bruising is symptomatic of a fracture to the temporal bone, and may a computed tomography (CT) scan for accurate diagnosis. Temporal boric fractures may rupture the eardrum, causing facial paralysis and sensorineural hearing loss,

[00228] Treatment of detected temporal bone fractures includes an antibiotic regimen to prevent meningitis, or an infection of brain tissue, In addition, surgery are performed to relieve any subsequent pressure on the facial nerve due to swelling or infection. Temporomandibular.JointDisease 1002291Some evidence exists for a relationship between temporomandibularjoint disease (TMD) and auris internal disorders. Anatomical studies demonstrate the possible involvement of the trigeminal nerve, where trigemminal innervation of the vascular system has been shown to control cochlear and vestibular labyrinth function. (Vass et al. Neuroscience (1998) 84:559-567). Additionally, projections of ophthalmic fibers of the trigeininal Gasser ganglion to the cochlea through the basilar and anterior inferior cerebellar arteries can play an important role in the vascular tone in quick vsaodilatatory response to metabolic stresses. e.g. intense noise. Auris interna diseases and symptoms, such as sudden hearing loss, vertigo and tinnitus, may originate front reduction of the cochlear blood flow due to the presence of abnormal activity in the trigeminal ganglion, for example from migrane or by the central excitatory effect originated in chronic or deep pain produced byTMD. 1002301Sinilarly, other researchers have found that the trigeminal ganglionalso innervates the ventral cochlcar nucleus and the superior olivary complex, which may interfere with authditory pathways leading to the auditory cortex where constant peripheral somatic signals from the opthahnic and mandibular trigenital peripheral innvervation occurs in TMD cases. (Shore et al. J.

Comp. Neurology (2000) 10:271-285). These somatosensoory and auditory system. interactions via the central nervous system may explain otic symptoms in the absence of existing disease in the ear, nose or throat.

[00231}Accordingly, forcefl muscle contractionsin TMD may elicit modulations in the neurological and auditory and equilibrium function. For example, the auditory and vestibular modulationsmayoccurasaresult of hyerptonicity andmuscular spasm,wvhich in turn irritates nerves and blood vessels that affect auris internal function by muscular trapping. Relief of the affected nerve or muscular contractions may act to relieve auditory or vestibular symptoms. Medications, including barbiturates or diazepam, may thus relieve auditory or vestibular dysfunction in TMD patients. UtricularDysunction

t002321 The utrile is one of the two otoliths found in the vestibular labyrinth. It is responsive to both gravity and linear acceleration along thehorizontal plane. Utricular dysfunction is a disorder caused by damage to the utricle.It is often characterized by subject's perception of tilting or imbalance.

Vertigo

[00233Vertigo is described as a feeling of spinning or swaying while the body is stationary. There are two types of vertigo. Subjective vertigo is the falsesensation ofmovement 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

[G02341While 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 in increased pressure on the cells of the inner ear which leads to the sensation of movement The most common cause of vertigo is benign paroxysmal positional vertigo, or BPPV. It can also be brought on by a head injury, or a sudden change of blood pressure. It is a diagnostic symptom of several diseases includingsuperior canal dehiscence syndrome. Vestibular Neuronitis

[002351Vestibular neuronitis, or vestibular neuropathy, is anacute, 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 may present as a single attack of vertigo, a series of attacks, or a persistent condition which diminishes over amatter of weeks. Symptoms typically include nausea, vomiting, and previous upper respiratory tract infections although there are generally no auditory symptoms. The first attack of vertigo usually severe, leading to nystagmus, a condition characterizedby flickering of the eyes involuntarily toward the affected side.Hearing loss does not usually occur.

[002361In 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 electronystamography, a method of electronically recording eye movements. Magnetic resonance imaging may also be performed to determine if other causes may play a role in the vertigo symptoms.

[002371Treatment of vestibular neuronitis typically involves alleviating 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 may include melizine, lorazepam, prochlorperazine, or scopolamine. Fluids and electrolytes may also be intravenously administered if the vomiting is severe. Corticosteroids, such as prednisilone, are also given if the condition is detected early enough.

[00238]Compositions disclosed herein comprising an antiviral agent can be administered for the treatment of vestibular neuronitis. Further, the compositions are administered with other agents that are typically used to treat symptoms of the condition, including antichoinergics antihistamines, benzodiazepines, or steroids. Treatment of vertigo is identical to Meniere's disease, and may

include meclizine, lorazepam, prochlorperazine or scopohnnine. Fluids and electrolytes may also be intravenously administered if the vomiting is severe.

[002391 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. 1002401There are several treatments for vestibular neuronitis. H1-receptor antagonists, such as dimenhydrinate, diphenhydraminemeclizine, and promuethazine, diminish vestibular stimulation and depress labyrinthine function through anticholinergie effects. Benzodiazepines, such as diazepan and lorazepamare 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 pathwaysFinally, corticosteroids (i. e prednisone) are prescribed to ameliorate the inflammation of the vestibular nerve and associated apparatus. Advantages of local otic administration 1002411To overcome the toxic and attendant side effects of systemic delivery, disclosed herein are methods and compositions for local delivery of therapeutic agents to auris media and/or auris interna structures. Access to, for example, the vestibular and cochlear apparatus will occur through theauris media including the round window membrane, the oval window/stapes footplate, the annular ligament and through the otic capsule/temporal bone.

f00242]In addition, localized treatment of the auris media and/or auris internal also affords the useof previously undesired therapeutic agents, including agents with poor PK profiles, poor uptake, low systemic release and/or toxicityissues. Because of the localized targeting of the otic agent formulations and compositions, as well as the biological blood barrier present in the auris internal the risk of adverse effects will be reduced as a result of treatment with previously characterized toxic or ineffective otic active agents, (e.g,immunomodulatory agents such as anti-TNF agents). Accordingly, also contemplated within thescope ofthe 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 oic agent.

[00243By specifically targeting the auris media or auris interna structures, adverse side effects as a result of systemic treatment are avoided. Moreover, by providing a controlled release otic agent formulation (e.g., immunomodulating agent or auris pressure modulator formulation) or composition to treat otic disorders, a constant, variable and/or extended source of an otic agent is provided to the individual or patient suffering from an otic disorder, reducing or eliminating the variability of treatment. Accordingly, one embodiment disclosed herein is to provide a formulation that enables at least one therapeutic agent to be released in therapeutically effective doses eitherat variable or constant rates such as to ensure a continuous release of the at least one agenL In some embodiments, the auris active agents disclosed herein are administered as an immediate release formulation or composition. In other embodiments, the auris active 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 active agent formulation is administered as both an immediate releaseand controlled release formulation, released either continuously orin a pulsatile manner, or variants of both. The release is optionally dependent on environmental or physiological conditions, for example, the external ionic enviroinent (see, e.g Oros* release system, Johnson &Johnson). t002441Also included within the embodiments disclosed herein is the use ofadditional auris media and/or auris internal agents in combination with the otic agent formulations and compositions disclosed herein. When used, such agents assist in the treatment of hearing or equilibrium loss or dysffinction as a result of an autoimmune disorder, including vertigo, tinnitus, hearing loss, balance disorders, infections, inflammatory response or combinations thereof. Accordingly, agents that ameliorate or reduce the effects of vertigo, tinnitus, hearing loss, balance disorders, infections, inflammatory response or combinations thereof are also contemplated to be used in combination with the otic agents described hereinincluding steroids, anti-emetic agents, local anesthetic agents, corticosteroids,chemotherapeutic agents, including cytoxan, azathiaprine ormethotrexate treatment with collagen, gamma globulin, interferons, copaxone, central nervous system agentsantibiotics, platelet-activating factor antagonists, nitric oxide synthase inhibitors and combinations thereof

100245]In addition, the auris-compatible pharmaceutical compositions or formulations included herein also include carriers, adjuvants, such as preserving, stabilizing, wetting or emuisifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. Such carriers, adjuvants, and other excipients will be compatible withthe environment in the auris media and/or auris internal. 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 pharmaceutical compositions or formulations disclosed herein are optionally targeted to distinct regions of the auris media and/or auris internal, 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 internal.

Treatment

is 1002461Provided herein are otic compositions suitablefor the treatment of any otic condition, disease or disorder (e.g., middle and/or inner ear disorder) described herein, comprising administration of an auris formulation described herein to an individual or patient in need thereof. The formulations 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 ocurrence of disease in individuals suspected ofhaving a disease orin 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.

[002471In some instances, because of their otic compatiblity and improved sterilitythe formulations described herein are safefor long-term administration. The auris compositions described herein have very low ototoxicity and provide a steady sustained release of a therapeutic agent for a period of at least one week, two weeks, three weeks or amonth. 1002481Provided herein are controlled release compositions and formulations to treat and/or prevent diseases associated with the ear, including the cochlea, the middle ear and inner ear, including autoinnune inner ear disorder (AIED), Mdnire's disease (endolymphatic hydrops). noise induced hearing loss (NIHL), sensorineural hearing loss (SNL) tinnnitus, otosclerosis, balance disorders, vertigo and the like.

[002491The etiology of several ear diseases or disorders consists of asyndrome of progressive hearing loss, including noise induced hearing loss and age-related hearing loss, dizzinessnausea, nystagmus, vertigo, tinnitus, inflammation, swelling, infection and/or congestion. These disorders may have many causes, such as infection, exposure to noise, injury, inflammation, tumors and/or adverse response to drugs or other chemical agents. Several causes ofhearing and/or equilibrium impairment are attributed to inflammation and/or an autoiimmune disorder and/or a cytokine mediated inflammatory response.

[00250]Provided herein are controlled release immunomodulator compositions and formulations to treat inflammation or infection of the auris media, including otitis media. A few therapeutic products are available for the treatment of AIED, including anti-TNF agents; however, systemic routes via oral, intravenous or intramuscular routes are currently used to deliver these therapeutic agents.

[002511Provided herein are controlled release aural pressure modulating compositions and tO formulations to treat fluid homeostasis disorders of the inner ear, including Meniere's Disease, endolymphatic hydrops, progressive hearing loss, including noise induced hearing loss andage related hearing loss, dizziness, vertigo,tinnitus and similar conditions.

[00252]Insome embodiments, the compositions provided herein are CNS modulating compositions and formulations to treat tinnitus, progressive hearing loss,including noise induced hearing loss and age-related hearing loss, and balance disorders. Balance disorders include benign paroxysmal positions vertigo, dizziness, endolymphatic hydrops7kinetosislabyrinthitis Mal de debarquement, Meniere's Disease, Meniere's Syndrome, myringitis, otitis media, Ramsay Hunt's Syndrome, recurrent vestibulopathy, tinnitus, vertigo, microvascular compression syndrome, utricular dysfunction, and vestibular neuronitis. A few therapeutic products are available for the treatment of balance disorders, including GABAA receptor modulators and local anesthetics.

[002531In some embodiments, the compositionsprovided herein are cytotoxic agent compositions and formulations for the treatment of autoimmune diseases of the ear, including autoimmune inner ear disease (AED). Also provided herein are controlled release cytotoxic agent compositions for the treatment of disorders of the auris media, including otitis media. The compositions disclosed herein are also useful for the treatment of cancer, particularly cancer of the ear. A few therapeutic products are available for the treatment of AIED, including certain cytotoxic agents. Particularly, the cytotoxic agents methotrexateand cyclophosphamide have been tested andare used forsystemic treatment of AIED. Also, thalidomide, while not currently administered Ior the treatment of AIED, has been used to treat Behget's disease, whichis often associated with AIED.

[002541In some embodiments, the compositions provided herein comprise auris sensory cell modulators for treating orameliorating hearing loss orreduction resulting from destroyed, stunted, malfunctioning damaged, fragile or missing hairs in the inner ear Further disclosed herein are controlled release auris sensory cell modulatingagent compositions and formulations to treat ototoxicity, excitotoxicity, sensorineural hearing loss, Meniere's Disease/Syndrome, endolymphatic hydrops, labyrinthitis, Raisay Hunt's Syndrome, vestibular neuronitis and microvascular compression syndrome.

1002551n some embodiments, the compositions provided herein are antimicrobial agent compositions and formulations for the treatment of otic disorders, including otitisexterna, otitis media. Ramsay Hunt syndrome, otosyphilis AIED. Meniere's disease, and vestibularneuronitis.

[002561In some embodiments, the compositions provided herein prevent, relieve, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals and/or the dysfunction of the mitochondria. 1002571 Also provided herein are controlled release ion channel modulating compositions and formulations to treat fluid homeostasis disorders of the inner ear, including Meniere's Disease, endolymphatic hydrops, progressive hearing loss, including noise inducedhearing loss and age related hearing loss, dizziness, vertigo, tinnitus and similar conditions. Systemic routes via oral, intravenous or intramuscular routes are currently used to deliver ion channel modulating therapeutic agents. Therapeutic Agents

[00258Notwithstanding any therapeutic agent used in the formulations described herein, the otie compositions described herein will have pH and osmolarity that is auris-acceptable. Anyotic composition described herein meets the stringent sterility requirements described herein and will be compatible with the endolymph and/or the perilymph. Pharmaceutical agents that are used in conjunction with theformulations disclosed herein include agents that ameliorate or lessen otic disorders, including auris internal disorders, and their attendant symptoms, which include but are not limited to hearing loss, nystagmus, vertigo, tinnitus, inflammationswelling, infection and

congestion. Otic disorders may have many causes, such as infection, injury, inflammation, tumors and adverse response to drugs or other chemical agents that are responsive to the pharmaceutical agents disclosed herein. A skilled practitioner would be familiar with agents that are useful in the amelioration or eradication of otic disorder; accordingly, agents which are not disclosed herein but are usefulfor the amelioration or eradication of otic disorders are expressly included and intended within the scope of the embodiments presented. In some embodiments, phannaceutically active metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of the otic agents disclosed herein that retain the ability of the parent antimicrobial agents to treat otic disorders are usefulin the formulations.

[002591Active ingredients or otic therapeutic agents include, but are not limited to, anti inflammatory agents, anti- anti-oxidants, neuroprotective agents, glutamate modulators, TNF-alpha modulators, interleukin I beta modulators,retinaldehyde modulators, notch modulators, gamma secretase modulators, thalidomide, ion and/or flulid (eg., water) homeostasismodulators, vasopressin inhibitors, inhibitors ofthe vasopressin-mediated AQP2 (aquaporin 2) system, transcriptional regulators of the innerear transcriptional regulatory network (including, eg, transcriptional regulators of estrogen-related receptor beta), inner ear hair cell growth factors,

- 54.

including BDNF (brain derived and NF-3,and other therapeutic modalities. Agents explicitly include an agonist of an otic target, a partial agonist of an otic target, an antagonist of an otic target, a partialantagonist of an otic target, an inverse agonist of an otic target, a competitive antagonist of an otic target, a neutral antagonist of an otic target, an orthostericantagonist ofan otic target, an allosteric antagonist of an otic target a positive alIosteric modulator of an otic target, or combinations thereof.

[00260 In addition, because the formulation is designed such that the active ingredient has limited or no systemic release, agents that produce systemic toxicities(e.g.,iver toxicity) or have poor PK characteristics (e.g. short half-life) are also optionally used. Thus, pharmaceutical agents which have to 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 drug in particular organs, tissues orsystems, through high levels needed to achieve efficacy, through the inability to be released through systemic pathways or through poor PK characteristics, are usefulin some embodiments herein. The formulations 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 aural pressure modulating 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 aural pressuremodulating formulation disclosed herein is 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 aural pressuremodulating formulations through use of a needle and syringe, a pump, a microinjection device, an in situ forming spongy material or any combination thereof.

[00261]In still other embodiments, application of any otic agent formulation described herein is targeted to the auris media through piercing of the intratynpanic membrane and applying the otic agent formulation directly to the auris media structures affected, including the walls of the tympanic cavity or auditory ossicles. By doing so, the auris active agent fornrulations disclosed herein are confined to the targeted auris media structure, and will not be lostfor example, through diffusion or leakage through the eustachian tube or pierced tympanic membrane. In some embodiments, the auris-compatible formulations disclosed herein are delivered to the auris externa in any suitable manner, including by cottonswab, injection or ear drops. Also, in other embodiments, the otic formulations described herein are targeted to specific regions of the auris external by applicationwith a needle and syringe, a pump, amicroinjection devicean in situ forming spongy material or any combination thereofFor example, in the case of treatment of otitis externa, antimicrobial agent fonnulations 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

[002621In some embodiments, agents which mayhave been previously rejected as, for example, an antimicrobial agent, may find use herein because of the targeted nature oftheembodiments which bypasssysteic effects, including toxicity and harmful side effects. By way of example only, onercept, a previously rejected anti-TNFagent due to toxicity and safetyissues, is useful as an anti TNF agent in sonie of the embodiments disclosed herein. Also contemplated within the scope of embodiments described herein is the administration ofhigher doses of pharmaceutical agents, for example agents that have dose limiting toxicities, compared to currently approved doses for such pharmaceutical agents 1002631Some pharmaceutical agents, either alone or in combination, are ototoxic.For example, some chemotherapeutic agents, including actinomycinbleomnycin, cisplatin, carboplatin and vincristine; and antibiotics, including erythromycin, gentamicinstreptomycin,dihydrostreptomycin, tobramycin, netilnicin, amikacin, neomycin, kanamycin, etiomycin, vancomycinmetronidizole, capreomycin, are mildly to very toxic, and may affect the vestibular and cochlear structures differentially.1However, in sone embodiments, the combination of an ototoxic drug, for example cisplatin, combination with an antioxidant is protective and lessen the ototoxic effects of the drug. Moreover, the localized application of the potentially ototoxic drug lessens the toxic effects that might otherwise occur through systemic application through the use of lower aniounts with maintained efficacy, or the use of targeted amounts for a shorter period of time. Accordingly, a skilled practitioner choosing a course of therapy for targeted otic disorder will have the knowledge to avoid or combine an ototoxic compound, or to vary the amount or course of treatment to avoid or lessen ototoxic effects.

[00264]In certain instances, pharmaceutical excipients, diluents or carriers are potentially ototoxic. For example, benzalkoniuni chloride, a common preservative, is ototoxic and therefore potentially harmful if introduced into the vestibular or cochlear structures In formulating a controlled release otic formulation,it is advised to avoid or combine the appropriate excipients, diluents or carriers to lessen or eliminate potential ototoxic components front the formulation, or to decrease the amount of such excipients, diluents or carriers. In some instances, the ototoxicity of the pharmaceutical agents, excipients, diluents, carriers, or formulations and compositions disclosed herein can be ascertained using an accepted animal model. See, e.T. Maritini, A., etal. Ann. N.Y Acad. Sci. (1999) 884:85-98. Optionally, a controlled release otic formulation includes otoprotective agents,such as antioxidants, alpha lipoic acid, calicum, fosfomycin or iron chelators, to counteract potential ototoxic effects that may arise from the use of specific therapeutic agents or excipients, diluents or carriers. 002651Other agents that are used in the embodiments disclosed herein, either alone or in combination with other auris internal agents, include anti-apoptotic agents, including caspases, JNK the insulindike growth factor (IGF) receptor, the epidermal growth factor (EGF) receptor, and/or the platlet-derived growth factor, In some embodiments, the growth factor is hepatocyte growth factor. 003701In some embodiments, the growth factoris an epidermal growth factor (EGF). In sone embodiments, the EGF is heregulin (HRO) In certain instancesHRG stimulates the proliferation of utricular sensory epithelium. In certain instances, 1RG-binding receptors are found in the vestibular and auditory sensory epithelium. 100371]In some embodiments, the growth factor is an insulin-like growth factor (GF). In some embodiments,the GF is GF-1 .Insome embodiments, theJGF-1 is mecasennin. In certain. instances,JGF- attenuates the damageinduced by exposure to an ainoglycoside. In certain instances, IE-1 stimulates the differentiation and/or maturation of cochlear ganglion cells, 100372]In some embodiments, the FGF receptor agonist is FGF-2. In some embodiments, the IGF receptor agonist is IGF Both the FGF and IGF receptors are found in the cells comprising the utricle epithelium. 1003731In some embodiments, the growth factor is hepatocyte growth factor (HGF). In some

[5 instances, HGF protects cochlear hair cells from noise-induced damage and reduces noise-exposure causedABR thresholdshifts,

[003741Also contemplated for use in the otic formulations described herein are growth factors including Erythropoietin(EPO), Granulocyte-colony stimulating factor (G-CSF). Granulocyte macrophage colony stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Insulin !0 like growth factor (IGF), Myostatin (GDF-8), Platelet-derived growth factor (PDGF), Thronbopoitin (TPO), Transforming growth factor alpha (TGF-),'Transforming growth factor beta (TE-f), Vascular endothelial growth factor (VEGF) or combinations thereof Neurotrophs

[003751In some embodiments, the growth factor is a neurotroph. In certain instances, neurotrophs are growth factors which prevent cells from initiatingapoptosis, repair damaged neurons and otic hair cells, and/or induce differentiation in progenitor cells. In some embodiments, the neurotroph is brain-derived neurotrophic factor (BDNF), iliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor (GDNF). neurotrophin-3, neurotrophin-4, and/or combinations thereof 100376] In some embodiments, the neurotroph is BDNF, In certain instances, BDNF isa neurotroph which promotes the survival of existing neurons (e.g. spiral ganglion neurons), and otic hair cells by repairing damaged cells, inhibiting the production of ROS, and inhibiting the induction of apoptosis. In certain embodimentsit also promotes the differentiation of neural and otic hair cell progenitors. Further, in certain embodiments, it protects the Cranial Nerve V1 from degeneration. In some embodiments, BDNF is administered in conjunction with fibroblast growth factor.

[00377]In some embodiments, the neurotroph is neurotrophin-3. In certain embodiments, neurotrophin-3 promotes the survival of existing neurons and otic hair cells, and promotes the differentiation of neural and otic hair cell progenitors. Further, in certain embodiments, it protects the VII nerve from degeneration.

[00378] In some embodiments, the neurotroph is CNTF In certain embodiments, CNTF promotes the synthesis of neurotransmitters and the growth of neuritis. In some embodiments, CNTF is administered in conjunction with BDNF.

[003791In some embodiments, the neurotroph is GDNF. In certain embodiments, GDNF expression is increased by treatment with ototoxic agents. Further, in certain embodiments, cells treated with exogenous GDNF havehigher survival rates after trauma then untreated cells. Immune System Cells

[00380]Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration of neurons and/or hair cells of the auris, and agents for treating or ameliorating hearing loss or reduction resulting from destroyed, stunted, malfunctioning, damaged, fragile or missing hairs in the inner ear. Accordingly, some embodiments incorporate the use of cells which participate in the repair of otic hair cells and neurons. In some embodiments, the cells which participate in the repair of otic hair cells andneurons are macrophages, ricroglia, and/ormicroglia-like cells. In certain instances, the concentration of macrophages and microglia increase in ears damaged by treatment with ototoxic agents. In certain instances, nicroglia like cells eliminate waste from the Organ of Corti and participate in the structural repair of hair cells following treatment with the ototoxic antibiotic neomycin. Ototoxic Agents 10038.11Contemplated for use with the formulations disclosed herein are agents that destroy neurons and/or otic hair cells. Accordingly, some embodiments incorporate the use of agents which fatally damage and/or induce apoptosis in the neurons and/or otic hair cells of the auris. in some embodiments, the agents which fatally damage and/or induce apoptosis in the neurons and/or otic hair cells of the auris are the aminoglycoside antibiotics (e.g.gentamicin, and anikacin), the macrolide antibiotics (e.g erythromycin), the glycopeptide antibiotics (e g. vancomycin), the loop diuretics (e.g. furosemide) salicylic acid, and nicotine. RetinoblastonaProteinModulation

[00382]Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration of neurons and/or hair cells of the auris, promote the growth of neurons and/or hair cells of the auris, and agents for treating or ameliorating hearing loss orreduction resulting from destroyed, stunted, malfunctioningdamaged, fragile or missing hairs in the inner ear. Further contemplated herein are agents that destroy neurons and/or otic hair cells. Accordingly, sonic embodinents incorporate the use of agents that modulate retinoblastoma protein (pRB). pRB is a member of the pocket protein family, It is encoded by the RB1 gene, In certain instances, it inhibits transition from G1 to S phase bybinding to and inactivating the E2f family of transcription factors.

In certain instances, it also regulates differentiation, and survival of hair cells. In certain instances, pRB knock-out mice demonstrate increased proliferation of hair cells.

[00383] In some embodiments, the agent that modulates one or more of the pRB is an agonist of pRB In some embodiments, the agent that modulates one or more of the pRB is an antagonist of pRBK In certain instances, a compound which agonizes or antagonizes pRB isidentified (e.g. by use of a high throughput screen). In some embodiments, a construct is designed such that a reporter gene is placed downstream of an £2F binding sequence. In some embodiments, theobinding sequence is TTTCGCGC, In some embodiments, the reporter gene is hiciferase, , CGFP, l4actamase or 0 galactosidase. In certain instances, E2f binds to the bindingsequence causing the transcription and expression ofthe reporter gene. In certain instances, an agonist of pRB causes an increase in the binding of pRB to E2f In certain instances, the increase in binding of pRB and E2f results in a decrease in the transcription and expression of the reporter gene. In certain instances, an antagonist of pRB causes a decrease in the binding of pRB to E2f. In certain instances, the decrease in binding of pRB and E2f results in a increase in the transcription and expression of the reporter gene. t5 1003841In some embodiments, the agent that modulates pRB is an siRNA molecule. In certain instances, thesiRNA molecule is as described herein. Salicylic acid 1003851Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration ofneurons and/or hair cells of the auris, and agents fortreating or ameliorating hearing loss or reduction resulting from destroyed, stunted, malfunctioning, damaged. fragile or missing hairs in the inner ear. Accordingly, some embodiments incorporate the use of salicylic acid. In certain instances, when administered before treatment with an aminoglycoside, it protects otic hair cells and spiral gangion neurons from aminoglycoside otatoxicity. Sodium Channel Blockers

[003861Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration of neurons and hair cells, and agents for treating or ameliorating hearing loss or reduction resulting from destroyed, stunted, malfunctioning, damaged, fragile or missing hairs in the inner ear. In certain instances, excitotoxicity causes the excessive opening of Na* channels. In certain instances, this results in excess Na ions entering the neuron. In certain instances, the excess influx of Na ions into the neuron causes the neuron to fire more often. In certain instances, this increasedfiring yields a rapid buildup of free radicals and inflammatory compounds. in certain instances, the free radicals damage the mitochondria, depleting the cell's energy stores. Further, in certain instances, excess levels of Na' ions activate excess levels of enzymes including, but not limited to, phospholipases, endonucleases, and proteases. In certain instances, the over-activation of these enzymes results in damage to the cytoskeleton, plasma membrane, mitochondria, and YNA of the neuron, Accordingly, some embodiments incorporate the use of agents which antagonize the opening of Na' channels. In some embodiments. sodium channel blockers are as described herein. Stem Cells and DifferentiatedAuris Sensory Cells 1003871Contemplatedfor use with the formulations disclosed herein are transplants of cells that supplement and/or replace the pre-existing neurons and/or hair cells of the auris In some embodiments, the agent is a stem cell In some embodiments, the agent is a partially or fully differentiated auris sensory cell, In some embodiments, the differentiated auris sensory cell is derived from a human donor. In some embodiments, the differentiated auris sensory cell is derived from a stem cell, the differentiation of which was induced under artificial (e.g. laboratory) 1.0 conditions. t00388]Stem cells are cells that possess the capacity to differentiate into multiple cell types. Totipotent stem cells can differentiate into embryonic cells or extraembryonic cells, Pluripotent cells can differentiate into cells of any of endoderm, nesodermn.,or ectoderm origin. Multipotent cells can differentiate into closely related cells (e.g hematopoietic stem cells). Unipotent cells can differentiate into only one type ofcell, but like other stem cells have the characteristic of self renewal. in some embodiments, the stem cell is totipotent, pluripotent, rultipotent, or unipotent. Further, stem cells canundergo mitotic division without themselves differentiating (i.e self renewal).

[00389] Embryonic stem (ES) cells are stem cells derived from the epiblast tissue of the inner cell mass of a blastocyst or earlier stage embryo. ES cells are pluripotent. In some embodiments, the stein cell is an ES cell. Adult stem cells (also known as somatic cells or genline cells) are cells isolated from a developed organism wherein the cells possess thecharacteristic of self-renewal, and the ability to differentiate into multiple cell types, Adult stem cells arepluripotent (for example, stem cells found in umbilical cord blood), multipotent or unipotent. Insome embodiments, the stem cell is an adult stem cell.

[00390]In some embodiments, a stem cell and/or a differentiated auris sensory cell is administered in combination with a differentiation stimulating agent. In some embodiments, the differentiation stimulating agent is a growth factor. In some embodiments, thegrowth factor is aneurotrophit (eg nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ornovel neurotrophin-l (NN~IT). in some embodiments, the growth factor is FGF, EGF, GF, PGF, or combinations thereof

[00391]In some embodiments, a stem cell and/or a differentiated auris sensory cell is administered to a subject in need thereof as a controlled release agent, In some embodiments, a stem cell and/ora differentiated auris sensory cell is administered to subject in need thereof as an immediate release agent (e.g. in a cell suspension) in combination with a controlled release auris sensory cell modulating agent.In some embodiments, a controlled release auris sensory cell modulating agent is a vector comprising an Atohl or BRN3 gene, an siRNA sequence targeting R131, a growth factor, or combinations thereof. 1003921In some embodiments, a stern cell and/or a differentiated auris sensory cell is administered to the cochlea or vestibular labyrinth. In some embodiments, a stem cell and/or a differentiated auris sensory cell is administered by via intratympanic injection, and/or a post-auricular incision. In some embodiments, a stem cell and/or a differentiated auris sensory cell is contacted with the Organ of Cortivestibulocochlear nerve, and/or crista ampullaris. Thyroid Hormone ReceptorAlodulation 1003931Contemplated for use with the formulations disclosed herein are agents that modulate the W0 degeneration of neurons and/or hair cells of the auris, promote the growth. of neurons and/or hair cells of the auris, and agents for treating or ameliorating hearing loss or reduction resulting from destroyed, stunted, malfunctioning, damaged, fragile or missing hairs in the inner ear. Accordingly, some embodiments incorporate the use of agents thatmodulateThyroid Honnone (TH) receptors. TheTH receptors are a family of nuclear hormone receptors. The family includes, but is not limited to TRa Iand TRp. In certain instances, TR knock-out mice demonstrate a decreased responsiveness to auditory stimuli, and a decrease in K current inhair cells.

[003941In some embodiments, the agentthat modulates one or more of the TH receptors is an agonist of the one or more TH receptors. In some embodiments, the agonist of one or more of the TH receptors is T(3,5,3'-triiodo-L-thyronine); KB-141 (35-dichloro-4-(4-hydroxy-3 isopropylphenoxy)phenylacetic acid); GC-1 (3,5-dimethyl-4-(4'-hydroxy-3-isopropylbenzyl) phenoxy acetic acid); GC-24 (3,5-dimethy-4-(4'-hydroxy-3-benzyl)benzylphenoxacetic acid); sobetirome (QRX-431); 4-OH-PCB106 (4-01H-2,3,3,4',5'-pentachlorobiphenyl); MB07811 ((2R.,4S)-4(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4 r -hydroxy-3 r isopropylbenzyl)phenoxy)methyl-2-oxido-[1,3,2-dioxaphosphonane); MB07344 (35-dimethyl-4 (4:r -hydroxy-3 z -isopropylbenzyl)phenoxy)mehylphosphonic acid); and combinations thereof. In certain instances, KB-141; GC1; sobetirome; and GC-24 are selective forTR TRPV.Modulation

[00395 Contemplated for use with the formulations disclosed herein are agents that modulate the degeneration of neurons and hair cells, and agents for treating or ameliorating hearing loss or reduction resulting from destroyed, stunted, malfunctioning, damaged, fragile or missing hairs in the inner car. Accordingly, some embodiments incorporate the use of agents thatmodulate TRPV receptors. TheTRPV (Transient Receptor Potential Channel Vanilloid) receptors are a family of non-selective ion channels permeable to calcium, amongst other ions. There are six members of the family: TRPV1-6. In certain instances, following treatment with kanamycin, TRPV 1 is upregulated. Additionally, in certain instances, antagonism of theTRPV 4 receptor makes micevulnerable to acoustic trauma, Further, in certain instances, capsaicin, an agonist of TRPV 1, prevents hyperlocomotion following an ischemic event.

[003961In some embodiments, the agent that modulates one or more of theTRPV receptors is an agonist of the one or more TRPV receptors. In some embodiments, the agonist of one or more of the TRPV receptors is capsaicin, resiniferatoxin, or combinations thereof. In sone embodiments TRPV modulating include the TRPV modulators disclosed in US application publications 2005/0277643, 2005/0215572, 2006/0194801, 2006/0205773, 2006/0194801, 2008/0175794, 2008/0153857, 2008/0085901,20080015183, 2006/0030618, 2005/0277646, 2005/0277631, 2005/0272931, 2005/0227986, 2005/0153984,2006/0270682, 2006/0211741, 2006/0205980, and 2006/0100490, and/or combinations thereof Sensory Hair Cell Restorative Agents 100397In some instances, immunomodulators and/or aural pressure modulators modulate the function of neurons and/or auris sensory cells.Therapeutic agents which assist in restoringsensory hair cel presence or function are also contemplated herein. These therapeutic agents assist in the treatment of hearing loss in patients, including sensoneuralhearing loss, presbycusis and hearing loss from excessive noise. Recent studies have demonstrated the use of insulin-like growth factor 1 (IGF-1) in the restoration of auditory function for noise-induced hearing loss patients. (Lee et al Otol Neurotol (2007) 28:976-981). Accordingly agents GF-1 IGF-l agonists or agentswhich upregulate the expression, production or function of IGF-i are optionally included with the formulations described herein. AdenosineModulators 1003981Adenosine is comprised of adenine attached to ribofuranose via afl-N9-glycosidic bond.In certain instances, adenosine is aninhibitory neurotransmitter. In certain instances, itfunctions as a ligand for four GPCRs - adenosine receptor A, adenosine receptor A2Aadenosine receptor A, and adenosine receptor A,in certain instances, the binding of adenosine toan adenosine receptor results in (either partially orfully) an anti-inflammatory effect, In certain instances, the binding of adenosine to an adenosine receptor results in (either partially or fully) vasodialation.In certain instances, it is produced in response to cellular damage (e.g., hypoxia, and ischemia), For example, depolarizationand asphyxia in theear induce the release of adenosine into perilymph where it exerts a protective effect,

[00399] Accordingly, in some embodiment adensoine modulators are used in thetreatment of cochlear and vestibular disorders. hi sonic embodiments, the adenosine modulator is ATI313 (4-(3 (6-aminoA-9-(5-cyclopropylearbamoyl-3,4-diliydroxytetrahydrofuran-2-yl)-9H-purin-2-yh)prop-2 ynyl)piperidine-1-carboxylic acid methyl ester); GW328267X ((2R,3R4S,5R)-2{6-amino-2-[(I. benzyl-2-hydroxyethyl)amino]-9H-purin-9-yl)-5-(2-ethyl-21-tetrazol-5-yl)tetrahydrofurn-3,4 diol); CGS 21680 hydrochloride (4-{2-[[6-Amino-9-(N-ethy-b-Dribofuranuronamindosyl)-9H purin-2-yl]aiino]ethyl]benzenepropanoic acid hydrochloride); CV 1808 (2 Phenylaminoadenosine); p-DITC-APEC (2-[4-2-[2-[(4-Isothiocyanatophenyl)thiocarbonylamino]e thylaminocarbonyl~ethyl]phenethylamino-5'-N-ethylcarbo xamidadenosine); SDZ WAG994 (N Cyclohexyl-2'-O-methyladenosine); CVT-3146 (regadenoson;1 -(9-(3,4-dihydroxy-5 (hydroxymethyl)oxolan-2-vi)-6-aminopurin-2-yl)pyrazol-4-yl)-N- methylcarboxamnide); ATL-146e (4-{3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2 ynyl} -cyclohexanecarboxylic acid methyl ester); 5'-n- Ethyl-carboxamidoadenosine; tecadenoson; CVT-510 (N-(3(R)-tetrahydrofuranyl)-6-aminopurine riboside); CCPA (2-ChloroN6 cyciopentyladenosine); CPA (N6T-Cyclopentyladenosine); GR 79236 (N-[(lS,2S)-2 Hydroxycycloperityl]adenosine); 2'-MeCCPA; PD 81723 ((2-Amino-4,5-dinethyl-3-thienyl)-[3 (trifluoromethyl)ph enylmethanone); PSB.36 (1-Butyl-8-hexahvdro2.,5-methanopentalen-3a(1H)~ yl)-3,7 -dihydro-3-(3-hydroxypropyl)-1Hpurine-2,6-dione); ribavirin; CHA (N6 cyclohexyladenosine); GW493838 (GSK); (-)-N6-(2-phenylisopropyl) adenosine; GW684067 ((2R.,3R4S,5R)-5-ethynyl-2-[6-tetrahydro-2H-pyran-4-ylanino)-9H-purin-9-viltetrahydrofuran-34 diol); CVT-3619 (2-(6-((2hydroxycyclopeniyl)aino)purin-9-yl)-5-((2 fluorophenythio)methyl)oxolane-3,4-diol); 2-C-1B-MECA (CF102;2-chloro (3-iodobenzyl)-5 N-methylcarbamoyladenosine); HEMADO; iB-MECA (CF101;N 6-(3-iodobenzyl)-5'-N methylcarbamoyladenosine); CP-532903 (N-(2,5-Dichlorobenzyl)-3taninoadenosine-5-N methylcarboxamide); CF502 (Can-Fite BioPharra) LJ-529 (2- chloro- N(6)-(3- iodobenzvl)- 5 N- methylcarbamoyl- 4- thioadenosine); BAA (8- butylaminoadenosine); 6-Amino-2-chloropurine riboside; 2-Chloroadenosine; NECA (5-N- ethylcarboxaidoadenosine); APNEA (N6-2-(4 aminophenyl)ethyladenosine); or combinations thereof. ModulatorsofAtoh 1

[00400] An additional sensory hair cell restorative agents are directed towards modulators to the products of theArch] (atonal; ATOH), NeWudrodand Neurogi genes. Atohl belongs to a family of basic Helix-Loop-Helix (bHLH) genes that are involved in cell fate determination across phyla and systemastypically being expressed in proliferating precursors. In mammals, at least three bHLH transcription factors are essential for sensory neuron development, inlcuding hair cells and. sensory neurons of the ear: Atohl, Neurod andNeurog]. Atoh], in particular, is essential for hair cell differentiation, and plays arole as a differentiation factor ofpostmitotic hair cells. Studies have also

shown that expression of Atoh1, in combination withBdn form afferent and efferent innervation In undifferentiated cells of epithelial origin.

[00401]Treatment of with ATOH protein supports the role of Atohl Iin sensory hair cell development, inducing the formation of new sensory hair cellsin cochlearstructures, and restoring hearing and balance function. Gene therapy using vectors inserted with theArahl gene further supports ATO's role in promoting and maintaining sensory hair cell function. Accordingly, one embodiment disclosed herein is the use ofATOH proteins or manipulation ofAtohl expression to induce sensory hair cell development inhearing and balance disorders.

[004021In additional embodiments,a neurotrophic growthfactor is administered to the auris interna via the formulations described herein to stimulate inner earhair cell neurotrophic growth factors. The damage caused to spiral ganglion neurons removes not only neural activity, but also neurotrophin support that is normally supplied by hair cells, the absence of which leads to cell death via apopotosis.

t004031In one embodiment, neurotrophicgrowth factor includes but is not limited to brain-derived neurotrophic fact, neurotrophin-3, glial-derived neurotrophic factor, neurotrophin-4/5,nerve growth factor, chilorphenylthio-cAMP (cptcAMP; a permeant cAMP analog), ciliary derived neurotrophic factor (CNTF) or combinations thereof.In another example, thesensory cell restorative agent is a brain-derived neutrophic factor (BDNF). In yet another example, the neurotrophic growthfactor is neurotrophin-3 (NT-3). In other examples, the neurotrophic growth factor is glial-derived neurotrophic factor (GODNF). In some examples, theneurotrophic growthfactor is a peptide or protein. In other embodiments,the neurotrophic growth factor stimulates or enhances spiral ganglion neuron survival. ERR/AR3B2 Antagonists

[004041Studies have also suggested a role for theorphan receptor estrogen related receptor /N3b2 in regulating endolymph production, thereby possibly playing a role inmediating cochlear and vestibular pressure in the endolymph fluid. (Chen et al. Dev, Cell (2007) 13:325-337). Accordingly, agents which antagonize ERRINr3b2 expression, protein production or protein function are contemplated as useful with the formulations disclosed herein, KCAQModulators

1004051Modulators of KCNQ are also contemplated within the scope of the embodiments disclosed herein. KCNQ proteins form postassium channels, which play a role by preventing accumulation of potassium in hair cells. Potassium concentrations are high in the endolymph, giving the endocochlear fluid a high positive potential, which in turn provides a large drive force for potassium entry into the hair celL KCNQ function is correlated with outer hair cell (OHC) survival; inhibition of KCNQ alters potassium homeostasis, resulting eventually in OHC degeneration. Accordingyly, treatment of the auris internal with KCNQ modulators, in some cases activators, is contemplated within the scope of the embodiments disclosed herein as useful in the maintenance of sensory hair cell function in both vestibular and cochlear structures, P2X Modulators

[00406Moduiators of P2X channel function are also contemplated within the scope of the embodiments, for use, for example, in auris intera disorders, such as cochlear inflammation and noise-induced hearing loss. P2X channels, which are gated by adenosine triphosphate, are present in a broad distribution of tissues, and are thought to play a rolein peripheral and central neuronal transmission, smooth muscle contraction and inflammation. Purine nucleotides are thought to play a role in cochlear disease, whereATP plays a cytotoxic role via both apoptosis andnecrosis due to the activation of P2X receptors. For example, chronic perfusion of the perilymphatic-space with ATP causes the proliferation of fibrous tissue and neoosterogenesis in the scala tympani. Moreover, noise exposure and hypoxia cause a significant elevantion ofATP concentration in the endolymphatic and perilyniphatic coiparments, which may represent an adaptive response of the cells to injury. 1004071Accordingly,one embodiment is the use of modulators of P2X in the treatment of cochlear and vestibulardisorders, including hearing and balance disorders. Antagonists and agonists to P2X channels include 1BzATP, TNP-ATPc,-meATP, A-317491. PPADS,NT279, meSurain, Reactive Blue Ii, RO-1, Adamantane aides, RO3 and 4,5-diarylinidazolines CNS modulating agents

[00408] In some instancesimmunomodulators and/or aural pressure modulators modulate central nervous system activity. Anticholinergics

[004091Contemplated for use withthe formulations disclosed herein are agents which ameliorate otic disorders, includingvestibular disorders and/or tinnitus, through local modulation of central nervous system (CNS) activity. Accordingly, some embodiments incorporate the use of agents which inhibit the release of the neurotransmitter acetylcholine in the CNS. Anticholinergic agents are substances which block acetylcholine in the central and the peripheral nervous system. They treat balance disorders by suppressing conduction in vestibular cerebellar pathways,thus increasing motion tolerance.

