Methods for preventing and treating hearing loss

Abstract

Acquired hearing loss due to chemotherapy or noise exposure is a major health problem and cisplatin chemotherapy often results in permanent hearing loss in cancer patients. However, there are no FDA-approved drugs for the treatment or prevention of cisplatin- or noise-induced hearing loss. In one aspect, the use of niclosamide, murolic acid, and ilesidomine as active agents to treat and prevent hearing impairment, and methods of using the compositions to treat and / or prevent hearing impairment or disorders are disclosed. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to limit the present application.

Description

[0001] Cross-reference to related applications: This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 050,568, filed July 10, 2020, pursuant to 35 U.S.C. §119(e) and 35 U.S.C. §111(a) (which is hereby incorporated by reference).

[0002] Statement regarding federally funded research or development:

[0003] This invention was developed with government support, based on licenses 1R43 DC018762, R01DC015444, and R01DC015010 granted by the National Institute of Deafness and Other Communication (NID) of the National Institutes of Health (NIH), license N00014-18-1-2507 granted by the Office of Naval Research, and license USARMC-RH170030 granted by the Department of Defense. The government holds certain rights to this invention. Background of the invention:

[0004] (1) Technical Field.

[0005] This invention relates to the therapeutic use of niclosamide for treating, inhibiting and / or preventing hearing loss.

[0006] (2) Background art, including information disclosed in accordance with 37 CFR 1.97 and 37 CFR 1.98.

[0007] Acquired hearing loss due to chemotherapy or noise exposure is a major health problem, and cisplatin-based chemotherapy often results in permanent hearing loss in cancer patients. However, there are no FDA-approved medications for treating or preventing cisplatin- or noise-induced hearing loss. Platinum-based chemotherapy is the standard of care for various types of cancer, including ovarian, lung, testicular, and head and neck cancers. Cisplatin is one of the most potent platinum compounds, causing permanent hearing loss in 40%–60% of cancer patients treated with it. One of the known mechanisms by which cisplatin damages auditory sensory cells is the formation of DNA adducts, leading to oxidative stress and apoptosis. To reduce cisplatin-induced damage to inner ear cochlear cells, various therapeutic strategies have been used in previous studies, including the use of antioxidants, anti-inflammatory agents, calcium channel blockers, kinase inhibitors, heat shock proteins, and thiols as chemical deactivators. For example, sodium thiosulfate (STS) has been shown to be effective in protecting hearing only in pediatric patients with localized hepatoblastoma receiving cisplatin chemotherapy; however, as a cisplatin chelator, it is not effective in preventing cisplatin-induced hearing loss (CIHL) in other cancer patients.

[0008] There is a need in the art for solutions to hearing loss caused by noise, antibiotics, cisplatin during chemotherapy, or aging. The described treatments are solutions to various problems in the art, such as narrow therapeutic windows and safety limits, and interference with the antitumor activity of cisplatin. Summary of the Invention

[0009] The present invention provides a method for preventing or treating hearing loss, the method comprising the steps of: administering an effective amount of a pharmaceutical composition containing a therapeutically active agent to an animal in need, wherein the therapeutically active agent comprises: niclosamide, ingenol, and elesclomol.

[0010] The subject matter of this invention also includes a composition for preventing or treating hearing loss by protecting inner ear cells from death, wherein the composition is an effective amount of an active agent selected from the group consisting of niclosamide, phorbol, and ilimismo or pharmaceutically acceptable salts thereof.

[0011] The subject matter of this invention also includes a medicine box made of: an active agent selected from the group consisting of: niclosamide, styraxol and irismo or pharmaceutically acceptable salts thereof; and one or more of: (A) at least one chemotherapeutic agent; at least one antibiotic inhibitor; and (C) instructions for use in preventing hearing loss. Attached image description:

[0012] The accompanying drawings, which are included in and form part of this specification, illustrate several aspects and, together with the description, serve to explain the principles of the invention.

[0013] Figure 1 This diagram illustrates how the compounds of the present invention were identified based on comparisons of datasets from drug screening association maps and pathway enrichment analyses, thereby revealing the drug's role in combating hearing loss. The transcriptomic profiles of cisplatin-resistant cancer cell lines and their parental cisplatin-sensitive cells were analyzed using the L1000CDS2 and GDA drug-gene interaction databases. Differentially expressed genes for each compound were then compared with those identified using KEGG pathway enrichment analysis based on the original GEO map to identify overlapping genes associated with cisplatin resistance. Of the initial 50 drugs, 30 were validated both in vitro and in vivo, with niclosamide emerging as the compound with the highest hit rate.

[0014] Figure 2A-2B Transcriptome analysis of cisplatin-resistant cancer cell lines reveals pathways and shared genetic targets associated with the identified drugs. A) GSEA analysis was used to analyze pooled cancer cell lineages available from the GEO database to identify enriched molecular pathways from the KEGG database. Upregulated pathways are shown in red on the left, while downregulated pathways are shown in blue on the right. The size of the circles correlates with the number of genes mapped to their respective FDR values. B) The gene expression profiles of each drug from the iLINCS database were compared with those differentially expressed genes identified by GSEA. Drugs were then graded using overlapping genes in the same direction.

[0015] Figure 3AF illustrates the protective effect of niclosamide against hearing loss. A) Lowest levels of caspase-3 / 7 activity in HEI-OC1 cells treated with cisplatin (50 μM) and the test compound. Raw caspase readings were normalized to caspase activity in cells treated with cisplatin / DMSO and those treated with 1% DMSO. Nicosamide was shown to reduce caspase activity to levels comparable to control cells at a dose of 4.4 μM. Data are shown as mean ± standard error (n = 3 wells per treatment). *P < 0.05 (one-way ANOVA). B) Highest protective levels in zebrafish treated with cisplatin and the test compound quantified by hair cell counts at the thalamus. Quantification of HC at SO3 (supraorbital line thalamus) and O1-2 (auricular line thalamus) revealed a significant reduction in cisplatin-induced damage in HC of zebrafish pretreated with 0.002 μM niclosamide (n = 5 to 8 per group, one-way ANOVA). C) Fluorescent staining of zebrafish thalamus treated with the medium (DMSO), cisplatin, and cisplatin + niclosamide (0.002 μM to 13.3 μM). D) Dose-response curve of niclosamide in HEI-OC1 cells with cisplatin exposure. E) Dose-response curve of niclosamide in HEI-OC1 cells without cisplatin exposure. F) Quantification of the highest protective levels in zebrafish treated with cisplatin and the experimental compound by hair cell count for each thalamus. Quantification of HC at SO3 (supraorbital line thalamus) and O1-2 (auricular line thalamus) revealed a significant reduction in cisplatin damage in HC of zebrafish pretreated with 0.002 μM niclosamide (n = 5 to 8 per group, one-way ANOVA).

