Methods and compositions for enhancing drug delivery into the inner ear

Abstract

Disclosed herein are methods and compositions for enhancing the delivery of therapeutics through the round window membrane into the inner ear; i.e., enhancing drug delivery to the inner ear.

Description

METHODS AND COMPOSITIONS FOR ENHANCING DRUG DELIVERY INTOTHE INNER EARCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 655,660 filed June 4, 2024, which is hereby incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under DC020005 and DC019960 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF DISCLOSURE

[0003] This disclosure relates to novel compositions that enhance the delivery of therapeutics to the inner ear and methods of use thereof.BACKGROUND OF DISCLOSURETechnical Background

[0004] Hearing loss affects nearly 20% of the global population. Several studies link hearing loss to the increased rate of dementia, Parkinson’s disease, depression, and more. To mitigate the detrimental effects of hearing loss, hearing aids, and cochlear implants are commonly used, with limited success. Cochlear implants have their limitations. Not only can cochlear implant users lose their residual hearing and face other surgical problems, but some fail to hear in noisy environments and lose the capacity to enjoy music. Hearing aid users also suffer from a lack, of normal auditory perception. Consequently, researchers are dedicated to developing treatments to restore hearing or prevent hearing loss.

[0005] A major obstacle to developing novel treatments for diseases and disorders of the ear is the lack of effective and minimally invasive procedures to deliver substances to the inner ear. Systemic delivery is challenging as only a fraction of the therapeutic passes through the blood labyrinth barrier of the inner ear and reaches the inner ear. Delivery via intracochlear and intratympanic procedures seems promising, but these procedures have major shortcomings. Intracochlear methods inject therapeutics directly into the cochlea during surgery, and they increase the risk of hearing loss through cerebrospinal andperilymph fluid leakage. Additionally, using intracochlear delivery methods results in large variability in the therapeutic outcomes since such surgeries are technically difficult and individual results are variable. Also, some intracochlear approaches suggested for inner ear therapy in small animals — e.g., through the semicircular canal are not feasible in humans. The intratympanic injection method is less invasive and causes minimal damage to the inner ear; intratympanic injection is used regularly to deliver steroids such as dexamethasone phosphate and gentamicin to treat idiopathic sudden sensorineural hearing loss and MeniSre's disease. However, intratympanic injection is less effective in delivering substances with large molecular weight, negative change, or hydrophilic properties to the inner ear, since the injected substances that fill the middle ear cavity must pass through a round window membrane (RWM) and / or an oval window membrane (OWM), with the RWM playing the predominant role.

[0006] Within this disclosure, we define the round window (RW) niche as a funnel- shaped depression in the middle ear that includes the RWM. When we refer to the lining cells of the niche we exclude the RWM lining from this term.

[0007] Thus, there is a need for the administration to the ear via simple delivery of therapeutic compositions into the middle ear and then passage through the round window membrane or oval window membrane.

[0008] The RWM exhibits two important roles in normal ear physiology: it is involved with the transmission of sound into the cochlea, and it serves as a barrier between the middle and inner ear (Paparella, Schachem, and Choo 1983; Goycoolea and Lundman 1997).

[0009] Previous studies have elucidated the complex structure of the RWM, revealing it to be a three-layered semipermeable membrane, comprising two epithelial layers enclosing a fibroblastic layer. The first and outer layer, facing the middle ear, consists of qnthelial cuboidal cells with apical, laterally placed tight junctions. The tight junctions prevent the passage of most molecules. This layer exhibits distinct features such as microvilli, tight junctions, abundant mitochondria, and a well-developed Golgi complex (Goycoolea and Lundman 1997; Goycoolea, Muchow, and Schachem 1988; Goycoolea 2001; Paparella, Schachem, and Choo 1983; Moatti, Silkstone, et al. 2023). The second layer is made of fibroblasts, collagen, and elastic fibers which contain blood and lymph vessels as well as nerve endings. The third layer consists of wide and flat inner epithelial cells, facing the inner ear.

[0010] As stated above, the RWM is only permeable to specific molecules, and while some substances and compositions can pass through the RWM, most therapeutics have low orno penetrance. The RWM is considered the major barrier that limits drugs from reaching the inner ear.

[0011] Interestingly, ongoing experiments demonstrate that factors like high osmolarity of drug solutions and drying of the middle ear including the RWM using suctioning can enhance drug passage into the inner ear perilymph by a factor of 2-3 and 10-15, respectively (Aa, Jj, and An 2008). These phenomena are also reported in organ systems endowed with cilia and mucus barriers, such as the nasal epithelium.

[0012] Investigations into drug kinetics reveal rapid clearance from the middle ear via mucociliary transport, much like the nasal cavity, with implications for drug absorption. In the nasal cavity, because of rapid clearance of mucus into the throat, inhaled drugs have a very short absorption window, which creates challenges for their effective delivery. It has been estimated that if the mucociliary transport rate follows first-order kinetics, the half-life for an inhaled drug is about 20 minutes (Gizurarson 2015). Similarly, Salt et al. also describes a rapid loss of drug from the solution in the middle ear at a similarly high rate (Salt et al. 2018), demonstrating a half-life of 28 minutes for dexamethasone phosphate and 75 minutes for gentamicin in guinea pigs. It was also found that CHIR99021 was lost from the middle ear guinea pigs with a 56.4 min halftime while VP A was lost with a 48.6 min halftime (McLean et al. 2021). Comparable measurements with samples from the human middle ear found that the losses were even faster, with halftimes of 10.6 min (CHIR99021) and 8.9 min (VP A). Taken together, these studies suggest the presence of mucociliary transport from the middle ear and in proximity to the RWM.

[0013] Controversial studies concerning ciliation distribution in the middle ear further underscore the need for a comprehensive understanding of these processes. One study delineated that the ventral portion of the middle ear cavity, near the Eustachian tube (ET) orifice and originating from the endoderm, exhibits ciliation, while the neural crest-derived epithelium lining the dorsal region lacks cilia (Thompson and Tucker 2013). Moreover, this work identified a transitional zone near the RWM with a combination of ciliated and non- ciliated epithelium, although the RWM itself is believed to be devoid of cilia. Conversely, another study suggested partial ciliation in the neural crest-derived epithelial cells lining the dorsal region (Luo et al. 2017). Furthermore, the ciliated epithelium was observed above the round and oval windows, speculating that this region may either represent a transformation of the neural crest-derived epithelium into a typical mucosal epithelium or an extension of the endoderm-derived region. Determining the precise boundaries between these two lineages remains challenging due to limitations in markers and lineage tracing tools.

[0014] In summary, the aforementioned studies demonstrate the exi stence of a mucociliary system in the lining of the middle ear and Eustachian Tube (Ohashi et al. 1985). This system plays a pivotal role in clearing the tympanic cavity from debris and excess fluids. Additionally, the activity of ciliary cells in the middle ear lining, including the round window niche region, bears a strong resemblance to that of the respiratory system.

[0015] Following the intratympanic injection of a therapeutic formulation into the middle ear, the mucociliary system effectively removes the injected therapeutic, typically delivered as a solution, via the Eustachian Tube (ET). Consequently, inhibiting the mucociliary system allows the injected drug to remain within the tympanic cavity and the round window niche area for an extended period. This enhancement in the drug’s bioavailability, in turn, facilitates the drug passage through the round window membrane and into the inner ear. Therefore, inhibitors of the mucociliary system, as part of an otic formulation, are capable of potentiating therapeutic delivery from the RW niche to the inner ear.SUMMARY OF THE DISCLOSURE

[0016] This disclosure relates to compositions that enhance the delivery of therapeutics to the inner ear and methods of use thereof.

[0017] In a first aspect, the present disclosure provides a method of delivering a therapeutic to the inner ear, the method including modulating the motility of cilia in the middle ear and round window niche and administering the therapeutic. Through the use of this method, passage of the therapeutic across the RWM and oval window, which separate the middle and inner ear, is more effective than when ciliary motility is not modulated.

[0018] In some embodiments of the first aspect, modulating ciliary motility involves inhibiting cilia motor activity. In some embodiments of the first aspect, inhibiting cilia motor activity is accomplished by contacting the cilia with an inhibitory compound.

[0019] In some embodiments of the first aspect, modulating ciliary motility and adm riiiinristering a therapeutic are performed sequentially. In some embodiments of the first aspect, modulating ciliary motility and administering a therapeutic are performed simultaneously.

[0020] In some embodiments of the first aspect, modulating ciliary motility is accomplished through modulating the activity of taste receptors. Such receptors are found in the round window membrane and the lining cells of the niche.

[0021] In a second aspect, the present disclosure provides a method of delivering a therapeutic to the inner ear, the method including modulating the activity of a taste receptorand administering a therapeutic. Through the use of this method, passage of the therapeutic across the RWM separating the middle and inner ear is more effective than when the activity of the taste receptor is not modulated.

[0022] In some embodiments of the second aspect, modulating the activity of the taste receptor involves inhibiting the taste receptor. In some embodiments of the second aspect, modulating the activity of the taste receptor involves activating the taste receptor.

[0023] In some embodiments of the second aspect, modulating the activity of the taste receptor is accomplished by contacting a taste receptor with a modulating agent. In some embodiments of the second aspect, the modulating agent is an inhibitor. In some embodiments of the second aspect, the modulating agent is an activator.

[0024] In a third aspect, the present disclosure provides an otic pharmaceutical formulation comprising a compound that modulates the motility of the cilia of the middle ear and round window niche.

[0025] In some embodiments of the third aspect, modulating ciliary motility involves inhibiting cilia motor activity. In some embodiments of the third aspect, inhibiting cilia motor acti vity is accomplished by contacting the cilia with an inhibitory compound.

[0026] In some embodiments of the third aspect, the formulation additionally includes a therapeutic for the inner ear. In some embodiments of the third aspect, the therapeutic is a small molecule. In some embodiments of the third aspect, the therapeutic is a biologic or an antibody, or an antigen-binding fragment thereof. In some embodiments of the third aspect, the therapeutic is a gene therapy.

[0027] In some embodiments of the third aspect, the formulation enhances passage of a therapeutic to the inner ear. In some embodiments of the third aspect, the enhanced passage is across the RWM separating the middle and inner ear. In some embodiments of the third aspect, the enhanced passage is across the oval window separating the middle and inner ear.

[0028] In some embodiments of the third aspect, modulating ciliary motility is accomplished through modulating the activity of taste receptors expressed on the surface of the cilia.

[0029] In a fourth aspect, the present disclosure provides an otic pharmaceutical formulation including a compound that modulates the activity of a taste receptor in the ear.

[0030] In some embodiments of the fourth aspect, modulating the activity of the taste receptor involves inhibiting the taste receptor. In some embodiments of the second aspect, modulating the activity of the taste receptor involves activating the taste receptor.

[0031] In some embodiments of the fourth aspect, modulating the activity of the taste receptor is accomplished by contacting the taste receptor with a modulating agent. In some embodiments of the fourth aspect, the modulating agent is an inhibitor. In some embodiments of the fourth aspect, the modulating agent is an activator.

[0032] In a fifth aspect, the present disclosure provides a method of enhancing therapeutic delivery to the inner ear, the method including modulating the motility of cilia in the middle ear and administering a therapeutic. Through the use of this method, delivery of a therapeutic to the inner ear is more effective than when ciliary motility is not modulated.

[0033] In some embodiments of the fifth aspect, modulating ciliary motility involves inhibiting cilia motor activity. In some embodiments of the fifth aspect, inhibiting cilia motor activity is accomplished by contacting the cilia with an inhibitory compound.

[0034] In some embodiments of the fifth aspect, modulating ciliary motility and administering a therapeutic are performed sequentially. In some embodiments of the fifth aspect, modulating ciliary motility and administering a therapeutic are performed simultaneously.

