Compositions and methods for preventing and treating hearing loss

Abstract

In one aspect, the use of compounds as active agents for the treatment and prevention of hearing impairment, and methods of using the compositions for the treatment and / or prevention of hearing impairment or disorder, are disclosed. In zebrafish and mice, the (E)-1-(3-(3, 4, 5-trimethoxyphenyl) acryloyl)-S, 6-dihydropyridine-2 (1H)-ketone and the derivative have an excellent protective effect on hearing loss induced by antibiotics, and the application of the (E)-1-(3-(3, 4, 5-trimethoxyphenyl) acryloyl)-S, 6-dihydropyridine-2 (1H)-ketone and the derivative has a good application prospect. In one aspect, (E)-1-(3-(3, 4, 5-trimethoxyphenyl) acryloyl)-S, 6-dihydropyridine-2 (1H)-one and derivatives are useful as therapies for the treatment and / or prevention of hearing loss. This abstract is intended as a scanning tool for purposes of retrieval in the particular art and is not intended to be limiting of the present invention.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS: This application claims the benefit under 35 USC §119(e) and 35 USC §111(a) of U.S. Provisional Patent Application Serial No. 63 / 078,571, filed on September 15, 2020, which is specifically incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT: This invention was made with government support under R01DC015010, R01DC015444, and R43DC019065 from NIH, N00014-18-1-2507 from Navy / ONR, and W81XWH-18-1-0442 from Army / MRMC. The government has certain rights in this invention. Background of the Invention (1) Technical field

[0001] The present invention relates to therapeutic uses of active agents, such as for treating, inhibiting, and / or preventing hearing loss. (2) Background Art, including information disclosed pursuant to 37 CFR 1.97 and 37 CFR 1.98.

[0002] More than 35 million Americans experience hearing loss. In mammals, hair cell damage is permanent. Antibiotic-induced hearing loss (AIHL) is a permanent side effect that often requires medical care. Hearing loss rates are ten times higher in newborns admitted to neonatal intensive care units than in newborns not admitted, often linked to antibiotic use. The spiral-shaped cochlea of ​​the inner ear is responsible for detecting sound. Inner hair cells lining the cochlea convert the mechanical vibrations of sound waves into chemical signals. These chemicals are then released from the hair cells and picked up by receptors on the auditory nerve fibers, which send electrical impulses to the brain. The inner ear cells lining the cochlea can be damaged by antibiotic use, leading to a condition known as sensorineural hearing loss.

[0003] At present, there is no clinically proven drug for treating hearing impairment (sensorenal and neural) or tinnitus associated with the inner ear, so there is a great need for drugs that can be used to prevent, alleviate or eliminate hearing impairment (or tinnitus). For individuals suffering from severe sensorineural hearing impairment, the most common remedy is a hearing aid, whose function is to amplify sound. Hearing aids are non-invasive and can improve the hearing of an individual. However, hearing aids are often very conspicuous and embarrassing for the wearer, and hearing aids cannot restore hearing to normal levels. In addition, hearing aids amplify sounds indiscriminately, sometimes amplifying sounds that the individual does not want to hear, such as ambient noise. There is a need in the art for a solution to hearing impairment caused by antibiotics. Summary of the Invention

[0004] The present invention provides a method for preventing or treating hearing loss, comprising the step of administering an effective amount of a pharmaceutical composition containing a therapeutically active agent to an animal or human in need thereof, wherein the therapeutically active agent comprises: piperlongumamide (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and its derivatives.

[0005] The present subject matter 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, wherein the active agent includes: piperlongumamide (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and its derivatives.

[0006] The subject matter of the present invention also includes a kit made from the following items: an active agent, wherein the active agent includes: piperlongumamide (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and its derivatives or pharmaceutically acceptable salts thereof; and one or more of the following: (A) at least one antibiotic; (B) at least one cancer drug; or (C) instructions for preventing hearing loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The accompanying drawings incorporated in and constituting a part of this specification illustrate several aspects and together with the description serve to explain the principles of the invention.

[0008] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0009] Figure 1 A is a diagram showing (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one (piper longumamide) having a structure represented by the formula shown. Figure 1 B is a diagram showing the formula of a derivative of piperlongumamide.

