WO2011049954A2

WO2011049954A2 - Compositions comprising wnt modulators or neurotoxins for the treatment of otic disorders

Info

Publication number: WO2011049954A2
Application number: PCT/US2010/053210
Authority: WO
Inventors: Jay Lichter
Original assignee: Otonomy, Inc.
Priority date: 2009-10-21
Filing date: 2010-10-19
Publication date: 2011-04-28
Also published as: WO2011049954A3

Abstract

Disclosed herein are compositions and methods for the treatment of otic diseases or conditions with otic agent compositions and formulations administered locally to an individual afflicted with an otic disease or condition, through direct application of these compositions and formulations onto or via perfusion into the targeted auris structure(s).

Description

COMPOSITIONS COMPRISING WNT MODULATORS OR NEUROTOXINS FOR THE TREATMENT OF OTIC DISORDERS

CROSS-REFERENCE

This patent application claims the benefit of U.S. Provisional Application Ser. No.

61/253,782 filed October 21 , 2009; U.S. Provisional Application Ser. No. 61 /255,379 filed October 27, 2009;US. Provisional Application. Ser. No. 61/255,780 filed October 28, 2009: U.S. Provisional Application Ser. No. 61/255,783 filed October 28, 2009; U.S, Provisional Application Ser. No. 61/297, 138 filed January 21, 20.10; U.S. Provisional Application Ser. No. 61/297,170 filed January 21, 2010; U.S. Provisional Application Ser. No. 61/366.677 filed July 22, 2010; all of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

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

SUMMARY OF THE INVENTION

[0002] Described herein are formulations, manufacturing methods, therapeutic methods, uses, and kits, tor the controlled release of desired agents to at least one structure or region of the ear. In some embodiments, formulations described herein comprise viscosity enhancing polymers in amounts that render the formulations substantially stable in an aqueous environment Such .as middle ear fluids, in other words, the formulations described herein are viscous and remain in extended contact with the middle ear; the formulations substantially do not wash away from the middle ear environment or drain out of the middle ear via the eustachian tube, in some embodiments, formulations described herein comprise viscosity enhancing polymers in amounts thai allow the formulations to gel at about body temperature. In other words, the formulations described herein gel upon contact with an auditory surface (e.g., the round window membrane in the inner ear) and remain in extended contact with the round window membrane thereby allowing for sustained delivery of active agents to the inner ear.

[0003] Provided herein, in some emodiments, are pharmaceutical .compositions suitable for use in the treatment of otic disorders by intratympanic administration on or near the round window membrane of the ear, which comprise an auris acceptable thermoreversible aqueous gel comprising of a polyoxypropylene and polyoxyethylene triblock copolymer and a WNT modulator such that sustained release of the WNT modulator across the round window membrane into the cochlea occurs for a period of at least 3 days.

[0004] In some embodiments, the composition has a pH between about 7.0 and about 8.0.

[0005] In some embodiments, sustained release is provided for a period of at least 5 days. In some embodiments, sustained release is provided for a period of at least 7 days. In some embodiments, sustained release is provided for a period of at least 10 days.

[0006] In some embodiments, the polyoxypropylene and polyoxyethylene triblock copolymer is present in the composition in an amount between about 14% and about 27% by weight of the composition. In some embodiments, the polyoxypropylene and

polyoxyethylene triblock copolymer is present in the composition in an amount between about 15% and about 21% by weight of the composition. In some embodiments, the polyoxypropylene and polyoxyethylene triblock copolymer is poloxamer 407.

[0007] In some embodiments, the composition comprises a suspension of multiparticulate WNT modulator. In some embodiments, the multiparticulate WNT modulator is essentially micronized WNT modulator.

[0008] In some embodiments, the WNT modulator is selected from 2-amino-4-[3,4- (methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine, or Cerberus. In certain embodiments, the WNT modulator is 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3- methoxyphenyl)pyrimidine.

[0009] In some of the above embodiments, the otic disorder treated with a WNT modulator is selected from Meniere's disease, sudden sensorineural hearing loss, noise induced hearing loss, age related hearing loss, vertigo and tinnitus.

[0010] Further provided herein are pharmaceutical compositions suitable for use in the treatment of otic disorders by intratympanic administration on or near the round window membrane of the ear, which comprise an auris acceptable thermoreversible aqueous gel comprising of a polyoxypropylene and polyoxyethylene triblock copolymer and a neurotoxin such that sustained release of the neurotoxin across the round window membrane into the cochlea occurs for a period of at least 3 days.

[0011] In some embodiments, the composition has a pH between about 7.0 and about 8.0. [0012] In some embodiments, sustained release is provided for a period of at least 5 days. In some embodiments, sustained release is provided for a period of at least 7 days. In some embodiments, sustained release is provided for a period of at least 10 days.

[0013] In some embodiments, the polyoxypropylene and polyoxyethylene triblock copolymer is present in the composition in an amount between about 14% and about 27% by weight of the composition. In some embodiments, the polyoxypropylene and polyoxyethylene triblock copolymer is present in the composition in an amount between about 15% and about 21% by weight of the composition. In some embodiments, the polyoxypropylene and polyoxyethylene triblock copolymer is poloxamer 407.

[0014] In some embodiments, the composition comprises a suspension of multiparticulate neurotoxin. In some embodiments, the multiparticulate neurotoxin is essentially micronized neurotoxin.

[0015] In some embodiments, the neurotoxin is selected from Botulinum Toxin Type A, erabutoxin, tetrodotoxin, batrachotoxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcic ludine, or PhTx3. In certain embodiments, the neurotoxin is Botulinum Toxin Type A.

[0016] In some of the embodiments described above, the otic disorder treated with a neurotoxin is selected from Meniere's disease, sudden sensorineural hearing loss, noise induced hearing loss, age related hearing loss, vertigo and tinnitus.

[0017] Further provided herein is the use of any formulation described above in the manufacture of a medicament for treatment of any otic disorder described herein.

BRIEF DESCRIPTION OF FIGURES

[0018] Figure 1. is an illustrative comparison of non-sustained release and sustained release formulations.

[0019] Figure 2 are illustrative predicted tunable releases of an otic agent from four compositions.

[0020] Figure 3 A-G illustrates the hemolysis in guinea pig red blood cells when exposed to serially diluted poloxamer solutions.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Provided herein are controlled release otic agent compositions and methods for treatment of inner and/or middle ear disorders comprising administration of formulations described herein into the inner ear, middle ear or the external ear. Provided herein are methods for treatment otic disorders, including, and not limited to, otitis externa, otitis media, otitis media with effusion (glue ear), cholesteatoma, sensorineural hearing loss, noise induced hearing loss, autoimmune inner ear disease (AIED), vertigo, Meniere's disease, otosclerosis or any other otic disorder.

[0022] Controlled release formulations for administration to the ear create several unrecognized problems not addressed by currently available treatment regimens. As 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. There is wide anatomical disparity between the ears of animals across species. A consequence of 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 for clinical approval.

[0023] The environment of the inner ear is an isolated environment. The endo lymph and the perilymph are static fluids and are not in contiguous contact with the circulatory system. 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. When the tympanic membrane is intact, the air of the middle ear is not in direct contact with the atmosphere outside the body. 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 and/or middle ear.

[0024] Formulations for administration to the middle ear create several problems not addressed by currently available treatment regimens. The middle ear is an air-filled cavity behind the ear drum (tympanic membrane) and includes the three ear bones or ossicles: the malleus (or hammer), incus (or anvil), and stapes (or stirrup). The three bones are arranged so that movement of the tympanic membrane causes movement of the malleus, which causes movement of the incus, which causes movement of the stapes. When the stapes footplate pushes on the oval window, it causes movement of fluid within the cochlea. The eustachian tube is connected tothe middle ear and is blocked in some instances due to effusion (e.g., during an active infection).

[0025] Due to the susceptibilty of the inner and/or middle 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 manufactured with low bioburden or 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.

[0026] In some instances, a disadvantage of liquid formulations is their propensity to drip into the eustachian tube and cause rapid clearance of the formulation from the ear. In certain embodiments, formulations described herein comprise viscosity enhancing polymers that form thickened liquids and/or gels that remain in contact with the target auditory surfaces (e.g., the middle ear surfaces, the round window membrane or the like) for extended periods of time. Auris formulations described herein avoid attenuation of therapeutic benefit due to drainage or leakage of active agents via the eustachian tube.

[0027] The current standard of care for auris formulations 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 extended release formulations that are thickened liquid formulations which adhere to auditory structures, 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 multiple intratympanic injections in individuals undergoing treatment for middle, and/or inner ear disease, disorder or condition. In certain instances, a reduced frequency of administration of intratympanic injections reduces the risk of permanent damage (e.g., perforation) to the ear drum. Accordingly, in some embodiments, the formulations described herein prolong residence time of an otic agent in the external, middle and/or inner ear.

[0028] Provided herein are otic formulations that are suitable for administration into the middle and/or inner ear and are formulated with a middle-ear and/or inner-ear compatible pH, ionic balance, and/or bioburden. In some embodiments, the auris-media-suitable compositions described herein are formulated with minimum excipients and thus reduce or eliminate irritation or toxicity in the environment of the ear.

Administration in the inner and/or middle ear

[0029] Auris formulations described herein are administered intratympanically, or into the ear canal, or in the vestibule of the ear. Localized administration in the ear allows an active agent to reach a target organ (e.g., inner and/or middle ear) and reduces or eliminates systemic accumulation of the active agent. In some instances, local administration into the inner and/or middle ear provides a higher therapeutic index for an active agent that would otherwise have dose-limiting systemic toxicity. In addition, localized treatment of the auris interna or media also affords the use of previously undesired therapeutic agents, including agents with poor pK profiles, poor uptake, low systemic release, and/or toxicity issues.

[0030] Accordingly, provided herein are otic formulations that are designed to be administered at a suitable temperature (e.g., a temparature close to room temperature, e.g., about 20 °C) that avoids incidence of vertigo that is associated with administration of cold formulations (e.g., formulations having a temperature at time of administration of below about room temparature). Further, the formulations comprise thermoreversible polymers that are biocompatible and/or otherwise non-toxic to the inner ear environment. In some embodiments, the thermoreversible gel is biodegradable and/or bioeliminated (e.g., the copolymer is eliminated from the body by a biodegradation process, e.g., elimination in the urine, the feces or the like).

[0031] In some embodiments, for application to the inner ear, the auris formulations described herein are administered (e.g., via intratympanic injection, as ear drops in the ear canal, direct perfusion during otic surgery) behind and/or through the tympanic membrane at or near the round window membrane and/or the ossicular chain. In some embodiments, sustained release formulations described herein are injected as a liquid into the tympanic cavityin the vicinity of the round window membrane and gel and/or form thickened liquids upon contact with auditory surfaces.

[0032] In some other embodiments, for application to the middle ear, the auris formulations described herein are administered (e.g., via intratympanic injection, as ear drops in the ear canal, direct perfusion during otic surgery) behind and/or through the tympanic membrane so that they are not in contact with the round window membrane and/or the ossicular chain. In some embodiments, sustained release formulations described herein are administered in the tympanic cavity, away from the round window membrane. In some embodiments, the formulations are deposited, by injection, on the walls of the middle ear and gel and/or form thickened liquids upon contact with auditory surfaces.

[0033] In other embodiments, the formulations are administered as a paint (e.g., the formulations are smeared on the walls of the tympanic cavity using a cotton-tipped stick). In some embodiments, the formulations are sprayed (e.g., as a fluid, a foam or the like) into the middle ear cavity (e.g., when the tympanic membrane has ruptured). In some embodiments, the formulations are administered on the auditory walls and not on auditory bones (e.g., the ossicles).

[0034] In some embodiments, formulations described herein are low viscosity liquid compositions suitable for administration as ear drops. Following administration, the formulations form thickened liquids and/or gels that do not wash away from the middle ear and/or the round window membrane and provide sustained release of active agents. By way of example, when formulations comprising viscosity-enhancing polymers are administered to an individual suffering from otitis media with effusion, the formulations do not wash away, and remain in contact with the walls of the middle ear preventing infection and/or further accumulation of mucus. In certain other embodiments, the formulations are deposited on auditory bones (e.g., as a treatment for otosclerosis).

[0035] Accordingly, provided herein are methods for localized administration of sustained release otic compositions in the middle ear and/or the inner ear hich allows an active agent to reach a target organ and reduces or eliminates systemic accumulation of the active agent. In some embodiments, direct administration into the ear avoids disadvantages associated with permeability of the tympanic membrane. In other words, the release of active agents from formulations described herein is not impeded by the diffusion barrier presented by the tympanic membrane.