[0016] Figures 4A-4FThis study demonstrates that niclosamide exhibits an antagonistic otoprotective effect against cisplatin in vivo. A) Compared with mice treated with cisplatin alone (Cis), niclosamide (Nic) showed a significantly reduced ABR threshold shift at 8, 12, and 32 kHz (n=8 per group, two-way ANOVA). B) Compared with mice treated with cisplatin alone, niclosamide showed a significantly reduced DPOAE threshold shift at 16 and 32 kHz (n=8 per group, two-way ANOVA). C, D) Wave I amplitude at baseline did not show differences in any of the four groups. Following cisplatin exposure, niclosamide was found to increase Wave I amplitude from 8 to 32 kHz compared with mice treated with cisplatin alone (n=8 per group, one-way ANOVA). E) Immunofluorescence images of the cochlea stained with myosin 6 (red) and DAPI (blue) in the 32 kHz region show the lowest level of hair cell loss when treated with both cisplatin and niclosamide. Nicosamide was shown to prevent cisplatin-induced hair cell loss. F) Quantitative analysis of outer hair cells (OHC) from immunofluorescence images showed that co-treatment with niclosamide conferred complete protection against cisplatin-induced hair cell loss (n=5 per group, Student's t-test). *P<0.05, data are shown as mean ± standard error across all figures.

[0017] Figures 5A-5F This study demonstrates the effect of niclosamide in preventing noise-induced hearing loss (NIHL). A) Nicosamide reduces NMDA excitotoxicity in the zebrafish neurata. Hair cell counts were quantified from zebrafish treated with 300 μM NMDA and 2 or 18.3 nM niclosamide. At both test doses, niclosamide significantly showed higher hair cell counts than zebrafish treated with NMDA alone. (n=5 per group, one-way ANOVA). B) Mice treated with niclosamide showed significantly reduced ABR threshold shifts at 8, 12, 32, 40, and 63 kHz in both noise-exposed and noise-treated groups compared to mice treated with saline plus noise. (n=8 per group, two-way ANOVA). C) There was no difference in DPOAE amplitude across all groups in the 10–80 dB SPL range. (n=8 per group, two-way ANOVA). D) Clonidamide-treated mice showed wave 1 amplitudes comparable to age-matched controls at 65–90 dB SPL and significantly higher wave 1 amplitudes than mice exposed to saline and noise at 80– and 90– dB SPL (n = 8 per group, one-way ANOVA). E) CtbP2 staining of inner hair cells showed that lonidamide prevented synaptic loss in noise exposure (CtbP2 is green). F) Quantification of CtbP2 spots (Y-axis) in each inner hair cell (x-axis) revealed that mice treated with lonidamide had significantly higher synapse counts than mice treated with the medium (n = 4 per group, Student's t-test). *P < 0.05. Data are shown as mean ± standard error in all figures.

[0018] Figure 6A -B shows the synergistic / cumulative otoprotective effects of niclosamide and ezetimibe. Three-dimensional contour plots (A) show the dose-response of neurothalamic hair cell protection in zebrafish treated with different concentrations of niclosamide (TT002) and ezetimibe, plotted as a synergistic distribution. Additionally, the Loewe synergistic and antagonistic scores (B) calculated for each dose combination indicated the highest synergistic activity when 0.66 nM niclosamide (TT002) was combined with 1.48 μM ezetimibe (n=5 per group). Other dose combinations showing synergistic effects are shown in dark blue boxes. Dose combinations with scores of 0 and 1 showed a cumulative effect. *P<5 x 10⁻²; **P<10⁻³, ***P<10⁻⁴ relative to control fish, one-sample t-test performed using Combenefit software 44. Data are shown as mean ± standard deviation. Ezetimibe is a cholesterol absorption inhibitor.

[0019] Figure 7 HPLC analysis of the in vitro interaction between niclosamide and cisplatin is shown.

[0020] Figure 8 The survival of non-small cell lung cancer cell line SHP77 and small cell lung cancer cell line A549 was shown in the presence or absence of cisplatin and 10 nM niclosamide. Nicosamide alone did not affect cisplatin-tumor killing activity.

[0021] Figures 9A-9B Dose-response curves of ilismo in the presence of cisplatin are shown. Caspase 3–7 activities were measured in HEI-OC1 cells treated with cisplatin (50 μM). Raw caspase readings were normalized to caspase activities in cells treated with cisplatin / DMSO and those treated with 1% DMSO. Ilismo was shown to reduce caspase activity to levels comparable to control cells at a dose of 40 μM. Data are shown as mean ± standard error (n = 3 wells per treatment). *P < 0.05 (one-way ANOVA). B) Quantification of the highest protective levels in zebrafish treated with cisplatin and ilismo by hair cell count at the neurothalamus. Quantification of HC at SO3 (supraorbital neurothalamus) and O1–2 (auricular neurothalamus) revealed a significant reduction in cisplatin-induced damage in zebrafish HC pretreated with 0.165, 1.48, and 13.3 μM ilismo.

[0022] Figures 10A-10BThe dose-response curves of megaphorol in the presence of cisplatin are shown. Caspase 3–7 activities were measured in HEI-OC1 cells treated with cisplatin (50 μM). Raw caspase readings were normalized to caspase activities in cells treated with cisplatin / DMSO and those treated with 1% DMSO. Irismoxol was shown to reduce caspase activity to levels comparable to control cells at a dose of 40 μM. Data are shown as mean ± standard error (n = 3 wells per treatment). *P < 0.05 (one-way ANOVA). B) The highest protective levels in zebrafish treated with cisplatin and megaphorol were quantified by hair cell counts at the neurothalamus. Quantification of HC at SO3 (supraorbital neurothalamus) and O1–2 (auricular neurothalamus) revealed a significant reduction in cisplatin-induced damage in zebrafish HC pretreated with 0.002, 0.0183, 0.165, and 1.48 μM megaphorol. Detailed Implementation

[0023] The invention can be more readily understood by referring to the following detailed description and examples included therein. Before disclosing and describing the compounds, compositions, articles, systems, devices, and / or methods of the invention, it should be understood that, unless otherwise stated, they are not limited to specific synthetic methods, or, unless otherwise stated, they are not limited to specific reagents, and therefore they are naturally variable. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, exemplary methods and materials are described hereafter.