[0035] In some embodiments of the fifth aspect, the therapeutic is a small molecule. In some embodiments of the fifth aspect, the therapeutic is a biologic or an antibody, or an antigen-binding fragment thereof. In some embodiments of the fifth aspect, the therapeutic is a gene therapy.

[0036] In some embodiments of the fifth aspect, modulating ciliary motility is accomplished through modulating the activity of taste receptors expressed on the surface of the cilia.

[0037] In a sixth aspect, the present disclosure provides a method of enhancing therapeutic delivery to the inner ear, the method including modulating the activity of a taste receptor and administering a therapeutic. Through the use of this method, delivery of a therapeutic to the inner ear is more effecti ve than when the activi ty of the taste receptor is not modulated.

[0038] In some embodiments of the sixth aspect, modulating the activity of the taste receptor involves inhibiting the taste receptor. In some embodiments of the sixth aspect, modulating the activity of the taste receptor involves activating the taste receptor.

[0039] In some embodiments of the sixth aspect, modulating the activity of the taste receptor is accomplished by contacting the taste receptor with a modulating agent. In some embodiments of the sixth aspect, the modulating agent is an inhibitor. In some embodiments of the sixth aspect, the modulating agent is an activator.

[0040] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 shows the presence of cilia, with polar 9+2 microtubule structures, on the porcine RWM and in the RW niche. FIG. 1A (panel (a)) shows an 8 μm-thick section of the inner ear derived from a newborn pig. This section, which was stained for acetylated tubulin (AC-tubulin), provides a detailed view of the porcine inner ear structure. The staining shows the ciliation of the RWM and lining cells of the niche. The adjacent image provides a higher magnification of the dashed box. Boxes A and B are magnified in the two images (below). FIG. 1B (panel (b)) shows an 8 μm-thick section, obtained a few sections deeper in the tissue. The staining shows the ciliation of the RWM and the lining cells of the niche. The adjacent image provides a higher magnification of the dashed box. Boxes A and B are magnified in the two images (below). FIG. 1C (panel (c)) shows a Transmission Electron Microscope (TEM) image of the outer epithelial layer of the RWM. The arrows show the presence of non-ciliated basal cells. FIG. 1D (panel (d)) shows the body of the cilia, made up of nine tubulin triplets. At the level of the basal foot, accessory proteins compose a triangular, electron-dense structure on TEM graphs, which indicates the orientation of the cilium as pointed out by the arrow in the zoomed-in box. FIG. 1E (panel (e)) shows a TEM micrograph of the basal body of cilia in two neighboring cells, revealing that cilia are oriented toward the same direction. FIG. 1F (panel (f)) shows a cross-section of the cilia with a 9+2 microtubule structure.

[0042] FIG. 2 shows the presence of acidic and neutral goblet cells on the lining cells of the niche. FIG. 2A (panel (a)) shows H&E staining of a porcine inner ear section (8 μm thick). The wine glass-shaped goblet cells are present on the lining cells of the niche, but not on the RWM. FIG. 2B (panel (b)) shows Alcian blue and Periodic acid-Schiff (PAS) staining of a porcine inner ear section (8 μm thick). The zoomed-in areas show the mix of acidic and neutral goblet cells present on the lining cells of the niche across the RWM, their secretion is readily accessible to the ciliated epithelium.

[0043] FIG. 3 shows OCT scans of the RW niche. In the presence of liquid, the flow is laminar. FIG. 3A (panel (a)) shows the middle ear cavity of a gerbil when dexamethasonefluorescein (DexF) was applied directly to the RW niche. The arrow indicates the direction of the OCT B-Scan. FIG. 3B (panel (b)) shows OCT B-Scans, at different time points after DexF application. The arrow at time t=0s shows the direction of OCT B-Scan. At t=0s, the RWM is at the normal state i.e., not bent. Both DexF and mucus can be detected in the RW niche; DexF has a higher contrast. Laminar flow is observed starting from t=66s. The arrows at t=88s indicate the flow direction in the RW niche toward the Eustachian tube (ET), located at the left side of the B-Scans. At t=780s, the majority of DexF is removed from the RW niche. FIG. 3C (panel (c)) shows the trajectory (5 frames) of the detected DexF particles in the RW niche recorded by OCT B-Scans at t=480s; the trajectory of the detected particles is color-coded. FIG. 3D (panel (d)) shows the velocity of the detected particles (2 frames). FIG. 3E (panel (e)) shows a simulation of the DexF concentration in the RW niche if the mucociliary transport of DexF follows first-order kinetics with a half-life (tizz) of 7 minutes as measured experimentally by the OCT scans. The DexF concentration for half times of 14 and 21 minutes is also provided. The concentration of the applied DexF is 800 μg / ml. By increasing the half-life of the DexF from 7 to 14 minutes, its concentration at the RW niche increases about 10-fold at 30 minutes.

[0044] FIG. 4. The RWM cells express taste receptor genes. FIG. 4A (panel (a)) shows the gene expression level of ciliary-related genes normalized to GAPDH, a housekeeping gene, measured by bulk RNA sequencing. The ciliary-related genes are categorized based on their functionality and location. The expression is shown for porcine RWM cells (epithelia and fibroblast cells), and human lung cells (ciliated and tuft cells, from Ref (Madissoon et al. 2023)) confirming the presence of ciliary-related genes required for ciliary function. FIG 4B shows the gene expression level normalized by GAPDH, measured by bulk. RNA sequencing, for taste receptors. The expression is shown for porcine RWM cells (epithelial and fibroblast cells) sorted using CD326, and human lung cells (ciliated and Tuft cells) confirming the presence of sweet and umami taste receptors of TAS1R1 and TAS1R3 in the porcine RWM.

[0045] FIG. 5. The ciliated cells at the porcine RW niche express taste receptors. FIG 5A (panel (a)) shows the expression of the bitter taste receptor, TAS2R4, in the ciliated cells (stained against Ac-Tubulin) of the porcine RW niche. FIG. 5B (panel (b)) and FIG. 5C (panel (c)) demonstrates that TAS2R4 is expressed in ciliated RWM cells and mucosal epithelial cells, respectively. FIG 5D (panel (d)) is a positive control that shows TAS2R4 staining in the porcine tongue, where bitter receptors are previously reported.

[0046] FIG. 6. Cilia inhibitors and bitter taste receptor antagonists increased the passage of dexamethasone fluorescein and quinine through the RWM in an ex-vivo model. FIG. 6A(panel (a)) shows that ciliary structures are present in the ex-vivo model. FIG. 6B (panel (b)) shows a schematic of the ex-vivo RWM chamber, as well as measurement of dexamethasone fluorescein (DexF; at 800 μg / ml) passage through the RWM explant in the absence / presence of other compounds. Different compounds, including bitter taste receptor antagonists such as: Gamma-aminobutyric acid (GABA), N, N-bis(carboxymethyl)-L-lysine (BCML), and cilia beat frequency inhibitors such as: ciliobrevin D (CD), Geldanamycin (GLD). All the compounds, except for low dosage of BCML, show a significant increase in the passage of DexF when ciliary beating is inhibited. Note that a threefold increase in BCML dosage facilitates DexF passage through the RWM. FIG. 6C, panel (c), demonstrates a significant increase in quinine (a highly bitter substance and agonist of TAS2R4; ImM) passage through the RWM following treatment with BCML, a TAS2R4antagonist (threefold BCML dose; i.e., 150 nM) 30 minutes prior to quinine exposure. Mixed effect analyses were used to assess significance. Significant improvement in DexF passage was observed with GLD, starting 2.5 hours after its addition.DETAILED DESCRIPTION OF THE INVENTION

[0047] It is to be understood that the particular aspects of the specification described herein are not limited to specific embodiments presented and can vary. It will also be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting. Moreover, particular embodiments disclosed herein can be combined with other embodiments disclosed herein, as would be recognized by a skilled person, without limitation.

[0048] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference in their entirety for all purposes.

[0049] Before describing the methods and compositions of the disclosure in detail, a number of terms will be defined. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, a reference to “a therapeutic target” means one or more therapeutic targets.

[0050] Throughout this specification, unless the context specifically indicates otherwise, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,” “includes,” and “including”) will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms “comprising,” “consisting essentially of,” and“consisting of" may be replaced with either of the other two terms, while retaining their ordinary meanings.

[0051] In some embodiments, percentages disclosed herein can vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated disclosure.

[0052] Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values herein that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0053] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. For example, “about 5%” means “about 5%” and also “5%.” The term “about” can also refer to ± 10% of a given value or range of values. Therefore, about 5% also means 4.5% - 5.5%, for example.

[0054] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”

[0055] As used herein, the term “modulating” refers to affecting a change in a property, function, pathways, or other aspect of a biological, biochemical, or chemical component of a living system. For example, modulating the activity of a receptor refers to causing said receptor’s activity to increase, decrease, or some combination thereof over time. Similarly, modulating a cellular or tissue component refers to causing a change in its structure or function relative to its state without, or prior to, the modulation.

[0056] As used herein, “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio or which have otherwise been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0057] As used herein, the terms “therapeutic amount,” “therapeutically effective amount” or “effective amount” can be used interchangeably and refer to an amount of a compound that becomes available through an appropriate route of administration to provide atherapeutic benefit to a patient for a disorder, a condition, or a disease. The amount of a compound that constitutes a “therapeutic amount,” “therapeutically effective amount” or “effective amount” will vary depending on the compound, the disorder and its severity, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art.

[0058] “Treating” or “treatment,” as used herein, covers the treatment of a disorder, condition, or disease described herein, in a subject, preferably a human, and includes: i. inhibiting a disease or disorder, i.e., arresting its development; ii. relieving a disease or disorder, i.e., causing regression of the disorder; iii. slowing progression of the disorder; and / or iv. inhibiting, relieving, ameliorating, or slowing the progression of one or more symptoms of the disease or disorder. For example, the terms “treating,” “heat,” or “treatment” refer to either preventing development or exacerbation of, providing symptomatic relief for, or curing a patient’s disorder, condition, or disease.

[0059] As used herein, the terms “patient,” “subject,” and “individual” can be used interchangeably and refer to an animal. For example, the patient, subject, or individual can be a mammal, such as a human to be treated for a disorder, condition, or a disease.

[0060] It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the methods and composition s as described herein or to imply that certain features are critical, essential, or even important to the structure or function of the subject matter recited in the claims.

[0061] This disclosure provides compositions that increase the passage of drugs through the round window membrane, which separates the middle and inner ear, and methods of use thereof, including for the enhancement of the delivery of therapeutics to the inner ear. The present disclosure contemplates a method of delivering a therapeutic to the inner ear, the method including modulating the motility of cilia in the ear and administering the therapeutic. In some embodiments, passage of the therapeutic across the round window membrane (RWM) separating the middle and inner ear is more effective using this method than when ciliary motility is not modulated. In some embodiments, passage of the therapeutic across the oval window membrane, which is located between the middle and inner ear, is more effective using tins method than when ciliary motility is not modulated.

[0062] In some embodiments, modulating ciliary motility involves inhibiting cilia motor activity including dynein-dependent microtubule gliding, ciliary transport, actin regulatory system, ciliogenesis, cytoplasmic dynein, ciliary beating pathways, disruption in intracellularATP production or transport. In some embodiments, this is accomplished pharmacologically. In some embodiments, inhibiting cilia motor activity is accomplished by contacting the cilia with an inhibitory compound. In some embodiments, the inhibitory compound is selected from the group consisting of ciliobrevin D, alvocidib (an inhibitor of ciliogenesis-associated kinase 1 [CILK1]), bromopride, droperiodol, sulpiride, tripelennamine hydrochloride, palonosetron hydrochloride, MK801 hydrogen maleate, tramadol hydrochloride, lorglumide sodium salt, thricolchicoside, GABA, dynarrestin, or salts and derivatives thereof. Ciliobrevin D inhibits Hedgehog (Hh) sigpaling, dynein-dependent microtubule gliding, and ATPase activity. Effectors of intracellular calcium and cyclic adenosine monophosphate (cAMP) levels can also be used to influence ciliary beating. Such effectors include molecules that influence the activity of phosphodiesterase (PDE1, PDE4) or dual PDE activation (PDE3 / 4), which relate to cAMP. Other targets for affecting ciliary action include Wnt signaling inhibitors, actin, and components of the actin regulatory system (Gizurarson 2015). Calcium-binding compounds may also be effective in interfering with arachidonic acid metabolism, guanylate cyclase, protein kinase, or intracellular ATP. Protein phosphorylation, cytoplasmic calcium, production of prostaglandins, nitric oxide, and cGMP also play a role in ciliary beating. Concurrent use of drugs such as non-steroidal anti-inflaatory drugs (NSAID) may have some effect on mucociliary clearance through their inhibiting effect on the enzyme cyclooxygenase that regulates arachidonic acid metabolism.