[0036] In some embodiments, the formulations described herein have a syringable viscosity. As used herein, a "syringable viscosity" is a viscosity that is low enough such that a pharmaceutical formulation described herein is a liquid that is capable of being administered (e.g., syringed) via narrow gauge needle or cannula or catheter using normal finger pressure (e.g., by a physician using normal finger pressure on the plunger of the syringe, such that the needle of the syringe can accurately and stably deliver the

pharmaceutical formulation at the targeted site (e.g., round window membrane of inner ear, sinonasal cavities or the like). Thus in some embodiments, formulations described herein are dispensed through a 18-31 gauge needle or cannula or catheter. In some embodiments, formulations described herein are dispensed through a 20-26 gauge needle or cannula or catheter. In some embodiments, formulations described herein are dispensed through a 25- 31 gauge needle or cannula or catheter. In some embodiments, formulations described herein are dispensed through a 27-31 gauge needle or cannula or catheter. In some embodiments, formulations described herein are syringable through a 27 gauge needle or cannula or catheter. In some embodiments, formulations described herein are syringable through a 29 gauge needle or cannula or catheter. In some embodiments, formulations described herein are syringable through a 31 gauge needle or cannula or catheter.

[0037] In some embodiments, an otic formulation described herein comprises between about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% and about 0.5%, 1%, 5%, 10%, 15%, 20% 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80% or 89% of a viscosity enhancing polymer. In some embodiments, a viscosity enhancing polymer is a thermoreversible polymer (e.g., a polyoxyethylene-polyoxypropylene triblock copolymer). In some embodiments, a viscosity enhancing polymer is a pH sensitive polymer. In some embodiments, a viscosity enhancing polymer is sensitive to concentration of ions (e.g., in some instances, alginates gel in presence of Ca⁺² ions).

[0038] In some embodiments, an auris-media or auris-externa compatible formulation described herein comprises between about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% and about 25%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of a

thermoreversible polymer. In some embodiments, the resulting formulation is a

thermoreversible gel, but it need not be thermoreversible; that is, depending on the amount of thermoreversible polymer, the resulting gel may be thermoreversible or not

thermoreversible. The classification "thermoreversible polymer" refers to polymers that are capable of forming thermoreversible gels in the range 15-42 degrees Celsius.

[0039] In some embodiments, a formulation described herein comprises at least about 14.0% and not more than about 25% of a thermoreversible polymer (e.g., polyoxyethylene- polyoxypropylene triblock copolymer) by weight of the composition. In some

embodiments, a formulation described herein comprises at least about 14.5% and not more than about 25% of a thermoreversible polymer (e.g., polyoxyethylene-polyoxypropylene triblock copolymer) by weight of the composition. In some embodiments, a formulation described herein comprises at least about 15% and not more than about 25% of a thermoreversible polymer (e.g., polyoxyethylene-polyoxypropylene triblock copolymer) by weight of the composition. In some embodiments, a formulation described herein comprises at least about 15% and not more than about 24% of a thermoreversible polymer (e.g., polyoxyethylene-polyoxypropylene triblock copolymer) by weight of the composition. In some embodiments, a formulation described herein comprises at least about 15% and not more than about 23% of a thermoreversible polymer (e.g., polyoxyethylene- polyoxypropylene triblock copolymer) by weight of the composition. In some

embodiments, a formulation described herein comprises at least about 15% and not more than about 22% of a thermoreversible polymer (e.g., polyoxyethylene-polyoxypropylene triblock copolymer) by weight of the composition. In some embodiments, a formulation described herein comprises at least about 15% and not more than about 21% of a thermoreversible polymer (e.g., polyoxyethylene-polyoxypropylene triblock copolymer) by weight of the composition. In some embodiments, a formulation described herein comprises at least about 16% and not more than about 21% of a thermoreversible polymer (e.g., polyoxyethylene-polyoxypropylene triblock copolymer) by weight of the composition.

[0040] In some embodiments, a formulation described herein comprises at least about 15% and not more than about 20% of a thermoreversible polymer (e.g., polyoxyethylene- polyoxypropylene triblock copolymer) by weight of the composition. In some

embodiments, a formulation described herein comprises at least about 15% and not more than about 19% of a thermoreversible polymer (e.g., polyoxyethylene-polyoxypropylene triblock copolymer) by weight of the composition. In some embodiments, a formulation described herein comprises at least about 15% and not more than about 18% of a thermoreversible polymer (e.g., polyoxyethylene-polyoxypropylene triblock copolymer) by weight of the composition. In some embodiments, a formulation described herein comprises at least about 15% and not more than about 17% of a thermoreversible polymer (e.g., polyoxyethylene-polyoxypropylene triblock copolymer) by weight of the composition.

[0041] In some of such embodiments, a thermoreversible polymer is a poloxamer. In some of such embodiments, the poloxamer is P407 (also known as PF-127, Pol-407, or Pluronic- 127). In some embodiments, a formulation comprising a W T modulator or neurotoxin described herein comprises at least about 14.5%, 15.0%, 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, or 18.0% and not more than about 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%, 20.0%, 21.0% or 25.0% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 14% and about 27% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 14.5% and about 25% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a W T modulator or neurotoxin described herein comprises between about 14.5% and about 21% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 14.5% and about 18% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 14.5% and about 16% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 14.5% and about 15.5% of PF-127 by weight of the composition.

[0042] In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 15.0% and about 25% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 15.0% and about 21% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 15% and about 18% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 15% and about 17% of PF-127 by weight of the composition. In some embodiments, a formulation comprising a WNT modulator or neurotoxin described herein comprises between about 15.0% and about 16.5% of PF 127 by weight of the composition.

[0043] By way of example, a buffered poloxamer 407 solution comprising between about 15-25% of poloxamer exhibits thermoreversible gelation properties and degrades in an aqueous environment.

[0044] In some embodiments, formulations described above that are suitable for treatment of middle ear conditions, have a gelation temperature between about 14 °C and about 42 °C and comprise between about 14% to about 27% of a thermoreversible polymer by weight of the composition. In some embodiments, the about 14% to about 27% of a thermoreversible polymer comprises a polyoxyethylene-polyoxypropylene triblock copolymer by weight of the composition.

[0045] In some embodiments, a formulation described herein has a gelation temperature of between about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, or 20 °C, and about 25 °C, 28 °C, 30 °C , 33 °C, 35 °C , 37 °C , 40 °C or 42 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 15 °C and about 40 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 15 °C and about 37 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 15 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 17 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 19 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 30 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 28 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 25 °C.

[0046] In some embodiments, the formulations described herein are free or substantially free of additional thickening agents. Examples of additional thickening agents include chitosan, or polyethylene glycol (PEG). In some embodiments, a formulation disclosed herein comprises less than about 5% by weight of chitosan. In some embodiments, a formulation disclosed herein comprises less than about 4% by weight of chitosan. In some embodiments, a formulation disclosed herein comprises less than about 3% by weight of chitosan. In some embodiments, a formulation disclosed herein comprises less than about 2% by weight of chitosan. In some embodiments, a formulation disclosed herein comprises less than about 1% by weight of chitosan. In some embodiments, a formulation disclosed herein comprises less than about 0.5% by weight of chitosan.

[0047] In some embodiments, the formulations described herein are free or substantially free of additional mucoadhesives. Examples of additional mucoadhesives include hyaluronic acid. In some embodiments, a formulation described herein comprises less than about 5% by weight of hyaluronic acid. In some embodiments, a formulation disclosed herein comprises less than about 4% by weight of hyaluronic acid. In some embodiments, a formulation disclosed herein comprises less than about 3% by weight of hyaluronic acid. In some embodiments, a formulation disclosed herein comprises less than about 2% by weight of hyaluronic acid. In some embodiments, a formulation disclosed herein comprises less than about 1% by weight of hyaluronic acid. In some embodiments, a formulation disclosed herein comprises less than about 0.5% by weight of hyaluronic acid.

[0048] In some embodiments, the formulations described herein are free or substantially free of additional preservatives and are suitable for administration to the ear. Additional preservatives do not include trace amounts of antioxidants (e.g., Butylated hydroxytoluene (BHT)) that stabilize thermoreversible polymers, and which are typically provided commercially with thermoreversible polymers. Examples of additional preservatives include benzethonium chloride, benzalkonium chloride, and thiomersal. In some embodiments, a formulation disclosed herein comprises less than about 50 ppm of each of benzethonium chloride, benzalkonium chloride, and thiomersal. In some embodiments, a formulation disclosed herein comprises less than about 25 ppm of each of benzethonium chloride, benzalkonium chloride, and thiomersal. In some embodiments, a formulation disclosed herein comprises less than about 20 ppm of each of benzethonium chloride, benzalkonium chloride, and thiomersal. In some embodiments, a formulation disclosed herein comprises less than about 10 ppm of each of benzethonium chloride, benzalkonium chloride, and thiomersal. In some embodiments, a formulation disclosed herein comprises less than about 5 ppm of each of benzethonium chloride, benzalkonium chloride, and thiomersal. In some embodiments, a formulation disclosed herein comprises less than about 1 ppm of each of benzethonium chloride, benzalkonium chloride, and thiomersal.

[0049] In some embodiments, the formulations described herein are free or substantially free of additional tonicity agents and are suitable for administration to the ear. Examples of additional tonicity agents include propylene glycol. Thus, in some embodiments, a formulation described herein is free or substantially free of propylene glycol. In some embodiments, a formulation disclosed herein comprises less than about 50 ppm of propylene glycol. In some embodiments, a formulation disclosed herein comprises less than about 25 ppm of propylene glycol. In some embodiments, a formulation disclosed herein comprises less than about 20 ppm of propylene glycol. In some embodiments, a formulation disclosed herein comprises less than about 10 ppm of propylene glycol. In some embodiments, a formulation disclosed herein comprises less than about 5 ppm of propylene glycol. In some embodiments, a formulation disclosed herein comprises less than about 1 ppm of propylene glycol. [0050] In some embodiments, the formulations described herein are free or substantially free of additional moisture retention agents and are suitable for administration to the ear. Examples of moisture retention agents include glycerin. Thus, in some embodiments, a formulation described herein is free or substantially free of glycerin. In some embodiments, a formulation disclosed herein comprises less than about 50 ppm of glycerin. In some embodiments, a formulation disclosed herein comprises less than about 25 ppm of glycerin. In some embodiments, a formulation disclosed herein comprises less than about 20 ppm of glycerin. In some embodiments, a formulation disclosed herein comprises less than about 10 ppm of glycerin. In some embodiments, a formulation disclosed herein comprises less than about 5 ppm of glycerin. In some embodiments, a formulation disclosed herein comprises less than about 1 ppm of glycerin.

[0051] The formulations described herein are substantially free of degradation products of the otic agent and/or the polymer components. As used herein, "substantially free of degradation products" means less than 5% by weight of the active agent and/or the polymer components are degradation products of the active agent and/or the polymer components. In further embodiments, the term means less than 3% by weight of the active agent and/or the polymer components are degradation products of the active agent and/or the polymer components. In yet further embodiments, the term means less than 2% by weight of the active agent and/or the polymer components are degradation products of the active agent and/or the polymer components. In further embodiments, the term means less than 1% by weight of the active agent and/or the polymer components are degradation products of the active agent and/or the polymer components.

[0052] In some embodiments, the formulations described herein are free or substantially free of additional common solvents and are suitable for administration to the ear. Examples of additional solvents include ethanol, propylene glycol, DMSO, N-Methyl-2-pyrrolidone, and cyclohexane. Thus, in some embodiments, a formulation described herein is free or substantially free of ethanol, propylene glycol, DMSO, N-Methyl-2-pyrrolidone, and cyclohexane. In some embodiments, a formulation disclosed herein comprises less than about 50 ppm of each of ethanol, propylene glycol, DMSO, N-Methyl-2-pyrrolidone, and cyclohexane. In some embodiments, a formulation disclosed herein comprises less than about 25 ppm of each of ethanol, propylene glycol, DMSO, N-Methyl-2-pyrrolidone, and cyclohexane. In some embodiments, a formulation disclosed herein comprises less than about 20 ppm of each of ethanol, propylene glycol, DMSO, N-Methyl-2-pyrrolidone, and cyclohexane. In some embodiments, a formulation disclosed herein comprises less than about 10 ppm of each of ethanol, propylene glycol, DMSO, N-Methyl-2-pyrrolidone, and cyclohexane. In some embodiments, a formulation disclosed herein comprises less than about 5 ppm of each of ethanol, propylene glycol, DMSO, N-Methyl-2-pyrrolidone, and cyclohexane. In some embodiments, a formulation disclosed herein comprises less than about 1 ppm of each of ethanol, propylene glycol, DMSO, N-Methyl-2-pyrrolidone, and cyclohexane.