[004101In some embodiments, the anticholinergic is glycopyrrolate, homatropine, scopolamine or atropine. In some embodiments, the anticholinergic is glycopyrrolate. In some embodiments,the anticholinergic is homatropine. In some embodiments, the anticholinergie is scopolamine, In some embodiments, the anticholinergic is atropine. Antihistanines 100411]Contemplated for use with the formulations disclosed herein are agents whichameliorate otie disorders, including vestibular disorders and/or tinnitus. through local modulation of central nervous system (CNS) activity. Accordingly, some embodiments incorporate the use of agents which block the action of neurotransmitters in the CNS. Histamineis aneurotransmitter in the CNS. Accordingly, some embodiments incorporate the use of agents which modulate histamine receptors (e.g. the H receptor,1Hr receptor, and/or the113 receptor). in some embodiments, anithistamines are as described herein, Calciwn Channel Blockers

[00412]Contemplated for use with the formulations disclosed herein are agents which ameliorate otic disorders, including vestibular disorders and/or tinnitus, through local modulation of central nervous system (CNS) activity. Accordingly, some embodiments incorporate the use of agents which block or antagonize Ca+ channels. Calcium channels are channels formed in the plasma membrane of neurons (amongst other cells) by integral membrane proteins. These channels conduct Ca through a cell's plasma membrane. In neurons, the flow of Ca is partly responsible for creating and propagating action potentials in neurons. It can also be responsible for the release of certain neurotranmtters.

[00413]Insome embodiments, the calcium channelantagonist is cinnarizine, flunarizine, or nimodipine. In some eibodiments, the calcium channel antagonist is cinnarizine.In some embodiments, the calcium channelantagonist is flunarizine. Income embodiments, the calcium channel antagonist is nitodipine. Other calciurn channel blockers include verapamil, ditiazem, onega-conotoxin, GVIA, amlodipine felodipinelacidipine, mibefradil, NPP (5Nitro2-(3 phenylpropylamino)benzoic Acid), flunarizine, and/or combinations thereof GABA Receptor Modulators

[004141Contemplated for use with the formulations disclosed herein are agents which ameliorate otic disorders, including vestibular disorders and/or tinnitus, through local modulation of central nervous system (CNS) activity. Accordingly, some embodiments incorporate the use of agents which modulate the action of GABA receptors in the CNS. GABA, ory-aminobutyric acid, is an inhibitory neurotransmitter in the CNS, It acts at inhibitory synapses of both pre- and postsynaptic neuronal processes The binding of GABA to its receptors (the GABAA receptor, the GABAB receptor, and the GABAc receptor) results in the opening of ion channels, and the flow of Cl into the cell and/or K' out oftheneuron, The result is hyperpolarization of the neuron. Accordingly, some embodiments incorporate the use of agents which increase or decrease the sensitivity of the GABA receptors, or activate the GABA receptors bymimicking GABA 100415]The benzodiazepine class of therapeutic agents are agonists of the GABA, receptor. When a benzodiazepine binds to the GABAA receptor it induces a conformational change which increases the affinity of GABA for its receptor. The result of the increasein the binding of GABA is an increase in the frequency with which the C1 channels in the neurons open. This causes hyperpolarization of the neural membrane. In some embodiments, the benzodiazepineis selected from the group consisting of: alprazolam, bromazepam, brotizolam, chlordiazepoxide, clonazepam, clorazepate, diazepai, estazolam., flunitrazepam, flurazepam, loprazolam, lorazepam, lormetazepam, idazolam, nimetazepam nitrazepam, oxazepam, prazepam, temazepam, triazolam or combinations thereof In some embodiments, the benzodiazepine is clonazepan diazepam, lorazepan, or combinations thereof in some embodiments, the benzodiazepine is diazepamt.

[004161 n some embodiments, the GABA receptor modulator is a loop diuretic. In some embodiments, the loop diuretic is furosemide, bumetanide, or ethacrynic acid. In some embodiments, the loop diuretic is furosemide. In some embodimentsthe loop diuretic is bumetanide. In some embodimentsthe loop diuretic is ethacrynic acid. Furosemidefor example, 5 binds to theii GABAA receptor and reversibly antagonizes CAIA-evoked currents of the a6, p2.and y2 receptors. By way of example only, useful loop diuretics include, but are not limited to, furosemide, bumetanide, and ethacrynic acid. 100417In some embodiments,the modulator of aGABA receptor is a GABA analogue. GABA analogues mimic GABA. Thus, when they bind to a GABA receptor, the receptor acts as though GABA is binding to it and the receptor is activatedIn some embodiments, the GABA analog is gabapentin, pregabalin, muscimol or baclofen. In some embodiments, the GABA analog is gabapentin. hn some embodiments, the GABA analog is pregabalin. In some embodiiients, the GABA analog is muscimol. In some embodiments, the GABA analogue is baclofen. Baclofen is an analogue of GABA which binds to and activates the GABA 8 receptor. Muscimol is also an analogue of GABA It agonizes the GABAA receptor, .eurotransmitterReuptake Inhibiuors

[00418] Contemplated for use with the formulations disclosed herein are agents which ameliorate otic disorders,including vestibular disorders and/or tinnitus, through local modulation of central nervous system (CNS) activity. Accordingly some embodiments incorporate the use of agents which inhibit the reuptake of neurotransmitters in the CNS. In some embodiments, the neurotransmitter reuptake modulator is an antagonist of a neurotransmitter reuptake target, partial agonistinverseagonist, neutral or competitive antagonist, allosteric antagonist, and/or orthosteric antagonist. Neurotransmitter reuptake inhibitors inhibit the reuptake of neurotransmitters into presynaptic cells of the CNS. This increases the concentration ofneurotransmitter available to stimulate post-synaptic cells of the CNS, t004191In some embodiments, the neurotransmitter reuptake inhibitors are tricyclicantidepressants. Tricyclic antidepressants work by inhibiting the re-uptake of the neurotransmitters norepinephrine and serotonin by pre-synaptic cells. This increases the level of serotonin and/or norepinephrine available to bind to the postsynaptic receptor In some embodimentslthe tricyclic antidepressant is 340 anitriptyline, nortriptyline, or trimipramine. In some embodiments, the tricyclic antidepressant is amitriptyline. insome embodiments, the tricyclic antidepressant isnortriptyline. In some embodiments, the tricyclic antidepressant is triipramine 100420 In some embodiments, the neurotransinitter reuptake inhibitor is a selective serotonin reuptake inhibitor. By inhibiting the reuptake of serotonin into the presynaptic cells, SSRs increase the extracellular level of serotonin. This increases the level of serotonin available to bind to the postsynaptic receptor. SSRTs are hypothesized to stimulate new neural growth within the inner ear.

In some embodiments, the selective serotonin reuptake inhibitor isfluoxetine, paroxetine, or sertraline. In some embodiments, the selective serotonin reuptake inhibitor is luoxetine, In some embodiments, the selective serotoninreuptake inhibitor is paroxetine.In some embodiments the selective serotonin reuptake inhibitor is sertraline,

[004211Contemplated for use with the formulations disclosed herein are agents that ameliorate otic disorders, including vestibular disorders and'or tinnitus, through local modulation of central nervous system (CNS) activity. Accordingly, some embodiments incorporate the use of agents that antagonize neurokinin receptors. There are at least three neurokinin receptors: NKi, NK2 and NK3. In certain embodiments, the binding of a ligand (e.g. a tachykinin peptide, substance P neurokinin A, and neurokinin B) to a neurokinin receptor induces the activation of phospholipase C, The activation of phospholipase C produces inositol triphosphate. In some embodiments, the neurokinin receptor is the NK Ireceptor, the NK2 receptor, the NK3 receptor, or combinations thereof. In some embodiments, the neurokinin receptor is the NK I receptor. In some embodiments, the antagonist of the NKl receptor is vestipitant

[00422]In some enibodinents, the SSRI inhibitor is administered in combination with a neurokinin receptor antagonist. In some embodiments, the SSRI is paroxetine and the neurokinin receptor is NKlIn some embodiments, the NK1 receptor antagonist is vestipitant. In certain embodiments, the co-adininistration of paroxetine and vestipitant treats, and/or the symptoms of tinnitus. Local Anesthetics

[00423}Contemplated for use with the formulations disclosed herein are agents which ameliorate otic disorders, including vestibular disorders and/or tinnitus, throughlocalmodulationofcentral

nervous system (CNS) activity.Accordingly, some embodiments incorporate the use of agents which decrease the rate of the depolarization and repolarization of neurons by, for example, blocking the Na channels in cell membranes.

[004241In some embodiments, the CNS modulator is a local anesthetic. In some embodiments, the local anesthetic is selected from the group consisting of- benzocaine carticaine cinchocaine, cyclomethycaine, lidocaine, prilocaine, propxycaine, proparacaine, tetracaine, tocainide, and trimecaine. In some embodiments, the local anesthetic is lidocaine. In some embodiments, the local anesthetic is tocainide, Sodiwn Channel Blockers

[00425]Contemplated for use with the formulations disclosed herein are agents which ameliorate otic disorders, including vestibular disorders and/or tinnitus, through local modulation of central nervous system (CNS) activity. Accordingly, some embodiments incorporate the use of agents which block or antagonize Na+ channels. Sodium channels are channels formed in the plasma membrane of neurons (amongst other cells) by integral membrane proteins, These channels conduct

Nat through a cell's plasma membrane. In neurons, the flow of Na' is partly responsible for creating and propagating action potentials in the neurons.

004261In some embodiments, the sodium channel blocker is carbamazepine, oxcarbazepine, phenytein, valproic acid, or sodium valproate, In some embodiments, the sodium channel blocker is carbamazepine, In some embodiments, the sodium channel blocker is oxcarbazepine. In some embodiments, the sodium channel blocker is phenytein. In some embodiments, the sodium channel blocker is vaIproic acid. In some embodiments, the sodium channel blocker is sodium valproate.

[00427 In some embodiments, the Na'channel blocker is vinpocetine ((3a,I6a)-Ebumameine-14 carboxylic acid ethyl ester); sipatrigine (2-(4-Methylpiperazin-1-lvi)-5(2,3,5-trichloropheny) pyrimidin-4-amine); amiloride (3,5-diamino-N-(aminoirninomethyl)-6-chloropyrazinecarbox amide hydrochloride); carbamazepine (5H-dibenzo[bflazepine-5-carboxamide); TTX (octahydro-12 (hydrymethyl)2-imino-59:7;10a-dimethan o-0aH-[1,3]dioxocino[6,5-d]pyrimidine 4,7,10,11,12-pen tol); RS100642 (1-(2,6-dimethyl-phenoxy)-2-ethylaininopropane hydrochloride); mexiletine ((1-(2,6-dimethylphenoxy)-2-aminopropane hydrochloride)) QX-314 (N-(2,6 Dimethylphenylcarbanioylmethyl)triethylammonium bromide); phenytoin (5,5 diphenylimidazolidine-2,4-dione); lamotrigine (64(2.3-dichlorophenyl)-1,2,4-triazine-3,5-diamine) 4030W92 (2,4-diamino-5-(2.3-dichlorophenyl)-6-fluoromethylpyrimidine); BWI003C87 (5-(23,5 2 trichlorophenyl) pyrimidine-24- 1.1 ethanesulphonate); QX-22( 4[26.dimethylphenyl)amino} N,N,N-trimethyl-2-oxoetha niminium chloride); ambroxol (trans-4-[[(2-Amino-35 dibromophenyl)methyljamino]cyclo hexanol hydrochloride); R56865 (N-[l-(4-(4 fluorophenoxy)butyl]-4-piperidinyl-N-methyl-2-benzo-thiazolamine); lubeluzole; ajmaline ((I7R,21alpha)-ajmalan-17,21-diol); procainamide (4-amno-N-(2-diethylaminoethyl)benzamide hydrochloride); flecainide; riluzoleor; or combinations thereof

[004281In some embodimentsagents which decrease the rate of the depolarization and repolarization of neurons by, for example, blocking the Na'"channels in cell membranes include local anesthetics. In some embodiments, the local anesthetic is selected from the group consisting of benzocaine, carticamne, cinchocaine, cyclomethycaine, lidocaine, prilocaine, propxycaine, proparacaine, tetracaine, tocainide, and trimecaine. In some embodiments, the local anesthetic is lidocaine. In some embodiments, the local anesthetic is tocainide, Thyrotropin,-ReleasingHormone 100429]Contemplated for use with theformulations disclosed herein are agents which ameliorate otic disorders, including vestibular disorders and/or tinnitus. through local modulation of central nervous system (CNS)activity. Accordingly, some embodiments incorporate the use of agents which modulate neurotransmitters. Thyrotropin-releasing hormone is a neurotransmitter which inhibits glutamate-induced excitation of neurons. In some embodiments, the CNS modulator is thyrotropin-releasing hormone,

Antimicrobial Agents f00430]Any antimicrobial agent useful for the treatment of otic disorders, e.g.,inflammatory diseases of the ear or cancer of theear, is suitablefor use in the formulations andmethods disclosed herein. In some einbodiments, the antimicrobial agent is-an antibacterial agent, an antifungal agent, an antiviral agent, an antiprotozoal agent, and/or an antiparasitic agent. Antimicrobial agents include agents that act to inhibit oreradicate microbes, including bacteria, fingi, viruses, protozoa, and/or parasites. Specific antimicrobialagents are used to combat specific microbes. Accordingly, a skilled practitioner would know which antimicrobial agentwould be relevant or useful depending on the microbe identified, or the symptoms displayed,

[00431lIn some embodiments, the antimicrobial agent is a protein, a peptide, an antibody, DNA, a carbohydrate, an inorganic molecule, or an organic molecule. I certain embodiments, the antimicrobial agents are antimicrobial small molecules. Typically, antimicrobial small molecules are of relatively low molecular weight, e.g. less than 1,000, or less than 600700, or between 300~700 molecular weight.

[00432Antibacterial agents include amikacin, gentarnicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin, geldanmycin, herbimycin, loracarbef, ertapenem, doripenem. imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, defprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceflibuten, ceftizoxime, ceftriaxone, cefepinie, cefobiprole teicoplanin, vancomycin, azithronycin clarithronycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectiromycin, aztreonam, ainoxicillinampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, meticillinnafeillin, oxacillin, penicillin, piperacillin, ticarcillan, bacitracin, colistin, polynyxin B ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin-norfloxacin, ofloxacin, trovfloxacin, rnafenide, prontosil, sulfacetamide, sulfarnethizole, sulfaniniilimde, sulfsalazine, sulfsioxazole trimethoprim, demeclocycline, doxycycline, minocycline, oxtetracycline, tetracycline, arsphenamine, chloramphenicol, clindamy'in, fincomycin, ethanbutol, fosfomycin; fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinuspristin./dalfopristin, rifampin, tinidazole, AL-15469A (Alcon Research), AL 38905 (Alcon Research) and combinations thereof 100433 Antiviral agents include acyclovir, famciclovir and valacyclovir. Other antiviral agents include abacavir, aciclovir; adfovir, amantadine, amprenavir, arbidol., atazanavir, artipla, brivdine, cidofovir, combivir, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, fomyirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, gardasilibacitabine, imunovir, idoxuridine, imiquinod, indinavir, inosine, integrase inhibitors, interferons, including interferon type III, interferon type U, interferon type 1 lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nuleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, proteaseinhibitors, reverse transcriptase inhibitors, ribavirin, rimiantadine, ritonavir, saquinavir, stavadine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tronantadine, truvada, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, and combinations thereof.

[004341Antifungal agents include amrolfine utenafine, naftifine, terbinafine, flucytosine, fluconazole, itraconazole ketoconazole, posaconazole, ravuconazole, voriconazole, clotrimazole, econazole, niconazole, oxiconazole, sulconazole, terconazole,tioconazole, nikkomycin Z. caspofungin, micafungin, anidulafungin, amphotericin B, liposonal nystastin, pimaricin, 1.0 griscofulvinciclopirox olamine, haloprogin, tolnafate, undecylenate. clioquinol, and combinations thereof

[00435]Antiparasitic agents include amitrazamoscanate, averiectin, carbadox diethylearbamizine, dimetridazole, diminazene, ivermectin, macrofilaricide, malathion., mitaban,oxamniquine permethrin, praziquante.prantel pamoate, selamectin, sodium stibogluconate, thiabendazole, and combinations thereof t004361In some embodiments, pharmaceutically active metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of the antimicrobial agents discussed above that retain the ability of the parent antimicrobial agents to treat otic disorders of the ear are also useful in the formulations disclosed herein, Free radical modulators 1004371In some instances, inunomodulators and/or aural pressure modulators relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals or the dysfunction of the mitochondria. Antioxidants 1004381Contemplated for use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due tofree radicals or the dysfunction of the mitochondria. Accordingly, some embodiments incorporate the use of agents which prevent andor ameliorate the damage caused by free radicals. In some embodiments, the agents which prevent and/or ameliorate the damage caused by free radicals is an antioxidant.

[004391Antioxidams, as disclosed herein, are also useful as protectants against ototoxic agents through the prevention of reactive oxygen species, neutralization of toxic products or blockage of the apoptosis pathway, Resveratrol (3,5,4'-Trihydroxystilbene), a representative example of an antioxidant, exerts its effects through variety of pathways, including the inhiition of MnSOD, which reduces superoxide to 1122 which inhibits free radical chain reactionsreducing superoxide levels in the cell. Moreover, resveratrol has been implicated inpreventing neuronal cell dysfunction and cell death. Otherantioxidants include but are not limited to vitamin E (tocopherol), vitamin C

(ascorbic acid), glutathione, lipoic acid, alpha lipoic acid, uric acid, carotenes, ubiquinol, melatonin, tocotrienols selenium, flavonoidspolyphenols, lycopene, lutein, lignan, butyl hydroxytoluene, cocnzyme QI0, salicylate, or combinations thereof

[00440]In certain embodiments, nitrones act synergistically with antioxidants, In certain embodiments, nitrones trap free radicals. in some embodiments, anitrone (e.g. alpha-phenyl-tert butylnitrone (PBN), allpurinol) is co-administered with an antioxidant. In certain embodiments, a nitrone co-administered with an antioxidant treats acute acoustic noise-induced hearing loss.

[004411n some embodiments, the antioxidant is N-acetyleysteine; vitamin E tocopherolss and tocotrienols); vitamin C; vitamin A; lutein: selenium glutathione: melatonin; a polyphenol; a carotenoid (e.g. lycopene, carotenes); coenzyme Q-10; Ebselen (2-phenyl-l,2-benzisoselenazol 3(21)-one (also called PZ 51 or DR3305); L-methionine; azulenyl nitrones (e.g, stilbazulenyl nitrone); L-(+)-Ergothioneine ((S)-a-Carboxy-2,3-dihydro-N,NN-trimethyi-2-thioxo-l imidazole4-ethanainiium inner salt); Caffeic Acid Phenyl Ester (CAPE); dimethylithiourea; dimethylsulfoxide; disufenton sodium (NXY-059: disodium 4[(Z-(tert-butyl

[5 oxidoazaniumylidene)methylbenzene-1,3-disulfonate); pentoxifylline; MTCI-86 (3-Methyl-l phenyl-2-pyrazolin-5-one);Amnbroxol (trans-4-(2-Amino-3,5-dibromnobenzylamino)cyclohexane HCl; U-83836E (()-2-((4-(2,6-di--Pyrrolidinyl-4-pyrimiidiyl)-I-piperzainyl)methyl)-3,4-dihydro 2,5,7,8-tetramethyl-2H1-lbenzopyran-6-ole2HCI); MITOQ (mitoquinone mesylate, Antipodean Pharmaceuticals); Idebenone (2-(10-hydroxydecyl)-5 6-dimethoxy-3-nethvl-cyclohexa-2,5-diene 1,4-dione); (t)-cyanidanol-3; or combinations thereof. Glutamate-ReceptorModulators 1004421Contemplated for use with the formulations disclosed herein are agents that modulate the production of free-radicalsand/or inhibit damage to themitochondria. Accordingly, some enibodiments incorporate the use of agents which modulate glutamate receptors. In some embodiments, the glutamate receptor is the AMPA receptor, the NMDA receptor, and/or a group I1 or III mOlu receptor. In some embodiments, a glutamate reptor modulator is as described herein. Iron Chelators

[00443]Contemplated for use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals or the dysfunction of the mitochondria. Accordingly, some embodiments incorporate the use of agents which prevent and/or ameliorate the damage caused by free radicals. In some embodiments, the agents which prevent and/or ameliorate the damage caused by free radicals is an iron chelator. The iron chelator, deferoxamine, prevents ototoxic damage to the earresulting from treatment with neomycin when it is coadministered with neomycin.

[004441Insome embodiments, the iron chelator is desferrioxamine (DFO); hydroxybenzy ethylene diamine; fullerenol-1, pyrrolidine dithiocarbamate; desferal; Yk-28 (5-[4-(2-hydroxyethyl) piperazine-1-ylnethyi]-quinoline-8-01); clioquinol; echinochrome; PIH (pyridoxal isonicotinoyl hydrazone); deferasirox;JIBED (NN bis (2hydroxybenzyl) ethylenediamine-N,N-diacetic acid); Sil (salicylaldehyde isonicotinoyl hydrazone); deferiprone; L1 (1,2-dimethy-3-hydroxy- 4 pyridone); Kojic acid (5-hydroxy-2-hydroxymethyl-4-pyronc); deferoxamine; 2,3 dihydroxybenzoate; or combinations thereof. MitochondrialAodulators 100445] Contemplated for use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of theauris due to free radicals or the dysfunction of the mitochondria. Accordingly, sonie embodiments incorporate the use of one or more agents that modulate the activity of the mitochondria. In some embodiments, the agent which modulates the activity of the mitochondria is acetylcarnitine; lipoic acid; or combinations thereof

Nitric Oxide Synthase modulators

1004461Contemplated for use with the compositions disclosed herein are agentsfor treating or ameliorating hearing loss or reduction resulting from destroyed, stunted, malfunctioning, damaged, fragile or missing hairs in the inner ear. Nitric oxide (NO) is a neurotransmitter. It is synthesized by multiple nitric oxide synthases (NOS) from arginine and oxygen. It is also derived from the reduction of inorganic nitrate. In certain instances, it induces vasodilation; thus,increasing blood flow. In certain instances, it increases cochlear bloodflow. In certain instances, NO damages blood .0 vessel walls. In certain instances, NO ameliorates vascular protein leakage in the cochlea, In certain instances, NO increases the sensitivity of hair cells. In certain instances, NO reacts with super-oxide to form thefree radical peroxynitrite. Accordingly. some embodiments incorporate the use of agents that modulate nitric oxide and/or nitric oxide synthase (NOS).

[00447In soni embodiments, the agent that modulates NO and/or NOS is an antagonist of NO or NOS. In some embodiments, the antagonist of NO and/or NOS is aminoguanidine; I-Amino-2 hydroxyguanidinep-toluensulfate; GED (guanidinoethydisulfide); bromocriptine mesylate; dexamethasone; SDMA (symmetric NNDimethyl-L-arginine);ADIMA (asymmetric N°N' Dimrethyl-L-arginine); L-NMMA (N-mnomethyl-L-arginine); L-NMEA (N"-monoethyl-L arginine); D-MMA (Nmonomethyl-D-arginine); L-NIL (Ns(1-Iminoethyl)-L-lysine hydrochloride); L-NNA (N(-initro-L-arginine); L-NPA (Ntpropyl-L-arginine); LNAME (N itro 5 L-arginine methyl ester dihydrochloride); L-VNIO (N( -- imtino-3-butenyl)--ornithine); diphenyleneiodonium chloride; 2-ethyl-2-thiopseudourea; haloperidol; L-NIO (L-N-(1 iminoethyl)ornithine); MEG (methyleegonidine); SMT(Smethylisothioureasulfate); SMTC (S methyl-L-thiocitrulline); 7-Ni (7-nitroindazole); nNOS inhibitor I((4)-N(4-Amino 5[aminoethyljaminopentyl)-N'-nitroguanidine); 13-PBITU (S,S-1,3-Phenylcne-bis(l;2 ethanediyl)-bis-isothiourea);I-thiocitrulline; TRIM (I-(2-trifluoromethylphenyl) imidazole); MTR

105 (S-thyisothiuronium diethylphosphate); BBS-1; BBS-2; ONO-1714 ((S,5S6R,7R)-7chloro 3-amino-Smethyl-2-azabicyclo[4.1.0]heptane hydrochIoride); GW273629 (3~[[2-[(1 ininoethyl)amino]ethyljsulphonyl}-L-alanine); GW 274150 ((S)-2-amino-(I-iminoethylamino)-5 thioheptanoic acid); PPA250 (3-(2,4-difluorophenyl)-642-[4(1H-imidazol-I-yhInethyl) phenoxyjethoxv}-2-phenylpyridine); AR-R17477 ([N-(42-((3-chlorophenyiethyl) amino) ethyl) phenyl)-2-thiophecarboxamidine dihydrochloride); AR-R18512 (N(2-methyl-12,3,4 tetrahydroisoquinoline-7-yl)-2-thiophenecarboxinildamide); spiroquinazolone; 1400W (N-{[3 (aminonethyl)phenyi]methyl]-ethanihidanide dihydrochloride); or combinations thereof.

[004481 In some embodiments, the agent that modulates NO and/or NOS is anagonist of NO and/or NOS, or a donor of NO, In some embodiments, the agonist of NOandor NOS, or donor of NO, is S-NC (S-nitrosocysteine); NTG (nitroglycerine); SNP (sodium nitropnisside); thapsigargin; vascular endothelial growth factor (VEGF); bradykini AIP; sphingosine-1-phosphate; estrogen; angiopoictin; acetylcholine: SIN-1 (3-morpholinosydnonimine); GEA 3162 (1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenv)-,ebloride): GEA 3175 (3-(3-chloro-2-nethylphenyl)-5-[[4 mcthylphenyl)sulphonyllamiino]-)hydroxide);( EA 5024 (1,2,34-oxatriazolium,5-amino-3 (30chlioro-2-methyl-phenyl)chloride); GEA 5538 (,2,34-Oxatriazolium,3-(3-chloro-2 methylphenyl)-5-{[[cyanomethylanino]carbonyl]amino]-hydroxide inner salt); SNAP (S-nitroso-N acetylpenicillamine); molsidomine; CNO-4 (1-[(45'-Bis(carboxy.methoxv)-2 nitrophenyl)methoxv]-2-oxo-3,3,diethyil--triazene dipotassium salt); CNO-5 ([1-(4',5' Bis(carboymcthoxy)-2-nitrophenyl)methoxy]-2-oxo-3,3-diethyl-1-triazine diacetoxynethyl ester); DEA/NO, IPA/NO, SPER/NO, SULFI/NO, OX/NODETA/NO; or combinations thereof, Sirtuin Modulators t004491The sirtuins (or Sir2 proteins) comprise class IllI of the histone deacetylases (RDACs) While they are classified as protein deacetylases some also function as mono-ADP ribosyltransferases. Each sirtuin protein has a homologous core sequence of 250 amino acids. This sequence is highly conserved over multiple species. Further, in order to catalyze the deacetylation of a protein, each sirtuin reqinres NAD* as a cofactor. There are seven members of the family: Sirtl, Sit2, Sirt3, Sirt4, Sirt, Sirt6, and Sir17. Sirtl and Sirt3 are protein deacetylases. Sirt2 is involved in mitosis f004501Agonism of Sirtl yields multiple benefits which have previously been identified in subjects undergoing caloric restriction. These benefits include, but are not limited to, decreased glucose levels and improved insulin sensitivity, increased mitochondrial activity, and decreased adiposity (due to the Sirt I mediated repression of.PPAR-y). Decreases in glucose levels and adiposity can contribute to the amelioration of presbycusis as diabetes and atherosclerosis are both factors which contribute to the developmentand progression of presbycusis.

[004511Sirtl can prevent apoptosis by deacetylating the pro-apoptotic genes p53 and Ku-70. Additional substrates for Siril include, but are not limited to, the transcription factors NxB, Fox0l, Fox03a, Fox04,Fox05; the transcription repressor Hiel; and Pgc-la., which regulates. among other cellular functions, adaptive thermogenesis, glucose metabolism, and triglyceride metabolism. Agonism of Sirt3 results in increased cellular respiration and a decrease in the production of reactive oxygenspecies (ROS).

[00452] The catalysis of deacetylation bysirtuins is NAD(nicotinamide adenine dinucleotide) dependent. Upon binding to an acetylated protein, the sirtuin hydrolyzes NAD by breaking the glycosidic bond between nicotinamide and ADP-ribose.The acetyl group of theacetylated proteins then transferred to ADP-ribose. At the completion of the reaction nicotinamide, the deacetylated protein, and 2'-O-acetyl-ADP-ribose are released.

[00453j Multiple compounds modulate the sirtuin catalyzed deacetylation of proteins. Administration of certain polyphenols suchas, but not limited to, stilbenes, chalcones flavones, isoflavones, flavanones, anthocyanidins, cateehins, results in the decrease of the Km of the deacetylation reaction. Further, as free nicotinamide antagonizes the deacetylation reaction, compounds which inhibit the binding of nicotinamide to sirtuins will also agonize the activity of sirtuins,

[00454] Administration of the sirtuin agonizing agent resveratrol (trans-35,4'-trihydroxystilbene) decreases apoptosis. It also increases glutamate uptake and thus ameliorates excitotoxicity Further, administration of resveratrol results in lower levels of reactive oxygen species (ROS) and thus ameliorates damage caused by ishemia, excitotoxicity, ototoxicity caused by cisplatinand aminoglycosides acoustic trauma and presbycusis. 1004551Contemplated for use with the formulations disclosedherein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals or the dysfunction of the mitochondria. Accordingly, some embodiments incorporate the use of one or more agents the modulate sirtuin catalyzed deacetylation reactions. Insome embodiments. the agent which modulates sirtuin catalyzed deacetylation reactions is a stilbene. In some embodiments, the stilbene is trans-stilbene, cis-stilbene, resveratrol, piceatannol, rhapontin, deoxyrhapontin, butein, or combinations thereof.

[00456In sonic embodiments, the stilbene is resveratrol .In some embodiments, the stilbene is an analog of resveratrol In some embodiments, the analog of resveratrol is SRT-501 (RM-I821) For additionalanalogs of resveratrol see U.S. Patent App. Pub.No. 2006/0276393, which is hereby incorporated by reference for this disclosure. 100457]Contemplated for use with the formulations disclosed herein areagents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals or the dysfunction of the mitochondria. Accordingly, some embodiments incorporate the use of one or more agents the modulate sirtuin catalyzed deacetylation reactions.In sone embodiments, the agent which nodulates sirtuin catalyzed deacetylation reactions is a chalcone, In some embodiments, the chalcone is chalcon; isoliquirtigen; butein; 4,2',4'-trihydroxychalcone;3,4,24%6£ pentahydroxychalcone; or combinations thereof f004581Contemplated for use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals or the dysfunction of the Mitochondria. Accordingly, some embodiments incorporate the use of one or more agents the modulate sirtuin catalyzed deacetylation reactions In some embodiments, the agent which modulates siruin catalyzed deacetylation reactions is a flavone. In some embodiments, the flavone is flavone, morin, fisetin; luteolin;quercetin; kaempferol; apigenin; gossypetin; myricetin; 6-hydroxyapigenin; 5-hydroxyflavone; 5,7,3'4,5-pentahydroxyflavone; 3,7.3,4',5' pentahydroxyflavone; 3,6,3'4'-tetrahydroxyflavone; 7,34'15-tetrahydroxyflavone; 3,6,2',41 tetrahydroxyflavone; 7,4'-dihydroxyflavone;7,8,3>4'tetrahydroxyflavone; 3.6,23' tetrahydroxyflavone; 4'-hydroxyflavone; 5-hydroxyflavone; 5,4'-dihydroxyflavone; 5,7 dihydroxyflavone; or combinations thereof. 1004591Contemplated for use withthe formulations disclosed herein areagents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals or the dysfunction of the mitochondria. Accordingly, some embodiments incorporate the use of one or more agents the modulate sirtuin catalyzed deacetylation reactions. In some embodiments, the agent which modulates sirtuin catalyzed deacetylation reactions is an isoflavone. In some embodiments, the isoflavone is daidzein, genistein, or combinations thereof. 100460] Contemplatedfor use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals or the dysfunction ofthe mitochondria, Accordingly, some embodiments incorporate the use of one or more agents the modulate sirtuin catalyzed deacetylation reactions. In some embodiments, the agent which modulatessirtuin catalyzed deacetylation reactions is a flavanone. In some embodiments, the flavanone is naringenin; flavanone; 3,5,7,3',4'-pentahydroxyflavanone; or combinations thereof.

[004611Contemplated for use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals or the dysfunction of themitochondria. Accordingly, some embodiments incorporate the use of one or more agents the modulate sirtuin catalyzed deacetylation reactions. In some embodiments, the agent which modulates sirtuin catalyzed deacetylation reactions is an anthocyanidin. In some embodiments, the anthocyanidin is pelargonidin chloride, cyanidin chloride, delphinidin.chloride, or combinations thereof

[004621 Contemplated for use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to freeradicals or the dysfunction of themitochondria.Accordingly, some embodiments incorporate the use of one or more agents the modulate sirtuin catalyzed deacetylation reactions. In some embodiments, the agent which modulates sirtuin catalyzed deacetylation reactions is a catechin. In someeibodiments, the catechin is( )-epicatechin (Hydroxy Sites: 3,5,7,3',4'); (-)-catcchin (Hydroxy Sites:3,573,4'; (-)gallocatechin (Hydroxy Sites: 3,5,7.3'A4',5') (+)-catechin (Hydroxy Sites: 3,5,7,34');( epicatechin (Hydroxy Sites: 3,5,7,3',4); or combinations thereof. 100463]Contemplated for use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris due to free radicals or the dysfunction of the mitochondria. Accordingly, some embodimentsincorporate the use of one or more agents that modulate the catalytic rate of sirtuin catalyzed deacetylation reactions. In some embodiments, the agent which modulates the catalytic rate of sirtuincatalyzed deacetylation reactions is dipyridainole, ZM 336372 (3-(dimethylamino)-N-[3-(4-hydroxybenzoyl)-amino]4-met hylphenyl]benzamide), camptothecin, coumestrol, nordihydroguaiaretic acid, esculetin, SRT-1720 (Sirris), SRT-1460 (Sirtris), SRT-2183 (Sirtris), or combinations thereof. 1004641Contemplated for use with the formulations disclosed herein are agents that relieve, prevent, reverse or ameliorate the degeneration of neurons and/or hair cells of the auris dueto free radicals or the dysfunction of the mitochondria. Accordingly, some embodiments incorporate the use of one or more agents the modulate sirtuin catalyzed deacetylation reactions.In some embodiments, the agent that modulates sirtuin catalyzed deacetylation reactions is a nicotinamide binding antagonistIn some embodiments, the nicotimramide binding antagonist is isonicotinamide or an analog of isonicotinamide. In some embodiments, the analog of isonicotinanide is p-l-5-methyl nicotinamid.e-2'-deoxyribose; p-D-l>5-methylnicotinamide-2-deoxyribofuranoside; P-. 4 ,5 dimethyl-nicotinamide-2'-de-oxyribose; orp-D-l'45-dimethl-nicotinamide-2'~ deoxyribofuranoside. For additional analogs of isonicotinamidesee U.S, Pat. Nos. 5,985,848; 6,066,722; 6,228,847; 6,492,347; 6,803,455; and U.S. Patent Publication Nos. 2001/0019823; 2002/0061898; 2002/0132783: 2003/0149261; 2003/0229033; 2003/0096830; 2004/0053944; 2004/0110772; and 2004/0181063, which are hereby incorporated by referencefor that disclosure. Ion channel modulators Potassium1aon ChannelMochlators

1004651Contemplated for use with the formulations disclosed herein are agents for treating or ameliorating hearing loss or reduction resulting from destroyed, stunted. malfunctioning, damaged fragile or missing hairs and neurons in the innerear. Accordingly, some embodiments incorporate the use of agents thatmodulate potassium ion concentrations. Insome embodiments, the agents that modulate potassium ion concentrations are agconists or antagonists of potassium ion channels. Potassiumionchannels are channelsthatregulate the flow of potassiumionsintoandoutofcells.In

the cochlea the transduction current through the sensory cells is carried by potassium ions and depends on the high concentration of potassium ions in the endolynph. Mutations in the genes encoding potassium channel protein result in both acquired and congenital hearing loss.

[004661The KCNQ family of potassium channels is a family of delayed rectifier voltage-gated potassium channelsfound in the cochlea. KCNQ Isubunits form potassium channels in vestibular dark cells and marginal cells of the stria vascularis. These channels regulate the level of potassium in endolymph. KCNQ4 subunits form channels hair cells. Mice with genes encoding KCNQ subunits knocked-out display a hearing loss during development, starting at four weeks of postnatal life.

[00467]In some embodiments, the agent that modulates a potassium channel is an agoinst of a potassium channel (e.g. a potassium channel opener). In some embodiments, the agonist of a potassium channel is nicorandil; minoxidil, levromakalim;i lemakalim; cromakalim;L-735,334 (14 hydroxy CAF-603 oleate); retigabine; flupirtine; BMS-204352 (3S)-(+)-(5-Chloro-2 methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-21-indole-2-one); DMP-543 (10,10 bis((2-fluoro-4-pyridinyl)methyl)-9(I1H)-anthracenone); or combinations thereof.

[00468] In some embodiments, the agent that modulates a potassium channel is an antagonist of a potassium channel (e.g. a potassium channel blocker). Income embodiments, theantagonist of a potassium channel is linopirdine; XE991 (10,10-is(4-pyridinymethyl)-9(101)-anthracenone); 4 AP (4-aminopyridine); 3,4-DAP (3,4-Diaminopyridine); E-4031 (4'~[[1-[2-(6-methyl-2 pyridyl)ethyl]-4-piperidinyllcarbonyl]-methanesulfonanilide); DIDS (4,4-diisothiocyanostilbene 2,2-disulfonic acid); Way 123,398 (N-methyl-N-(2methyl(1-methyl-H-benzimidazol-2 yl)aminoethyl)--((methylsulfonyl)amino)benzenesulfonamide iCl; CGS-12066A (7 Trifluorornethyl-4-(4-metbyl-I-piperazinyl)pyrrolo-(1.2-aiquinoxaline); dofetilide; sotalol; apamin; amiodarone; azimilide; bretylium; clofilium; tedisainil; ibutilide; sematilide; nifekalant; tamulustoxin and combinations thereof Pur/genicReceptor Modulators

[00469)Contemplated for use with the formulations disclosed herein are agents for modulating ion channels. Accordingly, some embodiments incorporate the use of agents that modulate the concentration of ions. In some embodiments, the agents that modulate the concentration of ions are agonists orantagonits of purigenic receptors.

[004701Purigenic receptors are a family of plasmamembrane-bound receptors. The family includes the P2X, P2Y, and P Ireceptors. The P2X receptors comprise ion channels. When ATP binds to the receptor the channel opens. The P2Y receptors comprise G-coupled protein receptors. The ligands for these receptors are ATP, ADP, UTP, UDP, UDPglucose.The P1 receptors comprise G-coupled protein receptors. The ligand for these receptors is adenosine. Purigenic receptors regulate ion homeostasis in the car. Endolymph, for examplerequires high potassium (K), low sodium (Na'), and low calcium (Ca ) ion levels for normal auditory transduction.

1004711In some embodiments, the agonist of a pmigenic receptor is ATP; ADP; UTP; UDP; UDP glucose; adenosine; 2-MeSAT; 2-MeSADP;4 imeATP; dATaS;KFPyS; Bz-ATP; MRS2703 (2 MeSADP with the beta-phosphate group blocked by a 1-(3,4dimethyloxypheny)eth-1-y phosphoester)); denufosol tetrasodium; MRS2365 ([[(R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio) 9H-purin-9 -yl]-2,3-dihydroxybicyclo[3. 1.0Jhex-i -yl]methyl diphosphoric acid mono ester trisodiumsalt); MRS 2690 (diphosphoricacid I-a-D-glucopyranosyl ester2-[(4'-nethylthio)uidin 5"yl] ester disodium salt); PSB 0474 (3-(2-xo-2-phenylethyl)-uridine-5'-diphosphate disodium salt) or combinations thereof.

[00472]In some embodiments, the antagonist of a purigenic receptor is A-317491 ((5-([(3 Phenoxybenzyl)[(IS)-1,2,3,4-tetrahydro-1-naphthalenyi]aminocarbonyl)-1,24 benzenctricarboxylic acid)); RO-3 (Roche). suramin; PPADS (pyridoxaliphosphate-6-azophenyl 24'-disulfonic acid); PPNDS (Pyridoxaldphosphate-6-(Ddaphthylazo-6nitro-4n'a-disulfbnate) tetrasodiumn salt); DIDS; pyridoxal-5-phosphatc;5(3-bromopheny)13-dihdro2H-benzofuro

[3,2-eJ-1,4-diazepin-2-one; cibacron blue; basilen blue; ivermeetin; A-438079 (3-[[5-(2,3 Dichlorophenyl)-IH-tetrazol-1 -yl]methyl]pyri dine hydrochloride); A-740003 ((N-(l {[(cyanoinino)(5-quinolinylamino) methyllamino}-2,2-dimethvlpropyl)-2-(3,4 dimethoxyphenyl)actamide):NF449(4,4,4'A'4'- (carbonylbis(imino-5.1,3 benzentriyibiscarbonyimino))tetrakis-henzene-,3-disulfonicacid); NF110 (para-44',4"',4'" (carbonylbis(imino-5,1,3-benzenetriylbis carbonylimino)))tetrakis-benzenesulfonic acid); MRS 2179 (2t.Deoxy-N6-niethyladenosine 3,5'bisphosphate tetrasodium salt); MRS 2211 (2[(2-chloro 5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde disodium salt); MRS 2279 ((R,2S4,5S)-4-[2-chloro-6-(methylamino)-9H1-purin-9-yl J-2 (phosphonooxy)bicyclo[31.0]hexane-I-methanol dihydrogen phosphate ester diammonium salt); MRS 2500 tetrasodium salt ((R,2S,4S,5S)-4~[2-1do-6-(methiamino)-9H-purin-9-yl]- 2 (phosphonooxy)bicyclo[3.1.0]hexane-l-methanol dihydrogen phosphate ester tetraammonium salt); NFl57 (8,8'-[carbonylbis[imino-3,1-phenylienecarbonylimino(4-fl uoro-3,1 phenylene)carbonylimino]]bis-1,35-naphthalene trisulfonic acid hexasodium salt); TNP-ATP; tetramethylpyrazine; Ipd[p-carboxymethylene ATP; py-chlorophosphomethylene.ATP; KN-62 (4

[(2)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo- 3-(4-phenyl-I-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester); NF023 (,8¶carbonybis(imino-3,1-phenylenecarbonylimino)]bis 1,3,5-naphthalene-trisuiphonic acid, hexasodium salt); NF279 (8,8'-[Carbonylbis(imino-4,1 phenylenecarbonylimino-4, - phenylenecarbonyliimino)ibis-1,3,5-naphthalenetrisulfonic acid hexasodium salt); spinorphin; or combinations thereof RNAi 1004731In some embodiments, where inhibition or down-regulationofatargetisdesired(e.g.genes encoding a component of a potassium channel, genes encoding a purigenic receptor), RNA interference is optionally utilized. In some embodiments, the agent that inhibits or down-regulates thetarget is an siRNA molecule In certain instancesthe siRNAmolecule is as described herein.