[0063] In some embodiments, modulating ciliary motility includes subjecting cilia to one or more of the factors listed in Table 1. In some embodiments, modulating ciliary motility includes contacting cilia with one or more of the compounds listed in Table 2. In some embodiments, modulating ci liary motility includes contacting cilia with one or more of the modulating agents listed in Table 3. Many of these compounds and their effects are disclosed in Gizurarson et al. (2015), “The Effect of Cilia and the Mucociliary Clearance on Successful Drug Delivery,” Biological and Pharmaceutical Bulletin 38 (4): 497-506, which is hereby incorporated by reference in its entirety.TABLE 1. Formulation factors affecting mucociliary clearanceTABLE 2. Compounds affecting mucociliary clearanceTABLE 3. Modulating agents affecting mucociliary clearance

[0064] In some embodiments, ciliary motility is modulated and the therapeutic is administered separately. In some embodiments, ciliary motility is modulated and the therapeutic is administered simultaneously. In some embodiments, ciliary motility is modulated for about 24 hours. In some embodiments, ciliary motility is modulated for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, ciliary motility is modulated for less than about 1 hour. In some embodiments, ciliary motility is modulated for more than about 24 hours. In some embodiments, the therapeutic is administered for about 24 hours. In some embodiments, the therapeutic is administered for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, the therapeutic is administered for less than about 1 hour. In some embodiments, the therapeutic is administered for more than about 24 hours.

[0065] In some embodiments, ciliary motility is modulated by modulating the activity of receptors expressed on the surface of the cilia. In some embodiments, ciliary motility is modulated by modulating the activity of taste receptors.

[0066] The present disclosure also contemplates a method of delivering a therapeutic to the inner ear, the method including modulating the activity of a taste receptor and administering the therapeutic. In some embodiments, the passage of the therapeutic across the round window membrane (RWM) separating the middle and inner ear is more effective using this method than when the activity of the taste receptor is not modulated. In some embodiments, the passage of the therapeutic across the oval window membrane, which is located between the middle and inner ear, is more effective using this method than when theactivity of the taste receptors is not modulated. In some embodiments, the taste receptor is selected from the group consisting of Type 1, Sweet and umami, TAS1R1-TAS1R3, Type 2, Bitter, TAS2R1-TAS2R64, ENaC salty receptor, aquaporin water receptors, and lipid receptors.

[0067] In some embodiments, modulating the activity of the taste receptor in volves inhibiting the taste receptor. In some embodiments, modulating the activity of the taste receptor involves activating the taste receptor. In some embodiments, modulating the activity of the taste receptor is accomplished by contacting the taste receptor with a modulating agent. In some embodiments, the modulating agent is an inhibitor. In some embodiments, the inhibitor is selected from the compounds listed in Table 4, Table 7, Table 10, Table 15, Table 18, Table 19, Table 20, Table 21, Table 23, and Table 24 below.

[0068] In some embodiments, the modulating agent is an activator. In some embodiments, the activator is selected from the compounds listed in Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, Table 14, Table 15, Table 16, Table 17, Table 18, Table 19, Table 20, Table 21, Table 22, Table 23, Table 24, and Table 25 below. In some embodiments, the modulating agent is an antagoni st of the taste receptor. In some embodiments, the modulating agent is an agonist of the taste receptor. In some embodiments, the modulating agent is an inverse agonist of the taste receptor. Many of these compounds are listed in Behrens (2023), Sigoillot et al. (2012) and Pydi et al. (2014), all of which are hereby incorporated by reference in their entirety.

[0069] In some embodiments, the modulating agent is lactisole, which modulates TAS1R3. Lactisole is a canonical antagonist of sweet taste receptor, selectively targeting to TAS1R3 subunit, a glucose-sensing receptor. Lactisole inhibits insulin secretion induced by glucose in mouse islets. In some embodiments, the modulating agent is FEMA 4774, which modulates TAS1R2 and TAS1R3. FEMA 4774 is a positive allosteric modulator of taste receptors TAS1R2 and TAS1R3, two subunits of the human sweet taste receptor. FEMA 4774 is also used as a sucrose sweetness enhancer. In some embodiments, the modulating agent is gurmarin. In some embodiments, the modulating agent is adenosine 5 -succinate. Adenosine 5 -succinate is a chemically AMP-related compound and potently inhibits denatonium benzoate / taste receptor activation of transducin.TABLE 4. TAS1R inhibitorsTABLE 5. TAS2R1 agonistsTABLE 6. TAS2R3 agonistsTABLE 7. TAS2R4 agonists, antagonists, and blockersTABLE 8. TAS2R5 agonistsTABLE 9. TAS2R7 agonistsTABLE 10. TAS2R8 agonists and antagonistsTABLE 11. TAS2R9 agonistsTABLE 12. TAS2R10 agonistsTABLE 13. TAS2R13 agonistsTABLE 14. TAS2R14 agonistsTABLE 15. TAS2R16 agonists and antagonistsTABLE 16. TAS2R20 agonistsTABLE 17. TAS2R30 agonistsTABLE 18. TAS2R31 agonists and antagonistsTABLE 19. TAS2R38 agonists and antagonistsTABLE 20. TAS2R39 agonists and antagonistsTABLE 21. TAS2R40 agonists and antagonistsTABLE 22. TAS2R41 agonistsTABLE 23. TAS2R43 agonists and antagonistsTABLE 24. TAS2R46 agonists and antagonistsTABLE 25. TAS2R50 agonists

[0070] In some embodiments, the activity of the taste receptor is modulated by inhibiting HSP90, as it affects eNOS production. In some embodiments, a HSP90 inhibitor is selected from the list in Table 26. Many of these inhibitors can be found in Magwenyane et al. (2022) and Li and Luo (2022), both of which are hereby incorporated by reference in their entirety.TABLE 26. HSP90 inhibitors

[0071] In some embodiments, the activity of the taste receptor is modulated for about 24 hours. In some embodiments, the activity of the taste receptor is modulated for about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, the activity of the taste receptor is modulated for less than about 1 hour. In some embodiments, the activity of the taste receptor is modulated for more than about 24 hours.

[0072] The present disclosure also contemplates a pharmaceutical formulation including a compound that modulates the motility of the cilia of the ear. In some embodiments, the pharmaceutical formulation is otic; i.e., administered directly into the ear. In some embodiments, the pharmaceutical formulation is oral; i.e., taken by mouth or ingested. In some embodiments, the pharmaceutical formulation is injectable. In some embodiments, the pharmaceutical formulation is topical. In some embodiments, the pharmaceutical formulation is administered enterally. In some embodiments, the pharmaceutical formulation is administered parenterally.

[0073] In some embodiments, modulating ciliary motility involves inhibiting cilia motor activity. In some embodiments, inhibiting cilia motor activity is accomplished by contactingthe cilia with an inhibitory compound. In some embodiments, the inhibitory compound is selected from the group consisting of ciliobrevin D, alvocidib (an inhibitor of ciliogenesis- associated kinase 1 [CILK1]), bromopride, droperiodol, sulpiride, tripelennamine hydrochloride, palonosetron hydrochloride, MK801 hydrogen maleate, tramadol hydrochloride, lorglumide sodium salt, thricolchicoside, GABA, dynarrestin, effectors of intracellular calcium and cyclic adenosine monophosphate (cAMP) levels, molecules that influence the activity of phosphodiesterase (PDE1, PDE4) or dual PDE activation (PDE3 / 4), Wnt signaling inhibitors, actin, components of the actin regulatory system, calcium binding compounds, non-steroidal anti-inflammatory drug (NSAID) drugs, cyclooxygenase inhibitors, and salts and derivatives thereof.

[0074] In some embodiments, modulating ciliary motility involves inhibiting cilia motor activity. In some embodiments, inhibiting cilia motor activity is accomplished by contacting the cilia with the pharmaceutical formulation. In some embodiments, the pharmaceutical formulation contains a compound selected from the group consisting of ciliobrevin D, alvocidib (an inhibitor of ciliogenesis-associated kinase 1 [CILK1]), bromopride, droperiodol, sulpiride, tripelennamine hydrochloride, palonosetron hydrochloride, MK801 hydrogen maleate, tramadol hydrochloride, lorglumide sodium salt, thricolchicoside, GABA, dynarrestin, effectors of intracellular calcium and cyclic adenosine monophosphate (cAMP) levels, molecules that influence the activity of phosphodiesterase (PDE1, PDE4) or dual PDE activation (PDE3 / 4), Wnt signaling inhibitors, actin, components of the actin regulatory system, calcium binding compounds, non-steroidal anti-inflammatory drug (NSAID) drugs, cyclooxygenase inhibitors, and salts and derivatives thereof.

[0075] In some embodiments, ciliary motility is modulated for about 24 hours. In some embodiments, ciliary motility is modulated for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, ciliary motility is modulated for less than about 1 hour. In some embodiments, ciliary motility is modulated for more than about 24 hours. In some embodiments, the therapeutic is administered for about 24 hours. In some embodiments, the therapeutic is administered for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, the therapeutic is administered for less than about 1 hour. In some embodiments, the therapeutic is administered for more than about 24 hours.

[0076] In some embodiments, the formulation additionally contains a therapeutic for the inner ear. In some embodiments, the therapeutic is a small molecule. In some embodiments, the therapeutic is a biologic or an antibody, or an antigen-binding fragment thereof. In some embodiments, the therapeutic is a gene therapy.

[0077] In some embodiments, the formulation enhances passage of a therapeutic to the inner ear. In some embodiments, the enhanced passage is across the round window membrane (RWM) separating the middle and inner ear. In some embodiments, the enhanced passage is across the oval window membrane separating the middle and inner ear.

[0078] In some embodiments, modulating ciliary motility is accomplished through modulating the activity of taste receptors expressed on the surface of the cilia.

[0079] The present disclosure also contemplates a pharmaceutical formulation including a compound that modulates the activity of a taste receptor in the ear. In some embodiments, the pharmaceutical formulation is otic; i.e., administered directly into the ear. In some embodiments, the pharmaceutical formulation is oral; i.e., taken by mouth or ingested. In some embodiments, the pharmaceutical formulation is injectable. In some embodiments, the pharmaceutical formulation is topical. In some embodiments, the pharmaceutical formulation is administered enterally. In some embodiments, the pharmaceutical formulation is administered parenterally.

[0080] In some embodiments, the taste receptor is selected from the group consisting of TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R8, TAS2R9, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R50, and TAS2R60.

[0081] In some embodiments, modulating the activity of the taste receptor involves inhibiting the taste receptor. In some embodiments, modulating the activity of the taste receptor involves activating the taste receptor.

[0082] In some embodiments, modulating the activity of the taste receptor is accomplished by contacting the taste receptor with a modulating agent. In some embodiments, the modulating agent is an inhibitor. In some embodiments, the inhibitor is selected from the compounds listed in Table 4, Table 7, Table 10, Table 15, Table 18, Table 19, Table 20, Table 21, Table 23, and Table 24 below. In some embodiments, the modulating agent is an activator. In some embodiments, the activator is selected from the group consisting of compounds that activate taste receptors. In some embodiments, the modulating agent is an antagonist of the taste receptor. In some embodiments, the modulatingagent is an agonist of the taste receptor. In some embodiments, the modulating agent is an inverse agonist of the taste receptor.