[0053] In some embodiments, the formulations described herein are free or substantially free of additional antiseptics that are commonly used to disinfect any component of an otic preparation and that are potentially ototoxic. Examples of additional antiseptics that are known to be ototoxic include acetic acid, iodine and merbromin. Additionally,

chlorhexidene, a commonly used antiseptic, is used to disinfect components of an otic preparation (including devices used to administer the preparation) is highly ototoxic in minute concentrations (e.g., 0.05%). Thus, in some embodiments, a formulation disclosed herein is free or substantially free of acetic acid, iodine, merbromin, and chlorhexidene. In some embodiments, a formulation disclosed herein comprises less than about 50 ppm of each of acetic acid, iodine, merbromin, and chlorhexidene. In some embodiments, a formulation disclosed herein comprises less than about 25 ppm of each of acetic acid, iodine, merbromin, and chlorhexidene. In some embodiments, a formulation disclosed herein comprises less than about 20 ppm of each of acetic acid, iodine, merbromin, and chlorhexidene. In some embodiments, a formulation disclosed herein comprises less than about 10 ppm of each of acetic acid, iodine, merbromin, and chlorhexidene. In some embodiments, a formulation disclosed herein comprises less than about 5 ppm of each of acetic acid, iodine, merbromin, and chlorhexidene. In some embodiments, a formulation disclosed herein comprises less than about 1 ppm of each of acetic acid, iodine, merbromin, and chlorhexidene.

[0054] Further, otic preparations require particularly low concentrations of several potentially-common contaminants that are known to be ototoxic. Other dosage forms, while seeking to limit the contamination attributable to these compounds, do not require the stringent precautions that otic preparations require. For example, the formulations described herein are free or substantially free of contaminants such as arsenic, lead, mercury, and tin. Thus, in some embodiments, a formulation disclosed herein is free or substantially free of arsenic, lead, mercury, and tin. In some embodiments, a formulation disclosed herein comprises less than about 50 ppm of each of arsenic, lead, mercury, and tin. In some embodiments, a formulation disclosed herein comprises less than about 25 ppm of each of arsenic, lead, mercury, and tin. In some embodiments, a formulation disclosed herein comprises less than about 20 ppm of each of arsenic, lead, mercury, and tin. In some embodiments, a formulation disclosed herein comprises less than about 10 ppm of each of arsenic, lead, mercury, and tin. In some embodiments, a formulation disclosed herein comprises less than about 5 ppm of each of arsenic, lead, mercury, and tin. In some embodiments, a formulation disclosed herein comprises less than about 1 ppm of each of arsenic, lead, mercury, and tin.

[0055] To prevent ototoxicity, otic agent pharmaceutical compositions or formulations disclosed herein are optionally targeted to distinct regions of the targeted auris structures, including but not limited to the tympanic cavity, vestibular bony and membranous labyrinths, cochlear bony and membranous labyrinths, the ear canal, and other anatomical or physiological structures located within the auris interna, auris media, or auris externa.

Certain Definitions

[0056] The term "auris-acceptable" with respect to a formulation, composition or ingredient, as used herein, includes having no persistent detrimental effect on the auris interna (or inner ear), auris media (or middle ear), and auris externa (or external 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 interna (or inner ear), auris media (or middle ear), and auris externa (or external ear) and is relatively or is reduced in toxicity to the auris interna (or inner ear), auris media (or middle ear), and auris externa (or external ear), i.e., the material is administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0057] As used herein, amelioration or lessening of the symptoms of a particular otic disease, disorder or condition by administration of a particular compound or pharmaceutical composition refers to any decrease of severity, delay in onset, slowing of progression, or shortening of duration, whether permanent or temporary, lasting or transient that is attributed to or associated with administration of the compound or composition.

[0058] "Thermoreversible polymers" or " thermosetting polymers"are polymers that undergo a reversible temperature-dependent phase transtion (e.g., a liquid to gel transition, a gel to liquid transition, or the like). Example of thermoreversible polymers that form thermoreversible gels include and are not limited to poloxamers (e.g., Pluronics F68^®, F88^®, and F108^®, F127^®, or the like) or any other thermosetting polymer described herein.

[0059] "Viscosity enhancing polymers" are polymers that increase viscosity of a formulation described herein so that the formulation forms a thickened liquid upon administration. In some embodiments, a viscosity enhanging polymer is a thermos ens itive polymer. In some embodiments, a thermosensitive polymer is not a thermoreversible polymer. In other embodiments, a thermosensitive polymer is a thermoreversible polymer. Suitable viscosity-enhancing polymers that are thermosensitive polymers include and are not limited to, hydrogels (e.g., chitosan), gelatin, hyaluronic acid, acrylic acid based polymers (e.g., Carbopol®), MedGel®, cellulose based polymers (e.g.,

carboxymethylcellulose), polymers comprising polyoxyethylene-polyoxypropylene triblock copolymers, poloxamers, or any other such polymer described herein. In some of such embodiments, the resulting formulation is a thermosensitive gel, but it need not be thermoreversible; that is, depending on the amount of thermosensitive polymer, the resulting gel may be thermoreversible or not thermoreversible.

[0060] The terms "effective amount" or "therapeutically effective amount," as used herein, refer to a sufficient amount of the active agent or otic agent (e.g., a W T modulator, a neurotoxin) 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 an otic agent disclosed herein is reduction and/or alleviation of the signs, symptoms, or causes of tinnitus or balance disorders. For example, an "effective amount" for therapeutic uses is the amount of otic agent, including a formulation as disclosed herein required to provide a decrease or amelioration in disease symptoms without undue adverse side effects. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. An "effective amount" of an otic agent disclosed herein is an amount effective to achieve a desired pharmacologic effect or therapeutic

improvement without undue adverse side effects. It is understood that "an effective amount" or "a therapeutically effective amount" varies, in some embodiments, from subject to subject, due to variation in metabolism of the compound administered, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. It is also understood that "an effective amount" in an extended-release dosing format may differ from "an effective amount" in an immediate release dosing format based upon pharmacokinetic and pharmacodynamic considerations.

[0061] As used herein, the term "otic agent" refers to active agents that treat, or reduce or ameliorate severity of any otic disorder described herein. Suitable "otic agents" include W T modulators, Botulinum Toxin Type A, or any other otic agent described herein. "Otic agents" may work by any suitable mechanism, including by being anti-inflammatory, toxic, cytostatic, immunomodulatory, anti-apoptotic and/or by being modulators of auditory hair cells or auditory nerve signalling.

[0062] The mean residence time (MRT) is the average time that molecules of an otic agent reside in an otic structure after administration of a dose.

[0063] A "prodrug" refers to an 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 therapeutically active form of the compound. To 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 is designed 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.

[0064] As used herein, the term "subject" is used to mean an animal, preferably a mammal, including a human or non-human. The terms patient and subject may be used

interchangeably.

[0065] The terms "treat," "treating" or "treatment," as used herein, include alleviating, abating or ameliorating a disease or condition, for example tinnitus, 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 stopping the symptoms of the disease or condition either prophylactically and/or therapeutically.

[0066] Other objects, features, and advantages of the methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only.

Diseases

[0067] Otic disorders, including auris interna, auris media, and auris externa disorders, produce symptoms which include but are not limited to hearing loss, nystagmus, vertigo, tinnitus, inflammation, swelling, infection, aural fullnes, ringing in the ear and/or congestion.

Otitis Externa (Swimmer's ear)

[0068] Otitis externa (OE) is an inflammation and/or infection of the external ear.

Symptoms of OE include otalgia, swelling, and otorrhea. If the condition progresses significantly, OE may cause temporary conductive hearing loss as a result of the swelling and discharge.

[0069] Any inflammation of the outer ear canal, such as infections, allergies, or skin conditions, can lead to otitis externa (swimmer's ear). Excessive moisture in the ear canal from showering or swimming alters the acidic environment of the ear canal, and allows for the invasion of bacteria or fungi with subsequent infection. Staphylococcus aureus, Pseudomonas aeruginosa or fungi are common causes of swimmer's ear.

[0070] In some embodiments, the treatment of OE with formulations disclosed herein encompasses the treatment of chronic otitis externa and granular myringitis, a specific form of OE characterized by chronic inflammation of the pars tensa of the tympanic membrane, chronic stenosing otitis externa characterized by repeated infections, and malignant or necrotizing external otitis, an infection involving the temporal and adjacent bones.

Otitis Media

[0071] Otitis media (OM) includes acute otitis media (AOM, mastoiditis), chronic otitis media, otitis media with effusion (OME, Glue Ear), recurrent acute otitis media (RAOM), chronic otitis media with effusion (COME), secretory otitis media, chronic secretory otitis media, and/or serous otitis media. Otitis media is caused by multifactorial and complex factors, including environmental, microbial and host factors. Symptoms usually involve hearing loss or aural fullness or transudate formation as a result of a rapid decrease in middle ear pressure relative to the atmospheric pressure.

Autoimmune Inner Ear Disease

[0072] In some embodiments, formulations described herein are suitable for treatment of Autoimmune inner ear disease (AIED), Meniere's Disease, Meniere's syndrome, endolymphatic hydrops or postural vertigo.

Sensorineural Hearing Loss, Noise Induced Hearing Loss and Excitotoxicityy

[0073] In some embodiments, formulations described herein are suitable for treatment of sensorineural hearing loss or noise induced hearing loss. Sensorineural hearing loss occurs when the components of the inner ear or accompanying neural components are affected, and may contain a neural (i.e., the auditory nerve or auditory nerve pathways in the brain are affected) or sensory component. Sensory hearing loss may be hereditary, or it may be caused by acoustic trauma (i.e. very loud noises), a viral infection, side effects of drugs or Meniere's disease. Noise induced hearing loss is caused by loud noises, for example, gun fire, loud music or other human-based noise. In some instances, excitotoxicity (caused by, for example, over activation of glutamate receptors) damages auris nerve cells and induces hearing loss. In some instances hearing loss is associated with damage to auris-sensory hair cells. In some embodiments, administration of any formulation described herein (e.g., a formulation comprising a WNT modulator or Botulinum Toxin Type A) reduces or ameliorates severity of hearing loss. By way of example, in some embodiments, administration of a formulation comprising Botulinum Toxin Type A described herein deadens auditory nerves and/or reduces sensitivity of hair cells, thereby reducing or eliminating vertigo and/or ringing in the ear.

Cholesteatoma

[0074] In some embodiments, formulations described herein are suitable for the treatment of cholesteatoma. Cholesteatoma is a type of skin cyst located in the middle ear. Symptoms associated with cholesteatoma include dizziness, drainage from the ear, hearing loss, pain or numbness in or around the ear.

Otosclerosis

[0075] Otosclerosis is localized bone remodeling within the otic capsule of the human temporal bone. In the early phase, lesions in the bony structures of the ear soften stable otic capsule bone ("active phase"). The active phase is followed by a reparative phase with bone deposition. Lamellar bone is replaced by woven spongiotic bone of greater thickness and vascularity. This spongiotic phase ("otospongiosis") produces symptoms of progressive sensorineural hearing loss, tinnitus, dizziness, and Meniere's syndrome. In further stages of the disease, the sclerotic phase is a phase of active demineralization of the bone around the inner ear, but also with a harder or sclerotic element to it. The abnormal bone growth fixates the stapes footplate to the oval window of the cochlea. This impairs movement of the stapes and therefore transmission of sound into the inner ear. Additionally the cochlear round window can also become sclerotic, and impair movement of sound pressure waves through the inner ear. The reparative and sclerotic phases of this disorder are referred to as otosclerosis.

Meniere 's Disease

[0076] Meniere's disease is a disorder of the inner ear which causes episodes of vertigo, ringing in the ears (tinnitus), a feeling of fullness or pressure in the ear, and fluctuating hearing loss. The disorder affects the inner ear including the labyrinth, which includes both the semicircular canals and the cochlea. An attack of Meniere's disease is preceded by fullness in one ear. Hearing fluctuation or changes in tinnitus may also precede an attack. In some instances, a Meniere's episode involves severe vertigo (spinning), imbalance, nausea and vomiting. By way of example, in some embodiments, administration of a formulation comprising Botulinum Toxin Type A described herein deadens auditory nerves and/or reduces sensitivity of hair cells, thereby reducing or eliminating vertigo and/or ringing in the ear.

[0077] Formulations described herein are useful for the treatment of other otic disorders including and not limited to Bullous myringitis, Eustachian tubal catarrh, Eustachian salpingitis, Labyrinthitis (e.g., serous labyrinthitis), facial nerve neuritis, Ramsay Hunt Syndrome, osteoreadionecrosis of the temporal bone, otosyphilis, cochleovestibular disorders, hereditary disorders, including Scheibe, Mondini -Michelle, Waardenburg's, Michel, Alexander's ear deformity, hypertelorism, Jervell-Lange Nielson, Refsum's and Usher's syndromes, and other diseases described in U.S. Appl. Nos. 12/427,663,

12/466,310, 12/472,034, 12/486,697, 12/493,61 1, 12/494,156, 12/500,486, 12/504,553, 12/506,091, 12/506,127, 12/506,573, 12/506,616, and 12/506,664, the disclosure of otic diseases described therein is incorporated herein by reference. Otic diseases that are not disclosed herein but are treated by admins itration of the formulations described herein are expressly included and intended within the scope of the embodiments described herein. Otic Agents

[0078] Provided herein are otic agent compositions and formulations that treat otic disorders and/or their attendant symptoms, including but not limited to infection, hearing loss, nystagmus, vertigo, tinnitus, inflammation, swelling, and congestion. Pharmaceutically active metabolites, salts, polymorphs, prodrugs, analogues, and derivatives of the otic agents disclosed herein that retain the ability of the parent otic agents to treat otic disorders are expressly included and intended within the scope of embodiments presented.

