Methods of treating inner ear fibrosis

Abstract

Disclosed herein are compositions and methods for preventing or attenuating the development of fibrosis and scarring, or for treating fibrosis and scarring that have resulted from implantation trauma, implantation and maintenance of a foreign body in the inner ear, such as a cochlear or vestibular implant, or from damage resulting from other injury to the inner ear, such as, for example, from electrical stimulation from an implant electrode.

Description

METHODS OF TREATING INNER EAR FIBROSISBACKGROUNDField[oooi] The present disclosure relates to treating inner ear fibrosis with PAI- 1 inhibitors, where the fibrosis is the result of tissue damage due to implantation of a cochlear or vestibular implant, the presence of a foreign body inside the inner ear, and / or repeated application of electrical stimulation to the inner ear tissues.Related Art

[0002] Medical devices have provided a wide range of therapeutic benefits to recipients over recent decades. Medical devices can include internal or implantable components / devices, external or wearable components / devices, or combinations thereof (e.g., a device having an external component communicating with an implantable component). Medical devices, such as traditional hearing aids, partially or fully-implantable hearing prostheses (e.g., bone conduction devices, mechanical stimulators, cochlear implants, etc.), pacemakers, defibrillators, functional electrical stimulation devices, and other medical devices, have been successful in performing lifesaving and / or lifestyle enhancement functions and / or recipient monitoring for a number of years.

[0003] The types of medical devices and the ranges of functions performed thereby have increased over the years. For example, many medical devices, sometimes referred to as “implantable medical devices,” now often include one or more instruments, apparatus, sensors, processors, controllers or other functional mechanical or electrical components that are permanently or temporarily implanted in a recipient. These functional devices are typically used to diagnose, prevent, monitor, treat, or manage a disease / injury or symptom thereof, or to investigate, replace or modify the anatomy or a physiological process. Many of these functional devices utilize power and / or data received from external devices that are part of, or operate in conjunction with, implantable components.SUMMARY

[0004] In one aspect, a pharmaceutical composition comprises a PAI-1 inhibitor for administration in a therapeutically effective amount to a subject following implantation of a cochlear or vestibular implant. The PAI-1 inhibitor prevents, attenuates or halts development of, or reduces, fibrosis of a subject’s inner ear tissues. The PAI- 1 inhibitor may be any of SK-216, TM 5441, TM 5275 sodium salt, annonacinone, AZ3976, embelin, and Tiplaxtinin. In one embodiment, the PAI-1 inhibitor is Tiplaxtinin.

[0005] In another embodiment an anti-inflammatory therapeutic agent is formulated in combination with the PAI-1 inhibitor. The anti-inflammatory may be any one or more of dexamethasone, prednisone, cortisone, hydrocortisone, and clodronate.. In another embodiment the anti-inflammatory agent is dexamethasone. In other embodiments the pharmaceutical composition includes one or more CNS stimulants such as amphetamine, armodafinil, atomoxetine, benzphetamine, caffeine citrate, dexmethylphenidate, diethylpropion, dextroamphetamine, lisdexamfetamine, methamphetamine, modafinil, oxybate, phendimetrazine, phentermine, pitolisant, solriamfetol, or methylphenidate.

[0006] In still other embodiments the pharmaceutical compositions are formulated as extended release compositions. In other embodiments, the pharmaceutical composition includes a second PAI-1 inhibitor. In some embodiments the composition is formulated for immediate release upon implantation of a medical device and with each electrical impulse delivered to the inner ear tissues.

[0007] In other embodiments, the anti-inflammatory therapeutic agent is co-formulated with at least the first PAI-1 inhibitor for administration to the subject.

[0008] In still other embodiments, a CNS stimulant is included in the composition with a PAI- 1 inhibitor to prevent, attenuate, halt or to reduce fibrosis of the inner ear following implantation of a cochlear implant or vestibular implant. In some embodiments the PAI-1 inhibitor is Tiplaxtinin. In other embodiments an anti-inflammatory agent, such as dexamethasone is included in the composition.

[0009] Another aspect is a method claim for preventing, attenuating, halting development of, or reducing the amount of fibrosis of the inner ear tissues after implantation of a medical device with an electrode. The method includes administering a PAI-1 inhibitor, such as Tiplaxtinin, TM 5441, TM 5275 sodium salt, SK-216, annonacinone, AZ3976, or embelin, which treats fibrosis resulting from the implant’s electrical stimulation in the inner ear. In another embodiment the PAI-1 inhibitor is Tiplaxtinin.

[0010] In some embodiments, two PAI-1 inhibitors are administered in combination. In other embodiments the medical device is a cochlear or vestibular implant.

[0011] In other embodiments the PAI-1 inhibitor, alone or in any combination with other therapeutic agents, is / are administered locally; in still other embodiments the PAI- 1 inhibitor,alone or in any combination with other therapeutic agents, is / are administered systemically; in other embodiments, the PAI- 1 inhibitor, alone or in any combination with other therapeutic agents, is / are administered in an extended release formulation. In still other embodiments, the PAI- 1 inhibitor, alone or in any combination with other therapeutic agents, is / are administered as released from a coating on the electrode of the medical device. In other embodiments the PAI- 1 inhibitor, alone or in any combination with other therapeutic agents, is / are administered as it is released from a coating on the electrode of the medical device following each electrical impulse.

[0012] In another method embodiment the combination of a second and / or third therapeutic agent with the PAI- 1 inhibitor(s) are administered; in some embodiments, the therapeutic agent may be any of dexamethasone, prednisone, cortisone, hydrocortisone, and clodronate. If the anti-inflammatory agent is dexamethasone, it is administered orally to an adult human subject in an amount between about 0.75 mg / kg to about 9 mg / kg daily, or optionally it is administered to an adult human subject via IV or IM in an amount between about 0.5 mg / kg to about 9 mg / kg daily.

[0013] In still other embodiments the anti-inflammatory agent is co-formulated with one or more PAI-1 inhibitors, and / or a CNS stimulant, selected from the groups provided above.

[0014] In still other method embodiments, a therapeutically effective amount of the PAI-1 inhibitor is administered to the subject in an amount reduced over at least about 1-6 months following implantation to 10% or less of an initial dose administered upon implantation. In certain method embodiments, the PAI-1 inhibitor is Tiplaxtinin, administered to a subject over at least about 1-6 months following implantation in a dose sufficient to maintain a its concentration between 100-300 ng / mL in perilymph fluid for at least 6 hours after administration.

[0015] In certain embodiments the PAI-1 inhibitor attenuates the development of or reduces the volume of fibrosis of the inner ear tissues by at least 10 percent. In some embodiments where not otherwise specified, the subject is a mammal such as a lab animal or d.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Disclosed embodiments are described in conjunction with the accompanying drawings.

[0017] FIG. 1A is a schematic diagram of a human ear.

[0018] FIG. IB is a schematic diagram of partial cut-away view of a human inner ear.

[0019] FIG. 1C is a schematic view of a cross sectional view of one turn of a canal of the cochlea of a human inner ear.

[0020] FIG. ID shows a human head and the location of a cochlear implant system.

[0021] FIG. IE is a profile of a human head, identifying the location of a cochlear implant system.

[0022] FIG. IF is a diagram of the cochlear implant system.

[0023] FIG. 1G is a diagram illustrating the inner connections among components of the cochlear implant system.

[0024] FIG. 2A is a photo of the human cochlea, while FIG. 2B is a photo of the top view of the human cochlea.

[0025] FIGs. 3A-3E show the results of in vitro assessment of Tiplaxtinin, a PAI-1 inhibitor, on reduction of fibrosis of the inner ear. FIG. 3A shows fibrosis in the inner ear, as demonstrated through immunochemistry. FIG. 3B shows the nearly complete reduction of fibrosis of the scala. FIGs. 3C and 3D show survival of the inner and outer hair cells (IHC and OHC, respectively), and FIG. 3E shows the over-expression (left) and under-expression (right) of genes using 5 pg / mL of Tiplaxtinin.

[0026] FIGs. 4A-4B show Tiplaxtinin clearance from plasma and perilymph, respectively over 24 hours. FIG. 4A shows the time-dependent, rapid clearance of Tiplaxtinin at various doses (1, 3, 10, 25, 50 and 100 pg / mL) in plasma following intraperitoneal (IP) injection over 24 hours. FIG 4B shows the time -dependent reduction of Tiplaxtinin in the perilymph at the same doses over 24 hours.

[0027] FIG. 5 shows the comparison of the effect before (control) and after transtympanic injection of Tiplaxtinin on auditory function in the ears of two rats.

[0028] FIGs. 6A-6B show the in vivo results of an assessment of Tiplaxtinin on ground impedance after electrical stimulation. FIG. 6A shows MP 1+2 impedances in the inner earsof rats following implantation and electrically stimulation. FIG. 6B shows impedance results following electrical stimulation and administration of Tiplaxtinin.

[0029] FIGs. 7A and 7B illustrate the experimental design associated with several of the examples concerning characterization of fibrosis, cell type, cellular structures, and cellular morphology based on the type of trauma experienced in the cochleae following insertion of an electrode array. The design encompasses both acute and chronic insertion trauma to the cochleae of rats (FIG. 7A). Acute insertion refers to insertion trauma (IT), which is insertion followed by removal of the electrode array from the cochlea. Chronic implantation refers to either (a) foreign body (FB) insertion, which is insertion into the cochlea of an electrode array, with no further stimulation (or removal); and (b) electrical stimulation (ES), which is electrical stimulation of an implanted electrode array by the sound processor. Within those treatments, cochleae were harvested at days 1, 4, 7, and 28 (DI, D4, D7 and D28, respectively) for immunohistochemistry and RT-qPCR analyses (FIG. 7B). Electrical stimulation (ES) treatment ran between D4-D28, after which fibrosis was quantified (“fibrosis invasion”) with cochlear clearing and lightsheet microscopy.

[0030] FIGs. 8A -8E show immunohistochemical images and FIG. 8F shows a graph depicting fibrosis invasion following cochlear implantation. FIGs. 8A and 8B show a fibrotic sheath (FS) encasing the electrode array (FS arrows). Cells expressing myofibroblastic aSMA labeling are shown in the fibrotic sheath (labeled aSMA with arrows). FIGs. 8D and 8E show additional fibrotic tissue in the scala tympani (ST), indicating cells expressing aSMA (FIG. 8D) and vimentin, a marker of fibrotic tissue (V, FIG. 8E). FIG. 8F is a graph showing the extent to which fibrosis developed in each of the ES (n=5), FB (n=6), IT (n=5), ES + Clodronate (n=5), and ES + Tiplaxtinin (n=5) treatment groups by D28.

[0031] FIG. 9A, 9B, 9C and 9D are immunohistochemical images depicting the effect of clodronate injection on macrophage invasion after cochlear implantation, with electrical stimulation (ES). Controls are shown in FIG. 9A. Macrophage depletion is shown with clodronate administration (FIG. 9B), the effect of implantation with electrical stimulation (FIG. 9C), and the effect of combination of electrical stimulation and clodronate administration (FIG. 9D). ST = scala tympani; SV = scala vestibuli; OC = organ of Corti; sg = spiral ganglion; si = spiral lamina.

[0032] FIG. 10 shows RT-qPCR data, modeling and clustering of genes across control, IT, FB and ES treatments (FIG. 10A, DI), (FIG. 10B, D4), (FIG. 10C, D7), and (FIG. 10D, D28),and their subsequence analyses (FIGs. 10E, Relative expression showing fold changes from DI through D28), FIG. 10F, Cluster 1, FIG. 10G, Cluster 2, FIG. 10H, Optimal number of clusters, FIG. 101, Cluster 3, and 10J, Cluster 4) at D1-D28. Data for FB and ES were combined as ES treatment began four days after implantation.

[0033] FIG. 11A and 11B depict gene ontology pathway analyses for RT-qPCR clusters grouped as Non-Differential Expression Cluster group (NDEC; cluster 4, FIG. 11A) and Differential Expression Cluster group (DEC; Clusters 1, 2 and 3, FIG. 11B) according to the effect of ES on gene expression.

[0034] FIGs. 12A - 121 show CCL3 immunohistochemical (maximum intensity projection) images depicting confocal stack of cochleae cryosection in controls (FIG. 12A), in the insertion trauma treatment group (FIG. 12B (DI), FIG. 12C (D4), FIG. 12D (D7) and FIG. 12E (D28)), and in the cochlear implantation treatment group (FIG. 12F (DI), FIG. 12G (D4), FIG. 12H (D7) and FIG. 121 (D28)).

[0035] FIGs. 13A - 131 show CCL12 immunohistochemical (maximum intensity projection) images depicting confocal stack of cochleae cryosection in controls (FIG. 13A), in the insertion trauma treatment group (FIG. 13B (DI), FIG. 13C (D4), FIG. 13D (D7) and FIG. 13E (D28)), and in the cochlear implantation treatment group (FIG. 13F (DI), FIG. 13G (D4), FIG. 13H (D7) and FIG. 131 (D28)).

[0036] FIGs. 14A - 14F show a variety of single cell RNA sequencing analyses. FIG. 14A shows a UMAP projection displaying localization of identified cell populations in the cochlear region; FIG. 14B shows a dot plot visualizing the key markers for each detected cell population. The dot size represents the percentage of cells expressing a marker and the cross hatching variations represent the average expression level. FIG. 14C shows a plot of the distribution (frequency) of various cell populations in the (untreated) controls, as well as in the ES, ES + Clodronate, and ES + Tiplaxtinin treatment groups. FIG. 14D shows a plot of cluster scores (1-4) on the integrated dataset in FIG. 14E. FIG. 14E shows a dot plot displaying the average score of each cluster through cell populations in controls versus the ES treatment group, broken down by cluster (1-4); and FIG. 14F shows a dot plot displaying the average score of each cluster through cell populations in the ES, ES + Clodronate, and ES + Tiplaxtinin treatment groups.

[0037] FIGs. 15A-15L show CD68 and IBA1 macrophage localization in immunohistochemical (maximum intensity projection) images depicting confocal stack ofcochleae cryosection in controls (FIG. 15A), and in the ES starting at D4 (FIG. 15B: D4; FIG. 15C: D7; and FIG. 15D D28), FB (FIG. 15E: DI; FIG. 15F D4; FIG. 16G D7; and FIG. 15H D28;) and insertion trauma (FIG. 15I D1; 15J:D4; FIG. 15K:D7; and FIG. 15L:D28) treatment groups, respectively.

[0038] FIGs. 16A-16L show CD 163 and IBA1 macrophage localization in immunohistochemical (maximum intensity projection) images depicting confocal stack of cochleae cryosection in controls (FIG. 16A), and in the ES starting at D4 (FIG. 16B: D4; FIG. 16C: D7; and FIG. 16D D28), FB (FIG. 16E: DI; FIG. 16F D4; FIG. 16G D7; and FIG. 16H D28;) and insertion trauma (FIG. 16I D1; 16J:D4; FIG. 16K:D7; and FIG. 16L:D28) treatment groups, respectively.

[0039] FIGs. 17A-17I show the macrophage transcriptomic response following electrostimulation and pharmacological treatments. FIG. 17A is a UMAP plot displaying localization of CD68+ expressing cells within the cochlear populations. FIG. 17B shows a dot plot illustrating CD68+ population top markers and their expression in the various identified cell populations. FIG. 17C shows a dot plot displaying clusters Cl through C4 average scores in macrophages across treatment groups. FIG. 17D shows a volcano plot displaying differentially expressed genes between the ES and controls (FDR < 0.1). FIG. 17E shows a heatmap of the average expression of differential expressed genes in the ES treatment group against controls (unstimulated macrophages) across all treatment groups. FIG. 17F shows a gene ontology analysis from differentially expressed genes, illustrating activated and suppressed functions in ES macrophage against unstimulated control. FIG. 17G shows a volcano plot showing differentially expressed genes in the Tiplaxtinin + ES treatment versus ES treatment groups. FIG. 17H shows a gene ontology analysis of Tiplaxtinin effect showing main activated and suppressed gene ontology terms. FIG. 171 shows a Gene set Enrichment Analysis (GSEA) analysis of main gene ontology terms displaying gene set enrichment in the Tiplaxtinin treated condition.DETAILED DESCRIPTION

[0040] Presented herein are compositions and methods for preventing or attenuating the development of fibrosis and scarring, or for treating fibrosis and scarring that have resulted from implantation trauma, implantation and maintenance of a foreign body in the inner ear, such as an implant, or from damage resulting from other injury to the inner ear, such as, for example, from electrical stimulation from an implant electrode. The disclosed compositionembodiments comprise pharmaceutical compositions comprising at least one inhibitor of plasminogen activator inhibitor (PAI). In some embodiments the PAI-1 inhibitor is Tiplaxtinin, also known as PAI-039 or tiplasinin (CAS No. 393105-53-8). Other PAI-1 inhibitor include SK-216, TM 5441, TM 5275 sodium salt, annonacinone, AZ3976, and embelin. Method embodiments comprise administration of a PAI-1 inhibitor to prevent, attenuate or halt development, or to reduce fibrosis. In still other method embodiments, the PAI- 1 inhibitor reduces fibrosis due to the tissue damage resulting from electrical stimulation at the site of an implanted device electrode.

[0041] The development of fibrosis is a common feature and appears to be a delayed consequence of the insertion trauma and / or a foreign body reaction after cochlear implantation. Encapsulation of the implant electrode is a normal healing process, but the response can lead to an excessive tissue proliferation into the cochlea, invading the cochlea and even forming new bone. Thus, fibrosis development following implantation poses deleterious prospects not only for long-term residual hearing preservation, but also for the efficiency of electrical stimulation by itself, at the same time increasing electrode impedances and the spread of fibrotic tissue into the cochlea. As a result cochlear fibrosis prevention is a major therapeutic target after cochlear implantation.

[0042] Currently the mechanisms responsible for fibrosis development after cochlear implantation are poorly understood. Recent evidence suggests that inflammation and epithelia- to-mesenchymal transition are responsible for invasion of inflammatory cells, tissue proliferation and extracellular matrix secretion. Advances in therapeutic interventions for fibrosis prevention are limited by the lack of knowledge about molecular and cellular mechanisms associated with fibrosis development following implantation. We have examined the effects of insertion trauma, foreign body reaction, and electrical stimulation on fibrosis development following implantation in a rat model. The expression of mRNA transcripts, with known involvement in fibrosis development in other tissues, immunolocalization of target proteins, immune cell characterization and fibrotic tissue invasion have been monitored following implantation to determine which pathways are preferentially involved during the development of fibrosis.

[0043] Presented here is also a proposed mechanism of action for development of fibrosis following implant in the inner ear.

[0044] More than five percent of the global population has experienced some degree of hearing loss, which can result from damage to inner ear structures and loss of auditory neurons or cochlear sensory cells. The etiology of hearing loss is varied and includes exposure to loud noise, physical injury, advancing age, genetic factors as well as disorders and diseases (W ang et al., 2018; Nyberg et al., 2019; Blanc et al., 2020).

[0045] To restore or improve hearing, implantable devices such as CI may be prescribed. The surgical implantation procedure, however, may lead to tissue damage (“insertion trauma”) in the implantation area, resulting in an inflammatory response and possibly temporary hearing loss. For the last decade, the cochlear implantation is used in patients with residual hearing. Unfortunately, long term results show that the patients progressively lose residual hearing after cochlear implantation. Although the improvement to the surgical procedure (soft surgery) may reduce the degenerative process, development cochlear fibrosis seems to be the main cause for the death of the remaining hair cells and auditory neurons.

[0046] Cochlear implantation is a validated and well accepted technique to restore auditory perception in patients suffering from inherited or acquired hearing loss. Electrode insertion into the cochlea can be compromised by the presence fibrosis tissues into the cochlea due to bacterial labyrinthitis (pneumococcal) (Nabili et al. Chronology of labyrinthitis ossificans induced by Streptococcus pneumoniae meningitis. Laryngoscope. 1999 Jun;109(6):931-5; Green et al. Labyrinthitis ossificans: histopathologic consideration for cochlear implantation. Otolaryngol Head Neck Surg. 1991 Mar;104(3):320-6), or immune labyrinthitis (Quaranta et al. Cochlear implants in systemic autoimmune vasculitis syndromes. Acta Otolaryngol Suppl. 2002;(548):44-8). Conversely, cochlear implantation itself cause the concurrence of fibrosis, which in turn provokes several deleterious side effects: i) fibrosis affects the survival of remaining hair cells and auditory neurons, and then compromises auditory rehabilitation, especially in case of electroacoustic stimulation (Choi & Oghalai. Predicting the effect of postimplant cochlear fibrosis on residual hearing. Hear Res. 2005 Jul;205(l-2): 193-200, Eshraghi. Prevention of cochlear implant electrode damage. Curr Opin Otolaryngol Head Neck Surg. 2006 Oct;14(5):323-8), ii) formation ofafibrotic layer around the electrode array compromises prevent correct electrode reinsertion in case of reimplantation (Rossi & Bisetti. Cochlear implant and traumatic lesions secondary to electrode insertion. Rev Laryngol Otol Rhinol (Bord). 1998; 119(5):317-22, Balkany et al. Cochlear reimplantation. Laryngoscope. 1999 Mar;109(3):351-5) and iii) fibrosis is responsible of electrodes' impedance shifts, and thus energy consumption increase.