[0010] Figure 2 Synthetic schemes for preparing the derivatives of the present invention are presented.

[0011] Figure 3 Synthetic schemes for preparing the derivatives of the present invention are presented.

[0012] Figure 4A A and B show synthetic schemes for preparing the derivatives of the present invention.

[0013] Figure 5A-C shows that piperlongumide protects zebrafish neuromasts from neomycin-induced hair cell death (A and B) and piperlongumide protects zebrafish neuromasts from gentamicin-induced hair cell death (C).

[0014] Figure 6 It is shown that piperlongum amide derivatives protect zebrafish neuromasts from excitotoxic damage. 5dpf Tg (Brn3c: mGFP) larvae were used for the experiment. Zebrafish were pretreated with kainic acid for one hour (300 μM) to simulate the excitotoxic damage induced by noise exposure, and then post-treated with piperlongum amide-25 for 2 hours (at concentrations of 0.001 μM, 0.1 μM, 10 μM, and 100 μM). Control animals were treated with DMSO (-) or piperlongum amide-25 (+) alone. The animals were then fixed and immunostained for GFP. Three neuromasts were assessed at the same anatomical position in each animal (n=5) for quantification. Data were plotted as mean + SD. Statistical testing was performed using one-way analysis of variance and Dunnett's post hoc test (P < 0.0001 compared to KA).

[0015] Figure 7 It is shown that piperlongumamide protects mouse embryonic fibroblasts (MEF) from damage by aminoglycoside antibiotics. MEFs were incubated with neomycin (100 μM) or gentamicin (8 μM) for 15 hours with or without piperlongumamide (0.1 nM-5 μM). Cell viability was assessed using an MTT (3-(4-, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assay (Thermo Fisher). Data were plotted as mean + SD. Statistical analysis was performed using one-way ANOVA and Dunnett's post hoc test (*P < 0.05, **P < 0.01 compared to Neo or GM, respectively).

[0016] Figure 8A(E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one preserves normal hair cell function in antibiotic-exposed mice (A) and B. C57B16 (Cdh23 corrected) (6-7 weeks old) were exposed to vehicle + kanamycin (Veh+Kan) (700 mg / kg bw twice daily for 14 days) (shown as circles with four interruptions); or (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one + kanamycin (PLM+Kan) and (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one (40 mg / kg bw once daily for 17 days) (shown as circles without interruptions). Shown are age-matched controls (age, shown as circles with three interruptions). Record the auditory brainstem response (ABR) thresholds of frequencies (4kHz, 5.6kHz, 8kHz, 16kHz, 22.6kHz, 32kHz, 45.2kHz, and 64kHz) before and after the treatment regimen. Compared with independent Kan-treated animals, when treating animals with PLM at 22.6kHz, we observed significant protection. Statistical analysis was performed with repeated measures analysis of variance (ANOVA) between the groups. B shows that in C57B16 mice over 17 days of treatment regimen, with and without kanamycin exposure, piperlongumamide (E) -1- (3- (3,4,5- trimethoxyphenyl) acryloyl) -S, 6- dihydropyridine -2 (1H) -one was non-toxic.

[0017] FIG9 shows that piperlongumamide (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and its derivatives reduce NF-kB activity in zebrafish.

[0018] Figure 10 Shown is the testing of the excitotoxicity of piperlongumamide derivatives in a zebrafish model.

[0019] Figure 11 It was shown that the piperlongumamide derivatives PG3, PG18, PG25, PG53 and PG54 exhibited better excitotoxicity than piperlongumamide in a zebrafish model. DETAILED DESCRIPTION

[0020] The present invention can be more easily understood by reference to the following detailed description of the present invention and the examples included therein. Before disclosing and describing the compounds of the present invention, compositions, products, systems, devices and / or methods, 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, so they can certainly vary. It should also be understood that the terms used herein are only used to describe the purpose of specific aspects and are not intended to be restrictive. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described.