[0079] The otic agent formulations disclosed herein are optionally targeted directly to otic structures where treatment is needed. Access to, for example, the vestibular and cochlear apparatus or tympanic cavity will occur through the auris media, auris interna and/or auris externa, including the round window and tympanic membrane, the oval window/stapes footplate, the annular ligament and/or through the otic capsule/temporal bone. In further or alternative embodiments, the auris formulations are capable of being administered via intratympanic injection, or via direct perfusion (e.g., during otic surgery). In other embodiments, the auris controlled release formulations are administered on or near the round window or the crista fenestrae cochleae through entry via post-auricular incision and surgical manipulation into or near the round window or the crista fenestrae cochleae area. Alternatively, the auris controlled release formulation is applied via syringe and needle, wherein the needle is inserted through the tympanic membrane and guided to the area of the round window or crista fenestrae cochleae. In some embodiments, otic agent formulations are delivered to the ear by injection, cotton swab or ear drops.

WNT modulators

[0080] In some embodiments, otic agents compatible with the formulations described herein include agents that modulate re-growth of damaged auris sensory hair cells. In some instances, modulation of the WNT pathway promotes morphogenesis and/or re-growth of damaged auris sensory hair cells. WNT signalling proteins include protein products encoded by genes such as WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT1 1, or WNT 16. Modulators of the WNT pathway include, and are not limited to, 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine, the signalling molecule Cerberus, or the like.

Neurotoxins

[0081] In some embodiments, otic agents compatible with the formulations described herein include neurotoxins for the treatment of otic nerve disorders. Such neurotoxins include venoms, channel agents and/or nerve agents including but not limited to Botulinum Toxin Type A (Botox®), erabutoxin, tetrodotoxin, batrachotoxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcicludine, PhTx3 or the like.

Vestibular suppressants

[0082] In some embodiments, otic agents compatible with the formulation described herein include vascular and/or vestibular suppressants. Examples of vestibular suppressants include and are not limited to anticholinergics, antihistamines, benzodiazepines, calcium channel antagonists, anti-emetics or the like. Examples of vestibular suppressants include and are not limited to meclizine, amytriptyline, droperidol, Lorazepam, Clonazepam,

Dimenhydrinate, Diazepam, or the like, and other vascular and/or vestibular suppressants described in U.S. Appl. No. 12/486,697, the vascular and/or vestibular suppressants described therein are incorporated herein by reference).

[0083] Other active agents that are compatible with the formulations described herein include and are not limited to otic agents described in U.S. Appl. Nos. 12/427,663,

12/466,310, 12/472,034, 12/486,697, 12/493,61 1, 12/494,156, 12/500,486, 12/504,553, 12/506,091, 12/506,127, 12/506,573, 12/506,616, and 12/506,664, the disclosure of otic agents described therein is incorporated herein by reference.

Imaging Devices

[0084] In some embodiments, any formulation described herein is used in combination with a mechanical or imaging device to monitor or survey the auris disorder (e.g., hearing loss). For example, magnetic resonance imaging (MRI) devices are contemplated within the scope of the embodiments described herein, wherein the MRI devices (for example, 3 Tesla MRI devices) are capable of evaluating disease progression (e.g., progression of Meniere's disease), and subsequent treatment with the otic formulations disclosed herein. In some embodiments, formulations described herein comprise Gadolinium-based dyes, iodine- based dyes, barium-based dyes, or the like and are used in the treatment of any otic disorder described herein and/or with any mechanical or imaging device or method described herein (e.g., a CAT scan). Such formulations allow for visualization of disease progression and/or formulation penetration in the ear and/or therapeutic effectiveness of the formulation. In certain embodiments, an imaging agent (e.g., gadolinium hydrate injection) is used in combination with three-dimensional real inversion recovery (3D-real IR) and/ three- dimensional fluid-attenuated inversion recovery (3D-FLAIR) magnetic resonance imaging (MRI), and/or any formulation described herein to evaluate disease severity (e.g., size of endolymphatic hydrops), formulation penetration into the inner and/or middle ear, and/or therapeutic effectiveness of the formulation. In some instances, a formulation described herein facilitates delivery of a sufficient amount of an imaging agent to the inner and/or middle ear and allows for visualization of disease progression and/or formulation penetration in the ear and/or therepautic effectiveness of the formulation.

[0085] In some embodiments, the compositions or devices described herein include a dye to help enhance the visualization of penetration of the formulation into the inner and/or middle ear. In some of such embodiments, dyes that are compatible with the auris-acceptable compositions described herein include and are not limited to Evans blue, Methylene blue, Isosulfan blue, Trypan blue, indocyanine green or the like.

Otic Surgery and Implants

[0086] In some embodiments, the pharmaceutical formulations, formulations described herein are used in combination with (e.g., implantation, short-term use, long-term use, or removal of) implants (e.g., cochlear implants). As used herein, implants include auris- interna, auris-media and/or auris-externa medical devices, examples of which include cochlear implants, hearing sparing devices, hearing-improvement devices, short electrodes, tympanostomy tubes, micro-prostheses or piston-like prostheses; needles; stem cell transplants; drug delivery devices; any cell-based therapeutic; or the like. In some instances, the implants are used in conjunction with a patient experiencing hearing loss. In some instances, the hearing loss is present at birth. In some instances, the hearing loss is associated with conditions such as AIED, glue ear, bacterial meningitis or the like that lead to osteoneogenesis and/or nerve damage with rapid obliteration of cochlear structures and profound hearing loss.

[0087] In some embodiments, administration of an otic formulation described herein in combination with an otic intervention (e.g., an intratympanic injection, surgery including a stapedectomy, a tympanostomy or the like, a medical device implant or a cell-based transplant) delays or prevents collateral damage to auris structures, e.g., irritation, inflammation and/or infection, caused by the external otic intervention (e.g., installation of an external device or surgery). In some embodiments, administration of an otic formulation described herein in combination with an implant allows for a more effective restoration of hearing loss compared to an implant alone. In some embodiments, administration of an otic formulation described herein in combination with an otic intervention reduces or eliminates post-surgical and/or post-implantation complications (e.g., inflammation, cell damage, infection, osteoneogenesis or the like). In some instances, perfusion of a surgical area with a formulation described herein reduces post-surgery or post- implantation recuperation time. In one aspect, formulations described herein, and modes of administration thereof, are applicable to methods of direct perfusion of the inner and/or middle ear compartments prior to otic intervention, during otic intervention, or after otic intervention, or a combination thereof.

Sterilization

[0088] Included within the embodiments disclosed herein are means and processes for sterilization of a pharmaceutical formulation disclosed herein for use in humans. The goal is to provide a safe pharmaceutical product, relatively free of infection causing microorganisms. The U. S. Food and Drug Administration has provided regulatory guidance in the publication "Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing" available at: http://www.fda.gov/cder/guidance/5882fnl.htm, which is incorporated herein by reference in its entirety.

[0089] As used herein, sterilization means a process used to destroy or remove

microorganisms and/or pyrogens that are present in a product or packaging. Available methods for the inactivation of microorganisms include, but are not limited to, the application of extreme heat, lethal chemicals, or gamma radiation.

[0090] Heat sterilization methods include the use of a saturated steam autoclave at a temperature of at least 121 °C, or dry heat sterilization (e.g., heating a dry powder for about 3 - 11 hours at internal powder temperatures of 130-140 °C, or for 1-2 hours at internal temperatures of 150-180 °C). Filtration sterilization is a method used to remove but not destroy microorganisms from solutions. [0091] In some embodiments, a formulation is subjected to terminal sterilization. In other words, the formulation that is autoclaved comprises the active agent and all the excipients. In other embodiments, all the excipients are subjected to heat sterilization and the active agent is sterilized separately; the active agent and the excipients are then mixed aseptically. In yet other embodiments, the active agent is sterilized separataely (e.g., dry-heat sterilized, irradiated, steam-sterilized) and the other excipients are sterile-filtered; the sterile active agent and the sterile-filtered solution are then mixed aseptically. In further embodiments, a sterile suspension of active agent in a solution comprising a thermosetting polymer is aseptically mixed with a second solution comprising a thermosetting polymer and optionally a second active agent.

[0092] In some embodiments, the formulations described above are prepared in a process wherein a first mixture comprising a higher concentration of an active agent (e.g., > 6%, >10%, >15% or >20% by weight of the formulation) and a viscosity enhancing polymer is prepared. In some of such embodiments, the higher drug concentration allows for heat sterilization of the first mixture while reducing or avoiding decomposition and/or aggregation and/or change in crystal form of the drug. The first mixture is heat sterilized. A second mixture comprising a viscosity enhancing polymer is prepared and heat sterilized and/or irradiated and/or fileter sterilized. The two mixtures are then mixed aspetically to provide a composition suitable for administration to the middle and/or inner ear.

[0093] In some instances, conventionally used methods of sterilization (e.g., heat treatment (e.g., in an autoclave), gamma irradiation, filtration) lead to irreversible degradation of polymeric components (e.g., thermosetting polymer components) and/or the active agent in the formulation. In some instances, sterilization of an auris formulation by filtration through membranes (e.g., 0.2 μιη membranes) is not possible if the formulation comprises thixotropic polymers.

[0094] Accordingly, provided herein are methods for sterilization of auris formulations that prevent degradation of polymeric components and/or the active agent during the process of sterilization. In some embodiments, the use of an appropriate thermosetting polymer in combination with a specific buffer and/or pH range for the formulation allows for high temperature terminal sterilization of formulations described herein with substantially low degradation of the therapeutic agent and/or the polymeric excipients. [0095] Any appropriate buffer is used depending on the otic agent used in the formulation. In some instances, since pK_a of TRIS decreases as temperature increases at approximately - 0.03/°C and pK_a of PBS increases as temperature increases at approximately 0.003/°C, autoclaving at 250°F (121°C) results in a significant downward pH shift (i.e. more acidic) in the TRIS buffer whereas a relatively much less upward pH shift in the PBS buffer and therefore much increased hydrolysis and/or degradation of an otic agent in TRIS than in PBS. Degradation of an otic agent and/or polymeric components is reduced by the use of an appropriate combination of a buffer and concentration of thermoreversible polymer.

[0096] In certain embodiments, any controlled release formulation described herein has less than about 100 colony forming units, less than about 60 colony forming units, less than about 50 colony forming units, less than about 40 colony forming units, or less than about 30 colony forming units of microbiological agents per gram of formulation. In some embodiments, the sterile formulations described herein are substantially free of microbes.

[0097] An additional aspect of the sterilization process is the removal of by-products from the killing of microorganisms. The process of depyrogenation removes such pyrogens from the sample. 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 E. coli LPS. Humans can develop a response to as little as 5 EU/kg of body weight. In certain embodiments, otic compositions described herein contain lower endotoxin levels (e.g. < 5 EU/kg of body weight of a subject, < 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 certain embodiments, the formulations described herein are substantially free of pyrogens.

pH and Practical Osmolarity

[0098] In some embodiments, an otic formulation disclosed herein is formulated to provide an ionic balance that is compatible with inner ear fluids (e.g., endolymph and/or perilymph) and/or middle ear fluids.

[0099] As used herein, "practical osmolarity/osmolality" or "deliverable

osmolarity/osmolality" means the osmolarity/osmolality of a formulation as determined by measuring the osmolarity/osmolality of the active agent and all excipients except the thermoreversible and/or viscosity enhancing polymer agent (e.g., polyoxyethylene- polyooxypropylene copolymers, or the like). The practical osmolarity of a formulation disclosed herein is measured by any suitable method, e.g., a freezing point depression method as described in Viegas et. al, Int. J. Pharm., 1998, 160, 157-162. In some instances, the practical osmolarity of a formulation disclosed herein is measured by vapor pressure osmometry (e.g., vapor pressure depression method) that allows for determination of the osmolarity of a formulation at higher temperatures. In some instances, vapor pressure depression method allows for determination of the osmolarity of a formulation comprising a thermoreversible polymer at a higher temperature such as for example the gelation temperature of the thermoreversible polymer.

[00100] In some embodiments, the osmolarity at a target site of action (e.g., the perilymph and/or middle ear) is about the same as the practical osmolarity (i.e., osmolarity of materials that cross or penetrate the round window and/or tympanic membrane) of a formulation described herein.