[0083] In some embodiments, the activity of the taste receptor is modulated for about 24 hours. In some embodiments, the activity of the taste receptor is modulated for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, the activity of the taste receptor is modulated for less than about 1 hour. In some embodiments, the activity of the taste receptor is modulated for more than about 24 hours.

[0084] The present disclosure also contemplates a method of enhancing therapeutic delivery to the inner ear, the method including modulating the motility of cilia in the ear and administering the therapeutic. In some embodiments, delivery of the therapeutic to the inner ear is more effective using this method than when ciliary motility is not modulated.

[0085] In some embodiments, modulating ciliary motility involves inhibiting cilia motor activity. In some embodiments, this is accomplished pharmacologically. In some embodiments, inhibiting cilia motor activity is accomplished by contacting the cilia with an inhibitory compound. In some embodiments, the inhibitory compound is selected from the group consisting of ciliobrevin D, alvocidib (an inhibitor of ciliogenesis-associated kinase 1 [CILK1]), bromopride, droperiodol, sulpiride, tripelennamine hydrochloride, palonosetron hydrochloride, MK.801 hydrogen maleate, tramadol hydrochloride, lorglumide sodium salt, thricolchicoside, GABA, dynarrestin, effectors of intracellular calcium and cyclic adenosine monophosphate (cAMP) levels, molecules that influence the activity of phosphodiesterase (PDE1, PDE4) or dual PDE activation (PDE3 / 4), Wnt signaling inhibitors, actin, components of the actin regulatory system, calcium binding compounds, non-steroidal anti-infk ry drug (NSAID) drugs, cyclooxygenase inhibitors, and salts and derivatives thereof.

[0086] In some embodiments, ciliary motility is modulated and the therapeutic is administered separately. In some embodiments, ciliary motility is modulated and the therapeutic is administered simultaneously. In some embodiments, ciliary motility is modulated for about 24 hours. In some embodiments, ciliary motility is modulated for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, ciliary motility is modulated for less than about 1 hour. In some embodiments, ciliary motility is modulated for more than about 24 hours. In some embodiments, the therapeutic is administered for about 24hours. In some embodiments, the therapeutic is administered for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, the therapeutic is administered for less than about 1 hour. In some embodiments, the therapeutic is administered for more than about 24 hours.

[0087] In some embodiments, the therapeutic is a small molecule. In some embodiments, the therapeutic is a biologic or an antibody, or an antigen-binding fragment thereof. In some embodiments, the therapeutic is a gene therapy.

[0088] In some embodiments, the formulation enhances passage of a therapeutic to the inner ear. In some embodiments, the enhanced passage is across the round window membrane (RWM) separating the middle and inner ear. In some embodiments, the enhanced passage is across the oval window membrane separating the middle and inner ear.

[0089] In some embodiments, ciliary motility is modulated by modulating the activity of receptors expressed on the surface of the cilia. In some embodiments, ciliary motility is modulated by modulating the activity of taste receptors expressed on the surface of the cilia.

[0090] The present disclosure also contemplates a method of enhancing therapeutic delivery to the inner ear, the method including modulating the activity of a taste receptor and administering the therapeutic. In some embodiments, delivery of the therapeutic to the inner ear is more effective using this method than when the activity of the taste receptor is not modulated.

[0091] In some embodiments, the taste receptor is selected from the group consisting of TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R8, TAS2R9, TAS2RI0, TAS2RI3, TAS2RI4, TAS2RI6, TAS2R19, TAS2R20, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R50, and TAS2R60.

[0092] In some embodiments, modulating the activity of the taste receptor involves inhibiting the taste receptor. In some embodiments, modulating the activity of the taste receptor involves activating the taste receptor.

[0093] In some embodiments, modulating the acti vity of the taste receptor is accomplished by contacting the taste receptor with a modulating agent. In some embodiments, the modulating agent is an inhibitor. In some embodiments, the inhibitor is selected from the group consisting of compounds that inhibit taste receptors. In some embodiments, the modulating agent is an activator. In some embodiments, the activator isselected from the group consisting of compounds that activate taste receptors. In some embodiments, the modulating agent is an antagonist of the taste receptor. In some embodiments, the modulating agent is an agonist of the taste receptor. In some embodiments, the modulating agent is an inverse agonist of the taste receptor.

[0094] In some embodiments, the acti vity of the taste receptor is modulated and the therapeutic is administered separately. In some embodiments, the activity of the taste receptor is modulated and the therapeutic is administered simultaneously. In some embodiments, the acti vity of the taste receptor is modulated for about 24 hours. In some embodiments, the activity of the taste receptor is modulated for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, the activity of the taste receptor is modulated for less than about 1 hour. In some embodiments, the activity of the taste receptor is modulated for more than about 24 hours. In some embodiments, the therapeutic is administered for about 24 hours. In some embodiments, the therapeutic is administered for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 hours. In some embodiments, the therapeutic is administered for less than about 1 hour. In some embodiments, the therapeutic is administered for more than about 24 hours.

[0095] In some embodiments, the therapeutic is a small molecule. In some embodiments, the therapeutic is a biologic or an antibody, or an antigen-binding fragment thereof. In some embodiments, the therapeutic is a gene therapy.

[0096] In some embodiments, the formulation enhances passage of a therapeutic to the inner ear. In some embodiments, the enhanced passage is across the round window membrane (RWM) separating the middle and inner ear. In some embodiments, the enhanced passage is across the oval window membrane separating the middle and inner ear.

[0097] In some embodiments, the activity of the taste receptor is modulated by modulating the activity of receptors expressed on the surface of the cilia. In some embodiments, the activity of the taste receptor is modulated by modulating the activity of taste receptors expressed on the surface of the cilia.Therapeutics

[0098] In some embodiments, a therapeutic or pharmaceutical formulation can include a pharmaceutically acceptable carrier, solvent, adjuvant, diluent, hydrogel, nanocarrier, macrocarrier, suspension, solution, or any combination thereof. The exact nature of the carrier, solvent, adjuvant, or diluent will depend upon the desired use for the composition and may range, for example, from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. The therapeutics of the present disclosure can be delivered through a variety of delivery methods.

[0099] Therapeutics and pharmaceutical formulations as described herein can be formulated as separate compositions that are given simultaneously or sequentially, or as a single composition. In certain embodiments, the therapeutics and pharmaceutical formulations of the present disclosure include one or more therapeutic agents.

[0100] Therapeutics and pharmaceutical formulations can take a form suitable (can be formulated) for virtually any mode of administration, including, for example, injection, transdermal, oral, topical, otic, systemic, nasal administration, inhalation, etc., Therapeutics and pharmaceutical formulations that can be delivered (e.g., are formulated to be administered) intravenously and / or into / onto the ear are contemplated herein.

[0101] The therapeutics described herein, or pharmaceutical formulations thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated (e.g., a therapeutically effective amount). By therapeutic benefit means eradication or amelioration of the underlying disorder being treated and / or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally can include halting or slowing the progression of the disease.

[0102] The amount of therapeutic or pharmaceutical formulation administered can be based upon a variety of factors, including, for example, the particular condition being treated, the mode of administration, whether the desired benefit is prophylactic and / or therapeutic, the severity of the condition being treated and the age and weight of the patient, the genetic profile of the patient, and / or the bioavailability of the particular therapeutic composition, etc.

[0103] Determination of an effective dosage of therapeutic(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages can be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of a therapeutic for use in animals may be formulated to achieve acirculating blood or serum concentration of the therapeutic composition that is at or above a half maximal effective concentration (EC50) of the particular therapeutic composition as measured in an in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular therapeutic via the desired route of administration is well within the capabilities of skilled arti sans. Initial dosages of therapeutic can also be estimated from in vivo data, such as animal models. A particularly useful animal model for measuring the permeability of a therapeutic through the round window membrane is disclosed in U.S. Patent Application No. 18 / 603,792, filed on March 13, 2024.

[0104] Dosage amounts of a therapeutic can be in the range of from about 0.0001 mg / kg / day, 0.001 mg / kg / day, or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higher or lower, depending upon, among other factors, the activity of the therapeutic, the bioavailability of the therapeutic composition or pharmaceutical formulation, other pharmacokinetic properties, the mode of administration and various other factors, including particular diseases being treated, the site of the disease within the body, the severity of the disease, the genetic profile, age, health, sex, diet, and / or weight of the subject. Dosage amount and interval may be adjusted individually to provide levels of the therapeutic which are sufficient to maintain a desired therapeutic effect. For example, a therapeutic can be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of therapeutic compositions may not be related to pl asma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.

[0105] Dosage amounts of a modulating agent can be in similar ranges to those for therapeutics and are similarly affected by various conditions. They can also be optimized without undue experimentation by those of ordinary skill in the art. The amount of modulating agent administered can be based upon a variety of factors, including, for example, the particular condition being treated, the mode of administration, whether the desired benefit is prophylactic and / or therapeutic, the severity of the condition being treated and the age and weight of the patient, the genetic profile of the patient, and / or the bioavailability of the particular therapeutic composition, etc.

[0106] Dosage amounts of a modulating agent can be in the range of from about 0.0001 mg / kg / day, 0.001 mg / kg / day, or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higheror lower, depending upon, among other factors, the activity of the modulating agent, the bioavailability of the composition or pharmaceutical formulation, other pharmacokinetic properties, the mode of administration and various other factors. Dosage amount and interval may be adjusted individually to provide levels of the modulating agent which are sufficient to maintain a desired effect. For example, a modulating agent can be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compositions may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.EXAMPLES

[0107] The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the disclosure in any way.MethodsAnimals

[0108] Yorkshire wild-type pigs (mixed gender) postnatal days P0, P20, and P40 were used. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at North Carolina State University, following the standards of the National Institute of Health and Committee on Care and Use of Laboratory Animals. An adult gerbil was used for the optical coherence tomography study. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Chicago University where the experiment took, place at Marine Biological Laboratory.Porcine inner ear dissection

[0109] Skin was removed and a large window was created on top of the porcine skull using a bone striker (Moatti et al. 2020; Moatti, Silkstone, et al. 2023; Moatti, Cai, et al.2023). The brain was removed to enable visualization of the inner ear from inside. Then, the bone surrounding the inner ear was further cut using the striker, and the porcine inner ear was dissected out of the bony skull using a bone cutter.Transmission electron microscopy (TEM)

[0110] For TEM investigation, the RWM tissues were removed using tweezers from the inner ears and fixed using 4% paraformaldehyde (PFA) and 1% glutaraldehyde in 0.1% sodium cacodylate at 4°C overnight The tissues were washed thrice in 0.1 M sodium cacodylate buffer and incubated with 2% osmium tetroxide for 1 hour, followed by three washes in 0.1 M sodium cacodylate buffer. Then, the tissues were dehydrated in 70% and 95% ethanol (EtOH) for 20-30 min at each step and twice in 100% EtOH for 30 min. The tissues were incubated with 2:1 and 1:1 ethanol: resin solution for 1 hour each, overnight in 1:1 ethanol: resin, 1:2 ethanol: resin for 1 hour, and in 100% EMbed-812 resin twice for 1 hour each time. Then, tissues were transferred to pure resin and incubated at 70°C overnight for resin embedding. Ultramicrotomy (using a Leica® UC7 ultramicrotome) was performed to obtain 85 nm-thick cross-sections. Lead citrate / uranyl acetate post staining of grids was performed. A Bio-TEM model HT7800 120 kV transmission electron microscope (Hitachi®) was used for imaging.Cryo-sectioning