[0047] In general, fibrosis is the final development of a multi-step inflammatory process resulting in abnormal and excessive collagen secretion after fibrocyte proliferation. Tissue repair consists of three overlapping stages: inflammation, granulation or proliferation, and tissue remodeling. During tissue remodeling, apoptosis removes myofibroblasts, and the deposited extracellular matrix (ECM) is remodeled to resemble the original tissue. Fibrosis occurs when inappropriate tissue remodeling results in excess ECM deposition due to inappropriate survival of cells or lack of ECM proteolytic degradation. The inflammation and enhanced TGFB signaling often present in fibrotic tissues causes cells to differentiate into fibroblasts via epithelial -to-mesenchymal transition (EMT). Chronic wounds feature dysregulated tissue remodeling with enhanced ECM degradation.

[0048] In the inner ear, the cells involved in the proliferative process are the mesothelial cells of the scala tympani and the scala vestibuli (Tinling et al. Fine structure histopathology of labyrinthitis ossificans in the gerbil model. Ann Otol Rhinol Laryngol. 2005 Feb; 114(2): 161- 66). Whereas, cellular and molecular mechanisms involved in fibrosis development are widely described for liver and lung (Wynn. Integrating mechanisms of pulmonary fibrosis. J Exp Med. 2011 Jul 4;208(7): 1339-50; Kisseleva & Brenner. Anti-fibrogenic strategies and the regression of fibrosis. Best Pract Res Clin Gastroenterol. 2011 Apr; 25(2):305- 17), data about cochlear fibrosis are currently not available.

[0049] Inner ear implants including CI also have been used for delivery of drugs to the inner ear, e.g., local delivery of a pharmaceutical composition to the middle or inner ear into perilymph fluid, for example, pumped from a refillable reservoir, or deposited as a gel in the middle ear at the round window for access through the round window, by cannula, by injection or, potentially, diffusion, particularly if the composition is formulated with a saline solution and / or the composition is prepared in nanoparticle form, or as a component of a coating on, or a delivery channel associated with, a CI electrode or other device component (Plontke et al., 2017; Wang et al., 2018; Mitchell et al. Engineering precision nanoparticles for drug delivery. Nature Reviews (2021) 20: 101-124). For example, glucocorticosteroids have been used to address inflammation and other issues in the auditory system following CI implantation and the resulting “insertion trauma.” Dexamethasone formulated in a polymer and coated on the electrodes of an implanted CI was shown to attenuate the loss of auditory sensory cells after exposure to tumor necrosis factor alpha (TNF-a); similarly, hearing loss induced by CI implantation could be reduced after administration of dexamethasone in a sustained release application. (Plontke et al., 2017)

[0050] . Increased impedance and fibrosis associated with implant trauma, as well as the migration of immune cells into the cochlea, were shown to be reduced following administration of dexamethasone delivered through coated electrodes (Id.; Wang et al., 2018).

[0051] An implantable cochlear device and system are described below that treats a patient using a control system, human intervention, combined with pharmaceutical compositions in methods for treatment of the inner ear with the goal of halting, reducing or attenuating the development of fibrosis at or around the site of implant and applied electrical stimulation. As noted, Cis may be surgically implanted into the cochlea to restore or improve hearing. Implantation may trigger an immune response by macrophages or other immune cells, recruiting additional immune cells (e.g., macrophages, NK cells, neutrophils, etc.) to the site of inflammation. Inflammation is an early step in tissue healing and reconstruction process, and when inflammation continues and excess ECM is produced, fibrosis ensues, as discussed further below.Anatomy of the human ear

[0052] FIG. 1A illustrates a human ear comprising an outer ear 101, a middle ear 105, and an inner ear 107. In a fully functional ear, outer ear 101 comprises an auricle 111 and an ear canal 131. An acoustic pressure or sound wave 103 is collected by auricle 111 and channeled into and through ear canal 131. Disposed across the distal end of ear canal 131 is a tympanic membrane 115 which vibrates in response to sound wave 103. This vibration is coupled to oval window or fenestra ovalis 117, which is adjacent round window 121, through the bones of the middle ear 105. The bones of the middle ear 105 comprise the malleus 113, the incus 109 and the stapes 111, collectively referred to as the ossicles 123. The ossicles 123 are positioned in the middle ear cavity 125 and serve to filter and amplify the sound wave 103, causing oval window 117 to articulate (vibrate) in response to the vibration of tympanic membrane 115. This vibration of the oval window 117 sets up waves of fluid motion of the perilymph within cochlea 140. Such fluid motion, in turn, activates tiny hair cells (not shown) inside of cochlea 140. Activation of the hair cells causes appropriate nerve impulses to be generated and transferred through the spiral ganglion cells (not shown) and auditory nerve 114 to the brain (also not shown) where they are perceived as sound.

[0053] The human skull is formed from a number of different bones that support various anatomical features. Illustrated in FIG. 1A is the temporal bone 135 which is situated at the side and base of the recipient’s skull (covered by a portion of the recipient’s skin / muscle / fat,collectively referred to herein as tissue). For ease of reference, the temporal bone 135 is referred to herein as having a superior portion 141 and a mastoid portion 143. The superior portion 141 comprises the section of the temporal bone 135 that extends superior to the auricle 111. That is, the superior portion 141 is the section of the temporal bone 135 that forms the side surface of the skull. The mastoid portion 143, referred to herein simply as the mastoid 143, is positioned inferior to the superior portion 141. The mastoid 143 is the section of the temporal bone 135 that surrounds the middle ear 105.

[0054] Relevant aspects of an example cochlea 140 are described below with reference to FIGs. 1B-1C. More specifically, FIG. IB is a perspective view of the cochlea 140 partially cut-away to display the canals and nerve fibers of the cochlea, while FIG. 1C is a cross- sectional view of one turn of the canals of the cochlea 140.

[0055] Referring first to FIG. IB, cochlea 140 is a conical spiral structure comprising three parallel fluid-filled canals or ducts, collectively and generally referred to herein as canals 182. Canals 182 comprise the tympanic canal 188, also referred to as the scala tympani 188, the vestibular canal 184, also referred to as the scala vestibuli 184, and the median canal 186, also referred to as the scala media 186. Cochlea 140 spirals about modiolus 192 several times and terminates at cochlea apex 194.

[0056] Portions of cochlea 140 are encased in a bony labyrinth / capsule and the endosteum (e.g., a thin vascular membrane of connective tissue that lines the inner surface of the bony tissue that forms the medullary cavity of the bony labyrinth). Spiral ganglion cells reside on the opposing medial side (the left side as illustrated in Fig. 1C) of cochlea 140. A spiral ligament membrane is located between lateral side of the spiral tympani 188 and the bony capsule, and between lateral side of scala media 186 and the bony capsule. The spiral ligament also typically extends around at least a portion of lateral side of scala vestibuli 184.

[0057] The fluid in the tympanic canal 188 and the vestibular canal 184, referred to as perilymph, has different properties than that of the fluid which fills scala media 186 and which surrounds organ of Corti 190, referred to as endolymph. The tympanic canal 188 and the vestibular canal 184 collectively form the perilymphatic fluid space of the cochlea 140. Sound entering a recipient’s auricle (not shown) causes pressure changes in cochlea 140 to travel through the fluid-filled tympanic and vestibular canals 188, 184. As noted, the organ of Corti 190 is situated on basilar membrane in the scala media 186 and contains rows of 16,000-20,000 hair cells (not shown) which protrude from its surface. Above them is the tectoral membranewhich moves in response to pressure variations in the fluid-filled tympanic and vestibular canals 188, 184. Small relative movements of the layers of membrane are sufficient to cause the hair cells in the endolymph to move thereby causing the creation of a voltage pulse or action potential which travels along the associated nerve fiber. Nerve fibers, embedded within the spiral lamina connect the hair cells with the spiral ganglion cells 183 which form auditory nerve 114. Auditory nerve 114 relays the impulses to the auditory areas of the brain (not shown) for processing.

[0058] In general, the basal region is the portion of the cochlea 140 located closest to the stapes (not shown in FIGs. IB and 1C) and extends to approximately the first turn of the cochlea (i.e., the region of the cochlea 140 between the cochlea openings, including the round and oval windows, the first cochlea turn). The apical region is portion of the cochlea 140 in proximity to the cochlear apex 194. More specifically, the cochlea 140 is generally a conical spiral structure (i.e., the spiral-like shape) and the apical region of the cochlea 140 is generally the last / fmal (i.e., most apical) 360 degrees of the cochlea.

[0059] There are a number of different types of devices in / with which embodiments of the present invention may be implemented. Merely for ease of description, the techniques presented herein are primarily described with reference to a specific device in the form of a cochlear implant (CI) system. However, it is to be appreciated that the techniques presented herein may also be partially or fully implemented by any of a number of different types of devices, including consumer electronic device (e.g., mobile phones), wearable devices (e.g., smartwatches), hearing devices, implantable medical devices, wearable devices, etc. consumer electronic devices, wearable devices (e.g., smart watches, etc.), etc. As used herein, the term “hearing device” is to be broadly construed as any device that delivers sound signals to a user in any form, including in the form of acoustical stimulation, mechanical stimulation, electrical stimulation, etc. As such, a hearing device can be a device for use by a hearing -impaired person (e.g., hearing aids, middle ear auditory prostheses, bone conduction devices, direct acoustic stimulators, electro-acoustic hearing prostheses, auditory brainstem stimulators, bimodal hearing prostheses, bilateral hearing prostheses, dedicated tinnitus therapy devices, tinnitus therapy device systems, combinations or variations thereof, etc.) or a device for use by a person with normal hearing (e.g., consumer devices that provide audio streaming, consumer headphones, earphones and other listening devices). In other examples, the techniques presented herein can be implemented by, or used in conjunction with, various implantable medical devices, such as vestibular devices (e.g., vestibular implants), visual devices (i.e.,bionic eyes), sensors, pacemakers, drug delivery systems, defibrillators, functional electrical stimulation devices, catheters, seizure devices (e.g., devices for monitoring and / or treating epileptic events), sleep apnea devices, electroporation devices, etc.

[0060] The anatomy of the ear of a rat or guinea pig, for example, is similar if not identical, to that of a human.Fibrosis and its development

[0061] Fibrosis is a common complication in many diseases and disorders such as cardiovascular disease, insulin resistance, pulmonary pathologies, liver disease, hepatitis, inflammatory bowel disease, Crohn’s disease, etc. It is a pathophysiological process that affects several different organs and shares a similar pattern of development in various tissues or organs, resulting in an accumulation of connective tissue, as extracellular matrix (ECM), and often leads to the dysfunction of tissue or organ affected. (Lis-Lopez, et al. Is the macrophage phenotype determinant for fibrosis development? Biomedicines (2021) 9: 1747.)

[0062] As in normal wound healing, fibrosis is initiated by tissue damage accompanied by the activation of the inflammatory response and the arrival of several types of blood cells to damaged tissue. Proliferative and remodeling phases normally follow. Where there is prolonged or persistent inflammation, overproduction or overactivation of cytokines such as Transforming growth factor-beta (TGFP) and the differentiation of resident fibroblasts and other cells into myofibroblasts produce an excessive amounts of ECM, which alters both the architecture and function of the tissue, and further enhances the release of pro-inflammatory cytokines, angiogenic factors, and chemokines.

[0063] The number of cells producing ECM is increased because of a variety of cell types that can switch their phenotype and become active myofibroblasts. These include fibroblasts, mesothelial cells, pericytes, and circulating fibrocytes, epithelial and endothelial cells, and macrophages. “Ml” macrophages begin as pro-inflammatory agents, infiltrating damaged tissue to remove dead cells, bacteria and foreign debris, and later change phenotype to promote anti-inflammatory effects as “M2” macrophages, taking up residence near and stabilizing newly forming blood vessels as fibroblasts and keratinocytes restore the layers of tissue and endothelial cells form the vasculature supplying blood to it. TGFp is a key player in the physiological repair of damaged tissue, collagen accumulation and the initiation of fibrogenesis, but others have important roles as well.

[0064] In earlier work (unpublished) we had evaluated gene regulation in inner ear tissues to identify potential gene targets for further pharmacological or cellular treatments in an in vivo model of cochlear implantation. Among a number of potential targets, we identified the serine protease inhibitor (serpin), plasminogen activator inhibitor- 1 (a / k / a Serpin-1 or PAI-1), which functions as the principal inhibitor of tissue plasminogen activator (tPA) and urokinase (uPA), the activators of plasminogen and, hence, fibrinolysis.

[0065] In normal tissue repair, a balance is struck, i.e., in tissue homeostasis, between proliferative (e.g., PAI-1) and proteolytic forces (e.g., tPA), but in fibrosis, proliferative activities dominate and eventually result in altered tissue or organ architecture, and in many cases lead to organ dysfunction. PAI-1 is one of the most important modulators of tPA and fibrogenesis / fibrinolysis.

[0066] PAI-1 is expressed in many cell types, and its role in cells varies depending on expression and activity levels. A variety of cytokines and growth factors, such as TGFp, insulin, as well as transcription factors regulate PAI-1 expression in a cell type- and physiologic condition-dependent manner. Further, complex interactions with other biological components may alter the molecular involvement of PAI- 1 , causing its effects to vary across tissues, organs and physiological conditions. For example, although elevated PAI-1 is associated with the development of fibrosis in general, in PAI- 1 -deficient mice, spontaneous cardiac-selective fibrosis can develop. In a model of lung fibrosis (in mice with PAI-1), when an inhibitor of PAI-1 is administered, fibrogenesis can be attenuated or halted. Further, data have also suggested that pharmacologic suppression of PAI-1 is tissue selective. For example, Tiplaxtinin, apotent, small molecule PAI-1 inhibitor (discussed below) protected against aortic remodeling but not cardiac fibrosis and hypertrophy when all three were induced by angiotensin II administration. (Weisberg et al. Pharmacological inhibition and genetic deficiency of plasminogen activator inhibitor- 1 attenuates angiotensin II / salt-induced aortic remodeling. Arterioscler Thromb Vase Biol. (2005) 25:365-371) Thus, the mechanisms by which PAI-1 functions in various tissues and conditions differ and whether a particular inhibitor will function sufficiently well or not at all in a particular tissue or under particular physiologic conditions, is not settled.Proposed mechanism of action by which cochlear fibrosis develops

[0067] The molecular mechanism(s) by which fibrosis develops in different tissues, or under different physiologic or pathophysiologic conditions, despite some common processes, are notfully understood. Insertion trauma, as we have reported here, is insufficient to trigger a significant fibrotic response but when an electrode array is inserted and stimulated, fibrosis is promoted. The mechanisms of fibrosis development has been clearly defined in the central nervous system, but sparce data are available for fibrosis development in the cochlea. Earlier work by our lab, some of which is discussed here, even suggests there are real differences within the tissues of the inner ear concerning the development of fibrosis. To this end, we provide a proposed mechanistic basis of fibrosis development in the cochlea with the goal of eventually unravelling workable treatment options for patients in need that will preserve their hearing and potentially be tailored to individual physiologic states.

[0068] Cochlear implantation and electrical stimulation promotes inflammatory reaction and fibrosis into the cochlea. Both RT-qPCR and scRNA seq analyses showed that macrophage invasion of the cochlea is an early key stage for fibrotic development inside the cochlea. Preventing macrophage invasion by Clodronate or modulating inflammatory phenotype is sufficient to prevent fibrosis outgrowth almost completely. The time window for therapeutic interventions and the intracellular pathways of macrophage activation remain to be investigated in order to develop effective and safe medications against cochlear fibrosis.

[0069] In the present study, foreign body insertion into the rat cochlea induced a fibrotic tissue invasion of 4.06% ± 1.51 of the total volume of the cochlea. Variability between others’ studies and ours may be explained by the diversity of foreign bodies used, including dedicated animal electrode arrays or surrogates like platinum wire or nylon with various lengths and flexibilities. After electrical stimulation, fibrosis occurs mainly around the electrode array in the basal turn of the cochlea, but can spread to the second turn in some cases. Our results also demonstrate that electrical stimulation is a potent fibrosis inducer when associated with a foreign body reaction (fibrosis evaluated at 7.64% ± 3.54 of total volume in our rat model after electrical stimulation).

[0070] In our model, we suggest that the canonical TGFp-Smad pathway is not a major effector of fibrosis. This hypothesis is further supported by the fact that the downstream protein Smad3 is found in supporting cells of the organ of Corti, auditory neurons and spiral ganglion supporting cells, but not in fibrotic tissues. Cochlear fibrosis may result from specific fibrotic processes different from other organs such as liver, lung, kidney or heart, and be due to the activation of Smad-independent pathways.

[0071] Analyses with scRNA seq showed that electrical stimulation is responsible for an increase of macrophages and neutrophils populations in the cochlea. Those results are consistent with RNA analyses using RT-qPCR since genes in the Clusters 1, 2 & 3 that were differentially expressed after electrical stimulation are also genes overexpressed in macrophagic population based on UMAP analyses.

[0072] The occurrence of fibrosis development and macrophagic activity in the cochlea was previously reported by others; indeed, overexpression of chemoattractants such as CCL3 and CCL12 using RT-qPCR or immunochemistry suggests that immune cell invasion could contribute to fibrosis development. ScRNA seq showed that macrophages display an overexpression of chemoattractant for immune cells such as Ccl2, Ccl4, Cxcll,Ccl7. This was further supported by immunohistological study that showed a massive macrophage infiltration in experimental conditions associated with fibrosis. We evaluated two subsets of macrophages, tissue resident macrophages (TRM) and monocyte-derived macrophages (MDM). TRM Ibal+ cells were located in the spiral ganglion and the stria vascularis during the entire experiment. A similar localization was found in normal control cochlea in our experiments and in humans. Some IBA1+ cells were found rarely in fibrotic tissues. Conversely, MDM were found in the cochlea in the stria vascularis and the scala tympani by D4, then in fibrotic tissues by D7 with a sustained expression in fibrotic tissues at D28 only in the ES condition. Most MDM were Ml phenotype with CD68+, with a limited number of M2 macrophages CD163+. It appears that the main entry point of MDM into the cochlea is the stria vascularis. In mature fibrosis, the persistence of macrophages has also been reported, but we found that they returned to their quiescent phenotype, with a filamentous elongated shape as described in other organs. The exact role of TRM in fibrosis development is not yet elucidated, since they do not seem to be usually found in fibrotic tissues in our experiments.

[0073] Cochlear invasion by MDM is found in other types of cochlea injury like noise trauma or aminoglycosides ototoxicity. Previously published reports indicate that macrophages play a subtle role in the balance between injury and repair in the cochlea. After a previous sensitization of the cochlea with an acoustic trauma associated with macrophagic invasion, a second noise exposure causes more sensory cell loss due to an overactivation of inflammatory response. Moreover, macrophage depletion by CSF1R inhibitor can protect cochlea from cisplatin injuries. Similar observations were made in case of electrode insertion after an acoustic trauma. Fibrotic tissue growth is shown by others to be higher in deafened cochlea after neomycin treatment than in normal hearing cochleae. This suggests that a previous activation ofmacrophages could be deleterious for sensory cell survival after cochlea injury. Conversely, the loss of TRM and MDM cells in B6.129P-Cx3crltmlLitt / J mice results in potentiation of noise induced hearing loss due to a loss a regulation of neutrophils invasion by macrophages. Similarly, the loss of fractline (CX3CLI), chemokine expressed in spiral ganglion neurons that is the ligand of CX3CRI, causes an increased loss of hair cells and auditory neurons an prolonged increase of cochlear leukocytes after noise trauma and aminoglycosides. The phenotyping of our macrophages shows that they display a Th-1 activated phenotype. Similarly to the proteomic level, transcriptional analyses demonstrated that electrically activated macrophages showed a specific phenotypic signature with an increase of cytotoxic markers, such as Camp, Defa5, RTl-Da, Np4, Ctsg, Hmgn2, suggesting that electrical stimulation was responsible for a phenotypic change towards an inflammatory Ml phenotype. Altogether, these data suggest that macrophages in our model have a pro-fibrotic effect. Moreover, immunohistochemistry demonstrated that anti-inflammatory macrophages CD 163+ were located in fibrotic tissues, especially after electrical stimulation. Similar results are observed in humans This suggests, in addition to our fibrotic tissue quantification and CD68 labeling on cochlear sections that electrical stimulation is responsible for a state of chronic macrophage activation compared to a passive electrode array. Promoting a phenotypic change of macrophages towards a M2 anti-inflammatory phenotype could be beneficial to prevent fibrosis with chronically implanted device as demonstrated in vitro by IL-4 immobilized PPy / Hep electrodes for electrocardiogram measurements.