[00101] The practical osmolality of an otic formulation disclosed herein is from about 100 mOsm/kg to about 1000 mOsm/kg, from about 200 mOsm/kg to about 800 mOsm/kg, from about 250 mOsm/kg to about 500 mOsm/kg, or from about 250 mOsm/kg to about 320 mOsm/kg, or from about 250 mOsm/kg to about 350 mOsm/kg or from about 280 mOsm/kg to about 320 mOsm/kg. In some embodiments, a formulation described herein has a practical osmolarity of about 100 mOsm/L to about 1000 mOsm/L, about 200 mOsm/L to about 800 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about 250 mOsm/L to about 320 mOsm/L, or about 280 mOsm/L to about 320 mOsm/L. In some embodiments, the practical osmolality is estimated as an additive combination of buffer osmolality and the osmolality of the supernatant of the gelled poloxamer in water.

[00102] In specific embodiments, the practical osmolality of a formulation described herein is measured in a cell-based assay. The osmolality experienced by red blood cells isolated from guinea pigs was determined as a function of the hemolysis index. RBCs were placed in poloxamer solutions of varying concentrations. 0.5 mL of 10% guinea pig red blood cells in saline was added into a 2.5 mL solution of poloxamer 407 in buffer. The resulting suspension was serially diluted and the hemolysis index of RBCs was recorded for each solution. The hemolysis index is defined as the ratio of absorbance of a sample at 540 nm to the absorbance of a 0.9% saline solution at 540 nm. A hemolysis index of 1 indicates that the "practical osmolality" experienced by the RBCs is suitable for inner ear administration. The RBCs are intact in media with a suitable practical osmolality (Figure 3). The osmolality of the poloxamer solution was also measured by freezing point depression method or vapor pressure methods. The practical osmolality of the formulation is measured using commercially available osmometers and the value is confirmed by the hemolysis assay.

[00103] Table 1 shows a comparison of osmolality as determined by the serial dilution cell- based assay and a direct measurement using freezing point depression or vapor pressure methods. The serial dilution method is predictive of practical osmolality that is compatible with the inner ear environment.

Table 1

FP: freezing-point osmometry; VP: vapor-pressure osmometry

a Sample preparation: 0.5 mL of 10% guinea pig red blood cells in saline was added into 2.5 mL of P407 in buffer solution

b Hemolysis Index is defined as the 540 nm Absorbance ratio of sample:0.9% saline

[00104] In some embodiments, useful formulations also include one or more pH adjusting agents or buffering agents. Suitable pH adjusting agents or buffers include, but are not limited to acetate, bicarbonate, ammonium chloride, citrate, phosphate, pharmaceutically acceptable salts thereof and combinations or mixtures thereof. 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 buffering system and/or the osmolarity of physiological fluids. In some embodiments, the pH of a formulation described herein is between about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 and about 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 1 1.0, 1 1.5, or 12.0. In some embodiments, the pH of a formulation described herein is between about 3.0 and about 12.0. In some embodiments, the pH of a formulation described herein is between about 4.5 and about 10.0. In some embodiments, the pH of a formulation described herein is between about 3.5 and about 8.5. In some embodiments, the pH of a formulation described herein is between about 5.5 and about 8.0. In some embodiments, the pH of a formulation described herein is between about 5.5 and about 9.0. In some embodiments, the pH of a formulation described herein is between about 6.5 and about 8.0. In some embodiments, the pH of a formulation described herein is between about 7.0 and about 7.8. In some embodiments, the pH of a formulation described herein is between about 7.0 and about 7.6. In some embodiments, the pH of a formulation described herein is between about 7.0 and about 7.4. In some embodiments, the pH of a formulation described herein is between about 7.4 and about 7.8.

[00105] In some embodiments, the formulations described herein have a pH and/or practical osmolarity as described herein, and have a concentration of active pharmaceutical ingredient between about 1 μΜ and about 10 μΜ, between about 1 mM and about 100 mM, between about 0.1 mM and about 100 mM, betwen about 0.1 mM and about 100 nM. In some embodiments, the formulations described herein have a pH and/or practical osmolarity as described herein, and have a concentration of active pharmaceutical ingredient between about 0.01% - about 40%, between about 0.01% - about 20%, between about 0.01% - about 10%, between about 0.01% - about 7.5%, between about 0.01% - 6%, between about 0.01 - 5%, between about 0.1% - about 40%, between about 0.1% - about 30%, between about 0.1% - about 20%, between about 0.1 - about 10%, or between about 0.1 - about 6% of the active ingredient by weight of the formulation. In some embodiments, formulations described herein have a pH and/or practical osmolarity as described herein, and have a concentration of active pharmaceutical agent between about 1% - about 40%, between about 5% - about 40%>, between about 10%> - about 40%>, betwen about 15%> - about 40%>, between about 10% - about 30%, between about 10% - 20%, betwen about 15% - about 25%, or between about 20% - 30%, of the active ingredient by weight of the formulation. In some embodiments, the formulations described herein have a pH and/or practical osmolarity as described herein, and have a concentration of active pharmaceutical ingredient between about 1 μg/mL and about 500 g/mL, between about 1 μg/mL and about 250 g/mL, between about 1 μg and about 100 μg/mL, between about 1 μg/mL and about 50 μg/mL, or between about 1 μg/mL and about 20 μg/mL of the active agent by volume of the formulation.

Tunable Release Characteristics

Particle Size

[00106] Size reduction is used to increase surface area and/or modulate formulation dissolution properties and/or to maintain a consistent average particle size distribution (PSD) (e.g., micrometer-sized particles, nanometer-sized particles or the like) for any formulation described herein. In some embodiments, any thickened formulation described herein comprises multiparticulates, i.e., a plurality of particle sizes (e.g., micronized particles, nano-sized particles, non-sized particles, colloidal particles); i.e, the formulation is a multiparticulate formulation. In some embodiments, any formulation described herein comprises one or more multiparticulate (e.g., micronized) therapeutic agents. In some embodiments, any formulation described herein comprises micronized therapeutic agents. Micronization is a process of reducing the average diameter of particles of a solid material. In some embodiments, the average diameter of particles in a micronized solid is from about 0.5 μιη to about 500 μιη. In some embodiments, the average diameter of particles in a micronized solid is from about 1 μιη to about 200 μιη. In some embodiments, the average diameter of particles in a micronized solid is from about 2 μιη to about 100 μιη. In some embodiments, the average diameter of particles in a micronized solid is from about 3μιη to about 50 μιη. In some embodiments, the use of multiparticulates of otic agent allows for extended and/or sustained release of the active agent from any formulation described herein compared to a formulation comprising non-multiparticulate or a water-soluble otic agent.

[00107] In specific embodiments, upon administration of a sustained release formulation comprising micronized active agent to an individual in need thereof, the micronized active agent particles serve as a depot for further extended release of the active agent even after the gel has eroded. In some of such embodiments, the micronized particles remain adhered to middle surfaces. Accordingly, in some embodiments, sustained release formulations suitable for methods described herein comprise substantially high concentrations of micronized active agent. In some of such embodiments, sustained release formulations are suspensions comprising micronized active agents.

[00108] In some instances, any particle in any formulation described herein is a coated or uncoated particle (e.g., a coated micronized particle, nano-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, complex 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/or fluidized bed jet mills).

[00109] In some embodiments formulations described herein comprise crystalline particles and/or isotropic particles. In some embodiments, formulations described herein comprise amorphous particles and/or anisotropic particles. In some embodiments, formulations described herein comprise therapeutic agent particles wherein the therapeutic agent is a free base, or a salt, solvate, cocrystal or prodrug thereof of a therapeutic agent, or any combination thereof.

[00110] As illustrated in Figure 2, compositions comprising multiparticulate (e.g., micronized) otic agents provide release of a therapeutically effective amount of otic agent over a longer period of time compared to compositions comprising non-particulate and/or water soluble otic agents. In some instances, the multiparticulate and/or less water-soluble otic agent provides a steady supply (e.g., +/- 20%) of active agent via slow

dissolution/release and serves as a depot for the active agent; such a depot effect increases residence time of the otic agent in the ear. In specific embodiments, selection of an appropriate particle size of the active agent (e.g., micronized active agent) and solubility of the otic agent is water, in combination with the amount of thermoreversible and/or viscosity enhancing polymer component in the composition, provides tunable release characteristics that allow for release of an active agent over a period of hours, days, weeks or months.

Otic agent release profile

[00111] In some embodiments, the MDT for an active agent from a formulation described herein is from about 30 hours to about 48 hours. In some embodiments, the MDT for an active agent from a formulation described herein is from about 30 hours to about 100 hours. In some embodiments, the MDT for an active agent from a formulation described herein is from about 30 hours to about 150 hours. In some embodiments, the MDT for an active agent from a formulation described herein is from about 30 hours to about 200 hours. A linear relationship between the formulations mean dissolution time (MDT) and the poloxamer concentration indicates that the otic agent is released due to the erosion of the polymer and not via diffusion. A non-linear relationship indicates release of otic agent via combination of diffusion and/or polymer degradation.

[00112] The MDT is inversely proportional to the release rate of an active agent from a composition or device described herein. Experimentally, the released otic agent is optionally fitted to the Korsmeyer-Peppas equation

where Q is the amount of otic agent released at time t, Q_a 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 release mechanism wherein n=l characterizes an erosion controlled mechanism. The mean 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 optionally calculated by:

[00113] In some embodiments, the MDT for an active agent from a formulation described herein is from about 30 hours to about 1 week. In some embodiments, the MDT for a formulation described herein is from about 1 week to about 6 weeks.

[00114] In some embodiments, the mean residence time (MRT) for an active agent in a formulation described herein is from about 20 hours to about 48 hours. In some

embodiments, the MRT for an active agent from a formulation described herein is from about 20 hours to about 96 hours. In some embodiments, the MRT for an active agent from a formulation described herein is from about 20 hours to about 1 week. In some embodiments, the MRT for an active agent from a formulation described herein is from about 1 week to about 6 weeks. [00115] In some embodiments, the mean dissolution time (MDT) for poloxamer from a formulation described herein is at least 6 hours. In some embodiments, the MDT for poloxamer from a formulation described herein is at least 10 hours. In some embodiments, the MDT for poloxamer from a formulation described herein is at least 24, 48, 60, 100, 150, 200 or 250 hours. The MDT is determined using techniques described herein in, for example, Example 6.

[00116] In certain instances, once drug exposure (e.g., concentration in the inner and/or middle ear) of a drug reaches steady state, the concentration of the drug in the inner and/or middle ear fluids stays at or about the therapeutic dose for an extended period of time (e.g., one day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, 3 weeks, 6 weeks, 2 months). In some embodiments, the steady state concentration of active agent released from a formulation described herein is about 5 to about 20 times the steady state concentration of an active agent released from a formulation that is not a viscosity enhanced formulation. In some embodiments, the steady state concentration of active agent released from a formulation described herein is about 20 to about 50 times the steady state concentration of an active agent released from a formulation that is not a viscosity enhanced formulation.

[00117] In some embodiments, any formulation described herein (e.g., a formulation comprising viscosity enhancing polymers) provides extended release of an otic agent for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 2 weeks, at least 4 weeks, at least 6 weeks, or at least 8 weeks. In other embodiments, any formulation described herein (e.g., a formulation that is substantially free of viscosity enhancing polymers) provides release of an otic agent for at least 3 hours, at least 5 hours, at least 8 hours, at least 12 hours, at least 18 hours or at least 24 hours.

[00118] In some embodiments, the Mean Residence Time (MRT) of an otic agent in the perilymph for any formulation described herein is between about 5, 7, 10, 15, 20, 24, 36, 48, 60, 70 or 80 hours and about 100, 200, 300, 400, 500 or 600 hours.

[00119] In some embodiments, where administration of a formulation described herein is amenable to direct perfusion of an auris structure or cavity, a composition described herein is a suspension of microparticulates or micronized otic agent and is substantially free of viscosity enhancing polymer components. In such instances, the composition provides essentially immediate release of an active agent. In some of such embodiments, the composition is useful in perfusion of otic structures, e.g., during surgery.

[00120] In some embodiments, a composition described herein isa suspension of microparticulates or micronized otic agent and is substantially free of thermoreversible polymer components. In some of such embodiments, the composition provides essentially immediate release of an active agent. In other embodiments, a suspension of

microparticulates or micronized otic agent that is substantially free of thermoreversible polymer components provides intermediate sustained release of active agent. For example, in certain other embodiments, a formulation comprising microparticulates or micronized otic agent that is substantially free of a thermoreversible polymer provides an extended sustained release of active agent. As used herein, immediate release of an active agent refers to substantially complete release of an otic agent from the formulation in less than about 5 hours. As used herein, intermediate sustained release refers to a shorter period of sustained release of an active agent from a formulation such as, for example, release of the active agent over between about 0.5, 1, 2, or 3 days and about 2, 3, or 4 days. As used herein, sustained release refers to extended release of an active agent from a formulation such as, for example, a sustained release of active agent over at least 2, 3, 5, 7, 14, 21, 28 days, or at least 1, 2, 3, 4, 5 or 6 months.