[0111] Inner ear tissues were fixed using 4% PFA. Tissues were decalcified in 350 mM EDTA-Naa for 7 days. Tissues were dehydrated using 10% sucrose solution for 0.5 hour, 20% sucrose solution for 1 hour, and 30% sucrose solution overnight. Then, tissues were embedded in a cryomold on dry ice and ethanol and transferred to -80°C overnight. At this point, tissues were cryo-sectioned into 8 μm sections.H&E staining

[0112] All materials were equilibrated to room temperature just prior to use and gently agitated. Tissues were cleaned from OCT embedding media and rehydrated in IX PBS for 1 minute 3 times followed by distilled (DI) water. The sections were stained in hematoxylin for 1 minute, washed with 3-5 changes of tap water, immersed in 0.02% ammonium hydroxide solution, washed with 3-5 changes of DI water, dehydrated with 95% EtOH for 30 seconds, counterstained in alcoholic eosin for 1 minute, washed with 3 changes of DI water, dehydrated through 3 changes of 95% EtOH and 2 changes of 100% EtOH (1 minute each), and cleared in 3 changes of xylene (1 minute each). Then, the slides were mounted in mounting media.Alcian blue and PAS staining

[0113] All materials were equilibrated to room temperature just prior to use and gently agitated. The sections were hydrated in DI water. The Acetic Acid Solution (3%) was applied to tissue section for 2 minutes. The excess Acetic Acid Solution (3%) was removed and without rinsing the Alcian Blue (pH 2.5) solution was applied for 15-20 minutes. The tissueswere rinsed for 2 minutes in running tap water followed by 2 changes of DI water. The Periodic Acid Solution was applied to the tissue section for 5 minutes. The slides were rinsed in 2 changes of DI water. The Schiff’s Solution was applied to the tissue section for 10-20 minutes. The slides were rinsed for 2 minutes in warm running tap water followed by 2 changes of DI water. The Hematoxylin (modified Mayer’s solution) was applied to tissue section for 2 changes of DI water. The tissues were dehydrated through graded alcohols. The tissues were cleared and mounted in mounting media.2D Immunohistochemistry stainin g

[0114] The sections were washed with lx PBS and blocked for 1 hour at 25 °C in blocking buffer (10% normal donkey serum, 1% Triton X-100, PBS). Afterward, sections were incubated with mouse anti-AC Tubulin (1 : 100) diluted in blocking buffer overnight at 4 °C, followed by 3 washes with lx PBS for 1 hour at 25 °C the next day. The sections were then incubated with donkey anti-mouse Alexa flour 647 (1 :250) antibody diluted in blocking buffer for 1 hour at 25 °C, followed by 3 washes with lx PBS. The sections were mounted in mounting media. Images were acquired using a confocal microscope.Intact RWM tissue dissection

[0115] Using a dental drill and 1 mm drill bit, we excised the RWM including some bone around it (to support the RWM) from the dissected inner ear tissues. The whole drilling procedure was done inside the PBS solution (Moatti, Silkstone, et al. 2023).RWM chamber fabrication

[0116] After the excision, the surrounding bone around the RWM was dried out using the sterile gas and glued into the bottom of a cut 0.5 mm Eppendorf tube using a dental cement. The whole construction was mounted on top of a transwell, without the mesh, in a 24 wellplate and immersed in a cell culture media (DMEM, 1% Antimycotic, 1% N2) overnight (150 μl on top of the RWM and 1.5 ml on the bottom). The next morning the media was changed (DMEM, 1% FBS, 1% Antimycotic, 1% N2) and the RWM chamber was ready for permeability measurements.Permeability measurements

[0117] To measure the permeability of substances, we mixed 150 μl of culture media (DMEM, 1% FBS, 1% Antibiotic Antimycotic, 1% N2) with the tested substance and placed the mixture in the top chamber. The top solution was redosed at the amount of 10 μl at each sampling point (7—9 times) to meet the conditions of a continuous infinite dosing. The bottom chamber was filled with 1.5 ml of cell culture media. To monitor the test substance passage, the cell culture media at the bottom of the chamber was sampled periodically. In eachsampling event, 100 μl of media was withdrawn from the bottom of the chamber. Before sampling, the media was pipetted up and down to mix and homogenize. After sampling the media, 100 μl of fresh media was added to the bottom chamber, this dilution in the media was compensated when calculating the concentrations (Moatti, Silkstone, et al. 2023). The purpose of replacing the solution was to simulate a static cell diffusion condition i.e., the concentration gradients are approximately static.AlamarBlue metabolic activity test

[0118] To perform a cell viability test, first, the explants were washed with PBS three times. Then 1 ml of fresh media containing 10% alamarBlue (Thermofisher, Dal 1025) was added to the chamber (top and bottom). The explants were placed in the incubator for 4-6 hours. The plate that covered the chamber was removed and absorbance was measured using a plate reader (Infinite M Plex from Tecan) at two wavelengths (570 nm and 600 nm). The percent reduction was calculated based on the vendor’s instructions, and this number was correlated to explant viability.Exposing the RWM in gerbil and OCT recording

[0119] Animal was anesthetized using intraperitoneal injections of ketamine (80 mg / kg) and xylazine (12 mg / kg), with no recovery allowed at the end of the experiments. Maintenance (1 / 4) doses of the anesthetic were given at intervals of between 10 and 60 min, as required to abolish pedal withdrawal reflexes. Animal was tracheotomized, but selfventilating. Core temperatures were maintained at 38 °C using a thermostatically controlled heating pad. The pinna and external meatus of the left ear was retracted and a 4 x 6 mm2wide opening was made into the postero-lateral bulla to expose the RWM and basal aspects of the cochlea, including the RW and the middle-ear ossicles (Fig 3a). Additional heating of the environment around the animals head was provided using a thermostatically controlled infrared lamp, such that the temperature at the edge of the open bulla was maintained at 34- 35 °C. All measurements were made under open-bulla conditions, but the cochlea itself was intact.

[0120] A spectral domain OCT system (Thorlabs Telesto TEL320C1, operating with a central wavelength of 1300 nm) was used for interferometric imaging and vibration measurements. The system provided cross-sectional (B-Scan) and axial images (A-Scans and C-scans). The sampling rate of the OCT recording system was 111.6 kHz. Each optical spectrum (A-Scan) had 2048 samples covering the ~210 nm bandwidth of the interferometer’s light source; each instantaneous axial image and vibration map correspondingly had 1024 spatial “pixels” covering the instrument’s ~3.5 mm depth-of-field(i.e., z-range). The optical recording system had an axial point spread function with a FWHM of ~6 μm, a lateral resolution (in the x-y plane) of 13 μm, and a linear operating range of >500 μm (all assessed in air, with a refractive index of 1). The theoretical axial resolution of our OCT system was ~2.9 μm in perilymph (with an assumed refractive index of 1.3); the numerical aperture of the imaging lens was 0.055, and the amount of light incident on the cochlea was ~3.7 mW.

[0121] Intra-cochlear images (B-Scans) were formed by scanning the OCT across a series of parallel, quasi-radial (but typically far from transverse) planes that sectioned the cochlear partition at different longitudinal positions (FIG. 3B; in this example, quasi-radial B-Scans were made at each of the positions indicated by the arrow). The spacing between the initial measurement planes was ~50-100 μm. Images were sequentially compensated for the oblique angles of incidence of the recording beam and for the refractive index of the intra-cochlear fluids, which was assumed to be 1.3.ResultsExample 1: The RW niche displays a gradient distribution of ciliated cells.

[0122] There are conflicting reports regarding whether the dorsal roof of the RWM is ciliated (Thompson and Tucker 2013; Luo et al. 2017). In this study, we utilized immunostaining to investigate the presence of ciliated cells in the porcine RW niche area. Inner ear sections, with a thickness of 8 μm, were stained with Acetylated (AC) Tubulin, a component found in cilia axonemes. FIG. 1A illustrates the Ac-Tubulin staining, which helps visualize the location of the cilia. The box in FIG. 1A is magnified in the figure to the right, displaying the RWM and the lining cells of the niche, both of which possess cilia. Boxes A. and B in FIG. 1A further confirm the presence of cilia on the RWM side. However, a gradual decrease in dilation is observed towards the bottom of the RWM tissue. FIG. 1B presents a deeper section along the cochlear duct, revealing more deciliation at the bottom section of the RWM, as evident in boxes A and B.

[0123] FIG. 1C presents a transmission electron microscope (TEM) image of the outer epithelial layer of the porcine RW niche, which feces the middle ear. Each ciliated cell is underpinned by one or two non-ciliated basal cells, indicated by arrows in FIG. 1C. Basal cells of a similar nature have been observed in the middle ear, trachea, and nasal mucosal epithelia. Some studies suggest that these basal cells act as progenitor cells for the ciliated cells (Cole et al. 2010; Musah, Chen, and Hoyle 2012; Rock et al. 2009; Schoch et al. 2004; Luo et al. 2017). FIGS. 1D-1F illustrate multiple cross-sections of cilia from the outerepithelial cells. These cross-sections reveal the 9 + 2 microtubule structure characteristic of each cilium. This 9 + 2 structure, commonly found in epithelial cells lining the trachea, oviduct, and brain, is associated with cell and fluid movement (Dutcher 1995). The orientation of each cilium can be determined at the basal foot, where an electron-dense triangle appears at the comer of the cilium ring in TEM images, as indicated by an arrow in FIG. 1D. As shown in FIG. 1E, TEM reveals that most cilia in the RW niche epithelium are oriented in the same direction. This suggests coordinated motility of the cilia, which would result in a laminar, directed flow (Kunimoto et al. 2012; Megan et al. 2015; Marshall and Kintner 2008; Luo et al. 2017). In cases where there is a change in direction, as is evident in the direction of the arrows in FIG. 1E, the change is gradual.Example 2: Goblet cells provide a mucus blanket for the ciliated epithelium of the RW niche

[0124] Goblet cells, characterized by their distinct wine glass-shaped morphology, play a role in forming a mucin blanket over the ciliated cells. FIG. 2A presents a section of the porcine inner ear, revealing cells with a distinct wine glass-shaped morphology in the zoomed-in area. To confirm that these are indeed goblet cells in the RW niche, Alcian blue periodic-acid-Schiff (PAS) staining was applied to the porcine inner ear sections, as shown in FIG. 2B. The Alcian blue PAS is intended for use in simultaneous histological visualization of sulfated and carboxylated acid mucopolysaccharides, sulfated and carboxylated sialomucins (glycoproteins), and neutral mucins produced by goblet cells. The zoomed-in area confirms the presence of both acidic and neutral mucins on the lining cells of the niche, but not on the RWM side. The mixture of acidic and neutral mucins is indicated by the mix of blue-mauve color.Example 3: The mucus in the RW niche has a coordinated directional flow with a circular pattern facilitated by ciliary motion.

[0125] To explore the mucociliary transport within the RW niche, we devised an in-vivo experiment using a gerbil model. We employed optical coherence tomography (OCT) to document the clearance of Dexamethasone fluoresceine (DexF) from the RW niche when it was directly applied to the RWM. Conducting such an experiment with a pig model would pose significant challenges due to the thicker skull and the extension of the mastoid bone along the middle ear canal. As depicted in FIG. 3A, once the bulla is opened, DexF is applied directly onto the RWM. The OCT B-scan is conducted along the arrow indicated in bothFIG. 3A and FIG. 3B. FIG. 3B presents a series of B-scan images captured at various time points. At time 0 S, no drug is applied, and the image displays a cross-section of the RWM in its normal state. Starting at 60 S, the RWM begins to bend, potentially due to the heavy load of the drug and mucus. The drug and mucus move via a laminar flow, as shown in the B- scans (note that DexF has a brighter contrast than the mucus, but this contrast is not due to its fluorescent properties). The direction of the flow is indicated by arrows at 88 S. The fluid, as pointed out, moves towards the ET on the left side of the B-scan image. By 780 S, most of the drug has been cleared from the RW niche area, and the RWM returns to its normal state, i.e., without bending.