Combination therapy

5 [00474]In certain embodiments, any otic active agent (e.g, an inmmunomodulator or an auris pressure modulator) is administered in combination with one orrmore of any other otic active agent described herein. In some embodiments, an otic agentis administered with ananti-emetic agent (e.g., when a balance disorder is accompanied by nausea). In some embodimentsan otic agent is administered in combination with one or more otoprotectant (e,g., when the administration of a Wt cytotoxic agent is accompanied by ototoxicity). In certain embodiments, an otic agent is administered in combination with, for example, an anti-emetic, an antimicrobial agent, a nitric oxide synthase inhibitor, an antioxidant, aneurotransmitter reuptake inhibitor, an otoprotectanta honeostasis modulator (e.g.,ion/ fluid (e.g.,water) homeostasis modulator) or the like.

AntiEmetic Agenrs/CentralNervousSystem Agents

[00475Anti-Emetic agents are optionally used in combination with any otic formulations disclosed herein. Anti-emetic agents include antihistanines and central nervous agents, including anti psychotic agents, barbiturates, benzodiazepines and phenothiazines. Other anti-emetic agents include the serotonin receptor antagonists, which include dolasetron, granisetron, ondansetron, tropisetron, palonosetron, and combinations thereof; dopamine antagonistsincluding domperidone, properidol haloperidol, chlorpromazine, promethazine, prochlorperazineand combinations thereof; cannabinoids, including dronabinol, nabilone, sativex, and combinations thereof; anticholinergics, including scopolamine; and steroids, including dexamethasone; trimethobenzamine emetrol, propofol, muscimol, and combinations thereof.

[00476]Optionally, Central Nervous System agents and barbiturates are useful in the treatment of nausea and vomiting symptoms that accompany an autoimmune otic disorder. When used, an appropriate barbiturate and/or central nervous system agent is selected to relieve or ameliorate speci icsymptoms without possible side effects, including ototoxicity. Moreover, as discussed above, targeting of the drugs to the round window membrane of the auris internareduces possible side effects and toxicity caused by systemic administration of these drugs. Barbiturateswhich act as a central nervous system depressantinclude allobarbital,alphenalamobarbital,aprobarbital, barnexaclone, barbital, brallobarbital, butabarbital, butalbital, butallylonal butobarbital, corvalol, crotylbarbital, cyclobarbital, cyclopal, ethallobarbital, febarbamate, heptabarbital, hexethal, hexobarbital, metharbital, methohexital, methylphenobarbital, narcobarbital, nealbarbital, pentobarbital, phenobarbital, primidone, probarbital, propallylonal, proxibarbital, reposal,

- Il1 - secobarbital, sigmodal, sodium thiopental, talbutal, thialbarbital, thianylal, thiobarbital, thiobutabarbital, tuinal valofane, vinbarbital, vinylbital, and combinations thereof. 100477]Other central nervous system agents which are optionally used in conjunction with the otic formulations disclosed herein include benzodiazepines or phenothiazines. Useful benzodiazepines include, but are not limited to diazepam, lorazepam, oxazepam, prazepam, alprazolam bromazepam chlordiazepoxide, clonazepam, clorazepate, brotizolam, estazolam, flunitrazepan. flurazepam, loprazolam, Iormetazepamn idazolam, nimetazepam, nitrazepam, ternazepam, triazolam, and combinations thereof.Examples of phenothiazines include prochlorperazine, chlorpromazine, promazine, trifiupromazine, levopromazine, methotrimepramazinemesoridazinethiroridazine, fluphenazine, perphenazine, flupentixol, trifluoperazine, and combinations thereof.

[004781 Antihistanmines, or histamine antagonists, act to inhibit the release or action of histamine. Antihistamines that target the HI receptorare useful, in the alleviation or reduction of nausea and vomiting symptoms that are associated with AlED other autoimmune disorders, as well as anti inflammatory disorders. Accordingly, some embodiments incorporate the use of agents which modulate histaminereceptors (e.g. the t ,receptor, 1H receptor, and/or the113 receptor).

[004791 Such antihistamines include, but are not limited to, meclizine, diphenhydramine., loratadine and quetiapine. Other antihistamines include mepyramine, piperoxan, antazoline, carbinoxamine, doxylamine, clemastine, dimenhydrinate, pheniramine, chlorphenamine, chlorpheniranmine, dexchlorphenirarnine, brompheniramine, triprolidine, cyclizine, chlorcyclizine, hydroxyzine, promethazine, alimemiazine, trimeprazine, cyproheptadine, azatadine, ketotifen, oxatomide and combinations thereof

[00480]In some embodiments, the H, receptor antagonist is meclizine hydrochloride. In some embodiments, the 1 receptor antagonist is promethazine hydrochloride, In some embodiments, the 1i receptor antagonist is dimenhydrinate. In some embodiments, the H, receptor antagonist is diphenhydramine. In some embodiments, the H1 receptor antagonist is cinnarizine. In some embodiments, the HI receptor antagonist is hydroxyzine pamoate. 100481}Antihistamines which target the 113receptor include, but are not limited to betahistine dihydrochloride Antimicrobial Agents

[00482] Antimicrobial agents are also contemplated as usefulwith the formulations disclosed herein. in some embodiments, the antimicrobial agent is as described herein. Corticosteroids

[004831Contemplated for use in combination with any otic formulation described herein (eg, aural pressure modulating fornulations. immunomodulator formulations described herein) are corticosteroid agents whichreduce or ameliorate symptoms or effects as a result of an autoimmune disease and/or inflammatory disorder, including AIED Such autoimmune response are a contributing factor to otic disorders such as Meniere's disease In some embodiments, corticosteroids modulate the degeneration of neurons and/or hair cells of the aurs, and agents for treating orameliorating hearing loss or reduction resulting from destroyed, stunted, ialfinctioning, damaged, fragile or missing hairs in the hiner ear. Accordingly, someembodiments incorporate the use of agents which protect otic hair cells from ototoxins.In some embodiments. the agent which protects otic hair cells from ototoxins is a corticosteroid. Such steroids include prednisolone, dexamethasone, dexamethasone phosphate, belomethasone, 21-acetoxypregnenolone, alclometasone, algestone, ameinonide.beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, diforasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinoloneacetonide, fluocinonide, fluocortin butyl. fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisoloneflurandrenolide,fluticasonepropionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, lotepredno etabonate, mazipredonemnedrysone, meprednisone, methylprednisolone, iometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival prednylidene, rimexolone, tixocortol, triaminolone, trianicinolone acetonide, triamcinolone benetonide, triancinolone hexacetonide and combinations thereof In certain instances, triamicinolone actenoide and dexamethasone protect oic hair cells from damage caused by the naturally occurring toxin 4hydroxy-2,3-nonenal (INE), which is produced in the inner ear in response to oxidative stress. Otoprotectants 1004841n some embodiments, any otic fonnulation described herein (e.g. auris sensory cell modulating agent formulations disclosed herein) further comprise otoprotectants that reduce, inhibit or ameliorate the ototoxicity of agents such as chemotherapeutic agents and/or antibiotics as described herein, or reduce, inhibit or ameliorate the effects of other environmental factors, including excessive noise and the like. Examples of otoprotectants include, and are not limited to, thiols and/or thiol derivatives and/or phannaceutically acceptable salts, or derivatives (e.g. prodrugs) thereof (eg., D-methionine, L-methionine, ethionine, hydroxyl iethionine, methioninol. anifostine, mesna (sodium 2-sulfanylethanesulfonate), a mixture of D and L methionine, normnethionine, honomethionine, S-adenosyl-n-ethionine),diethyldithiocarbamate, ebselen (2-phenyl-1,2 benzisoselenazol-3(2)-one), sodium thiosulfate, AM-I 11 (a cell permeable JNK inhibitor, (LaboratoiresAuris SAS)), leucovorin, leucovorin calcium, dexrazoxane, piracetam Oxiracetan, Aniracetan, Prainracetarn, Phenylpiracetam (Carphedon), Etiracetam. Levetiracetam, Nefiracetani, Nicoracetain, Rolziracetam, Nebracetam, Fasoracetan Coluracetan, Dimiracetam., Brivaracetam, Seletracetam, Rolipramand or combinations thereof Otoprotectants allow for the administration of chemotherapeutic agents and/or antibiotics at doses that are higher than maximal toxic doses; the chemotherapeutic agents and/or antibiotics would otherwise be administered at lower doses due to ototoxicity. Otoprotectants when optionally administered by itself, also allow for the anelioration, reduction or elimination of the effect of environmental factors that contribute to loss of hearing and attendant effects, including but not limited to noise-induced hearing loss and tinnitus.

[004851The amount of otoprotectant in any formulation described herein on a rnole:mole basis in relation to the ototoxic chemotherapeutic agent (e.g. cis platin) and/or an ototoxic antibiotic (eg. gentanicin) is in the range of from about 5:1 to about 200:1, fromabout 5:1 to about 100:1, or from about 5:1 to about 20:1. The amount of otoprotectant inany formulation described herein on amolar basis in relation to the ototoxic chemotherapentic agent (e.g, cis platin) and/or an ototoxic antibiotic (e.g. gentamicin) is about 50:1, about 20:1 or about 10:1. Any the auris sensory cell modulating agent formulation described herein comprises from about 10 ng/mL to about 50 mg/inL, front about 20 mg/mL to about 30 mg/mL, or from about 25mg/mL of otoprotectant. ChenotherajpeuticAgents

[004861Chemnotherapeuctic agents are also contemplated for use with the formulations disclosed herein. Chemotherapeutic agents act by killing cancer cells or microorgaisms, and may include antineoplastic agents that target cancer or malignant cells. Some chemotherapeutic agents, either alone or in combination, are also ototoxic.For example, cisplatin is a known cochleotoxic agent. However, use of csplatin in combination with antioxidants are protective and lessen the ototoxic effects of the chemotherapeutic agent. Moreover, the localized application of the cytotoxic drug may lessen the ototoxic effects that might otherwise occur through systemic application through the use of lower amounts with maintained efficacy, or the use of targeted amounts for a shorter period of time. Accordingly, a skilled practitioner choosing a course of therapy for tumor growth will have the knowledge to avoid or combine an ototoxic compound, or to vary the amount or course of treatment to avoid or lessen ototoxic effects. 1004871Chemotherapeuticagents that are used in combination with theformulations disclosed herein include, for example, but are not limited to adriamycin, imidazole carboxamide, cyclophosphamide, mechlorethamine, chloranmbucil melphalan, daunorubicin, doxorubicin, epirubicinidarubicin, mitoxanthrone, valrubicin, paclitaxel, docetaxel, etoposide, teniposide, tafluposide, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine,fluorouracil, gemeitabine, mercaptopurinemnethotrexate, tioguanine. bleomycin, carboplatin, cisplatin, oxaliplatin, all-trans retinoic acidvinblastine, vincristine, vindesine, vinorelbine.and combinations thereof. Hiomeostasismodulators

[00488]Homeostatis modulators are contemplated as useful with the formulations described herein. Homeostasis modulators include ionand fluid (e.g. water) homeostasis modulators. In some instances, homeostasis modulators include Na/K-ATLPase modulators, ENaC modulators, vasopressin receptor modulators, diuretics or the likeas described herein. NWKA TPaseModulators

[00489]Na/K-ATPase modulators are contemplated for use with the forulations disclosed herein. Cochlear homeostasis is dependent on the electrolyte composition of the endolymph, which is regulated by an active exchange of Na and K+ via a ATPase. Examples of Na/K-ATPase modulators include, and are not limited to, nimodipine (a sodium-potassium adenosine triphosphatase stimulator), ouabain, and furosenide.

00490]Presented below (Table 1) are examples ofactiveagents contemplated for use with the formulations disclosed herein. 1004911Active Agents (including pharmaceutically acceptable salts of these active agents) for Use with the Formulations DisclosedfHerein (TABLE 1) Auris Condition Therapeutic Agent Benign Paroxysmal Positional Vertigo Diphenhydramine Benign Paroxysmal Positional Vertigo> Lorazepam __

Benign Paroxysmal Positional Vertigo Meclizine Benitn Paroxysmal Positional Vertigo Oldansetron Haring Loss Estrogen Estogen and progesterone Hearing Loss (E+P) Hearing Loss Folic acid Lactated Ringerswith Hearing Loss 0.03% Ofloxacin Hearing Loss Methotrexate Methylprednisolone sodium Hearing Loss succinate Hearing Loss N-acetvl cysteine Meniere s Disease Betahistine Meniere's Disease Sildenafil Middle Ea Effision Pneumonococcal vaccine Otitis Externa Diclofenac sodium; dexote Otitis ExtenaAcute AL-Il5469A/AL-38905 Otitis Media Amoxicillin/clavulanate Otitis Media Amoxycillin Otitis Media Chlorpeniramine maleate Otitis Media Domase alfa Otitis Media Echinacea purpurea Otitis Media -aropenem medoxonil Otitis Media Levofloxacin Otitis Media PNCRM9 Otitis Media Pneumococcal vaccine

Auris Condition Therapeutic Agent Otitis Media Telithromycin Otitis Media Triamcinolone acetonide Otitis Media Zmax Otitis Media with Effusion Lansoprazole Otitis Media, Acute ALI5469A; AL 8905 Otitis Media, Acute Amoxicillin Otitis Media, Acute Amnoxicillin-clavulanate Otitis Media, Acute Azithromycin Otitis Media, Acute Azithromycin SR Otitis Media, Acute Cefdinir Otitis Media, Acute Hyland's earache drops Otitis Media, Acute Montelukast Otitis Media, Acute Pneumonococcal vaccine Otitis Media, Acute with Typanostormy Tubes AL-15469A/AL38905 Sulfamethoxazole Otitis MediaChronic trimethoprim Otitis Media Suppuradve Azithromycin Otitis Media Suppurative Telithromycin Otosclerosis Acetylcysteine Ototoxicitv Aspirin Tiiitus Acamprosate Tinmitus Gabapentin Tinnitus _Modafinil.

Tinnitus Neramexane Tinnitus Neramnexaner mesylate Tinnitus Piribedil Timtus Vardenafil Tinnitus Vestipitant - Paroxetine Tinnitus Vestiplitant Tinnitus Zinc sulfate

Devices

[00492]Also contemplated herein are the use of devices for the delivery of the pharmaceutical formulations disclosed herein, or alternatively for the measurement or surveillance of thefunction of the aurs formulations disclosed herein. For example, in one embodiment pumps, osmotic devices or other means of mechanically delivering pharmaceuticalformulations are used for the delivery of the pharmaceutical formulations disclosed herein. Reservoir devices are optionally used with the pharmaceutical drug delivery unitsand reside either internally along with thedrug delivery unit, or externally of the auris structures.

[00493]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(MR) devices are specifically contemplated within the scope of the embodiments, wherein the MR devices (for example, 3 Tesla MRI devices) are capable of evaluting Meniere Disease progression and subsequent treatment with the pharmaceutical formulations disclosed herein. See, Carfrae et at Laryngoscope 118:501-505 (March 2008). Whole body scanners, or alternatively cranial scanners, are contemplated, as well as higher resolution(7Tesla, 8 Tesla, 9.5 Tesla or I1 Tesla forhumans) are optionally used in MRI.scanning.

General Methods of Sterilization

[004941Provided herein are otic compositions that ameliorate or lessenotic disorders described herein. Further provided herein are methods comprising the administration of said otic compositions, In some embodiments, the compositions are sterilized. Included within the embodiments disclosed herein are means and processes for sterilization of a pharmaceutical composition disclosed herein for use in humans. The goal is to provide a safe phannaceutical product, relatively free of infection causing micro-organisms. The U. S. Food and Drug Administration has providedregulatory guidance in the publication "Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing" availableat:http://wwwifdagov/cder/guidance/5882fl.htm, which is incorporated

herein by reference in its entirety. No specific guidelines are available for safe pharmaceutical products for treatment of the inner ear.

100495]As used herein, sterilization means a process used to destroy or remove microorganisms that are present in a product or packaging. Any suitable method available for sterilization of objects and compositions is used. Available methods for the inactivation of microorganisms include, but are not limited to, the application of extreme heat, lethal chemicals, or gamma radiation, In some embodiments isa process for the preparation of an otic therapeutic formulation comprising subjecting the formulation to a sterilization method selected from heat sterilization, chemical sterilization, radiation sterilization or filtration sterilization. The method used depends largely upon the nature of the device or composition to be sterilized. Detailed descriptions of many methods of sterilization are given in Chapter 40 of Remington: The Science and Practice of Pharmacy published by Lippincott, Williams & Wilkins, and is incorporated by reference with respect to this subject matter. Sterilization by Heat

[00496]Many methods are available for sterilization by the application of extreme heat. One method is through the use of a saturated steam autoclave. In this method, saturated steam at a temperature of at least 1.21 °C is allowed to contact the object to be sterilized. The transfer of heatiseither directly to the microorganism, in the case of an object to be steriized, or indirectly to the microorganism by heating the bulk of an aqueous solution to be sterilized. This method is widely practiced as it allows flexibility, safety and economy in the sterilization process.

[00497]Dry heat sterilization is a method which is used to kill microorganisms and perform depyrogenation at elevated temperatures. This process takes place in anapparatus suitable for heating HEPA-filtered microorganism-free air to temperatures of at least 130-180 °C for the sterilization process and to temperatures ofat least 230-250 °Cfor the depyrogenation process. Water to reconstitute concentrated or powdered formulations is also sterilized by autoclave, Chemical Sterilization 1004981Chemical sterilization methods are an alternative for products that do not withstand the extremes of heat sterilization. In this method,a variety of gases and vapors with germicidal properties, such as ethylene oxide, chlorine dioxide, fornaldehyde or ozone are used as the anti apoptotic agents.The germicidalactivity of ethylene oxide, for example, arises from its ability to serve as a reactive alkylating agent. Thus, the sterilization process requires the ethylene oxide vapors to make direct contact with the product to be sterilized. RadiationSterilization 1004991One advantage of radiation sterilization is the ability to sterilize many types of products without heat degradation or other damage, The radiation commonly employed is beta radiation or alternatively, gamma radiation from a "Co source. The penetrating ability of gamia radiation allows its use in the sterilization of many product types, including solutions, compositions and heterogeneous mixtures. The germicidal effects of irradiation arisefrom. the interaction of gamma radiation with biological macromolecules. This interaction generates charged species and free radicals, Subsequent chemical reactions, such as rearrangements and cross-linking processes, result in the loss of normal function for these biological macromolecules. The formulations described herein are also optionally sterilized using beta irradiation. Filtration

1005001Filtration sterilization is a method used to remove but not destroy microorganisms from solutions.Membrane filters are used to filter heat-sensitive solutions, Such filters are thin, strong, homogenous polymers of mixed cellulosic esters (MCE) polyvinylidene fluoride (PVF; also known. as PVDF), or polytetrafluoroethylene (PTFE) and have pore sizes ranging from 0.1 to 0.22 pm. Solutions of various characteristics are optionally filtered using different filter membranes. For example,PVF and PTFE membranes are well suited to fltering organic solvents while aqueous solutions are filtered through PVF or MCE memibranes.Filter apparatus are available for use on many scales ranging from the single point-of-use disposable filter attached to a syringe up to commercial scale filters for use in manufacturing plants. The membrane filters are sterilized by autoclave or chemical sterilization. Validation ofmembrane filtration systems is performed following standardized protocols (Microbiological Evaluation of Filters for Sterilizing Liquids, Vol 4, No. 3. Washington, D.C: Health Industry Manufacturers Association, 1981) and involve challenging the membrane filter with a known quantity (ca. 1O'em) ofunusually small microorganisms, such as Brevundimonas diminuta (ATICC 19146).

[005011Pharmaceutical compositions are optionally sterilized by passing through membrane filters. Formulations comprising nanoparticles (US.Pat No. 6,139,870) or multilamellar vesicles (Richard etal. International Journal of Pharmaceutics (2006), 312(1-2):144-50) are amenable to sterilization

by filtration through 0.22 pm filters without destroying their organized structure.

[005021In. some embodiments, the methods disclosed here comprise sterilizing the formulation (or components thereof) by means of filtration sterilization. In anotherembodiment the aurisacceptable otic therapeutic agent formulation comprises a particle wherein the particle formulation is suitable for filtration sterilization. In a further embodiment said particle formulation comprises particles of less than 300 nn in size, of less than 200 ur in size, of less than 100mn in size. I1 another embodiment the auris-acceptable formulation comprises a particle formulation wherein the sterility of the particle is ensured by sterile filtration of the precursor component solutions. In another embodiment the auris-acceptable formulation comprises a particle formulation wherein the sterility of the particle formulation is ensured by low temperaturesterile filtration. In a further embodiment, said low temperature sterile filtration occurs at a temperature between 0 and 30 °C, or between 0 and 20 °C or between 0 and 10 °C, or between 10 and 20 'C, or between20 and 30 'C, In another embodiment is a process for the preparation of an auris-acceptable particle formulation comprising: filtering the aqueous solution containing the particle formulation at low temperature through a sterilizationfilter; lyophilizing the sterile solution; and reconstituting the particle formulation with sterile water prior to administration.

[00503]In specific embodiments, filtration and/or filling procedures are carried out at about 5°C below the gel temperature (Tgel) of a formulation described herein and withviscosity below a theoretical value of 100P to allow for filtration in a reasonable time using a peristaltic pump, 100504 In another embodiment the auris-acceptable otic therapeutic agent formulation comprises a nanoparticle formulation wherein the nanoparticle formulation is suitable for filtration sterilization. In a further embodiment thenanoparticle formulation comprises nanoparticles of less than 300 nm in size, of less than 200 mn in size, or of less than 100 nm in size. In another embodiment the auris acceptable formulation comprises a microsphere formulation wherein the sterility of themicrosphere is ensured by sterilefiltration of the precursor organic solution and aqueous solutions. In another embodiment the antis-acceptable formulation comprises a thermoreversible gel formulation wherein the sterility of the gel formulation is ensured by low temperature sterile filtration Ina further embodiment, the low temperature sterile filtration occurs at a temperature between 0 and 30 C, or between 0 and 20 °C or between 0 and 10 °C, or between 10 and 20 C, or between 20 and 30 C. In another embodiment is a process for the preparation of an auris-acceptable thernoreversible gel formulation coiiprising: filtering the aqueous solution containing the thermoreversible gel components at low temperature through a sterilization filter; lyophilizing the sterile solution; and reconstituting the thermoreversible gel formulation with sterile water prior toadministration. 100505In certain embodiments, the active ingredients are dissolved i a suitable vehicle (eig, a buffer) and sterilized separately (e.g. by heat treatment, flotation, gamma radiation); the remaining excipients (e.g fluid. gel components present in auris formulations) are sterilized in a separate step by a suitable method (e.g. filtration and/or irradiation of a cooled mixture of excipients); the two solutions that were separately sterilized are then mixed aseptically to provide a final auris formulation. (005061In some instances, conventionally used methods of sterilization (e.g., heat treatment (eg.,in an autoclave), gamma irradiation, filtration) lead to irreversible degradation of polymeric components (e.g., thermosetting, gelling ormucoadhesive polymer components) and/or the active agent in the fnnulation. in some instances, sterilization of an auris formulation by filtration through membranes (e.g., 0.2 pM membranes) is not possible if the formulation comprises thixotropic polymers that gel during the process of filtration. 1005071Accordingly, provided herein are methods for sterilization of auris forrmulations that prevent degradation of polymeric components (e.g., thermosetting and/or gelling and/or macoadhesive polymer components) and/or the active agent during the process of sterilization. In some embodiments, degradation of the active agent (e.g., any therapeutic otic agent described herein) is reduced or eliminated through the use of specific p1 ranges for buffer components and specific proportions of gelling agents in the formulations. In some embodiments, the choice of an appropriate gellling agent and/or thermosetting polymer allows for sterilization of formulations described herein by filtration. Income embodiments, the use of an appropriate thermosetting polymer and an appropriate copolymer (e.g, a gellling agent) in combination with a specific pH range for the formulation allows for high temperature sterilization of formulations described with substantially no degradation of the therapeutic agent or the polymeric excipients. An advantage of the methods of sterilization provided hereinis that, in certain instances, the formulations are subjected to terminal sterilization via autoclaving without any loss of the active agent and/or excipients and/or polymeric components during the sterilization step and arerendered substantially free ofmicrobes and/or pyrogens. Microorganisms

[00508]Provided herein are auris-acceptable compositions that ameliorate or lessen otic disorders described herein. Further provided herein are methods comprising the administration ofsaid otic compositions. In some embodiments, the compositions are substantially free of microorganisms. Acceptable sterility levels are based on applicable standards that define therapeutically acceptable otic compositions, including but not limited to United States Pharmacopeia Chapters <1111> et seq. For example, acceptable sterility levels include 10 colony fonning units (ciu) per gram of formulation, 50 cfu per gram of formulation. 100 efu per grarn of formulation, 500 cfu per gram of formulation or 1000 efu per grain of formulation. In addition, acceptable sterility levels include the exclusion of specified objectionable microbiological agents. By way of example, specified objectionable microbiological agents include but are not limited to Escherichia coli (E. coli), Salmonella sp.,Pseudomonas aeruginosa (P. aeruginosa) and/or other specific microbial agents,

1005091 Sterility of the auris-acceptable otic therapeutic agent formulation is confirmed through a sterility assurance program in accordance with United States Pharnacopeia Chapters <61 ><62> and <71>. A key component of the sterility assurance quality control, quality assurance and validation process is the method of sterility testing. Sterility testing, by way of example only, is performed by two methods. The first is direct inoculation wherein a sample of thecomposition to be tested is added to growth medium and incubated for a period of time up to 21 days. Turbidity of the growth medium indicates contamination.Drawbacks to this method include the small sampling size of bulk materials which reduces sensitivity, and detection of microorganism growth based on a visual observation.An alternative method is membrane filtration sterility testing. In this method, a 1.5 volume of product is passed through a small membrane filter paper. The filter paper is then placed into media to promote the growth of microorganisms. This method has the advantage of greater sensitivity as the entire bulk product is sampled.The commercially available Millipore Steritest sterility testing system is optionally used for determinations by membrane filtration sterility testing. For the filtration testing of creams or ointments Steritest filter system No. TLHVSL210 are used. For the filtration testing of emulsions or viscous products Steritest filter system No. TLAREM210 or TDAREM210 are used. For the filtration testing of pre-filled syringes Steritest filter system No. TTHASY210 are used. For the filtration testing of material dispensed as an aerosol or foam Steritest filter system No. TTVA210 are used. For the filtration testing of soluble powders in ampoules or vials Steritest filter system No. TTHADA210 or TTHADV210 are used. 1005101Testing for E. coli and Salnonella includes the use of lactose broths incubated at 30 - 35 C for 24-72 hours, incubation in MacConkey and/or EMB agars for 18-24 hours, and/or the use of Rappaport medium. Testing for the detection of P. aeruginosa includes the use of NAC agar. United States Phannacopeia Chapter <62> further enumerates testing procedures for specified objectionable microorganisms.

[005111In certain embodiments, anycontrolled release formulation described herein has less than about 60 colony forcing units (CFU), less than about 50 colony forming units, less than about 40 colony forming units, or less than about 30 colony fonning units ofmicrobial agents per gram of ftrnulation. In certain embodiments, the otic formulations described herein are formulated to be isotonic with the endolymph and/or the perilymph. Endotoxins

[005121Provided herein are otic compositions that ameliorate or lessen otic disorders described herein. Further provided herein are methods comprising the administration of said otic compositions, In some embodiments, the compositions are substantially free of endotoxins. Anadditional aspect of the sterilization process is the removal of by-productsfrom the killing ofmicroorganisms (hereinafter, "Product") The process of depyrogenation removes pyrogens from the sample, Pyrogens are endotoxins or exotoxins which induce an immune response. An example of an endotoxin is the hpopolysaccharide (LPS) molecule found in the cell wall of gram-negative bacteria. While sterilization procedures such as autoclaving or treatment with ethylene oxide kill the bacteria, the LPS residue inducesa proinflammatory immune response, such as septic shock. Because the molecular size of endotoxins can vary widely, the presence of endotoxins is expressed in"endotoxin units" (EU). One EU is equivalent to 100 picograms of R coli LPS. Humans can develop a response to as little as 5EU/kg of body weight. The sterility is expressed in any units as recognized in theart, In certain embodiments, otic compositions described herein contain lower endotoxin levels(eg. e<4 EU/kg of body weight of a subject) when compared to conventionally acceptable endotoxin levels (e.g. 5 EU/kg of body weight of a subject).In some embodiments, the auris-acceptable otic therapeutic agent formulation has less than about 5 EU/kg of body weight of a subject. In other embodiments, the auris-acceptable otic therapeutic agent formulation has less thanabout 4 EU/kgof body weight of a subject. In additional embodiments, the auris-acceptable otic therapeutic agent formulation has less than about 3 EU/kg of body weight of a subject, In additional embodiments, the auris-acceptable otie therapeuticagent formulation has less than about 2 EU/kg of body weight of a subject;

[0051311n some embodiments, the auris-acceptable otic therapeutic agent formulation has less than about 5 EU/kg of formulation. In other embodiments, the awis-acceptable otic therapeutic agent formulation has less than about 4EU/kg of formulation. In additional embodiments, the auris acceptable otic therapeutic agent formulation has less than about 3 EU/kg of formulation. In some embodiments, the auris-acceptable otic therapeutic agent formulation has less than about 5 EUkg Product. In other embodiments, the auris-acceptable otic therapeutic agent formulation has less than about 1 EU/kg Product. In additional embodiments, the auris-acceptable otic therapeutic agent formulation has less than about 0.2 EU/kg Product. In some embodiments, the auris-acceptable otie therapeutic agent formulation has less than about 5 EU/g of unit orProduct. In other embodiments, the auris-acceptable otic therapeutic agent formulation has less than about 4 EU/ g of unit or Product. In additional embodiments, the auris-acceptable otic therapeutic agent formulation has less than about 3 EU/g of unit or Product. In some embodiments, the auris-acceptable otic therapeutic agent formulation has less than about 5 EU/mng of unit or Product. In other embodiments, the auris acceptable otic therapeutic agent formulation has less than about 4 EU/ mg of unit or Product. In additional embodiments, the auris-acceptable otic therapeutic agent formulation has less than about

3 EU/mg of unit or Product. In certain embodiments, otic compositions described herein contain from about1toabout 5 EU/mL of formulation in certain embodiments, otic compositions described herein contain from about 2 to about 5 EU/mL of formulation, from about 3 to about 5 EU/mL of formulation, or from about 4 to about 5 EU/mL of formulation.

[005141In certain embodiments, otic compositions described herein contain lower endotoxin levels (e.g. < 0,5 EU/mL of formulation) when compared to conventionally acceptable endotoxin levels (e.g., 0.5 EU/mL of formulation). In some embodiments, theauris-acceptable otic therapeutic agent formulation has less than about 0.5 EU/mL of formulation. In other embodiments, the auris acceptable otic therapeutic agent formulation has less than about 0.4 EU/mLI of fmulation. In additional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 0.2 EU/nIL of formulation. 1005151Pyrogen detection, by way of example only, is performed by several methods. Suitable tests for sterility include tests described in United States Pharmacopoeia (ULSP) <71> Sterility Tests (23rd edition, 1995).The rabbit pyrogen test and the Linulus amebocyte lysate test are both specified in the United States Pharmacopeia Chapters <85> and <151> (USP23/NF 18, Biological Tests, The United States Pharmacopeial Convention, Rockville, MD,1995). Alternative pyrogen assays have been developed based upon the monocyte activation-cytokine assay. Uniform cell lines suitable for quality control applications have been developed and have demonstrated the ability to detect pyrogenicity in samples that have passed the rabbit pyrogen test and the Limulus amebocyte lysate test (Taktak et al. .Pharm, Phannacol. (1990), 43:578-82). In an additional embodiment, the auris acceptable otic therapeutic agent formulation is subject to depyrogenation. In a further embodiment, the process for the manufacture of the auis-acceptable otic therapeutic agent formulation comprises testing the formulation for pyrogenicity.In certain embodiments, the formulations described herein are substantially free of pyrogens. pH and Osmolarity

1005161The main cation present in the endolynph is potassium. In addition the endolyrmphhas a high concentration of positively charged amino acids. The main cation present in the perilymph is sodium, In certain instances, the ionic composition of the endolymph and perilymph regulate the electrochemical impulses of hair cells, In certain instances, any change in the ionic balance of the endolymph or perilymph results in a loss of hearing due to changes in the conduction of electrochemical impulses along otic hair cells In some embodiments, a composition disclosed herein does not disrupt the ionic balance of the perilymphIn some embodiments, a composition disclosed herein has an ionic balance that is the same as or substantially the same as the periymph. In some embodiments, a composition disclosed herein does not disrupt the ionic balance of the endolymph. In some embodiments, a composition disclosed herein has an ionic balance that is the same as or substantially the same as the endolymph. In some embodiments, an otic fonnulation described herein is formulated to provide an ionic balance that is compatible with innerear fluids (i.e., endolymph and/or perilymph).

1005171The endolymph and the perilymph have a pH that is close to the physiological pH of blood. The endoiynph has a pH range of about 7.24.9; the perilymph has a pH range of about 7.2 - 7.4. The in situ pH of the proximal endolymph is about 7.4 while the p of distal endolymph is about 7.9. 100518] In some embodiments, the pH of a composition described herein is adjusted (e.g, by use of a buffer) to anendolymph-compatible pH range of about 7.0 to 8.0 and a preferred pH range of about 7.2 --- 7.9. In some embodiments, the pH of the auris formulations described herein is adjusted (e.g., by use of a buffer) to a perilyniph --compatible p11 of about 7.0 7.6, and a preferred pH range of about 72-74. 100519 In some embodiments, useful formulations also include one or more pH adjusting agents or buffering agents. Suitable p1 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.

[00520]In one embodiment, when one or more buffers are utilized in the formulations of the present disclosure, they are combined, e.g., witha 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 body's natural bufferingsystem. In some embodiments, from about 5 mM to about 200mM. concentration of a buffer is present in the gel formulation. In certain embodiments, from about a 20 mM to about a 100 mM concentration of a buffer is present. another embodiments, the concentration of buffer is such that a p1 of the formulation is between 3 and 9, between 5 and 8, or alternatively between 6 and 7. In other embodiments, the pH of the gel formulation is about 7. 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 another embodiment the buffer is a sodium acetate buffer having a pH of about 5.5 to about 6.0. In a further eTibodinment the buffer is a sodium acetate buffer having a pH of about 6.0 to about 6.5 Inone embodiment the buffer is a sodium citrate buffer having a pH of about 5,0 to about 8.0. In another embodiment the

bufferis a sodium citrate buffer having a p1 of about 5,5 to about 7.0. In one embodiment the buffer is a sodium citrate buffer having a p-I of about 6.0 to about 6.5. 100521]In some embodiments, the concentration of buffer is such that a pH of the formulation is between 6 and 9, between 6 and 8, between 6 and 7.6, between 7 and 8. In other embodiments, the pH of the gel formulation is about 6.0, about 6.5, about 7 or about 7.5. n one mnbodinient is a buffer such as tris(bydroxymethyl)am-inomethane, bicarbonate, carbonate or phosphate atslightly basic p1 In one embodiment, the buffer is a sodium bicarbonate buffer having a pH of about 7.5 to about 8.5. In another embodiment the buffer is a sodium bicarbonate buffer having a pH iof about 7.0 to about 8.0. In a further embodiment the buffer is a sodium bicarbonate buffer having a p1- of about 6.5 to about 7.0 In one embodiment the buffer is a sodium phosphate dibasic buffer having a pH of 5 about 6. to about 9.0. Inanother embodiment the buffer is a sodium phosphate dibasic buffer having a pH of about 7.0 to about 8.5. In one embodiment the buffer is a sodium phosphate dibasic buffer having a p1 of about 7.5 to about 8.0. 100522] Ione embodiment, diluents are also used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide p1 control to or maintenance) are utilized as diluents in the art including, but not limited to a phosphate buffered saline solution.

[005231Ina specific embodiment the p1 of a composition described herein is between about between about 6.0 and about 7.6,between 7 and about 7.8, between about 70 and about 6, between about 72 and about 7.6, or between about 72 and about 74. In certain embodiments the p1- of a composition described herein is about about 6,0.about 6.5, about 7.0,about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, or about 7.6. In some embodiments, the p1 of any formulation described hereinis designed to be compatible with the targeted oticstructure (e.g., endolymph perilymph or the like). 1005241In some embodiments, any gel formulation described herein has a p-I that allows for sterilization (e.g, by filtration or aseptic mixing or heat treatment and/orautoclaving (eg., terminal sterilization)) of a gel formulation without degradation of the otic agent or the polymers comprising the gel. In order to reduce hydrolysis and/or degradation of the otic agent and/or the gel polymer during sterilization, the buffer pH is designed to maintain pH of the formulation in the 7-8 range during the process of sterilization. 2 [00525]In specific embodiments, any gel formulation described herein has a pi that allows for terminal sterilization (e.g. by heat treatment and/or autoclaving) of a gel formulation without degradation of the otic agent or the polymers comprising the gel. For example, in order to reduce hydrolysis and/or degradation of the otic agent and/or the gel polymer during autoclaving, the buffer p1 is designed to maintain pH of the formulation in the 7-8 range at elevated temperatures. Any appropriate buffer is used depending on the otic agent used in the formulation, In some instances, since pKa of TRIS decreases as temperature increases at approximately -003/°C and pK, of PBS increases as temperature increases at approximately 0.003/°C, autoclaving at 250F (121°C) results in a significant downward pH shift (.e, more acidic) in theTRiS buffer whereas a relatively much less upward pH shift in the PBS buffer and thereforemuch increased hydrolysis and/or degradation of an otic agent inTRIS than in PBS. Degradation of an otic agent is reduced by the use of an appropriate combination of a buffer and polymeric additives (e.g. P407, CMC) as described herein.

[005261Insome embodiments, a p-Iof between between about 6.0 and about 7.6, between about 7 and about 7.8, between about 7.0 and about 7.6, betweenabout 7.2 and 7.6, between about 7.2 and about 7.4 is suitable forsterilization (e.g, by filtration or aseptic mixing or heat treatment and/or autoclaving (e.g. terminal sterilization)) of auris formulations describedherein.in specific embodiments a formulation pH of about 6.0, about 6,5, about 70, about 7. 1, about 7.2, about 7.3, about 7.4,about 75., or about 7.6 is suitable for sterilization (e.g, by filtration or aseptic mixing or heat treatment and/or autoclaving (e.g.,terminal sterilization)) of any composition descibed herein.

[005271In some embodiments, the formulations described herein have a p1 between about 3 and about 9, or between about 4 and 8, or between about 5 and 8, or betweenabout 6 and about 7. or between about 6.5 and about 7, or between about 5.5 and about 7.5, or between about 7.1 andabout 7.7, and have a concentration of active pharmaceutical ingredient between about 0.1 mM and about 100 mM. In some embodiments, the fornulations described herein have a pH between about 5 and about 8, or between about 6 and about 7 or between about 6.5 and about 7, or between about 55 and about 75, or betweenabout 7.1 and about 77,andhave a concentration of active pharmaceutical ingredient between about I and about 100mM. In. some embodiments, the formulations described herein have a pH between about 5 and about 8, or between about 6 and about 7, or between about 6.5 and about 7, or between about 5.5 and about 7.5, or between about 7.1 and about 7.7 and have a concentration of active pharmaceutical ingredient between about 50 and about 80 mM. In some embodiments, the concentration of active pharmaceuticalingredient between about 10 and about 100 mM. In other embodiments, the concentration of active pharmaceutical ingredient between about 20 and about 80 nM. In additional embodiments, the concentration of active pharmaceutical ingredient between about 10 and about 50 mM.

[005281In some embodiments, the formulations have a pH1as described herein, and include a thickening agent (i.e, a vicosity enhancing agent) such as, by way of non-limiting example, a cellulose based thickening agent described herein. In some instancesthe addition of a secondary polymer (e.g., a thickening agent) and a p-I offormulation as described herein, allows for sterilization of a formulation described herein without any substantial degradation of the otic agent and/or the polymer components in the otic formulation. In some embodiments, the ratio of a thermoreversible poloxamer to a thickening agent in a formulation that has apH as described herein, is about 40:1, about 35:1, about 30:1, about 25:1,about 20:1, about 15:1 or about101.For example, in certain embodiments, a sustained and/or extended release fonnulation described herein comprises a combination of poloxamer 407 (pluronic F127) and carboxymethylcellulose (CMC) in a ratio of about 40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 15:1. or about 10:1. In some embodiments, the amount of thermoreversible polymer in any formulation described herein is about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% of the total weight of the formulationIn some embodinents, the amount of therinoreversible polymer in any formulation, described herein is about 14%. about 15%, about 16%, about 17%, about 18%, about 19% about 20%, about21%, about 22%, about 23%,about 24% or about 25% of the total weight of the formulation. in some embodiments, the amount of thickening agent (e.g., a gelling agent) in any formulation described herein is about 1%, 5%, about 10%, or about 15% of the total weight of the formulation. In some embodiments, the amount of thickening agent (e.g, a gelling agent) in any formulation described herein is about 0.5% about 1%,about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%,about 4%, about 4.5%, or about 5% of the total weight of the formulation.

[00529In some embodiments, the pharmaceutical formulations described herein are stable with respect to pH over a period of any of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about I week, at least about 2 weeks, at least about 3 weeksat least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about I month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months. or at least about 6months. in other embodiments, the formulations described herein are stable with respect to pH over a period of at least about I week. Also described herein are formulations that are stable with respect topH over a period of at least about I month TonicityAgents

[00530In general, theendolymphhas a higher osmolality than the perilynph For example, the endolymphhas an osmolality of about 304 m0smlkg 120 while the penlymph has an osmolality of about 294 mOsm/kg H20. In sone embodiments, auris compositions described herein are formulated to provide an osmolarity of about 250 to about 320 mM (osmolality of about 250 to about 320 m0sm/kg 1120) ; and preferably about 270 to about 320 mM (osinolality of about 270 to about 320 m0sm/kg H 2 0 ). inspecific embodiments,osmoarit/osmolalityofthe present formulations is adjusted, for example, by the use of appropriate salt concentrations (e.g, concentration of potassium salts) or the use of tonicity agents which renders the formulations endolynph-compatible and/or perilymph-compatible (i.e. isotonic with the endolymph and/or perilymph In some instances, the endolymph-conrpatible andor perilymph-compatible formulations described herein cause minimal disturbance to the environment of the inner ear and cause minimum discomfort (e.g., vertigo and/or nausea) to amannal upon administration.

[005311In some embodiments, any formulation described herein is isotonic with the perilymph. Isotonic formulations are provided by the addition of a tonicity agent. Suitable tonicity agents include, but are not limited to any pharmaceutically acceptable sugar, salt or any combinations or mixtures thereof, such as, but not limited to dextrose, glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.

[005321Useful auris compositions includeone or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or anunonium cations and chloride, citrateascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammoniuni sulfate.

[00533In further embodiments, the tonicity agents are present in an amount as to provide a final osmolality of an otic formulation of about 100 mOsm/kg to about 500 mOsm/kg, from about 200 mOsm/kg to about 400 mOsmkg, from about 250 mOsm/kg to about 350 mOsm/kg or from about 280 mOsm/kg to about 320 mOsni/kg In someembodiments, the formulations described herein have a osmolarity of about 100 mOsm/ to about 500 mOs/L about 200 mOsnL to about 400 mOsmL, about 250 mOsn/L to about 350 mOsm/L, or about 280 mOsn/L to about 320mOnsm/L In some embodiments, the osmolarity of any formulation described herein is designed to be isotonic with the targeted otic structure (e.g., endolymph, perilymph or the like).