[00121] The release profile of an otic agent from a solution or suspension or gel formulation is tunable as described above. Accordingly, in certain embodiments, a suspension of microparticulates or micronized otic agent provides intermediate sustained release or extended sustained release. In certain embodiments, a composition comprising a thermoreversible polymer and microparticulate or micronized otic agent provides intermediate sustained release or extended sustained release. In certain embodiments, a solution of an otic agent provides immediate release or intermediate sustained release.

[00122] In certain instances, once drug exposure (e.g., concentration in the endo lymph or perilymph) of a drug reaches steady state, the concentration of the drug in the endolymph or perilymph stays at or about the therapeutic dose for an extended period of time (e.g., one day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, 3 weeks, 6 weeks, 2 months). In some embodiments, the steady state concentration of active agent released from a controlled release formulation described herein is about 5 to about 20 times the steady state concentration of an active agent released from a formulation that is not a controlled release formulation. In some embodiments, the steady state concentration of active agent released from a controlled release formulation described herein is about 20 to about 50 times the steady state concentration of an active agent released from a formulation that is not a controlled release formulation.

[00123] In specific embodiments, any formulation described herein provides extended release of an otic agent for at least 7 days, at least 10 days, at least 2 weeks, at least 4 weeks, at least 6 weeks, or at least 8 weeks.

Pharmaceutical Formulations

[00124] Provided herein are pharmaceutical formulations that include at least one active agent and a pharmaceutically acceptable diluent(s), excipient(s), or carrier(s).

Viscosity Enhancing Polymers

[00125] Viscosity enhancing polymers form thickened liquids and/or gels when incorporated in aqueous solutions. In some embodimeents, viscosity enhancing polymers are composed of polyoxypropylene and polyoxyethylene block copolymers and form thermoreversible gels when incorporated into aqueous solutions. These polymers have the ability to change from the liquid state to the gel state at temperatures close to body temperture, therefore allowing useful formulations that are applied to the targeted structure(s). The liquid state-to- gel state phase transition (gelation temperature) is dependent on the polymer concentration, buffer concentration and the ingredients in the solution. In some embodiments, a thermoreversible gel suitable for compositions described herein is an aqueous gel comprising of a polymer of polyoxypropylene and polyoxyethylene.

[00126] Poloxamer is a synthetic block polymer of ethylene oxide and propylene oxide. Poloxamer 407 (PF-127, P407) is a thermoreversible polymer composed of

polyoxyethylene-polyoxypropylene copolymers. Other poloxamers include 124, 188 (F-68 grade), 237 (F-87 grade), and 338 (F-108 grade). Aqueous solutions of poloxamers are stable in the presence of acids, alkalis, and metal ions. PF-127 (or P407)is a commercially available polyoxyethylene-polyoxypropylene triblock copolymer of general formula E106 P70 El 06, with an average molar mass of 13,000. The polymer can be further purified by suitable methods that will enhance gelation properties of the polymer. It contains approximately 70% ethylene oxide, which accounts for its hydrophilicity. It is one of the series of poloxamer ABA block copolymers, whose members share the chemical formula shown below.

[00127] Poloxamers are available in several types, and with varying molecular weights ranging from about 2000 to about 15000. The a-hydro-co-hydroxypoly(oxyethylene)_a poly(oxypropylene)b poly(oxyethylene)_a block copolymers comprise varying ratios of a b as shown below:

[00128] In certain embodiments, a thermoreversible gel formulation described herein comprises a poloxamer. In specific embodiments, a thermoreversible gel formulation described herein comprises PF-127 (or P407). When placed in contact with the body, such a gel preparation will form a semi-solid structure and a sustained release depot. Furthermore, poloxamers (e.g., PF-127 and P407) have good solubilizing capacity, low toxicity, and are biocompatible.

[00129] In an alternative embodiment, the thermoreversible gel and/or viscosity enhancing polymer comprises a PEG-PLGA-PEG triblock copolymer (Jeong etal, 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 PLGA copolymer ranges from about 1 : 1 to about 20: 1. The resulting coploymers are soluble in water and form a free-flowing liquid at room temperature, but form a gel at body temperature.

[00130] ReGel® is a tradename of MacroMed Incorporated for a class of low molecular weight, biodegradable block copolymers having reverse thermal gelation properties as described in U.S. Pat. Nos. 6,004,573, 6, 1 17949, 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, 09/559,799 and 10/919,603. The biodegradable drug carrier comprises ABA-type or BAB-type triblock copolymers or mixtures thereof, wherein the A-blocks are relatively hydrophobic and comprise biodegradable polyesters or poly(orthoester)s, and the B-blocks are relatively hydrophilic and comprise polyethylene glycol (PEG), said copolymers having a hydrophobic content of between 50.1 to 83% by weight and a hydrophilic content of between 17 to 49.9% by weight, and an overall block copolymer molecular weight of between 2000 and 8000 Daltons.

[00131] In some embodiments, other gel formulations and/or viscosity enhancing polymers are useful depending upon the particular active agent, other pharmaceutical agent or excipients/additives used, and as such are considered to fall within the scope of the present disclosure. For example, other commercially-available glycerin-based gels, glycerin-derived compounds, conjugated, 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 pharmaceutical formulations described herein. In some embodiments, bioacceptable gels include, but are not limited to, alginate hydrogels SAF®-Gel (ConvaTec, Princeton, N.J.), Duoderm® Hydroactive Gel (ConvaTec), Nu-gel ©(Johnson & Johnson Medical,

Arlington, Tex.); Carrasyn®(V) Acemannan Hydrogel (Carrington Laboratories, Inc., Irving, Tex.); glycerin gels Elta® Hydrogel (Swiss-American Products, Inc., Dallas, Tex.), K-Y® Sterile (Johnson & Johnson), gelatin hydrogels, chitosan, silicon-base gels (e.g., Medgel®) or the like. Other thermosensitive and/or bioacceptable gels suitable for compositions described herein include acrylic acid-based polymers (e.g., Carbopol®), cellulose based polymers (e.g., hydroxypropylmethyl cellulose, carboxymethyl cellulose, or the like), alkyl aryl poly ether alcohol-based polymer (e.g., Tyloxapol®), or the like.

Gelation temperature

[00132] In one embodiment, a pharmaceutical formulation described herein is a liquid at about room temperature. In certain embodiments, the pharmaceutical formulation is characterized by a phase transition between about room temperature and about body temperature (including an individual with a serious fever, e.g., up to about 42 °C). In some embodiments, the phase transition occurs between at least about 1 °C below body temperature and body temperature, between at least about 2 °C below body temperature and body temperature, between at least about 3 °C below body temperture and body temperature, between at least about 4 °C below body temperature and body temperature, between at least about 6 °C below body temperature and body temperature, between at least about 8 °C below body temperature and body temperature, between at least about 10 °C below body temperature and body temperature, between at least about 15 °C below body temperature and body temperature, or between at least about 20 °C below body temperature and body temperature.

[00133] In one embodiment, administration of any formulation described herein at about room temperature (e.g., between about 18 °C to about 28 °C) reduces or inhibits vertigo associated with intratympanic administration of cold (e.g., temperature below about 18 °C) otic formulations. In some embodiments, the pharmaceutical formulations described herein are liquids (viscosity of less than about 500 cP) at about room temperature and are administered (injected or perfused) as liquids at or about room temperature.

[00134] In some embodiments, a formulation described herein has a gelation temperature of between about 5 °C, 10 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, or 20 °C, and about 25 °C, 28 °C, 30 °C , 33 °C, 35 °C , 37 °C , 40 °C or 42 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 5 °C and about 42 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 10 °C and about 42 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 14 °C and about 42 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 14 °C and about 40 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 14 °C and about 37 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 14 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 16 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 18 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 42 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 37 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 35 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 30 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 28 °C. In some embodiments, a formulation described herein has a gelation temperature of between about 20 °C and about 25 °C.

[00135] Since the polymer systems of thermoreversible gels dissolve more completely at reduced temperatures, methods of solubilization include adding the required amount of polymer to the amount of water to be used at reduced tempertures. 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. In some embodiments, the dissolution is carried out a temperature between about 10 °C and about 20 °C. The mixture is stirred or shaken to bring about a more rapid dissolution of the thermoreversible polymer. In some instances the active agent and/or other pharmaceutically active agent is suspended if it is insoluble in water. The pH/osmolarity of the formulation is modulated by the addition of appropriate buffering agents.

Viscosity

[00136] In some embodiments, a formulation described herein contains a thermoreversible polymer sufficient to provide a viscosity of between about 10,000 and about 1,000,000 centipoise. In some embodiments, a formulation described herein contains a

thermoreversible polymer sufficient to provide a viscosity of between about 50,000 and about 1,000,000 centipoise. In some embodiments, a formulation described herein contains a thermoreversible polymer sufficient to provide a viscosity of between about 150,000 and about 1,000,000 centipoise. In some embodiments, a formulation described herein contains a thermosetting polymer sufficient to provide a viscosity of between about 50,000 and about 600,000 centipoise. In some embodiments, a formulation described herein contains a thermoreversible polymer sufficient to provide a viscosity of between about 100,000 and about 500,000 centipoise. In some embodiments, a formulation described herein contains a thermoreversible polymer sufficient to provide a viscosity of between about 150,000 and about 400,000 centipoise. By way of example, a thermoreversible polymer concentration of about 15.5% in a composition described herein provides an apparent viscosity of about 270,000 cP. By way of example, a thermoreversible polymer concentration of about 16% in a composition described herein provides an apparent viscosity of about 360,000 cP. By way of example, a thermoreversible polymer concentration of about 17% in a composition described herein provides an apparent viscosity viscosity of about 480,000 cP. [00137] In some embodiments, the formulations described herein are low viscosity formulations at body temperature. In some embodiments, a low viscosity formulation described herein provides an apparent viscosity of from about 100 cP to about 10,000 cP.

[00138] The viscosity is measured at a shear rate of 0.31 s^"1 using a cone/plate viscometer ( Brookfield DVII + Pro viscometer with a CP50 spindle at 0.08 rpm as a reference).

Buffers

[00139] In some embodiments, formulations described herein comprise buffers. In one embodiment is a buffer such as acetate or citrate buffer at slightly acidic pH. In one embodiment the buffer is a sodium acetate buffer having a pH of about 4.5 to about 6.5. In one embodiment the buffer is a sodium citrate buffer having a pH of about 5.0 to about 8.0, or about 5.5 to about 7.0.

[00140] In an alternative embodiment, the buffer used is tris(hydroxymethyl)aminomethane, bicarbonate, carbonate or phosphate at slightly basic pH. In one embodiment, the buffer is a sodium bicarbonate buffer having a pH of about 6.5 to about 8.5, or about 7.0 to about 8.0. In another embodiment the buffer is a sodium phosphate dibasic buffer having a pH of about 6.0 to about 9.0.

[00141] In some embodiments, the concentration of the buffer component is adjusted to bring the practial osmolarity of any formulation described herein within a biocompatible range.

Solvents

[00142] In some embodiments, in a formulation described herein, the solvent is water. In some embodiments, a formulation described herein comprises a mixture of solvents (e.g., a mixture of water and an alcohol, or the like). In some embodiments, in a formulation described herein the solvent is a mixture of ethanol and water.

Additional excipients

[00143] In some embodiments, a formulation described herein further comprises additional biocompatible excipients. Example of additional excipients include agents for imaging and/or visualization, alkyl saccharides (e.g., dodecyl maltoside, or the like), hyaluronic acid, (including and not limited to Hyalastine®, Hyalectin®, Hyaloftil®), and/or partial esters and/or salts thereof (e.g., barium salt of hyaluronic acid, or any other salt of hyaluronic acid described in WO/1998/017285, salts described therein are incorported herein by reference), hyaluronidase (e.g., PH-20 (Halzoyme)) or any other excipient that modulates release profile and/or stabilty and/or permeability and/or drug uptake and/or bioavailability and/or toxicity and/or immunogenicity and/or gelation characteristics of any formulation described herein. In some embodiments, a formulation described herein comprises a mucoadhesive (e.g., an acrylic acid based polymer, hyaluronic acid or any other auris-compatible mucoadhesive). In some embodiments, a formulation described herein comprises a permeability enhancer (e.g., hyaluronic acid, dodecyl maltoside or any other auris- acceptable permeability enhancer). Additional excipients are described in U.S. Appl. Nos. 12/427,663, 12/466,310, 12/472,034, 12/486,697, 12/493,611, 12/494, 156, 12/500,486, 12/504,553, 12/506,091, 12/506, 127, 12/506,573, 12/506,616, and 12/506,664, the disclosure of excipients described therein is incorporated herein by reference.