[0021] While aspects of the invention may be described and claimed within specific statutory categories, such as the systems statutory category, this is for convenience only, and one skilled in the art will understand that each aspect of the invention may be described and claimed within any statutory category. Unless expressly provided otherwise, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a particular order. Thus, when a method claim does not specifically provide in the claim or specification that the steps are to be limited to a particular order, it is in no way intended that an order be inferred in any respect. This applies to any possible non-explicit basis for interpretation, including matters of logic regarding the arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0022] 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 as 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 program that are intended to prevent hearing loss due to noise or aging by protecting inner ear cells from death, as well as hearing loss caused by chemotherapy or antibiotic-induced hearing loss. Therapeutic agents refer to chemicals used to treat or alleviate a disease condition or ailment.

[0023] 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 as 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 program that are intended to prevent hearing loss due to noise or aging by protecting inner ear cells from death, as well as hearing loss caused by chemotherapy or antibiotic-induced hearing loss. Therapeutic agents refer to chemicals used to treat or alleviate a disease condition or ailment.

[0024] Now refer to Figure 1, showing piperlongamide (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one. Piperlongamide is an alkaloid extracted from Piper longum Linn that exhibits anti-atherosclerotic, anxiolytic, antidiabetic, antidepressant, antibacterial, antiplatelet aggregation, anxiolytic, and anti-inflammatory properties. Piperlongamide prevents the production of tumor necrosis factor-α and interleukin-6. It is also known as Piplartine, 5,6-dihydro-1-[(2E)-1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propen-1-yl]-2(1H)-pyridinone, Piplartine, 5,6-dihydro-1-(1-oxo-3-[3,4,5-trimethoxyphenyl]-trans-2-propenyl)-2[1H]-pyridinone.

[0025] Various derivatives of piperlongum amide have also shown efficacy as therapies for treating and / or preventing hearing loss. The general structure of derivatives within the scope of the present invention is shown. More specifically, acryloyl derivatives are contemplated. The acryloyl group is an enone form having the structure H2C=CH-C(=O)-; it is an acyl group derived from acrylic acid.

[0026] The derivatives of piperlongum amide are formed by the following structure: The wavy bonds indicate E and Z isomers. N=1, 2, 3, 4, 5, 6 R=H, alkyl, hydroxyl, thiol, halogen, acid, ester, amide, amine, substituted alkyl chain, substituted cyclic alkyl ring, heterocycle, piperidine, cyclohexanol, heteroaromatic ring, substituted heteroaromatic ring, cycloaliphatic ring, heterocycloaliphatic ring. In another aspect, the derivative of piper longum amide is formed by the following structure: The wavy bonds indicate E and Z isomers. N=0, 1, 2, 3, 4 R═H, alkyl, alkoxy, hydroxy, thiol, halogen, acid, ester, amide, amine, substituted alkyl chain, substituted cyclic alkyl ring, heterocycle, piperidine, cyclohexanol, heteroaromatic ring, substituted heteroaromatic ring, cycloaliphatic ring, heterocycloaliphatic ring. As used herein, term " alkyl " is a branched or unbranched saturated hydrocarbon radical with 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl etc. Alkyl groups can be cyclic or acyclic. Alkyl groups can be branched or unbranched. As described herein, for example, alkyl groups can be replaced by one or more groups including but not limited to alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, sulfo-oxo or mercaptan. A " low alkyl " group is an alkyl group containing from one to six (for example, one to four) carbon atoms. The term alkyl group can also be C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, etc., up to and including C1-C24 alkyl. Cyclic compound (or cyclic compound) is the term for a compound in the chemical field, wherein one or more series of atoms are connected to form a ring. The size of the ring may vary from three to many atoms, and includes instances in which all atoms are carbon (i.e., carbocycles), no atoms are carbon (inorganic cyclic compounds), or both carbon and non-carbon atoms are present (heterocyclic compounds).

[0027] The basic structure of the derivatives of piperlongum amide is shown in Figure 1 B and Table 1 is:

[0028] R1 = 2-furyl, 2-phenylthio, phenyl 3-methoxyphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 4-chloro, 4-nitro, 2,2-difluoro-1,3-benzodicyclopentadiene, 2-naphthyl; and R2 = N-linked β-, γ-, δ-, or ε-lactam.