[00534]In some embodiments, the formulations described herein have a p1 and osmolarity as described herein, and have a concentration of active pharmaceutical ingredient between about I1 M

and about 10 M,between about 1 mM and about 100 mM, between about 0.1imM and about 100 mM, betwen about 0.1 mM and about 100 nM, In some embodiments, the formulations described herein have a pH and osmolarity as described herein, and have a concentrationof active pharmaceutical ingredient between about 0.2 - about 20%., between about 0.2 about 10%, between about 0.2 about 7.5%, between about 02-5%, between about 0.2--- about 3%, between about 0.1-- about 2% of the active ingeredient by weight of the formulation. In some embodiments, the formulations described herein have a pH and osmolarity as described herein, and have a concentration of active pharmaceutical ingredient between about 0.1--- about 70 mg/ni, between about Im -n about 70 mg/mL, between about I ng about 50 mg/mL,, between about I mg/nitL and about 20 mg/mL, between about I mg/mL to about 10 mg/mL, between about 1 mg/mLto about 5 mg/mL, or between about 0.5 mg/ni to about 5 mg/m. of the activeagent by volume of the formulation.

Particlesize

[00535] Size reduction is used to increase surface area and/or modulate formulation dissolution properties. it is also used to maintain a consistent average particle size distribution (PSD) (e.g, micrometer-sized particles, nanometer-sized particles or the like) for anyfonnulation described herein. In some instances, any formulation described herein comprises mulitparticulates, i.e., a plurality of particle sizes (e.g., micronized particles, nano-sized particles, non-sized particles) i.e, the formulation is a multiparticulate formulation. In soni embodiments, any formulation described herein comprises one or more multiparticulate (e.g., micronized) therapeutic agents. Micronization is a process of reducing the average diameter of particles of a solid material. Micronized particles are from about micrometer-sized in diameter to about picometer -sized in diameter. In some embodiments, the use of muitiparticulates (e,g., micronized particles) of an otic agent allows for extended and/or sustained release of the otic agent from any formulation described herein compared to a formulation comprising non-mutiparticulate (e,g, non-micronized) otic agent, In some instancesformulations containing multiparticulate (e.g. micronized) otic agents are ejected from a 1mL syringe adapted with a 27G needle without any plugging or clogging.

[00536] In some instances, any particle in any formulation described herein is a coated particle (e g., a coated micronized particle) and/or a microsphere and/or a liposomal particle. Particle size reduction techniques include, by way of example, grinding, milling (e~g.air-attrition milling (jet milling), ball milling), coacervation, high pressure homogenization, spray drying and/or supercritical fluid crystallization. In some instances, particles are sized by mechanical impact (e.g., by hammer mills, ball mill and/or pin mills). In some instances, particles are sized via fluid energy (e.g., by spiral jet mills, loop jet mills, and/orfluidized bedjet mills). In some embodiments formulations described herein comprise crystalline particles. In sone embodiments, formulations described herein comprise amorphous particles, In some embodiments, formulations described herein comprise therapeutic agent particles wherein the therapeutic agent is a free base, or a salt, or a prodrug of a therapeutic agent, or any combination thereof

[00537 In sone instances, a combination of an otic agent and a salt of the oticagent is used to prepare pulsed release otic agent formulations using the procedures described herein. In some formulations, a combination of a micronized otic agent (and/or salt or prodrug thereof) and coated particles (e,g., nanoparticles, liposomes, microspheres) is used to prepare pulsed release otic agent formulations using any procedure described herein. Alterntaively, a pulsed release profile is achieved by solubilizing up to 20% of the delivered dose of the otic agent (e.g., micronized otic agent, or free base or salt or prodrug thereof multiparticulate otic agent, or free base or salt or prodrug thereof) with the aid of cyclodextrins, surfactants (e.g., poloxamers (407, 338, 188), tween (80 60, 20,8 1),PEG-hydrogenated castor oil, cosolvents like N-methyl-2-Pyrrolidone or the like and preparing pulsed release fornulations using any procedure described herein. 1005381Insone specific embodiments, any otic formulation described herein comprises one or more micronized otic agents. In some of'such embodiments, anmcronized otic agent comprises micronized particles, coated (e.g, with an extended release coat) micronized particles, or a combination thereof In some of suchembodiments, a micrnized otic agent comprisingmicronized particles, coated micronized particles, or a combination thereof comprises an otic agent as a free base, a salt, a prodrug or any combination thereof. Controlled Release Otic Formulations

[005391In certain embodiments, any controlled release otic formulation described herein increases the exposure of an otic agent and increases the Area Under the Curve (AUC) in otic fluids (e.g. endolyiph and/or perilymph) by about 30%, about 40% about 50%,about 60%, about 70%, about

80% or about 90% compared to a formulation that is not a controlled release oti formulationIn certain embodiments, any controlled release otic formulation described herein increases the exposure of an otic agent and decreases the Ca in otic fluids (e.g., endolymph and/or perilymph) by about 40%, about 30%, about 20%, or about 10%,compared to a formulation that is not a controlled release otic formulation. In certain embodiments, any controlled release otic formulation described herein alters (eg.reduces) the ratio of C. to C;jcompared to a formulation that is not a controlled release otic formulation. In certain embodiments, the ratio of C to Cais 10:1, 9:1, 8:1, 7:1, :1, :, 4:1.3:1,2:1 or 1:1. in certain embodiments, any controlledrelease otic formulation described herein increases the exposure of an otic agent and increases the length of time that the concentration of an otic agent is above C 1 iby about 30%, about 40%, about 50%,about 60% about 70%, about 80% or about 90% compared to a formulation that is not a controlled release otic formulation. In certain instances, controlled release formnulations described herein delay the time to Cw. In certain instances, the controlled steady release of a drug prolongs the time the concentration of the drug will stay above the Ci n some embodiments, auris compositions described herein prolong the residence time of a dmg in the inner ear. In certain instances, once drug exposure (e.g. concentration in the endolymph or perilynph) of a drug reaches steady state, the concentration of the drug in the endolymph or perilynph stays at or about the therapeutic dose for an extended period of time (e.g., one day, days, 3 days, 4 days, 5 days, 6 daysor I week). 1005401The otic formulations described herein deliver an active agent to the external, middle and/or inner ear, including the cochlea and vestibular labyrinth, Local otic delivery of the auris compositions described herein allows for controlled release of active agents to auris structures and overcomes the drawbacks associated with systemic administration (e.g. low bioavailability of the drug in the endolymph or perilymph, variability in concentration of the drug in themiddleand/or internal ear). 1005411Controlled-release options include gel formulations, liposomes, cyclodextrins, biodegradable polymers, dispersable polymners, emulsions, microspheres or microparticles, hydrogels (e.g., a self-assembling hydrogel displaying thixotropic properties that also functions as an absorption enhancer; including instances in which the penetration enhancer is a surfactant comprising an alkyl-glycoside and/or a saccharide alkyl ester), other viscous media, paints, foams, in situ. forming spongy materials, xerogels, actinic radiation curable gels, liposomes, solvent release gels, nanocapsules or nanospheres, and combinations thereof; other options or components include mucoadhesives, penetration enhancers, bloadhesives, antioxidants, surfactants. buffering agents, diluents, salts and preservatives To the extent viscosity considerations potentially limit the use of a syringe/needle delivery system, thernoreversible gels or post-administration viscosity-enhancing optionsare also envisioned, as wellas alternative delivery systems, including pumps, microinjection devices and the like.

[005421in one embodiment of the auris-acceptable aural pressure modulating formulations described hereinthe aural pressure modulator is provided in a gel formulation, also referred to herein as "auris acceptable gel formulations," "auris interna-acceptable gel formulations""auris gel formulations" or variationsthereof All of the components of the gel formulation must be compatible with the auris internal. Further, the gelformulations provide controlled release of the aural pressure modulator to the desired site within the auris internal; in some embodiments, the gel formulation also has an immediate or rapid release component for delivery of the aural pressure modulator to the desired target site,

1005431Provided herein,in sonic embodiments, are auris fornulations that comprise thernoreversible gelling polymers and/or hydrogels. In some instances, the formulations are liquid at or below room temperature but gel at body temperatures. In sonic instances, intratympanic injection of cold formulations (e.g., formulation with temperatures of <20 'C) causes a dramatic change in the imer ear environment and causes vertigo in individuals undergoing treatment for inner ear disorders. Preferably; the formulations described herein are designed to be liquids that are administered at or near room temperature and do not cause vertigo or other discomfort when administered to an indivdual or patient. f00544]In some embodiments, the formulations are bimodal formulations and comprise an miiediate release component and an extended release component. In sone instances, bimodal formulations allow for a constant rate of release of an immediate release component (multiparticuiate agent (e.g, micronized active agent) from the gelled polymer and a constant rate of release of an extended release component (e.g., an encapsulated active agent that serves as a depot for extending the release of an active agent). In other embodiments, the otic compositions described herein are administered as a controlled release formulation, released either continuously or in a pulsatile manner, or variants of both. In still other embodiments, the active agent formulation is administered as both an immediate release and controlled release formulation, released either continuously or in a pulsatile manner, or variants of both.lIn certain embodiments, the fonnulations comprise penetration enhancers that allowfor delivery of the active agents across the oval window or the round window of the ear. 1005451In some embodiments, the auris gel formulations are biodegradeable. In other embodiments, the uris gel formulationsinclude a mucoadhesive excipient to allow adhesion to the extemal mucous membrane of the round window. In yet other embodiments, the auris gel formulations include a penetration enhancer excipient; in further embodiments, the auris gel formulation contains a viscosity enhancing agent, In other embodiments, the auris pharmaceutical fornulations provide an auris-acceptable microsphere or microparticle; in still other embodiments, theauris

pharmaceutical formulations provide an auris-acceptable liposome, in yet other embodiments, the auris pharmaceutical formulations provide an auris-acceptable paint, foam or xerogel. in other embodiments, the auris pharmaceutical formulations provide an auris-acceptable in situ fonning spongy material. Further embodiments includea thermoreversible gel or actinic radiation curable gel in the auris pharmaceuticals formulation, such that upon preparation of the gel at room temperature or belowthe formulation is a fluid, but upon application of the gel into or near the auris interna and/or auris media target site, including the tympanic cavity, round window membrane or the crista fenestrac cochleae, the auris-pharmaceutical formulation stiffens or hardens into a ge-like substance. Some embodiments include the use of a combination of a mucoadhesive and a thermoreversible gel in any otic formulation. described herein

100546]1The formulations disclosed herein alternatively encompass an otoprotectant agent in addition to the at least one active agent and/or excipients, including but not limited to such as antioxidants, alpha lipoic acidcalicum, fosforycin or iron chelators, to counteract potential ototoxic effects that nayarise from the use of specific therapeutic agents or excipients, diluents or carriers.

[00547]One aspect of the embodiments disclosed herein is to provide a controlled release aura pressure modulating auris-acceptable composition or formulation for the treatment of fluid homeostasis disorders, The controlled release aspect of the compositions and/or formulations disclosed herein is imparted through a variety of agents, including but not limited to excipients, agents or materials that are acceptable for use in the auris interna or other otic structure. By way of example only, such excipients, agents or materials includeau auris-acceptable polymer, annauns acceptable viscosity enhancing agent, an auris-acceptable gel, an aunis-acceptable microsphere, an auris-acceptable hydrogen, an auris-acceptable liposome, an auris-acceptable nanocapsule or nanosphere, an auris-acceptable themoreversible gel, or combinations thereof.

[005481Thus, provided herein are pharmaceutical compositions that include at least one auris therapeutic agent and auris-acceptable diluent(s), excipient(s), and/or carrier(s).In some embodiments, the pharmaceutical compositions include other medicinal or pharmaceutical agents, carriers, adiuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. hi other embodiments, the pharmaceutical compositions also contain other therapeutic substances. Auris-Acceptable Gel Formulations

[005491Gels, sometimes referred to as jellies, have been defined in various ways.For example, the United States Pharmacopoeia defines gels as semisolidsystems consisting of either suspensions made up of small inorganic particles or large organic molecules interpenetrated by a liquid. Gels can further consist of 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. Single-phase gels are usually prepared from synthetic macromolecudes (e.g., Carbomer@) or fromnatural 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.

[005501Gels can also be classified as being hydrophobic or hydrophilic. The bases of a hydrophobic gel usually consists of a liquid paraffin with polyethylene or fatty oils gelled with colloidal silica, or aluminum or zinc soaps.l I contrast, the bases of hydrophobic gels usually consists of water, glycerol, or propylene glycol gelled with a suitable gelling agent (e.gtragacanthstarch, cellulose derivatives, carboxyvinylpolyners, and/or manesium-aluminum silicates).

[005511In certain embodiments, the rheology of the gel fbnnulation is pseudo plastic, plastic, thixotropic, or dilatant. Thermoreversible Gels

[005521Polymers composed of polyoxypropylene and polyoxyethylene are known to foml 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 temperture, therefore allowing useful topical formulations. The liquid state-to-gel state phase transition is dependent on the polymer concentration and the ingredients in the solution.

[005531 "ReGefl'M"is a tradename of MacroMed Incorporated for a class oflow 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 drug carriers disclosed in pending U.S. patent application Ser. Nos 09/906,041, 09/559799 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 poly(ortho ester)s, and the B-blocks are relativelyhydrophilic 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 datons. The drug carriers exhibit water solubility at temperatures below normal mamnalian 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 poly (ortho ester), in which the polyester is synthesized from monomers selected from the group consisting of D,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lacticacid, L-lactic acid, glycolide, glycolic acid, -caprolactone, a-hydroxyhexanoic acid, y- butyrolactone, y-hydroxybutyric acid, S-valerolactone,5-hydroxyvaleric acid, hydroxybutyric acids, malic acid, and copolymers thereof and having an average molecularweight 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.

[005541Additional biodegradable thermoplastic polyesters include AtriGeIT (provided by Atrix Laboratories, Inc.)andor those disclosed, e.g. in U.S. Patent Nos. 5,324,519; 4,938763; 5,702,716; 5,744,153; and 5,990,194; wherein the suitable biodegradable thermoplastic polyester is disclosed as a thermoplastic polymer. Examples of suitable biodegradable thermoplastic polyesters include polylactides, polyglycolides, polycaprolactones, copolymers thereof terpolyners thereof, and any combinations thereof In sonm such embodiments, the suitable biodegradable thermoplastic polyester is a polylactide, a polyglycolide, a copolymer thereof, a terpolymer thereof, or a combination thereof. In one embodiment, the biodegradable thermoplastic polyester is 50/50 poly (DL-lactide-co-glycolide) having a carboxy terminal group; is present in about 30 wt. % to about40 wt. % of the composition; and has an average molecular weight of about 23,000 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 composition; and has an average molecular weightofabout15,000toabout24,000.In

further or alternative embodiments, the terminal groups of the poly(DL-lactide-co-glycolide) are either hydroxyl, carboxyl, or ester depending upon themethod of polymerization. Polyondensation of lactic or glycolic acid provides a polymer with terminal hydroxyl. and carboxyl groupsRing opening polymerization of the cyclic lactide 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 1-dodecanol provides a polymer with one hydroxyl group and one ester tenninal groups. Ring-opening polymerization of the cyclic monomers with a diol such as 1,6-hexanediol or polyethylene glycol provides a polymer with only hydroxyl terminal groups.

[005551Additional embodiments include Poloxamer thermoreversible copolymers. Poloxamer 407 (PF-127) is a nonioni surfactant composed of polyoxyethylene-polyoxypropylene copolyrners Other commonly used poloxainers include 188 (F-68 grade), 237 (F-87 grade), 338 (F-I08 grade). Aqueoussolutions of poloxamersare stable in the presence of acids, alkalis, and metal ionsPF-127 is a commercially available polyoxyethylene-polyoxypropylene triblock copolymer of general formula E106 P70 El06, with an average molar mass of 13,000. It contains approximately 70% ethylene oxide, which accounts for its hydrophilicity. It is one of the series of poloxamer ABA block copolynmers, whose members share the chemical formula shown below. hydrophilic hydrophiic

H O-CH2-CHO-CH-CH2X'0-CHCH OH a CH3 b a

hydrophobic

[005561P-Fl27 is of particular interest since concentrated solutions (>20% w/w) of the copolymer are transforned from low viscosity transparent solutions to solid gelson heating to body temperature.Thisphenomenontherefore,suggests that when placed in contact with the bodythe

gel preparation will for a semi-solidstructure and a controlled release depot. Furthermore, PF-1227 has good solubilizing capacity, low toxicityand is, therefore, considered a good medium for drug delivery systems.

[00557)In an alternative embodiment, the thermogel is a PEG-PGLA-PEG triblock copolymer (Jeong metal Nature (1997), 388:860-2; Jeong etal, J. Control. Release (2000) 63:155-63; Jeong etal, Adv, Drug Delivery Rev. (2002) 54:37-51). The 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 PGLA copolymer can range from about 1:1 to about 20:1 The resulting coploymers are soluble in waterand forma free-flowing liquid at room temperature but form a hydrogel at body temperature, A commercially available PEGI-PGLA-PEG triblock copolymer is RESOMER RGP t50106 manufacutred by Boehringer Ingelheim, This material is composed of a PGLA copolymer of 50:50 poly(DL-lactide-co-glycolide) and is 10% w/w of PEG and has a molecular weight of about 6000. 1005581In some embodiments, a suitable combination of gelling agents and a thermoreversible gel is utilized in the controlled release formulations described herein. Suitable gelling agents for use in preparation of the gel formulation include, but are not limited to celhuloses, cellulose derivatives., cellulose ethers (e.g., carboxymethylcellulose, ethycellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropyhmethylcellulose, hydroxypropylcellulose, methylcellulose), guargum xanthangum,locust bean gum, alginates (e.g., alginic acid), silicates, starch, tragacanth, carboxyvinyl polymers, carrageenan, paraffin, petrolatum and any combinations ormixtures thereof In some other embodiments, hydroxypropylnethyiclellulose (Methocel) is utilized as the gelling agent. In certain embodiments, the thickening agents (i.e., viscosity enhancing agents) described herein are also utilized as the gelling agent for the gel formulations presented herein. 100559]Suitable combinations of thermoreversible gels with a thickening agent include, by way of non-limiting example, a combination of poloxamer thermoreversible copolyrners with cellulose based thickening agents described herein. In some instances, the addition of a secondary polymer (e.g., a thickening agent) introduces a diffusional barrierand reduces the rate of release of the eti agent. An appropriate thickening agent (e.g., a cellulose based polymer, e.g., CMC polymer) is selected based on the viscosity ofa 2% solution of the secondary polymer (e.g., CMC); the selected secondary polymer (e.g.,CMC) provides a 2% polymer solution with viscosity less than 15,000 cP. In specific formulations, the selected secondary polymer (e.g, CMC) provides a 2% polymer solution with viscosity from about 4,000 cP to about 10,000 cP.In some embodiments, the ratio of a thermoreversible poloxamer to a gelling agent isabout 50: 1, about 40:1, about 35:1, about 30:1, about 25:1, about 20:1. about 15:1 or about 10:1. For example,in certain embodiments, a controlled release formulation described herein comprises a combination of poloxamer 407 (pluronic F127) and carboxymethyleellulose (CMC) in a ratio of about about 50:1, 40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 15:1 or about 10:1. Hydrogels 1005601 Chitosan glycerophosphate (CGP) is a biodegradable matrix for the formation of hydrogels. CGP has been shown to be suitable for local delivery ofdexamethasone to theinnerear, where 50% of the active agent was released after 24 hours, followed by a linear decline over 5 days of perilymph drug levels.In some embodiments, CGP is used as a biodegradable viscosity enhancing agent or gelling agent for controlled release of active agents from the formulations disclosed herein. In certain embodiments, when CGP is used as a viscosity enhancing agent or gelling agent, the compositions further comprise liposoes. Liposomes are added to further control the release of active agents from thefornulations disclosed herein, whether they be hydrophobic or hydrophilic antimicrobial small molecules. 1005611n some embodiments, other gel formulations are also contemplated to be useful depending upon the particular embodiment, and as such are considered to fall within the scope of the present disclosure. For example, other currently commercially-available glycerin-based gels, glycerin derived compounds, coniugated, or crosslinked gels, matrices,hydrogels, and polymers, as well as gelatins and their derivatives. alginates, and alginate-based gels, and even various native and synthetic hydrogel and hydrogel-derived compounds are all expected to be useful in the formulations described herein. In some embodiments, gels include, but are not limited to, alginate hydrogels SAF-Gel (ConvaTe, Princeton, NJ), Duodern Hydroactive Gel (ConvaTec),Nu-gel (Johnson & Johnson Medical, Arlington, Tex); Carrasyn (V) Acemannan Hydrogel (Carington Laboratories, Inc. Irving Te,); glycerin gels EtaHydrogel (Swiss-/Aerican Products, Inc., DallasTex.) and K-Y Sterile (Johnson & Johnson). In one embodimenta sterilized solution of sodium alginate is mixed with a sterilized solution of an auris-compatible calcium salt, the therapeutic agent(s), and a polysaccharide. Upon admixing, a gel is formed in a desired amount of time having a desired viscosity, In further embodiments, biodegradable biocompatible gels also represent compounds present in formulations disclosed and described herein. In some embodiments, a hardening agent (e.g., giutaraldehyde, formaldehyde) is added to a biodegradable hydrogel gel Contemplated for use in fonaulations described herein are biodegradable hydrogels comprising, by way of example, 0.1, 0.5,1, 1.5, 2 2.5, 3,35,4 4.5,5, 6, 7,89, 10, 15, 20,25, 30, 40, 50, 60, 70 80 90 or 100 mM glutaraldehyde (e.g., agelatin gel and/or a glycerin gel and/ora chitosan hydrogel comprising 10 mM glutaraldehyde). In further embodiments, biodegradable biocompatible gels also represent compounds presenting auris-acceptable formulations disclosed and described herein. For examples, of formulations and their characteristics see Table 1.

1005621In.some formulations developed: for administration to a mammal, and for compositions formulated for human administration, the gel comprises substantially all of the weight of the composition. In other embodiments, the gelcomprises as much as about 98% or about 99% of the composition by weight. In a further embodiment, this is desirous when a substantially non-fluid, or substantially viscous formulation is needed In a further embodiment, when slightly less viscous, or slightly more fluid formulations are desired, the biocompatible gel portion of the formulation comprises at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, or even at least about 80% or 90% by weight of the compound. Of course, all intermediate integers within these ranges are contemplated to fall within the scope of this disclosure, and in some embodiments, even more fluid (and consequently less viscous) gel compositions are formulated, such as for example, those in which the gel or matrix component of the mixture comprises not more than about 50% by weight, not more than about 40% by weight, not more than about 30% by weight, or even those than comprise not more than about 15% or about 20% by weight of the composition,

[005631If desired, the gels may also contain preservatives, cosolvents, suspending agents, viscosity enhancing agents, ionic-strength and osinolality adjustors and other excipients inaddition to buffering agents. Suitable water soluble preservatives which are employed in the drug delivery vehicle are sodium isulfite, sodium thiosulfate, ascorbate, benzalkoniurn chloride, chorobutanol, thimerosal, parabensbenzyl alcohol, phenylethanol and others. These agents are present, generally in amounts of about 0.001% to about 5% by weight and, preferablyin the amount of about 0.01 to about 2% by weight.

[005641Suitable 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 tromethamine (TRIS). These agents are present in amounts sufficient to maintain the p11 of the system at 7,4+0.2 and preferably, 7.4 As such, the buffering agent can be as much as 5% on aweight basis of the total composition. 1005651(Cosolvents can be used to enhance drug solubility, however, some drugs are insoluble. These can often be suspended in the polymer vehicle with the aid of suitable suspending or viscosity enhancing agents. 1005661Since the polymer systems of the thernioreversible gel dissolve more completely at reduced temperatures, the preferred methods of solubilization are to add the required amount of polymer to the amount of water to be used. 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 can be stirred or shaken to bringabout a more rapid dissolution of the polymer. The active pharmaceutical ingredient and various additives such as buffers, salts, and preservatives can subsequently be added and dissolved. In some embodiments the pharmocologically active substance is suspended if it is insoluble in water.'The pH ismodulated by the addition of appropriate bufferingagents.

[00567In certain embodiments the polymer systems of the thenmoreversible gets are designed to remain liquids up to temperatures of about 15-- 25 C, about 18 -22 C, or about 20 C. In some instances, the fomuationsdescribed herein are manufactured under conditions such that the temperature of the manufacturing room is maintained below 25°C to retain the temperature of a polymer solution at about 25°C, about 23C, about 21i, or about 19°C. In certain instances, the fonulations described herein are manufactured under conditions such that the temperature of a manufacturing room is maintained at about 19°C. In some of such instances, the temperature of the polymer solution is maintained at or below about 19'C up to 3 hours of the initiation of the manufacturing, without the need to chill/cool the container. In someinstances, the temperature of thesolution is maintained at or below about 19°C up to 3hours of the initiation of the manufacturing by use of ajacketed container for the polymer solution. Auris-Acceptable Aclinic Radiation Curable Gel 100568 In other embodiments, the gel is an actinic radiation curable gel, such that following administration to or near the targeted auris structure, use of actinic radiation (or light, including UV light, visible light, or infrared light) the desired gel properties arefonmed. By way of example only, fiber optics are used to provide the actinic radiation so as to form the desired gel properties.In some embodiments, thefiber optics and the gel administration device form a single unit, In other embodiments, thefiber optics and the gel administration device are provided separately Auris-Acceptable Solvent Release Gel

[005691In some embodiments, the gee is asolventrelease gel such that the desired gelproperties are formed after administration to or near the targeted auris structure, that is, as the solvent in the injected gel formulation diffuses out the gel, a gel having the desired gel properties is formed, For example, a formulation that comprises sucrose acetate isobutyrate, a pharaceutically acceptable solvent, one or more additives, and the auris therapeutic agent is administered at or near the round window membrane: diffusion of the solvent out of the injected foulation provides a depot having the desired gel properties, For example, use of a water soluble solvent provides a high viscosity depot when the solvent diffuses rapidly out of the injected formulation. On the other hand use of a hydrophobic solvent (e.g..benzyl benzoate) provides a less viscous depot. One example of an auris acceptable solventrelease gel formulation is the SABERTM Delivery System marketed by DURECT Corporation,

[00570jPresented below are examples of potential controlled release excipients:

KEmeormaion Eamedarcrsts Chitosan glycerophosphate I unable degradationofmatrixin vitro (CGP) • tunableVP2 modulator release in vitro: e.g.,50 % of drug ------ ............ --------released after 24 hrs --- - -

0 biodegradable * compatible with drug delivery to the inner ear suitable for macromolecules and hydrophobic drugs PEG-PLGA-PEG triblock * tunable high stability: e.gmaintains mechanical integrity > 1 polymers month in vitro tunable fast release of hydrophilic drugs: e.g., 50 % of drug released after 24 hrs, and remainder released over ~ 5 days tunable slow release of hydrophobic drugs: e.g.~ 80 % released after 8 weeks * biodegradable * subcutaneous injection of solution: e.g., gel forms within seconds and is intact after I month PE0-PPO-PEO triblock * Tunable sot-gel transition temperature: e.g., decreases with copolymers (e.g., Pluronic increasing F127 concentration or Poloxameres) (e.g F1127) Chitosan glycerophosphate - CGP formulation tolerates liposomes: e.g., up 15uMnL with drug-loaded liposomes liposomes. liposomes tunably reduce drug release time (e.g.. up to 2 weeks in vitro), increase in liposome diameter optionally reduces drug release kinetics (e.g.,liposome size between 100 and 300 nm) release parameters are controlled by changing composition of _ P§ PI'---------------------------------------- -__ -- --

Auris internaMucoadhesiveLcipients 100571]Mcoadhesive characteristics may also be imparted to the gel or other auris-interna formulations disclosed herein, including a thermoreversible gel, by incorporation of nucoadhesive carbomers, such as Carbopol 934P, to the omposition (Majithiya etal, AAPS PharmSciTech (2006), 7(3), p. Fl; EP0551626). 1005721The term 'inucoadhesion' is commonly used for materials that bind to the mucin layer of a biological membrane. To serve as mucoadhesive polymers, the polymers should possess some general physiochenical features such as predominantly anionic hydrophilicity with numerous hydrogen bond forming groups, suitable surface property for wetting mucus/mucosal tissue surfaces and sufficient flexibility to penetrate the mucus network. Income embodimentsmucoadhesive forulations describedierein adhere tothe round window and/or the oval window and/or any inner ear structure.

005731Mucoadhesive agents including, but not limited to, at least one soluble polyvinylpyrrolidone polymer (PVP); a water-swellable, but water-insoluble, fibrous, cross-linked carboxy-functional polymer; a crosslinked poly(aciylic acid) (e.g. Carbopol 947P); acarbomer homopolymer; a carbomer copolymer; a hydrophilic polysaccharide gun, naltodextrin, a cross-linked alignate gum gel, a water-dispersible polycarboxylated vinyl polymer, at least two particulate components selected from the group consisting of titanium dioxide, silicon dioxide, and clay, or a mixture thereof. The mucoadhesive agent are used in combination with a viscosity increasing excipient, or are used alone to increase the interaction of the composition with a mucosal layer. In one non limiting example, the mucoadhesive agent is maltodextrin and/or an alginate gum. Those of ordinary skill in the art will recognize that the mucoadhesive character imparted to the composition should be at a level that is sufficient to deliver an effective amount of the composition to, for example the mucosal membrane of theround window in an amount that may coat the mucosal membrane, and thereafter deliver the composition to the affected areas, including by way of example only, the vestibular and/or cochlearstructures of the auris interna.Those of ordinaryskill in the art can determine the mucoadhesive characteristics of the compositions provided herein, and may thus determine appropriate ranges. One method for detennining sufficient mucoadhesiveness may include monitoring changes in the interaction of the composition with a mucosal layer, including but not limited to measuring changes in residence or retention time of the composition in the absence and presence of the excipient.

[00574Mucoadhesive agents have been described, for example, in US, Patent Nos.6,638,521, 6,562,363, 6,509,028 6,348,502,6,319,513, 6,306,789, 5,814,330, and 4,900,552, each of which is hereby incorporated by reference in its entirety. 1005751In one non-liniting example, the mucoadhesive agent is maltodextrin. Maltodextrin is a carbohydrate produced by the hydrolysis of starch that are derived from corn, potato, wheat or other plant products, Maltodextrin are used either alone or in combination with other mucoadhesive agents to impart mucoadhesive characteristics on the compositions disclosed herein In one embodinent, a combination ofmaltodextinand a carbopol polymer are used to increase the mucoadhesive characteristics of the compositions disclosed herein.

[00576]In another non-limiting example, a nucoadhesive agent can be, for example, at least two particulate components selected from titanium dioxide, silicon dioxide, and clay, wherein the composition is not further diluted with any liquid prior to administration and the level ofsilicon dioxide,if present, is from about 3% to about 15%, by weight of the composition. Silicon dioxide, if present, are selected from the group consisting of furmed silicon dioxide, precipitated silicon dioxide, coacervated silicon dioxide, gel silicon dioxide, and mixtures thereof. Clay, if present, are kaolin minerals, serpentine minerals, smectites, illite or a mixture thereof. For example, clay can be laponite, bentonite, hectorite, saponite, montmorillonites or a mixture thereof Stabilizers 1005771In one embodiment, stabilizers are selected from, for example, fatty acids, fatty alcohols, alcohols, long chain fatty acid esters, long chain ethers, hydrophilic derivatives offatty acids, polyvinyl pyrrolidones, polyvinyl ethers, polyvinyl alcohols,hydrocarbons, hydrophobic polymers, moisture-absorbing polymers, and combinations thereof, income embodiments, amide analogues of stabilizers are also used. In a further embodiment, the chosen stabilizer changes the hydrophobicity of the formulation (e.g., oleic acid, waxes), or improves the mixing of various components in the formulation (e.g., ethanol), controls the moisture level in the formula (e.g. PVP or polyvinyl pyrrolidone), controls the mobility of the phase (substances with melting points higher than room temperature such as long chain fatty acids, alcohols, esters, ethers, arnides etc. or mixtures thereof' waxes), and/or improves the compatibility of the formula with encapsulating materials (e.g., oleic acid or wax). In another embodiment some of these stabilizers are used as solvents/co-solvents (e.g., ethanol). Ina further embodiment, stabilizers are present in sufficient amount to inhibit the degradation of theactive pharmaceuticalingredient. Examplesof such stabilizing agents, include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0,001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate,(k) cyclodextrins, (1) penitosan polysulfate and otherheparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof. 100578]Additional useful aunis-acceptable formulations include one ormore anti-aggregation additives to enhance stability of otic formulations by reducing the rate of protein aggregation. The anti-aggregation additive selected depends upon the nature of the conditions to which the otic agents, for example anti-TNF antibodies are exposed. For example, certain formulations undergoing agitation and thermal stress require a different anti-aggregation additive than a formulation undergoing lyophilization and reconstitution. Useful anti-aggregation additives include, by way of example only, urea, guanidinium chloride,simple amino acids such as glycine or argininesugars, polyalcoholspolysorbates polymers such as polyethylene glycol and dextrans, alkyl saccharides, suchas alkyl glycoside, and surfactants. 1005791Other useful fonnulations include one or more antioxidants to enhance chemical stability where required. Suitable antioxidants include, by way of example only, ascorbicacid and sodium metabisulfite. In one embodiment, antioxidants are selected from metal chelating agents, thiol containing compounds and other general stabilizing agents.

[005801Still other useful compositions include one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic 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, octox'nol 40. 1005811 some embodiments, the pharmaceutical formulations described herein are stable with respect to compound degradation over a period of any of at least about I day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 daysat least about I week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months. at least about 5 months. or at least about 6 months. In other embodiments, the formulations described herein are stable with respect to compound degradation over a period of at least about 1 weekAlso described herein are formulations that are stable with respect to compound degradation overa period of at least about I month,

1005821In other embodiments, an additional surfactant (co-surfactant) andor buffering agent is combined with one or more of the phannaceutically acceptable vehicles previously described herein so that the surfactant and/or buffering agent maintains the product at an optimal pH for stability. Suitable co-surfactants include, but arenot limited to: a) natural and synthetic lipophilic agents, e.g, phospholipids. cholesterol, and cholesterol fatty acid esters and derivatives thereof; b) nonionic surfactants, whichinclude for example, polyoxyethylenefatty alcohol esters, sorbitan fatty acid esters (Spans), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene (20) sorbitan monooleate (Tween 80), polyoxyethylene (20) sorbitan monostearate (Tween 60), polyoxyethylene (20) sorbitan ionolaurate (Tween 20) and other Tweens, sorbitan esters, glycerol esters, e.g., Myj and glycerol triacetate (triacetin), polyethylene glycols, cetyl alcohol, cetostearyl alcohol, stearyl alcohol,polysorbate 80, poloxamers, poloxamines, polyoxyethylene castor oil derivatives (e.g., Cremophor R1440, CremphorA25, CremphorA20, Crenophor 5 EL) and other Creinophors, sulfosuccinates, alkyl sulphates (SLS); PEG glyceryl fatty acid esters such as PEG-8 glyceryl caprylate/caprate (Labrasol), PEG-4 glyceryl caprylate/caprate (Labrafac Hydro WL 1219), PEG-32 glyceryl laurate (Gelucire 444/14), PEG-6 glyceryl mono oleate (Labrafil M 1944 CS) PEG6 glvceryl linoleate (Labrafil M 2125 CS); propylene glycol mono- and di-fatty acid esters, such as propylene glycol laurate, propylene glycol caprylate/caprate; Brij* 700, ascorbyl-6palmitate, stearylamine, sodiumlauryl sulfate, polyoxethyleneglycerol triiricinoleate, and any combinations or mixtures thereof; c) anionic surfactants include, but are not limited to, calcium carboxymethylcellulose, sodium carboxymethylcelhdlose, sodium sulfosuccinate, dioctyl, sodium alginate, alkyl polyoxyethylene sulfates, sodium auryl sulfate, triethanolamine stearate, potassium launrate, bile salts, and any combinations or mixtures thereof; and d) cationic surfactants such as quarternary ammonium compounds, benzalkonium chloride, cetyltrimethylaminonium bromide, and lauryldimethylbenzyl-ammonium chloride, 100583]In a further embodiment, when one or more co-surfactants are utilized in the formulations of the presentdisclosure, they are combined. e.g, with a pharmaceutically acceptable vehicle and is present in the final formulation, e.g., in an amount ranging from about 0.1%to about20%, from about 0,5% to about 10% In one embodiment, the surfactant has an HLB value of 0 to 20. In additional embodiments, the surfactant has an HLB value of 0 to 3, of 4 to 6, of 7 to 9, of 8 to 18, of 13 to 15, of 10 to 18. Preservatives

[005841In some embodiments, an auris controlled release formulation described herein is free of preservatives. In soni embodiments, a composition disclosed herein comprises a preservative. Suitable auris-acceptable preservatives for use ina composition disclosed herein include, but are not limited to benzoic acid, boric acid, p-hydroxybenzoates, benzyl alcohol, lower alkyl alcohols (e.g., ethanol, butanol or the like), quaternary compounds, stabilized chlorine dioxide, mercurials, such as merfen and thiomersal, mixtures of theforegoing and the like. Suitable preservatives for uise with a formulation disclosed herein are not ototoxic. In some embodiments, a formulation disclosed herein does not include a preservative that is ototoxic. In some embodiments, a formulation disclosed herein does not include benzalkonium chloride or benzethoniurn chloride. 100585 In certain embodiments, any controlled release formulation described herein has an endotoxin level of less than 05 EU/kg. less than 0.4 EUkg or less than 0.3 EU/kg. In certain embodinents any controlled release formulation described herein has less than about 60 colony forming units (CFU), has less than about 50 colony formingunits, has less than about 40 colony forming units, has less than about 30 colony forcing units ofmicrobial agents per gram of formulation. In certain embodiments, any controlled release formulation described herein is substantially free of pyrogens, 1005861In a further embodiment, the preservative is, by wayof example only, anantimicrobial agent, within the formulation presented herein. In one embodiment, the formulation includes a preservative such as by way of example only, methyl paraben. In another embodiment, themethyl paraben is at a concentration of about 0.05% to about 1.0% about 0.1% to about 0.2%. In a further embodiment, the gel is prepared by mixing water,inethylparaben, hydroxyethylcellulose and sodium citrate In a fAther embodiment, the gel is prepared bymixing water, methylparaben, hydroxyethyellulose and sodium acetate. In a further embodimentthe mixture is sterilized by autoclaving at 120 C for about 20 minutes, and tested for pH, methylparaben concentration and viscosity before mixing with the appropriate amount of the active pharmaceutical ingredient disclosed herein, In certain embodiments, the preservative employed in any auris-compatible formulation described herein is an antioxidant (e.g., butyl hydroxytoluene (BHT) or the like, as described herein). In certain embodiments, an antioxidant preservative is non-toxic and/or non irritating to the inner ear environment. Carriers

1005871Suitable carriers for use in a fonnulation described herein include, but are not limited to, any pharmaceutically acceptable solvent, For example, suitable solvents include polyalkylene 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 pharmaceutically acceptablesurfactant and solvent.

1005881 Insome embodiments, other excipients include, sodium stearyl fumarate, diethanolamine cetyl sulfate, isostearate, polyethoxylated castor oil, benzalkonium chloride, nonoxyl 10, octoxynol 9, sodium lauryl sulfate, sorbitan esters (sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, sorbitan tristearate, S sorbitan laurate, sorbitan oleate, sorbitan palmitate, sorbitan stearate, sorbitan dioleate, sorbitan sesqui-isostearatesorbitansesquistearate, sorbitan tri-isostearate), lecithins, phospholipids, phosphatidyl cholines (c8-cl8), phosphatidylethanolamines (c8-cl8), phosphatidylglycerols (cS c18), pharmaceutical acceptable salts thereof and combinations or mixtures thereof. 100589In further embodimentsthe carrier is polyethylene glycol. Polyethylene glycol is available in 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.

[005901In 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 polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 (Tween 80) and any combinations or mixtures thereof. In. further embodiments, polysorbate 80 is utilized as the pharmaceutical acceptable carrier. 100591]In one embodiment, water-soluble glycerin-based thickenedformulations utilized in the preparation of pharmaceutical delivery vehicles that comprise at least one active pharmaceutical ingredient contains at least about 0.1% of the water-soluble glycerin compound or more. in some embodiments, the percentage of active pharmaceutical ingredient 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 Insome embodiments, the amount of the compound(s) in each therapeutically useful formulation 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 administrationproduct shelf life, as well as other pharmacological considerations are contemplated herein and the preparation of such pharmaceutical formulations is presented herein. SuspendingAgents

[00592]In one embodiment is a active pharmaceutical ingredient in a pharmaceutically acceptable thickened formulation wherein the formulation comprises at least one suspending agent. 1005931In one embodiment, at least one cytotoxic agent is included in a pharmaceutically acceptable enhanced viscosity formulation wherein the formulation further comprises at least one suspending agent, wherein the suspending agent assists in imparting controlled release characteristics to the frmulation. In some embodiments, suspending agents also serve to increase the viscosity of the auris-acceptable cytotoxic agent formulations and compositions.

[00594]Suspending agentsincludeby exampleonly, compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30,vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxyethylcellulose, methycellulose, hydroxypropyimethyieeliulose, hydroxymethylcellulose acetate stearate polysorbate-80, hydroxyethylcellulose, sodium alginate, gums. such as, eig., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e g., sodium carboxymethylcellulose, methyleelulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polvethoxylated sorbitantmonolaurate, povidone and the like. In some embodiments, useful aqueous suspensions also contain one or more polymers as suspending agents, Useful polymers include water-soluble polymers such as cellulosic polymers, e.g, hydroxypropyl methylcelhulose, and water-insoluble polymers such as cross-linked carboxyl containing polymers.