[0126] In order to gain insights into mucociliary transport and the properties of mucus, we tracked the path of DexF particles at various time intervals using the Trackpy algorithm (Allan et al. 2014). The algorithm was set to consider only spherical particles that could be traced over five consecutive frames, captured at a rate of 30 fps. The color-coded trajectory (spanning five frames) and velocity (between two frames) of the particles are depicted in FIG. 3C and FIG. 3D, respectively. The laminar flow with circular motion is clearly visible in both the trajectory and velocity plots. This pattern has been observed previously in airliquid interface cultures of human airway cells, where the coordinated ciliary motion transports mucus in a circular pattern (Matsui et al. 1998; Tarran et al. 2005; Button and Boucher 2008; Zhang et al. 2009; Sears, Yin, and Ostrowski 2015).

[0127] Subsequently, we computed the clearance rate from the RW niche. Under the assumption that the mucociliary transport of DexF adheres to first-order kinetics, the clearance down to the ET can be represented by the equation where C denotesthe concentration of the applied drug, t represents time, and t1 / 2represents the half-life of the drug(Gizurarson 2015). Thus, the end product of this equation will be where Ctis aremaining dose of the drug in the RW niche at time t and C0is the administrated dose of the drug. From the OCT recording, the half-life (t1 / 2) is 7 min. If the half-life is increased to 2x and 3x, the simulated clearance rate will decrease as represented in FIG. 3E.Example 4: Bulk RNA sequencing has revealed the expression of ciliary genes in RWM cells

[0128] To investigate whether genes associated with ciliary formation and maintenance are expressed in the RWM, we performed bulk RNA sequencing on epithelial cells derived from the RWM. Using flow cytometry, we separated the RWM cells into epithelial cells (CD326+) and non-epithelial cells (CD326-). After sorting, the cells were plated and cultured for three weeks prior to RNA sequencing. FIG. 4A displays the gene expression levels of genes associated with motile cilia in CD326+ / - cells (Patir et al. 2020). The expression levels were compared to the gene expression levels of ciliated and tuft cells derived from human lung tissue (Madissoon et al. 2023). The RWM cells express genes that are associated with motile cilia, albeit at a lower expression level compared to human airway-ciliated cells.

[0129] Our study revealed that RWM cells express genes for taste receptors, specifically the sweet TAS1R3 and umami TAS1R3, as illustrated in FIG. 4B. Furthermore, we conducted a comparison of gene expression between RWM cells, and the ciliated and tuft cells found in human lungs. This comparison is significant as both cell types contribute to local innate immunity and substance removal via taste receptors (F. Li 2013; Yamamoto and Ishimaru 2013; An and Liggett 2018; Shah et al. 2009; Carey, Lee, and Cohen 2016).

[0130] The RWM cells do not evidently express the genes for bitter taste receptors. Given that in tuft cells, the sweet, umami, and bitter taste receptors collectively modulate the immune response, we sought to further explore the presence of the bitter receptor in RWM cells. To this end, we employed immunostaining against TAS2R4, which is a bitter recqjtor 4. As depicted in FIGS. 5A-5C, the entire RW niche area tested positive for the TAS2R4 protein. For comparison, we have included sections of the porcine tongue, shown in FIG. 5D, as a positive control.

[0131] We also found the AQP5 gene, which is responsible for chemosensory detection of water or hypoosmotic fluids in RWM cells (Gilbertson, Baquero, and Spray- Watson 2006). These osmotic sensors and transduction of hypoosmotic stimuli involves water influx through aquaporins followed by activation of volume-regulated anion channels (causing cell depolarization). Water is also shown to evoke taste responses (Galindo- Cuspinera et al. 2006). This receptor could be potentially used in drug formulation to improve the passage across the RWM.

[0132] Traces of ENaC (channel subunit γ) gene, Na+or salty taste recqjtor,(Bachmanov and Beauchamp 2007) were also detected in RWM cells. The ENaC channel also plays a role in the surface liquid layer, in opposition, playing a role in mucociliary transport. Inhibition of ENaC is shown to improve mucociliary function in cystic fibrosisairways (Reihill et al. 2016). The activation of this receptor should play a role in enhancing drug transport by decreasing the liquid layer.Example 5: Inhibition of ciliary beating improves DexF passage across the RWM.

[0133] Subsequently, we hypothesized that by inhibiting cilia beating, we could enhance drug delivery to the inner ear. This enhancement could be achieved by prolonging the duration that the injected drug formulation remains in the middle ear, and therefore in proximity to the RWM. To test this, we suppressed the ciliary function using Ciliobrevin D (CD) and evaluated the passage of DexF through the RWM using an ex-vivo porcine RWM chamber (Moatti, Silkstone, et al. 2023). CD is a small molecule that acts as an inhibitor of Dynein (Firestone et al. 2012; Roossien, Miller, and Gallo 2015). The ex-vivo RWM chamber, encompassing the R WM tissue and its surrounding bony structure, is depicted in FIG. 6A. The microscopic image captured at the conclusion of the experiment, as shown in FIG. 6A, reveals the presence of cilia upon closer inspection. This confirms that the cilia were preserved throughout the dissection and testing procedures.

[0134] FIG. 6B presents a diagram of the ex-vivo chamber, which is similar to a transwell. Dexamethasone fluorescein (DexF) is introduced into the upper section of the chamber, and its transit through the RWM is gauged by taking samples from the lower section, which is separated from the upper section by the RWM. While the removal process through the ET cannot be exactly replicated, the ex-vivo setup enables us to establish a correlation between the degree of ciliary movement inhibition and the enhancement in drug passage. We administered DexF both with and without CD and monitored drug passage across the chamber at various time points. The concentration of DexF in the lower chamber is depicted in FIG. 6B. A significant enhancement in drug passage was observed after 22 hours. Prior studies have indicated that CD induces a temporary inhibition of ciliary motor function after approximately 20 hours, which accounts for the observed improvement in drug passage only after 22 hours (Sainath and Gallo 2015; Choi et al. 2019).Example 6: Inhibition of taste receptors improves DexF passage across the RWM.

[0135] Taste family 2 receptors, also referred to as TAS2Rs, are recognized for their role in increasing the cilia beat frequency in the airways (Kuek et al. 2022). Consequently, inhibiting TAS2R4, which is expressed in the RW niche, could potentially decrease the drug clearance from the tympanic cavity. Prior research has indicated that GABA and BCML function as bitter blockers, serving as antagonists and inverse agonists of TAS2R4,respectively (Pydi et al. 2014). It is speculated that endogenous GABA might be playing a key role in regulating the function of TAS2R4 in human leukocytes, airways, trachea, colon, gut, and heart (Pydi et al. 2014). GABA is also recognized as the first bitter blocker to have potency in the lower nanomolar range. BCML has shown inverse agonist activity.

[0136] We tested the effect of both blockers on the passage of DexF using the ex-vivo chamber. After 22 hours, when using GABA but not BCML (50 nM) significant improvement in DexF was reported (FIG. 6B). When increasing the concentration of BCML three times (150 nM) DexF passage was enhanced through the RWM similarly to GAB A. This result suggests an interaction between glucocorticoids such as DexF and the bitter taste receptor targeted by TAS2R4 inhibitors. The interaction was further tested by examining the passage of quinine, an additional bitter drug and TAS2R4 agonist, through the RWM with or without BCML treatment (150 nM; FIG. 6C). As shown in FIG. 6C, pretreatment with the TAS2R4 antagonist BCML for 30 minutes significantly increased the passage of quinine (1 mM) through the RWM compared to controls, quinine exposed without BCML treatment. The impact of GABA and BCML inhibition on bitter taste receptors beyond TAS2R4 has not been explored extensively. Therefore, it remains uncertain whether DexF interacts with other taste receptor groups, and not only TAS2R4.Example 7: HSP90 inhibition modulates bitter receptors leading to higher passage across the RWM.

[0137] Considering the wide range of ligands that interact with TAS2Rs, it is improbable that a single universal blocker capable of targeting all 25 human TAS2Rs could be developed. Thus, we looked at the heat shock protein 90 (HSP90) inhibition which is found to be integral to all TAS2R-stimulated NO (nitric oxide) production (Carey et al. 2022). HSP90 has been shown previously to function as a natural immune system regulator by enhancing endothelial NO synthase (eNOS) activation downstream of TAS2R signaling, thereby influencing NO production without disrupting initial Ca2+signaling. These studies imply that HSP90 holds significance in bolstering antibacterial innate immunity within the airways, potentially offering a valuable target for managing conditions like chronic rhinosinusitis, asthma, or cystic fibrosis. In this study, we focused on the inhibition of HSP90 as a means to enhance drug passage across the RWM. We examined the impact of Geldanamycin, known as an HSP90 inhibitor (Takaki et al. 2007), and administered it in conjunction with DexF, as depicted in FIG. 6B. Remarkably, it became apparent that thepassage of DexF across the RWM was enhanced just 0.5 hours post-exposure, and showed significant improvement by the 2.5-hour mark.Discussion

[0138] We have shown that RWM is partially, and the lining cells of the niche are fully ciliated. This is the first time that RWM is shown to have a ciliary structure. The RWM also shows gradual deciliation from the bottom as it moves inside the cochlear duct. This has an important implication for intratympanic drug delivery to the inner ear. This fact explains the previous reports showing that high viscosity solution injected increased RWM permeability and drying the RWM increased permeability (Aa, Jj, and An 2008).

[0139] We have confirmed the mucus secretary cells' presence on the lining cells of the niche (besides the middle ear goblet cells), which provides mucus when an external substance is placed on the RWM. This provides quick access to remove the injected drugs from the RW niche area.

[0140] We also revealed the directional flow towards the ET with a rotational pattern in the RW niche facilitated by the RWM ciliated epithelium via in-vivo OCT recordings. It is shown previously in the lung epithelium that the coordinated beat of motile cilia creates a horizontal flow across their tips (Satir and Sleigh 1990). In human airway culture, the cilia coordinate their motion to transport mucus in a continuous circular pattern as well (Matsui et al. 1998; Tarran et al. 2005; Button and Boucher 2008; Zhang et al. 2009; Sears, Yin, and Ostrowski 2015). We observe similar behavior here, confirming the presence of motile cilia in the RW niche. Since there are no reports of mucus-secreting cells present on the lining cells of the round window (RW) niche in gerbils, we cannot attribute the observed mucociliary transport solely to the round window niche. The middle ear might also be aiding in this process. The mucociliary transport in the RW niche being captured in vivo could explain the quick removal of drugs delivered via intratympanic injections, both in animal models and humans.

[0141] The mucociliary characteristics that are revealed from the OCT recording could also aid us in designing the pharmacological, physical, mechanical, microbacterial, and chemical characteristics of the drug being administered. The gained knowledge from other organs with mucociliary characteristics, such as the nasal cavity and lung, can be translated to this organ, which opens up new opportunities to improve intratympanic delivery efficiency. For example, if the formulation can be adjusted to have mucoadhesive properties, the clearance pattern will change as shown in FIG. 4E. If the RWM is being dried and the muco-ciliary functions are haltered for the period of drug applications for example, 5-7 min, a similar scenario to increasing half time from 7 to 14 min would be created. The half-time of 14 min on the graph after 30 min of drug administration shows roughly 10x higher concentration than the half-time of 7 min. That could explain the 1 Ox increase in the drug presence in the perilymph of the inner ear in guinea pigs when RWM was dried during drug administration (Aa, Jj, and An 2008). This finding could help us evaluate different factors that affect the rate of mucociliary transport.

[0142] We also used CD to temporarily inhibit the movement of the cilia and investigate its effect on the RWM permeability for DexF, we observed that after 24 hours of CD addition, when the ciliary motion is inhibited, the passage is increased. Although the removal processes cannot be understood in this ex-vivo RWM chamber that lacks ET clearance, it provides us with a tool to investigate the role of ci liary motion versus mucociliary removal effect on the drug passage across RWM.