[0024] While aspects of the invention may be described and claimed in specific statutory categories (such as the systems statutory category), this is merely for convenience, and those skilled in the art will understand that every aspect of the invention may be described and claimed in any statutory category. Unless expressly stated otherwise, it is never intended that any method or aspect set forth herein require its steps to be performed in a specific order. Therefore, when a method claim does not specifically specify in the claims or description that the steps are to be limited to a specific order, it is never intended to infer the order in any way. This applies to any possible non-explicit basis of interpretation, including logical questions concerning the arrangement of steps or operational procedures, simple meanings derived from grammatical organization or punctuation, or the number or type of aspects described in the description.

[0025] In one aspect, the compounds can be used as a therapy for treating and / or preventing hearing loss. In various aspects, the compounds and compositions of the present invention can be administered in the form of pharmaceutical compositions formulated according to a intended method of administration. The compounds of the present invention are defined as therapeutic agents in a treatment regimen or procedure intended to prevent hearing loss due to noise or age-related causes by protecting inner ear cells from death, as well as hearing loss induced by chemotherapy or antibiotics. A therapeutic agent is defined as a chemical substance used to treat or alleviate symptoms or pain of a disease.

[0026] Now for reference Figure 1 and 2A -B, based on comparisons of datasets from drug screening association maps and pathway enrichment analyses, compounds are identified to reveal their effects against hearing loss. This method was developed to obtain drug candidates from a diverse chemical space, thus covering a wide range of biological pathways and avoiding bias associated with focusing on previously reported pathways. The resulting compounds were compared with ototoxic agents (cisplatin), noise, or antibiotic exposure (cisplatin). Figure 5A / D), noise ( Figure 5B ) and antibiotics ( Figure 5C The gene expression transcriptome profiles of at least one of several cell lines or mouse strains treated with one of the compounds shown overlap with those of at least one of several cell lines or mouse strains treated with one of the compounds. Specifically, the datasets sought conform to NIHL-resistant and NIHL-sensitive mouse strains (129SvJ and CAST). Specifically and additionally, the datasets sought conform to cisplatin-resistant and sensitive cancer cell lines, the HEI-OC1 cell line, and in vivo mouse cochlear single-cell RNA seq with and without cisplatin treatment. Similarly, transcriptome interference of antibiotic treatment-related damage in the organ of Corti of newborn mice exposed to gentamicin is also used. Compounds that overlap with the computational results of these datasets include, but are not limited to, niclosamide, styraxol, and irismo.

[0027] Now for reference Figure 2A -B, Transcriptome analysis of cisplatin-resistant cancer cell lines revealed pathways and common gene targets associated with the identified drugs. A) GSEA analysis was used to analyze pooled cancer cell lineages available from the GEO database to identify enriched molecular pathways from the KEGG database. Upregulated pathways are shown in red on the left, while downregulated pathways are shown in blue on the right. The size of the circles correlates with the number of genes mapped to their respective FDR values. B) The gene expression profiles of each drug from the iLINCS database were compared with those differentially expressed genes identified by GSEA. Drugs were then graded using overlapping genes in the same direction.

[0028] Niclosamide, a previously FDA-approved drug, has been widely used since 1982 to treat tapeworm infections, demonstrating excellent protective effects against cisplatin-induced hearing loss in zebrafish and mice when administered prophylactically. Niclosamide also showed protective effects against phycocyanin-induced hair cell loss in zebrafish and against noise-induced hearing loss in mice. In summary, these data suggest that niclosamide can be reused as an otoprotective agent against cisplatin and noise-induced damage. In one aspect, niclosamide can be used as a therapy for the treatment and / or prevention of hearing loss. In various aspects, the compounds and compositions of the present invention can be administered in the form of pharmaceutical compositions formulated according to the intended method of administration. The compounds of the present invention are defined as therapeutic agents in a treatment regimen or procedure designed to prevent hearing loss due to noise or age-related causes by protecting inner ear cells from death, as well as hearing loss induced by chemotherapy or antibiotics. A therapeutic agent is defined as a chemical substance used to treat or alleviate symptoms or pain of a disease.

[0029] Niclosamide was shown to prevent hair cell apoptosis. Through the presented models and data, niclosamide was identified as effective against hair cell loss in animals. The models revealed properties essential for otoprotective compounds, such as high efficacy against hair cell loss, relatively low toxicity, lack of metal chelation, and blood-brain barrier permeability. Niclosamide was shown to exhibit high efficacy and high affinity in mouse and zebrafish models used to demonstrate its protective effect against hair cell loss. The lateral line thalamus of zebrafish is a valuable model for testing the protective effect of compounds against cisplatin toxicity in vivo, as their HC is believed to be homologous to HC in the mammalian inner ear and is readily accessible to the drug in vivo. Teitz et al., J. Exp. Med. [Journal of Experimental Medicine] 2; 215(4):1187-1203 (2018) have shown that mouse models involving HEI-OC1 can effectively validate the therapeutic use of compounds against hearing loss caused by cisplatin, noise, antibiotics, and aging. Teitz et al., J. Exp. Med. 2; 215(4):1187-1203 (2018).

[0030] The compounds and compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, pharmaceutical compositions can be formulated for topical or systemic administration, such as by instillation or injection into the ear, inhalation (e.g., into the ear), intravenous administration, topical administration, or oral administration. Niclosamide can be synthesized by a variety of methods known in the art. (See Jayaprakash, V. et al., *Medicinal Chemistry of Neglected and Tropical Diseases: Advances in the Design and Synthesis of Antimicrobial Agents*. United States, CRC Press, 2019. p. 348. RC961. M467.)