[00595[In one embodiment, the present disclosure provides compositions comprising a therapeutically effective amount of an active phanrmaceutical ingredient in a hydroxyethyl cellulose gel. Hydroxyethyl cellulose (HEC) is obtained as a dry powder which can be reconstituted in water or an aqueous buffer solution to give the desired viscosity (generally about 200 cps to about 30,000 eps, corresponding to about 0.2 to about 10% HEC). In one embodiment the concentration of HEC is between about 1% and about 15%,about I% and about 2%, or about L5% and about 2% 100596In some embodiments, the formulations include excipients, other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifing agents, solution promoters, and salts, In some embodiments, the excipients, carriers, adjuvants, are useful in fonning a pharmaceutically acceptable thickened formulation. Income embodiments, the thickened fannulation comprises a stabilizer. In another embodiment the formulation comprises a solubilizer, In a further embodiment the formulation comprises an antifoaming agent.In yet a further embodiment, the formulation comprises an antioxidant. In yet anotherembodiment, the formulation comprises a dispersing agent. In one embodiment, the formulation comprisesa surfactant. In yet another embodiment, the formulation comprises a wettingagent, iscosity Enhancing Agents 10597In one embodiment is a thickened formulation comprising at least one active pharmaceutical ingredient and a viscosity agent. Also described herein are controlled release formulations comprising an aural pressure modulating agent and a viscosity enhancing agent. Suitable viscosity enhancing agents include by way of example only, gelling agents and suspending agents. In one embodiment, the enhanced viscosity formulation does not include a pharmaceutically acceptable buffer. In other embodiments, the enhanced viscosity formulation includes a pharmaceutically acceptable buffer. Sodium chloride or other tonicityagents are optionally used to adjust tonicity, if necessary. 1005981Described hereinare formulations comprising an active pharmaceutical ingredient and a thickening agent. Suitable thickening agents include by way of example only, gelling agents and suspending agents. In one embodiment, the thickened formulation does not include a pharmaceutically acceptable buffer.In another embodiment, the thickened formulation includes a pharmaceutically acceptable buffer. 1005991By way of example only, the auris-acceptable viscosity agent include hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone (PVP: povidone) carboxymethvl cellulose, polyvinyl alcohol, sodium chondrointin sulfate, sodium hyaluronate. Other viscosity agents that are used in pharmaceutical compositions described herein include, but are not limited to, acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium. stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthancellulose, microcrystalline cellulose (MCC), ceratonia, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose; maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gumxnthumgum,polyethyleneglycol(e.g.PEG200 4500), gum tragacanthe thyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether/maleic anhydride copolymer (PVMMA),poly(ethoxyethyl methacrylate),poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyi-cellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, Splenda@ (dextrose, maltodextrin and sucralose) or combinations thereof. I specific embodiments, the viscosity-enhancing excipient is a combination of methylcellulose (MC) and CMC. In another embodiment, the viscosity-enhancingagent is a combination of carboxymethylated chitosan, or chitin, and alginate, The combination of chitin and alginate with the CNS modulators disclosed herein acts as a controlled release formulation, restricting the diffusion of the CNS modulator front the formulation, Moreover, the combination of carboxymethylated chitosan and alginate is optionally used to assist in increasing the permeability ofany active agent described herein through the round window membrane.

[00600J1none embodiment, the phannaceutically acceptable thickened formulation comprises at least one gelling agent. In one embodiment, the pharmaceutical formulation is a thickened formulation comprising at least one active pharmaceutical ingredient wherein the compound is utilized at a concentration of about 0.005 m.g to about 5 mg per gram of gelling agent.in another embodiment is an active pharmaceutical ingredient utilized at a concentration of about I mg to about 5 mg per gram of gelling agent In another embodiment is anactive pharmaceutical ingredient utilized at a concentration of about 0.005 mg to about 0.05 mg per gram of gelling agent. In another embodiment is an active pharmaceutical ingredient utilized at a concentration of about 0.05rg to about 0.5 mg per gram of gelling agent. In another embodiment is an active pharmaceutical ingredient utilized at a concentration of about 05 mg to about 5 mg per grain of gelling agent. In another embodiment is an active pharmaceutical ingredient utilized at a concentration of about 0.1 mg to about 5 mg per gram of gelling agent.

[006011in some embodiments is a thickened formulation comprising from about 0.1 mM and about 100 mM of an active pharmaceutical ingredient, a pharmaceutically acceptable viscosity agentand water for injection, the concentration of the viscosity agent in the water being sufficient to provide a thickened formulation with a final apparent viscosity from about 100 to about I,000,000 cP. In certain embodimentsthe viscosity of the gel is in the range from about 100 to about 500,000 cP about 100 cP to about 1,000 cP, about 500 cP to about 1500 P, about 1000 c to about 3000 cP, about 2000 cP to about 8,000 ePabout 4,000 cP to about 10,000 cP, about 10,000 cP to about 50,000 ci In further embodiments, the auris gel formulation contains a viscosity enhancing agent sufficient to provide a viscosity of between about 500 and 1,000,000 centipoise, between about 750 and 1,000,000 centipoise; betweenabout 1000 and 40,000 centipoise; between about 2000 and 35,000 centipoise; between about 3000 and 30,000 centipoise; between about 4000 and 25,000 centipoise; between about 5000 and 20,000 centipoise; or between about 6000 and 15,000 centipoise.

[006021In other embodiments, when an evenmore 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 pharmaceutical ingredientIn highly concentrated samples, the biocompatible thickened formulation comprises at least about 65%, at least about 75%, at least about 85%,at least about 90% or at least about95% or morebyweight ofthe active pharmacutical ingredient.

[006031In some embodiments, the viscosity of any formulation described herein is designed to provide an optimal rate of release from a otic compatible gel. In sone specific embodiments, a formultion viscosity of at least 700 cP (e.g., at 20°C, i.e, at2 degrees below Tgel, measured at a shearrate of 0.6 sa) substantially decreases the release rate of ano ite agent from a gel, i.e. substantially increases the mean dissolution time (MDT) of an otic agent. In specific embodiments, the rate of release of an otic agent from a formulation described herein is modulated by the incorporation of a secondary polymer. In specific embodiments, water soluble polynmer, (eg., cellulose based polymers (e.g., sodium carboxymethylcellulose), or poloxamer or the like) is incorporated as a secondary polymer for modulation of the release rateand/or mean dissolutiontime of an otic agent front aformulation described herein. In some instances, the concentration and grade of polymers is selected by the use of graphs shown in Figures 2 and 3 for commonly available water soluble polymers. 100604In some instancesa combination of polymers (e g, a poloxamer and a cellulose based polymer) provides a viscosity that is greater than the viscocity of a formulation comprising a single polymer (e.g.a poloxamer). In specific embodiments, a combination of a poloxamerand a cellulose based polymer (e.g- sodium carboxymethylcellulose) provides a composition of viscosity above 500 cP, above 300 cP or above 100 P. 1006051In one embodiment the thickened formulation described hereinis not a liquid at room temperature. In other embodiments, the thickened formulation formulation described herein is a liquid at room temperature. In some embodiments, the viscosity of the gel formulations presented herein are measured by any means described herein. For example, in some embodiments, an LVDV IICP Cone Plate Viscometer and a Cone Spindle CPE-40 is used to calculate the viscosity of the gel formulation described herein. In other embodiments, a Brookfield (spindle and cup) viscometer is used to calculate the viscosity of the gelformulation described herein. In some embodiments, the viscosity ranges referred to herein are measured at room temperature. In other embodiments, the viscosity ranges referred to herein are measured at body temperature. In certain embodiments, the thickened formulation is characterized by a phase transition between room temperture and. body temperature. In some embodiments, the phase transition occurs at 10 C below body temperture, at 2 °C below body temperture at 3 °C below body temperature, at 4 °C below body tempertureat 6 °C below body temperture, at 8 °C below body temperature, at 10 C below body temperture 1006061In some embodiments, the gel formulations are designed to be liquids at or about room temperature. In some instances, intratyrnpanic injection of cold formulations (e.g., formation with temperatures of< 20 0) causes vertigo. in some embodiments, the gel formulations are infected as liquidsat temperatures of about 15 ° to about 25C, about 18 ° to about 22 °, or about 20 C

[00607]In some instances, auris-acceptable gel formulations do not require the use of a thickening agent. Such gel formulations incorporate at least one pharmaceutically acceptable buffer. Inone aspect is a gel formulation comprisingan active pharmaceutical ingredient and a pharmaceutically acceptable buffer. in another embodiment, the pharmaceutically acceptable excipient or carrier is a gelling agent.

Auris-Acceptable PenetrationEnhancers 100608In.another embodiment the fonnulation further comprises one or more penetration enhancers. Penetration into biological membranes can be enhanced by the presence of penetration enhancers. Penetration enhancers are chemical entities that facilitate transport of coadministered substances across biological membranes. Penetration enhancers can be grouped according to chemical structure. Surfactants, both ionic and non-ionic, such as sodium lauryl sulfate, sodium laurate, polyoxyethylene-20-cetyl ether, laureth-9, sodium dodecylsulfate, dioctyl sodium sulfosuccinate, polyoxyethylene-9-lauryl ether (PLE), Tween 80, nonylphenoxypolyethylene (NP POE) polysorbates and the like. function as penetration enhances, Bile salts (such as sodium glycocholate sodium deoxycholate, sodium taurocholate, sodium taurodihydrofusidate, sodium glycodihydrofusidate and the like), fatty acids and derivatives (such as oleicacid, caprylic acid, mono- and di-glycerides, lauric acids, acylcholines, caprylic acids, acylcarnitines, sodium caprates and the like), chelating agents (such as EDTA, citric acid, salicylates and the like), sulfoxides (such as dimethyl sulfoxide (DMSO), decylmethyl sulfoxide and the like), and alcohols (suchas ethanol, isopropanol, propylene glycol, polyethylene glycol, glycerol, propanediol and the like) also function as penetration enhancers. In addition, the peptidelike penetration enhancers described in U.S. Patent Nos.7,151,191, 6,221,367 and 5,714,167, herein incorporated by references for such disclosure, are contemplated as an additional embodiment. These penetration enhancers are amino acid and peptide derviatives and enable dg absorption by passive transcellular diffusion without affecting the integrity of membranes or intercellular tight junctions. In some embodiments, a penetration enhancer is hyaluronie acid.

[00609)In some embodiments, the auris acceptable penetration enhancer is a surfactant. In some embodiments, the auris acceptable penetration enhancer is a surfactant comprising an alkyl glycoside and/or a saccharide alkyl ester. As used herein, an "alkyl-glycoside" means a compound comprising any hydrophilic saccharide (e.g. glucose, fructose, sucrose, maltose, or glucose) linked to a hydrophobic alkyl In some embodiments, the auris acceptable penetration enhancer is a surfactant comprising an alkyl-glycoside wherein the alkyl-glycoside comprises asugar linked to a hydrophobic alkyl (e.g., an alkyl comprising about 6 to about 25 carbon atoms) by an amide linkage, an amine linkage, a carbamnate linkage, an ether linkage, a thioether linkage, an ester linkage, a thioester linkage, a glycosidic linkage, a thioglycosidic linkage, and/or a ureide linkage. In some embodiments, the auris acceptable penetration enhancer is asurfactant comprising hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecylb dodecyl-, tridecyl-, tetradecyl, pentadecyl- hexadecyl-, heptadecyl and octadecyl a- orfi-D-maltoside; hexyl-, heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl, hexadecyl- heptadecyl-, and octadecyl f- or p-Dglucoside; hexyl-, heptyl-, octyl-, nonyl-,decyl-, undecyl- dodecy-, tridecyl-, tetradecyl, pentadecyl-, hexadecyl-, heptadecyl-, and octadecyl a- or p-D-sucroside; hexyl-, heptyl-, octyl-, dodecyl-, tridecyl-, and tetradecyl-fD-tiomaioside; heptyl- or octyl-1-thio-a- or fiD- glucopyranoside; alkyl thiosucroses; alkyl maltotriosides;long chain aliphatic carbonic acid aides of sucrose P amino-alkyl ethers;derivatives of palatinose or isomaltamine linked by an amide linkage to analkyl chain and derivatives of isomaltamine linked by urea to an alkyl chain; long chain aliphatic carbonic acid ureides of sucrose n-amino- alkyl ethers and long chain aliphatic carbonic acid aides of sucrose 1- amino-alkyl ethers. In some embodiments, the auris acceptable penetration enhancer is a surfactant comprising an alkyl-glycoside wherein the alkyl glycoside is maltose, sucrose, glucose, or a combination thereof linked by a glycosidic linkage to an alkyl chain of 916 carbonatoms (e.g., nonyl-, decyl-, dodecyl- and tetradecyl sucroside; nonyl-,decyl-, dodecyl- and tetradecyl glucoside; and nonyl-, decyl-, dodecyl- and tetradecyl malloside) In some embodiments, the auris acceptable penetration enhancer is a surfactant comprising an alkyl-glycoside wherein the alkyl glycoside is dodecylmaltoside, tridecyhnaltoside, and tetradecyhmaltoside.In some embodiments, theauris acceptable penetration enhancer is a surfactant comprising an alkylglycoside wherein the alkyl glycoside is tetradecyl- I-D-raltoside. Income embodimentsthe aunis acceptable penetration enhancer is a surfactant comprising an aikyl-glycoside wherein the alkyl-glycoside is a disaccharide with at least one glucose. in some embodiments, the auris acceptable penetration enhancer is a surfactant comprising a-D-glucopyranosyl-p-glycopyranoside n-Dodecyl-4-O-oa D glucopyranosyl-f-eglycopyranoside, and/or n-tetradecyl-4-O-a- D-glucopyranosyl-p glycopyranoside. In some embodiments, the auris acceptable penetration enhancer is a surfactant comprising an alkyl-glycoside wherein the alkyl-glycoside has a critical miscelle concentration (CMC) of less than about ImM in poure water orin aqueous solutions. In some embodiments, the auris acceptable penetration enhancer is a surfactant comprising an alkyl-glycoside wherein an oxygen atom within the alkyl-glycoside is substituted with a sulfuratom. In some embodiments, the auris acceptable penetration enhancer is a surfactant comprising an alkyl-glycoside wherein the alkylglycoside is the § anomer. Insome embodiments, the auris acceptable penetration enhancer is a surfactant comprising an alkyl-glycoside wherein the alkylglycoside comprises 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99,1%, 99,5%, or 999% of the panomer. 1006101Incertain instances, the penetration enhancing agent is a hyaluronidase In certain instances, a hyaluronidase is a human or bovine hyaluronidase. In some instances, a hyaluronidase is a human hyaluroidase (e.g, hyahironidase found in human sperm, PH20 (Halozyme),Hyelenex@ (Baxter International, Inc.)). In some instances, a hyaluronidase is a bovine hyaluronidase (e.g., bovine testicular hyaluronidase, Amphadase@ (Amphastar Pharmaceuticals), Hydase@ (PrimaPharm, Inc). In some instances, a hyluronidase is an ovine hyaluronidase, Vitrase@ (ISTA Phanaceuticals). In certain instances, a hyaluronidase described herein is a recombinant hyaluronidase. Insome instances, ahyaluronidase described herein is a humanized recombinant hyaluronidase. In sone instances, a hyaluronidase described herein is a pegylated hyaluronidase (e.g., PEGPH20 (Halozyme)). Foamsand Paints

[006111In some embodiments, the auris therapeutic agents disclosed herein are dispensed as an auris-acceptable paint. As used herein, paints (also known as film formiers) are solutions comprised of a solvent, a monomer or polymer, an active agent, and optionally one or more pharmaceutically acceptable excipients. After application to a tissue, the solvent evaporates leaving behind a thin coating comprised of the monomers or polymers, and the active agent. The coating protects active agents and maintains them inanimmobilized state at the site of application. This decreases the amount of active agent which are lost and correspondingly increases the amount delivered to the subject. By way of non-limiting example, paints include collodions (e.g. Flexible Collodion, USP), and solutions comprising saccharide siloxane copolymers anda cross-linkingagent. Collodions are ethyl ether/ethanolsolutions containing pyroxylin (a nitrocellulose) After application, the ethyl ether/ethanol solution evaporates leaving behind a thin film ofpyroxylin. In solutions comprising saccharide siloxane copolymers, the saccharine siloxane copolymers form the coating after evaporation of the solvent initiates the cross-linking of the saceharide siloxane copolymers. For additional disclosures regarding paints, see Remington: The Science and Practiceof Pharmacy which is hereby incorporated in its entirety. The paints contemplated for useherein, are flexible such that they do not interfere with the propagation of pressure waves through the ear. Further, the paints are applied as a liquid (i.e. solution, suspension. or emulsion), a semisolid (i.ea gel, foam, paste, or jelly) or an aerosol.

[006121 In some embodiments, the auris therapeutic agents disclosed herein are dispensed as a controlled-release foam. Examples of suitable foamable carriers for use in the compositions disclosed herein include, but are not limited to alginate and derivatives thereof, carboxymetthylcellulose and derivatives thereof, collagen, polysaccharides, including, for example, dextran, dextran derivatives, pectin, starch, modified starches such as starches having additional carboxyl and/or carboxamide groups and/or having hydrophilic side-chains, cellulose and derivatives thereof, agar and derivatives thereof, such as agar stabilised with polyacrylanmide polyethylene oxides, glycol methacrylates, gelatin, gums such as xanthum, guar, karaya, gellan, arabic, tragacanth and locust bean gum, or combinations thereof Also suitable are the salts of the aforementioned carriers, for example, sodium alginate. The formulation optionally further comprises a foaming agent, which promotes the formation of the foamincluding asurfactant or external propellant. Examples of suitable foaming agents include cetrimide, lecithin, soaps, silicones and the like. Commercially available surfactants such as Tween@i are also suitable. Auris-Acceptable In-Situ FormingSpongy Material 1006131Also contemplated within the scope of the embodiments is the use of a spongy material, formed in situ in the auris intema or aurismedia. In some embodiments, the spongy material is formed from hyaluronic acid or its derivatives. The spongy material is impregnated with a desired auris therapeutic agent and placed within the auris media so as to provide controlled release of the auris therapeutic agent within the auris media, or in contact with the round window membrane so as to provide controlled release of the auris therapeutic agent into the auris intema. In some embodimentsthe spongy material is biodegradable. Cyclodexirinfirmuations

[006141n a specific embodiment, the formulation alternatively comprises a cyclodextrin. Cyclodextrins are cyclic oligosaccharides containing 6, 7, or 8 glucopyranose units, referred to as a

cyclodextrin, f-cyclodextrin. ory-cyclodextrin respectively. Cyclodextrins have been found to be particularly uscfil in pharmaceutical fonnulations. Cyclodextrins have a hydrophilic exterior, which enhances water-soluble, and a hydrophobic interior which forms a cavity. In an aqueous environment, hydrophobic portions of other molecules often enter the hydrophobic cavity of cyclodextrin to fonn inclusion compounds. Additionally, cyclodextrins are also capable of other types of nonbonding interactions with molecules that are not inside the hydrophobic cavity. Cyclodextrins have threefree hydroxyl groups for each glucopvranose unit, or 18 hydroxyl groups on f-cyclodextrin, 21 hydroxyl groups on -cyclodextrin, and 24 hydroxyl groupsony cyclodextrin. One or more of these hydroxyl groups can be reacted with any of a number of reagents to form a large variety of cyclodextrin derivatives. Some of the more common derivatives of cyclodextrin are hydroxypropyl ethers, sulfonates, and sulfoalkylethers. Shown below is the structure of -cyclodextrin and the hydroxypropyl--cyclodextrin (HPpCD)

RO 07--O

R ROS O0R P OR OR O R=H RO RO P-cyclodextrin RO OR RO R =CH2 CH(OH)CHj R R hydroxypropyli-cyclodextrn OR O R RP 0 0 oO

RO OR 1006151The use of cyclodextrins in pharmaceutical compositions is well known in the art as cyclodextrins and cyclodextrin derivatives are oftenused to improve the solubility of a drug, Inclusion compoundsare involved in many cases of enhanced solubility; however other interactions between cyclodextrins and insoluble compounds can also improve solubility. Hydroxypropyl-

cyclodextrin (HPDCD) is commercially available as a pyrogen free product. I is anonhygroscopic white powder that readily dissolves in water. HP3CD is thennally stable and does not degradeat neutral pH. Thus, cyclodextrins improve the solubility ofa therapeutic agent in. a composition or formulation. Accordingly, in some embodimets, cyclodextrins are included to increase the solubility of the auris-acceptable otic agents within the formulations described herein. In other embodiments, cyclodextrins inaddition serve as controlled release excipients within the formulations described herein.

[00616]Preferred cyclodextrin derivatives for use include oxcyclodextrin3-cyclodextriny cyclodextrin, hydroxvethyl p-cyclodextrin, hydroxypropyl y-cyclodextriansulfated -cyclodextrin, suifated r-cyclodextrins sulfobutyi ether -cyclodextrin, j00617]1The concentration of the cyclodextrin used in the compositions and methods disclosed herein can vary according to the physiochemical properties., pharmacokinetic properties, side effect or adverse events, formulation considerations,or other factors associated with the therapeutically active agent, or a salt or prodrug thereof The properties of other excipients in a composition may also be important. Thus, the concentration or amount of cyclodextrin used in accordance with the compositions and methods disclosed herein can vary. 1006181In certain embodiments,the formulation further comprise a suitable viscosity agent, suchas hydroxypropyl methyleellulose, hydroxyethyl cellulose, polyvinylpyrrolilidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondrointin sulfatesodium hyaluronate etc. as a dispersant, if necessary. A nonionic surfactant such as polysorbate 80, polysorbate 20, tyoxapol, Crenophor, HCO 40 etc. is optionally used. In certain embodiments, the preparations optionally contain a suitable buffering system, such as phosphate, citrate, borate, tris, etc, and pH regulators such as sodium hydroxide and hydrochloric acid also are optionally used in the formulations of the inventions. Sodium chloride or other tonicity agents are also used to adjust tonicity, if necessary. Auris Acceptable Microspheresand Nanospheres

[006191Otic agents and/or other pharmaceutical agents disclosed herein are optionally incorporated within controlled release particles, lipid complexes, liposomes, nanoparticles, nicrospheres, nanocapsules or other agents which enhance or facilitate the localized delivery of the otic agent. In some embodiments, a single thickened formulation is used, in which at least one active pharmaceutical ingredient is present, while in other embodiments, a pharmaceutical forulation that comprises a mixture of two or more distinct thickenedformulations is used, in which at least one active pharmaceutical ingredient is present. In some embodiments, combinations of sols, gels and/or biocoinpatible matrices are also employed to provide desirable characteristics of the thickened formulations, In certain embodiments, the thickened formulation compositions are cross-linked by one or more agents to alter or improve the properties of the composition.

[006201Microspheres have been described in the following references, whichare incorporated herein by reference: Luzzi, L. A., J Pharm. Psy. 59:1367 (1970); US Pat. No. 4,530,840; Lewis, D. H. "Controlled Release of Bioactive Agents from Lactides/Glycolide Polymers" in Biodegradable Polymers as Drug Delivery Systems, Chasin, M. and Langer R. eds., Marcel Decker (1990); U.5 Pat. No, 4,675,189; Beck et al, "Poly(lactic acid) and Poly(lactic acid-co-glycolic acid)

Contraceptive Delivery Systems," in Long Acting Steroid Contraception, Mishell, D. R., ed., Raven Press (1983); U,. Pat No. 4,758;435; U.S.Pat, No. 3,773,919; U.S. Pat.No. 4,474,572; G. Johns et al. "Broad Applicability of a Continuous Formation Process," Drug Delivery Technology vol, 4 (Jan/Feb. 2004), each of which is hereby incorporated by reference for such disclosure, Examples of protein therapeutics formulated as microspheres include: U.S. Pat. No. 6,458,387; U.S. Pat. No. 6,268,053; U.S. Pat, No. 6,090,925; U.S. Pat. No, 5,981,719; and U.S. Pat. No. 5578,709, and are herein incorporated by reference for such disclosure. 100621jMicrospheres usually have a spherical shapealthough irregularly-shaped microparticles are possible.The microspheres vary in size, ranging from subnicron to 1000 micron diameters. Preferably, submicron to 250 micron diameter microspheresare desirable, allowing administration by injection with a standard gauge needle. The microspheres can thus be prepared by any method which produces incrospheres in. a size range acceptable for use in an injectable composition. injection are accomplished withstandard gauge needles used for administering liquid compositions.

[00622]Suitable examples of polymeric matrix materials include poly(glycolicacid), poly-d,-lactic acid, poly-i-lactic acid, copolymers of the foregoing, poly(aliphatic carboxylic acids). copolyoxalates, polycaprolactone, polydioxonene, poly(orthoearbonates)poly(acetals), poly(lactic acid-caprolactone), polyorthoesterspoly(glycolic acid-caprolactone), polydioxonene, polyanhydrides, polyphosphazines, and natural polymers including albumin, casein, and some waxes, such as, glycerol mono-and distearate, and the like. Various commercially available poly (lactide-co-glycolide) materials (PLGA) are used in the method disclosed herein. For example, poly (dl-lactic-co-glycolic acid) is commercially available from Boehringer-Ingelheim as RESOMER RG 503 H. This product has amole percent composition of 50% latide and 50% lycolide. These copolymers are available in a wide range of molecular weights and ratios of lactic acid to glycolic acid. A preferred polymer for use is poly(d,1-lactide-co-lycolide), It is preferred that the molar ratio oflactide to glycolide in such a copolymer be in the range of from about 95:5 to about 50:50 In other embodiments. PLGA copolymers with polyethylene glycol (PEG) aresuitable polymeric matrices for the formulations disclosed herein, For example, PEG-PLGA-PEG block polymers are biodegradable matrices for gel formation that provide high mechanical stability of the resulting gel. Mechanical stabilities ofgels using PEG-PLGA-PEG block polymers have been maintained for more than one month in vitro. In some embodiments. PEG-PLGA-PEG block polymers are used to control the release rate of cytotoxic agents with different physical properties. Particularly, in somc embodiments, hydrophilic cytotoxic agents are released more quickly, e.g., approximately 50% of drug release afer 24 hours, the remainder released over approximately 5 days, whereas hydrophobic agents are released more slowly, e.g., approximately 80% after 8 weeks. 1006231he molecular weight of the polymeric matrix material is of sonic importance. The molecular weight should be high enough so that it forms satisfactory polymer coatings, i.e., the polymer should be a good film former. Usually, a satisfactorymolecular weight is in the range of 5.000 to 500,000 daltons. The molecular weight of a polymer is also important from the point of view that molecular weight influences the biodegradation rate of the polymer. For a diffusional mechanism of drug release, the polymer should remain intact until all of the drug is released from the microparticles and then degrade. The drug can also be released from the microparticles as the polymeric excipient bioerodes. By an appropriate selection of polymeric materials amicrosphere formulation can be made such that the resulting microspheres exhibit both diffusional release and biodegradation release properties. This is useful in affordingmultiphasicrelease patterns.

1006241A variety of methods areknown by which compounds can be encapsulated in microspheres. In these methodsthe active pharmaceutical ingredientis generally dispersed or emulsified.using stirrersagitators. or other dynamic mixing techniques, in a solvent containing wall-forming material. Solvent is then removed from the microspheres, and thereafter the microsphere product is obtained. 1006251In one embodiment, controlled release formulations are made through the incorporation of the otic agents and/or other pharmaceutical agents into ethylene-vinyl acetate copolymermatrices. (See U.S PatentNo. 6,083,534, incorporated herein for such disclosure). In another embodiment, otic agents are incorporated into poly (lactic-glycolic acid) or polyIactic acid microspheres, In yet another embodiment, the otic agents are encapsulated into alginate microspheres. (See U.S Patent No. 6,036,978, incorporated hereinfor such disclosure), Biocompatible methacrylate-based polymers to encapsulate the otic agents or compositions are optionally used in the formulations and methods disclosed herein.A wide range of methacrylate-based polymer systems are commercially available, such as the EUDRAGITpolymers marketed by Evonik. One useful aspect of methacrylate polymers is that the properties of the forniulation are varied by incorporating various co-polymers. For example. poly(acrylic acid-co-methylmethacrylate) microparticles exhibit enhanced mucoadhesion propetes as the carboxylic acid groups in the poly(acryic acid)form hydrogen bonds with mucin (Park etal Phan.Res (1987) 4(6):457-464). Variation of the ratio between acrylic acid and methylmethacrylate monomers serves to modulate the properties of the co-polymer. Methacrylate-based microparticles have also been used in protein therapeutic formulations (Naha et al, Journal of Microencapsulation 04 February, 2008 (online publication)). In one embodiment, the enhanced viscosity auris-acceptable formulations described herein comprises otic agent incrospheres wherein the microspheres are formedfrom a methacrylate polymer or copolymer. In an additional embodiment, the enhanced viscosity formulation described herein comprises otic agent microspheres wherein the microspheres are nucoadhesive, Other controlled release systems, including incorporation or deposit of polymeric materials or matrices onto solid or hollow spheres containing otic agents, are also explicitly contemplated within the embodiments disclosed herein.

The types of controlled release systems available without significantly losing activity of the otic agent are determined using the teachings, examples, and principles disclosed herein

[006261 An example of a conventional microencapsulation process for pharmaceutical preparations isshown in U.S. Pat. No. 3,737,337, incorporated herein by reference. The substances to be encapsulated or embedded are dissolved or dispersed in the organic solution of the polymer (phase A), using conventional mixers, including (in the preparation of dispersion) vibrators and high-speed stirrers, etc. The dispersion of phase (A), containing the core material in solution or in suspension, is carried out in the aqueous phase (B), again using conventional mixerssuch as high-speed mixers, vibration mixers, or even spray nozzles, in which case the particle size of the incrospheres will be determined not only by the concentration of phase (A), but also by the emulsate or microsphere size. With conventional techniques for the microencapsulation of active pharmaceutical ingredients, the microspheres formwhenthesolventcotaining an active agent and a polymer is emulsified or dispersed in an immiscible solution by stirring, agitating, vibrating, or sone other dynamic mixing technique, oftenfor a relatively long period of time. f00627] Conventional methods for the construction of inicrospheres are also described in US. Pat No. 4,389,330andU.SPat.No. 4,530,840,incorporated herein by reference. The desired agent is dissolved or dispersed in an appropriate solvent. To the agent-containing medium is added the polymeric matrix material in an amount relative to the active ingredient which gives a product of the desired loading of active agent. Optionally, all of the ingredients of themicrosphere product can be blended in the solvent medium together. Suitable solvents for the agent and the polymeric matrix material include organic solvents such as acetone, halogenated hydrocarbons such as chloroform, methylene chloride and the like, aromatic hydrocarbon compounds, halogenated aromatic hydrocarbon compounds, cyclic ethers, alcohols, ethyl acetate and the like, 100628jIn some embodiments, the controlled-release auris-acceptable mnicrospheres are combined in a controlled-release auris-acceptable increased-viscosity formulation, including a gel

[00629]A suitable controlled-release auris-acceptable microsphere example for use with the auis acceptable therapeutic agents disclosed herein includes CHRONIJECT a PLGA-based controlled release injectable drig delivery system. Chroniject microspheres are useful for both hydrophobic and hydrophilic auris therapeutic agents, with achieved durations of release ranging from as short as 1 week to as long as1 year. Release profiles for the microspheres are achieved by modifying polymerand/or process conditions, with initial release or burst of the auris therapeutic agent also available.'The manufacturing process is adaptable to aseptic conditions, allowing direct therapeutic use of the manufactured product. Chroniject manufacturing processes are described in U.S. Patent Nos. 5,945,126; 6,270,802 and 6,3361,798, each of which is hereby incorporated by reference for such disclosure.

[006301 The mixture of ingredients in the solvent is emulsified in a continuous-phase processing medium; the continuous-phase medium being such that a dispersion of microdroplets containing the indicated ingredients is formed in the continuous-phase medium. Naturally, the continuous-phase processing medium and the organic solventmust be immiscible, and most commonly is water although nonaqueous media such as xylene and tolueneand synthetic oils and natural oils can be used. Usually, a surfactant is added to the continuous-phase processing medium to prevent the rnicroparticles from agglomerating and to control the size of the solvent microdroplets in the emulsion. A preferred surfactant-dispersing medium combination is a 1 to 10 wt. % poly vinyl alcohol in water mixture. The dispersion is forced by mechanical agitation of the mixed materials. t0 An emulsion can also be formed by adding small drops of theactive agent-wall forming material solution to the continuous phase processingimedium. The temperature during the formation of the emulsion isnot especially critical but can influence the sizeand quality of themicrospheres and the solubility of the drug in the continuous phase. It is desirable to have as little of the agent in the continuous phase as possible. Moreover, depending on the solvent and continuous-phase processing medium employed, the temperature must not be too low or the solvent and processing medium will solidify or the processing medium will become too viscous for practical purposes, or too high that the processing mediumwill evaporate, or that the liquid processing medium will not be maintained. Moreover, the temperature of the medium cannot be so high that the stability of the particular agent being incorporated in the microspheres is adversely affected. Accordingly, the dispersion process can be conducted at any temperature which maintains stable operating conditions, which preferred temperature being about 30 °C to 60 °C depending upon the drug and excipient selected.

[006311The dispersion which is formed is a stable emulsion and from this dispersion the organic solvent immiscible fluid can optionally be partially removed in the first step of the solvent removal process The solvent can easily be removed by coinnon techniques such as heating, the application of a reduced pressure or a combination of both. The temperature employed to evaporate solvent from the microdroplets is not critical, but shouldnot be that high that it degrades the agent employed in the preparation of a given microparticle, nor should it be so high as to evaporate solvent at such a rapid rate to cause defects in the wall forming material. Generally, from 5 to 75%, of the solvent is removed in the first solvent removal step.

[00632]After the first stage, the dispersed nicroparticles in the solvent immiscible fluid medium are isolated from the fluid medium by any convenient means of separation. Thusfor example, the fluid can be decanted from the microsphere or the microsphere suspension can be filtered, Still other, various combinations of separation techniques can be used if desired,

[006331Following theisolation of the microspheres from the continuous-phase processing medium, the remainder of the solvent in the microspheres is removed by extraction. In this step, the mnicrospheres can be suspended in the same continuous-phase processing medium used in step one, with or without surfactant, or inanother liquid. The extraction medium removes the solvent from the inicrospheres and yet does not dissolve the microspheres. During the extraction, the extraction medium with dissolved solvent can optionally be removed and replaced with fresh extraction medium. This is best done on a continual basis, Obviously, the rate of extraction medium replenishment of a given process is a variable which can easily be determined at the time the process is performed and, therefore, no precise limits for the rate must be predetermined. After the majority of the solvent has been removed from the microspheres, the microspheres are dried by exposure to air or by other conventional drying techniques suchas vacuum drying, drying over a desiccant, or the like This process is very efficient in encapsulating the agent since core loadings of up to 80 wt. %, preferably up to 60 wt, % are obtained.

[00634] Alternatively, controlled release microspheres containing an active pharmaceutical agent can be prepared through the use of static mixers. Static or motionless mixers consist of a conduit or tube in which is received anumber of static mixing agents. Static mixers provide homogeneous mixing ina relatively short length of conduit, and in a relatively short period of time. With static mixers, the fluid moves through the mixer, rather than some part of the mixer, such as a blade., moving through the fluid. 1006351A static mixer can be used to create an emulsion. When using a static mixer to form an emulsion, severalfactors determine emulsion particle size, including the density and viscosity of the various solutions or phases to be mixed, volume ratio of the phases, interfacial tension between the phases, static mixer parameters (conduit diameter; length of mixing element; number of mixing elements), and linear velocity through the static mixer. Temperature is a variable because it affects density, viscosity, and interfacial tension. The controlling variables are linear velocity, sheer rate, and pressure drop per unit length of static mixer.

[00636] In order to create microspheres containing an active pharmaceutical agent, an organic phase and an aqueous phase are combined.The organic and aqueous phases are largely or substantially immiscible, with the aqueous phase constituting the continuous phase of the emulsion. The organic phase includes an active pharmaceutical agent as well as a wall-forning polymer or polymeric matrix material, The organic phase can be prepared by dissolving an active pharmaceuticalagent in an organic or other suitable solvent, or by fonning a dispersion or an emulsion containing theactive agent, The organic phase and the aqueous phase are pumped so that the two phases flow simultaneously through a static mixer, thereby forming an emulsion which comprises microspheres containing the active pharmaceutical agent encapsulated in the polymeric matrix material. The organic and aqueous phases are pumped through the static mixer into a large volume of quench liquid to extract or remove the organic solvent, Organic solventare removed from the microspheres while they are washing or beingstirred in the quench liquid. After the microspheres are washed in a quench liquid, they are isolated, as through a sieve, and dried.

[006371 The process whereby microspheres are prepared using a staticnixer is optionally carried out for a variety oftechniques used to encapsulate active agents. The process is not lirnited to the solvent extraction technique discussed above, but can be used with other encapsulation techniques. For examplethe process can also be used with a phase separation encapsulation technique, To do so, an organic phase is prepared that comprises an active pharmaceutical agent suspended or dispersed in a polymer solution. The non-solvent second phase is free from solvents for the polymer and active agent. A preferred non-solvent second phase is silicone oil. The organic phase and the non-solvent phase are pumped through astatic mixer into a non-solvent quench liquid, such as heptane. The semi-solid particles are quenched forcomplete hardening and washing. The process of microencapsulation may also include spray drying, solvent evaporation, a combination of evaporation and extraction, and melt extrusion.

[00638]In another embodiment, the microencapsulation process involves the use of a static mixer with a singlesolvent.'This process is described in detail in U.S. application Ser, No. 08/338,805, herein incorporated by reference. An alternative process involves the use of a static mixer with co solvents. In this process for preparing biodegradable microspheres comprising a biodegradable polymeric binderand an active pharmaceutical agent, a blend of at least two substantially non-toxic solventsfree of halogenated hydrocarbons, is used to dissolve both the agent and the polymer. The solvent blend containing the dissolved agent and polymer is dispersed in an aqueous solution to form droplets. The resulting emulsion is then added to an aqueous extraction medium preferably containing at least one of the solvents of the blend, whereby the rate of extraction of each solvent is controlled, whereupon the biodegradable microspheres containing the pharmaceutically active agent are forced. The process has the advantages that less extraction medium is required because the solubility of one solvent inwater is substantially independent of the other and solvent selection is increased, especially with solvents that are particularly difficult to extract. 1006391Nanoparticles arem aterial structures of about 100 nm or less in size. One use of nanoparticles in pharmaceutical formulations is the formation of suspensions as the interaction of the particle surface with solvent is strong enough to overcome differences in density. Nanoparticle suspensions can be sterilized as the nanoparticles are small enough to be subjected to sterilizing filtration (US. 6,139.870) Nanoparticles comprise at least one hydrophobic, water-insoluble and water-indispersible polymeror copolymer emulsified in a solution or aqueous dispersion of surfactants, phospholipids or fatty acids. The active pharmaceutical ingredient are introduced with the polymer or the copolyner into the nanoparticles. 1006401Lipid nanocapsules act as controlled release structures, as well for penetrating the round window membrane and reaching auris interna targetsis also contemplated herein. See Zou et al. J Biomed. Material Res.,online pub. (April 24, 2008). Lipid nanocapsules are forced by emulsifying 1. 028 g capric and caprylic acid triglycerides (LABRAFAC WL 1349; avg. mw 512), 0.075 g soybean lecithin (LIPOID S75-3; 69% phosphatidylcholine and other phospholipids) 0.846 g surfactant (SOLUTOL 185),mixture of polyethylene glycol 660 hydroxystcarate and free polyethylene glycol 660; 0.089 g NaCI and 2.962 g water. The mixture is stirred at room temperature to obtain an oil emulsion in water. After progressive heating at a rate of 4 °C/nin under magnetic stirring, a short interval of transparency should occur close to 70 °C and the inverted phase (water droplets in oil) obtained at 85 °C.Three cycles of coolingand heating is then applied between 85 C and 60 C at the rate of 4 C/min, and a fast dilution in cold water at a temperature close to 0V to produce asuspension of nanocapsules. To encapsulate auris intera active agents, the agent are added just prior to the dilution with cold water.

[006411Agents mayalso be inserted into the lipid nanocapsules by incubation for 90 minutes with an aqueous micellar solution of the auris interna active agent. The suspension, is then vortexed every 15 minutes, and then quenched in an ice bath for 1 minute

[00642] Suitable surfactants are, by way of example, cholic acid or taurocholic acid salts. Taurocholic acid, the conjugate forced from cholic acid and taurine,is a fully metabolizable sulfonic acid surfactant.An analog of taurocholic acid, tauroursodeoxycholic acid (TUDCA), is a naturally occurring bile acid and is a conjugate of taurine and ursodeoxycholic acid (UDCA). Other naturally occurring anionic (e.g., galactocerebroside sulfate), neutral (e.g., lactosylceramide) or zwitterionic surfactants (e.g., sphingomyelin, phosphatidyl choline, palmitoylc arnitine) could also be used to prepare nanoparticles, 100643] The phospholipids are chosen, by way of example, from natural, synthetic or semi-synthetic phospholipids; lecithins (phosphatidylcholine) such as, for example, purified egg or soya lecithins (lecithin E 100, lecithin E80 and phospholipons, for example phospholipon 90), phosphatidylethanolanine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, dipaliitoylphosphatidylcholine, dipalmitoylgiycerophosphatidylcholine, diniyristoylphosphatidylcioline. distearoylphosphatidycholine and phosphatidie acid or mixtures thereof are used more particularly.

[00644] The fatty acids are chosen from, by way of example, from lauric acid, mysristic acid, palmitic acid, stearic acid, isostearic acid, arachidic acid, behenic acid. oleic acid, myristoleic acid, palmitoleic acid, linoleic acid, alpha-linoleic acid, arachidonic acid, eicosapentaenoic acid, crucic acid, docosahexaenoic acid, and the like.

[00645]Suitable surfactants can preferably be selected from known organic and inorganic pharmaceuticalexcipients. Such excipients include various polymers, low molecular weight oligomers, natural products, and surfactants, Preferred surface modifiers include nonionic and ionic surfactants. Two or more surface modifiers can be used in combination.

[006461Representative examples of surfactants include cetyl pyridiniui chloride, gelatin, casein, lecithin (phosphatides), dextran, glycerol, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcoholcetomacrogol emuIsifying wax, sorbitan esters, polyoxycthylenc alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters; polyethylene glycols, dodecyl trimethyl ammonium bromide, polyoxyethylenestearates, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, carboxyniethylcellulose calcium, hydroxypropyl cellulose (HPC,I-IPC-SL, and HPC-L), hydroxypropyl methylcellulose (HPMC), carboxymethycellulose sodium, methylcellulose, hydroxyethyleelluiose, hydroxypropylcelhilose, hydroxypropylmethyl-cellulose phithalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), 4-(1,1,3,3-tetaanethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol, superione, and triton), poloxamers, poloxamnines, a charged phospholipid such as dimyTistoyl phophatidyl glycerol., dioctylsulfosuccinate (DOSS); Tetronic 1508, dialkylesters of sodium sulfosuccinic acid.Duponol P; Tritons X-200, Crodestas F I10,p-isononylphenoxypoly-(glycidol), Crodestas SL-40,RTM, (Croda, Inc.); and SA90H14CO, which is CH 11CIt (CON(CI)-CL (COIH)4 (Ci1)2 (Eastman Kodak Co.); decanoylN methylghicamide; n-decyl p-D-glucopyranoside; n-decyl f-D-maltopyranoside; n-dodecyl p-D glucopyranoside; n-dodecyl fi-D-maltoside; heptanoyl-N-methylglucamide; n-heptyl--D glucopyranoside; n-heptyl p-D-thioglucoside; n-hexyl §-D-glucopyranoside; nonanoyl-N methylglucamide; n-noyl§-D-gluopyranoside; octanoy-N-methylglucarmide; n-octylp-D glucopyranoside; octyl p-D-thioglucopyranoside; and the like.

[00647Most of these surfactants are known pharmaceutical excipients and are described in detail in the handbook of Pharmaceutical Excipients, published jointly by theAmerican Pharmaceutical Association and'The Pharmaceutical Society of Great Britain (The Pharmaceutical Press, 1986), specifically incornporated by reference.