[0143] The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation, or limitations that are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments claimed. Thus, it should be understood that although the present description has been specifically disclosed by embodiments, optional features, modification, and variation of the concepts herein disclosed may be resorted to by those skilled in the area and that such modifi cations and variations are considered to be within the scope of these embodiments as defined by the description and the appended claims. Although some aspects of the present disclosure can be identified herein as particularly advantageous, it is contemplated that the present disclosure is not limited to these particular aspects of the disclosure.

[0144] Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0145] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any elements) can be removed from the group.

[0146] It should be understood that, in general, where the disclosure, or aspects of the disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

[0147] ReferencesAa, Mikulec, Hartsock Jj, and Salt An. 2008. “Permeability of the Round Window Membrane Is Influenced by the Composition of Applied Drug Solutions and by Common Surgical Procedures.” Otology & Neurotology : Official Publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology 29 (7). https: / / doi.org / 10.1097 / MAO.0b013e31818658ea.An, Steven S., and Stephen B. Liggett. 2018. “Taste and Smell GPCRs in the Lung: Evidence for a Previously Unrecognized Widespread Chemosensory System.” Cellular Signalling, In Honor of Bob Lefkowitz, 41 (January): 82-88. https: / / doi.Org / 10.1016 / j.cellsig.2017.02.002.Bachmanov, Alexander A., and Gary K. Beauchamp. 2007. “Taste Receptor Genes.” Annual Review of Nutrition 27:389. https: / / doi.org / 10.1146 / annurev.nutr.26.061505.111329.Behrens, Maik. 2023. “Taste 2 Receptors in GtoPdb v.2023.1.” IUPHAR / BPS Guide to Pharmacology CITE 2023 (1). https: / / doi.Org / 10.2218 / gtopdb / F117 / 2023.l.Button, Brian, and Richard C. Boucher. 2008. “Role of Mechanical Stress in Regulating Airway Surface Hydration and Mucus Clearance Rates.” Respiratory Physiology & Neurobiology, Respiratory Biomechanics, 163 (1): 189-201. https: / / doi.org / ! 0.1016 / j .resp.2008.04.020.Carey, Ryan M., Benjamin M. Hariri, Nithin D. Adappa, James N. Palmer, and Robert J. Lee. 2022. “HSP90 Modulates T2R Bitter Taste Receptor Nitric Oxide Production and Innate Immune Responses in Human Airway Epithelial Cells and Macrophages.” Cells 11 (9): 1478. https: / / doi.org / 10.3390 / cellsll091478.Carey, Ryan M., Robert J. Lee, and Noam A. Cohen. 2016. “Taste Receptors in Upper Airway Immunity,” July, https: / / doi.org / 10.1159 / 000445137.Choi, Brian K. A., Philippe M. D’Onofiio, Alireza P. Shabanzadeh, and Paulo D. Koeberle. 2019. “Stabilization of Primary Cilia Reduces Abortive Cell Cycle Re-Entry to Protect Injured Adult CNS Neurons from Apoptosis.” Edited by Rafael Linden. PLOS ONE 14 (8): e0220056. https: / / doi.org / 10.1371 / joumal.pone.0220056.Cole, Brook B., Russell W. Smith, Kimberly M. Jenkins, Brian B. Graham, Paul R. Reynolds, and Susan D. Reynolds. 2010. “Tracheal Basal Cells: A FacultativeProgenitor Cell Pool.” The American Journal of Pathology 177 (1): 362-76. https: / / doi.org / 10.2353 / ajpath.2010.090870.Dutcher, Susan K. 1995. “Flagellar Assembly in Two Hundred and Fifty Easy-to-Follow Steps.” Trends in Genetics 11 (10): 398-404. https: / / doi.org / 10.1016 / S0168- 9525(00)89123-4.Firestone, Ari J., Joshua S. Weinger, Maria Maldonado, Kari Barlan, Lance D. Langston, Michael O’Donnell, Vladimir I. Gelfand, Tarun M. Kapoor, and James K. Chen. 2012. “Small-Molecule Inhibitors of the AAA+ ATPase Motor Cytoplasmic Dynein.” Nature 484 (7392): 125-29. https: / / doi.org / 10.1038 / naturel0936.Galindo-Cuspinera, Veronica, Marcel Winnig, Bernd Bufe, Wolfgang Meyerhof, and Paul A. S. Breslin. 2006. “A TAS1R Receptor-Based Explanation of Sweet ‘Water-Taste.’" Nature 441 (7091): 354-57. https: / / doi.org / 10.1038 / nature04765.Gilbertson, Timothy A., Arian F. Baquero, and Kristina J. Spray-Watson. 2006. “Water Taste: The Importance of Osmotic Sensing in the Oral Cavity.” Journal of Water and Health 4 (SI): 35-40. https: / / doi.org / 10.2166 / wh.2006.0042.Gizurarson, Sveinbjom. 2015. “The Effect of Cilia and the Mucociliary Clearance on Successful Drug Delivery.” Biological and Pharmaceutical Bulletin 38 (4): 497-506. https: / / doi.org / 10.1248 / bpb.bl4-00398.Goycoolea, Marcos V. 2001. “Clinical Aspects of Round Window Membrane Permeability Under Normal and Pathological Conditions." Acta Oto-Laryngologca 121 (4): 437- 47. https: / / doi.Org / 10.1080 / 000164801300366552.Goycoolea, Marcos V., and Lars Lundman. 1997. “Round Window Membrane. Structure Function and Permeability: A Review.” Microscopy Research and Technique 36 (3): 201-11. https: / / doi.org / 10.1002 / (SICI)1097-0029(19970201)36:3<20I::AID- JEMT8>3.0.CO;2-R.Goycoolea, Marcos V., David Muchow, and Patricia Schachem. 1988. “Experimental Studies on Round Window Structure: Function and Permeability.” The Laryngoscope 98 (S44): 1-20. https: / / doi.org / 10.1288 / 00005537-198806001-00002.Hegan, Peter S., Eric Ostertag, Aron M. Geurts, and Mark S. Mooseker. 2015. “Myosin Id Is Required for Planar Cell Polarity in Ciliated Tracheal and Ependymal Epithelial Cells.” Cytoskeleton 72 (10): 503-16. https: / / doi.org / 10.1002 / cm.2I259.Kuek, Li Eon, Derek B. McMahon, Ray Z. Ma, Zoey A. Miller, Jennifer F. Jolivert, Nithin D. Adappa, James N. Palmer, and Robert J. Lee. 2022. “Cilia Stimulatory and Antibacterial Activities of T2R Bitter Taste Receptor Agonist Diphenhydramine: Insights into Repurposing Bitter Drugs for Nasal Infections.” Pharmaceuticals 15 (4): 452. https: / / doi.org / 10.3390 / phl5040452.Kunimoto, Koshi, Yuji Yamazaki, Tomoki Nishida, Kyosuke Shinohara, Hiroaki Ishikawa, Toshiaki Hasegawa, Takeshi Okanoue, et al. 2012. “Coordinated Ciliary Beating Requires Odf2-Mediated Polarization of Basal Bodies via Basal Feet." Cell 148 (1): 189-200. https: / / doi.Org / 10.1016 / j.cell.201I.10.052.Li, Feng. 2013. “Taste Perception: From the Tongue to the Testis.” Molecular Human Reproduction 19 (6): 349-60. https: / / doi.org / 10.1093 / molehr / gat009.Li, Zi-Nan, and Ying Luo. 2022. “HSP90 Inhibitors and Cancer: Prospects for Use in Targeted Therapies (Review).” Oncology Reports 49 (1): 6. https: / / doi.org / 10.3892 / or.2022.8443.Luo, Wenwei, Hong Yi, Jeannette Taylor, Jian-dong Li, Fanglu Chi, N. Wendell Todd, Xi Lin, Dongdong Ren, and Ping Chen. 2017. “Cilia Distribution and Polarity in the Epithelial Lining of the Mouse Middle Ear Cavity.” Scientific Reports 7 (March): 45870. https: / / doi.org / 10.1038 / srep45870.Madissoon, Elo, Amanda J. Oliver, Vitalii Kleshchevnikov, Anna Wilbrey-Clark, Krzysztof Polanski, Nathan Richoz, Ana Ribeiro Orsi, et al. 2023. “A Spatially Resolved Atlas of the Human Lung Characterizes a Gland-Associated Immune Niche.” Nature Genetics 55 (1): 66-77. https: / / doi.org / 10.1038 / s41588-022-01243-4.Magwenyane, Ayanda M., Samuel C. Ugbaja, Daniel G. Amoako, Anou M. Somboro, Rene B. Khan, and Hezekiel M. Kumalo. 2022. “Heat Shock Protein 90 (HSP90) Inhibitors as Anticancer Medicines: A. Review on the Computer-Aided Drug Discovery Approaches over the Past Five Years.” Computational and Mathematical Methods in Medicine 2022 (May): 2147763. https: / / doi.org / 10.1155 / 2022 / 2147763.Marshall, Wallace F, and Christopher Kintner. 2008. “Cilia Orientation and the Fluid Mechanics of Development.” Current Opinion in Cell Biology, Cell structure and dynamics, 20 (1): 48-52. https: / / doi.Org / 10.1016 / j.ceb.2007.l 1.009.Matsui, Hirotoshi, Barbara R. Grubb, Robert Tarran, Scott H. Randell, John T. Gatzy, C. William Davis, and Richard C. Boucher. 1998. “Evidence for Periciliary Liquid Layer Depletion, Not Abnormal Ion Composition, in the Pathogenesis of Cystic Fibrosis Airways Disease.” Cell 95 (7): 1005-15. https: / / doi.org / 10.1016 / S0092- 8674(00)81724-9.McLean, Will J., Ashley S. Hinton, Jenna T. J. Herby, Alec N. Salt, Jared J. Hartsock, Sam Wilson, David L. Lucchino, et al. 2021. “Improved Speech Intelligibility in Subjects With Stable Sensorineural Hearing Loss Following Intratympanic Dosing of FX-322 in a Phase lb Study.” Otology & Neurotology 42 (7): e849. https: / / doi.org / 10.1097 / MA0.0000000000003120.Moatti, Adele, Yuheng Cai, Chen Li, Kristen D. Popowski, Ke Cheng, Frances S. Ligler, and Alon Greenbaum. 2023. “Tissue Clearing and Three-Dimensional Imaging of the Whole Cochlea and Vestibular System from Multiple Large-Animal Models.” STAR Protocols 4 (2): 102220. https: / / doi.Org / 10.1016 / j.xpro.2023.102220.Moatti, Adele, Yuheng Cai, Chen Li, Tyler Sattler, Laura Edwards, Jorge Piedrahita, Frances S. Ligler, and Alon Greenbaum. 2020. “Three-Dimensional Imaging of Intact Porcine Cochlea Using Tissue Clearing and Custom-Built Light-Sheet Microscopy.” Biomedical Optics Express 11 (11): 6181-96. https: / / doi.org / 10.1364 / BOE.402991.Moatti, Adele, Dylan Silkstone, Taylor Martin, Keith Abbey, Kendall A Hutson, Douglas C Fitzpatrick, Carlton J Zdanski, Alan G Cheng, Frances S Ligler, and Alon Greenbaum. 2023. “Assessment of Drug Permeability through an Ex Vivo Porcine Round Window Membrane Model.” iScience 26 (6): 106789. https: / / doi.Org / 10.1016 / j.isci.2023.106789.Musah, Sadiatu, Jing Chen, and Gary W. Hoyle. 2012. “Repair of Tracheal Epithelium by Basal Cells after Chlorine-Induced Injury." Respiratory Research 13 (1): 107. https: / / doi.org / 10.1186 / 1465-9921-13-107.Ohashi, Yoshihiro, Yoshiaki Nakai, Shoko Kihara, Kenichi Maruoka, Hiroshi Ikeoka, and Yoshinori Uemura. 1985. “The Ciliary Activity of the Middle Ear Lining-Functional and Morphological Observation.” Auris Nasus Larynx 12 (January): S 123-25. https: / / doi.org / 10.1016 / S0385-8146(85)80122-X.Paparella, Michael M., Patricia A. Schachem, and Young Bin Choo. 1983. “The Round Window Membrane: Otological Observations.” Annals of Otology, Rhinology & Laryngology 92 (6): 629-34. https: / / doi.org / 10.1177 / 000348948309200619.Patir, Anirudh, Amy M. Fraser, Mark W. Barnett, Lynn McTeir, Joe Rainger, Megan G. Davey, and Tom C. Freeman. 2020. “The Transcriptional Signature Associated with Human Motile Cilia.” Scientific Reports 10 (1): 10814. https: / / doi.org / 10.1038 / s41598-020-66453-4.Pydi, Sai P., Tyler Sobotkiewicz, Rohini Billakanti, Rajinder P. Bhullar, Michele C. Loewen, and Prashen Chelikani. 2014. “Amino Acid Derivatives as Bitter Taste Receptor (T2R) Blockers*.” Journal of Biological Chemistry 289 (36): 25054-66. https: / / doi.org / 10.1074 / jbc.M114.576975.Reihill, James A., Brian Walker, Robert A. Hamilton, Timothy E. G. Ferguson, J. Stuart Elbom, M. Jackson Stutts, Brian J. Harvey, Vinciane Saint-Criq, Siobhan M. Hendrick, and S. Lorraine Martin. 2016. “Inhibition of Protease-Epithelial Sodium Channel Signaling Improves Mucociliary Function in Cystic Fibrosis Airways.” American Journal of Respiratory and Critical Care Medicine 194 (6): 701-10. https: / / doi.org / 10.1164 / rccm.201511-2216OC.Rock, Jason R., Mark W. Onaitis, Emma L. Rawlins, Yun Lu, Cheryl P. Clark, Van Xue, Scott H. Randell, and Brigid L. M. Hogan. 2009. “Basal Cells as Stem Cells of the Mouse Trachea and Human Airway Epithelium.” Proceedings of the National Academy of Sciences 106 (31): 12771-75. https: / / doi.org / 10.1073 / pnas.0906850106.Roossien, Douglas, Kyle Miller, and Gianluca Gallo. 2015. “Ciliobrevins as Tools for Studying Dynein Motor Function.” Frontiers in Cellular Neuroscience 9. https: / / www.frontierein.org / ffllicles / 10.3389 / fiicel.2015.00252.Sainath, Rajiv, and Gianluca Gallo. 2015. “The Dynein Inhibitor Ciliobrevin D Inhibits the Bidirectional Transport of Organelles along Sensory Axons and Impairs NGF- Mediated Regulation of Growth Cones and Axon Branches.” Developmental Neurobiology 75 (7): 757-77. https: / / doi.org / 10.1002 / dneu.22246.Salt, Alec N., Jared J. Hartsock, Fabrice Piu, and Jennifer Hou. 2018. “Dexamethasone and Dexamethasone-Phosphate Entry into Perilymph Compared for Middle Ear Applications in Guinea Pigs.” Audiology & Neuro-Otology 23 (4): 245-57. https: / / doi.org / 10.1159 / 000493846.Satir, P., and M. A. Sleigh. 1990. “The Physiology of Cilia and Mucociliary Interactions.” Annual Review of Physiology 52 (1): 137-55. https: / / doi.org / 10.1146 / annurev.ph.52.030190.001033.Schoch, Kelly G., Adriana Lori, Kimberlie A. Bums, Tracy Eldred, John C. Olsen, and Scott H. Randell. 2004. “A Subset of Mouse Tracheal Epithelial Basal Cells Generates Large Colonies in Vitro.” American Journal of Physiology-Lung Cellular and Molecular Physiology 286 (4): L631-42. https: / / doi.org / 10.1152 / ajplung.00I12.2003.Sears, Patrick R., Wei-Ning Yin, and Lawrence E. Ostrowski. 2015. “Continuous Mucociliary Transport by Primary Human Airway Epithelial Cells in Vitro.” American Journal of Physiology - Lung Cellular and Molecular Physiology 309 (2): L99-108. https: / / doi.org / 10.1152 / ajplung.00024.2015.Shah, Alok S., Yehuda Ben-Shahar, Thomas O. Moninger, Joel N. Kline, and Michael J. Welsh. 2009. “Motile Cilia of Human Airway Epithelia Are Chemosensory.” Science 325 (5944): 1131-34. https: / / doi.org / 10.1126 / science.1173869.Sigoillot, Maud, Anne Brockhoff, Wolfgang Meyerhof, and Loic Briand. 2012. “Sweet- Taste-Suppressing Compounds: Current Knowledge and Perspectives of Application.' Applied Microbiology and Biotechnology 96 (3): 619-30. https: / / doi.org / 10.1007 / s00253-012-4387-3.Takaki, Eiichi, Mitsuaki Fujimoto, Takashi Nakahari, Shigenobu Yonemura, Yoshihiko Miyata, Naoki Hayashida, Kaoru Yamamoto, et al. 2007. “Heat Shock Transcription Factor 1 Is Required for Maintenance of Ciliary Beating in Mice.” The Journal of Biological Chemistry 282 (51): 37285-92. https: / / doi.org / 10.1074 / jbc.M704562200.Tarran, Robert, Brian Button, Maryse Picher, Anthony M. Paradiso, Carla M. Ribeiro, Eduardo R. Lazarowski, Liqun Zhang, et al. 2005. “Normal and Cystic Fibrosis Airway Surface Liquid Homeostasis: THE EFFECTS OF PHASIC SHEAR STRESSAND VIRAL INFECTIONS*.” Journal of Biological Chemistry 280 (42): 35751-59. https: / / doi.org / 10.1074 / jbc.M505832200.Thompson, Hannah, and Abigail S. Tucker. 2013. “Dual Origin of the Epithelium of the Mammalian Middle Ear.” Science (New York, N.Y.) 339 (6126): 1453 56. https: / / doi.org / ! 0.1126 / science.1232862.Yamamoto, Kurumi, and Yoshiro Ishimaru. 2013. “Oral and Extra-Oral Taste Perception.” Seminars in Cell & Developmental Biology, Semaphorins & Taste Perception, 24 (3): 240-46. https: / / doi.Org / 10.1016 / j.semcdb.2012.08.005.Zhang, Liqun, Brian Button, Sherif E. Gabriel, Susan Burkett, Yu Yan, Mario H. Skiadopoulos, Yan Li Dang, et al. 2009. “CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium.” PLOS Biology 7 (7): 61000155. https: / / doi.org / 10.1371 / joumal.pbio.1000155.