[0074] If both macrophage invasion and fibrosis were observed in our model, the role of macrophage in fibrotic development is yet to be demonstrated as this macrophage invasion could be a simple marker, but not an inducer of, fibrotic reaction. In our experimental model, depletion of both MDM and TRM by Clodronate resulted in a decreased amount of fibrosis. This depletion was confirmed on cochlear sections and at a cellular level using scRNA seq, where cell death markers were overexpressed in the myeloid population and resulted in a reduction of myeloid cell proportion in the cochlea. Similar effects of Clodronate have already been shown in other models such liver or lung fibrosis.

[0075] We aimed at regulating macrophage inflammatory functions using Tiplaxtinin. Tiplaxtinin is an inhibitor of PAI- 1 function. In our model PAI-1 was found in fibrotic tissues and cells lining the scalatympani and vestibuli at 1 month after implantation. The role of PAI - 1 in tissue remodeling is critical since it regulates proteolytic activities of macrophages through uPA / uPAR activation. PAI-1 deficient mice can display an excessive lung fibrotic phenotypeafter bleomycine induction or an increased wound healing. Conversely pharmacological blockade of PAI-1 reduce fibrosis in pulmonary, liver and cardiac models of fibrosis. Moreover, PAI-1 has been shown to promote monocyte migration and M2 polarization of macrophages in cancer and can be blocked by Tiplaxtinin. In animals treated by gel application of Tiplaxtinin at the time of the surgery prior to electrical stimulation, we observed a dramatic reduction of fibrotic tissue development. Similar findings were reported by others who used a perfusion of tPA, which is inhibited by PAI-1, during cochlear implant surgery of guinea pigs. Fibrosis was reportedly reduced from 31.7 to 27.5% of total volume. However, this effect is lesser than we observed in our experiment, and suggests that PAI-1 inhibitor Tiplaxtinin has other targets than plasminogen activators.

[0076] ScRNA seq experiments showed that Tiplaxtinin treatment modified the transcriptional signature of macrophages with a strong downregulation of genes associated with macrophages cytotoxicity by comparison with the electrically stimulated condition only as shown by Gene Ontology pathways analyses. Unlike Clodronate, Tiplaxtinin treatment was not responsible for a reduction or monocyte / macrophages population into the cochlea. Tiplaxtinin treatment resulted in a shift of B cell / neutrophils proportion into the cochlea compared to the control cochlea, favoring the neutrophils. No previously published data reported the analyses of scRNA seq in macrophages after Tiplaxtinin treatment. Other papers report that Tiplaxtinin treatment reduces macrophage polarization, proinflammatory macrophage-Ml -phenotype, including CD 11c, IL-6, and MCP-1 in renal and fat induced fibrosis. Altogether , these data support the fact that PAI- 1 inhibition prevents fibrosis after cochlear implantation through an inhibition of the inflammatory phenotype of cochlear macrophages.Pharmaceuticals used to prevent or treat fibrosis

[0077] Pharmaceuticals other than PAI-1 inhibitors have been used to treat inner ear conditions, including fibrosis, in combination with a CI, with or without electrical stimulation. See, for example, US Pat. Application 2007 / 198063A1, which describes the combination of CI and an anti-scarring drug or combination of drugs that prevent or reduce scarring or fibrosis. No PAI-1 inhibitors are suggested for use.

[0078] PAI-1 inhibitors, however, have been used in a variety of tissues other than the inner ear to treat fibrosis, including in animal models of thrombosis. See, for example, US Pat. No. 9,527,878, which describes compounds for treatment of diseases associated with ECM accumulation, e.g., renal fibrosis, chronic obstructive pulmonary disease, polycystic ovary syndrome, restenosis, renovascular disease and organ transplant rejection. Inhibition of PAI-1, such as from monoclonal antibodies, has been shown to be effective in decreasing fibrin deposition in thrombotic animal models. Similarly, both Cesari et al. and Ghosh & Vaughn summarize the use of PAI-1 inhibitors used in various fibrotic conditions. (Cesari et al. Plasminogen activator inhibitor- 1 (PAI-1): a key factor linking fibrinolysis and age-related subclinical and clinical conditions. Cardiovcisc Ther. (2010) 28(5):e72-e91; Ghosh & Vaughn. PAI-1 in tissue fibrosis. J Cell Physiol. (2012) 227(2):493-507) Further, Ghosh & Vaughn discuss the varied roles of PAI-1 in several pathophysiologic conditions in which fibrosis develops, noting that PAI-1 expression and activity vary depending on cell type and physiologic or pathologic milieu. This suggests that deciphering the mechanism(s) of action of the development of fibrosis and of the role of PAI-1 in its development in the inner ear, which have not been examined to date, may be key to applying a workable pharmaceutical approach to treating and reducing fibrosis in the inner ear, and ultimately, the prevention of hearing loss.

[0079] Tiplaxtinin, also known as tiplasinin or PAI-039 (CAS No. 393195-53-8), is a smallmolecule PAI-1 inhibitor, among a variety of PAI-1 inhibitors including, e.g., SK-619, SK- 216, TM 5441, TM 5275 sodium salt, annonacinone, AZ3976, embelin, and others. Tiplaxtinin is a highly selective PAI-1 inhibitor. (Elokdah, et al. Tiplaxtinin, a novel, orally efficacious inhibitor of plasminogen activator inhibitor- 1: design, synthesis and preclinical characterization. J Med Chem. 2004, 47, 3491-94).

[0080] Tiplaxtinin has been used, for example, to treat kidney or lung fibrosis or thrombosis in cardiac / vascular conditions. See, for example, US Pat. Application 2015 / 0164874A1 describes its use in idiopathic pulmonary fibrosis. Herman, et al. and Baxi et al. both discuss its successful use in a rat model of thrombosis (Hennan, et al. Effect of Tiplaxtinin (PAI-039), an orally bioavailable PAI-1 antagonist, in a rat model of thrombosis. J Thromh Haemost. (2008) 6: 1558-1564; Baxi et al. Dose-dependent thrombus resolution due to oral plasminogen activator inhibitor (PAI)-l inhibition with Tiplaxtinin in a rat stenosis model of venous thrombosis. Thromh Haemost. (2008) 99:749-58). Similarly, Ma & Fogo, in their discussion of the use of PAI-1 inhibitors for treating kidney fibrosis, also discuss Tiplaxtinin use, in particular, to attenuate fibrosis development in cardiac tissue (Ma & Fogo. PAI-1 and kidney fibrosis. Front Biosci . (2016) 14:2028-41). Elokdah et al. (2004) similarly demonstrated the attenuating effect that Tiplaxtinin has on vascular occlusion by slowing by more than five-fold the average time to vascular occlusion in a rat model of carotid thrombosis.

[0081] To date, neither Tiplaxtinin, nor PAI- 1 inhibitors in general, have been reportedly used for treating fibrosis of the inner ear.Pharmaceutical compositions and their preparation

[0082] Single compounds or molecules as well as the combinations of active and nonactive compounds including biologically active molecules and compounds, together with all other components that may be required to formulate a particular form (tablet, liquid, gel, etc.) for a particular mode of administration to deliver a therapeutically effective amount are collectively and generally referred to in this disclosure as “pharmaceutical compositions.” Tire term “compound’’ or “agent’’ may also refer to any single component that comprises a pharmaceutical composition. Pharmaceutical compositions or “compositions,” as that term is used, are administered according to disclosed method embodiments to treat fibrosis of the inner ear and, specifically to prevent or attenuate the development of fibrosis, may act to reduce inflammation and / or otherwise reduce fibrosis in the area and its surrounding tissues. If inflammation is the result of injury at the site of implantation of a medical device such as a CI, whether from insertion trauma, the resulting foreign body in the inner ear, or from electrical stimulation by a CI electrode in preferred method embodiments, the attenuation of inflammation and the development of fibrosis over the days and weeks that ensue is a primary goal.

[0083] In some disclosed method embodiments, one or more pharmaceutical compositions are administered to treat fibrosis of the inner ear following medical device implantation. In some embodiments, the methods are applied in a clinical setting; in others, a research setting.

[0084] In the disclosed composition embodiments, pharmaceutical compositions may comprise any single or combination of the following: biological substances, bioactive substances, conjugated or fusion molecules or compounds, viral and non-viral vectors, natural, synthetic and recombinant molecules, antibodies and antibody fragments, etc., pharmaceutical agents / active pharmaceutical ingredients (APIs) including commercially available versions of the same, genes, nucleases, endonucleases, nucleic and ribonucleic acids such as messenger RNA (mRNA), siRNA and miRNA, naked DNA, DNA vectors, oligonucleotides, antisense polynucleotides, peptides, polypeptides, proteins including binding proteins, anti-oxidants, and signalling compounds that promote recovery and resolution, other chemicals, ions, and other molecules used to modulate inflammation within the body of individual, any of which may beadministered in the form of nanoparticles. A person of ordinary skill in the art will appreciate that each if these substances can be generated by methods known in the art.

[0085] Conjugated molecules can be administered for treating a wide variety of disorders and diseases. These molecules are characterized by having a cell-permeable (or -penetrating) component that facilitates delivery of another, linked component, a molecule or compound with biological activity, to intracellular or intranuclear sites of action where they may elicit a variety of effects, including, e.g., among other things, the regulation of gene expression through interference with post-transcription processes. Further, numerous types of vectors can be used to deliver and express one or more therapeutic molecules in target cells.

[0086] In certain method embodiments for treating fibrosis of the inner ear, the concentration of inflammatory cytokines is decreased by administering pharmaceutical compositions comprising a corticosteroid such as dexamethasone. Such compositions may be systemically administered in an amount sufficient to provide a suitable, final dexamethasone concentration in the perilymph. One or more of other anti-inflammatory agents that may be included in the disclosed composition or method embodiments include prednisone, cortisone, hydrocortisone, and clodronate.

[0087] The amount of composition included within therapeutically active formulations according to the disclosed embodiments is an effective amount for affecting the desired outcome, i.e., treatment of inner ear fibrosis. The concentrations or activities of compounds or compositions measured in blood or other fluids is known in the art. See, e.g., Rafai et al.

[2017] Tietz textbook of clinical chemistry and molecular diagnostics (6thed.) St. Louis: Elsevier.

[0088] The choice of pharmaceutically acceptable carrier, excipient or diluent may be selected based on the composition and the intended route of administration, as well as standard pharmaceutical practice. Such compositions may comprise any agents that may aid, regulate, release, or increase entry into the body compartment, tissue, intracellular or intranuclear target site, such as binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), or other agents. Nonlimiting examples include polymers and silicones. Administration of an implant of a formulation for the sustained release may also be used to obtain prolonged exposure and action, and in some embodiments may be, e.g., a liquid, gel, or solid implant or may be in the form of particles, including nanoparticles. The term "sustained release" refers to formulations from which the composition is released at a slow rate allowing for a longer period of exposure at active concentrations. Intramuscular and subcutaneous administration generallyinvolves injection of the pharmaceutical formulation until it forms a deposit from which the composition can be released in a sustained fashion. A refillable reservoir or deposit (or depot) may also be used, e.g., in the middle ear, from which the formulation is dispensed. Further, a number of technologies have been developed to permit sustained release of formulations injected into the ear for transfer through the round window membrane, including implantation of an osmotic or digital minipump (Wimmer et al., 2004); hyaluronic acid liposomal gel (El Kechai et al., 2016); a film-forming agent and microspheres containing, e.g., corticosteroids (Dormer et al., 2019) a combination nanotechnology-hydrogel delivery system (Li etal., 2017).

[0089] A carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., including solutions, gels, films, particles, powders, creams, ointments, and lotions, some of which may be further formulated as sustained release preparations.

[0090] To prepare the pharmaceutical compositions according to the disclosed embodiments, a therapeutically effective amount of one or more of the compounds or compositions according to the disclosed embodiments are preferably intimately admixed with a pharmaceutically acceptable carrier, diluent, or excipient, according to conventional pharmaceutical compounding techniques to produce a dose. The term "pharmaceutically acceptable carrier diluent or excipient" refers to any substance, not itself a therapeutic agent, used as a carrier or vehicle, or nonactive component of the composition for administration to an individual, or added to a pharmaceutical composition to improve its handling or storage properties, or to permit or facilitate formation of a unit dose of the composition, and that does not produce unacceptable toxicity or interaction with other components in the composition.

[0091] In some embodiments, the active compound such as a PAI-1 inhibitor or Tiplaxtinin specifically, or a combination of PAI- 1 inhibitor and at least one additional therapeutic agent, such as an anti-inflammatory agent, a CNS stimulant, or a second PAI-1 inhibitor, is included with the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a recipient a therapeutically effective amount for the desired indication, without causing serious toxic effects or being cleared from the system before it can reach its intended site of action.

[0092] In some embodiments, the pharmaceutical compositions may optionally include at least one additional therapeutic agent, such as a CNS stimulant, such as amphetamine, armodafmil, atomoxetine, benzphetamine, caffeine citrate, dexmethylphenidate, diethylpropion, dextroamphetamine, lisdexamfetamine, methamphetamine, modafinil, oxybate, phendimetrazine, phentermine, pitolisant, solriamfetol, or methylphenidate. The skilled artisanwill understand that CNS stimulants may be present in any therapeutically effective dose appropriate for the subject and relative to the subject’s body weight for the desired indication.

[0093] In some embodiments, the pharmaceutical compositions may optionally include at least one additional therapeutic agent, such as an anti-inflammatory agent, e.g., dexamethasone. Other anti-inflammatory agents include, but are not limited to: prednisone, cortisone, hydrocortisone, and clodronate.. Anti-inflammatory agents may be present in the pharmaceutical compositions in any therapeutically effective dose appropriate for the subject and relative to the subject’s body weight and for the desired indication. For example, in some embodiments in connection with implantation of a CI or VI, dexamethasone is administered orally in a therapeutic amount to an adult human subject, ranging from about 0.75 to about 9 mg daily. In other embodiments, dexamethasone may be administered parenterally (IV or IM) in a therapeutic amount to an adult human subject, ranging from about 0.5 to about 9 mg daily. In other embodiments in which a young child or an animal under veterinary care is the subject, for example, a person of ordinary skill would adjust the therapeutic amount accordingly based on appropriate dosages for the weight of the child or animal subject. In certain embodiments, the anti-inflammatory therapeutic agent is co-formulated with the PAI-1 inhibitor and / or other therapeutic agent for administration to the subject.

[0094] In still other embodiments the pharmaceutical compositions are formulated as extended release compositions. In other embodiments, the pharmaceutical composition includes a second PAI- 1 inhibitor, also formulated for extended release, for example, to cover a period in which electrical stimulation of the inner ear tissue will take place. In some embodiments the composition is formulated for immediate release upon implantation of a medical device and with each electrical impulse delivered to the inner ear tissues. In other embodiments, the PAI- 1 inhibitor is administered through release from at least a first coating on an at least first electrode of the medical device. In still other embodiments, the extended release formulation comprises at least one PAI-1 inhibitor, an anti-inflammatory agent and / or a CNS stimulant.

[0095] Pharmaceutical compositions comprising PAI-1 inhibitors can be prepared according to a general protocol provided for Tiplaxtinin in Example 6 (with sterile, deionized water) and Example 7 (with sterile 0.9% saline solution). A person of ordinary still would understand additional, acceptable substitutions that would not alter the basic formulation or its use.

[0096] Briefly, in some embodiments, the pharmaceutical composition is prepared as follows. First, the PAI-1 inhibitor stock solution, starting at a concentration of (25 mg / pL), should bestored at -20°C in DMSO. Pluronic gel (20%) is prepared by mixing 4 g of Pluronic powder with 20 mL of sterile, deionized water using a magnet in the solution; it should be shaken for about 3 hours at 4°C until a homogenous and transparent solution is obtained. At 4°C the solution is a liquid; at room temperature, it is a gel. The pharmaceutical composition with the PAI-1 inhibitor is then prepared as follows with a dilution of 1 / 1000 in Pluronic gel made in two steps. First, a 1: 10 dilution is prepared in Pluronic gel by combining 5pL of Tiplaxtinin with 45 pL Pluronic gel (Pluronic F-127, Sigma) in a sterile Eppendorf pipette; the mixture is then vortexed for 30 seconds and maintained on ice. Next, a 1: 100 dilution is prepared from the first step, again in Pluronic gel by adding 10 pL of the PAI-1 inhibitor to 990 pL of Pluronic gel in a second sterile Eppendorf pipette. In other embodiments, 0.9% sterile saline solution is substituted for the sterile, deionized water.Modes of administration

[0097] Approaches to the delivery of pharmaceutical compositions include but are not limited to localized administration / delivery approaches where the pharmaceutical compositions are initially delivered at or near a target location within the recipient. The goal of localized deli very is that only the target location, and possibly a small amount of surrounding tissue, is exposed to the pharmaceutical composition. Thus, pharmaceutical compositions are delivered at the specific target location and may remain in a proximity of the target location. In some embodiments, the pharmaceutical composition may be introduced into the body via an outlet of the catheter, which is positioned at the target location within the recipient. An example of such localized delivery-' of a pharmaceutical composition may occur, for example, by inserting in the femoral artery' of the thigh a catheter and guiding it to a specific location where the composition will be released to act locally, such as in the arterial supply to the cochlea, or into the cranium. Another example is injecting a liquid or gel composition into the middle ear for absorption through the round window membrane into the inner ear labyrinth. In some embodiments, the composition may be locally deposited in the middle ear in the form of a gelbased or liquid-based composition, allowing transport from the delivery site to the site of action. In other embodiments, delivery approaches may include intra-cochlear delivery introduced via a cochlear implant electrode array, or by injection into the cochlea.

[0098] In some embodiments, the pharmaceutical composition may be delivered locally by injection or infusion into the labyrinthine artery or cochlea / vestibule supply artery, where microparticles or nanoparticles accumulate at an appropriate site, e.g., they may gather in themicrocapilliaries or at the more permeable (i.e., leaky) blood labyrinth barrier (BLB), or blood perilymph barrier (BPB), or blood brain barrier (BBB). In other embodiments, the compositions can be formulated as nanoparticles and may, in fact, be able to cross these barriers when the composition is positioned, injected or otherwise has entered the body at or near the barriers or through the blood.

[0099] In some embodiments, the PAI-1 inhibitor is administered locally through injection, cannula or from the CI electrode itself, for example, to ensure a therapeutic concentration of the inhibitor in the perilymph. In the case of Tiplaxtinin, in some embodiments the concentration would range between about 100 ng / mL and 300 ng / mL of perilymph fluid at the time of or following implantation, or at or following application of electrical stimulation.[ooioo] In other circumstances, pharmaceutical compositions may be delivered to a recipient using a systemic administration approach. With systemic delivery, the pharmaceutical composition is introduced into the circulatory system of the recipient so that the entire body of the recipient is exposed to the pharmaceutical composition(s). Systemic administration of pharmaceutical compositions can take place via, e.g., enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion, or implantation).[ooioi] Systematic and localized modes of pharmaceutical compositions administration have their advantages, as well as drawbacks that may limit its use for certain recipients, disorders, diseases, etc. The goal of localized administration is to direct the release of the composition to only the target location; a small amount of surrounding tissue may be exposed to the composition’s effect. The fact that the pharmaceutical composition is delivered at or near the target location is advantageous in that, e.g., a relatively high concentration of the composition may be administered to the target location as the rest of the recipient’s body has limited exposure to it. Localized administration suffers from the problem that certain areas of a recipient’s body are difficult to access in a manner that allows for the direct delivery of the pharmaceutical composition.