[006481 The hydrophobic, water-insoluble and water-indispersible polymer or copolymer are chosen from biocompatible and biodegradable polymers, for example lactic or glycolic acid polymers and copolymers thereof, or polylactic/polyethylene (or polypropylene) oxide copolymers, preferably with molecular weights of between 1000 and 200000, polvhydroxybutyric acid polymers, polylactones of fatty acids containing at least 12 carbon atoms, or polyanhydrides 1006491In one embodiment, the nanoparticles are suitable forusewith hydrophobic active principles. Theactive principles which can be used are chosen from the major classes of medicaments for use in human or veterinary medicine. In some embodiments, the active principles are chosenfrom principles for use in the cosmetics or agrifood industry orsports medicine or from diagnostic agents. By way of example, active principles which are of interest in the pharmaceutical industry are chosen. in a non-limiting manner, from antirheumatic, non-steroidal anti-inflammatory (e g. NSAIDs) analgesic, antitussive and psychotropic agents, steroids, barbiturates, antimicrobial, antiallergenic, antiasthmatic, antispasmodic, antisecretory and cardiovascular agents, ceebral vasodilators, cerebral and hepatic protective agents, therapeutic agents of the gastrointestinal tract, anticancer or antiviral agents vitamins, contraceptives, vaccines, etc,

[00650] The nanoparticles are obtained by the technique of evaporation of solvent, from an aqueous dispersion or solution of phospholipids and of an oleic acid salt into which is added an immiscible organic phase comprising the active principle and the hydrophobic, water-insoluble and water indispersible polymer or copolymer. The mixture is pre-enmulsified and then subjected to homogenization and evaporation of the organic solvent to obtain an aqueous suspension of very small-sized nanoparticles f00651] A variety of methods can be employed to fabricate nanoparticles. These methods include vaporization methods, such as free jet expansion, laser vaporization, spark erosion, electron explosion and chemical vapor deposition; physical methods involving mechanical attrition (e.g., "pearimilling" technology, Elan Nanosystems),super critical C02 and interfacial deposition following solvent displacement. In one embodiment, the solvent displacement method is used, The size of nanoparticles produced by this method is sensitive to the concentration of polymer in the organic 1i solvent; the rate of mixing; and to the surfactant employed in the process. Continuous flow mixers can provide the necessary turbulence to ensure small particle size. One type of continuous flow mixing device that can be used to prepare nanoparticles has been described (Hansen et al. J Phys Chem 92, 2189-96, 1988). In otherembodiments, ultrasonic devices, flow through homogenizers or supercritical CO2 devices are used to prepare nanoparticles. 100652JIf suitable nanoparticle homogeneity is not obtained on direct synthesis, then size-exclusion chromatography can be used to produce highly uniform drug-containing particles that arc freed of other components involved intheir fabrication. Size-exclusion chromatography (SEC) techniques, such as gel- filtration chromatography, can be used to separate particle-bound drug from free drug or to select a suitable size range of drug-containing nanoparticles. Various SEC media, such as Superdex 200, Superose 6, Sephacryl 1000 are commercially available and are readily employed by persons of skill in the art for the size-basedfactionation of mixture, Additionally, nanoparticles can be purified by centrifugationmembrane filtration and by use of other molecular sieving devices, crosslinked gels/naterials and membranes.

[00653] Liposomes or lipid particles may also be employed to encapsulate the otic agent formulations or compositions. Phospholipids that are gently dispersed in an aqueous medium form multilayer vesicles with areas of entrapped aqueous media separating the lipid layers. Sonication, or turbulent agitation, of these multilayer veiscles results in the formation of single layer vesicles, commonly refered to as liposomes, with sizes of about 10-1000 nra.These liposomes havemany advantages as drug carriers They are biologically inert, biodegradable, non-toxic and non-antigenic Liposones can be formed in various sizes and with varying compositions and surface properties.

Additionally, they are able to entrap a wide variety of small molecule drugs and release the drug at the site of liposone collapse,

[006541Suitable phospholipids for use in the present compositions are, for example, phosphatidyl cholines, ethanolamines and serines, sphingomyelins, cardiolipins, plasnalogens, phosphatictic acids and cerebrosides, in particular those which are soluble together with piroxicami n non-toxic, pharmaceutically acceptable organic solvents. Preferred phospholipids are, for example, phosphatidyl choline, phosphatidyl ethanoinine, phosphatidyl serine, phosphatidylinositol, lysophosphatidyl choline, phosphatidyl glycerol and the like,and fixtures thereof especially lecithin, eg. soya lecithin.The ainount of phospholipid used in the present formulation can range from about 10 to about 30%, preferably from. about 15 to about 25% and in particular is about 20% 100655] ipophilic additives are employed advantageously to modify selectively the characteristics of the liposomes. Examples of such additives include, for example, stearylanine, phosphatictic acid, tocopherol, cholesterol, cholesterol hemisuccinate and lanolin extracts. The amount of lipophilic additive used can range from 0.5 to 8%. preferably from 1.5 to 4% and in particular is about 2%. Generally, theratio of the amount of lipophilic additive to theamount of phospholipid ranges from about 1:8 to about 1:12 and in particular is about 1: 10. Said phospholipid, lipophilic additive and the active ingredient piroxicam are employed in conjunction with a non-toxic, pharmaceutically acceptable organic solvent system which can dissolve said ingredients. Said solvent system not only must dissolve the active pharmaceutical ingredient completely, but it also has to allow the formulation of stable single bilayered liposomes. The solvent system comprises dimethylisosorbide and tetraglycol (glycofurol, tetrahydrofurfuryl alcohol polyethylene glycol ether) in an amount of about 8 to about 30%. In said solvent system, the ratio of the amount of dimethylisosorbide to the amount of tetraglycol can range from about 2:1 to about 1:3, in particular from about 1:1 to about 1:2.5 and preferably is about 1:2. The amount of tetraglycol in the fial composition thus can vary from 5 to 20% in particular from 5 to 15% and preferably is approximately 10%.The amount of dimethylisosorbide in the final composition thus can range from 3 to 10%, in particular from 3 to 7% and preferably is approximately 5%. {00656] The term "organic component" as used hereinafter refers to mixtures comprising said phospholipid,,lipophilic additives and organic solvents.

[00657The active pharmaceutical ingredient is dissolved in the organic component. It is advantageous to use micronized forms of the active ingredient to facilitate its dissolution. The amount of active ingredient in the final formulation ranges from 0.1 to 5.0%. In addition, other ingredients such asanti-oxidants are added to the organic component. Examples include tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, ascorbyl palmitate. ascorbyl oleate and the like

[00658j1The aqueous component of the present fornimlation comprises mainly water and may contain various additives such as electrolytes, buffer systems, preservatives and the like. Suitable electrolytesinclude metal salts, in particular alkali metal and earth alkaline metal salts such as, for example, calcium chlorides, sodium chloride, potassium chloride, preferably sodium chloride. The concentration of the electrolytes may vary over a wide range and depends on the nature and the concentration of each of the ingredients in the final formulation and should be sufficient to stabilize the liposomal membranes.In the present composition the amount of sodium chloride can range from 0,05 to 0.2%. Buffer systems comprise mixtures of appropriate amounts of an acid such as phosphoric, succinic, or preferably citric acid, and a base, in particular sodium hydroxide. Said t0 buffer systems should maintain the pH of the formulation within the range of 3 to 9, alternatively within therange or 6 to 8 or between the range of 5 to7. Preservatives which can be employed in the present composition to prevent degradation by microorganisms may comprise benzoic acid, methylparaben and propylparaben.

[00659]Uiposomal formulations are optionally prepared by (a) heating the phospholipid and the organic solvent system to about 60-80 °C in a vessel, dissolving the active ingredient, then adding any additional formulating agents, and stirring the mixture until complete dissolution is obtained; (b) heating the aqueous solution to 90-95 °C ina second vessel and dissolving the preservatives therein, allowing the mixture to cool and then adding the remainder of the auxiliary formulating agents and the remainder of the water, and stirring the mixture until complete dissolution is obtained; thus preparing the aqueous component (c) transferring the organic phase directly into the aqueous component, while homogenizing the combination with a high perfonnance mixingapparatus, in particular a high-shear mixer; and (d) adding a thickener to theresulting mixture while further homogenizing. Preferably, the aqueous component is placed in a suitable vessel which can be equiped with a homogenizer and homogenization is effected by creating great turbulence during the injection of the organic component. Any mixing means or homogenizer which exerts high shear forces on the mixture are employed. Generally, a mixer capable of speeds from about 1,500 to 20,000 rpm, in particular from about 3,000 to about 6,000 rpm are employed. Suitable thickening agents for use in process step (d) are for example, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyleellulose or mixtures thereof, cellulose derivatives being preferred. The amount of thickening agent depends on the nature and the concentration of the other ingredients and in general ranges from about 0.5 to 1.5%, and in particular is approximately 15%. In order to prevent degradation of the materials used during the preparation of the liposimal formulation, it is advantageous to purge all solutions with an inert gas such as nitrogen or argon, and to conduct all steps under an inert atmosphere. Liposomes prepared by the above described method usually contain most of the active ingredient bound in the lipid bilayer and separation of theliposomes from unencapsulated material is not required.

Auris-Acceptable Lipid Formulations 1006601In someembodiments, the drug delivery formulation is a lipid-based formulation. In some embodiments, the lipid-based drug delivery formulation is a lipid emulsion (e.g,microemulsions and oil-in-water emulsions), a lipid vesicle (e.g.. liposomes liosomes micelles and transfersomes) or a combination thereof In some embodiments, thelipid-based drug delivery fomiulation is a lipid vesicle wherein the lipid vesicle is a liposome. In some embodiments, the lipid-based drug delivery formulation is a phospholipid-based formulation. in some embodiments, the lipid-based drug delivery formulation is a phospholipid-based formulation wherein the natural or synthetic phospholipid isphosphaidylethanolamine,phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, lysophospholipids. egg or soybean phospholipid, or a combination thereof The phospholipid is optionally salted or desalted, hydrogenated or partially hydrogenated, natural, synthetic, or semisynthetic. In some embodiments, the lipid-based drug delivery fornulationis a phospholipid-based formulation (e.g., hydrogenated ornonhydrogenated phospholipids. lecithins, phosphatidyl cholines (C8-Cl8), phosphatidylethanolamines (8-C18), phosphatidylglycerols (C8-Cl8)) wherein the phospholipid is phospholipon 90H(1,2-dia-cyl-SN glycero-3-phospbatidyl choline), egg phospholipids P123, Lipoid E80; Phospholipon 8H@, 80G®, 90H@ and I00H@, or combinations thereof.

[00661]In some embodiments, the lipid-based drug delivery formulation comprisesawater-soluble

preservative (i.e., a component that prevents microbes from substantially growing and multiplying), income embodiments, the lipid-based drug delivery fornulation comprises a water-soluble preservative wherein the preservative is a benzethonium salt (eg., benzethonium chloride), benzoic acid, and/or a benzylkonium salt (e.g., bezylkonim1 chloride). As used herein, water soluble means that the component has a solubility in water from about 100Pg/TL (0.01%) to about 0.01 mg/mL (0,1%)

[006621In some embodiments, the lipid-based drug delivery formulation comprises a lipid soluble anti-oxidant anti-oxidant. In some embodiments, the lipid-based drug delivery fornulation comprises vitamin E.

[006631In some embodiments, the lipid-based drug delivery formulation comprises less than about 2% w/wless than about 15%, less than about LO%, less than about 0,5%, or less than about 0.25% of a viscosity enhancing agent, 1006641In some embodiments, the lipid-based drug delivery formulation has a viscosity of at least about 10,000 centipoise, at least about 20,000 centipoise. at least about 30,000 centipoise, at least about 40,000 centipoise, at least about 50,000 centipoise at least about 60,000 centipoise, or at least about 70,000 centipoise, all at 58° C, without the presence of anymethyl-cellulose or other viscosity enhancing agents. In some embodiments, the lipid-based drug delivery formulation comprises oleyl alcohol to enhance the transmembrane penetration.

t006651In some embodiments, the lipid-based drug delivery formulation comprises a penetration enhancer (eg., a low molecular weight alcohol (e.g.., ethanol, oleyl alcohol), alkyl methanol sulphoxidesN-methyl-2-pyrrolidone, fatty amines (eg..oleylamine), fattyacids (e.g., olic acid, palmitoleic acid, linoleic acid, myristate acid), gluconic acid (the hexonic acid derived from glucose by oxidation of the aldehydegroup at C- to a carboxyl group) and its derivatives, such as gluconolactone (especially, glucono-D-lactone, a chelating agent produced by the oxidation of glucose), azone and propylene glycol, singly or in combination). In some embodiments, the lipid based drug delivery formulation comprises a penetration enhancer wherein the penetration enhancer is propylene glycol, either alone or in up to a 1:1 ratio with another enhancer, such as oleic acid or ethanol. In some embodimentsthe lipid-based drug delivery formulation comprises a penetration enhancer wherein the penetration enhancer is gluconolatone (e.g., glucono-D-lactoe), either alone or in up to a 1: ratio with another enhancer, such as propylene glycol. 1006661In some embodiments, the lipid-based drug delivery formulation comprises about 25% v/v or less of any one or more chemical penetration enhancer(s), most preferably from about 2% to 15% vI', although the exact formulation will vary depending onthe presence andamountsofexcipients, preservatives, water, p-i modulators, and the like included therein.

[006671In some embodiments, prepared liposomes loaded with the aural pressure modulators herein are gently mixed with viscosity. mucosal adhesives or absorption penetration enhancers. For example, aural pressure modulators loaded into liposomes are mixed with a chitosan glycerophosphate composition, allowing in situ gelling of the composition at intemal body temperatures of approximately 37 'C. The liposome size are optionally increased or decreased to modulate the release kinetics of the controlled release particles, In additional aspects, release kinetics are altered by changing the lipid composition of the liposones as described above. 100668The formulations described herein areadminstered in any suitable form, By way of non limiting examples, the formulations are administered as otic drops, as intratympanic injections, as foamsor as otic paints The formulations are administeredvia canula and/or injection, via a drop dispenser, as a spray in the ear canal, or as a paint via a cotton tipped stick, Controlled Release Kinetics

[006691 The goal of every drug delivery technique is to deliver the proper amount of drug to thesite faction at the right timeto obtain a therapeutic benefit.In general, controlled release drug formulations impart control over the release of drug with respect to site of release and time of release within the body. As discussed herein, controlled release refers to any release other than solely immediate release. In some instances, controlled release is delayed release, extended release, sustained release and/or pulsatile release (e.g., a combination of extended releaseand immediate release) or a combination thereof. Many advantagesare offered by controlled release. First, controlled release of a pharmaceutical agent allows less frequent dosing and thus minimizes repeated treatment. Second, controlled release treatment results in more efficient drug utilization and less of the compound remains as a residue Third, controlled release offers the possibility of localized drug delivery by placement of a delivery device or formulation at the at the site of disease. Still further, controlled release offers the opportunity to administer and release two or more different drugs, each having a unique release profile, or to release thesame drug at different rates or for different durations, by means of a single dosage unit. 100670 In a specific embodiment the formulations described herein provide a therapeutically effective amount of at least one active pharmaceutical ingredient at the site of disease with no systemic exposure. In an additional embodiment the frnulations described herein provide a therapeutically effectiveamount of at least one active pharmaceutical ingredient at the site of disease with no detectable systemic exposure.

[00671] The formulation are designed to provide drig delivery over a desired period of time, including periods up to severalweeks. As such, the patient will not need repeated administration of the drug, or at the least, fewer and less frequent administration of the drug.

1006721Drugs delivered to the auris internal have commonly been administered systemically via oral, intravenous or intramuscular routes, However, systemic administration for pathologies local to the auris intema increases the likelihood of systemic toxicities and side effects and creates a non productive distribution of drug in which high levels drug are found in the serum and correspondingly lower levels are found at the auns interna, 1006731 1one embodiment, the formulations disclosed herein additionally provides an immediate release of an otic agent from the formulation, or within 1 minute, or within 5 minutes, or within 10 minutes, or within 15 minutes, or within 30 minutes, or within 60 minutes or within 90 minutes. In other embodiments, a therapeutically effective amount of at least one otic agent is released from the formulation immediately, or within 1 minute, or within 5 minutes, or within 10 minutes, or within.15 minutes, or within 30 minutesor within 60 minutes or within 90minutes In certain embodiments the formulation comprises an aurispharnaceutically acceptable gel formulation providing immediate release of at least one otic agent. Additional embodiments of the formulation may also include an agent that enhances the viscosity of the formulations included herein.

[006741An immediate or rapid release option includes use of different viscosity-enhancing polymers, multi-component gels and nanospheres (or sub-micron spheres). Inaddition, the microspheres are optionally coated with an immediate-release component and a controlled-release component. 100675]In certain embodiments the formulation comprises a gel formulation providing immediate release of at least one active pharmaceutical ingredient, Additional embodiments of the formulation may also include a thickener that thickens the formulations included herein. In otherembodiments the thickened comprises a liposomal formulation providing immediate release of at least one active pharmaceutical ingredient. In certain other embodiments the formulation comprises a cyclodextrin containingorulation providing immediate release of at least one active pharmaceutical ingredient, In additional embodiments the fonnulation comprises a microsphere formulation providing immediate release of at least one active pharmaceutical ingredient. In additional embodiments the formulation comprises a nanoparticle fonnulation providing immediate release of at least one active pharmaceutical ingredient. 100676]In other or further embodiments, the formulation provides a controlled release formulation of at least one otic agent. In certain embodiments, diffusion of at least one otic agent from the formulation occurs for a time period exceeding 5 minutes, or 15 minutes; or 30 minutes, or 1 hour, Sor 4 hours, or 6 hours, or 12 hours, or 18 hours, or I dayor 2 days, or 3 days, or 4 days, or 5 days., or 6 days, or 7 days, or 10 days, or 12 days, or 14 days, or 18 days, or21 days. or 25 days, or 30 days or 45 days, or 2 months or 3 months or 4 months or 5 months or 6 months or 9 months or I year. In other embodiments, a therapeutically effective amount of at least one otic agent is released from theformulation for a time period exceeding 5 minutes, or 15 minutes, or 30 minutes, or 1 hour, or 4 hours, or 6 hours, or 12 hours, or 18 hours, or I day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 10 days, or 12 days. or 14 days, or 18 days, or 21 days, or 25 days, or 30 days, or 45 days, or 2 months or 3 months or 4 months or 5 months or 6 months or 9 months or I year. 100677 In other embodiments, the formulation provides both an immediate release and an extended release formulation of an otic agent. In yet other embodiments, the formulation contains a 0.25:1 ratio, or a 0.5:1 ratio, or a 1:1 ratio, or a 1:2 ratio, or a 1:3, or a 1:4 ratio, or a 1:5 ratio, ora 1:7ratio, or a 1:10 ratio, or a 1. 15 ratio, or a1:20 ratio of immediate release and extended release formulations. In a further embodiment the formulation provides an immediate release of a first otic agent and an extended release of a second otic agent or other therapeutic agent. In yet other embodiments, the formulation provides an irimediate release and extended release formulation of at least one otic agent, and at least one therapeutic agent. In some embodiments, the foniUlation provides a 0251 ratio, or a 0.51 ratio, or a 1:1 ratio, or a 1:2 ratio, or a 1:3, or a 1:4 ratio, or a 1:5 ratio, or a 1:7 ratio, ora 1:10 ratio, or a 1: 15 ratio, ora 1:20 ratio of immediate release and extended release formulations of a first otic agent and second therapeutic agent, respectively, 1006781In a specific embodiment the formulation provides a therapeutically effective amount of at least one otic agent at the site of disease with essentially no systemic exposure. In an additional embodiment the formulation provides a therapeutically effective amount of at least one otic agent at the site of disease with essentially no detectable systemic exposure, In other embodiments, the formulation provides a therapeutically effective amount of at least one otic therapeutic agent at the site of disease with little or no detectable detectablesystemic exposure.

[006791In some instances, upon administration (e.g., intratympanicinjection) of a conventional otic formulation (e.g, DSP in a buffer), the concentration of a drug in the the perilymph of an individual will rise sharply (C, at about 1-2 hours) and then taper off (Figure 1) to below C ,, In some instances, administration of an otic formulation described herein lowers the ratio of Cmx to Cm,,., and provides a larger Area Under the Curve (AUC) with a prolonged PK profile based on the C (Figure 1). In certain instances, controlled release formulations described herein delay the time to Cm, In certain instances, the controlled steady release of a drug prolongs the time the concentration

of the drug will stay above the minimum therapeutic concentration (i.e., Ci), Insome instances, controlled release of an otic agent provided by the fori-nulations described herein allows for release of an otic agent at concentrations greater than C,, for a period of at least 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 3 weeks orI month. In some embodiments, auris compositions described herein prolong the residence time of a drug in the inner ear. In certain instances, once drug exposure (e.g.,concentration in the perilymph) of a drug reachessteady state, theconcentrationof the dug in the perilymph stays at or about the therapeutic dose for an extended period oftime (eg., one day, 2 days, 3 days, 4 days,5 days, 6 days, orI week), In some embodiments, otic formulations decribed herein increase the bioavailability and/orsteady state levels of a drug in auris structures (e.g, in inner ear and/or the endolymph and/or the perilyiph).

t006801In some instances, upon administration of a controlled release auris formulation described herein (e.g, a formulation comprisingan anti-inflammatory agent (e.g., an anti-TNF agent)), drug concentrations relative to the binding constants of one or more otic receptors (e.g., corticoid receptors, NMDA receptors, glutamate receptors or the like, or any combination thereof) are relevant in determining a biologically meaningful PK profile or theminimum concentration of an active agent required for a therapeutic effect. In some instances, upon administration of a controlled release auris formulation described herein, drug concentrationsrelative to the binding constants of two receptors, such as, by way of example only, mineracorticoid receptor (MR) and glucocorticoid receptor (CR), are relevant in determining the C j,,or the biologically most meaningful PK profile. In some instances, for example, a drug saturates a first receptor (e.gGR) first, then saturates a second receptor (e.g,MR), and there is therapeutic benefit even when the first receptor is saturated and the second receptor is not yet saturated. Insome instances, the drug concentration for saturation the second receptor is about the same as the Cz. In some of such instances, for example, a next dose is administered when drug concentration drops below saturation levels of the second receptor and/or the C,,, (Figure 1).

[00681]The combination of immediate release, delayed release and/or extended release otic compositions or formulations are combined with other pharmaceutical agents, as well as the excipients, diluents, stabilizers, tonicity agents and other components disclosed herein, As such, depending upon the otic agent used, the thickness or viscosity desired. or the mode of delivery

-169.- chosen, alternative aspects of the enbodinents disclosed herein are combined with the immediate release, delayed release and/or extended release embodimnents accordingly. 1006821in certain embodiments, the pharmacokinetics of the otic formulations described herein are determined by injecting the formulation on or near the round window membrane of a test animal (including by way of example, a guinea pig or a chinchilla). At a determined period of time(e,g.,6 hours, 12 hours, .1day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days for testing the pharmacokinetics of a formulation over a 1 week period), the test animal is euthanized and the inner ear removed and tested for the presence of the otic agent. As needed, the level of otic agent is measured in other organs. In addition, the systemic level of the otic agent is measured by withdrawing a blood sample from the test animal. in order to determine whether the formulation impedes hearing, the hearing of the test animal is optionally tested. t006831 Alternativelyan inner ear is provided (as removed from a test animal) and the migration of the otic agent is measured. As yet another alternative, anin vitro model of a round window membrane is provided and the migration of the otic agent ismeasured. Modes of Otic Administration t00684iProvided herein are modes of treatment for otic compositions that ameliorate or lessen otic disorders described herein. Drugs delivered to the inner ear have been administered systenically 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 drug in which high levels of drug are found in the serum and correspondingly lower levels are found at the inner ear. 100685Provided herein are methods comprising the administration of said auris compositions on or near the round window membrane via intratympanic infection Insome 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 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 composition disclosed herein is then deposited on or near the round window orcrista fenestrae cochleae for localized treatment. In other embodiments, a 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 composition disclosed herein is applied at or near the round window membrane via a microinjection device. In yet other embodiments, a composition disclosed herein is applied inthe tympanic cavity. In some embodiments, a composition disclosed herein is applied on the tymnpanic membrane, In still other embodiments, a composition disclosed herein is applied onto or in the auditory canal. The formulations 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 described herein are useful in surgical procedures including, by way of non-limiting examples, cochleostomy, labyrinthotomy, mastoidectomy, stapedectomy, endolymphatic c sacculotomy or the like,

[006861 InltraympaUcInjecions 1006871in some embodimentsa 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, xylocaine). In some embodiments, the anterior-superior and posterior-inferior quadrants of the tympanic membrane are anesthetized. 1006881In 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 tympanic 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 CO laser). In one embodiment the delivery system is a syringe and needleapparatus that is capable of piercing the tympanic membrane and directly accessing the round window membrane or cristafenestrae cochleae of the auris intena, 1006891In one embodiment, the needle is a hypodermic needle used for instant delivery of the gel formulation. The hypodermic needle are a single use needle or a disposable needle.In some embodiments, a syringe are used for delivery of the pharmaceutically acceptable gel-based 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 anotherembodiment, the hypodermic syringe is a single usesyringe. In a further embodiment, the glass syringe is capable of being sterilized. in yet a further embodiment, the sterilization occurs through an autoclaveIn another embodiment, the syringe comprises a cylindrical syringe body wherein the gel formulation isstored before use.In other embodiments, the syringe comprises a cylindrical syringe body wherein the pharmaceutically acceptable otic gel-based compositions as disclosed herein is stored before use which conveniently allowsfor mixing with a suitable pharmaceutically acceptable buffer.In other embodiments, the syringe may contain other excipients, stabilizers, suspending agents, diluents or a combination thereof to stabilize or otherwise stably store the otic agent or other pharmaceutical compounds contained therein. 1006901In 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 ofthe auris acceptable otic gel 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 internaIn other embodiments, the delivery system comprises multiple syringes, each syringe of the multiple syringes contains at least one component of the gel 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 atleast one reservoir wherein the at least one reservoir comprises an otic agent, ora pharmaceutically acceptable buffer, or a viscosity enhancing agent, such as a gelling 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.

[006911In some embodiments, a needle punctures the posterior-inferior quadrant of the tyrnpanic membrane. In some embodiments, the needle is wider than a 18 gauge needle. In another embodiment, the needle gauge is from 18 gauge to 30 gauge. In a further embodiment, the needle is a 25 gauge needle. Depending upon the thickness or viscosity of a composition disclosed herein, the gauge level of the syringe or hypodennic needle are varied accordingly. In sonic embodiments, the formulations described herein are liquids and can be administered via narrow gauge needles or cannulae (e.g.,22 gauge needle, 25 gauge needle, or cannula),minimizing damage to the tympanic membrane upon administration. In some embodiments, the formulations described herein gel upon contact with auditory surfaces and/or at body temperature; there is no need for patients to lie on their sidewhile the otic agent takes effect. Theformulations described herein are administered with minimal discomfort to a patient.

[006921In some embodiments, an otoendoscope (e.g, about 1.7 mmin 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. 1006931In some embodiments, a composition disclosed herein is injected onto the round window membrane. In some embodiments, 0.4 to 0.5 cc of a composition disclosed herein is injected onto the round window membrane. 1006941In soni embodiments, the tympanic membrane puncture is left to heal spontaneously. In some embodiments, a paper patch myringoplastyis performed by a trained physician. In some embodiments, a tympanoplasty is performed by a trained physician. In some embodimentsan 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

[00695]In some embodiments, a composition disclosed herein is administered to the inner ear. In some embodiments, a composition. disclosed herein is administered to the inner ear via anincision in the stapes footplate. In some embodiments, a composition disclosed herein is administered to the

- 172-.

cochlea via a cochleostomy. In some embodiments, a composition disclosed herein is administered to the vestibular apparatus (e.gsemicircular canals or vestibule). 1006961 In some embodiments, a composition disclosed herein is applied via syringe and needle. In other embodiments, a composition disclosed herein is applied via microcatheters implanted into the patient. in some embodiments, a composition disclosed herein isadministered via a pump device In still further embodiments, a composition disclosed herein is applied via a icroinjection device. In some embodiments, a composition disclosed herein is administered via a prosthesis, a cochlear implant, a constant infusion pump, or a wick. 100697 In some embodiments, the delivery device is an apparatus designed for 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 drug 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 forsuch disclosure. US.Patent Application No. 08/874,208, which is incorporated herein by reference for such disclosure,describes a surgical method for implantinga fluid transfer conduit to deliver therapeutic agents to the inner ear. U.SPatentApplication Publication 2007/0167918, which is incorporated herein by reference for such disclosure, further describes a combined otic aspirator and medication dispenser for intratympanic fluid sampling and nedicament application. Dosage

1006981The compositions containing the compound(s) described herein are administered for prophylactic and/or therapeutic treatments. In therapeutic applications, the compositions are administered to a patient already suffering from a disease, condition or disorder, in an amount sufficient to cure or at least partially arrest the symptoms of the disease, disorder or condition. Amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapythe patient's health status and response to the drugs, and the judgment of the treating physician.

[006991 The amount of a given agent that will correspond to such an amount will vary depending upon factors such as the particular compound, disease condition and its severity, but can nevertheless be routinely determined in manner know in theart according to the particular circumstances surrounding the case, including, e.gthe specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated In general, however, doses employed for adult human treatment will typically be in the range of 0,02-50 mg per administration, preferably 1-15 mg per administration, The desired dose may conveniently be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals. Frequency ofadministration

[007001in the case wherein the patient's condition does not improve. upon the doctor's discretion the administration of the compounds are administered chronically, that is, for an extended period of time, including throughout the duration of the patient's life in order to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition. 00701]1n the case wherein the patient's status does improve, upon the doctor's discretion the administration of the compounds are given continuously; alternativelythe dose of drug being administered are temporarily reduced or temporarily suspended for a certain length of time (i~ea "drug holiday"). The length of the drug holiday can vary betw een 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days days, 10days, 12 days, 15 days, 20 t0 days 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days 350 days, and 365 days. The dose reduction during a drug holiday are from 10%-100%, including by way ofexample only 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%,55%,60%,65%, 70%, 75% 80%, 85%, 90%, 95%,and 100%. f00702]Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. Patients can, however, require intennittent treatment on a long-term basis upon any recurrence of symptoms. 100703]In some erbodiments, the inital administration is of a particular formulation and the subsequent administration is of a different formulation or active phannaceutical ingredient. Kits and Other Articles of Manufacture 100704 The disclosure also provides kits for preventing, treating or ameliorating the symptoms of a diseases or disorder in a mammal Such kits generally will comprise one or more of the pharmaceutically acceptable gel-based compositions as disclosed herein, and instructions for using the kit. The disclosure also contemplates the use of one or more of the formulations, in the manufacture of mtedicaments for treating, abatingreducing, or ameliorating thesymptoms of a disease, dysfunction, or disorder in a mammal,such as a human that has, is suspected of having, or at risk for developing an auris intenia disorder. 100705 In some embodiments, a kit disclosed herein comprises a needle that can penetrate a tympanic membrane and/or a round window, In some embodiments, a kit disclosed herein further comprises a hydrogel with a penetration enhancer (e,g, an alkylglycoside and/or a saccharide alkyl ester).

[00706JIn some embodiments, kits include a carrier, package, or container that is compartmentalized to receive one ormore containers such as vialstubes, and the like, each of the container(s) including one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In other embodiments, the containers are formed from a variety of materials such as glass or plastic

[007071 The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products presented herein. See, e.g., U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bagsvials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.A wide array of formulations of the compounds and compositions provided herein are contemplated as are a variety of treatments for any disease, disorder, or condition that would benefit by extended release administration of a therpeutic agent to theauris intema..

1007081In some embodiments, akit will typically includes one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and/or devices) desirable from a commercial and user standpoint for use of a formulation described herein. Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with. instructions for use. A set of instructions will also typically be included. 1007091In a further embodiment, a label is on or associated with the container. In yet a further embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In other embodiments a label is used to indicate that the contents are to be used for a specific therapeutic application.in yetanother embodiment, a label also indicates directionsfor use of the contents, such as in the methods described herein. 007101In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. In another embodiment, the pack for example contains metal or plastic foil, such as a blister pack. In a further embodiment, the pack or dispenser device is accompanied by instructions for administration. In yet a further embodiment, the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. In another embodiment, such notice, for example, is the labeling approved by the US Food and Drug Administration for prescription drugs, or the approved product insert In yet another embodiment, compositions containing a compound provided herein fonnulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. EXAMPLES Example I... Preparation of a Thermoreversible Gel anti-INF Formulation Ingredient Quantity (mgg of _________________________ formulation) Adalimumab 10.0 methylparaben_-1.0 HPMC 10.0 Poloxamier 407 180.0 | TIS HC buffer (0. 1 M) 789.0

[007111Adalimumabis supplied in 40 mg/0.8 mL pre-filled glass syringes containing approximately 4.93 mg sodium chloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22tug dibasic sodium phosphate dihydrate, 024 mg sodium citrate, 1.04 mg citric acidmonohydrate, 0,6 mg mannitol, 0.8 mg polysorbate 80 and water. All mixing vessels are siliconized or otherwise treated to prevent adalinumab from adhering to the vessel walls. f00712] A I0 g batch of gel formulation containing 1.0% ofadalimumab is prepared by suspending 1. 80 g of Poloxamer 407 (BASF Corp.) in 5.00 g of TRIS HC buffer (0,1 N) and the components are mixed under agitation overnight at 4 'C to ensure complete dissolution. The hydrox)propyhnethylcellulose (100,0 mg),inethylparaben (10 mg)and additional TRIS HCI buffer (0.1 M) (2.89 g) is added and further stirring allowed until complete dissolution is observed. Adalimumab (100 mg) is added and mixed to maintain activity, The mixture ismaintained below room temperature until use. Examples 2 - 10 100713}Thennoreversible gel formulations comprising VP2 antagonist lixivaptan, diazepan, methotrexateanioxicillin, AMN082 SRT-501 Neramexane, JB004/A, KCNQ modulator Retigahine, are prepared using a procedure similar to the procedure in Example . In further examples, thermoreversible gel formulations comprising micronized VP2 antagonist lixivaptan, diazepam, methotrexate, amoxicillin, AMN082, SRT-501, Neramexane,1B004/A, KCNQ modulator Retigabine, are prepared using a procedure similar to the procedure in Example 1

ExampLeI1 -Preparation of a Mucoadhesive. Thermoreversible Gel Calcineurin Inhibitor Formulation Ingredient Quantity (mg/g of taeY~mu~ . formulation) tacrolimus 10,0 methylparaben 1.0 HPMC 10 Carbopol 934P 2,0 Poloxamer 407 180.0 TRIS HCI buffer (01 M) 787.0

1007141A 10-gbatch of mucoadhesive, gel formulation containing 1,0% of anti-TNF agent is prepared bysuspending 20.0 mg of Carbopol 934P and 180 g of Poloxanier 407 (BASF Corp.) in 5.00 g ofTRISFHCi buffer (0. 1M) and the components are mixed under agitation overnight at 4 C to ensure complete dissolution, The hydroxypropylmethylcellulose (100.0 mg), methylparaben (10 mg) and additional'TRIS HCl buffer (01M) (287 g) areadded and further stirring allowed until complete dissolution is observed. Tacrolimus (100 g) is added and mixed while maintaining activity. The mixture is maintained below room temperature until use.

Examlcs 12 - 18

[00715jMucoadhesive thermoreversible gel formulations comprising diazepan, AMN082, D methionineGanciclovir, SRT501 neonyin, the KCNQ modulator.XE-991 are prepared using a procedure similar to the procedure in Examplel ifurther examples, mucoadhesive thermoreversible gel formulations comprising micronized diazeparnAMN082, D-methionine Ganciclovir, SIT-501, neonycin, the KCNQ modulator XE-991 are prepared using a procedure similar to the procedure in Example11.

Example 19Pre ation of a Mucnalhesive-based TACE Inhibitor Fornulation Ingredient I Quantity (mggof _______________________ ornulation) BMS-561392 10.0 parafhn oil 200 trihvdroxvstearate 10 cetyl dimethicon copolyol 30 water qs ad 1000 phosphate buffer p7.4 ,_ qs pH 7.4

[007161Thecream-type formulation is first prepared by gently mixing I S-561392 with an organic solvent. Asecond system is prepared by mixing paraffin oil, trihydroxystearate and etyl dimethicon copolyol with warming to 60 °C, Upon cooling to room temperature, the lipid system is mixed with the aqueous phase for 30 minutes. Examples 20 - 25

[007171Mucoadhesive-based formulations lidocaine.Cl, niethotrexate, benzthine penicillin G, piceatannol, cyclophosphamide and CNQX are prepared using a procedure similar to the procedure in Example19,

Example 26-- Preparation of a Mucoadhesive. Thermoreversible Gel IKK Inhibitor Forniulation Ingredient Quantity(mg/gof ____________________ ormulation) BMS-345541 10.0 mehylparaben 1.0 Poloxamer 407 180.0 Carbopol 934P 2.0 TRIS HCi buffer (0.1 M) 317

[00718]The Carbopol 934P and Poloxamer 407 (BASF Corp.) is first suspended in the TRIS HCI buffer (0.1 M) and the components are mixed under agitation overnightat 4 °C to ensure complete dissolution. Themethylparaben is added and further stirring allowed until complete dissolution is observed. The BMS-345541 is mixed in while retainingactivity. The mixture is maintainedbelow room temperature until use. Examples 27 - 28

[00719Mucoadhesive thermoreversible gel formulation comprising methotrexate, alpha lipoic acid is prepared using a proceduresimilar to the procedure in Example 26

007201Viscosity determinations of the pharmaceutical compositions described herein are performed at room temperature and 37 °C and are made using a Brookfield (spindle and cup) viscometer at 20 rpm.

Example 29 - Preparation ofan Enhanced Viscosity Mucoadhesive Controlled ReleaseAntiTNF formulation {00721] Poly (lactic-glycolicacid) (PLGA) microspheres, containing anti-TNF binding protein, are prepared by a modified solvent evaporation method using a double emulsion. (See U.S. Patent No. 6,083,354 incorporated by reference forsuch disclosure Cohen et aL Pharn. Res. (199]) 8713 720). Briefly, anti-TNF binding protein (TBP solution or powder of'TBP and bovine serum albumin (TBPI solution added to BSA powder in double distilled water; freeze dried into powder and sieved to give particles of sizesranging from 75 un to 250-425 nmis dissolved in double distilled water. PELGA is separately dissolved in methylene chloride, A mixture of the PLGA and TBPI is probe sonicated (model VC-250, Sonic & Materials Inc.) for 30 see to form the first inner emulsion (W 1 /0). The emulsion is then poured, under vigorous mixing using a magnetic bar, into 2 mL aqueous it polyvinylalcohol (PVA) saturated with methylene chloride to fonn the second emulsion ((Wl/0)W2). The resulting double emulsion is subsequently poured into 200 mL of 0.1% PVA and continuously stirred for 3 hrat room temperature until most of the methylene chloride evaporates, leaving solid microspheres. The microspheres are collected by centrifugation (1000 g for 10 m),sized using sieves with apertures of 100t m and freeze dried (16 hr, Freeze Dryer, Lab Conco) into a powder. The microspheres are mixedinto the enhanced viscositymucoadhesive formulation of Example 19.

Exanle 30.-Preparation ofLiposomal VP2 Antagonist Fornation

Ingredient - Quantity (mggof Lixiapn 5.0 soya lecithin 200.0 cholesterol 20,0 tetragycol dimethylisosorbide J100.0 50.0 niethylparaben 2.0 propyparaben 0.2. BHT 0.1 sodium chloride 1. HPMC 15.0 sodium hydroxide 0.6 citric acid LO purified water, USP 603.6

[007221Heat the soya lecithin, tetraglycol and dimethyl isosorbide to about 70-75 'C. Dissolve the lixivaptan, cholesterol and butylated hydroxytoluene in the heated mixture. Stir until complete dissolution is obtained. Heat about one third of the water to 80-95 'C in a separate vessel and dissolve the preservatives nethylparaben and propylparaben in the heated water while stirring Allow thesolution to cool. to about 25 °C and then add the disodium edetate, sodium chloride, sodium hydroxide and citric acid. Addthe remainder of the water and stir to obtain a complete solution. Transfer the organic mixture into the aqueousmixture by means of a vacuum, while homogenizing the combination with a high-shear mixer until ahomogeneous product is obtained. Add the hydroxypropyl methyleellulose into the biphasic mixture by means of a vacuum while homogenizing with a mixer. The homogenizer is a Silverson high-shear mixer operating at approximately 3000 rpm.. Single bilayered liposomes are formed. The white lipogel cream is ready for use

Exanile 31 - Preparation of a VP2 Antagonist Nanoprticle Fornulation 1007231750amg (15 mgimLtheoretical) of a diblock copolymer consisting of the combination of a poly(d,1-lactic acid) of mass 30 kD and ofa polyethylene glycol of mass 2kD (PLA-PEG) and 250 mg(5n g/mLtheoretical)of tolvaptanisdissolved in20 ilof ethyl acetate (solution.A). 175 nagof lecithin E80 and 90 nig of sodimn oleate is dispersed in 50 iL of 5% w/v glucose solution (solution B). Solution A is emulsified in solution B with an Ultra-turrax stirrer and the pre-emulsion is then introduced into a Microfluidizer 110 S.RTM. type homogenizer for 10 minutes at 10 °C. The volume of emulsion recovered is about 70 mL (70 g). The ethyl acetate is removed using a rotary evaporator at reduced pressure (100 mm of mercury) to a suspension volume of about 45 ml (45 g)

[00724]Nanoparile fonulation of a KCNQ modulator flupirtine is prepared using a procedure similar to the procedure in Example 31,

Example 32 - Preparation of a 5% Cyclodextrin VP2 Antagonist Formulation 1007251To a suitable 150 mL glass vessel is added tolvaptan (5,0 g), sterile 2% dibasic sodium phosphate dodecahydrate solution (9.0 g) and hydroxypropyl-cyclodextrin (50 g) The resulting mixture is stirred until a clear solution is formed. To this solution is added sterile 2% polysorbate 80 solution (5 g), sterile% stock HPMC 2910 (EM) solution (2.5 g) and 5% sterile sodium chloride solution ( Iig), and stirring is continued until homogeneous. Sterile water for injection is added to get to 95% of batch size."The solution is stirred at room temperature for 30rin and pH is adjusted to 7.2, Finally, water for injection is added to get a Enal batch size of 100 g.

Example 33 Preparation of a 5% Cyclodextrin KCN Modulator mucoadhesive thernoreversible S gelCfomulation 1007261 A. 5%CD solution of Flupirtine is prepared according to the procedure in Example 32 and added to the mucoadhesive thermoreversible gel formulation of Example 1 1.

Example 34--- Preparation of a 50% VP2 Antagonist 95:5 dlPLGA Microspherei ornulation 1007271Twenty-five grars (25 g) of 95:5 d-PLGA and 25 g of OPC-31260 are codissolved in 196 g ethyl acetate in an Erlemeyer flask at 52 °C. The drug/polymer solution is added to a 1000 mL glass jacketed reactor containing 550 g of 5% aqueous polyvinyl alcohol containing 9.7 2 of ethyl acetate. Reactor contents are stirred with an overhead stir motor and the temperature is maintained at 52 C. by a circulatingbath. The emulsion size is monitored by light microscopy and the stirring is stopped when the particle size is found to be inthe desired size range (less than 300 microns), usually after about 2 minutes. The stir speed is reduced to avoid further size reduction of the sterilized emulsion. After stirring for a total of 4minutes, the reactor contents are pressure transferred into 40 liters of water at 12 °C. After stirring for 20minutes the hardenedmnicrospheres are isolated and the product then transferred into 20 liters of water at 12 'C. After approximately 3 hours, the second wash is transferred onto a sievestack composed of 25, 45, 90, 150, and 212 micron openings. The product on the sieves is washed with copious amounts of cold water to separate the different sizes of microspheres. After drying on the sieves overnight, the different fractions are collected and drying was continued under vacuum at room temperature. Formulations with other drug levels are prepared by simply adjusting the polymer/drug ratio.