[0031] The properties of the pharmaceutical compositions used for administration depend on the route of administration and can be readily determined by those skilled in the art. In all respects, the pharmaceutical compositions are sterile or sterilizable. The therapeutic compositions characterized in this invention may contain carriers or excipients, many of which are known to those skilled in the art. Excipients that may be used include buffers (e.g., citrate buffers, phosphate buffers, acetate buffers, and bicarbonate buffers), amino acids, urea, alcohols, ascorbic acid, phospholipids, polypeptides (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water, and glycerol. The nucleic acids, polypeptides, small molecules, and other modulating compounds characterized in this invention can be administered via any standard route of administration. For example, administration may be parenteral, intravenous, subcutaneous, or oral. Modulating compounds can be formulated in various ways depending on the corresponding route of administration. For example, liquid solutions can be prepared for administration by instillation into the ear, for injection, or for ingestion; gels or powders can be prepared for ingestion or topical application. The methods used to prepare such formulations are well known and can be found, for example, in Remington's Pharmaceutical Sciences, 18th edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1990.

[0032] In all respects, the disclosed pharmaceutical compositions include the disclosed compounds (including pharmaceutically acceptable salts thereof) as active ingredients, pharmaceutically acceptable carriers, and optional other therapeutic components or adjuvants. The compositions of the present invention include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, but the most suitable route in any given case will depend on the specific host and the nature and severity of the condition to which the active ingredient is administered. The pharmaceutical compositions can be conveniently presented in unit dosage forms and prepared by any method well known in the pharmaceutical field.

[0033] In various aspects, the pharmaceutical compositions of the present invention may include a pharmaceutically acceptable carrier and a compound of the present invention or a pharmaceutically acceptable salt thereof. The compounds of the present invention or pharmaceutically acceptable salts thereof may also be included in the pharmaceutical composition in combination with one or more other therapeutically active compounds.

[0034] In preparing compositions for oral dosage forms, any convenient pharmaceutical medium may be used. The pharmaceutical compositions of the present invention comprise, as the active ingredient, the compound of the present invention (or a pharmaceutically acceptable salt thereof), a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The compositions of the present invention include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration; however, in any given case, the most suitable route will depend on the specific host and the nature and severity of the condition to which the active ingredient is administered. The pharmaceutical compositions can be conveniently presented in unit dosage forms and prepared by any method well known in the pharmaceutical field.

[0035] The pharmaceutical compositions of the present invention, suitable for parenteral administration, can be prepared as solutions or suspensions of the active compound in water. Suitable surfactants, such as hydroxypropyl cellulose, may be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof in oil. Furthermore, preservatives may be included to prevent harmful microbial growth.

[0036] Pharmaceutical compositions of the present invention suitable for injectable applications include sterile aqueous solutions or dispersions. Alternatively, the compositions may be in the form of sterile powders for ad hoc preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be an easily injectable and effective fluid.

[0037] The following description is intended to illustrate how those skilled in the art can prepare and evaluate the claimed methods, compounds, compositions, articles, and / or apparatus, and is not intended to limit the scope of the invention.

[0038] In various ways, these compounds (such as niclosamide) can be used in combination with one or more other drugs in the form of a pillbox to prevent, control, improve hearing loss, or reduce the risk of hearing loss when other drugs are known to damage hearing.

[0039] Now for reference Figure 3 A and Figure 3 DE, a compound, protects mouse inner ear cell lines from cisplatin toxicity. House Ear Institute-Organ of Corti 1 (HEI-OC1) cells (House Research Institute) were cultured at 33°C and 10% CO2 in high-glucose Durbecco modified Eagle medium (Life Technologies, USA) supplemented with 10% fetal bovine serum, as previously described (Kalinec et al., 2003). Cells were seeded at 8,000 cells / well in 96-well plates and allowed to adhere overnight. Reference now. Figure 3 A. For drug screening, HEI-OC1 cells were pretreated with 30 drug candidates at concentrations ranging from 2 nM to 40 μM one hour prior to cisplatin administration. The cisplatin dose (50 μM) was based on our previously published dose-response curves. Cells were co-incubated with cisplatin and drug candidates for another 19 hours prior to Caspase-Glo 3 / 7 assays (Promega, Madison, WI), as previously described. Additionally, cells treated with DMSO only and kenpaullone were used as positive controls to validate our results. The DMSO concentration in the drug formulations was adjusted to 0.1% v / v, and it was confirmed that 0.5% DMSO had no effect on cell death kinetics (Hall et al., 2014). Results were run in triplicate and normalized to controls containing only cisplatin and only culture medium. The percentage of caspase activity was used to determine the relative protective effect of each compound. Luminescent detection representing caspase activity in each well was obtained using a Cytation hybridization multimode reader (Biotek, Winooski, VT, USA). The percentage of cells protected was calculated using the caspase 3 / 7 readings and the following formula:

[0040]

[0041] Now for reference Figure 3DE illustrates the dose-response curves of niclosamide in HEI-OC1 cells with and without cisplatin exposure. Caspase activity was measured using a caspase-Glo 3 / 7 assay, and the results were calculated as a percentage of protection as an indicator of cell survival / viability. The percentage of protection for each compound at the test dose was then plotted to illustrate the dose-response curves, and the IC50 was calculated. 50 HEI-OC1 cells treated with niclosamide achieved 0% caspase activity or complete protection at a dose of approximately 4.4 μM. Furthermore, the calculated IC50 value of niclosamide was... 50 The relative concentration was 280 nM. Niclosamide has a wide therapeutic window, showing some level of protection over more than 80% of the tested dose range. Mice administered niclosamide did not exhibit weight loss or abnormal behavior at the maximum IP dose approved by the IACUC. Mice treated with a combination of cisplatin (30 mg / kg / single injection) and niclosamide did not exhibit general toxicity compared to mice treated with cisplatin alone. In the following examples, niclosamide (5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxy-benzamide, purity ≥95%) was used in all experiments (Cayman Chemical, USA).

[0042] The protective effect of niclosamide in HEI-OC1 cells was compared with that of tamparone (a known CDK2 inhibitor that enhances cell survival by reducing cisplatin-induced mitochondrial ROS production). The effect of tamparone in HEI-OC1 cells was previously characterized in Teitz et al., J. Exp. Med. [Journal of Experimental Medicine] 215(4):1187-1203 (2018). This comparison demonstrated that niclosamide exhibited a level of protection against cisplatin damage comparable to that of tamparone in HEI-OC1 cells, and better protection than four other benchmark compounds (sodium thiosulfate, ebuselenol, dexamethasone, and N-acetylcysteine).