[0102] In some embodiments, the PAI-1 inhibitor is administered systemically to ensure a therapeutic concentration of the inhibitor in the perilymph. In the case of Tiplaxtinin, in some embodiments concentration would range between about 100 ng / mL and about 300 ng / mL of perilymph fluid at the time of or following implantation or at or after application of electrical stimulation.

[0103] In the context of the inner ear, localized administration may not pose the risk of systemic toxicity, but it may be more difficult as the inner ear, and in particular, the apical region of a recipient’s cochlea, is difficult to access. Moreover, injecting a pharmaceutical composition into the cochlea may cause the loss of residual hearing due to disruption of the BLB or other tissue barrier, e.g., through destruction of hair cells, forming an opening the cochlea that changes the cochlear dynamics, and other effects. In some embodiments, administration of a pharmaceutical composition to the perilymph or endolymph compartments may include any of the following known methods: (i) via injection or deposition of the composition at the round window membrane, or through a cochleostomy to the scala tympani; (ii) through a direct cochleostomy to the scala media; (iii) using a combination of those two pathways; (iv) implanting a CI with drug-eluting electrodes (through a polymer, silica, silicone or other coating, or delivered through a separate delivery cannula associated with the electrode, or other similar delivery channel); and (v) a less invasive, newer approach: systemic transfer of, e.g., a gene (Wang et al., 2018).

[0104] As referenced above, a drawback of systemic administration is that almost all tissues of the body are exposed to the pharmaceutical compositions administered in this way. Accordingly, systemically administered pharmaceutical compositions must be relatively harmless to the body, and further, must be administered to achieve a low enough concentration to prevent inducing unwanted side effects outside of the intended target location. However, systemic administration is easier to administer than is local administration, especially to the middle or the inner ear areas, and has the potential reach nearly all tissues if a whole-body approach is needed.

[0105] The choice of systemic or local administration must be informed by the pharmacokinetics of the pharmaceutical composition being administered. A person of ordinary skill will appreciate the impact pharmacokinetics will have on the mode of administration, the dose, the timing, and duration of treatment. Several excellent reviews and discussions are available regarding local (middle / inner ear) pharmacokinetics of various pharmaceutical agents (see, e.g., Plontke et al., 2017; Salt 2005; Nyberg et al 2019; Salt and Plontke 2018; Rybak et al., 2019; and McCall et al., Drug delivery for treatment of inner ear disease: current state of knowledge. Ear and Hearing, vol. 31(2): 156-65, 2010.(doi: 10.1097 / AUD.0b013e3181c351f2).

[0106] In the context of the inner ear, systemic administration may be problematic as noted, as it is difficult to deliver pharmaceutical compositions in therapeutically effective concentrationsneeded for the cochlea (or perilymph fluid) without inducing unacceptable toxic levels at other areas of the body (e.g., the pharmacokinetics limits the ability to deliver the drug to a specific location, such as high in the apex of the cochlea). That is, systemic administration of pharmaceutical compositions to the inner ear may require such low concentrations (to prevent systemic toxicity) as to render the pharmaceutical compositions largely ineffective for inner ear treatment.

[0107] Some of the methods presented herein are primarily described with reference to a specific implantable medical device system, namely, a cochlear implant system. However, a person of ordinary skill in the art would appreciate that the methods disclosed also may be used with other types of implantable medical devices or implantable medical device systems. For example, the methods presented herein may be used with other auditory prosthesis systems and related devices. The methods and pharmaceutical compositions disclosed herein may also be used with tinnitus therapy devices, vestibular devices (e.g., vestibular implants), visual devices (i.e., bionic eyes), sensors, pacemakers, drug delivery systems, defibrillators, functional electrical stimulation devices, catheters, seizure devices (e.g., devices for monitoring and / or treating epileptic events), sleep apnea devices, electroporation devices, and others.

[0108] The pharmaceutical compositions of the embodiments or modifications thereto can be administered in any number of ways, e.g., including without limitation, by any one or more of the following: (1) inhalation; (2) atopical lotion, solution, cream, ointment or dusting powder; (3) by use of a transdermal patch; (4) orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules, micro- or nanoparticles, either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents; (5) for ophthalmic or auditory disorders and diseases, they may be formulated as drops or a solution, gel or other form for injection or use in a reservoir; (6) for parenteral administration (any route of administration that does not involve absorption via the gastrointestinal tract), they may be administered intravenously, intramuscularly, intracavemosally or subcutaneously, or inhaled or other intranasal administration in the form of a sterile aqueous solution or injectable implant or (micro- / nano-) particles, or inhalable substances that may contain other substances, e.g., with adequate salt or monosaccharide content to make the solution isotonic with blood or with substances that allow sustained release, such as micro- or nano-particles or a gel; and (7) for enteral administration (entry to the circulatory system via absorption through the gastrointestinal tract) of the compositions may be administered in the form of tablets, gels, solutions, lozenges, semi-solids and any otheredible or insertable forms, which can be formulated in a conventional manner according to known methods in the art.

[0109] In disclosed embodiments, any suitable mechanism of delivery may be used to systemically introduce the pharmaceutical composition for treatment of inner ear fibrosis. In one embodiment, localized release of a systemically circulating pharmaceutical composition is shown, using as a reference, a cochlear implant 100 and cochlea 140, as detailed with reference to FIGs. 1A-1G. As noted elsewhere, other devices may be used in keeping with the totality of this disclosure.Doses and schedules

[0110] One of ordinary skill in the art will recognize that in the disclosed embodiments a therapeutically effective amount provided in the composition, as well as the dosing, and timing of dosing will depend on the condition being treated and its severity, the pharmaceutical composition and its pharmacokinetics, the mode of administration, and other factors such as weight and condition of the patient, and the judgment of the prescribing caregiver, among other considerations.[oom] In general, a therapeutically effective amount of the pharmaceutical compositions in dosage form usually ranges from less than about 0.001 mg / kg subject body weight to about 2.5 g / kg subject body weight on a per hour, day or other time period basis, regardless if those amounts are delivered in a single dose or apportioned over multiple periods of administration in the specified period. In the most preferred embodiments, pharmaceutical formulations according to the present invention are administered in a suitable carrier in amounts ranging from about 1 pg / kg to about 100 mg / kg per hour, day or per other period, again, regardless if those amounts are delivered in a single dose or apportioned over multiple periods of administration in the specified period. In some embodiments, the pharmaceutical compositions comprise not only a therapeutically effective amount at least one PAI-1 inhibitor, but also a therapeutically effective amount of an anti-inflammatory agent and / or a CNS stimulant.

[0112] In the disclosed, preferred embodiments, a therapeutically effective amount of the pharmaceutical composition in dosage form is, based on an hourly, daily or other period basis, and dependent on the route of administration, among other things, usually less than about 0.001 mg / kg body weight, less than about 0.025 mg / kg, less than about 0.050 mg / kg, less than about 0.075 mg / kg, less than about 0.100 mg / kg, less than about 0.15 mg / kg, less than about 0.20mg / kg, less than about 0.25 mg / kg, less than about 0.30 mg / kg, less than about 0.35 mg / kg, less than about 0.40 mg / kg, less than about 0.45 mg / kg, less than about 0.50 mg / kg, less than about 0.55 mg / kg, less than about 0.60 mg / kg, less than about 0.65 mg / kg, less than about 0.70 mg / kg, less than about 0.75 mg / kg, less than about 0.80 mg / kg, less than about 0.85 mg / kg, less than about 0.90 mg / kg, less than about 0.95 mg / kg, less than about 1.0 mg / kg, less than about 2.0 mg / kg, less than about 3.0 mg / kg, less than about 4.0 mg / kg, less than about 5.0 mg / kg, less than about 6.0 mg / kg, less than about 7.0 mg / kg, less than about 8.0 mg / kg, less than about 9.0 mg / kg, less than about 10.0 mg / kg, less than aboutl l .O mg / kg, less than about 12.0 mg / kg, less than about 13.0 mg / kg, less than about 14.0 mg / kg, less than about 15.0 mg / kg, less than about 16.0 mg / kg, less than about 17.0 mg / kg, less than about 18.0 mg / kg, less than about 19.0 mg / kg, less than about 20.0 mg / kg, less than about 25.0 mg / kg, less than about 30.0 mg / kg, less than about 35.0 mg / kg, less than about 40.0 mg / kg, less than about 45.0 mg / kg, less than about 50.0 mg / kg, less than about 55.0 mg / kg, less than about 60.0 mg / kg, less than about 65.0 mg / kg, less than about 70.0 mg / kg, less than about 75.0 mg / kg, less than about 80.0 mg / kg, less than about 85.0 mg / kg, less than about 90.0 mg / kg, less than about 95.0 mg / kg, less than about 100 mg / kg, less than about 120.0 mg / kg, less than about 140.0 mg / kg, less than about 160.0 mg / kg, less than about 180.0 mg / kg, less than about 200.0 mg / kg, less than about 250.0 mg / kg, less than about 300.0 mg / kg, less than about 350.0 mg / kg, less than about 400.0 mg / kg, less than about 600.0 mg / kg, less than about 800.0 mg / kg, less than about 1.0 g / kg, less than about 1.5 g / kg, less than about 2.0 g / kg, less than about 2.5 g / kg, less than about 2.75 g / kg, ranges between about 0.10 mg / kg to about 1.25 mg / kg, ranges between about 0.10 mg / kg to about 0.50 mg / kg, ranges between about 0.20 mg / kg to about 0.40 mg / kg, ranges between about 0.20 mg / kg to about 0.60 mg / kg, ranges between about 0.30 mg / kg to about 0.70 mg / kg, ranges between about 0.40 mg / kg to about 0.80 mg / kg, ranges between about 0.50 mg / kg to about 0.90 mg / kg, ranges between about 0.60 mg / kg to about 1.0 mg / kg, ranges between about 0.90 mg / kg to about 1.10 mg / kg, ranges between about 1.00 mg / kg to about 1.30 mg / kg, ranges between about 1.10 mg / kg to about 1.5 mg / kg, ranges between about 1.25 mg / kg to about 1.75 mg / kg, ranges between about 1.5 mg / kg to about 2.0 mg / kg, ranges between about 1.75 mg / kg to about 2.25 mg / kg, ranges between about 2.0 mg / kg to about 2.5 mg / kg, ranges between about 2.0 mg / kg to about 5.0 mg / kg, ranges between about 3.0 mg / kg to about 7.0 mg / kg, ranges between about 5.0 mg / kg to about 10 mg / kg, ranges between about 10.0 mg / kg to about 20 mg / kg, ranges between about 20.0 mg / kg to about 30.0 mg / kg, ranges between about 30.0 mg / kg to about 40 mg / kg, ranges between about 40.0 mg / kg to about 50.0 mg / kg, ranges between about 50.0 mg / kg to about 60.0 mg / kg, ranges between about 60.0 mg / kg to about70.0 mg / kg, ranges between about 70.0 mg / kg to about 80.0 mg / kg, ranges between about 80.0 mg / kg to about 90.0 mg / kg, ranges between about 90.0 mg / kg to about 100.0 mg / kg, ranges between about 100.0 mg / kg to about 110.0 mg / kg, ranges between about 110.0 mg / kg to about 120.0 mg / kg, ranges between about 120.0 mg / kg to about 130.0 mg / kg, ranges between about 130.0 mg / kg to about 140.0 mg / kg, ranges between about 140.0 mg / kg to about 150.0 mg / kg, ranges between about 150.0 mg / kg to about 160.0 mg / kg, ranges between about 160.0 mg / kg to about 170.0 mg / kg, ranges between about 170.0 mg / kg to about 180.0 mg / kg, ranges between about 180.0 mg / kg to about 190.0 mg / kg ranges between about 190.0 mg / kg to about 200.0 mg / kg, ranges between about 200.0 mg / kg to about 250.0 mg / kg, ranges between about 250.0 mg / kg to about 300.0 mg / kg, ranges between about 300.0 mg / kg to about 350.0 mg / kg, ranges between about 350.0 mg / kg to about 400.0 mg / kg, ranges between about 400.0 mg / kg to about 450.0 mg / kg, ranges between about 450.0 mg / kg to about 500.0 mg / kg, ranges between about 500.0 mg / kg to about 550.0 mg / kg, ranges between about 550.0 mg / kg to about 600.0 mg / kg, ranges between about 600.0 mg / kg to about 650.0 mg / kg, ranges between about 650.0 mg / kg to about 700.0 mg / kg, ranges between about 700.0 mg / kg to about 750.0 mg / kg, ranges between about 750.0 mg / kg to about 800.0 mg / kg, ranges between about 800.0 mg / kg to about 850.0 mg / kg, ranges between about 850.0 mg / kg to about 900.0 mg / kg, ranges between about 900.0 mg / kg to about 950.0 mg / kg, ranges between about 950.0 mg / kg to about 1.0 g / kg, ranges between about 1.0 g / kg to about 1.2 g / kg, ranges between about 1.2 g / kg to about 1.4 g / kg, ranges between about 1.4 g / kg to about 1.6 g / kg, ranges between about 1.6 g / kg to about 1.8 g / kg, ranges between about 1.8 g / kg to about 2.0 g / kg, ranges between about 2.0 g / kg to about 2.25 g / kg, ranges between about 2.25 g / kg to about 2.5 g / kg, ranges from about 2.5 g / kg to about 2.75 g / kg, ranges from about 2.75 g / kg to about 3.0 g / kg of body weight. Typically, a physician or other caregiver licensed to prescribe pharmaceuticals will determine the actual dosage most suitable for an individual subject, and it will vary by composition and other factors, as referenced above.

[0113] With respect to the amount of PAI-1 inhibitor in the disclosed pharmaceutical compositions as noted above, a stock solution of the selected PAI-1 inhibitor may be prepared according to the protocol provided in Example 6 or 7, and the dilutions altered to accommodate each particular situation including the route of administration. Based on the materials and methods described in those examples, in general, a dose will range between about 50pL to about 800 pL on a per kg body weight basis. Specifically an amount may be selected from the following per kg body weight: between about 50pL to about 150 pL; between about 125pL toabout 225pL; between about 200pL to about 300pL; between about 175pL to about 275pL; between about 250pL to about 350 pL; between about 325pL to about 425pL; between about 400pL to about 500 pL; between about475pL to about 575 pL; between about 550pL to about 650 pL; between about 625pL to about 725 pL; and between about 700pL to about 800 pL. A person of ordinary skill in the art will be able to determine an appropriate amount of the prepared gel or solution, such as for Tiplaxtinin in Examples 6 and 7, which may be administered to a subject, depending on the subject, the mode of administration, and the situation in which the composition is being administered.

[0114] Regardless of the subject, the dosage and the route of administration, the goal is to achieve a target concentration of the PAI-1 inhibitor between about 100 ng / mL and about 300 ng / mL of perilymph fluid between about 1 and about 6 hours or longer following administration.

[0115] With respect to the amount of anti-inflammatory agent used in the disclosed pharmaceutical compositions, a person of ordinary skill would be able to determine an appropriate amount based on accepted dosages for an individual subject’s needs and the context in which the agent is being administered. Based on an acceptable dosage of dexamethasone administered orally or through IV or IM injection to an adult human, a person of ordinary skill would be able to modify such amounts for a particular subject and a particular context. For an adult human, oral daily dosing will range generally between about 0.75 mg to about 9 mg, between about 0.75 mg to about 2 mg, between about 1 mg to about 3 mg, between about 1.5 mg to about 5 mg, between about 3 mg to about 6 mg, between about 4 mg to about 7 mg, between about 5 mg to about 8 mg, and between about 6 mg to about 9 mg. If administration is provided through an intraperitoneal route (e.g., through IV or IM modes), daily dosing may range between about 0.5 mg to about 9 mg, between about 0.5 mg to about 2 mg, between about 1 mg to about 3 mg, between about 1.5 mg to about 5 mg, between about 3 mg to about 6 mg, between about 4 mg to about 7 mg, between about 5 mg to about 8 mg, and between about 6 mg to about 9 mg. A person of ordinary skill will be able to make appropriate adjustments to these ranges based on the subject, e.g., maturity, age, species if not human, etc., the mode of administration and the context in which the agent is administered, either as part of a composition, co-formulated with a PAI-1 inhibitor, or in a separate composition.

[0116] With respect to the amount of CNS stimulant used in the disclosed pharmaceutical compositions, a person of ordinary skill would be able to determine an appropriate amountbased on accepted dosages for an individual subject’s needs and the context in which the agent is being administered. Such stimulants are available in various dosage forms (e.g., oral suspensions, chewable tablets, capsules, liquids and patches and other forms).

[0117] All ranges and ratios discussed here can and necessarily do describe all values, subranges and subratios therein for all embodiments, and all such subranges and subratios also form part and parcel of the disclosed embodiments. Any listed range or ratio can be easily recognized as sufficiently describing and enabling the same range or ratio being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. each range or ratio discussed herein can be readily broken down into a lower third, middle third and upper third, etc. Alternatively, a range of 1 to 50 is understood to include any number, fraction or combination of numbers from the group of positive numbers between about 0 and up to and including about 50.

[0118] In some embodiments two or more pharmaceutical compositions may be administered simultaneously; in other embodiments two or more pharmaceutical compositions may be administered serially. In still other embodiments two or more pharmaceutical compositions can be administered each through a different mode of administration (e.g., systemic, intracochlear, locally to the middle ear).

[0119] In certain method embodiments for treating fibrosis of the inner ear, the pharmaceutical composition is administered once daily; in other embodiments, the compound is administered twice to six times daily; in yet other embodiments, the compound is administered once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once per year. In other embodiments, the composition is administered on an irregular basis, or in response only to electrical stimulation of the tissues. In still other embodiments, the formulation is administered on an as-needed basis, as determined through the closed control system.

[0120] In some embodiments, methods comprise the pharmaceutical formulation being administered for treating inner ear fibrosis, which administration will extend for time periods of about 1 to 24 hours, 1 to 4 days, 3 to 6 days, 5 to 8 days, exceeding one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, 9 months, one year, two years, three years, four years, or five years, ten years, or fifteen years; or for example, any time period range in hours, days, months or years in which the low end of the range is any time period between 1 hour and 2 years, 2 to 10 years, 15 years, and the upperend of the range is between 15 days and 20 years (e.g., between 4 weeks and 15 years, between 6 months and 20 years). In some cases, it may be advantageous for the formulations of the disclosed embodiments and their modifications to be administered for the life of the patient.

[0121] In some embodiments the amount of the PAI- 1 inhibitor administered is reduced over a period of 1-6 months following implantation to 10% or less of an initial dose administered upon implantation.

[0122] Otic conditions typically are treated by administering multiple doses of drops or injections over several days and up to two weeks, sometimes with multiple doses administered daily. Pharmaceutical compositions of the embodiments disclosed herein may be readministered at any desired frequency (e.g., daily, weekly, etc.) to achieve a suitable therapeutic effect. In some embodiments, the composition may be delivered with a cochlear implant or during the cochlear implantation procedure. In other embodiments, the compositions may be delivered with any other type of middle or inner ear implant or device, or as a component of a known control system.

[0123] A person of ordinary skill in the art will appreciate that the dosing amount or interval may need to be adjusted according to the needs of individual patients or subjects. In some embodiments involving longer intervals of administration, compositions may be administered in a form appropriate for sustained release, including the formation of depots for such release.

[0124] In preferred embodiments, the pharmaceutical compositions remains effective for at least one hour, two hours, four hours, six hours, eight hours, ten hours, 12 hours, at least 1 day, at least 3 days, at least 1 week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, and one year, and the schedule for administration of the composition may be maintained during that period as necessary up to at least two years, at least three years, at least four years, or at least five years, ten years, fifteen years, twenty years, or for the remainder of the subject's life.Treating fibrosis of the inner ear using a medical device and administering a pharmaceutical composition comprising a PAI-1 inhibitor

[0125] In some method embodiments, pharmaceutical compositions may be administered ultimately to treat conditions, disorders and diseases arising from or involving dysfunction relating to fibrosis of the inner ear. The embodiments of disclosed methods and pharmaceutical compositions for treating fibrosis of the inner ear may further be used in any known, broadermethod to monitor or treat a recipient of a CI or other medical device implanted in the inner ear.