Claims

What is claimed is:

1. A method of delivering a therapeutic to the inner ear comprising: modulating the cilia of the ear and administering the therapeutic.

2. The method of claim 1, wherein modulating the cilia of the ear comprises inhibiting cilia motor activity.

3. The method of claim 2, wherein inhibiting cilia motor activity is accomplished by contacting the cilia with an inhibitory compound.

4. The method of claim 3, wherein the inhibitory compound is selected from the group consisting of ciliobrevin D, alvocidib (an inhibitor of ciliogenesis-associated kinase 1 [CILK1]), bromopride, droperiodol, sulpiride, tripelennamine hydrochloride, palonosetron hydrochloride, MK801 hydrogen maleate, tramadol hydrochloride, lorglumide sodium salt, thricolchicoside, GABA, dynarrestin, effectors of intracellular calcium and cyclic adenosine monophosphate (cAMP) levels, molecules that influence the activity of phosphodiesterase (PDE1, PDE4) or dual PDE activation (PDE3 / 4), Wnt signaling inhibitors, actin, components of the actin regulatory system, calcium binding compounds, non-steroidal anti-inflammatory drug (NSAID) drugs, cyclooxygenase inhibitors, and salts and derivatives thereof.

5. The method of any one of claims 1-4, wherein modulating the cilia and administering the therapeutic are performed sequentially.

6. The method of any one of claims 1-4, wherein modulating the cilia and administering the therapeutic are performed simultaneously.

7. The method of any one of claims 1-6, wherein modulating the cilia is accomplished through modulating taste receptors expressed on the surface of the cilia.

8. A method of delivering a therapeutic to the inner ear comprising: modulating a taste receptor and administering the therapeutic.

9. The method of claim 8, wherein modulating the taste receptor comprises inhibiting the taste receptor.

10. The method of claim 8, wherein modulating the taste receptor comprises activating the taste receptor.

11. The method of any one of claims 8-10, wherein modulating the taste receptor is accomplished by contacting the taste receptor with a modulating agent.

12. The method of any one of claims 8-11, wherein the taste recqptor is selected from the group consisting of TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R8, TAS2R9, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R50, and TAS2R60.

13. The method of claim 11, wherein the modulating agent is selected from the group consisting of GABA and the compounds listed in Tables 4-26.

14. An otic pharmaceutical formulation comprising a compound that modulates the activity of the cilia of the ear.

15. The formulation of claim 14, wherein modulation of the cilia inhibits cilia motor activity.

16. The formulation of any one of claims 14-15, wherein the compound is selected from the group consisting of ciliobrevin D, alvocidib (an inhibitor of ciliogenesis-associated kinase 1 [CILK1]), bromopride, droperiodol, sulpiride, tripelennamine hydrochloride, palonosetron hydrochloride, MK.801 hydrogen maleate, tramadol hydrochloride, lorglumide sodium salt, thricolchicoside, GABA, dynarrestin, effectors of intracellular calcium and cyclic adenosine monophosphate (cAMP) levels, molecules that influence the activity of phosphodiesterase (PDEl, PDE4) or dual PDE activation (PDE3 / 4), Wnt signaling inhibitors, actin, components of the actin regulatory system, calcium binding compounds, non-steroidal anti-inflammatory drug (NSAID) drugs, cyclooxygenase inhibitors, and salts and derivatives thereof.

17. The formulation of any one of claims 14-16, further comprising a therapeutic for the inner ear.

18. The formulation of claim 17, wherein the therapeutic is a small molecule.

19. The formulation of claim 17, wherein the therapeutic is a biologic, an antibody, or an antigen-binding fragment thereof.

20. The formulation of claim 17, wherein the therapeutic is gene therapy.

21. The formulati on of any one of claims 14-20, wherein the formulation enhances the passage of a therapeutic to the inner ear.

22. The formulation of claim 21, wherein the enhanced passage is across the round window membrane (RWM) or the oval window.

23. An otic pharmaceutical formulation comprising a compound that modulates a taste receptor in the ear.

24. The formulation of claim 23, wherein modulation of the taste receptor comprises inhibiting the taste receptor.

25. The formulation of claim 23, wherein modulation of the taste receptor comprises activating the taste receptor.

26. The formulation of any one of claims 23-25, wherein modulating the taste receptor is accomplished by contacting the taste receptor with a modulating agent.

27. The formulation of any one of claims 23-26, wherein the taste receptor is selected from the group consisting of TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R3, TAS2R4, TAS2R5, TAS2R7, TAS2R8, TAS2R9, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R50, and TAS2R60.

28. The formulation of claim 26, wherein the modulating agent is an inhibitor selected from the group consisting of inhibitory compounds listed in Table 4, Table 7, Table 10, Table 15, Table 18, Table 19, Table 20, Table 21, Table 23, Table 24, and Table 26.

29. The formulation of claim 26, wherein the modulating agent is an activator selected from the group consisting of activating compounds listed in Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, Table 14, Table 15, Table 16, Table 17, Table 18, Table 19, Table 20, Table 21, Table 22, Table 23, Table 24, and Table 25.

30. A method of enhancing therapeutic delivery to the inner ear, comprising: modulating the cilia of the ear and administering the therapeutic.

31. A. method of enhancing therapeuti c delivery to the inner ear, comprising: modulating a taste receptor and administering the therapeutic.

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