Dosing Methods

[00144] The formulations described herein are administered via transtympanic injection or via non-transtympanic approach to the inner and/or middle ear. The injection volume varies based on concentration of the otic agent and/or the release profile of the otic agent. In additional embodiments, the formulation described herein is administered onto the round window membrane via surgical approach to the round window membrane comprising modification of the crista fenestrae cochleae. In some embodiments, for certain formulations described herein, injection volume is between about 25 μΐ., to about 400 \iL. In some embodiments, for certain formulations described herein, injection volume is between about 5 μϊ_^ to about 40 μ In some embodiments, for certain formulations described herein, injection volume is between about 1.5 μΐ., to about 10 μ In some embodiments, formulations described herein are administered as otic drops. In some embodiments, formulations described herein are perfused in auditory structures.

Frequency of Administration

[00145] In some embodiments, a compositon disclosed herein is administered to an individual in need thereof once. In some embodiments, a compositon disclosed herein is administered to an individual in need thereof more than once. In some embodiments, a composition described herein is administered as otic drops between 2 to 4 times a day. In some embodiments, a composition described herein is administered as otic drops once a day. In some embodiments, a composition described herein is administered as otic drops and/or injection once a day, every two days, every three day, every four days, every five days, every six days, once a week, every two weeks, every three weeks, or once a months. [00146] The number of times a composition is administered to an individual in need thereof depends on the discretion of a medical professional, the disorder, the severity of the disorder, and the individuals's response to the formulation. In some embodiments, a formulation described herein is administered as prophylactically, therapeutically or as a chronic treatment over an extended perior of time.

[00147] In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the otic agent compounds may be given continuously; alternatively, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 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 may be from 10%- 100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[00148] Once improvement of the patient's otic conditions has occurred, a maintenance otic agent dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is optionally reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms.

Kits/Articles of Manufacture

[00149] In some embodiments, otic formulations described herein are manufactured as ready to use single component solutions that are administered to an individual in need thereof. In other embodiments, otic formulations described herein are manufactured as multi- component kits comprising dry-heat sterilized multiparticulate (e.g., micronized, nanoparticles, non-sized particles) otic agent powder, a medium for reconstitution of the dry powder (e.g., sterile water or buffer or saline) and/or a solution comprising the

thermoreversible and/or viscosity enhancing polymer and a buffer. The dry powder is reconstituted with the sterile medium and/or the solution comprising the thermoreversible and/or viscosity enhancing polymer and buffer just prior to administration of the otic formulation to an individual in need thereof. EXAMPLES

Example 1 - Preparation of a Thermoreversible Gel comprising W T modulator

[00150] A 10-g batch of formulation containing a WNT modulator is prepared. 74 mg of sodium chloride USP (Fisher Scientific.) is dissolved with 8.2g of sterile filtered DI water and the pH is adjusted to 7.4 with 1 M NaOH. The buffer solution is chilled down and 3.0 g of poloxamer 407 (BASF Corp., containing approximately 100 ppm of BHT) is sprinkled into the chilled PBS solution while mixing, solution is mixed until all the poloxamer is dissolved. The poloxamer is sterile filtered using a 33mm PVDF 0.22μιη sterile syringe filter (Millipore Corp.) and delivered to 2 mL sterile glass vials (Wheaton) in an aseptic environment, the vials are closed with sterile butyl rubber stoppers (Kimble) and crimped sealed with 13 mm Al seals (Kimble). 20 mg of micronized 2-amino-4-[3,4- (methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine is placed in separate clean depyrogenated vials, the vials are closed with sterile butyl rubber stoppers (Kimble) and crimped sealed with 13 mm Al seals (Kimble), vials are dry heat sterilized (Fisher Scientific Isotemp oven) for 7 hours at 140°C. Before administration for the experiments described herein, 1 mL of the cold poloxamer solution is delivered to a vial containing 20 mg of sterile micronized 2-amino-4- [3 ,4-(methylenedioxy)benzyl-amino] -6-(3 - methoxyphenyl)pyrimidine using a 21G needle (Becton Dickinson) attached to a 1 mL sterile syringe (Becton Dickinson), suspension mixed well by shaking to ensure homogeneity of the suspension. The suspension is then withdrawn with the 21G syinge and the needle is switched to a 27 G needle for administration.

Example 2 - Preparation of a Thermoreversible Gel Botulinum Toxin Type A Composition

[001511 16% poloxamer 407 NF in 50mM TRIS buffer: Weigh 0.4518 g of sodium chloride (Fisher scientific) + 0.6034 g of tromethamine (Fisher scientific) dissolve with 82 g of DI water, then add 850 of 5 N HC1 to adjust pH to 7.5. Weigh 67.3 g of above buffer, cool down buffer then sprinkle 12.8 g of poloxamer 407 NF (Spectrum chemicals) while mixing. Mix until a clear translucid solution is obtained. Filter-sterilize the solution using a 0.2μιη sterilizing filter.

[00152] Onabotulinumtoxin A is reconstituted in 16% P407 in 50 mM TRIS buffer described above and sterile filtered.

Example 3 - Dissolution testing

[00153] Dissolution is performed at 37°C in snapwells (6.5 mm diameter polycarbonate membrane with a pore size of 0.4 μιη). 0.2 mL of the formulation is placed into snapwell and left to harden, then 0.5 mL of 0.9% saline 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 active agent concentration by UV at 215 nm using an Evolution 160 UV/Vis spectrophotometer (Thermo Scientific).

Quantitation is performed against an external calibration standard.

Example 4 Effect of pH on degradation products for autoclaved 17% poloxamer 407NF/ 2% otic agent in PBS buffer

[00154] A stock solution of a 17% poloxamer 407/ 2% otic agent is prepared by dissolving 351.4 mg of sodium chloride (Fisher Scientific), 302.1 mg of sodium phosphate dibasic anhydrous (Fisher Scientific), 122.1 mg of sodium phosphate monobasic anhydrous (Fisher Scientific) and an appropriate amount of an otic agent with 79.3 g of sterile filtered DI water. The solution is cooled down in a ice chilled water bath and then 17.05 g 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.

[00155] 17% poloxamer 407/ 2% otic agent in PBS pH of 5.3. Take an aliquot

(approximately 30mL) of the above solution and adjust the pH to 5.3 by the addition of 1 M HC1.

[00156] 17% poloxamer 407/ 2% otic agent in PBS pH of 8.0. Take an aliquot

(approximately 30mL) of the above stock solution and adjust the pH to 8.0 by the addition of 1 M NaOH.

[00157] A PBS buffer (pH 7.3) is prepared by dissolving 805.5 mg of sodium chloride

(Fisher Scientific), 606 mg of sodium phosphate dibasic anhydrous (Fisher Scientific), 247 mg of sodium phosphate monobasic anhydrous (Fisher Scientific), then QS to 200g with sterile filtered DI water.

[00158] 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.

[00159] One 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-sterilmatic autoclave (settings, slow liquids) and sterilized at 250°F for 15 minutes. After the autoclave the samples are left to cool down to room temperature and then placed in refrigerator. The samples are homogenized by mixing the vials while cold.

[00160] Appearance (e.g., discoloration and/or precipitation) is observed and recorded. HPLC analysis is performed using an Agilent 1200 equipped with a Luna CI 8(2) 3μιη, lOOA, 250x4.6 mm column) using a 30-80 acetonitrile gradient (1-lOmin) of (water - acetonitrile mixture containing 0.05%TFA), for a total run of 15 minutes. Samples are diluted by taking 30μΙ. 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.

Example 5 Effect of buffer type on the degradation products for formulations containing poloxamer 407NF after heat sterilization (autoclaving).

[00161] A TRIS buffer is made by dissolving 377.8 mg of sodium chloride (Fisher

Scientific), and 602.9 mg of Tromethamine (Sigma Chemical Co.) then QS to lOOg with sterile filtered DI water, pH is adjusted to 7.4 with 1M HC1.

Stock solution containing 25% Poloxamer 407 solution in TRIS buffer:

[00162] Weigh 45 g of TRIS buffer, chill in an ice chilled bath then sprinkle into the buffer, while mixing, 15 g of poloxamer 407 NF (Spectrum Chemicals). The mixture is further mixed until all the poloxamer is completely dissolved.

[00163] A series of formulations is prepared with the above stock solution. An appropriate amount of otic agent (or salt or prodrug thereof) and/or otic agent as

micronized/coated/liposomal particles (or salt or prodrug thereof) is used for all experiments.

Stock solution (pH 7.3) containing 25% Poloxamer 407 solution in PBS buffer:

[00164] PBS buffer described above is used. Dissolve 704mg of sodium chloride (Fisher Scientific), 601.2 mg of sodium phosphate dibasic anhydrous (Fisher Scientific), 242.7 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.

[00165] A series of formulations is prepared with the above stock solution. An appropriate amount of otic agent (or salt or prodrug thereof) and/or otic agent as

[00166] One 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-sterilmatic 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 homogenize the samples.

[00167] HPLC analysis is performed using an Agilent 1200 equipped with a Luna CI 8(2) 3μιη, lOOA, 250x4.6 mm column) using a 30-80 acetonitrile gradient (1-lOmin) of (water - acetonitrile mixture containing 0.05%TFA), for a total run of 15 minutes. Samples are diluted by taking 30μΙ_^ of sample and dissolving with 1.5mL of a 1 : 1 acetonitrile water mixture. Purity of the otic agent in the autoclaved samples is recorded. The stability of formulations in TRIS and PBS buffers is compared.

[00168] Viscosity measurements are performed using a Brookfield viscometer RVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31 s^"1), equipped with a water jacketed temperature control unit (temperature ramped from 15-34°C 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. Only formulations that show no change after autoclaving are analyzed.

Example 6: In vitro comparison of relase profile.

[00169] Dissolution is performed at 37°C in snapwells (6.5 mm diameter polycarbonate membrane with a pore size of 0.4 μιη), 0.2 mL of a gel and/or thickened formulation described herein is placed into 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 every hour (0.1 mL withdrawn and replace with warm buffer). Samples are analyzed for otic agent concentration by UV at 245 nm against an external calibration standard curve. Pluronic concentration is analyzed at 624 nm using the cobalt thiocyanate method. Relative rank- order of mean dissolution time (MDT) as a function of %P407 is determined. A linear relationship between the formulations mean dissolution time (MDT) and the P407 concentration indicates that the otic agent is released due to the erosion of the polymer and not via diffusion. A non-linear relationship indicates release of otic agent via combination of diffusion and/or polymer degradation.

[00170] The MDT is inversely proportional to the release rate of an active agent from a composition or device described herein. Experimentally, the released otic agent is optionally fitted to the Korsmeyer-Peppas equation:

where Q is the amount of otic agent released at time t, Qa 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 release mechanism wherein n=l characterizes an erosion controlled mechanism. The mean 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 optionally calculated by:

[00171] Alternatively, samples are analyzed using the method described by Li Xin-Yu paper [Acta Pharmaceutica Sinica 2008,43(2):208-203] and Rank-order of mean dissolution time (MDT) as a function of %P407 is determined. Example 7 - Application of an Enhanced Viscosity Otic agent Formulation onto the Round Window Membrane

[00172] A formulation according to Example 1 is prepared and loaded into 5 ml siliconized glass syringes attached to a 15-gauge luer lock disposable needle. Lidocaine is topically applied to the 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 otic agent formulation applied directly onto the round-window membrane.

Example 8 - Application of an Enhanced Viscosity Otic agent Formulation onto the middle ear [00173] A formulation according to Example 1 is prepared and loaded into 5 ml siliconized glass syringes attached to a 15-gauge luer lock disposable needle. Lidocaine is topically applied to the tympanic membrane, and the needle tip is guided into place over the tympanic membrane. The otic agent formulation is applied by piercing the anterior inferior portion of the tympanic membrane.

Example 9 - In vivo testing of Intratvmpanic Injection of otic agent formulation in a guinea

Eig.