[0126] The disclosed embodiments and modifications may be extended to treat age-related hearing loss or temporary cochlear implant-related hearing loss, e.g., by switching off or modulating the immune system. The embodiments disclosed for methods of treating inner ear fibrosis can be used prophylactically, to prevent, reduce or delay the progression of hearing loss or other auditory disorders associated fibrosis or other inner ear function. In other disclosed embodiments pharmaceutical compositions may be used alone or in combination with treatments or components of treatments for other conditions, disorders, or diseases of the inner ear.

[0127] In some disclosed embodiments, methods and pharmaceutical compositions are used as a component in one or more treatment regimens targeting a combination of fibrosis of the inner ear and fibrosis in other tissues. For example, disclosed the compositions and methods for treating fibrosis of the inner ear can be used in combination with treatments in subjects at risk of developing, or in subjects having, a condition known to be associated with fibrosis development in other tissues (e.g., kidney, lung, heart, etc.), which conditions may enhance the risk of developing fibrosis of the inner ear following implantation trauma, electrical stimulation or other inner ear tissue damage.

[0128] In some method embodiments, humans are treated for inner ear fibrosis according to essentially a similar protocol used in rodents, as described in Example 5 below, particularly with respect to administration of the pharmaceutical composition comprising Tiplaxtinin. Humans share a similar anatomy of the inner ear with rodents such as rats and guinea pigs; hence, the basics of the implantation process are similar if not the same.

[0129] Briefly, a medical device such as a CI or VI is implanted in a human ear under general anesthesia in a hospital or clinic., with at least one electrode extending through an opening the surgeon makes in the mastoid bone behind the ear.

[0130] The tympanic bulla is opened, and then the round window is opened with a needle to permit insertion of the one or more electrodes into the cochlea via the round window. In an alternative embodiment, the tympanic bulla and round window are accessed in an endoscopic procedure. (Marchioni et al. Endoscopic cochlear implant procedure. Eur Arch Otorhinolaryngol. (2013) DIO 10.1007 / s00405-013-2490-4) In other embodiments a CI insertion tool may be used. Hussong et al. An automated insertion tool for cochlear implants:another step towards atraumatic cochlear implant surgery. I nt. J CARS (2010)5 : 163-71) In one embodiment, the Tiplaxtinin-containing composition, prepared as described with 0.9% sterile saline solution in Example 7, and at least 100 pL of the composition is injected using a pipette or, optionally, a syringe to fdl the bulla, or more optionally, a catheter or other device. Once the bulla is filled, the round window opening is sealed with muscle or other tissue, and the skin is sutured. In other embodiments, the Tiplaxtinin- or other PAI-1 inhibitor-containing composition is prepared as described but is administered to the patient orally or through an intraperitoneal mode. In other embodiments such oral or intraperitoneal or other mode of administration would follow application of each electrical impulse, and the amount of PAI- 1 inhibitor administered is reduced over a period of 1-6 months to 10 percent or less of the initial dose administered upon implantation. In yet other embodiments, the composition is administered through an IV or IM route. In still other embodiments, any other route of administration identified herein as long as, accounting for the particular formulation, the administered dosage results in the therapeutic concentration of Tiplaxtinin to be maintained between 100 and 300 ng / mL of perilymph fluid. For all treatment methods used in humans or other animals in clinical or research settings, any dosage level or schedule of administration of the composition with or without electrical stimulation as described elsewhere herein may be used provided the therapeutic concentration of Tiplaxtinin or other PAI-1 inhibitor is maintained between about 100 ng / mL and about 300 ng / mL of perilymph fluid, or in a concentration sufficient to achieve the desired result.

[0131] The final part of the implantation process involves positioning an implantable component ((112) as shown in FIG. ID) in, for example, a pocket between the scalp / muscle and bone behind the ear. The component receives information from an external speech processor that is worn on the outer scalp ((106), as shown in FIG. ID). The surgeon then closes the incisions and the patient is allowed to recover from anesthesia.

[0132] In some embodiments, methods are provided for preventing, attenuating or halting the development of, or reducing, fibrosis of the inner ear tissues in a subject having an electrical medical device with at least a first electrode implanted in the inner ear. Such embodiments include the method of administrating a therapeutically effective amount of a first therapeutic agent, such as a PAI-1 inhibitor. In some embodiments the PAI-1 inhibitor is Tiplaxtinin. In other embodiments the PAI-1 inhibitor is selected from among other PAI-1 inhibitors: TM 5441, TM 5275 sodium salt, SK-216, annonacinone, AZ 3976 and embelin. The PAI-1inhibitor is capable of preventing, slowing, or halting the development of fibrosis, or reducing the volume of fibrosis at the site of implantation and electrical stimulation.

[0133] In the disclosed method embodiments, the PAI-1 inhibitor attenuates the development of fibrosis of the inner ear tissues of the subject receiving treatment by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0134] In the disclosed method embodiments, the PAI- 1 inhibitor reduces an amount of fibrosis existing in the inner ear tissues of the subj ect receiving treatment by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0135] In some embodiments, the methods, pharmaceutical compositions and their modifications may be used in tandem with a control system known in the art (see below) that is partially or fully automated to treat inner ear fibrosis. In some embodiments, the implanted device may be used as a component in a known control system that describes the monitoring or treatment of a CI recipient. In other embodiments the pharmaceutical formulation may be used in such a control system, for example the system disclosed in US20210001113 Al for addressing hearing loss of CI recipients.

[0136] In some embodiments, the known control system in which the disclosed embodiments are used may be fully automated. In some embodiments, a single implanted medical device may be employed to treat fibrosis of the inner ear and may have multiple internal components hard wired for intra-device communication. In other embodiments, wireless technology may link various components of the device to a computer or other control device for communication of collected data. In still other embodiments a combination of hardwiring and wireless technologies may be used. In some embodiments the method of treating fibrosis of the inner ear may be implemented in a clinical or hospital setting; in other embodiments, the method may be implemented remotely.

[0137] In some embodiments the medical device used to treat fibrosis of the inner ear provides progress reports on demand for the current procedure or longer-term progress, which can provide an historical record of the recipient’s progress for the patient’s electronic medical record.Cochlear implant system

[0138] FIGs. ID, IE, IF, and 1G are diagrams illustrating an example of a cochlear implant system 102 with which aspects of the methods presented herein can be implemented. The cochlear implant system 102 comprises an external component 104 and an implantable component 112. In the examples of FIGs. 1D-1G, the implantable component is sometimes referred to as a “cochlear implant” or “CI.” FIG. ID is a schematic diagram illustrating the implantable component 112 implanted in the head of a recipient, while FIG. IE is schematic drawing of the external component 104 worn on the head of the recipient. FIG. IF is another schematic view of the cochlear implant system 102, while FIG. 1G is a block diagram illustrating further details of the cochlear implant system 102. For ease of description, FIGs. 1D-1G will generally be described together.

[0139] As noted, cochlear implant system 102 includes an external component 104 that is configured to be directly or indirectly attached to the body of the recipient and an implantable component 112 configured to be implanted in the recipient. In the examples of FIGs. 1D-1G, the external component 104 comprises a sound processing unit 106, while the implantable component 112 includes an internal coil 114, a stimulator unit 142, and an elongate stimulating assembly 116 configured to be implanted in the recipient’s cochlea.

[0140] In the example of FIGs. 1D-1G, the sound processing unit 106 is an off-the-ear (OTE) sound processing unit, sometimes referred to herein as an OTE component, that is configured to send data and power to the implantable component 112. In general, an OTE sound processing unit is a component having a generally cylindrically shaped housing 127 and which is configured to be magnetically coupled to the recipient’s head (e.g., includes an integrated external magnet 150 configured to be magnetically coupled to an implantable magnet 152 in the implantable component 112). The OTE sound processing unit 106 also includes an integrated external coil 108 that is configured to be inductively coupled to the implantable coil 114.

[0141] It is to be appreciated that the OTE sound processing unit 106 is merely illustrative of the external devices that could operate with implantable component 112. For example, in alternative examples, the external component may comprise a behind-the-ear (BTE) sound processing unit or a micro-BTE sound processing unit and a separate external. In general, a BTE sound processing unit comprises a housing that is shaped to be worn on the outer ear of the recipient and is connected to the separate external coil assembly via a cable, where the external coil assembly is configured to be magnetically and inductively coupled to theimplantable coil 114. It is also to be appreciated that alternative external components could be located in the recipient’s ear canal, worn on the body, etc.

[0142] FIGs. 1D-1G illustrate an arrangement in which the cochlear implant system 102 includes an external component. However, it is to be appreciated that the disclosed embodiments and modifications may be implemented in cochlear implant systems having alternative arrangements. For example, embodiments presented herein can be implemented by a totally implantable cochlear implant or other totally implantable medical device. A totally implantable medical device is a device in which all components of the device are configured to be implanted under skin / tissue of a recipient. Because all components are implantable, a totally implantable medical device operates, for at least a finite period of time, without the need of an external device / component. However, an external component can be used to, for example, charge the internal power source (battery) of the totally implantable medical device.

[0143] Returning to the specific example of FIGs. 1D-1G, FIG. 1G illustrates that the OTE sound processing unit 106 comprises one or more input devices 153 that are configured to receive input signals (e.g., sound or data signals). The one or more input devices 153 include one or more sound input devices 118 (e.g., microphones, audio input ports, telecoils, etc.), one or more auxiliary input devices 119 (e.g., audio ports, such as a Direct Audio Input (DAI), data ports, such as a Universal Serial Bus (USB) port, cable port, etc.), and a wireless transmitter / receiver (transceiver) 120. However, it is to be appreciated that one or more input devices 153 may include additional types of input devices and / or less input devices (e.g., the wireless transceiver 120 and / or one or more auxiliary input devices 119 could be omitted).

[0144] The OTE sound processing unit 106 also comprises the external coil 108, a charging coil 151, a closely-coupled transmitter / receiver (transceiver) 122, sometimes referred to as or radio-frequency (RF) transceiver 122, at least one rechargeable battery 159, and a processing module 124. The processing module 124 comprises one or more processors 157 and a memory device (memory) 126 that includes sound processing logic 128 and sensing logic 155. The sensor may provide measurements in terms of the standard cochlear implant impedance measurement, NRT measurement, or ECochG measurement. The memory device 126 may comprise any one or more of: Non-Volatile Memory (NVM), Ferroelectric Random Access Memory (FRAM), read only memory (ROM), random access memory (RAM), magnetic disk storage media devices, optical storage media devices, flash memory devices, electrical, optical, or other physical / tangible memory storage devices. The one or more processors 157 are, forexample, microprocessors or microcontrollers that execute instructions for the sound processing logic 128 and / or sensing logic 155 stored in memory device 126.

[0145] The implantable component 112 comprises an implant body (main module) 134, a lead region 136, and the intra-cochlear stimulating assembly 116, all configured to be implanted under the skin / tissue (tissue) of the recipient. The implant body 134 generally comprises a hermetically-sealed housing 138 in which RF interface circuitry 140 and a stimulator unit 142 are disposed. The implant body 134 also includes the intemal / implantable coil 114 that is generally external to the housing 138, but which is connected to the transceiver 140 via a hermetic feedthrough (not shown in FIG. 1G). As shown in FIG. 1G, the stimulator can comprise sensing hardware 133, as described further below.

[0146] As noted, stimulating assembly 116 is configured to be at least partially implanted in the recipient’s cochlea. Stimulating assembly 116 includes a plurality of longitudinally spaced intra-cochlear electrical stimulating contacts (electrodes) 144 that collectively form a contact or electrode array 146 for delivery of electrical stimulation (current) to the recipient’s cochlea.

[0147] Stimulating assembly 116 extends through an opening in the recipient’s cochlea (e.g., cochleostomy, the round window, etc.) and has a proximal end connected to stimulator unit 142 via lead region 136 and a hermetic feedthrough (not shown in FIG. 1G). Lead region 136 includes a plurality of conductors (wires) that electrically couple the electrodes 144 to the stimulator unit 142. The implantable component 112 also includes an electrode outside of the cochlea, sometimes referred to as the extra-cochlear electrode (ECE) 139.

[0148] As noted, the cochlear implant system 102 includes the external coil 108 and the implantable coil 114. The external magnet 150 is fixed relative to the external coil 108 and the implantable magnet 152 is fixed relative to the implantable coil 114. The magnets fixed relative to the external coil 108 and the implantable coil 114 facilitate the operational alignment of the external coil 108 with the implantable coil 114. This operational alignment of the coils enables the external component 104 to transmit data and power to the implantable component 112 via a closely-coupled wireless link formed between the external coil 108 with the implantable coil 114. In certain examples, the closely-coupled wireless link is a radio frequency (RF) link. However, various other types of energy transfer, such as infrared (IR), electromagnetic, capacitive and inductive transfer, may be used to transfer the power and / or data from an external component to an implantable component and, as such, FIG. 1G illustrates only one example arrangement.

[0149] As noted above, sound processing unit 106 includes the processing module 124. The processing module 124 is configured to convert received input signals (received at one or more of the input devices 153) into output signals for use in stimulating a first ear of a recipient (i.e., the processing module 124 is configured to perform sound processing on input signals received at the sound processing unit 106). Stated differently, the one or more processors 157 are configured to execute sound processing logic 128 in memory 126 to convert the received input signals into output signals 145 that represent electrical stimulation for delivery to the recipient.

[0150] As noted, FIG. 1G illustrates an embodiment in which the processing module 124 in the sound processing unit 106 generates the output signals. In an alternative embodiment, the sound processing unit 106 can send less processed information (e.g., audio data) to the implantable component 112 and the sound processing operations (e.g., conversion of sounds to output signals 145) can be performed by a processor within the implantable component 112. That is, the implantable component 112, rather than the sound processing unit 106, could include a processing module that is similar to processing module 124 of FIG. 1G.

[0151] Returning to the specific example of FIG. 1G, the output signals 145 are provided to the RF transceiver 122, which transcutaneously transfers the output signals (e.g., in an encoded manner) to the implantable component 112 via external coil 108 and implantable coil 114. That is, the output signals are received at the RF interface circuitry 140 via implantable coil 114 and provided to the stimulator unit 142. The stimulator unit 142 is configured to utilize the output signals to generate electrical stimulation signals (e.g., current signals) for delivery to the recipient’s cochlea via “stimulation channels,” where each stimulating channel comprises one or more of the electrodes 144. In this way, cochlear implant system 102 electrically stimulates the recipient’s auditory nerve cells, bypassing absent or defective hair cells that normally transduce acoustic vibrations into neural activity, in a manner that causes the recipient to perceive one or more components of the received sound signals.

[0152] FIGs. 2A is a photo illustrating a perspective of a cochlea 140, while FIG. 2B is a photo illustrating a top view of the cochlea 140. The photos of FIGs. 2A and 2B have been annotated to show the location / path (hatched sections in sequence) of a stimulating assembly 116 within the cochlea 140.EXAMPLESExample 1: Assessment of Tiplaxtinin as a potential antifibrotic drug in vitro.

[0153] Tiplaxtinin was evaluated in vitro to assess its antifibrotic effect. After an intraperitoneal overdose injection of sodium pentobarbital, neonatal Wistar rats (P1-P2) weredecapitated. The half-head was dissected in chilled slicing solution (4° C) to remove the temporal bones. Because the slicing procedure damages the cochlea, the morphology of the sensory epithelium was protected by infusing a gel within the perilymph-containing scalae. This was achieved by infusing a solution of artificial perilymph (AP) containing 35% pluronic gel at 4°C via a glass micropipette inserted in the round window niche, with the infused solution flowing out of the cochlea through the opened oval window. The cochlea was then carefully oriented to obtain planar slices in the modiolar plane that radially transected the three scalae. The whole cochlea slicing was done in a slicing solution at room temperature (which solidifies the pluronic gel), and several slices of 250-300 pm thickness were obtained. Cochlear slices were then transferred to the chilled oxygenated slicing solution to dissociate the pluronic gel from the cochlear tissue and laid to recover for 15 min. The slice preparations were then placed in 6-well culture plates containing 2000 pl / well of culture medium (BD Falcon, Franklin Lakes, NJ). They were cultured in Dulbecco's modified Eagle's medium (DMEM) which contained either 6g / l D-glucose, 4 mM L-glutamine, 10 mg / ml N-2 supplement, and 30 U / l penicillin alone (control condition) or also with 300 pM DEX, with Ara-C at a concentration of 100 pM, or with PAI-1 inhibitor at a concentration of 5 pg / ml (experimental conditions).

[0154] Quantification of cochlear fibrosis: After 5 days of culture, the cochlear slice samples were incubated with calcein AM (1 / 500; Life Sciences) for 45 minutes to label living cells, including proliferating fibrocytes and fibroblasts (LIVE / DEAD® Viability / Cytotoxicity Kit for cells, Life Sciences). Pictures of labeled slices were taken with an epifluorescence microscope to evaluate the surface of the cochlear scalae filled with fibrotic tissue.

[0155] Organ of Corti explants: Organ of Corti explants were obtained after harvesting neonatal (P0-P2) Wistar rat temporal bones as described above. The otic capsule was dissected, and the stria vascularis, Reissner's membrane, and modiolus were gently removed from the organ of Corti. The organ of Corti explants were carefully uncoiled and kept in a 6-well culture plate containing 2000 pl / well of the same culture medium (see above) either alone or with the addition of 300 pM of DEX or 100 pM of Ara-C, or 5 pg / ml of PAI- 1 inhibitor.

[0156] Quantification of hair cell survival: Hair cell survival was assessed in 5-day cultured organ of Corti explants. The specimens were fixed with 4% paraformaldehyde in PBS and processed for immunochemistry. After several PBS rinses, the explants were pre-incubated in 0.1% Triton X100 diluted in PBS and in 30% normal goat serum in PBS, then they were incubated with a rabbit polyclonal antibody raised against myosin 7a (Proteus Biosciences, Ramona, CA, 1 / 250) to identify inner (IHC) and outer hair cells (OHC), and with a mousemonoclonal antibody directed against neurofilament-200 kD (Sigma, Saint-Louis, MO, 1 / 500) to label primary auditory neurons. Goat antibody anti -rabbit IgGs conjugated to Alexa Fluor 488 and anti-mouse IgGs conjugated to Alexa Fluor 647 (Life Sciences, 1 / 1000) were used as secondary antibodies. After further rinses in PBS, the explants were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and observed using a Zeiss LSM510 confocal microscope (Carl Zeiss MicroImaging GmbH, Gottingen, Germany). Cell counts were assessed over a 2 mm length of the organ of Corti explant from the basal turn in the region corresponding to 6 to 8 mm from the apex. Hair cells were considered to be absent if myosin 7a- labelled cell bodies were missing.

[0157] Tiplaxtinin does not interfere with intracellular pathways that are also involved in cell survival such as TNF pathway, for example. Thus, by targeting the epithelial -mesenchymal transition (EMT), we hoped to reduce side effects on cell survival or physiology.Example 2: Assessment of Tiplaxtinin systemic and local exposure in vivo.

[0158] All experimental procedures were conducted with 4-week-old female rats (weight ~ 150-200g), with a minimization of animal’s number used and of their suffering. Adult female Wistar rats (rattus norvegicus) were purchased from Janvier Laboratories (Le Genest-Saint- Isle, France) and were housed in facilities accredited by the French Ministry of Agriculture and Food (D-34-172-36, 20 May 2021) with food and water ad libitum. Animal care and handling followed the animal welfare guidelines of the ‘Institut National de la Sante et de la Recherche Medicale’ (INSERM), under the approval of the local Ethical Committee. (#18016- 2018121111279084 v5).

[0159] Quantification of Tiplaxtinin in biologic fluids after systemic and local (perilymph) in vivo in a rat following implantation of an electrode array. Adult female Wistar rats (weight ~ 150 g) have been used in these experiments. Animals (n=5 or 6 for each group) were exposed either to single dose IP of Tiplaxtinin (Img / kg, 3mg / kg, or 10 mg / kg), or to Pluronic gel (Poloxamer 405) with a Tiplaxtinin concentration of 25, 50 or 100 pg / ml. The gel was given through a transtympanic injection under general anesthesia. Plasma and perilymph samples were collected 1 hour (only plasma), 6 hours and 24 hours after the injection. Sample were then analyzed in triplicate using HPLC followed by mass spectroscopy. In vivo preliminary studies had shown that Tiplaxtinin can reach the perilymphatic fluid and achieve a therapeuticconcentration through the injection at the round window, whereas Tiplaxtinin is unable to cross the blood-perilymph-brain barrier in its current form.