[0029] Table 1Table 1

[0030] Can be achieved through Figures 2-4A The piperlongum amide derivatives of Table 1 were synthesized from the compounds described in Table B. The piperlongum amide derivatives were derived from the following reaction scheme:

[0031] Now refer to Figure 3Semisynthetic optimization of natural products is a method that leads to FDA-approved drugs with significant clinical activity. To optimize the therapeutic potential of PG, we propose the following medicinal chemistry plan to further improve the otoprotective and biopharmaceutical properties of PG.

[0032] Synthetic analogs focused on para-substituted aromatic compounds to amplify the SAR on the aromatic ring, and we proposed two main aromatic series, i) mono- and polysubstituted benzene rings and ii) unsubstituted or substituted heteroarenes.

[0033] In Scheme 1, briefly, commercially available 3-aryl-α,β-unsaturated carboxylic acids are converted to acid chlorides with oxalyl chloride. Various lactams are activated with n-butyl lithium and reacted with acids. 3-aryl-α,β-unsaturated carboxylic acids are converted to acid chlorides with oxalyl chloride. Various lactams are activated with n-butyl lithium and reacted with acid chlorides to produce the final products. R1 = 3-methoxy, 3-trifluoromethyl, 4-trifluoromethyl, 4-cyano, 4-nitro, 4-chloro, 3,4,5-trimethoxy, 2,2-difluoroacetal ( Figure 2 ).

[0034] To synthesize piperlongumamide derivatives with optimized aromatic groups i) mono- and polysubstituted benzene rings, and ii) unsubstituted or substituted heteroaromatics, Scheme 2 ( Figure 2 ): The synthesis of β-aryl-α,β-unsaturated carboxylic acids is followed by the reaction of Scheme 1 ( Figure 2 ).

[0035] In Scheme 2, β-aryl-α,β-unsaturated carboxylic acids are synthesized according to Scheme 2. Briefly, an aryl or heteroaryl aldehyde is reacted with methyl 2-acetic acid bromide under Wittig conditions. Saponification of the methyl ester yields the β-aryl-α,β-unsaturated carboxylic acid. We plan to synthesize PG analogs with at least 50-75 unique aromatic groups.

[0036] To synthesize the piperlongumamide derivatives with optimized linker groups i) saturated amide linker, ii) reduced alkyl linker, iii) β-aryl-α,β-unsaturated sulfonamide, and iv) saturated sulfonamide, Scheme 3 ( Figure 4A ) and 4( Figure 4B ):

[0037] In the case of piperidine amide (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one analogs according to Scheme 1 ( Figure 2 ) and after reduction according to Scheme 3, a saturated amide linker and an alkyl linker group are generated. According to Scheme 4 ( Figure 4B) will synthesize β-aryl-α,β-unsaturated sulfonamides. In Scheme 4, 2-bromomethylsulfonyl chloride can react with various lactams in the presence of n-butyllithium to produce sulfonamides.

[0038] Compounds are shown to prevent hair cell apoptosis. The presented models and data identify compounds that can combat hair cell loss in animals. The models reveal properties essential for otoprotective compounds, such as high efficacy against hair cell loss and relatively low toxicity. Compounds are shown to have high efficacy and high affinity in mouse and zebrafish models used to demonstrate protection against hair cell loss. Zebrafish lateral line neuromasts are a valuable model for testing compounds for protection against hearing loss in vivo because their HCs are thought to be homologous to HCs in the mammalian inner ear and are easily accessible to drugs in vivo. Teitz et al., J. Exp. Med. 2; 215(4): 1187-1203(2018). Mouse models involving embryonic fibroblast viability have proven effective in validating the therapeutic use of compounds against hearing loss caused by cancer treatments such as cisplatin, noise, antibiotics, and aging. Teitz et al., J. Exp. Med. 2; 215(4): 1187-1203(2018).

[0039] 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, for example, by instillation or injection into the ear, insufflation (such as into the ear), intravenous, topical or oral administration. The compounds can be synthesized by a variety of methods known in the art.