[00174] Female guinea pigs (Charles River) weighing 200-300 g, of approximately 6-8 weeks of age are used (N = 4 per group). Prior to any procedure, animals are anesthetized using a combination of xylazine (10 mg/kg), ketamine (40 mg/kg) and acepromazine (0.75 mg/kg) for up to an hour via the intramuscular route. If needed, an intraoperative booster is administered intraperitoneally representing one-tenth of the original dose. Intratympanic injection - Each animal is positioned so that the head is tilted at an angle to favor injection towards the round window niche. Briefly, under visualization with an operating microscope, 50 μΐ of formulations comprising 0 to 50% otic agent and varying concentrations of PF-127 are admininstered to the animals. The formulations are injected using a 27G or 30G needle through the tympanic membrane into the superior posterior quadrant behind which the round window niche is located. During the procedure and until recovery, animals are placed on a temperature controlled (40 °C) heating pad until consciousness is regained at which time they are returned to the vivarium. Perilymph sampling procedure - The skin behind the ear of anesthetized guinea pigs is shaved and disinfected with povidone-iodine. An incision is then made behind the ear, and muscles are carefully retracted from over the bulla. A hole is drilled though the bulla using a dental burr so that the middle ear is exposed and accessible. The cochlea and the round window membrane are visualized under a stereo surgical microscope. A unique microhole is hand drilled through the bony shell of the cochlea (otic capsule) adjacent to the round window. Perilymph (5 μΐ) is then collected using a microcapillary inserted into the cochlear scala tympani. Plasma and CSF collection methods - Blood is collected by cardiac puncture into heparin coated tubes. To collect the cerebrospinal fluid (CSF), a small skin incision is made just posterior to the cranial vertex. The skin is then retracted, and the trapezius muscle scraped off the occipital bone. A small hole is then drilled through the bone. The dura is cut with a sharp scalpel and a micropipette inserted to collect blood-free CSF (50 μΐ). Analytical method

[00175] Determination of otic agent concentrations is performed using high pressure liquid chromatography (HPLC) combined with mass spectrometry detection (MS). The limit of detection of the method is 1.0 ng/ml. Samples (perilymph, plasma and CSF) are extracted by liquid-liquid extraction using dichloromethane:hexane:MTBE (1 : 1 : 1 v/v/v). The organic portion is then dried and the extracts reconstituted with a watenmethanol solution (1 : 1, v/v). The samples are analyzed by reversed phase HPLC (1100 series, Agilent) using an Atlantis dC18 column maintained at 40 °C. The mobile phase is nebulized using heating nitrogen in a Z-spray source/interface and the ionized compounds detected using MS/MS (Tandem quadrupole mass spectrometer, Quattro Ultima, Waters). Peak heights of an otic agent are determined using MassLynx software (Waters). The calibration curves are obtained by fitting the peak height ratios of analyte / internal standard and the standard concentrations to a suitable equation using MassLynx. Sample otic agent concentrations are then interpolated using the equations derived from the calibration curves.

Data Analysis

[00176] Pharmacokinetic parameters are calculated using conventional noncompartmental pharmacokinetic methods. The apparent clearance (CL app) is calculated as the ratio between the administered intratympanic dose and the exposure (AUC). Thus the injection volume and the concentration of an otic agent and poloxamer in a formulation are tested to determine optimal parameters for preclinical and clinical studies.

Example 10 - In vivo testing of Intratympanic Injection of otic agent formulation in sheep

[00177] Female sheep (Buckham Sheep Farm, Kalamazoo, MI) weighing 50-65kg, of approximately 2-4 years of age are used (N=l, 2 ears per group). Prior to any procedure, animals are anesthesized using a combination of xylazine (0.22 mg/kg), glycopyrrolate (0.01 mg/kg) and ketamine (15 mg/kg) administered IM in addition to isoflurane by inhalation. Intratympanic injection - Each intubated animal is immobilized and placed laterally in reverse trendelenburg position, with the rostrum slightly elevated to ensure access to the round window. Following ear cleaning (using Otocalm and warm saline), and under otoscopic visualization, 600 μΐ of formulations comprising 0 to 50% otic agent and PF-127 are admininstered to the animals. The formulation are injected using a 25G or 27G needle through the tympanic membrane into the posterior inferior quadrant towards the round window niche. After dosing, the animal is left on an incline with its head up for approximately 30min to allow the dosing solution to settle into the tympanic cavity.

Procedure is then repeated for the opposite ear. Perilymph sampling procedure - The animal is intubated and placed in lateral recumbency. A post-auricular skin incision is made and the post-auricular vein located and ligated. Cautery is performed to expose the bulla and temporal bone. The middle ear is accessed using a nitrogen powered drill and a round- tipped bur. The middle ear ossicles are pushed to the side, with care taken to avoid damaging the round window membrane. Using a 0.5-1 mm round-tipped burr, a hole is hand drilled into the basal turn of the cochlea until the bone is thin enough to pierce with a modified sewing needle. Perilymph (50μ1) is then collected using Hamilton syringe connected to a 28-32G needle inserted into the cochlear scala tympani. Plasma and CSF collection methods - Blood is collected from the jugular vein into heparin coated tubes. To collect the cerebrospinal fluid (CSF), a small skin incision is made over the cisterna magna and a 22G needle inserted to sample blood-free CSF (500 μΐ).

[00178] The samples are analysed as described above. The gel elimination time course for each formulation is determined. A faster gel elimination time course of a formulation indicates lower mean dissolution time (MDT). Thus the injection volume and the concentration of an otic agent and poloxamer in a formulation are tested to determine optimal parameters for preclinical and clinical studies.

Example 11 - In vivo extended release kinetics

[00179] A cohort of 21 guinea pigs (Charles River, females weighing 200-300g) is intratympanically injected with 50 μϊ_^ 15 - 17% Pluronic F-127 formulation buffered at 280 mOsm/kg and containing 1.5% to 35% otic agent by weight of the formulation. Animals are dosed on day 1. The release profile for the formulations is determined based on analysis of the perilymph and/or the middle ear fluids. Example 12 - Evaluation of W T modulator formulation in an AIED Animal Model

Methods and Materials

Induction of Immune Response

[00180] Female albino National Institutes of Health-Swiss mice (Harlan Sprague-Dawley, Inc., Indianapolis, Inc.) 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 (associated KLH) is dissolved in PBS and injected subcutaneous ly in the back of the animal (0.2 mg emulsified in Freund's complete adjuvant). The animals are given a booster (0.2 mg KLH in Freund's incomplete adjuvant, and then injected ten weeks later with 0.1 mg KLH in 5 μΐ PBS (pH 6.4) 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 μιη hole is drilled through the cochlear capsule into the scala tympani of the lateral basal turn. KLH or PBS control is slowly injected using a Hamilton syringe coupled with a plastic tube to a glass micropipette 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

[00181] KLH and control mice are sorted into two groups (n = 10 in each group). The otic agent formulation of Example 1 is applied to the round window membrane of one group of animals. Control formulation containing no otic agent is applied to the second group. The otic agent and control formulations are reapplied three days after the initial application. The animals are sacrificed after the seventh day of treatment.

Analysis of Results

Electrophysiologic Testing

[00182] The hearing threshold for the auditory brainstem response threshold (ABR) to click stimuli for each ear of each animal is initially measured and 1 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 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=512) and displayed. Threshold is defined as the stimulus level between the record with no visibly detectable response and a clearly identifiable response.

Histochemical analysis

[00183] Animals 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 7.2) for 14 days (4°C). After decalcification, temporal bones are immersed sequentially in increasing concentrations (50%, 75%, 100%) of optimal cutting temperature (OCT) compound

(Tissue-Tek, Miles Inc., Elkhart, IN), snap-frozen (-70°C), and cryostat-sectioned (4 μιη) parallel to the modiolus. Sections are collected for hematoxylin and eosin (H&E) staining and immunohistochemical analysis.

[00184] The severity of inflammation is 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 inflammation, and a score of 5 indicates that all cochlear turns had severe infiltration of inflammatory cells.

Example 13 - Evaluation of Botulinum Toxin Type A formulation in an AIED Animal Model

[00185] The formulation from Example 2 is also tested using a similar procedure as described above.

Example 14 - Evaluation of W T Modulator Administration in Meniere's Patients

[00186] Study Objective

[00187] The primary objective of this study will be to assess the safety and efficacy of a WNT modulator in ameliorating Meniere's Disease in human subjects.

[00188] Study Design

[00189] This will be a Prospective, Randomized, Double-blind, Placebo-controlled, Multicenter, Phase IB Study comparing WNT modulator administration to placebo in the treatment of Meniere's disease in patients with unilateral disease. Approximately 100 subjects will be enrolled in this study. Each group will receive either a single dose of a sustained release WNT modulator formulation from Example 1 or placebo treatment. [00190] Subjects who do not complete the study will not be replaced. Patients receiving the study drug will be administered a gel formulation directly onto the subjects' round window membrane and monitored for 3 months. Each patient will receive a vestibular and hearing evaluation before the treatment and every two weeks after administration of the study drug.

[00191] Primary Outcome Measures:

[00192] The primary objective of this study is to evaluate the safety and tolerability of two ascending doses of the W T modulator relative to placebo. Safety assessments will be performed for 3 months post single intratympanic injection of the WNT modulator or placebo.

[00193] Secondary Outcome Measures:

[00194] The secondary objective of this study is to evaluate the clinical activity of two doses of a WNT modulator relative to placebo. Change in baseline for vertigo frequency will be evaluated. The impact of tinnitus on activities of daily living will be measured. Hearing loss in the affected ear will be measured by audiometric examination. Quality of life will be measured by patient reported questionnaire. Severity of vertigo episodes will be measured by the patient reported vertigo score.

Example 15 - Clinical Trials of Botulinum Toxin Type A in Tinnitus Patients

[00195] Study Objective

[00196] The primary objective of this study will be to assess the safety and efficacy of

Botulinum Toxin Type A compared with that of placebo in ameliorating tinnitus symptoms in afflicted patients.

[00197] Study Design

[00198] Approximately 100 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 Botulinum Toxin Type A delivered in a thermoreversible gel, or controlled release placebo formulation. Route of Administration will be intratympanic injection.

[00199] Primary Outcome Measure

[00200] 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).

[00201] Secondary Outcome Measures [00202] 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).

[00203] 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 embodiments 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

1. A pharmaceutical composition suitable for use in the treatment of otic disorders by intratympanic administration on or near the round window membrane of the ear, which comprises an auris acceptable thermoreversible aqueous gel comprising of a polyoxypropylene and polyoxyethylene triblock copolymer and a W T modulator such that sustained release of the WNT modulator across the round window membrane into the cochlea occurs for a period of at least 3 days.

2. The composition of claim 1, wherein the composition has a pH between about 7.0 and about 8.0.

3. The composition of claim 1 , wherein sustained release is provided for a period of at least 5 days.

4. The composition of claim 1, wherein sustained release is provided for a period of at least 7 days.

5. The composition of claim 1, wherein sustained release is provided for a period of at least 10 days.

6. The composition of claim 1, wherein the polyoxypropylene and polyoxyethylene triblock copolymer is present in the composition in an amount between about 14% and about 27% by weight of the composition.

7. The composition of claim 1, wherein the polyoxypropylene and polyoxyethylene triblock copolymer is present in the composition in an amount between about 15% and about 21% by weight of the composition.

8. The composition of claim 1, wherein the polyoxypropylene and polyoxyethylene triblock copolymer is poloxamer 407.

9. The composition of claim 1, wherein the composition comprises a suspension of multiparticulate WNT modulator.

10. The composition of claim 1, wherein the multiparticulate WNT modulator is

essentially micronized WNT modulator.

1 1. The composition of any one of claims 1-10, wherein the WNT modulator is

2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine, or Cerberus.

12. The composition of any one claims 1-10, wherein the W T modulator is 2-amino-4- [3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine.

13. The composition of claim 1, wherein the otic disorder is selected from Meniere's disease, sudden sensorineural hearing loss, noise induced hearing loss, age related hearing loss, vertigo and tinnitus.

14. A pharmaceutical composition suitable for use in the treatment of otic disorders by intratympanic administration on or near the round window membrane of the ear, which comprises an auris acceptable thermoreversible aqueous gel comprising of a polyoxypropylene and polyoxyethylene triblock copolymer and a neurotoxin such that sustained release of the neurotoxin across the round window membrane into the cochlea occurs for a period of at least 3 days.

15. The composition of claim 14, wherein the composition has a pH between about 7.0 and about 8.0.

16. The composition of claim 14, wherein sustained release is provided for a period of at least 5 days.

17. The composition of claim 14, wherein sustained release is provided for a period of at least 7 days.

18. The composition of claim 14, wherein sustained release is provided for a period of at least 10 days.

19. The composition of claim 14, wherein the polyoxypropylene and polyoxyethylene triblock copolymer is present in the composition in an amount between about 14% and about 27% by weight of the composition.

20. The composition of claim 14, wherein the polyoxypropylene and polyoxyethylene triblock copolymer is present in the composition in an amount between about 15% and about 21% by weight of the composition.

21. The composition of claim 14, wherein the polyoxypropylene and polyoxyethylene triblock copolymer is poloxamer 407.

22. The composition of claim 14, wherein the composition comprises a suspension of multiparticulate neurotoxin.

23. The composition of claim 14, wherein the multiparticulate neurotoxin is essentially micronized neurotoxin.

24. The composition of any one of claims 14-23, wherein the neurotoxin is Botulinum Toxin Type A, erabutoxin, tetrodotoxin, batrachotoxin, maurotoxin, agitoxin, charybdotoxin, margatoxin, slotoxin, scyllatoxin, hefutoxin, calciseptine, taicatoxin, calcicludine, or PhTx3

25. The composition of any one of claims 14-23, wherein the neurotoxin is Botulinum Toxin Type A.

26. The composition of claim 14, wherein the otic disorder is selected from Meniere's disease, sudden sensorineural hearing loss, noise induced hearing loss, age related hearing loss, vertigo and tinnitus.