[0043] Now for reference Figure 3 BC and Figure 3F represents the highest level of protection in zebrafish treated with cisplatin and the experimental compound, quantified by hair cell counts at the lateral line thalamus. Experimental zebrafish (Danio rerio) juveniles were obtained by pairing with adult fish raised at Creighton University using standard methods approved by the Institutional Animal Care and Use Committee. The lateral line thalamus of zebrafish is a valuable system for testing the protective efficacy of compounds against cisplatin toxicity in vivo because their HC is considered homologous to HC in the mammalian inner ear and is readily accessible to drugs. We used Tg(pou4f3:mGFP) expressing membrane-binding GFP in HC. Experimental fish were housed in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 nM MgSO4, pH 7.2) at 28.5 °C. Animals were cryo-anesthetized after drug treatment and before fixation. The examined nerve thalamus SO3 and O1-2 are part of the cranial system and include the ototrichum, middle thalamus, and tectal thalamus.

[0044] For screening, Tg(brn3c:GFP) larvae with a post-fertilization day (dpf) of 5 were pre-incubated for 1 hour with 41 drug candidates at concentrations of 0.002, 0.018, 0.165, 1.48, and 13.3 μM, followed by co-incubation with 400 μM cisplatin for 4 hours. Subsequently, the animals were transferred to E3 water for 5 hours and fixed overnight in 4% paraformaldehyde (PFA) (26). Neural thalamus HCs were immunolabeled with anti-otoferlin (HCS-1, DSHB) and anti-GFP (NB100-1614, Novus Biologicals). These two labels were used to detect and count neural thalamus HCs to reduce the chance of losing some HCs after treatment, as we previously noted that incubation with the compounds affected GFP expression, making it more difficult to detect under a fluorescence microscope. The auricular, median, and tectal nerve thalamus were identified, and HC values ​​at SO3 (supraorbital nerve thalamus) and O1-2 (auricular nerve thalamus) were manually counted using a Zeiss AxioSkop 2 fluorescence microscope with a 40x oil objective. The efficacy and potency of the compounds were then evaluated, with the highest-rated compounds exhibiting higher protective activity at lower concentrations.

[0045] Quantitative analysis of HC at SO3 (supraorbital line thalamus) and O1-2 (auricular line thalamus) revealed a significant reduction in cisplatin-induced damage in HC from zebrafish pretreated with 0.002 μM niclosamide (n = 5 to 8 per group, univariate ANOVA). References Figure 3F, Quantification of HC at SO3 (supraorbital nerve thalamus) and O1-2 (auricular nerve thalamus) in zebrafish at multiple doses (n = 5 to 8 per group, one-way ANOVA). *P < 0.05, data are shown as mean ± standard error (n = 5 per group). *P < 0.05, data are shown as mean ± standard error in all figures.

[0046] Now for reference Figure 3 B. The numbers were normalized to those of zebrafish treated only with 300 μM cisplatin (as 0% protection) or culture medium (DMSO; as 100% protection) to generate the percentage of protection for each compound. Drug candidates were graded based on the most effective dose for preventing cisplatin-induced damage to the neurothalamus HC. Figure 2B Of the top 42 compounds tested, niclosamide was found to be the most potent drug candidate, providing the highest level of protection (approximately 50%) at the lowest concentration tested (0.002 μM). Figure 3 B). The maximum protective effect was achieved at 0.002 μM, indicating that this is a suitable concentration for human use. This concentration is approximately 9,000 times lower than the Cmax (18 μM) after oral administration of 2,000 mg in humans.

[0047] Now for reference Figure 3 C. Fluorescent staining of the neurotumor of zebrafish treated with niclosamide. Nicosamide was shown to reduce hair cell loss in zebrafish treated with cisplatin at concentrations ranging from 0.002 to 13.3 μM. GFP is shown in green, and otodermal protein is shown in red (n = 3 per drug dose, scale bar = 20 μm).

[0048] Now for reference Figure 4A -F, Niclosamide attenuates cisplatin-induced hearing loss in FVB / NJ mice. Mice: The procedure used with mice was approved by the Institutional Animal Care and Use Committee (IACUC) of Creighton University. For functional assessments, including cisplatin and noise exposure experiments, 5- to 7-week-old FVB / NJ mice, mixed males and females, were used in the experiments from Jackson Laboratory (Bar Harbor, Maine, USA). For cisplatin and noise studies, FVB / NJ mice were treated with 10 mg / kg niclosamide via intraperitoneal (IP) injection. Niclosamide was dissolved in physiological saline (0.9% NaCl solution) in 1% DMSO and vortexed several times before injection. Treatment began 24 hours before cisplatin or noise exposure, once daily for 3 days. Cisplatin was administered via IP injection at a dose of 30 mg / kg, divided into two daily doses. Before and for 7 days before and after cisplatin treatment, animals were given 1 mL of warm sterile saline subcutaneously twice a day to prevent dehydration.

[0049] Now for reference Figure 4A ABR testing was performed 2–3 days before and 5 days after cisplatin exposure. Experimental groups included: cisplatin and 1% DMSO, cisplatin and niclosamide, niclosamide only, and an age-matched control receiving saline. For noise testing, auditory testing was performed 2–3 days before noise exposure (examples described below), on day 1 post-exposure to monitor transient threshold shift (TTS), and on day 14 post-exposure to monitor transient threshold shift (PTS). Experimental groups included: noise exposure and saline, noise exposure and niclosamide, niclosamide only, and an age-matched control. After the final auditory function measurements, mice were euthanized, and cochleas were collected for morphological evaluation. Based on the results of FVB / NJ ABR / DPOAE threshold variance and power analysis, ten FVB / NJ mice (n=5 of each sex) were used in each group in this study.

[0050] To investigate the effects of drugs on auditory function in mammals, we first treated 5–7 week old FVB / NJ mice with four drug candidates: niclosamide (10 mg / kg / day), phorbol (0.3 mg / kg / day), and irismo (5 mg / kg / day) for 4 consecutive days (IP) to monitor drug safety. All test doses were determined based on previously published data showing the maximum non-toxic dose administered intraperitoneally (IP). At the given doses, no mice showed signs of reduced weight loss or pain and distress. Mice treated with niclosamide at 20 mg / kg / day (IP) for 4 consecutive days showed signs of pain and distress, including arched backs and a 20%–30% weight loss; therefore, 10 mg / kg / day was the maximum dose used in this study.