[0160] FIG. 4A HPLC data shows the time-dependent, rapid clearance of Tiplaxtinin at the various doses (1, 3, 10, 25, 50 and 100 pg / mL) in plasma following intraperitoneal (IP) injection over 24 hours; it was undetectable by 24 hours regardless of the dosage, and no Tiplaxtinin was found in the perilymph. FIG 4B shows the time -dependent reduction of Tiplaxtinin locally, in the perilymph at the same doses over 24 hours, demonstrating a maintenance of the concentration in the perilymph at 25, 50 and 100 pg / mL, setting the therapeutic concentration of Tiplaxtinin between 100 and 300 ng / mL of perilymph.Example 3: Assessment of the safety of Tiplaxtinin gel viatranstympanic injection on auditory function.

[0161] We evaluated Tiplaxtinin ’s safety on auditory function following transtympanic injection (100 ug / mL) of Tiplaxtinin gel in the ears of two rats (four ears). Auditory brainstem responses (ABRs) were assessed under anesthesia in an anechoic and shielded soundproof room one month after surgery. The animal’s core temperature was regulated at 38 ± 1°C by a thermal regulated heating blanket. ABRs were recorded via an electrode positioned at the vertex and a reference placed in the animal’s neck musculature on the side of the tested ear, and the ground electrode on the opposite side. ABR acquisitions and stimulations were controlled by a digital interface created in Lab VIEW (National Instruments, Austin, TX) with a capture card containing two input and two output channels (PXI-4461 , National Instruments) .

[0162] Sounds were delivered by a high frequency speaker (JBL 2426H, Northridge, CA) placed 10 cm from the ear tested. They consisted of pure tone frequencies (4, 8, 12, 16, 24 and 32 kHz ) modulated by a trapezoidal envelope (0.5 ms rise and fall time, 9 ms plateau, 11 presentations per second, alternating phase). For each frequency, the acoustic stimulation was delivered from 0 to 100 dB SPL in 5 dB steps, with contralateral masking. The signals were averaged 500 times. FIG. 5 shows the comparison before (control) and one month after Tiplaxtinin injection on auditory function. No threshold shift was observed one month after treatment, indicating that Tiplaxtinin does not cause hearing loss, consisting with in vitro data.Example 4: Assessment of Tiplaxtinin administration on ground impedance in vivo.

[0163] We evaluated the effect of Tiplaxtinin in vivo on ground impedance. Animals were housed in special cages with ad-libitum access to water and standard food pellets placed on the cage, and were allowed to move freely in their cages during electrical stimulation.

[0164] For electrophysiological recordings and electrical stimulation, the 11 -pin connector was plugged into a cochlear implant CI 24RE emulator (Cochlear Limited, Australia). Immediately after surgery, electrodes' impedances and electrical nerve compound action potential (eCAP) were measured with Custom sound software (Cochlear Custom Sound V5.2 Programming software, Cochlear Ltd, Australia ) to verify the proper functioning of the cochlear implant. Impedances measurements were conducted in monopolar (MP) 1 + 2 mode (which measures the electrical impedance between one intracochlear electrode and both monopolar extracochlear electrode), and eCAPs (electrical Compound Action Potentials) were measured with Neural Response Telemetry (NRT) using the auto-NRT function or custom stimulation and recording parameters if auto-NRT was not capable of determining eCAPs thresholds. eCAPs are the electrophysiological responses elicited by the synchronous activation of the primary auditory neurons in response to an electrical stimulus. In this experimental setup, electrodes with impedances at or below 35 kOhm were considered as functional. Conversely, electrodes with impedances higher than 35 kOhms were unable to be stimulated as they were considered to exceed safe limits defined by the Shannon equation. Data of electrode arrays with more than two functional electrodes were considered for analyses. The CI processor was programmed with Custom Sound suite 5.2 software. The C-levels (comfort level) were set-up to the value of NRT threshold for each electrode and T-levels were set-up 10 current units below C-levels. Cochlear electrical stimulation was delivered via the emulator connected to a sound processor (Cochlear Nucleus CP800 sound processor, Cochlear Limited, Australia). Animals were stimulated 4 hours a day with a 70 dB SPL white noise with a modulated amplitude of 100% at a rate of 4 Hz, 5 days a week. The electrical stimulation started 4 days after implantation. Impedances and NRT measurements were carried out weekly to verify proper electrode functioning and to adjust the stimulation C- and T-levels to NRT thresholds changes overtime as described before.

[0165] FIG. 6A (control) shows MP 1+2 impedances in rats implanted and electrically stimulated as a further validation of the electrical stimulation protocol. FIG. 6B shows the impedance results following both electrical stimulation and administration of Tiplaxtinin. With validation of the electrical stimulation protocol completed, we assessed fibrosis development.Example 5: Experimental design and the assessment of fibrosis development

[0166] FIG. 7A-7B depict the experimental protocol and various experimental treatments used: Insertion Trauma (IT) where the electrode array was inserted and removed from the cochlea, Foreign Body (FB) where the electrode array was inserted without further stimulation, and finally Electrical Stimulation (ES) where the electrode array was electrically stimulated by the sound processor (FIG. 7A). In those groups, cochleae were harvested at Days 1, 4, 7 and 28 (DI, D4, D7 and D28, respectively) for immunohistochemistry and RT-qPCR (FIG. 7B). All groups were analyzed at D28 for fibrosis invasion with cochlea clearing and lightsheet microscopy. One month following implantation of an electrode, we administered Tiplaxtinin (25 ng / mL) to the round window in the inner ears of rats, and evaluated fibrosis development.

[0167] Rats were anesthetized with an intraperitoneal injection of Zoletil 100 (30 mg / kg Virbac, Carros, France) and 2% Rompun (2 mg / kg, Bayer, Leverkusen, Germany), and an incision was made at the level of the scalp; the subcutaneous tissues and the pericranium were freed and cauterized to have a direct contact between the skull bone and the pad. A 10-pin connector was secured on the rat skull with dental cement and a 1.5 mm length self-tapping screws. Two extra-cochlear electrodes were placed in a subcutaneous pocket located behind the scalp incision. The tympanic bulla was exposed and opened using a retroauricular approach. After opening of the bulla, the round window membrane was opened using the tip of a 30 gauge needle. The electrode arrays were inserted into the cochlea via the round window. Tiplaxtinin gel (100 pl) was injected with a p200 pipette to fill the bulla. The round window niche was sealed with a piece of muscle and the skin was sutured. A glucose solution (1 mL) was administered to assist animal recovery, and 0.3 mL of Teramycine®, (O. lmL / Kg) were injected subcutaneously to prevent infections. Examples 6 and 7 below provide the materials and methods for preparing the Tiplaxtinin gel with sterile water (Ex. 6) or 0.9% sterile saline solution (Ex. 7). In earlier work (unpublished) we had quantified fibrotic tissue in cochleae after various types of tissue damage, i.e., after insertion (insertion trauma), with a foreign body (e.g., a CI), and electrical stimulation in an effort to determine what type of injury actually led to fibrosis development. We observed that only actual implantation, not insertion trauma itself, contributed to cochlear fibrosis, which increased even more with the addition of electrical stimulation. Insertion trauma alone did not induce fibrosis. Not only did we find that electrical stimulation increases the amount of fibrosis that developed after implantation, but it also induced an earlier development of fibrosis compared to the trauma of implantation and maintenance of a foreign body in the inner ear tissue. The earlier work also demonstrated thatimplant trauma alone did not induce fibrosis as having the implant remain in the tissue followed by the application of electrical stimulation.

[0168] Further, in-vivo studies in rats revealed PAI-1 offers improved protection inner (IHC) and outer hair cells (OHC) and a larger effect size to suppress fibrotic tissue in comparison with other reference compounds such as DEX (dexamethasone) or Aracytine (Ara). See FIG. 3B-3D. Q-PCR analyses show that Tiplaxtinin significantly reduced the expression of several targets that had been previously defined in vivo-. Serpin-1 (PAI-1), Smad 3 & 7, PDGFb; it also decreased the expression of fibrotic markers such as Acta2, and MMP9. See FIG. 3E.Methods for further in vivo experiments.

[0169] In the electrical stimulation groups (with or without additional treatments), a 11 -pin connector was secured on the rat skull by dental cement and 1.5 mm length self-tapping screws in addition to electrode array insertion performed as described earlier.

[0170] In the group receiving Clodronate for monocyte / macrophages depletion, animal were injected with liposomal Clodronate (Clodronate liposomes 2 ml / Kg; SKU: C-005, Liposoma research ref et concentration) using tail intravenous injection performed the day of the surgery and 4 days after the surgery.

[0171] In the group receiving Tiplaxtinin treatment, a gel formulation (Tiplaxtinin - PAI 039 - AXON MEDCHEM) formulated in thermosensitive Pluronic Fl 72 gel, (25pg / ml) was applied once in the tympanic bulla directly after electrode array insertion. See also Examples 6 and 7 below.

[0172] To assess the appropriate positioning of the electrode array into the basal turn of the scala tympani using this round window approach, microCT scan imaging (Skyscan 1076® Brucker microCT, Kartuizersweg, Belgium) was performed in 2 additional animals after euthanasia and collection of implanted temporal bones. The imaging used was X-ray source 20-100kV spot size <5 pm 10W, X-ray detector 4000x2672 pixels 12bit. A multiplanar reconstruction was applied to the native images using the manufacturer software Nrecon® (Brucker microCT), producing a 18 pm isotropic 3D image. FIG. 3A shows and example light sheet microscopy 3D image of an implanted cochlea. The newly formed fibrosis tissue is clearly visible in the area the electrode array was implanted. These 3D images were used to quantify fibrosis in an earlier study (unpublished).Histological evaluation of fibrosis and macrophages on cochlear cryosections

[0173] Cochleae of implanted rats have been harvested at DI, D4, D7 or D28 after surgery under deep anesthesia. After fixation in a solution of phosphate buffered saline (PBS, 10 mM phosphate buffer, pH 7.4, NaCl 0.9%) containing 4% paraformaldehyde, the cochleae were decalcified in 10% ethylene -diamine -tetraacetic acid solution (EDTA, Sigma, Saint-Louis, MO, USA) for 7 days. They were cryoprotected in PBS with 20% sucrose (8 hours, RT), transferred overnight at RT to a 1 : 1 mixture of PBS / 20% sucrose and OCT Compound (Tissue- Tek, Tokyo, Japan). Finally the samples were quickly frozen at -80°C in a model block filled with OCT Compound. The cochleae were then cut parallel to the mid modiolar axis into 12-14 pm thick sections using a cryostat (CM3050S, Leica, Vienna, Austria).

[0174] After several PBS rinses, the sections were pre-incubated in 0. 1% Triton X100 diluted in PBS and in 10% normal goat serum in PBS, then they were incubated with primary antibodies to label either hair cells, neurons, or Vimentin, E-cadherin, CCL3, CCL12, SERPINE1, Smad3, TGFpi, aSMA, CD68, CD163 or IBA1. IgGs conjugated to Alexa Fluor (Life Sciences, 1 / 1000) were used as secondary antibodies.Whole cochlea clearing and image analyses

[0175] Cochleae were harvested 1 month after surgery as described above. After decalcification in 10% ethylene-diamine-tetraacetic acid solution (EDTA, Sigma, Saint-Louis, MO, USA), bone surrounding the cochlea was removed and samples were incubated with 0,3% triton XI 00 and normal goat serum diluted in phosphate buffered saline (PBS IX) for 3 hours. Then, immunolabeling was performed on the whole cochlea using a combination of 3 primary antibodies for 2 days at room temperature. Myosin 7A and Na,K-ATPase antibodies have been used to label hair cells and auditory neuron fibers respectively, and Vimentin antibodies to label extracellular matrix of fibrotic tissue.

[0176] At the end of incubation, samples were washed three times in PBS-T under agitation and were then incubated with secondary antibodies overnight and then washed three times with PBS-T. The cochleae were dehydrated in serial dilutions of ethanol (50%, 70% and 100% ) for 6 hours each under agitation at 4°C. Finally, ethanol was removed and cochleae were immersed in Ethyl cinnamate (Sigma) 6h at RT. Sample were imaged in Ethyl cinnamate with Ultramicroscope II (LaVision Biotec, Bielefeld, Germany), using an Olympus MVPLAPO 2x lens (NA: 0,5) attached to a stereomicroscope (MVX10 with Zoom body 0,63X to 6,3X) and the cochleae were imaged with a 2.5X magnification. Images (.ome.tiff) generated with Ultramicroscope II were converted in Imaris file (.ims) using Imaris File converter (File Converter 9.2) and visualized and treated in 3D view with Imaris suite 9.9 software (Imaris software, Oxford). Fibrotic tissue labeled with Vimentin was detected and a volume (pm3) was generated by intensity thresholding using surface creation tool (Imaris software). The total volume of the cochlea was delimited manually by detection of the otic capsule. A comparison of fibrotic tissue growth into cochleae was performed between conditions (insertion trauma,foreign body, electrical stimulation, electrical stimulation + clodronate, and electrical stimulation + Tiplaxtinin).

[0177] FIGs. 8A - 8E provide an example of the immunohistochemical panels resulting from this type of analysis, providing a qualitative evaluation of cochlear fibrosis using immunochemistry on cryosections of cochleae harvested about one month after implantation. FIGs. 8A-8E show the development of fibrotic tissue in the area of the electrode array and provide further information on the effect of the treatments on macrophage infiltration. Twentyeight days after cochlear implantation, a fibrotic sheath can be seen surrounding the electrode array (FIG. 8A-8B). Cells expressing myofibroblastic aS MA labeling were found in this fibrotic sheath (FIG. 8A), whereas no epithelial cell marker such as CadE (FIG. 8B) was observed. Moreover, additional fibrotic tissue was observed in the scala tympani (ST) (white arrow, FIG. 8D-8E). This loose tissue included cells expressing aSMA (FIG. 8D) and vimentin (FIG. 8E). A quantification was performed on whole cleared cochleae using lightsheet microscopy and vimentin as a marker of fibrotic tissue (FIG. 8C). The fibrotic proliferation into the cochlea was evaluated 28 days after surgery in electrical stimulation (ES, n=6), foreign body (FB, n=4), insertion trauma (IT, n=5), electrical stimulation + Clodronate treatment (ES + Clodronate, n=5), and electrical stimulation + Tiplaxtinin treatment (ES + Tiplaxtinin, n=5). IT condition promoted a weak fibrotic proliferation compared to FB treatment. If a foreign body was sufficient to induce cochlear fibrosis, the addition of an electrical stimulation (ES treatment) increased the fibrosis invasion dramatically. This effect was counterbalanced either by monocyte / macrophage cell ablation using Clodronate treatment or macrophage function modulation by Tiplaxtinin treatment. aSMA : a-smooth actin, Myo7A : myosin 7A (labelling of hair cells), NF-200 : neurofilament 200 (labelling of auditory neurons), E-Cad : E-Cadherin, oc: organ of Corti, ST ; Scala Tympani. FIG. 8F provides a quantitative evaluation of fibrosis development using whole cleared cochleae, showing that rats administered Tiplaxtinin locally demonstrated after one month a significant reduction in the development or maintenance fibrotic tissue compared to those that received electrical stimulation only. Almost no fibrotic tissue growth took place after IT treatment (0.78% ± 0.65 (n=4)), while a significant tissue outgrowth was observed in FB and ES treatments (4.06% ± 1.51 (n=6) and 7.64 % ± 3.54 (n=5) compared to the IT treatment (ANOVA p<0.0001; FB vs IT, p=0.0162, and ES vs. IT, p <0.0001).Example 6: Preparation of a Tiplaxtinin gel using sterile, deionized water

[0178] A Tiplaxtinin solution was prepared from 25 mg / pL Tiplaxtinin (PAI 039, CAS 393105-53-8) (Axon Medchem B.V.). Stock was stored at -20°C in DMSO. Pluronic gel (20%) was prepared by mixing 4 g of Pluronic powder with 20 mL of sterile, deionized water using a magnet in the solution; it was shaken for about 3 hours at 4°C until a homogenous and transparent solution was obtained. At 4°C the solution is a liquid; at room temperature, it is a gel.

[0179] The pharmaceutical composition with Tiplaxtinin was prepared as follows, with a dilution of 1 / 1000 in Pluronic gel made in two steps. First, a 1: 10 dilution was prepared in Pluronic gel by combining 5pL of Tiplaxtinin with 45 pL Pluronic gel (Pluronic F-127, Sigma) in a sterile Eppendorf pipette; the mixture was vortexed for 30 seconds and then maintained on ice. Next, a 1 : 100 dilution was prepared from the first step, again in Pluronic gel by adding 10 pL Tiplaxtinin to 990 pL of Pluronic gel in a second sterile Eppendorf pipette.Example 7: Preparation of a Tiplaxtinin gel using saline

[0180] A Tiplaxtinin solution was prepared from 25 mg / pL Tiplaxtinin (PAI 039, CAS [393105-53-8]) (Axon Medchem B.V.). Stock was stored at -20°C in DMSO. Pluronic gel (20%) was prepared by mixing 4 g of Pluronic powder with 20 mL of sterile saline (0.9% sodium chloride) using a magnet in the solution; it was shaken for about 3 hours at 4°C until a homogenous and transparent solution was obtained. At 4°C the solution is a liquid; at room temperature, it is a gel.

[0181] The pharmaceutical composition with Tiplaxtinin was prepared as follows, with a dilution of 1 / 1000 in Pluronic gel made in two steps. First, a 1: 10 dilution was prepared in Pluronic gel by combining 5pL of Tiplaxtinin with 45 pL Pluronic gel (Pluronic F-127, Sigma) in a sterile Eppendorf pipette; the mixture was vortexed for 30 seconds and then maintained on ice. Next, a 1 : 100 dilution was prepared from the first step, again in Pluronic gel by adding 10 pL Tiplaxtinin to 990 pL of Pluronic gel in a second sterile Eppendorf pipette.Example 8: Evaluation of fibrotic tissue and cells following clodronate injection and ES.

[0182] The effect of Clodronate on macrophage recruitment and population in fibrotic tissue and on the population of CD68+ and IBA1+ cells in with and without ES. Rats were implanted with cochlear electrodes as previously described in Example 5. Clodronate, a commonly usedanti-osteoporotic drug and is known to kill monocytes and macrophages, has been found to have anti-inflammatory properties. Clodronate was administered systemically as indicated in Example 5; most macrophage activity is observed between 4 to 7 days, and after 7 days following implantation.

[0183] FIGs. 9A-9D provide immunohistochemical images of fibrotic tissue after clodronate and / or ES, with identification of and localization of particular types of cells. CD68+ and IBA1+ cells are labeled as such. Hair cells and neurons are labeled PV; nuclei are labeled DAPL From DO to D4 (FIG. 9B) after Clodronate treatment, macrophage content in fibrotic tissue was depleted, no CD68+ cells were found, and a dramatic reduction in IBA1+ cells was observed. After ES (FIG. 9C), an increase of IBA1+ and CD68+ cells was observed in the spiral ligament (si), the stria vascularis (sv) and the spiral ganglion (sg). When a clodronate treatment was applied with ES (FIG. 9D), some IBA1+ cells were observed in the sv, the si and the sg with a very limited number of CD68+ cells. Fibrotic tissue was no longer observed, and no additional macrophages had been recruited to the region in which electrical stimulation had taken place. Thus, clodronate appears to neutralize the damaging effect of ES on fibrosis development.

[0184] To determine if macrophage invasion was a cause or a consequence of cochlear fibrosis, we compared the cochleae of electrically stimulated animals with or without macrophage depletion using liposomal clodronate. In naive animals (non implanted) treated by clodronate, TRM were almost completely depleted as shown in FIG. 9B. Few IBA1+ cells were observed in the stria vascularis and the spiral ganglion. After electrical stimulation and clodronate treatment, we could observe a decrease in CD68+ and CD 163+ cells when compared with ES condition (FIG. 9C & 9D) at D7. Those observations are further supported by scRNA seq data (FIG. 14C) that show the proportion of macrophages is reduced in ES + clodronate compared to ES condition alone at D7. The comparison of total fibrosis outgrowth in the whole cochlea, the volume of fibrosis was significantly reduced in the ES+Clodronate group versus ES group (FIG. 8F, 2.08+ 1.30 vs 7.64 % ±3.54 respectively, n=5 each, p=0.0002).