Exaple 35 100728]Microspheres comprising KCNQ modulator XE-991 are prepared using a procedure similar to the procedure in Example 34.

Example 36 -Preparation of a 50% VP2 Antagonist 65:35 d,-PLGA Microsphere Formulation

[007291Microspheres are produced by themethod of Example 34 except that a different biodegradable polymer matrix was utilized. A 65:35 d-PLGA polymer was used in place of the 95:5 polymer indicated in Example 34.

Exanle 37 Preparation of a Mucoadhesive, Cyclodextrin-based VP2 Antagonist Formulation

Ingredient Quantity (mg/g of formulation) Lxivaptan 20.0 IP@CD 500 prplene glycol so parafin oil 200 trihydroxystearate 10 cetyl dimethicon copolyol 30 water qs ad 1000 phosphate buffer pH 7A qs pH 7,4 j00730]The cream-type formulation is prepared by solubilizing lixivaptan with propylene glycol and this solution is added to a suspension of HP@CD in water. A second system is prepared by mixing paraffin oil, trihydroxystearate and cetyl dirmethicon copolyol with warming to 60 'C. Upon cooling to room temperature, the lipid system is mixed with the aqueous phase in a homogenizer for 30 minutes.

Example 38 Preparation of a Cyclodextrin-containing Thermoreversible Gel 2.5% VP2 Autagonist Formulation Ingredient Quantity (mg/g of ______________________ formulation) 5% CD solution 500.0 methylparaben 1.0 Poloxamer 407 180.0 TRIS -HClbuffer (0.1 M) 317,0

[00731] The Poloxamer 407 (BASF Corp.) is suspended in the TRIS H10 buffer (0 1M) and the componentsare mixed under agitation overnight at 4 °C to ensure complete dissolution. The cyclodextrin solution from Example 4 and methylparaben is added and further stirring allowed until complete dissolution is observed. The mixture ismaintained below room temperature until use. I5 Example 39- Preparation of a Cyclodextrin-containing Mucoadhesive, Thermoreversible Gel VP2 Antagonist Formulation Ingredient Quantity (mg/g of ________ _______________ formulation) 5% CD solution 500.0 methylparaben 1.0 Poloxamer 407 180.0 L Carb11 934P 2.0 R C1buffer (0. 1 M) 317.0 1007321The Carbopol 934P and Poloxamer 407 (BASF Corp.) is suspendedin the TRIS ICl buffer (0. 1 M) and the components are mixed under agitation overnight at 4 °C to ensure complete dissolution, The cyclodextrin solution front Example 32 and methylparaben is added and further stirring allowed until complete dissolution is observed. The mixture is maintained below room temperature until use. Examples 40 100733]A cyclodextrin containing thermoreversible gel formulation comprising 2.5% KCNQ modulator flupirtine is prepared using a procedure similar to the procedure in Example 38.

Example 41 -Preparation of a Gel VP2 Antagonist Formulation

f formulation) SR-121463 2(0( chitosan 2040 ilycerophosphate disodium 80.0 water 880

[007341A 5 ml solution of acetic acid is titrated to a pH of about 40 The chitosan is added to achieve a pH of about 5.5. The SR-121463 is then dissolved in the chitosan solution. This solution is sterilized by filtration. A 5 mL aqueous solution of glycerophosphate disodium is also prepared and sterilized. The two solutions are mixed and within 2 hat 37° Cthe desired gel is formed. Examples42 - 49

[007351Gel formulations comprising vestipitant, gabapentin, thalidomide, carbanazepine gentamicin, SRT-2183 and P2X modulator A-317491 are prepared using a procedure similar to the procedure in Example 41

Example Prparation ofa GelLposome VP? Antagonist Formulation ingredIent Quantiy SR-121463 20.0in/ Liposomes 15 umol/mL Chitosan-Glyerop osphate 1000mgg

[007361The liposomes are prepared in the presence of the VP2 antagonist SR-I21463 by the reversed-phase evaporation method, where lipids in chloroform or chloroform-methanol (2:1, v/v) are deposited on the sides of a tube by evaporation of the organic solvent. The lipid film is redissolved in diethyl ether and the aqueous phase (pH 7.4 300 mOsm/kg) containing 20mM lepes and 144 mI NaCl is added. The mixture is sonicated to obtain a homogeneous emulsion, and then the organic solventis removed under vacuum. The preparation is extruded to obtain the required liposome size and free components removed by size-exclusion chromatography using a Sephadex G 50 column (Amersham Phanmacia Biotech, Uppsala, Sweden).

[007371>o prepare the chitosan-giyerophosphateformulation, a 5 ml. solution of acetic acid is titrated to a pH of about 4.0, The chitosan is added toachievea p-I of about 5.5.This solution is sterilized by filtration. A 5 mL aqueous solution of glycerophosphate disodium is also prepared and sterilized, The two solutions are mixed and within 2 h at 37° C, and the desired gel is formed. The chitosan-glycerophosphate solution is gently mixed with the liposomes at room temperature.

Example 51 Preparation of aKCNO Modulator Nanoparticle Formulation

[007381750 mg (15 mg/mL theoretical) of a diblock copolyner consisting of the combination of a poly(d,i-lactic acid) of mass 30 kD and of a polyethylene glycol ofmass 2 kD (PLAPEG)and 250 mg (5 mg/mL theoretical) of flupirtine is dissolved in 20 mL of ethyl acetate (solution A). 175 mg of lecithin E80 and 90 mg of sodium oleate is dispersed in 50 nL of 5% w/v glucose solution (solution B). Solution A is emulsified in solution B with an Ultra-turrax stirrer and the pre-emulsion is then introduced into a Microfluidizer 110 S;RTM. type homogenizer for 10 minutes at 10 °C The volume of emulsion recovered is about 70nL (70 g). The ethyl acetate is removed using a rotary evaporator at reduced pressure (100 mm of mercury) to a suspension volume of about 45 mL (45 g).

Example 52. Preparationof a Mucoadhiesive, Thermoreversible Gel AL-15469A/AL-38905 Formulation Ingredient Quantity(mg/gof formulation) AL-15469A 25.5 AL-38905 25,5 methylparaben 2.55 Hyvpromellose 25.5 Carbopol 934P 5.1 Poloxamer 407 459 TRIS HCI buffer (0.1 M) 2006.85

[00739]Both AL-5469A and AL-38905 aresupplied as solids. They are rehydrated in water to a final molarity of10mM. 1007401A 10-g batch of mucoadhesive gel formulation containing 1.0% of AL-15469A and 1% of AL-38905 is prepared by first suspending Poloxamer 407 (BASF Corp.) and Carbopol 934P inITRIS HCl buffer (0.1 M), The Poloxamer 407, Carbopol 934Pand TRIS are mixed under agitation overnight at 4 °C to ensure completedissolution of the Poloxamer 407 and Carbopol 934P in the TRIS.'The hypromelloseniethylparaben and additional TRISHClbuffer(0.1 M)is added. The composition is stirred until dissolution is observed. TheAL-15469Aand AL-38905 solutions are added and the composition is mixed until a homogenous gel is produced. The mixture is maintained below room temperature until use.

Example 53 - Preparation of a Hydrogel-based Vestipitant/Paroxitene Formulation Ingredient Quantity (mg/g of formulation) Vestipitant 10,0 Paroxetine 10.0 paraffin oil 200.0 trihydroxystearate 10.0 cetyl dimethicon copolyol 30.0 water qs ad 1000 phosphate buffer pH 74 qs pP 7.4

[00741]Both Vestipitant and Paroxitene are supplied as solids. A solution of Vestipitant is prepared by gently mixing Vestipitant with water until it is dissolved. A solution of Paroxitene is prepared by gently mixing Paroxitene with water-until it is dissolved.

[00742) Then, the oil base is prepared by mixing paraffin oil, trihydroxystearate and cetyl dimethicon copolyol at temperatures up to 60 °C The oil base is cooled to room temperature and the Vestipitant and Paroxitene solutions are added. The two phases are mixed until a homogenous monophasic hydrogel is forced.

Example 54 -Preparation of Liposomal JB004/A Modulator Formation hIgredient Quantity (mg/gof cream') JB004/A 2m soya lecithin 100.0 cholesterol 10.0 tetraglycol 50.0 dAnethlisosorbide 25,0 methylparaben 1.0 propiylparaben U --- BHT 0.05 sodium chloride 0,5 HPMC 7.5 sodium hydroxide 0.3 citric acid .5 purified water, USP 302.55 100743]Heat the soya lecithin, tetraglycol and dimethyl isosorbide to about 70-75 °C. Dissolve the JB004/A, cholesterol and butylated hydroxytoluene in the heated mixture. Stir until complete dissolution is obtained.Heat about one third. of the water to 80-95 °C in a separate vessel and dissolve the preservatives methylparaben and propylparaben in the heated water while staining Allow the solution to cool to about 25 °C and then add the disodium edetate, sodium chloride, sodium hydroxide and citric acid. Add the remainder of the water and stir to obtain a complete solution. Transfer the organic mixture into the aqueous mixture by means of a vacuum, while homogenizing the combination with a high-shear mixer until a homogeneous product is obtained Add the hydroxypropyl methylcellulose into the biphasic mixture by means ofavacumn while homogenizing with a mixer. The homogenized is a Silverson high-shear mixer operating at approximately 3000 rpm. Single bilayered liposomes are forned. The white lipogel cream is ready for use,

Examples 55 - 56

[00744]Liposomal preparations ofAMN082, KCNQ modulatorretigabine are prepared usinga procedure similar to the procedure in Example 54.

Example 57::-Cotrolled/Immediate Release Antimicrobial Formulation Ingredient Quantity(mg'g of formulation) PLA Microspheres comprising 15 -30% Benzathine penicillin G

Propylene Glycol 30 Glycerin 20 Methylcellulose 20 (METHOCEL@ A4M) Benrzathine penicillin G 10 Water s ad 1000

1007451PLA (poly(L-lactide)) microspheres comprising benzathine penicillin G are prepared by adding sufficient PLA to 100 mL dichloromethane to produce a 3% wt/vol solution. 1.29 g benzathine penicillin G is added to the solution with mixing. The solution is then added dropwise to 2 L distilled water containing 0.5% wt/vol poly(vinyl alcohol) with stirring to produce an oil/water emulsion. Stirring is continued for a sufficient period to allow evaporation of the dichloromethane and the formation of solid microspheres, Microspheres are filtered, washed with distilled water, and dried until no weight loss is observed

1007461The immediate release portion of the formulation is prepared by generating a 2% methyleellulose solution in a water/propylene glycol/glycerin solvent system under stirring. Benzathine penicillin G is added to the solution while stirring is continued to yield a1% benzathine penicillin G low-viscosity gel. The appropriate amount ofmicrospheres comprising benzathine penicillin G is then mixed with the low-viscosity gel to yield a combination controlled/innediate release benzathine penicillin G ofic formulation.

-ample58- Preparation of a Cvclosporine Thermoreversible Gel Formulation comprising a penetration enhancer Ingredient Quantity(mg/g of formulation) Cvclosporme 100 Sodin citrate 1.25 Sodium ascorbate 0.8 1yaluronidase P1120 10 Poloxamer 407 15 Water qs ad 1000 Phospha~te bfferft pH 4 , qs pHT74

[007471The liquid formulation is prepared by mixing micronized cyclosporine andhyaluronidase P1120 with a buffer to form a first solution, A second system is prepared by mixing poloxamer 407, sodium citrate, and sodium ascorbate in water with warming to 60 °C, The first solution is added to the second system and mixed well,

Example 59- Preparation of a SB656933 Thermoreversible Gel Formulation. compising a penetration enhancer Ingredient Quantity(1g/g of formulation)

SB656933 10.0 Sodium citrate 1.25 Sodium ascorbate 0.' Dodecyl maitoside 10 Poloxamer 407 15 Carboxymethyl cellulose 5 Water qs ad 1000 Phosphate buffer pH 7.4 qs H7.4

[007481The liquid formulation is prepared by mixing SB656933 and dodecyl maltoside with a buffer toform a first solution. A second system is prepared by mixing poloxamer 407, carboxymethyl cellulose, sodium citrate, and sodium ascorbate in water with warming to 60 °C, The first solution is added to the second system and mixed well. The solution is autoclaved at 120 C for 2 hours.

aimple 60lPreparation of a JB004/A Thermoreversible Gel Formulation for visualization Ingredient Quantity (mg/gof I formulation) JB004/A 10.0 Sodium citrate 1.25 Sodium ascorbate 0.8 Evans blue 2 Poloxamer 407 15 Carboxyrnethyl cellulose 5 Water qs ad 1000 Phosphate bufferpH 7.4 qs pH 7.4 -- I

[00749]The liquid formulation is prepared by mixing J1004/Aand Evans blue with a buffer to form a first solution, A second system is prepared by mixing poloxamer 407, carboxyethyl cellulose, sodium citrate. and sodium ascorbate in water with warming to 60 °C The first solution is added to the second system and mixed well. The solution is autoclaved at 120 ° for 2 hours.

ExampIe 61 Effect ofpH on degradation products for autoclaved 17% Voloxamer 407NF/2% otic acent in PBS buffer

[007501A stock solution of a 17% poloxamer 407/2% otic agent is prepared by dissolving 3514 mg of sodium chloride (Fisher Scientific), 302.1 mg of sodium phosphate dibasic anhydrous (Fisher Scientific),.122.1 mg of sodium phosphatemonobasic anhydrous (Fisher Scientific) and an appropriate amount of an otic agent with 793 g of sterilefiltered DI water. The solution is cooled down. in a ice chilled water bath and then 17.05g of poloxamer 407NF (SPECTRUM CHEMICALS) is sprinkled into the cold solution while mixing. The mixture is further mixed until the poloxamer is completely dissolved. The pH for this solution is measured.

[00751]17% poloxamer 407/ 2% otic agent in PBS p1 of 5.3. Take an aliquot (approximately 30mL)of the above solution and adjust the pH to 53 by the addition of 1 M HCL

[00752117% poloxamer 407/22% otic agent in FBS pH of 8.0. Take an aliquot (approximately 30nL) of the above stock solution and adjust the pF1 to 8.0 by the addition of 1 M NaOH. 1007531A PBS buffer (p-I7.3) is prepared by dissolving 805.5 mg of sodium chloride (Fisher Scientific), 606 ing of sodium phosphate dibasic anhydrous (Fisher Scientific), 247 rg of sodium phosphate monobasic anhydrous (Fisher Scientific),then QS to 200g with sterile filtered DI water, (007541 A 2% solution of an otic agent in PBS pH 7.3 is prepared by dissolving an appropriate amount of the otic agent in the PBS buffer and QS to 10 g with PBS buffer. 1007551One rL samples are individually placed in 3mL screw cap glass vials (with rubberlining) and closed tightly. The vials are placed ina Market Forge-sterilmatic autoclave (settings, slow liquids) and sterilized at 250T for 15 minutes. After the autoclave the samples are left to cool down to room temperatureand then placed in refrigerator The samples are homogenized bymixing the vials while cold.

[007561Appearance (e.g., discoloration and/or precipitation) is observed and recorded. HIPLC analysis is performed using an Agilent 1200 equipped with a Luna C]8(2) 3im, I1A, 250x46 mm column) using a 30-80 acetonitrile gradient (1 10min) of (water -acetonitrile mixture containing 0.05%TFA), for a totalrun of 15 minutes. Samples are diluted by taking 30pL of sample and dissolved with 1.5mL of a 1:1 acetonitrile water mixture. Purity of the otic agent in the autoclaved samples is recorded.

[007571In general the formulation should not have any individual impurity (e.g., degradation product of otic agent) of more than 2% and more preferably not more than one percent. in addition, the formulationshouldnot precipitate during storage or change in color after manufacturing and storage. 007581Formulations comprising VP2 antagonist lixivaptan, diazepam, methotrexate, amoxicillin, AMN082, SRT-501, Neramexane,1JB004/A, KCNQ modulator Retigabine, and tacrolinus, prepared according to the procedure in Example 61, are tested using the above procedure to determine the effect of pH on degradation during the autoclaving step. Exanie 62 Effect of autoclaving on the release profile and viscosity of a 17% poloxamer 407NF 2% otic agent in PBS.

[007591An aliquot of the samplefrom example 61 (autoclaved and not autoclaved) is evaluated for release profile and viscosity measurement to evaluate the impact of heat sterilization on the properties of the gel.

[007601Dissolution is performed at 37C insnapwells (6.5 mm diameter polycarbonate membrane with a pore size of 0.4 pm). 0.2 mL of gel is placed into snapwell and left to harden, then 0.5mL.is placed into reservoir and shaken using a Labline orbit shaker at 70 rpm. Samples are taken every hour (0.1 mL withdrawn and replace with warm buffer). Samples are analyzed for poloxamer concentration by UV at 624 nm using the cobaltthiocyanate method, against an external calibration standard curve. In brief"20gL of the sample is mixed with 1980pL of a 15mM cobalt thiocyanate solution and absorbance measured at 625 nm, using a Evolution 160 U/Vis spectrophotometer (Thermo Scientific).

[007611 Thereleased otic agent is fitted to the Korsmeyer-Peppas equation

where Q is the amount of otic agent released at time ts Q, is the overall released amount of otic agent, k is a release constant of the nth order, n is a dimensionless number related to the dissolution mechanism and b is the axis intercept, characterizing the initial burst releasemechanism wherein n=1 characterizes an erosion controlled mechanism, Thetmean dissolution time (MDT) is the sum of different periods of time the drug molecules stay in the matrix before release, divided by the total number of molecules and is calculated by:

100762]Viscositymeasurements areperformed using a Brookfield viscometer RVDV-I1+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of0.31 s1), equipped with a waterjacketed temperature control unit (temperature ramped from 15-34°C at 6 °C/min). Tgel is defined as the inflection point of the curve where the increase in viscosity occurs due to the sot-gel transition. 100763]Formulations comprising VP2 antagonist lixivaptan, diazepam,nmethotrexate, amoxicillin, AIN082S SRT-501 Neramexane, JB004/A, KCNQ modulator Retigabine, and tacrolimus, prepared according to the procedure in Example 61, are tested using the above procedure to determine the effect of autoclaving on the release profile, Tgel and viscosity of the formulations, Example63 Effect of addition of a secondary pioymer on the degradation products and viscosity of a formulation containing 2%otic aaent and 17% voloxamer 407NF after heat sterilization (autoeCaIYg.h

[007641Solution A. A solution of pH 7.0 comprising sodium carboxymethylcellulose (CMC) in PBS buffer is prepared by dissolving 178.35 mg of sodium chloride (Fisher Scientific), 300.5 mg of sodium phosphate dibasic anhydrous (Fisher Scientific), 126.6 mg of sodium phosphate monobasic anhydrous (Fisher Scientific) dissolved with 78.4 of sterile filtered DI water, then 1 g of Blanose 7M65 CMC (Hercules, viscosity of 5450cP @ 2%) is sprinkled into the buffer solution and heated to aid dissolution, and the solution is then cooled down. 1007651A solutionof pH 7.0 comprising 17% poloxamer 407NF/l% CMC/2% otic agent in PBS buffer is trade by cooling down S 1g of solution A in a ice chilled water bath and then adding an appropriate amount of an otic agent followed by mixing, 1.74g ofpoloxamer 407NF (Spectrum Chemicals ) is sprinkled into the cold solution while mixing. The mixture is furthermixed until all the poloxamer is completely dissolved.

1007661Two mL of the above sample is placed in a 3rL screw cap glass vial (with rubber lining) and closed tightly. The vial is placed in a Market Forgesterilmatic autoclave (settings, slow liquids) and sterilized at 250°F for2$ minutes. After autoclaving the samples left to cool down to room temperature and then placed in refrigerator. The sample is homogenized by mixing while the vials are cold, t00767]Precipitation or discoloration are observed after autoclaving. HPLC analysis is performed using an Agilent 1200 equipped with a Luna Cl8(2) 3pm, 100A, 250x4.6 mm column) using a 30 80 acetonitrile gradient (1-10min) of (water -acetonitrile mixture containing 0.05%TFA), for a total run of 15 minutes. Samples are diluted by taking 30gL ofsample and dissolving with 1.5mL of a 1:1 acetonitrile water mixture, Purity of the otic agent in the autoclaved samples is recorded. 1007681Viscosity measurements are performed using a Brookfield viscometer RVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shearrate of0.31 s), equipped with a waterjacketed temperature control unit (temperature ramped from 15-34°C at 1 .6 C/mini). Tgel is defined as the inflection point of the curve where the increase in viscosity occurs due tothesol-gel transition 100769]Dissolution is performed at 37CC for the non-autoclaved sample in snapwells (6.5r m diameter polycarbonate membrane with a pore size of 0.4 n), 02 ml.. of gel is placed into snapwell and left to harden, then 0.5 ml is placed into reservoir and shaken using a Labline orbit shaker at 70 rpm Samples are taken every hour (0.1 mL withdrawn and replaced with warm buffer). Samples are analyzed for otic agent concentration by UV at 245 nm, against an external calbration standard curve.

[00770]Formulations comprising VP2 antagonist lixivaptan, diazepam, methotrexate, amoxicillin, AMN082. SRT-501 Neramexane, JB004A.KCNQ modulator Retigabine, and tacrolimus, prepared according to the procedure in Example 63, are tested using the above procedure to determine the effect addition of a secondary polymer on the degradation products and viscosity of a formulation containing 2% otic agent and 17% poloxamer 407NF after heat sterilization (autoclaving). Example 64 Effect ofbuffer type on the degradation products for formulations containing poloxamer 407NF after heat sterilization (autoclavingt f00771]A TRIS buffer is made by dissolving 377.8 mg of sodium chloride (Fisher Scientific), and 602.9mg of Tromethamine (Sigma Chemical Co.) then QS to IOg with sterile filtered DI water, pI is adjusted to 7.4 with IM 14C Stock solution containing 25% Poloxamer 407 solution in TRIS buffer:

[00772]Weigh 45 g of TRIS buffer, child in an ice chilled bath then sprinkle into the bufferwhile mixing, 15 of poloxamer 407 NF (Spectrum Chemicals). The mixture is father mixed until all the poloxameris completely dissolved.

1007731 A series of formulations is prepared with the above stock solution. An appropriate amount of otic agent (orsalt or prodrug thereof) and/or otic agents microized/coated/liposomal particles (or salt or prodrug thereof) is used for all experiments, Stock solution (p173) containing 25% Poloxamer 407 solution in PBS buffer: f007741PBS buffer fromexample 61 is used. Dissolve 704mg of sodium chloride (Fisher Scientific), 601.2 ing of sodium phosphate dibasic anhydrous (Fisher Scientific), 2427 mg of sodium phosphate monobasic anhydrous (Fisher Scientific) with 140.4 g of sterile filtered DI water. The solution is cooled down in an ice chilled water bath and then 50g of poloxamer 407NF (SPECTRUM CHEMICALS) is sprinkled into the cold solution while mixing, The mixture is further mixed until the poloxamer is completely dissolved

[007751A series of formulations is prepared with theabove stock solution.Anappropriteamount of otic agent (or saltor prodnig thereof)and/or otic agent as micronized/coated/liposomal particles (or salt or prodrug thereof) is used for all experiments.

[00776] Tables 1 and 2 list samples prepared using the procedures described in Example 64. An I4 appropriate amount of otic agent is added to each sample to provide a final concentration of 2% otic agent in the sample. Table L Preparation of samples containing TRIS buffer

Sample PH 25% Stock TRIS Buffer Solution(g) (g). 20%P407/2 otic agent/TRIS 7.45 8.01 1 82 18%P407/2 oticagent/TRIS 7.45 7.22 2.61 16%P407/2 otic agent/RIS 745 6.47 3.42 18%P4072 otic agent/TRTS 7.4 7 18 2.64 4% otic agent/iRIS 7,5 9.7 2% otic agent/TRIS 7,43 5 1% otic agent TRS 7-35 5 2% otic agent /TRIS suspension ) 74 4.9

Table 2. Preparation of samples containing PBS buffer (p of 7.3)

Sample 25% Stock Solution PBS Buffer (g) in PBS (g) - - 0%P407/2 otic agent'PBS 8.03 1 82 1S%P407/2 otic agentIBS 7.1 2.63 16%P407/2 otic agent /PBS 6,45 3 44 18%P407/2 otie agentP PBS - 2163 2%tic agent PBS 9

1007771OTe mL samples are individually placed in 3mL screw cap glass vials (with rubber lining) and closed tightly. The vials are placed in a Market Forge-sterimatic autoclave (setting, slow liquids) and sterilized at 250°F for 25 minutes. After the autoclaving the samples are left to cool down to room temperature. The vials are placed in the refrigerator and mixed while cold to homgenize the samples.

[007781HPLC analysis is performed using an Agilent 1200 equipped with aLuna C18(2) 3m. 100A, 250x4.6 mm column) using a 30-80 acetonitrile gradient (1-10min) of (water -acetonitrile mixture containing 0:05%TFA), fora total run of 15 minutes Samples are diluted by taking 30uL of sample and dissolving with 1.5mL of a 1:1 acetonitrile water mixture. Purity of the otic agent in the autoclaved smplesisrecorded.Thestabilityofformulations inTRIS and PBS buffers is compared.

[007791Viscosity measurements are performed using a Brookfield viscometer RVDV-4P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31 s-), equipped with a water jacketed temperature control unit (temperature ramped from 15-34'C at 1 6 °C/min), Tgelis defined as the inflection point of the curve where the increase in viscosity occurs due to the sol-gel transition. Only formulations that show no changeafterautoclaving areanalyzed.

[007801Formulations comprising VP2 antagonist fixivaptan, diazepam, methotrexate, amoxicillin, AMN082,SRT-501, Neramexane. JB004/A, KCNQ modulator Retigabine, and tacrolimus, prepared according to the procedure in Example 64, are tested using the above procedure to detemine the effect addition of a secondary polymer on the degradation products and viscosity of a formulation containing 2% otie agent and 17% poloxaner 407NF after heat sterilization (autoclaving). 1007811Formulations comprising micronized VP2 antagonist lixivaptan, diazepam, methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A. KCNQ modulator Retigabine, and tacrolimus, prepared according to the procedure in Example 64, are subjected to autoclaving Stability of formulations containing micronized otic agent is compared to solution counterparts. Exampile65: Pulsed release otic formulations

[007821A combination of neramexane and neramexane hydrochloride (ratio of 1:1) is used to prepare a pulsed release otic agent formulation using the procedures described herein. 20% of the delivered dose of neramexane is solubilized in a 17% poloxamer solution of example 61 with the aid of beta-cyclodextrins, The remaining 80% of the otic agent is then added to the mixture and the final formulation is prepared using any procedure described herein.

[00783]Pulsed release fonulations comprising VP2 antagonist lixivaptan, diazepam, methorexate, amoxicillin AMN082, SRT-50I, JB004/A. KCNQ modulator Retigabine, and tacrolimus prepared according to the procedures and examples described herein are tested using procedures described herein to determine pulse release profiles. Example 66 Preparation of a 17%poloxamer 407/2% otic avent/78 pum Evans blue in PBS

[00784] A Stock solution of Evans Blue (5.9mg/niL) in PBS buffer is prepared by dissolving 5.9mg of Evans Blue (Sigma Chemical Co) with I mL of PBS buffer (from example 61).

[00785] A Stock solution containing 25% Poloxamer 407 solution in PBS buffer from example 64 is used in this study, An appropriate amount of an otic agent is added to the stock solution from example 64 to prepare formulations comprising 2% of an otic agent (Table 3). Table 3. Preparation of poloxamer 407 samples containing Evans Blue __ ___ ___ ------- ------- Sample ID 25% P4Oin PBS Buffer (g) Evans Blue PBS (g) Solution (pt) 17%P407/2 oticagent /EB 13.6 6 265 20%P407/2 otic agent/EB 16,019 3.62 265 25%P407/2 otic agent/EB 19.63 265

[007861Formulations comprising VP2 antagonist lixivaptan, diazepan, methotrexate, amoxicillin, AMN082, SRT501, Neramexane, JB004/A, KCNQ modulator Retigabine, and tacrolimus are prepared according to the procedure in Example 66 and are sterile filtered through 0.22tm PVDF syringe filters (Millipore corporation), and autoclaved. 00787 The above formulations are dosed to guinea pigs inthe middle ear by procedures described herein and theability of formulations to gel upon contact and the location of the gel is identified after dosingand at 24 hours after dosing. Example67: Terminal sterilization of poloxamer 407 formulations with and without a visualization d

[00788117% poloxamer407/ 2% otic agent/ in phosphate buffer, pH 7.3: Dissolve 709mg of sodium chloride (Fisher Scientific), 742 mgof sodium phosphate dibasic dehydrate USP (Fisher Scientific), 251.1 mg of sodium phosphate monobasic monohydrate USP (Fisher Scientific)andan appropriate amount of an otic agent with 158.1 g of sterile filtered DI water. The solution is cooled down in an ice chilledwater bath and then 34.13g of poloxamer 407NF (Spectrum chemicals) is sprinkled into the cold solution while mixing.The mixture is further mixed until the poloxamer is completely dissolved.

[00789}17% poloxamer407/ 2% otic agent/ 59ppm Evans blue in phosphate buffer: Take two mL of the 17% poloxamer4o7/ 2% otic agent/ in phosphate buffer solution and add 2mL of a 5.9 ng/L Evans blue (Sigma-Aldrich chemical Co) solution in PBS buffer. 100790125% poloxamer407/ 2% otic agent/ in phosphate buffer: Dissolve 330.5mg of sodium chloride (Fisher Scientific), 334.5 mg of sodium phosphate dibasic dehydrate USP (Fisher Scientific), 125.9mg of sodium phosphate monobasic monohydrate USP (Fisher Scientific)and an appropriate amount of an otic agent with 70.5 g of sterile filtered DI water. 100791]The solution is cooled down in an ice chilled water bath and then 25. g of poloxaner 407NF (Spectrum chemicals) issprinkled into the cold solution while mixing. The mixture is further mixed until the poloxamer is completely dissolved.

[00792125% poloxamer407/ 2% otic agent/59ppm Evans blue in phosphate buffer: Take two mL of the 25% poloxamer407/2% otic agent/ in phosphate buffer solution and add 2 mL of a 5.9 mg/nl. Evans blue (Sigma-Aldrich chemical Co) solution in PBS buffer. 1007931Place 2mL. of frnnulation into a 2mL glass vial (Wheaton serum glass vial)and seal with 13 mm butyl str (kimble stoppers) and crimp with a 13 mmaluminuin seal.The vialsare placed in a Market Forge-sterilmatic autoclave (settings, slow liquids) and sterilized at 250'F for 25 minutes After the autoclaving the samples are left to cool down to room temperature and then placed in refrigeration. The vials are placed in the refrigerator and mixed while cold to honogenize the samples. Sample discoloration or precipitation after autoclaving is recorded

[007941 HPLC analysis is performed using an Agilent 1200 equipped with a Luna Cl8(2) 3ym. 1OA, 250x4.6 mm column) using a 30-95 methanol:acetate buffer pH 4 gradient (I-6min), then isocratic for 11 minutes, for a total run of 22 minutes. Samples are diluted by taking 30pL of sample and dissolved with 0.97mL of water. The main peaks are recorded in the table below. Purity before autoclaving is always greater than. 99% using this method.

[00795]Viscosity measurements are performed using a Brookfield visconeter RVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31 s'), equipped with a waterjacketed temperature control unit (temperature ramped from 15-34C at 1.6 C/min). Tgel is defined as the inflection point of the curve where the increase in viscosity occurs due to the sol-gel transition

[00796jFormulations comprising VP2 antagonist lixivaptan, diazepam, methotrexate, amoxicillin, AMN082,SRT-501 Neramexane, 1B004/A, KCNQ modulator Retigabine, and tacrolimus prepared according to the procedure in Example 67, are tested using the above procedures to determine stability of theformtulations. Example 68: In vito comparison of release profile.

[007971Dissolution is performed at 37C in snapwells (6.5 mm diameter polycarbonate membrane with a pore size of 0.4 i) 0.2mL of a gel formulation described herein is placedinto snapwell and left to harden, then 0.5 ml buffer is placed into reservoir and shaken using a Labline orbit shaker at 70 rpm. Samples are taken ever' hour (0.1 mL withdrawn and replace with warm buffer). Samples are analyzed for otic agent concentration by UV at 245nm against an external calibrationstandard curve. Pluronic concentration is analyzed at 624 nm using the cobalt thioyanate method. Relative rank-order of mean dissolution time (MiT) as a function of %P407 is determined.. A linear relationship between the formulations mean dissolution time (MDT) and theP407 concentration indicates that the otic agent is released due to the erosion of the polymer gel (poloxamer) and not via diffusion, A non-linear relationship indicates release of otic agent via a combination of diffusion and/or polymer gel degradation.

[007981Alternatively, samples are analyzed using the method described by Li Xin-Yu paper [Acta Pharnaceutica Sinica 200843(2):208-203] and Rank-order of mean dissolution time (MDT) as a function of %P407 is determined,

[007991Formulations comprising VP2 antagonist lixivaptan, diazepamn, methotrexate, amoxicillin, AMNO82, SRT-501, Neramexane, JB004/A,KCNQ modulator Retigabine, and tacrolimus.prepared according to the procedures described herein, are tested using the above procedure to determine the release profile of the otic agents. Example 69; In vitro comarison of gelationtemerature 1008001The effect of Poloxamer 188 and an otic agent on the gelation temperature and viscosity of Poloxamer 407 formulations is evaluated with the purpose ofmanipulating the the gelation temperature.

[008011A 25% Poloxamer 407 stock solution in PBS buffer and the PBS solution from example 64 are used. Poloxamer I88NF from BASFis used. An appropriate amount of otic agent is added to the solutions described in Table 4 to provide a 2% formulation of the otic agent. Table 4 Preparation of samples containing poloxamer 407/poloxamer 188 Sample 25%P407 Stock Poloxamer 188 PBS Buffer Solution (g) (mg) (g) 16%P407/10%PI88 3.207 501 1.3036 i7%P407/l0%P188 3,4089 500 1.1056 18%P407/10%P188 3.6156 502 0.9072 19%P407/10%P188 3. 8183 500 07050 20%P407/10%PI88 4.008 501 0.5032 20%P407/5%P188 4.01 256 0.770

[00802JMeandissolution time, viscosity and gel temperature of the above formulationsare measured using procedures described herein.

[008031] An equation is fitted to the data obtained and can be utilized to estimate the gelatin temperature of F127/F68 mixtures (for 17-20% F127 and 0-10% F68).

T,=-1.8( F 27)+1.3(%F68)+53 1008041An equation is fitted to the data obtained and can be utilized to estimate the Mean Dissolution Time (hr) based on the gelation temperature of Fl27/F68 mixtures (for 17-25% F27 and 0-10% F68). using results obtained in example 67 and 69 MDT = -0.2 -)+ 8

[008051Formulations comprising VP2 antagonist lixivaptan, diazepam, methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A KCNQ modulator Retigabine, and tacrolimus are prepared by addition of an appropriate amount of otic agents to the solutions described in Table 4 The gel temeparatureof the formulations is determined using the procedure described above. Example 70: Determination of temperature range for sterile filtration

[008061The viscosity at low temperatures is measured to help guide the temperature range at which the sterile filtration needs to occur to reduce the possibility of clogging.

[008071Viscosity measurements are performed using a Brookfield viscometer RVDV-1I+P with a CPE-40 spindle rotated at 1, 5 and 10 rpm (shear rate of 7.5, 37.5 and 75 s-), equipped with a water jacketed temperature control unit (temperature ramped from 0-25° at 1.6C/min),

[00808] The Tgel of a 17% Pluronic P407 is determined as a function of increasing concentration of otic agent. The increase in Tgel for a 17%pluronic formulation is estimated by: ATgff0.93[% otic agent] 100809]Formulations comprising VP2 antagonist lixivaptan, diazepan, methotrexate, amoxicillin, AMN082, SRT501, Neranexane, JB004/A, KCNQ modulator Retigabine, and tacrolimus, prepared according to procedures described herein, are tested using the above procedure to determine the temperature range for sterile filtration. The effect of addition of increased amounts of otic agent on the Tgel, and the apparent viscosity of the formulations is recorded. Example71: Determination ofmanufacturing conditions

Table 5. Viscosity of potential formulations at manufacturing / filtration conditions. Apparent Viscosiy (cP) Sample 5°C below Tgel 20°C Temperature (@1QOCP Placebo 52 cP @17°C 120 cP 19C 17%P407/2% otic 90 cP @iI8°C 147 185°C agent

17%P407/6% otic 142 cP @ 22°C 105 cP 19.7°C agent Viscosity measured at a shearrate of 37.5 s 10081.01An 8 liter batch of a 17% P407 placebo ismanufactured to evaluate the manufacturing/filtration conditions. The placebo is manufactured by placing 6.4 liters of DI water i a 3 gallon SS pressure vessel, and left to cool down in the refrigerator overnight. The following morning the tank was taken out (water temperature 5°C, RT 18°C) and 48g of sodium chloride, 29.6 g of sodiumphosphate dibasic dehydrate and 10 g of sodium phosphate monobasicmonohydrate is addedand dissolved with anoverhead mixer (IKA RW20O@41720 rpm) Half hour later, once the buffer is dissolved (solution temperature 8°C, RT I8°C) ,1.36kg of poloxamer 407 NF (spectrum chemicals) is slowly sprinkled into the buffersolution in a 15 minute interval (solution temperature

12C, RT 18°C), then speed is increased to 2430 rpm. After anadditional one hour mixing, mixing speed is reduced to 1062 rpm (complete dissolution).

[00811 The temperature of the room is maintained below 25°C to retain the temperature of the solution at below 19°C, The temperature of the solution is maintained at below 19'C up to 3 hours of the initiation of the manufacturing, without the need to chill/cool the container.

[008121Three different Sartoscale (Sartorius Stedim) filters with a surface area of 17.3 cm'are evaluated at 20 psi and 14'0 of solution 1) Sartopore2,0.2tm5445307HlS-FF(PES), flowrateofI6nL/mmi

2) Sartobran P, 0.2gm 5235307HS-FF (cellulose ester), flow rate of 12mL/min

3) Sartopore2XLI, 02um 54453071S-FF (PES), flow rate of 15mLnmin

100813]Sartopore 2 filter 5441307H4-SS is used, filtration is carried out at the solution temperature using a 0.450.2pm Sartopore 2 150sterile capsule (Sartorius Stedim) with a surface area of 0.015m2 at a pressure of 16psiFlow rate is measured at approximately 100 mL/min at 1 6 psi, with no change in flow rate while the temperature is maintained in the 6.5-14°C range. Decreasing pressure and increasing temperature of the solution causes a decrease in flow rate due to an increase in the viscosity of thesolution. Discoloration of thesolution is monitored during the process. Table 6. Predicted filtration time for a 17%poloxamer 407 placebo at a solution temperature range of 6.5-140C using Sartopore 2, 0.2pim filters at a pressure of 16 psi of pressure. Filter Size (M) Estimated flow rate Time to filter 81 ( /n(estimated) Sartopore 2, size 4 0,015 100 mL/min 80 Min Sartopore 2, size 7 0.05 330 mLImin 24 min Sartopore2, size 8 0.1 670 mL/min 12

008141ViscosityTgel and UNVis absorption is check before filtration evaluation. Pluronic UV/Vis spectra are obtained by a Evolution 160 UVNis (Thermo Scientific). A peak in the range of 250-300 nm is attributed to BHT stabilizer present in the raw material (poloxamer) Table 7lists physicochemical properties of the above solutions before and after filtration. Table 7. Physicochemical properties of 17% poloxamer 407 placebo solution before and after filtration Sample Tgel (°C) Viscosity @ 19°C Absorbance @ 274 nm (cP) Before filtration 22 100 0.3181 After filtration 22 100 0.3081 Viscosity measured a shear rate of37.5 s

[008151The above process is applicable for manufacture of 17% P407 formulations, and includes temperature analysis of the room conditions. Preferably, a maxinium temperature of 19°C reduces cost of cooling the container during manufacturing.In some instances, a jacketed container is used to further control the temperature of the solution to ease manufacturing concerns. Example72in vitro Release of otic agent from an autoclavedimicronized samIle

[00816117%poloxamer 407/1.5% otic agent in TRIS buffer: 250:8 ng of sodium chloride (Fisher Scientific), and 302.4mg of Tromethamine (Sigma Chenmical Co.) is dissolved in 39.3g ofsterile filtered DI water, pH is adjusted to 7.4 with IM HC, 4.9 g of theabove solution is used and an appropriate amount of micronized otic agent is suspended and dispersed well. 2 niL of the formulation is transferred into a 2 ml glass vial (Wheaton serum glass vial) and sealed with 13 mm butyl styrene (kimble stoppers) and crimped with a 13 mm aluminum seal.. The vial is placed in a Market Forge-sterilmatic autoclave (settings, slow liquids) and sterilized at 250°F for 25 minutes. After the autoclaving the sample is left to cool down toroom temperature. The vial is placed in the refrigerator and mixed while cold to homogenize the sample. Sample discoloration or precipitation after autoclaving is recorded.

[008171Dissolution is performed at 37C in snapwells (6,5 mm diameter polycarbonate membrane with a pore size of 0.4m), 0.2 mL of gel is placed into snapwel and left to harden, then 05 ml PBS buffer is placed into reservoir and shaken using aLabline orbit shakerat 70 rpm. Samples are taken every hour [0.1 mL withdrawn and replaced with warm PBS buffer containing 2% PEG-40 hydrogenated castor oil (BASF) to enhance otic agent solubility). Samples are analyzed for otic agent concentration by UV at 245nm againstan external calibration standard curve. The release rate is compared to other formulations disclosed herein.-MDT time is calculated for each sample.

[00818] Solubilization of otic agent in the 17% poloxamer system is evaluated by measuring the concentration of the otic agent in thesupematant after centrifuging samples at 15,000 rpm for 10 minutes using an eppendorf centrifuge 5424. Otic agent concentration in the supernatantis measured by UV at 245nm against an external calibration standard curve. 100819]Fonnulations comprising micronized ofic agents VP2 antagonist lixivaptan, diazepam, methotrexate, amoxicillin, AMN082, SRT-501, Neramexane, JB004/A, KCNQ modulator Retigabine, and tacrolinus, prepared according to the procedures described herein, are tested using the above procedures to determine release rate of the otic agent from each formulation. Example 73 Release rate or MDT and viscosity offormulation containing sodnn carboxymethyl cellulose. 100820117% poloxamer 407/2% otic agent/1% CMC (Hercules Blanose 7M): A sodium carboxymethyleellulose (CMC) solution (pH 7.0) in PBS buffer is prepared by dissolving 205.6 mg of sodium chloride (Fisher Scientific), 372.1 mg of sodium phosphate dibasic dihydrate (Fisher Scientific), 106.2 mg of sodiur phosphate monobasic monohydrate (Fisher Scientific) in 78.1 of sterile filtered DI water. I g of Blanose 7M CMC (Hercules, viscosity of533cP @ 2%) is sprinkled into the buffer solution and heated to ease solution, solution is then cooled down and 17.08 g poloxamer 407NF (Spectrun Chemicals ) is sprinkled into the cold solution while mixing.A foniulation comprising 17% poloxamer 407NT/l% CMC/2% otic agent in PBS buffer is made adding/dissolving an appropriate amount of otic agent to 9.8 g of the above solutionand mixing until all the oticagent is completely dissolved.