[0051] Baseline auditory brainstem response (ABR) was measured in 16 mice divided into three groups receiving phorbol, ilimex, and niclosamide. Mice received the aforementioned drug doses, followed by 30 mg / kg cisplatin (IP; 4 mice per group) on day 2 post-treatment. Daily monitoring showed that mice receiving phorbol and ilimex experienced weight loss exceeding 20%, and we observed a mortality rate of 40%–60% in these mice by day 5 post-treatment. Mice treated with cisplatin alone showed a weight loss of 10%–15%. However, mice treated with niclosamide showed a weight loss of less than 10%, and all survived the second ABR test on day 5 post-cisplatin injection.

[0052] Now for reference Figure 4BThe distortion product otoacoustic emission (DPOAE) thresholds in cisplatin-treated, niclosamide-treated, control, and cisplatin-niclosamide-treated mice were measured at frequencies ranging from 8 to 40 kHz. On day 5 post-cisplatin injection, the DPOAE threshold in the control group was significantly lower than that in the cisplatin-treated group. Two-way ANOVA and subsequent post-hoc Tukey tests showed statistically significant differences between cisplatin-niclosamide-treated mice and the cisplatin-only group at 16 kHz and 32 kHz.

[0053] Now for reference Figure 4C -D, mean wave-1 amplitudes at 8, 16, 32, and 40 kHz were measured in control and niclosamide-treated mice before and on day 5 after cisplatin injection. ABR wave-1 amplitude represents the overall activity of the cochlear nerve. A two-way ANOVA (group x frequency) was used to compare amplitudes of 85 dB SPL stimulation between groups at baseline ABR measurements; no group differences were detected (P > 0.05). On day 5 after cisplatin injection, two-way ANOVA revealed a significant two-way interaction of group x stimulation levels, and a post-hoc Tukey test revealed that the cisplatin-niclosamide-treated group had higher amplitudes at all test frequencies of 85 dB SPL compared to the cisplatin-only group. Figure 4C D).

[0054] Now for reference Figure 4E -2F, representative sample of mouse HC at 32kHz (the most protected frequency region shown by ABR measurement). Figure 4E Quantitative data of HC count were presented in... Figure 4F One-way ANOVA revealed a significant group effect (P<0.05). Post-hoc tests at each frequency revealed that the cisplatin-niclosamide group had more HC survival than the cisplatin group at the 30 kHz region. These data confirm that niclosamide protects OHCs from cisplatin damage. Nicosamide protects zebrafish from NMDA-induced hair cell loss. Application of N-methyl-d-aspartate (NMDA) to zebrafish neurata HCs was also used to mimic the glutamate excitotoxicity associated with noise exposure (Katie S. Kindt and Lavinia Sheets, 2018). HC counts following zebrafish exposure to 300 μM NMDA resulted in hair cell loss. However, pretreatment with 2 nM and 18 nM niclosamides before cisplatin exposure significantly increased HC survival compared to cisplatin-DMSO-exposed zebrafish larvae.

[0055] Referring now to Figure 5, niclosamide has a protective effect against NIHL in vivo in zebrafish and FVB / NJ mice. CIHL and NIHL share mechanical commonalities. Referring now to... Figure 5ATo test whether niclosamide protects HC from excitotoxic trauma, a zebrafish model simulating noise damage was used. Zebrafish were exposed to the ionic glutamate receptor agonist N-methyl-D-aspartate (NMDA) (previously shown to induce progressive HC loss in zebrafish lateral line organs with or without niclosamide (Sheets, 2017)). 5-dpf larvae were pre-incubated with 300 μM NMDA for 50 min, followed by incubation with 2 nM and 18.3 nM niclosamide for 2 h.

[0056] Now for reference Figure 5B -F, mice were injected with 10 mg / kg niclosamide once daily for four consecutive days: one day before noise exposure (8-16 kHz at 105 dB SPL), on the day of noise exposure, and two days after noise exposure. Control animals received the medium injections according to the same schedule.

[0057] Now for reference Figure 5B The noise-induced ABR threshold shift was obtained by subtracting the pre-exposure threshold from the retained threshold. Daily two-way ANOVA revealed a significant main effect in the day 1 group. Tukey's multiple comparison test revealed that on day 14, the niclosamide-noise exposure group had a lower threshold shift than the noise exposure group across all test frequencies from 8 kHz to 63 kHz.

[0058] Now for reference Figure 5C DPOAE amplitude was measured in mice at f2 frequencies ranging from 10 to 80 dB SPL. For the noise-niclosamide group, DPOAE amplitude was significantly higher than that in the noise-saline group on day 15 post-noise exposure. Two-way ANOVA (group x frequency) was used to compare pre-exposure amplitude with day 15 amplitude. ANOVA revealed no significant two-way group x frequency interaction, suggesting that OHC function is similar across all groups, and that the protective effect of niclosamide against noise may be due to prevention of synaptic disorders.

[0059] Now for reference Figure 5D Mean wave-I amplitudes at 10, 20, 28.3, and 40 kHz were measured on day 15 post-noise exposure. A two-way ANOVA (group x stimulus level) was used to compare amplitudes at 10–90 dB SPL stimulus intensities between groups during the pre-noise test; no group differences were detected. On day 15, only 60–90 dB SPL stimulus levels were used because many subjects did not respond below 60 dB SPL. The two-way ANOVA revealed a significant interaction between group x stimulus levels (P < 0.001). Tukey later revealed that the niclosamide-noise group had higher amplitudes at 80 and 90 dB SPL compared to the noise exposure group. Figure 5DThe results of wave-I amplitude analysis showed that the cochlear nerve activity in the noise-niclosamide group was comparable to that in the age-matched control group, and there were no statistically significant differences between the groups.

[0060] Now for reference Figure 5E To assess protection of the ribbon synapse, cochlear samples were immunostained with CtBP2 (one of the most abundant proteins in the synaptic ribbon body) (Kujawa SG et al. 2006). A representative sample of mouse ribbon synapses at 16 kHz is shown. Now refer to Figure 5F The outer hair cells of the ribbon synapses were counted. An independent samples t-test at 16 kHz revealed that the niclosamide-noise group had more synaptic ribbon survival than the saline-noise group. OHCs were also counted in the same cochlea. An independent samples t-test at 32 kHz revealed a similar proportion of surviving OHCs in the niclosamide-noise group compared to the saline-noise treated animals. CtbP2 ribbon counting was performed using the 16 kHz frequency region because ribbons have been shown to be more abundant in this frequency region.