[0185] The role of macrophages was further investigated by Tiplaxtinin application in addition to ES. Despite the fact that Tiplaxtinin did not reduce the proportion of macrophages compared to ES treatment (FIG. 14C), histological evaluation on cleared cochleae showed a drastic reduction of fibrotic tissues, even more than in the Clodronate conditions. Altogether those data suggest that macrophage invasion is required for fibrotic development after cochlearimplantation, but Tiplaxtinin experiments also show that fibrosis can be blocked even in the presence of macrophages.Example 9: RT-qPCR, modeling and clustering analyses

[0186] To investigate the molecular pathways activated in the different treatments (IT, FB and ES), RNA was extracted from whole cochleae to perform RT-qpCR analyses, conducted at D 1 , D4, D7 and D28. We used an 84-gene panel and opted for a machine learning algorithm to classify genes according to their profiles of evolution according to timing and other experimental conditions.

[0187] Animals were euthanized by decapitation under deep anesthesia with Euthasol®(140mg / kg, Dechra veterinary products ). Rats cochleae have been harvested 1 day, 4 days, 7 days and 1 month (28 days) after surgery. They were transferred into the vial containing RNA later solution (Ambion, Cambridge, UK) and stored at -80°C until further processing. For RNA extraction, the cochleae were pooled (3 cochleae by sample), ground and homogenized using magnetic beads (Magnalyser, Roche, Switzerland). The lysate was prepared for RNA extraction using the TRIzol-based kit provided by Ambion according to manufacturer’s instructions. The yield and the quality of extracted mRNA was controlled using Nanodrop spectrophotometer (Thermofisher, Waltham, MA, USA). Samples were then processed for cDNA synthesis using RT2 first strand kit (Qiagen, Hilden, Germany). The reactions were performed with 0.5 pg RNA according to the manufacturer's instructions. The conditions of reactions were: 42°C for 5 min, 42°C for 15 min, and the reaction was stopped at 95°C for 5 min. Then, the samples were diluted 1:5 for utilization in RT-qPCR and stored at - 80°C until qPCR reactions.RT-qPCR analysis

[0188] Pre-coated plates RT2 profiler (PARNZ-120, Qiagen) were used for qPCR analyses. Ten pl of cDNA was used for each reaction in addition to the qPCR mix. qPCR reactions were performed in triplicate for each condition and each timepoint (control, DI, D4, D7, D28) on Roche Lightcycler 480. qPCR was performed using RT2 qPCR SYBR Green Master Mix (Qiagen). All qPCR performed using SYBR Green were conducted at 50°C for 2 min, 95 °C for 10 min, and then 40 cycles of 95°C for 15 s and 60°C for 1 min. The specificity of the reactions was verified by melt curve analysis. Normalization of data was performed by selecting 2 genes (Actb and Ldha) from a panel of 6 housekeeping genes. Data are expressedas Fold-Change. Fold-Change (2A(- DD CT)) is the normalized gene expression (2A(- DCT)) in the Test Sample divided by the normalized gene expression (2A(- DCT)) in the Control Sample. Fold-change values less than one indicate a down-regulation, and the fold-change values more than one indicate an upregulation.

[0189] FIGs. 10A-10J summarize RT-qPCR data across control (FIG. 10A), IT (FIG. 10B), FB (FIG. 10C) and ES treatments (FIG. 10D), and their subsequent modeling and clustering analyses (FIG. 10E-10J). RT-qPCR analyses results for the 84-gene panel in IT (black dots), FB (triangles) and ES treatment conditions (squares). FB and ES groups’ D 1 data are combined since the ES began only four days after implantation (FIG. 10C). Fold-changes Averaged fold-changes (triplicate) for each gene are plotted as single points with error bars (SD). The gray zone (centered on the value of 1-fold change) references the 0.5-2 fold change zone that was not considered as clinically significant compared to control. Values less than 0.5 (inner zone of circles) or greater than 2 (outer zone of circles) indicate under- or over-expression of the gene, respectively. Individual results are listed in Table 1.

[0190] Evolution of fold-change Evolutions of Fold-change along with time and conditions were modeled with a statistical adjustment on each gene based on a mathematical model of linear spline with two fixed knots (knotl: D4; knot 2: D7) (FIG. 10E). A K-means algorithm was used to classify genes into an optimized number of clusters based on R-squared values for each gene and for each condition (FIG. 10H). An optimal number of four clusters was determined. Evolution of gene expression differs in Clusters 1, 2 & 3 from Cluster 4. In the first three Clusters (FIGs. 10F, 10G and 101), we observed an increase of average gene expression with the ES by comparison with the IT and FB conditions. Gene expression in Cluster 4 (FIG. 10J) does not seem to be modified by the experimental conditions and is only slightly elevated at D28. Differences between the FB and the IT conditions were found at DI for Cluster 1; DI, D7 and D28 for Cluster 2 and at all time points for Cluster 3. Gene that can be found in Cluster 1 are: Cavl, Ccl3, Ctgf, Den, Il lb, Itgal, Itga2, Itgb8, Lox, Plat, Pig, Tgfb2 and Tgif; Cluster 2 :Ccll2 and Timpl; Cluster 3: Colla2, Col3al and Mmp3. Other genes of the panel are included in Cluster 4, except those removed from the analyses as explained before.

[0191] A reduction of matrix dimension was performed by modeling gene expression changes as a function of time and condition (IT, FB and ES) by a spline function (FIG. 10E). The distribution of residues (R- squared) after modeling was used to assess the model adjustment. 84% of genes evaluated in IT condition, 85.4% of genes in FB condition and 91.5% of genes in ES condition have a R-squared superior to 0.7, meaning that this modeling was accurateenough to depict gene variations. Six out of 84 genes (Ifhg, 1110, 1113, 114, Mmpl, and Thbsl) were not considered for further analyses since they showed no over or under-expression at any time point or any condition. A clustering was performed using the K-means algorithm on the parameters of the fitted spline and found an optimal number of 4 clusters (FIG. 10H). The clusters are described in FIGs. 10F, 10G, 101 and 10J. Note that the cluster line shows the average level of expression of all genes included in the cluster. However, Fold-change values may vary among genes in the same cluster but the trend of variation over time and amongst experimental conditions remains the same. The raw value of averaged gene expression in a cluster must not be considered by itself as it depends on the number of genes included in the cluster and of their respective level of expression. Gene expression levels at D 1 are the same for FB and ES conditions since electrical stimulation starts at D4 only.Example 10: single cell RNA (scRNA) sequence analysis

[0192] scRNA sequence analysis was performed to identify cell populations in cochlear fibrotic tissues as well as to identify key markers within certain cell populations to better understand fibrotic tissue development following implantation, and how Tiplaxtinin may affect such development.

[0193] Cochleae were dissected and gently ground, then incubated in solution the mix digestion containing RPMI 1640, Glutamax (Gibco, 61870-010), 10% FBS (Gibco, 10500- 064, heat-inactivated), Collagenase IV (1 mg / mL) (Gibco, 17104-019) and DNase I (0.03 mg / mL) (Sigma, DN-100MG). Enzymatic digestion of the cochlea was performed by incubating the tube in a thermomixer at 900-950 rpm for 1 hour at 37°C. The tissue was triturated and filtered using a 40-pm strainer and 1 ml of FACS buffer PBS containing 4% FBS (Gibco, 10500-064, heat-inactivated) was added. The dissociated cells were centrifuged at 500 x g for 5 min at 4°C and the supernatant was removed carefully. The pellet was resuspended in 200 pL of FACS buffer and the viability of the cells was tested using viability dye eFluor 780 (eBiosciences 65-0865-14).

[0194] Library construction was performed using Chromium Next GEM Single Cell 3' v2 Reagent Kits (10X genomics) following the instruction manuals. Briefly, the dissociated cells were loaded into a Chromium Controller (10X Genomics) and the encapsulated cells were lysed individually. Reverse transcription within each oil droplet was performed and the cDNAs were amplified. Next-generation sequencing was performed using an Illumina HiSeq 4000system (Illumina, San Diego, CA) and the sequence reads were aligned to rat reference genome (STAR) using the Cell Ranger pipeline (10X Genomics).

[0195] scRNAseq analysis was performed in Rstudio, using Seurat pipeline from Satija lab. Data pre-processing and filtering was carried out based on UMI number higher than 500 and smaller than 40,000, while maintaining a mitochondrial gene ratio smaller than 10%. Genes with 0 counts in all cells were also excluded from the analysis. Doublet exclusion was then performed using doubletfinder tool. Matrices were then integrated using the canonical correlation analysis (CCA) method implemented in Seurat. Dimensional reduction was performed first by executing Principal Component Analysis (PCA) then Uniform Manifold Approximation and Projection (UMAP), using the first 20 principal components (PCs). Clustering was then performed using the Seurat FindNeighbors function with a resolution of 1 allowing the emergence of 29 clusters which were then joined through an extensive marker research. Cell type identification was performed on the integrated object by a combined strategy using the FindMarkers function in Seurat, literature based markers, as well as geneset scoring of list of markers in other murine cochlear scRNAseq datasets, allowing for the identification of 10 major cell types (doi.org / 10.1073 / pnas.2221744120). We excluded the erythrocytes from our datasets for the analysis, since their presence can alter the proportions of other populations and hide the global data structure, both of which can easily fluctuate with harvesting conditions. Population characterization was performed using FindAllMarkers function run in the control dataset since classical markers can be hidden by electrostimulation induced genes (FIG. 14B).

[0196] The Geneset scoring method was then used to explore the scope of gene clusters from bulk data by the AddModule Score Seurat function allowing score building from a list of genes for every cell in the datasets. Cluster scores were then determined, and explored taking into account the average score in a cell population throughout our conditions as well as the percentage of cells displaying the score. Separation of macrophages into subsets was performed on top of cell annotation by cd68+ status, a well known myeloid marker positive expression with a Z-score >1. Differential expression testing was then performed on subsets of cells among the controls and different treatment groups using Model-based Analysis of Single Cell Transcriptomics (MAST) package (McDavid A, Finak G, Yajima M (2024). MAST: Model-based Analysis of Single Cell Transcriptomics. R package version 1.30.0, github.com / RGLab / MAST / ). DE analysis was first performed on ES versus control group macrophages, followed by the ES+Tiplaxtinin treatment group versus the ES treatment groupmacrophages. Genes were considered differentially expressed (DEGs) if they displayed an FDR of 10% and a log2Fold change of 0.5. Volcano plots were generated with EnhancedVolcano package. Gene set Enrichment analysis (GSEA) was then performed on the DEGs using the gseGO function from 4.2.2 version of clusterProfiler package.

[0197] FIGs. 14A-14F provide results of the scRNA seq analyses. Single cell RNA sequencing data were generated from the control group as well as from the ES, ES + Tiplaxtinin and ES + Clodronate treatment groups according to the methods described above. Because our previous results pointed out immune cells as possible effectors of fibrotic reactions, we explored our cell populations at a transcriptomic level. FIG. 14A demonstrates the localization of various cell populations involved in immune responses (macrophages, monoctyes, neutrophils, B cells, T cells, NK cells, Mast cells, etc.), when fibrosis is visualized around an implanted electrode array.

[0198] Cell populations were evaluated for cell composition, as shown in FIG. 14B, a dot plot of the average expression of key markers in a variety of cell populations shown in FIG. 14A. The data show an enrichment in immune cell populations containing macrophages, monocytes, neutrophils and lymphocytes, displaying unique markers.

[0199] Although the control and treatment samples exhibited similar cell compositions, cell populations vary among controls and treatment groups. For example, after ES, there is a strong decrease of B cells (73 to 34 %), and an increase of neutrophils, macrophages and monocytes, as shown in FIG. 14C, indicating shows the distribution (frequency) of various cell populations in the controls, and in ES, ES + Clodronate, and ES + Tiplaxtinin treatment groups. With the administration of Clodronate with ES, the macrophage population dropped from about 13.5% to 7%, which was apparently sufficient to block fibrosis development. The NK population also dropped precipitously from about 7.5% to 0.75%. Likewise, the administration of Tiplaxtinin with ES strongly reduced NK cells from about 7.5% to 0.14%. In contrast, Tiplaxtinin induced the production of neutrophils.

[0200] We examined the potential cell source for the transcriptomic profiles that we had observed with gene clusters by performing gene set scoring using the four different clusters on the scRNA seq data sets. Gene clusters 1-3 displayed differences among the treatment groups at day 7 (D7) were highly expressed in myeloid cell populations (e.g., mast cells, neutrophils, monocytes, macrophages, etc.), while cluster 4 was scattered among the cell populations. See FIG. 14D, showing the distribution of cell populations broken down by cluster (1-4), and FIG.14E showing gene expression in controls versus the ES treatment groups, broken down by cluster (1-4). In addition, clusters land 2 displayed a strong score in myeloid populations. In general, higher cluster scores were observed with ES treatment alone, but decreased when Tiplaxtinin or Clodronate were administered. See FIGs. 14E and 14F, showing gene expression in those same cell types in ES versus ES + Clodronate, versus ES + Tiplaxtinin treatment groups, again, broken down by cluster (1-4).Example 11: Statistical analyses and deep learning clustering

[0201] Statistical analyses were performed with R® software. Volumes of fibrotic tissues in cleared cochleae were compared between conditions (IT, FB, ES, ES+Clodronate, ES+Tiplaxtinin) using Welch’s ANOVA followed by post-hoc Tukey’s test. A p-value <0.05 was considered as significant. Comparison of impedances and NRT values along with time was performed using two-way ANOVA (electrode number X time) followed by post-hoc Tukey’s test. A p-value <0.05 was considered as significant.

[0202] Quantitative PCR analyzes Fold Change (FC) values were compared between timepoints (DI, D4, D7 and D28) for different conditions as follows: electrical stimulation group (ES) vs control group (C), insertion trauma group (IT) vs C, foreign body group (FB) vs IT, and ES vs FB. Those comparisons were made to determine the specific effect of each condition against others. Assuming that the animals with electrical stimulation also have an insertion trauma and a foreign body reaction inside the cochlea, the comparison of ES samples was made against the FB condition that also includes the effects of an insertion trauma. Then the comparison ES vs FB depicts the specific effect of the electrical stimulation only. Same applies for FB and IT conditions to determine the specific effect of foreign body reaction against insertion trauma only. The comparison ES vs control group was given to provide the global insight of the changes observed in the “human-like” situation. The p-values were calculated for all the conditions (IT, FB and ES) using ANOVA followed by a post-hoc False Discovery Rate (FDR) correction for multiple analysis. Statistical significance was set at an alpha level of 0.05. For results reporting, we considered only genes with a significant Fold Change (FC) difference and FC values >2 or <0.5. Indeed, usual variations of qPCR results are within this range and values close to 1 (even with a statistical difference) were not considered as biologically relevant.

[0203] In a second step of the analyses, we investigated the gene expression profde along with time in the three different experimental treatment groups (IT, FB and ES) and compared those profdes to determine: (i) if several genes can be grouped according to similarities in their expression profiles; and (ii) if those groups are similar or not in the different experimental conditions (IT, FB and ES). This permitted searching for genes whose changes of expression are more specific to electrical stimulation or to foreign body reaction. A reduction of dimensions of the matrix of results was performed by modeling RT-qPCR gene expression changes along with time. The average FC values (3 replicates), calculated at 4 different time points for each condition (IT, FB and ES) and for each gene, were statistical adjusted, based on a mathematical model of linear spline with two fixed knots (D4 and D7) (Truncated power basis).

[0204] Mathematical model:FC= B0+Blt+B2 (t-4)+ +B3 (t-7)+

[0205] For each gene, B0, B 1 , B2, B3 were calculated and a clustering on these spline parameters was carried out to classify genes in IT, FB and ES conditions. K-means algorithm was used to execute clustering.Example 12: Gene ontology pathway analyses for RT-qPCR Clusters

[0206] The genes identified by the clustering of RT-qPCR data were used for further pathways analyses. Genes showing a similar profile of expression between FB, ES and IT conditions were analyzed separately from genes displaying different expression profiles. Then, 2 analyses were performed using the ShinyGO 0.76 website (bioinformatics.sdstate.edu) using Gene Ontology Biological Process database. FDR (False Discovery Rate) was calculated based on nominal p-value from the hypergeometric test. Fold Enrichment is defined as the percentage of genes analyzed belonging to a pathway, divided by the corresponding percentage in the background. For each gene input, FDR was calculated and cutoff p-values used were 0.05. The top 20 biological process pathways were then reported based on their Fold Enrichment and their statistical significance (displayed as -LoglO FDR). Thus, the highest -LoglO FDR values correspond to the lowest corresponding p-value.

[0207] Clusters were grouped in Non-Differential expression group (NDEC; Cluster 4) and Differential Expression Cluster group (DEC) including Clusters 1, 2 and 3 according to the effects of ES on gene expression. Genes included in the NDEC group (FIG. 11 A), are involvedin multiple biological processes such as vascular and epithelial cell development, proliferation and migration. In the DEC group (FIG. 11B), numerous genes involved in inflammatory and immune processes (neutrophil and granulocyte chemotaxis) were expressed. Pathways not relevant with this model, such as embryogenesis, are shown in a lighter shade of gray.Example 13: CCL3 and CCL12 immunohistochemistry in the implanted cochlea

[0208] Methods of preparing the immunohistochemical images are described above in Example 5. FIGs 12 and 13 show the maximum intensity projection image of a confocal stack of cochlea cryosection in controls (FIG. 12A and FIG. 13A), in insertion trauma group (FIG. 12B-E and FIG. 13B-E) and in the cochlear implantation with electrical stimulation group (FIG. 12F-I and FIG. 13F-I) at different timepoints after surgery: 1 day (FIG. 12B and 13F; FIG. 13B and 13F), 4 days (FIG. 12C and 13G; FIG. 13C and 13G), 7 days (FIG. 12D and 13H; FIG. 13D and 13H), and 28 days (FIG. 12E and 131; FIG. 13 E and 131). CCL3 in FIG. 12, and CCL12 in FIG. 13 are identified as such (CCL3 or CCL12) with arrows or arrowheads; hair cells and neurons are labeled anti-Myosin 7A (Myo 7A), nerve fibers are labeled anti NF 200 (NF 200), and nuclei are labeled Hoescht. The white arrow indicates fibrotic tissue in the scala tympani (ST). Fibrotic tissues are observed in the ST 28 days after implantation. In the cochleae of controls, CCL3 is observed in the supporting cells of the organ of Corti (OC), but there is no CCL12 expression, with a non-specific labeling in the tectorial membrane. After implantation, CCL3 is expressed shortly in the stria vascularis (SV) at DI and also in fibrotic tissues and CCL 12 positive cells are observed at the border of the ST, the bm and the SM. This labeling decreases along with time except for the implanted condition where CCL 12 positive cells are observed in the fibrotic tissues in addition to the bm. Scale bar 100 pm. ST: scala tympani, SV: scala vestibuli, SM: scala media, sg: spiral ganglion, sv: stria vascularis, bm: basilar membrane, oc: organ of Corti.

[0209] Thus, we considered separately genes from Differential Expression Clusters (Clusters 1, 2 & 3, referred to DEC later on), and Non-Differential Expression Clusters (Cluster 4, referred to NDEC later on). Those genes were analyzed and sorted using Gene Ontology Biological Processes bioinformatics. The top 20 biological processes with significant FDR were retrieved for both DEC & NDEC (FIGs. 12A-12B). In the NDEC group (FIG. 12A), main biological pathways identified included tissue proliferation (cell migration, cellular proliferation and differentiation), and vascularization development. In the DEC group (FIG.12B), the most represented biological processes included immune cell chemotaxis (neutrophils and granulocyte), vascularization development (angiogenesis, morphogenesis, blood vessel development), and response to growth factors.

[0210] Altogether, these data suggest that cellular proliferation and neovascularization are processes common to IT, FB, and ES. Conversely, inflammatory and immune reactions are more specific of FB and ES, with an upregulation of gene expression generally higher with ES than FB as shown in DEC. As those analyses were performed on whole cochleae and might be non specific of fibrotic process, assuming that other mechanisms such as hair cell loss for example may occur, we analyzed the tissue expression of selected proteins of interest on cochlear sections to determine if their localization was compatible with a fibrotic process developing into the scalae.