[00821117% poloxamer 407/2% otic agent/0.5% CMC (Blanose 7M65): A sodium carboxymethyleellulose (CMC) solution (pH7.2) in PBS buffer is prepared by dissolving 257 ng of sodium chloride (Fisher Scientific) 375 mg of sodium phosphate dibasic dihydrate (Fisher Scientific) 108 nig of sodium phosphate monobasic monohydrate (Fisher Scientific) in 78.7g of sterilefiltered DI water. 0.502 g of Blanose 7M65 CMC (Herculesviscosity of 5450cP @ 2%) is sprinkled ito the buffer solution and heated to ease solution, solution is then cooled down and 17,06 g poloxamer 407NF (Spectrum Chemicals ) is sprinkled into the cold solution while mixing. A 17% poloxamer 407NF/l% CMC/2% otic agent solution in PBS buffer is made adding/dissolving an appropriate amount of otic agent to 9,8 g of the above solution, andmixing until the otic agent is completely dissolved. 100822]17% poloxamer 407/2% otic agent/0.5% CMC (Blanose 7119): A sodium carboxymethylellulose (CMC)solution (pH73) in PBS buffer is prepared by dissolving 256.5 mg of sodium chloride (Fisher Scientific), 374rmg of sodium phosphate dibasic dihydrate (Fisher Scientific), 107 mg of sodium phosphate monobasic monohydrate(Fisher Scientific) in 78.6g of sterilefiltered Dxwater, then 0.502 g of Blanose 7H9 CMC (Hercules, viscosity of 5600cPl I%) is sprinkled to the buffer solution and heated to ease solution, solution is then cooled down and 17,03 g poloxamer 407NF (Spectrum Chemicals) is sprinkled into the cold solution while mixing. A 17% poloxaner 407NF/l% CMC/2% otic agent solution in PBS buffer ismade adding/dissolving an appropriate amount of otic agent to 9.8 of the above solution, and mixing until the otic agent is completely dissolved.

[00823]Viscosity measurements are performed using a Brookfield viscometer RVDV-HP with a CPE40 spindle rotated at 0.08rpm (shear rate of 06s), equipped with a water jacketed temperature controlunit (temperature rampedfrom 10-34C at 1.6"C/min). Tgel is defined as the inflection point of the curvewhere the increase in viscosity occurs due to the sol-gel transition.

[00824]Dissolution is performed at 37C in snavpwells (6.5 mm diameter polycarbonate membrane with a pore size of 0.4 im) 0.2 nL of gel is placed into snapwell and left to harden, then 0.5 mL PBS buffer is placed into reservoir and shaken using a Labline orbit shaker at 70 rpm. Samples are taken every hour, 0. 1 mL withdrawn and replaced with warmPBS buffer. Samples are analyzedfor otic agent concentration by UVat 245nn against an external calibration standard curve. The release rate is compared to the formulation disclosed in example 63,MDT time is calculated for each of the above formulations. 100825Formulations comprising VP2 antagonist lixivaptan, diazepam, methotrexate, amoxicillin, AMN082, SRT-501,NeramexaneJ B004/A, KCNQ modulator Reigabine, and tacrolimus, prepared according to procedures described above, are tested using the above procedures to determine relationship between releaserate and/or mean dissolution time and viscosity of fomulation containing sodium carboxymethyl cellulose. Any correlation between the mean dissolution time (MDT) and theapparent viscosity (measuredat 2°C below the gelation temperature) is recorded. Example 74 - Application of a Enhanced Viscosity Calcineurin Inhibitor Fornulation onto the Round Window Membrane

[00826]A formulation according to Example I is prepared and loaded into 5 nL siliconized glass syringes attached to a 15-gauge luer lock disposable needle. Lidocaine is topically applied tothe tympanic membrane, and a small incision made to allow visualization into the middle ear cavity. The needle tip is guided into place over the round window membrane, and the immunomodulator formulation applied directly onto the round-window membrane.

Examples 75 89 100827Enhanced Viscosity AL-15469A/AL-38905 Formulation of Example 52, cytotoxic agent methotrexate formulation of Example 4, AMN082 formulation of Example 13, Antimicrobial gentamicin formulation of Example 46, SR-50 ffrnulation of Example 16 are tested using a procedure similar to the procedure in Example 61

Emnle 90- Evaluation of a Calcineurin Inhibitor Formulation in an AIED Annial Model Methods and Materials Induction ofInmune Resonse

[008281Female albino National Institutes of HealthSwiss mice (Harlan Sprague-Dawley Inc. Indianapolis, ne.) weighing 20 to 24 g are used. Keyhole limpet hemocyanin (KLH; Pacific Biomarine Supply Co., Venice, CA) is suspended in phosphate-buffered saline (PBS) IpH 6.4), dialyzed aseptically against PBS and centrifuged twice. The precipitate (associatedKH11) is dissolved in PBS and injected subcutaneously in the back of the animal (0.2mg emulsified in Freund's complete adjuvant). The animals are given a booster (0.2 mg KLH in Freund's incomplete adjuvantand then injected ten weeks later with 0.1 mg KLH in 5 pl PBS (p116.4)through a microhole drilled through the cochlear capsule. The cochlea is approached using an operating microscope and sterile technique. A postauricular incision is made, and a hole is drilled into the bullae to allow good visualization of the promontory of the cochlear basal turn, stapedial artery, and

round window niche.The stapedial artery is cauterized and removed, and a 25 pm hole is drilled through the cochlear capsule into the scala tympani of the lateral basal tum. KLH or PBS control is slowly injected using a Hamilton syringe coupled with a plastic tube to a glass micapipette filled with the antigen or control The hole is sealed with bone wax after injection, and excess fluid is removed. Only one cochlea per animal is treated with KLH.

Treatment 1008291KLH and control mice are sorted into two groups (n = 10 in each group). Calcineurin inhibitorformulation of Example 11 containing tacrolirnus is applied to the round window membrane of one group of animals. Control formulation containing no tacrolimus is applied to the second group. The calcineurin inhibitor and control fornAations are reapplied three days after the initial application, The animals are sacrificed after the seventh day of treatment.

Analysis of Results ElectraphysiologicTesting 100830] The hearing threshold for the auditory brainsteni response threshold (ABR) to click stimuli for each ear of each animal isinitially measured and I week after the experimental procedure. The animals are placed in a single-walled acoustic booth (Industrial Acoustics Co, Bronx, NY, USA.) on a heating pad. Subdermal electrodes (Astro-Med Inc. Grass Instrument Division, West Warwick RI, USA) were inserted at the vertex (active electrode), the mastoid (reference), and the hind leg (ground). Click stimuli (0.1 millisecond) are computer generated and delivered to a Beyer DT 48, 200 Ohm speaker fitted with an ear speculum for placementin the external auditory meatus. The recorded ABR is amplified and digitized by a battery-operated preamplifier and input to a Tucker Davis Technologies ABR recording system that provides computer control of the stimulus, recording, and averaging functions (Tucker Davis Technology, Gainesville, FL, USA). Successively decreasing amplitude stimuli are presented in 5-dB steps to the animal, and the recorded stimulus locked activity is averaged (n=512) and displayed, Threshold is defined as the stimulus level between the record with no visibly detectable response and a clearly identifiable response.

Histochenicalanalysis 1008311Animals are anesthsized and sacrificed via intracardiac perfusion of heparinized warm saline followed by approximately 40 mL periodate-lysine-paraformaldehyde (4% paraformaldehyde final concentration) fixative. Right-side temproal bones are immediately removed and decalcified with buffered 5% ethylenediamine tetra-acetate (pH 72) for 14 days (4°C), After decalcification, temporal bones are immersedsequentially in increasing concentrations (50% 75%, 100%) of optimal cutting temperature (OCT) compound (Tissue-Tek, Miles Ine., Elkhart, IN), snap-frozen(

70 °C), and cryostat-sectioned (4 m) parallel to the modiolus. Sections are collected for hematoxylin and eosin (H&E) stainingand immunohistochemical analysis.

[008321The severity of inflanmationis assessed according to the amount of cellular infiltration of the scala tympani, and an unbiased score is given to each cochlea. A score of 0 indicates no inflanunation, and a score of 5 indicates that all cochlear turns had severe infiltrationof inflammatory cells. Examples 91-92 1008331Mucoadhesive thermoreversible gel formulation comprising Etanercept prepared. according to Example 26, mucoadhesive thermoreversible gel formulation comprising antimicrobial ganciclovir of example 15 are evaluated in anAIED animal model usinga procedure similar to the procedure in Example 67. Example 93 - Evaluation of a Calcineurin Inhibitor Formulation in an Otitis Media Animal Model Induction f Otitis Media

[00834}Healthy adult chinchillas weight 400 to 600 g with normal middle ears, ascertained by otoscopy and tympanometry are used for these studies, Eustachian tube obstruction is performed 24 hours before inoculation to prevent the inoculunfrom flowing out of the eustachian tube. One milliliter of type 3 S.pneunoniae strain at 4-h-log phase (containing approximately 40 colony forming units (CFU)) is placed directly into both middle ear hypotympanic bullae of the chinhillas Control miceare inoculated with one milliliter sterile PBS.

Treatment

[008351S pneumoniae inoculated and control mice are sorted into two groups (n = 10 in each group), A calcineurin inhibitor formulation of Example 2 containing tacrolimus is applied to the walls of the tympanic cavity of one group of animals. Control formulation containing no tacrolimus is applied to the second group. The anti-TNF and control formulations are reapplied three days after the initial application. The animals are sacrificed after the seventh day of treatment. Analysis of Results

[00836]Auris media ear fluid (MEF) is sampled at 1, 2 6, 12, 24, 48 and 72 hours after pneumoccal inocualtion. Quantitative MTF cultures are performed on sheep blood agar, with the quantitation threshold set at 50 CFULInflammatory cells are quantitated with ahernocytorneter, and differential cell enumeration performed with Wright's staining.

Examples 94 - 95

[008371Mucoadhesive thermoreversible gel formulation comprising methotrexate of Example 27 and thermoreversible gel formulation comprising amoxicillin of Example 5 are evaluated in an Otitis Media animal model using a procedure similar to the procedure in Example 68

Example 96 - AIED Clinical Trials using TACE Inhibitor Formulations {00838 Tenadult patients are selected due to initial steroid responsiveness followed by recurrence of hearing loss when steroids are tapered or after completion of steroidtreatment.The'TACE inhibitor formulationof Example 3 containing 0.3 mg of BMS-561392 is administered to each patiet's round window membrane through piercing of the tympanic membrane. Reapplication of the TACE inhibitor formulations is performed 7 days after the initial application, and again at 2 and 3 weeks of treatment.

100839]Hearing evaluations consisting of pure tone audiometry (250-8000 Hz) and speech testing useig dissyllabic word lists in French are administered to each patient. Testing is carried out both before the application of the TACE inhibitor formulation and at 1, 2,3 and 4 weeks post-initial treatment. Example 97 - Evaluation of VP2 Antagonist.Formulations in an Endoivmppatic Hydrops Animal Model

[008401The following procedure is used to determine the efficacy of the thermoreversible gel formulation of lixivaptan as prepared in Example 2.

Materials and Methods 100841.Thirty-five Hartley guinea pigs with a positive Preyer's reflex and weighing about 300 g are used. Five animals, which serve ascontrols (normal eargroup), are fed for 5 weeks with neither operation nor treatment, and the remaining 30 serve as experimental animals. All experimental animals received electro-cauterization of the endolymphatic sac (Lee et al., Acta Otolaryngol (1992) 112:658-666 Takeda et aL, Equilib. Res. (1993) 9:139-143), Four weeks after surgery, these animals are divided into three groups of non-infusion hydropic ears, vehicle-treated hydropice ars and lixivaptan-treated hydropic cars, consisting of 10 animals each. The group of non-infusion hydropic ears receive no treatment except for electro-cauterization of the endolymphatic sac. In the groups of vehicle-treated hydropic ears and Iixivaptan-treated hydropic ears, the thermoreversihle gel formulation is applied to the round window membrane. One week afteradrninistration of the composition, all animals are sacrificed for assessment of the changes of the endolymphatic space. Allanimals are left undisturbed and freely moving in individual cages in a quietroom throughout the period, except during experimental procedures.

[00842] To assess the changes to the endolymphatic space, all animals are transcardially perfused with physiologicalsalinesolutionunder deep anesthesia by a peritoneal injection of pentobarbital and fixation is performed with 10% fornalin, The left temporal bones are removed and postfixed in 10% formalin solution for 10 days ormore. Thereafter, they are decalcified with 5% trichloroacetic acid for 12 days and dehydrated in a graded ethanol series. Theyare embedded in paraffin and celloidin. The prepared blocks are cut horizontally into 6 gm sections. The sections are stainedwith henatoxylin and cosin and observed under a light microscope. Quantitative assessment of changes of the endolymphatic space is performed according to the method ofTakeda (Takeda et al., Hearing Res. (2003) 182:9-18).

Exanle 98

100843]KCNQthermureversible gel formulation of retigabineaspreparedin. Example 10 is tested in an Endolymphatic Hydrops Animal Model using a procedure similar to the procedure of Example 72, Example 99 - Evaluation of LixivptaAdministration inMeniere's Patients Study Objective

[00844] The primary objective of this study will be to assess thesafety and efficacy of Lixivaptan (100 mg) in ameliorating Meniere's Disease in human subjects,

Methods Study Design

1008451This will be a phase 3, multicentre, double-blind, randomised, placebo-controlled, parallel group study comparing lixivaptan administration (100 mg) to placebo in the treatment of endolymphatic hydrops. Approximately 100 subjects will be enrolled in this study, and randomised (1:1) to1 of 2 treatment groups based on a randomisation sequence prepared by the sponsor. Each group will receive either 100 mg lixivaptan +meclizine or meclizine treatment alone.

[008461Subjects who do not complete the study will not be replaced. All patients will receive daily meclizine treatment for 8 weeks. Patients receiving the study drug (Lixivaptan 100 mg ormatching placebo) will be administered a gel formulation directly onto the subjects' round window membrane for 8 weeks. Each patient will receive a vestibular and hearing evaluation before each treatment with meclizine and the study drug. Example 100 - Clinical Trials of Vestipitant/Paroxitene in Tinnitus Patients

Study Objective

[00847] The primary objective of this study will be to assess the safety and efficacy of Vestipitant/Paroxitene compared with that of placebo in ameliorating tinnitus symptoms in afflicted patients. Study Design 1008481 This will be a phase 3, multicentre, double-blind, randomised, placebo-controlled, three-arm study comparing Vestipitant/Paroxitene to placebo in the treatment of tinnitus. Approximately 100 subjects will be enrolled in this study, and randomised (1:1) to I of 3 treatment groups based on a randomisation sequence prepared by sponsor. Each group will receive 280 mg Paroxitene/350 mg Vestipitant delivered in a thernoreversible gel, or controlled release placebo formulation. Release of Vestipitant/Paroxitene is controlled release and occurs over 14 days. Route ofAdministration will be intratympanic infection. Primary Ontcome Measure

[008491Visual Analog Scales (VAS) to measure the change in tinnitus loudness as perceived at the moment of the measurement at 2hrs after dosing (orat any other time point vs. pre-dose baseline).

Secondary Outcome Measures

[008501]VAS to measure tinnitus pitch, distress and anxiety. Pure Tone Audiometry

& Psychoacoustic assessment. Sleep & Tinnitus questionnaires. Safety, tolerability and after pharmacokinetics of drug. [ Time Frame: perceived at themoment of the measurement at 2 hrs dosing (or at any other time point vs.pre-dose baseline). Inclusion Criteria

[00851]Patients are included if they meet any of the following criteria: * Male or female subjects with a diagnosed tinnitus.

• Subject with TI severity grade of 3 or 4. • Subjects willing to restrict alcohol intake. * Women of childbearing potential who abstain from intercourse OR agree to birth control. Women of non-childbearing potential.

Exchision Criteria

[00852]Patients will be excluded if they meet any of the following criteria: * Subject with THI severity grade = 5 or less than or equal to 2.

* Subject with pathologic level of anxiety or depression.

• Subject with no audiogram deficit and with normal hearing. * Subjects that do not respond to the lidocaine infusion test or show a large variability in pre infusion values. * Subjects with any serious medical disorder or condition that would preclude the administration of Vestipitant or Paroxetine. N Existence of any surgical ormedical. condition which might interfere with the PK of the drug. • Subjects with hepatic impairment or a history of liver dysfunction * Subjects with renal impairment.

a Subjects positive for lIV, hepatitis C or hepatitis B. a Subjects with abnormal laboratory, ECG or physical examination findings. & Subjects who are not euthyroid.

30 • Subjects with a history of hepaticcardiac, renal, neurologic, cerebrovascular, metabolic or pulmonary disease. • Subjects who have had a myocardial infarction.

* Subjects with a history of seizure disorders. Subjects with history of cancer.

35 * Subjects with a history of drug or other allergy,

* Subjects positive for drug use and/or a history of substance abuse or dependence.

0 Subjects who have taken psychotropic drugs or antidepressants within specified time frames, Medication or foodstuff (e.g. grapefruit or grapefruit juice) which is known to interfere with liver enzymes. * The subject had a non-psychotropic medication with a serotonergic mechanism of action. * Subjects who have recently used an investigational drug or recently participated in a trial. * Subjects who have exhibited intolerance to NK Iantagonists or SSR1s. • Women who have a positive pregnancy test. * Female subjects who intend to get pregnant or male subjects who intend to father a child within the next 4 weeks following the last study drug administration in the study. * Subjects, who have donated a unit of blood or more within the previous month or who intend to donate blood within one month of completing the study. Example 101 - Clinical Trials of Neramexane in Tinnitus Patients

Study Objective

[O0853]The primary objective of this study will be to assess the safety and efficacy of Nerarnexane compared with that of placebo in ameliorating tinnitus symptoms in afflicted patients. Study Design three-arm

[00854]This will be a phase 3, multicentre, double-blind, randomised, placebo-controlled, study comparing Neramexane to placebo in the treatment of tinnitus. Approximately 250 subjects will be enrolled in this study, and randomised (1:1) to I of 3 treatment groups based on a randomisation sequence prepared by sponsor. Each group will receive 300mg Neramexane delivered in a thermoreversible gel, or controlled release placebo formulation. Release of Neramexane is controlled release and occurs over 14 days. Route of Administration will be intratympanic injection. Primary Outcome Measure

[00855]Visual Analog Scales (VAS) to measure the change in tinnitus loudness as perceived at the moment of the measurement at 2 hrs after dosing (or atany other time point vs. pre-dose baseline). Secondary Outcome Measures

[00856]VAS to measure tinnitus pitch, distress and anxiety. Pure Tone Audiometry &

Psychoacoustic assessment. Sleep & Tinnitus questionnaires. Safety, tolerability and pharmacokinetics of drug. [ Time Frame: perceived at the moment of the measurement at 2 hrs after dosing (or at any other time point vs. pre-dose baseline). Inclusion Criteria 35 [00857]Patients are included if they meet any of thefollowing criteria: • Male or female subjects with a persistent, subjective, uni or bi-lateral tinnitus tinnitus. • Subjects willing to restrict alcohol intake.

* Women of childbearing potential who abstain from intercourse OR agree to birth control. * Women of non-childbearing potential.

Lvclusion Crteria

[008581Patients are excluded if they meet any of the following criteria: a Intermittent or pulsatile tinnitus • Subject with pathologic level of anxiety or depression.

0 Subject with noaudiogram deficit and with normal hearing. * Subjects that do not respond to the lidocaine infusion test or show a large variability in pre infusion values. * Existence of any surgical or medical condition which might interfere with the PK of the drug. * Subjects with hepaticimpairment or a history of liver dysfunction. * Subjects with renal impairment.

15. Subjects positive for HI!, hepatitis C or hepatitis B. * Subjects with abnormal laboratory, ECG or physical examination findings. * Subjects who are not euthyroid.

* Subjects with a history of hepatic, cardiac, renal, neurologic, cerebrovascular, metabolic or pulmonary disease.

* Subjects who have had a myocardial infarction. * Subjects with a history of seizure disorders.

* Subjects with history of cancer. * Subjects with a history of drug or other allergy.

* Subjects positive for drug use and/or a history of substance abuse or dependence.

• Subjects who have taken psychotropic drugs or antidepressants within specified time frames. • Medication or foodstuff (e.g. grapefruit or grapefruit juice) which is known tointerfere with liver enzymes. * Subjects who have recently used an investigational drug or recently participated in a trial.

* Women who have a positive pregnancy test. • Female subjects who intend to get pregnant or male subjects who intend to fathera child within the next 4 weeks following the last study drug administration in the study. * Subjects, who have donated a unit of blood or more within the previous mohth or who intend to donate blood within one month of completing the study. Example 102 - Clinical Trials of AL-15469A/AL-38905 in Acute Otitis Externa Patients

Study Objective

[00859]The primary objective of this study will be to assess the safety and efficacy of AL 15469A/AL-38905 compared with that of placebo in ameliorating Acute Otitis Externa symptoms in afflicted patients.

Study Design

[00860]This will be a phase 3, multicentre, double-blind, randomised, placebo-controlled, three-arm study comparing.AL-15469A/AL-38905 (100 mg and 200 mg) to placebo in the treatment of tinniws, Approximately 1500 subjects will be enrolled in this study. and randomised (1:1) to 1 of 3 treatment groups based on a randomisation sequence prepared by sponsor. Each group will receive 100 mg controlled release AL-15469A/AL-38905, 200 mg controlled releaseAL- 5469A/AL-38905 or controlled release placebo formulation. Primary Outcome Measures:

[00861]Clinical cure [ Time Frame: Day 3 and Day 12

Secondary Outcome Measures:

[00862]Microbiological success [ Time Frame: Day 12]

Inclusion Criteria:

[00863]Patients must be at least 6 months of age or older. Further, patients must have a clinical diagnosis of AOE based on clinical observation and of presumed bacterial origin. Additionally, patients must demonstrate a minimum combined score of4 in at least I affected ear at the Day I exam for tenderness, erythema, and edema,

Exclusion Criteria:

[00864]Patients will be excluded if they meet any of the following criteria:

* Duration of pretherapy signs or symptoms of AOE greater than four (4) weeks.

• Presence of a tympanostomy tube or perforated tympanic membrane in the treated ear(s). Patients with a history of tympanic membrane perforation should not be enrolled unless the absence of a current perforation is confirmed at Visit I prior to enrollment. * Clinically diagnosed chronic suppurative otitis media acute otitis media, acute otorrhea in patients with tympanostomy tubes, or malignant otitis externa.

* Known or suspected ear infection of fungal or mycobacterial origin. * Prior otologic surgery within 6 months of study entry. Seborrheic dermatitis or other skin conditions of the external auditory canal. * Current or prior history of an immunosuppressive disorder (eg., HIV positive) or current immunosuppressive therapy (e.g.,cancer chemotherapy) or known acute or chronic renal disorders 35 or active hepatitis.

* Diabetic patients (controlled or uncontrolled) based upon assessment by Investigator. * Any systemic disease or disorder, complicating factor or structural abnormality that would negatively affect the conduct or outcome of the study [e.g, cleft palate (includingrepairs), Downs Syndrome, and cranial facial reconstruction. * Any current known or suspected infection (other than AOE) requiring systemic antimicrobial therapy. * Use of prohibited medications or inadequate washout of any medication listed in protocol.

* Concomitant use of topical or oral analgesics (i.e., NSAIDs and aspirin products) which

may have anti inflammatory effects. Patients on low dose aspirin therapy (81 mg per day) at the time of enrollment are enrolled and continue the low dose aspirin during the study Use of acetaminophen ("Tylenol") is permitted during the trial.

Example 103 - Clinical Trials of JB004/A in Meniere's Disease Patients

Study Objective

[00865]The primary objective of this study will be to assess the safety and efficacy of JB004/A compared with that of placebo in ameliorating tinnitus symptoms in Meniere's Disease afflicted patients, Study Design

[00866]This will be a phase 3,multicentre, double-blind, randomised, placebo-controlled, three-arm study comparing JB004/A to placebo in the treatment of tinnitus. Approximately 250 subjects will be enrolled in this study, and randomnised (1:1) to I of 3 treatment groups based on a randomisation sequence prepared by sponsor. Each group will receive 300 mg J13004/A delivered in a thermnoreversible gel, or controlled release placebo formulation. Release of JB004/A is controlled release and occurs over 30 days. Route of Administration will be intratympanic injection. Primary Outcome Measure

[00867]Visual Analog Scales (VAS) to measure the change in tinnitus loudness as perceived at the moment of the measurement at 2 hrs after dosing (or at any other time point vs. pre-dose baseline). Alternatively, audiometry is used in the healthy ear to match the tone of the tinnitus in the affected ear. Secondary Outcome Measures

[008681VAS to measure tinnitus pitch, distress and anxiety. Pure Tone Audiometry &

Psychoacoustic assessment. Sleep & Tinnitus questionnaires. Safety, tolerability and pharmacokinetics of drug. Time Frame:perceived at the moment of the measurement at 2 hrs after dosing (or at any other time point vs. pre-dose baseline). Inclusion Criteria

[008691Patients are included if they meet any of the following criteria:

* Male or female subjects diagnosed with a tinnitus. * Subjects willing to restrict alcohol intake.. * Women of childbearing potential who abstain from intercourse OR agree to birth control.

* Women of non-childbearing potential.

Exclusion Criteria

[00870Patients are excluded if they meet any of the following criteria: * Intermittent or pulsatile tinnitus

* Subject with pathologic level of anxiety or depression. * Subject with no audiogram deficit andwith normal hearing. * Subjects that do not respond to the lidocaine infusion test or show a large variability in pre infusion values. * Existence of any surgical or medical condition which might interfere with the PK of the drug.

* Subjects with hepatic impairment or ahistory of liver dysfunction. * Subjects with renal impairment.

* Subjects positive for HIV, hepatitis C or hepatitis B

* Subjects with abnormal laboratory, ECG or physical examination findings, * Subjects who are not euthyroid. or • Subjects with a history of hepatic, cardiac, renal, neurologic, cerebrovascular, metabolic pulmonary disease. * Subjects who have had a myocardial infarction.

* Subjects with a history of seizure disorders.

* Subjects with history of cancer.

* Subjects with a history of drug or other allergy. * Subjects positive for drug use and/or a history of substance abuse or dependence.

* Subjects who have taken psychotropic drugs or antidepressants within specified time frames. • Medication or foodstuff (e.g. grapefruit or grapefruit juice) which is known to interfere with 30 liver enzymes. * Subjects who have recently used an investigational drug or recently participated in a trial. * Women who have a positive pregnancy test.

* Female subjects who intend to get pregnant or male subjects who intend to father a child within the next 4 weeks following the last study drug administration in the study.

Subjects, whohave donated a unit of blood or -more within the previous month or who intend to donate blood within one month of completing the study. Example 104 - Evaluation of Alpha LipoicAcdin anEarly OnseAge-Related Hearinu Impairment DBA-Mouse Model

[00871]DBA mice are administered an alpha-lipoic acid formulation of Example 3 directly onto the round window membrane, beginning 2,4 or 8 weeks after birth. The hearing threshold for the auditory brainstem response threshold (ABR) to click stimuli for each ear ofeach animal is initially measured and on a weekly basis during and after the experimental procedure. The animals are placed in a single-walled acoustic booth (Industrial Acoustics Co Bronx, NY, USA) on a heating tO pad. Subdermal electrodes (Astro-Med Inc. Grass Instrument Division. West Warwick, RI, USA) were inserted at the vertex (active electrode), the mastoid (reference), and the hind leg (ground). Click stimuli (0 1 millisecond) are computer generated and delivered to a Beyer DT 48,200 Ohm speaker fitted with an ear speculum for placement in the external auditory meatus. The recorded AIR is amplified and digitized by a battery-operated preamplifier and input to a Tucker-Davis Technologies ABR recording system that provides computer control of the stimulus, recording, and averaging functions (Tucker Davis Technology, Gainesville FL. USA). Successively decreasing amplitude stimuli are presented in 5-d1B steps to the animaland therecorded stimulus-locked activity is averaged (n=512) and displayed. Threshold is defined as the stimulus level between the record with no visibly detectable response and a clearly identifiable response.

Example 105 - Evaluation ofDiazepam in an drops Animal Model Methods and Materials Induction ofEndolymphatic Hydrops

[00872)Female albino National Institutes of Health-Swiss mice (Harlan Sprague-Dawley, Inc., Indianapolis, Inc.) weighing 20 to 24 g are used. Artificial endolymph is injected into the cochlear duct. Treatrment

[00873]The endolymphatic mice and control mice are sorted into two groups (n = 10 in each group). The CNS modulating formulation of Example 5 containing diazepam is applied to the round window membrane of one group of animals. Control formulation containing no diazepam is applied to the second group. The CNS modulating and control formulations are reapplied three days after the initial application. The animals are sacrificed after the seventh day of treatment. Analysis of Results ElectrophysiologicTesting 100874]The hearing threshold for the auditory brainstem response threshold (ABR) to click stimuli for each ear of each animal is initially measured and I week after the experimental procedure. The animals are placed in a single-walled acoustic booth (Industrial Acoustics Co, Bronx, NY, USA) on a heating pad. Subdenral electrodes (Astro-Med, Inc. Grass Instrument Division,.West Warwick,

RI USA) were inserted at the vertex (active electrode), the mastoid (reference), and the hind leg (ground). Click stimuli (0.1 millisecond) are computergenerated and delivered to a Beyer DT 48, 200 Ohm speaker fitted with an ear speculurn for placementin the external auditory meatus. The recorded ABR is amplified and digitized by a battery-operated preamplifier and input to a Tucker Davis Technologies ABR recording system that provides computer control of the stimulus, recording, and averaging functions (Tucker Davis Technology, Gainesville, FL, USA), Successively decreasing amplitude stimuli are presented in 5-dB steps to the animal, and the recorded stimulus locked activity is averaged (n=512) and displayed. Threshold is defined as the stimulus level between the record with no visibly detectable response and a clearly identifiable response.

Example 106 - Clinical Trial of Diazepam as a Treatment for Tinnitus

[00875]Active Ingredient: Diazepam

[00876]Dosage: 10 ng delivered in 10 L of a thermoreversible gel. Release of diazepam is controlled release and occurs over thirty (30) days.

[00877}Route of Administration: Intratympanic injection

[008781Treatment Duration: 12 weeks

[008791Methodology * Monocentric

Prosepective

' Randomized Double-blind Placebo-controlled * Parallel group

Adaptive

[008801Inclusion Criteria 0 Male and female subjects betweenthe 18 and 64 years of age.

0 Subjects experiencing subjective tinnitus.

0 Duration of tinnitus is greater than 3 months. 0 No treatment of tinnitus within 4 weeks.

[00881] Evaluation Criteria * Efficacy (Primary) Total score of the Tinnitus Questionnaire

* Efficacy (Secondary) Audiometric measurements (mode, frequency, loudness of the tinnitus, pure tone 35 audiogram, speech audiogram) Quality of Life questionnaire

Safety Treatment groups were compared with respect to incidence rates of premature termination, treatment-emergent adverse events, laboratory abnormalities, and ECG abnormalities,

[00882]StudyDesian

100883]Subjects are divided into three treatment groups. The first group is the safety sample The second group is the intent-to-treat (ITT) sample. The third group is the valid for efficacy (VfE) group.

[00884For each group, one half of subjects to be given diazepam and the remainder to be given placebo.

[008851Statistical Methods

[00886]The primary efficacy analysis is based on the total score of the Tinnitus Questionnaire in the T'T sample. The statistical analysis is based on an analysis of covariance (ANCOVA) with baseline as covariant and the last observation carried forward value as dependent variable. Factor is "treatment."The homogeneity of regression slopes is tested. The analysis is repeated for the VfE sample.

[00887]Audiometric measurements (mode, frequency, loudness of the tinnitus, pure tone audiogram. speech audiogram) as well as quality of life are also analyzed via the aforementioned model. The appropriateness of the model is not tested. Values are exploratory and are not adjusted for multiplicity.

Example 107-Evaluation of Ctotoxic Acent Formulationsin an Ear CancerAnimal Model

[00888}Cytotoxic agent formulations are tested in an ear cancer animal model, described in Arbeit, J. M., et al. Cancer Res. (1999), 59: 3610-3620. A cohort of K14PV16 transgenic mice is divided into control/untreated and test/treated mice groups for comparison of the effect of cytotoxic agent formulation administration on the development ofear cancer. The cytotoxic agent methotrexate formulation of Example 4 is administered to the ear of the test mice group starting at age 4 weeks. The chemopreventive effect of the cytotoxic agent formulation is assessed by sacrificing treated mice at age 8, 16, and 32 weeks, and comparing the number of lesions and histopathological and phenotypic markers (papillomatosis, dermal inflammatory cell infiltration, corneal parakeratosis, etc.) at the various stages of neoplastic progression to control mice of the same age. The effectof cytotoxic agent formulations on the progression of established, late-stage neoplasiais assessed by administering the cytotoxic agent formulation of Example 4 to K14--PV16 transgenic mice starting at age 28 weeks. The mice are sacrificed at age 32 weeks, and the effect of the cytotoxic agent 35 formulation is assessed by comparing the number of lesions and histopathological and phenotypic markers to control mice of the same age.

Example 108 - AIED Clinical Trials using Cytotoxic Agent Formulations

[00889)Ten adults patients are selected due to initial steroid responsiveness followed by recurrence of hearing loss when steroids are tapered or after completion of steroid treatment The cytotoxic agent formulation of Example 4 containing methotrexate is administered to each patient's round window membrane through piercing of the tympanic membrane. Reapplication of the cytotoxic agent formulations is performed 7 days after the initial application, and again at 2 and 3 weeks of treatment.

[00890]1Hearing evaluations consisting of pure tone audiometry (250-8,000 Hz) and speech testing using dissyllabic word lists in French are administered to each patient. Testing is carried out both before the application of the cytotoxic agent formulation and at 1, 2,3 and 4 weeks post-initial treatment.

Example 109 - Evaluation of N-acetylcysteine.(NAC) in a Cisplatin-Induced OtotoxicityvMouse Model Methods and Materials Induction of Orotoxicity

[00891]Twelve Harlan Sprague-Dawley mice weighing 20 to 24 g are used. Baseline auditory brainstem response (ABR) at 4-20mHz is measured. The mice are treated with cisplatin (6mg/kg of body weight). The cisplatin is delivered to the aorta by IV infusion. Treatment

[0892]The control group (n=10) are administered saline following administration of the cisplatin. The experimental group (n=10) are administered NAC (400 mg/kg of body weight) following administration of the cisplatin, Analysis of Results ElectrophysiologicTesting

[00893]The hearing threshold for the auditory brainstem response threshold (ABR) to click stimuli for each ear of each animal is initially measured and I week after the experimental procedure. The animals are placed in a single-walled acoustic booth (industrial Acoustics Co, Bronx, NY, USA) on a heating pad. Subdermal electrodes (Astro-Med, Inc. Grass Instrument Division, West Warwick, RI USA) were inserted at the vertex (active electrode), the mastoid (reference)and the hind leg (ground) Click stimuli (0.1 millisecond).are computergenerated and delivered to a Beyer DT 48, 200 Ohm speaker fitted with an ear speculum for placement in the external auditory meatus. The recorded ABR is amplified and digitized by a battery-operated preamplifier and input to a Tucker Davis Technologies ABR recording system that provides computer control of the stimulus, recording, and averaging functions (Tucker Davis Technology, Gainesville, FL, USA). Successively decreasing amplitude stimuli are presented in 5-dB steps to the animal, and the recorded stimulus locked activity is averaged (n=5 12) and displayed. Threshold is defined as the stimulus level between the record with no visibly detectable response and a clearly identifiable response. Example I 10

[00894}L(+-Ergothioneine was tested in a cisplatin-induced ototoxicity mouse model using a procedure similar to the procedure in Example 84. Example I l - Evaluation of AMN082 on Cisplatin-Induced Ototoxicity Study Objective

[008951The primary objective of this study will be to assess the safety and efficacy of AMN082 (100 mg) compared with that of placebo in preventing Cisplatin-induced ototoxicity. Methods Study Design

[00896]This will be a phase 3multicentre, double-blind, randomised, placebo-controlled, parallel group study comparing AMN082 (100 mg) to placebo in the treatment of Cisplatin-induced ototoxicity. Approximately 140 subjects will be enrolled in this study, and randomised (1:1) to 1 of 2 treatment groups based on a randomisation sequence prepared by sponsor. Each group will receive either AMNO82 100 mg or placebo.

[00897]Subjects who do not complete the study will not be replaced. Patients will receive weekly chemotherapy (cisplatin at a dose of 70 mg/m for 7 veeks and daily radiation. Following chemotherapy, patients wil receive the study drug (AMN082 500 mg or matching placebo) administered as a gel formulation directly onto the subjects' round window membranefor 8 weeks.

[00898]Each patient will receive a hearing evaluation before each treatment with Cisplatin. Two to four weeks after the final dose of Cisplatin, each patient will receive ahearing evaluation. Pre treatment audiogram will be compared with the post treatment audiogram to determine the degree of cisplatin-induced ototoxicity. Patients will thereafter receive a hearing evaluation at 4-week intervals concomitant with the AMN082 treatment. Main Criteria b Inclusion

[00899]Male or female outpatients aged between 18 and 75 years receiving chemotherapy with Cisplatin. Patients expected to receive a minimum of 3 rounds of chemotherapy. If a subject becomes pregnant during the study, she will be immediately withdrawn and no studymedication will be administered. Exclusion Criteria 100900]Patients who have had middle ear surgery. Patients who have active external or middle ear disease Patients who have preceding pure tone average of >40 dB HL. Example 112 - Evaluation of Antimicrobial Agent Formulations in an Otitis Externa Animal Model

[00901]Otitis externais induced in 20 Sprague-Dawley rats using a plastic pipette to aggravate the tissue of the ear canal. All of the rats develop OEwithin one day. The antimicrobial formulation of

Example 17 containing neomycin is administered to the ears of half of the rats using a needle and syringe, while the remaining rats receive the same formulation without the neomycin. The ear canal tissue is observed for redness and swelling that characterizes the condition, Light microscopy is used to analyze biopsy samples from the rats. Example 113 - Clinical Trial of Antimicrobial Agent Formulations for the Treatment of Otosyphilig

[00902]Patients selected for the study present symptoms of cochleovestibular dysfunction and positive syphilis serology. Patients are divided into two groups, a test group receiving intratympanic administration of the formulation of Example 57 in conjunction with an intramuscular (IM) injection of 2.4 million units of benzathine penicillin G (the recommended treatment for syphilis), and a control group receiving only the carrierandmicrospheres of the otic formulation of Example 57 in conjunction with an IM injection of 2.4 million units of benzathine penicillin G. Patients are monitored for improvement of hearingtinnitus, vertigo, and nystagmus following administration of the active agents. The primary outcome of the trial is improvement of cocheovestibular function at the 6 month post-treatment visit. The outcome for patients receiving the formulation of Example 6 and the recommended therapy is compared to the outcome for patients receiving only the carrier for the otic formulation and the recommended therapy in order to determine the efficacy of localized delivery of an antimicrobial agent formulation for the treatment of otic symptoms of syphilis. Example 114 - Clinical Trials of KCNQ Modulator in Vertigo Patients Study Objective 2 [00903]The primary objective of this study will be to assess the safety and efficacy of retigabine compared with that of placebo in ameliorating vertigo symptomsin afflicted patients. Methods Study Design

[00904]This will be a phase 3, multicentre, double-blind, randomised, placebo-controlled, three-arm study comparing retigabine (100 mg and 200 mg) to placebo in the treatment of vertigo symptoms. Approximately 150 subjects will be enrolled in this study, and randomised (1:1) to I of 3 treatment groups based on a randomisation sequence prepared by sponsor. Each group will receive 200 mg controlled release retigabine, 400 mg controlled release retigabine or controlled release placebo formulation.

[009051After a I-week baseline phase, patients from each group will be randomized to a 16 week double treatment period (8-week treatment followed by an 8-week maintenance period) Primary efficacy will be measured as a percentage change in the frequency and intensity of vertigo symptoms, including dizziness, loss of balance and incidence of nystagmus after treatment as compared to baseline measurements.

[00906]While preferred embodiments of the present invention have been shown and described herein, such embodiments are provided by way of example only.Various alternatives to the embodinents described herein are optionally employed in practicing the inventions. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims (14)

1. An intratympanic composition for the treatment or prevention of hearing loss, wherein the intratympanic composition comprises an auris sensory cell modulator and an auris acceptable gel, wherein the auris sensory cell modulator is a neurotrophin, wherein the auris acceptable gel is a thermoreversible gel comprising from 14% to 19% by weight of a poloxamer, and wherein the poloxamer is poloxamer 407.

2. The intratympanic composition of claim 1, wherein the auris acceptable gel is capable of being injected by a narrow gauge needle or cannula through the tympanic membrane to an area on or near the round window membrane.

3. The intratympanic composition of claim 1 or claim 2, wherein the composition has an osmolarity of from about 100 mOsm/L to about 500 mOsm/L.

4. The intratympanic composition of any one of claims I to 3, wherein the neurotrophin is present at a concentration of about 0.1 mg/mL to about 5 mg/mL.

5. The intratympanic composition of claim 4, wherein the neurotrophin is present at a concentration of about 0.5 mg/mL to about 5 mg/mL.

6. The intratympanic composition of any one of claims 1 to 5, wherein the composition has a pH between 7.0 and 8.0.

7. The intratympanic composition of any one of claims 1 to 6, wherein the neurotrophin is selected from brain-derived neurotrophic factor (BDNF) or neurotrophin-3.

8. The intratympanic composition of any one of claims 1 to 7, wherein sustained release of the auris sensory cell modulator into the cochlea occurs for a period of at least 5 days after a single administration.

9. The intratympanic composition of any one of claims 1 to 8, wherein the composition provides a therapeutically effective amount of neurotrophin in the inner ear to prevent cells from initiating apoptosis.

10. The intratympanic composition of any one of claims I to 9, wherein the composition provides a therapeutically effective amount of neurotrophin in the inner ear to repair damaged neurons and otic hair cells.

11. The intratympanic composition of any one of claims 1 to 10, wherein the composition provides a therapeutically effective amount of neurotrophin in the inner ear to induce differentiation in progenitor cells.

12. The intratympanic composition of any one of claims I to 11, wherein the auris acceptable gel comprises from about 15 wt% to about 18 wt% of Poloxamer 407.

13. A method of treating or preventing hearing loss in a subject, the method comprising administering a therapeutically effective amount of an intratympanic composition to the subject, wherein the intratympanic composition comprises an auris sensory cell modulator and an auris acceptable gel, wherein the auris sensory cell modulator is a neurotrophin, wherein the auris acceptable gel is a thermoreversible gel comprising from 14% to 19% by weight of a poloxamer, and wherein the poloxamer is poloxamer 407.

14. Use of an intratympanic composition in the manufacture of a medicament for treating or preventing hearing loss in a subject, wherein the intratympanic composition comprises an auris sensory cell modulator and an auris acceptable gel, wherein the auris sensory cell modulator is a neurotrophin, wherein the auris acceptable gel is a thermoreversible gel comprising from 14% to 19% by weight of a poloxamer, and wherein the poloxamer is poloxamer 407.