[0061] Now for reference Figure 6A -B, 5 dpf zebrafish were incubated with a medium (DMSO), cisplatin alone at a concentration of 300 μM, cisplatin and ezetimibe (0.002 μM to 13.3 μM), cisplatin and niclosamide (0.02 nM to 18.3 nM), or cisplatin and a combination of ezetimibe / niclosamide. After 6 hours, the animals were transferred to freshwater for 1 hour, then sacrificed and treated as described above for immunohistochemistry and HC counting. To further elucidate whether niclosamide synergistically works with the Nrf2 activator to protect zebrafish HC from cisplatin damage, we tested the extent of HC damage in the presence of niclosamide and the Nrf2 activator ezetimibe.

[0062] Now for reference Figure 6A Niclosamide and ezetimibe exhibited synergistic / cumulative otoprotective effects. A three-dimensional contour plot (A) shows the dose-response for neurothalamic hair cell protection in zebrafish treated with different concentrations of niclosamide and ezetimibe, plotted as a synergistic distribution. Additionally, the Loewe synergistic and antagonistic scores (B) calculated for each dose combination indicated the highest synergistic activity when 0.66 nM niclosamide was combined with 1.48 μM ezetimibe (n=5 per group). Other dose combinations showing synergistic effects are shown in dark blue boxes. Dose combinations with scores of 0 and 1 showed a cumulative effect. *P<5x10⁻²; **P<10⁻³, ***P<10⁻⁴ relative to control fish, one-sample t-test performed using Combenefit software 44. Data are shown as mean ± standard deviation.

[0063] Ezetimibe alone showed higher HC counts per thalamus at 1.48 μM, while niclosamide alone showed higher counts at concentrations above 2 nM. However, the combination of the two compounds, niclosamide and ezetimibe, showed significantly higher hair cell counts at much lower doses (n=5 per group, one-way ANOVA). Zebrafish were treated with a combination of niclosamide (range .02–18.3 nM) and EZ (range .0183–13.3 μM).

[0064] Apart from Figure 6A -B, but not shown, 5dpf zebrafish were incubated for 6 hours with the medium, niclosamide (2 nM to 1.48 μM), and dimethyl maleate (DEM) as a positive control, to evaluate the effect of niclosamide on ggcsh (a downstream target of Nrf2). Ggcsh has previously been identified as a downstream target of Nrf2 in zebrafish (Sheets, 2017). After incubation, total RNA was isolated from whole fish and processed to measure the induced expression of the Nrf2 downstream gene ggcsh. ggcsh expression was significantly reduced in zebrafish treated with niclosamide in the range of 0.002–1.48 μM (n = 5 per group, one-way ANOVA). Additionally, Nrf2a morpholine oligonucleotides were prepared, and zebrafish morpholine mutants were then incubated with cisplatin and / or niclosamide. Zebrafish eggs were injected with 4.5 ng of heteromorpholine cyclic oligonucleotide or nrf2a morpholine cyclic oligonucleotide (5'-CATTTCAATCTCCATCATGTCTCAG SEQ NO:1). At 3 dpf, uninjected morpholine mutant animals were exposed to the vector, cisplatin (300 μM), cisplatin + niclosamide 18.3 nM, or niclosamide alone for 6 hours. After 1 hour of recovery, the animals were fixed and treated for immunohistochemistry. Due to the lack of suitable antibodies to detect Nrf2 protein in zebrafish, Nrf2a KD was confirmed by analyzing the expression of glutathione S-transferase P1 (gstp1) (a downstream target). Zebrafish morpholine cyclic oligonucleotides demonstrated that nrf2a knockdown reduced the otoprotective effect of niclosamide. Uninjected random control zebrafish showed significantly higher hair cell counts after co-treatment with cisplatin and niclosamide, while nrf2a knockdown zebrafish showed similar hair cell counts to those treated with cisplatin alone (n=5 per group, one-way ANOVA). These results demonstrate that the protective effect of niclosamide against cisplatin ototoxicity in zebrafish requires Nrf2.

[0065] Now for reference Figure 7HPLC analysis confirmed the absence of interaction between niclosamide and cisplatin: chemically bound drug-drug interactions could negatively impact cancer treatment. A simple explanation for niclosamide's protective effect against CIHL is its direct inactivation of cisplatin, similar to several otoprotective agents used in clinical trials (STS, ebuselenol, etc.). To investigate any potential interactions between niclosamide and cisplatin, we developed an in vitro HPLC method. This method uses the following parameters: HPLC: Shimadzu Prominence-i LC-2030C, column: Agilent Eclipse Plus C18, PN-959961-902, 5% ACN, 95% water: 0–2 min; ACN continuously increased to 95%: 2–12 min; held at 95% ACN: 12–17 min; ACN continuously decreased to 5%: 17–20 min; flow rate: 1 mL / min; temperature 37°C; run time: 20 min. Other parts of the method can be prepared using a 1 mg / ml stock solution (prepared in a suitable solvent and mixed to produce 1:1 and 1:10 ratios of cisplatin and niclosamide in the final injection solution) as well as pure cisplatin and niclosamide. Results showed no interaction between niclosamide and cisplatin (no third peak) at several dose ratios of niclosamide and cisplatin. Our in vitro results are consistent with their synergistic interaction with cisplatin and its protective effect against noise-induced damage in an RCC xenograft model.

[0066] Now for reference Figure 8In vitro models of the non-small cell lung cancer cell line SHP77 and the small cell lung cancer cell line A549 were incubated with or without cisplatin and 10 nM niclosamide. Cells were fed in cultures using Matrigel as previously described in Kelley and Driver, Curr. Protoc. Neurosci. [Handbook of Laboratory Neuroscience] Chapter (4); Unit (4); 34.1-10 (2010). One day after cell culture, cells were pre-incubated for 1 hour at 37°C in 5% Co2 with or without the test compound in DMEM growth medium (12430-054; GIB CO Life Technologies, containing 1% FBS [16000-044; GIB CO Life Technologies] and 50 μg / ml ampicillin). This was followed by incubation for 24 hours at 37°C with or without the test compound in the growth medium with 50 μM cisplatin (479306; Sigma-Aldrich). The 50 μM cisplatin concentration was used for explant assays as it consistently showed a reduction of approximately 40% in outer hair cells in the mouse cochlea after 24 hours. The cochlea was fixed in 4% PFA and actin was stained with Alex Fluor 568 phalloidin to determine HC activity. The cochlea can also be stained using DAPI, FM1-43 dye uptake, and immunohistochemical staining with known HC markers (including parvalbumin and myosin 7a). The cochlea can be imaged using confocal microscopy. Two 160 μm regions in the central ring can be photographed, and the number of intact HC cells can be counted.