[0211] Among immune cell markers in the DEC group, we searched for CCL3 and CCL12 expression in implanted cochleae. Expression of CCL3 (MCP-1), a protein secreted by macrophages and recruiting immune cells including monocytes / macrophages, was found in the supporting cells of the organ of Corti in normal conditions (FIG. 12A) and early after insertion trauma or implantation (FIG. 12B and 12F). CCL3 positive cells were found in the stria vascularis at DI (FIG. 12F), and in fibrotic tissues at D28 (FIG. 121). Expression of CCL12 (MCP-5), a chemoattractant for eosinophils, lymphocytes and monocytes, was found in the mesothelial cells lining the scala tympani as early as DI after insertion trauma or cochlear implantation (FIG. 13B and 13F). This expression was stronger at D4 (FIG. 13C and 13G), and then decreased along with time except in implanted cochlea where CCL12 positive cells are observed in fibrotic tissues at 1 month (FIG. 131). CCL12 was not expressed in the control cochlea (FIG. 13A). Note that CCL3 and CCL12 localizations were not different in FB and ES groups (data not shown).

[0212] We also investigated the immunolocalization of TGFpi and Smad3 to investigate the TGFp signaling pathway. TGFpi expression is restricted to the supporting cells of the organ of Corti in control, insertion trauma and implanted condition. At D28, TGFpi positive cells are observed in fibrotic tissues. Downstream activation of Smad3 is observed in the supporting cells of the organ of Corti, and in hair cells and in auditory neurons. No Smad3 expression is observed in mesothelial cells of the different scala or in fibrotic tissues. Those results support the idea that fibrotic development into the cochlea is not promoted by canonic TGFp activation, and that activation of immune cells may act by a different pathway. (Data not shown.)

[0213] Example 14: CD68+ and IBA1+ macrophage localization in the implanted cochlea

[0214] Methods of preparing the immunohistochemical images are described above in Example 5. FIGs. 15A-15L show the maximum intensity projection images of a confocal stack of cochleae cryosections in controls (FIG. 15A), in the ES group (FIG. 15B-15D), in the FB group (FIG. 15E-15H), and in the IT group (FIG. 15I-15L), as indicated at different time points after surgery: DI (FIG. 15E and FIG 151); D4 (FIGs. 15B, 15F and 15J); D7 (FIGs. 15C, 15G and 15K); and D28 (FIGs. 15D, 15H and 15L). Note that DI for FB and ES are not different since the electrical stimulation started at D4. CD68+ and IBA1+ cells are labeled as such. Hair cells and neurons are labeled PV; and nuclei are labeled DAPI. Chronic implantation with or without electrical stimulation induces fibrosis development (white arrow) around electrode array (FIGs. 15D and 15H) at D28. In controls (FIG. 15A), IBA1+ cells are present in stria vascularis (sv) (arrowhead) and in spiral ganglion (sg), with an increase at D4 in all treatment groups (FIGs. 15B, 15F and 15J). This overexpression remains elevated at D7 in the sv and the scala tympani (ST) only in implanted animals (FIGs. 15C and 15G) and prolonged at D28 in the sv and the fibrotic tissues in the ES group (FIG. 15D). A very limited number of CD68+ cells was observed in spiral ganglion (sg) in the control condition (FIG. 15A). CD68+ cells are observed in the sv as early as DI in all treatment groups (FIGs. 15E and 151) and in the ST only in the FB / ES conditions (FIG. 15E). CD68+ cells are found in the ST in ES group (FIG. 15B) and FB group (FIG. 15G). At D28, CD68+ cells remain present in the fibrotic tissues and the sg in ES group (FIG. 15D) but not in FB group (FIG. 15H). Immunolabeling in the sv decreases in all conditions by D7 (FIGs. 15C, 15G and 15K). The white arrow indicates fibrotic tissue in ST; arrowheads show IBA1+ or CD68+ labeling. Scale bar 100 pm. ST: scala tympani, SV: scala vestibuli, SM: scala media, sg: spiral ganglion, sv: stria vascularis, bm: basilar membrane, oc: organ of Corti, si: spiral ligament.

[0215] Therefore, differences observed in RT-qPCR analyses elicited by the electrical stimulation seemed to be mainly due to macrophages at a cellular level as shown by scRNA seq. To confirm this hypothesis of macrophage involvement during the fibrotic process,immunolabeling of tissue resident macrophages (TRM, IBA1+, CD68+), and monocyte derived macrophages (MDM, IBA1- CD68+) was performed to assess macrophagic activity in tissue at different timepoints. In control animals, TRM cells are found mainly in the spiral ganglion and stria vascularis (FIG. 15A). TRM cells increase in the stria vascularis at D4 in all conditions (FIG. 15B, 15F and 15J) and then decrease more rapidly in IT group than in FB or ES group (FIG. 15C, 15D, 15G, 15H, 15K, 15L). In addition TRM cells are located in the scala tympani in ES and FB conditions (FIG. 15B and 15G) and remain in fibrotic tissue at D28 only in ES group (FIG. 15D). MDM macrophages are observed as soon as DI in the scala tympani and the stria vascularis in the FB / ES condition (FIG. 15E) and IT condition (FIG. 151). MDM cells expression remains located in the stria vascularis in IT group (FIG. 15 J) and then decreases rapidly, whereas expression is sustained in scala tympani and spiral ganglion in FB (FIG. 15F and 15G) and ES conditions (FIG. 15B and 15C). Those cells remain in fibrotic tissues only in ES conditions at D28 (FIG. 15D).

[0216] Example 15: CD163+ and IBA1+ macrophage localization in the implanted cochlea

[0217] Methods of preparing the immunohistochemical images are described above in Example 5. FIGs. 16A-16L show the maximum intensity projection images of a confocal stack of cochleae cryosections in controls (FIG. 16A), in the ES group (FIG. 16B-16D), in the FB group (FIG. 16E-16H), and in the IT group (FIG. 16I-16L), as indicated at different time points after surgery: DI (FIG. 16E and FIG 161); D4 (FIGs. 16B, 16F and 16J); D7 (FIGs. 16C, 16G and 16K); and D28 (FIGs. 16D, 16H and 16L). Note that DI for FB and ES are not different since the electrical stimulation started at D4. CD163+ and IBA1+ cells are labeled as such. Hair cells and neurons are labeled with PV. Nuclei are labeled DAPI . In control condition (FIG. 16A), few CD 163+ cells are observed in the stria vascularis (sv) and the spiral ganglion (sg). AtD4, CD 163+ cells are observed in scala tympani (ST) only in ES group (FIG. 16B). In a further stage of fibrotic development, fewer CD 163+ cells are located in the ST of FB group (FIG. 16G) than in ES group (FIG. 16C) by comparison with IBA1+ labeling that is found in both conditions. At D28, CD163+ cells are observed only in fibrotic tissues of the ES group (FIG. 16D). The white arrow indicates fibrotic tissue in the ST; and arrowheads refer to IBA1+ or CD163 labeling. Scale bar 100 pm. ST: scala tympani, SV: scala vestibuli,SM: scala media, sg: spiral ganglion, sv: stria vascularis, bm: basilar membrane, oc: organ of Corti, si: spiral ligament.

[0218] To refine their phenotyping and detect macrophages involved in angiogenesis and tissue remodeling, CD163+ immunolabeling was used to identify M2c macrophages. CD163+ cells are transiently observed in the stria vascularis by DI in IT, FB and ES conditions (FIG. 16E and 161). Conversely, a sustained CD163+ expression was noticed in scala tympani and fibrotic tissues, with a stronger CD163+-cell expression in the ES group (FIG. 16B, 16C and 16D) than in the FB group (FIG. 16F, 16G and 16H). Altogether, our data suggest that macrophages recruited during the fibrotic processes are both MDM and TRM. An increase of macrophages was transiently observed in the stria vascularis and the spiral ganglion early after the initial injury and then macrophages migrated into the scala tympani and the fibrotic tissues. Macrophages are numerous in case of fibrotic tissue growth and their numbers seem to be higher in the ES group than in the FB group. The shape of macrophages, which is an indicator of activation state, changed between time points. In control cochleae, CD68+, IBA1+ and CD 163+ have an elongated shape similar to quiescent macrophages. At D7, the shape of those cells found in the scala tympani is rounded, suggesting an activated phenotype. Finally, at D28, most macrophages return back to their original phenotype of elongated cells in fibrotic tissues.

[0219] Example 15: additional scRNA analyses, with a focus on macrophages

[0220] Methodology relating to scRNA seq analyses is included in Example 10 above.

[0221] Because macrophages are known to play a key role in tissue repair and in some pathological contexts, and because we observed in our scRNA seq analysis that macrophages play a critical role in transcriptomic dynamics after ES, we examined the macrophage population in greater depth.

[0222] First, we filtered the macrophage population based on cd68+ expression, a well -known myeloid marker. (FIG. 17A) We next characterized the macrophage population based on the expression of key markers: cd68, Mpegl, Clqb, PtprO, etc. (FIG. 17B) Bulk cluster scores were then calculated on the filtered population; stronger scores were observed upon ES, which were then attenuated with the administration of Clodronate or Tiplaxtinin.

[0223] To determine the ES transcriptomic signature on the macrophage population, differential expression analysis was performed in controls and in the ES treatment group, andthe average expression levels compared. Further, gene ontology (GO) analysis was performed on the gene set to explore functional enrichment within the gene set. (See FIGs. 17D, 17E and 17F) Among the up-regulated genes, several chemokines (Ccl2, Ccl4, Cxcll and Ccl7) were observed to support GO terms such as neutrophil and leukocyte chemotaxis, suggesting a potential role of macrophages into recruiting immune populations upon ES. This observation may be consistent with other observations indicating a strong enrichment of immune populations, especially myeloid and neutrophils. An increase in cytotoxic gene expression, e.g., Camp, Defa5, RT-Da, No4, Ctsg, and Hmgn2, was also observed, suggesting stronger cytotoxicity of macrophages upon ES. (See FIG. 17F)

[0224] Finally, we examined the effect Tiplaxtinin treatment may have on macrophage function to provide support for the role macrophages play in fibrosis development. Several models describe the mechanism(s) by which Clodronate affects myeloid cell population viability, but the mechanisms of action of Tiplaxtinin are not as well understood. To further characterize the macrophage population including its effects following ES and the combination of ES and Tiplaxtinin, gene expression was compared in the ES and ES + Tiplaxtinin treatment groups. The expression of cytotoxic genes was significantly downregulated with the administration of Tiplaxtinin (FIG. 17G), suggesting that Tiplaxtinin treatment may attenuate the cytotoxic effects macrophages have following ES (FIGs. 17H-17I).

[0225] Thus, we see that ES is responsible for an increase of macrophages and neutrophil populations in the fibrotic tissue surrounding the implanted cochlea. These results are consistent with RT-qPCR analyses, as genes in the Clusters 1, 2 and 3 that were differentially expressed after ES are also genes overexpressed in macrophagic populations based on UMAP analyses. Tiplaxtinin administration appears to attenuate the ES-induced rise in cytotoxic gene expression in macrophages within the fibrotic tissue having a cochlear implant.

[0226] As should be appreciated, while particular uses of the technology have been illustrated and discussed above, the disclosed technology can be used with a variety of devices in accordance with many examples of the technology. The above discussion is not meant to suggest that the disclosed technology is only suitable for implementation within systems akin to that illustrated in the figures. In general, additional configurations can be used to practice the processes and systems herein and / or some aspects described can be excluded without departing from the processes and systems disclosed herein.

[0227] This disclosure described some aspects of the present technology with reference to the accompanying drawings, in which only some of the possible aspects were shown. Other aspects can, however, be embodied in many different forms and should not be construed as limited to the aspects set forth herein. Rather, these aspects were provided so that this disclosure was thorough and complete and fully conveyed the scope of the possible aspects to those skilled in the art.

[0228] As should be appreciated, the various aspects (e.g., portions, components, etc.) described with respect to the figures herein are not intended to limit the systems and processes to the particular aspects described. Accordingly, additional configurations can be used to practice the methods and systems herein and / or some aspects described can be excluded without departing from the methods and systems disclosed herein.

[0229] According to certain aspects, systems and non-transitory computer readable storage media are provided. The systems are configured with hardware configured to execute operations analogous to the methods of the present disclosure. The one or more non-transitory computer readable storage media comprise instructions that, when executed by one or more processors, cause the one or more processors to execute operations analogous to the methods of the present disclosure.

[0230] Similarly, where steps of a process are disclosed, those steps are described for purposes of illustrating the present methods and systems and are not intended to limit the disclosure to a particular sequence of steps. For example, the steps can be performed in differing order, two or more steps can be performed concurrently, additional steps can be performed, and disclosed steps can be excluded without departing from the present disclosure. Further, the disclosed processes can be repeated.

[0231] Although specific aspects were described herein, the scope of the technology is not limited to those specific aspects. One skilled in the art will recognize other aspects or improvements that are within the scope of the present technology. Therefore, the specific structure, acts, or media are disclosed only as illustrative aspects. The scope of the technology is defined by the following claims and any equivalents therein.

[0232] It is also to be appreciated that the embodiments presented herein are not mutually exclusive and that the various embodiments may be combined with another in any of a number of different manners.

Claims

CLAIMSWhat is claimed is:

1. A pharmaceutical composition administered in a therapeutically effective amount to a subject following implantation of a cochlear or vestibular implant, comprising: at least a first plasminogen activator inhibitor- 1 (PAI-1) inhibitor, wherein the PAI-1 inhibitor is formulated to be delivered to damaged tissues of the subject’s inner ear, and wherein the PAI-1 inhibitor prevents, attenuates, or halts development of, or reduces, fibrosis of a subject’s inner ear following implantation.

2. The composition of claim 1 , wherein the first PAI- 1 inhibitor is selected from the group consisting of SK-216, TM 5441, TM 5275 sodium salt, annonacinone, AZ3976, embelin, and Tiplaxtinin.

3. The composition of claim 2, wherein the first PAI-1 inhibitor is Tiplaxtinin.

4. The composition of any of claims 1-3, further comprising an anti-inflammatory therapeutic agent formulated in combination with the first PAI-1 inhibitor.

5. The composition of claim 4, wherein the anti-inflammatory agent is selected from the group consisting of dexamethasone, prednisone, cortisone, hydrocortisone, and clodronate.

6. The composition of claim 5, wherein the anti-inflammatory agent is dexamethasone.

7. The composition of any of claims 1-6 further comprising a CNS stimulant.

8. The composition of claim 7, wherein the CNS stimulant is selected from the group consisting of amphetamine, armodafinil, atomoxetine, benzphetamine, caffeine citrate, dexmethylphenidate, diethylpropion, dextroamphetamine, lisdexamfetamine, methamphetamine, modafinil, oxybate, phendimetrazine, phentermine, pitolisant, solriamfetol, and methylphenidate.

9. The composition of any of claims 1-8, wherein the first PAI-1 inhibitor is formulated as an extended-release composition.

10. The composition of any of claims 1-9, wherein the first PAI-1 inhibitor is formulated in combination with a second PAI-1 inhibitor.

11. The composition of any of claims 1-10, wherein at least the first PAI-1 inhibitor is formulated for release upon implantation of the medical device and with each electrical impulse delivered to the inner ear tissues.

12. The composition of any of claims 1-11, wherein the anti-inflammatory therapeutic agent is co-formulated with at least the first PAI-1 inhibitor for administration to the subject.

13. A composition comprising a PAI-1 inhibitor and a CNS stimulant for use in the prevention, attenuation, or halting of development of, or reducing, fibrosis following implantation of a cochlear or vestibular implant in a subject’s inner ear, wherein the CNS stimulant is selected from the group consisting of amphetamine, armodafinil, atomoxetine, benzphetamine, caffeine citrate, dexmethylphenidate, diethylpropion, dextroamphetamine, lisdexamfetamine, methamphetamine, modafinil, oxybate, phendimetrazine, phentermine, pitolisant, solriamfetol, and methylphenidate.

14. The composition of claim 13, wherein the PAI-1 inhibitor is Tiplaxtinin.

15. The composition of claim 13 or claim 14 further comprising an anti-inflammatory agent.

16. The composition of claim 15, wherein the anti-inflammatory agent is dexamethasone.

17. A method for preventing, attenuating or halting development of, or reducing, fibrosis of the inner ear tissues in a subject having an electrical medical device with at least a first electrode implanted in the inner ear, comprising: administration of a therapeutically effective amount of a first therapeutic agent to the subject, wherein the first therapeutic agent is a first plasminogen activator inhibitor- 1 (PAI-1) inhibitor,and wherein the PAI- 1 inhibitor prevents, slows , halts or reduces development of fibrosis as a result of tissue damage from both implantation and repeated delivery of electrical impulses through at least the first electrode.

18. The method of claim 17, wherein the PAI-1 inhibitor is selected from the group consisting of Tiplaxtinin, TM 5441, TM 5275 sodium salt, SK-216, annonacinone, AZ3976, and embelin.

19. The method of claim 18, wherein the PAI-1 inhibitor comprises Tiplaxtinin.

20. The method of any of claims 17-19 further comprising a second therapeutic agent administered in combination with the first PAI-1 inhibitor, each in a therapeutically effective amount to the subject, wherein the second therapeutic agent is a second PAI-1 inhibitor.

21. The method of any of claims 17-20, wherein the medical device is a cochlear implant or vestibular implant.

22. The method of claim 21, wherein the medical device is a cochlear implant.

23. The method of claim 21, wherein the medical device is a vestibular implant.

24. They method of any of claims 17-23 further comprising local administration of the PAI-1 inhibitor.

25. The method of any of claims 17-24, wherein the PAI-1 inhibitor is administered systemically.

26. The method of any of claims 17-24, wherein the PAI-1 inhibitor is administered in an extended-release manner.

27. The method of any of claims 17-23, wherein the PAI-1 inhibitor is administered via release from at least a first coating on the first electrode of the medical device.

28. The method of any of claims 17-27, wherein the PAI-1 inhibitor is administered via release upon implantation of the medical device and with each electrical impulse delivered to the inner ear tissues.

29. The method of any of claims 20-28, further comprising the administration of a therapeutically effective amount of a second therapeutic agent, wherein the second therapeutic agent comprises an anti-inflammatory therapeutic agent selected from the group consisting of dexamethasone, prednisone, cortisone, hydrocortisone, and clodronate.

30. The method of claim 29, wherein the anti-inflammatory therapeutic agent comprises dexamethasone, wherein the dexamethasone is administered orally to an adult human subject in a therapeutically effective dose between about 0.75 mg to about 9 mg, or optionally, is administered parenterally to an adult human subject in a therapeutically effective dose between about 0.5 mg to about 9 mg daily.

31. The method of claim 29 or claim 30, wherein the anti-inflammatory therapeutic agent is co-formulated with one or more additional PAI-1 inhibitors and / or a CNS stimulant for administration to the subject, wherein the CNS stimulant is selected from the group consisting of amphetamine, armodafmil, atomoxetine, benzphetamine, caffeine citrate, dexmethylphenidate, diethylpropion, dextroamphetamine, lisdexamfetamine, methamphetamine, modafinil, oxybate, phendimetrazine, phentermine, pitolisant, solriamfetol, or methylphenidate.

32. The method of any of claims 17-31, wherein a therapeutically effective amount of the PAI-1 inhibitor is administered to the subject upon implantation and / or following application of each electrical stimulus in a dose sufficient to maintain a therapeutic concentration of PAI- 1 of between about 10 ng / mL and about 1000 ng / mL of perilymph fluid for about 1 to 6 hours.

33. The method of any of claims 17-32, wherein the PAI-1 inhibitor is administered in a therapeutically effective amount to the subject, and wherein an amount of the PAI-1 inhibitor administered is reduced over a period of at least about 1-6 months following implantation to 10% or less of an initial dose administered upon implantation.

34. The method of any of claims 17-33, wherein the PAI-1 inhibitor is Tiplaxtinin, and wherein an amount of the Tiplaxtinin is administered over a period of at least about 1 to about 6 months to the subject following each application of electrical stimulation in a dose sufficient to maintain a therapeutic concentration of Tiplaxtinin of between about 100 ng / mL and about 300 ng / mL in perilymph fluid for at least 6 hours after administration.

35. The method of any of claims 17-34, wherein the PAI-1 inhibitor attenuates development of fibrosis of inner ear tissues of the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

36. The method of any of claims 17-35, wherein the PAI-1 inhibitor reduces an amount of fibrosis of inner ear tissues of the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

37. The method of any of claims 17-29 and 31-36, wherein the subject is a mammal.

38. The method of claim 37, wherein the mammal is a laboratory animal.

39. The method of claim 37, wherein the mammal is a human.

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