[0067] To compare the explant assays with niclosamide, the known benchmark compound kemparone can be tested under a similar procedure as described above. The following modifications can be used in the comparative assays: (1) a filter (Millicell, PICM03050; Millipore) instead of Matrigel in a 6-well culture plate, with 1 ml of culture medium solution inside and outside the filter; (2) a P3 FVB mouse strain; and (3) a dose-response model based on cisplatin at 50, 100, 150, and 200 μM, with a 150 μM cisplatin dose consistently showing approximately 40% loss of outer hair cells over 24 hours. (Teitz et al., J. Exp. Med. [Journal of Experimental Medicine] 215(4):1187-1203 (2018)). Co-treatment with niclosamide and cisplatin can be further characterized by treating explants with different concentrations of cisplatin at different time points. For example, 150 μM cisplatin can be used with or without the test compound, and the above measurements can be performed after 48 hours of incubation.

[0068] Cisplatin elution assays can be used to confirm the role of CIHL and the test compound in mitigating this reaction. For example, various concentrations of cisplatin can be removed after 90 minutes of incubation (an estimated time for cisplatin to remain in the inner ear after IP injection), and the above measurements can be performed after different incubation periods.

[0069] Now for reference Figure 9A Ilismo prevents CIHL. Caspase activity was measured using a caspase-Glo 3 / 7 assay, and the results were calculated as a percentage of protection as an indicator of cell survival / viability. The percentage of protection for each compound at the test dose was then plotted to illustrate the dose-response curve, and the IC50 was calculated. 50 HEI-OC1 cells treated with ilismo achieved 20% caspase activity at a dose of approximately 40 μM. Additionally, the calculated IC50 of niclosamide... 50 Relatively low, at 0.02 μM ( Figure 9A ).

[0070] Now for reference Figure 9B Ilisimole prevented cisplatin ototoxicity in zebrafish at multiple doses (n = 5–8 per group, one-way ANOVA). *P < 0.05, data are shown as mean ± standard error (n = 5 per group). *P < 0.05, data are shown as mean ± standard error in all figures. Zebrafish were incubated with 0.002, 0.018, 0.165, 1.48, and 13.3 μM ilisimole for 1 hour, followed by co-incubation with 400 μM cisplatin for 4 hours. Ilisimole showed protective effects against CIHL at doses of 0.165, 1.48, and 13.3 μM.

[0071] Now for reference Figure 10A Giant salicylates prevented CIHL. Caspase activity was measured using a caspase-Glo 3 / 7 assay, and the results were calculated as a percentage of protection as an indicator of cell survival / viability. The percentage of protection for each compound at the test dose was then plotted to show dose-response curves, and the IC50 was calculated. 50 HEI-OC1 cells treated with phorbolone achieved 30% caspase activity at a dose of approximately 40 μM. Furthermore, the calculated IC50 of phorbolone was... 50 Relatively low, at 1.54 μM ( Figure 10A ).

[0072] Now for reference Figure 10BMegaphoryl alcohol prevented cisplatin ototoxicity in zebrafish at multiple doses (n = 5–8 per group, one-way ANOVA). *P < 0.05, data are shown as mean ± standard error (n = 5 per group). *P < 0.05, data are shown as mean ± standard error in all figures. Zebrafish were incubated with 0.002, 0.018, 0.165, 1.48, and 13.3 μM megphoryl alcohol for 1 hour, followed by co-incubation with 400 μM cisplatin for 4 hours. At doses of 0.002, 0.0183, 0.165, and 1.48 μM, megphoryl alcohol showed protective effects against CIHL.

[0073] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference.

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[0130] Although the invention has been described in detail with reference to the illustrated embodiments, these details are not intended to limit the scope of the invention as defined in the appended claims. Embodiments of the invention for which proprietary rights or privileges are claimed are defined as follows: sequence list <110> Hearing Therapy Co., Ltd. <120> Methods for preventing and treating hearing loss <130> 30006.0005A <150> 63 / 050,568 <151> 2020-07-10 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic zebrafish (Danio rerio) <400> 1 catttcaatc tccatcatgt ctcag 25

Claims

1. Use in an effective amount of a pharmaceutical composition in the preparation of a medicament for the prevention of hearing loss in mammals in need, wherein the pharmaceutical composition comprises a therapeutically active agent, wherein the therapeutically active agent is niclosamide or a pharmaceutically acceptable salt thereof, the use including protecting inner ear cells from death caused by noise or a chemotherapeutic agent, wherein the chemotherapeutic agent is cisplatin.

2. The use as claimed in claim 1, wherein the step of administering an effective amount of the pharmaceutical composition to a mammal at risk of ototoxicity occurs prior to exposure to ototoxic damage.

3. The use as claimed in claim 1, wherein the step of administering an effective amount of the pharmaceutical composition to a mammal at risk of ototoxicity occurs 24 hours prior to exposure to ototoxic damage.

4. Use of an effective amount of the pharmaceutical composition in the preparation of a medicament for the prevention of hearing loss in mammals at risk of ototoxicity, wherein the pharmaceutical composition comprises a therapeutic active agent, wherein the active agent is niclosamide or a pharmaceutically acceptable salt thereof, wherein the ototoxicity risk is caused by a chemotherapeutic agent or noise exposure, wherein the chemotherapeutic agent is cisplatin.

5. The use as claimed in claim 4, wherein the step of administering an effective amount of the pharmaceutical composition to a mammal at risk of ototoxicity occurs prior to exposure to ototoxic damage.

6. The use as claimed in claim 4, wherein the step of administering an effective amount of the pharmaceutical composition to a mammal at risk of ototoxicity occurs 24 hours prior to exposure to ototoxic damage.

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