[0040] The properties of the pharmaceutical composition for administration depend on the mode of administration and can be easily determined by one of ordinary skill in the art. In various aspects, the pharmaceutical composition is sterile or sterilizable. The therapeutic compositions characterized in the present invention may contain carriers or excipients, many of which are known to those skilled in the art. Excipients that can 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 regulatory compounds characterized in the present invention can be administered by any standard route of administration. For example, administration can be parenteral, intravenous, subcutaneous, or oral. The regulatory compound can be formulated in various ways according to the corresponding route of administration. For example, a liquid solution can be prepared for administration by dropping into the ear, for injection, or for ingestion; a gel or powder can be prepared for ingestion or topical application. Methods for preparing such formulations are well known and may be found, for example, in Remington's Pharmaceutical Sciences, 18th ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990.

[0041] In various aspects, the disclosed pharmaceutical compositions include as active ingredients the disclosed compounds (including one or more pharmaceutically acceptable salts thereof), pharmaceutically acceptable carriers, and optionally other therapeutic ingredients 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 nature and severity of the disease to which the specific host and active ingredient are administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any method well known in the pharmaceutical field.

[0042] In various aspects, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier and a compound of the present invention or a pharmaceutically acceptable salt of the compound. The compound of the present invention or its pharmaceutically acceptable salt may also be included in the pharmaceutical composition in combination with one or more other therapeutically active compounds. The pharmaceutical carrier used may be, for example, a solid, liquid or gas.

[0043] The pharmaceutical composition of the present invention includes a compound of the present invention (or a pharmaceutically acceptable salt thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more other therapeutic agents or adjuvants. The present composition includes compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, but the most suitable approach in any given case will depend on the nature and severity of the illness to which the specific host and active ingredient are administered. The pharmaceutical composition can be conveniently presented in unit dosage form and prepared by any method known in the pharmaceutical field.

[0044] 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 hydroxypropylcellulose, can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof in oils. In addition, preservatives can be included to prevent the harmful growth of microorganisms.

[0045] Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. In addition, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be an effective fluid for easy injection.

[0046] In various aspects, the compounds of the present invention can be used in combination with one or more other drugs in the form of a kit to prevent, control, ameliorate, or reduce the risk of hearing loss when other drugs are known to damage hearing, such as antibiotics. Certain antibiotics, particularly aminoglycoside antibiotics (such as gentamicin, streptomycin, and neomycin), have hearing-related side effects that are most common in people with kidney disease or pre-existing ear or hearing problems.

[0047] Now refer to Figure 5A-C, (E) -1- (3- (3,4,5-trimethoxyphenyl) acryloyl) -S, 6-dihydropyridin-2 (1H) -one (piper longum) protects zebrafish lateral line neuromasts from aminoglycoside-induced hair cell loss in vivo. Zebrafish lateral line neuromast HC counts are commonly used as an in vivo model for screening otoprotectants. Zebrafish (Danio rerio) experimental larvae were obtained from adult fish pairs raised at Creighton University using standard methods approved by the Institutional Animal Care and Use Committee. The fish used were Tg (pou4f3: mGFP) expressing membrane-bound GFP in HC. The experimental fish were maintained at 28.5°C in E3 medium (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2 and 0.33nM MgSO4, pH 7.2). The animals were anesthetized by freezing after drug treatment and before fixation. The neuromasts examined, SO3 and O1-2, are part of the cranial system and include the otic, median, and opercular neuromasts. The zebrafish lateral line neuromasts are a valuable system for testing compounds for protection against aminoglycoside toxicity in vivo, as their HCs are thought to be homologous to those in the mammalian inner ear and are readily accessible to drugs.

[0048] For screening, 5-day postfertilization (dpf) Tg(brn3c:GFP) juveniles were preincubated with 10 nM, 10 nM, 1 μM, 10 μM, 100 μM, and 300 μM (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one for 1 h and subsequently incubated with 200 μM neomycin (Neo) for 30 min (as described in the previous section). Figure 5A and B), or incubated with 100 μM gentamicin (GM) for 1 h (as Figure 5C DMSO, Neo alone, and GM alone were used as controls for their respective experiments ( Figure 5A and C).

[0049] Subsequently, the animals were transferred to E3 water for 5 hours and fixed overnight in 4% paraformaldehyde (PFA). Neuromast HCs were immunostained with anti-otoferlin (HCS-1, DSHB) and anti-GFP (NB100-1614, Novus Biologicals). These two markers were used to detect and count neuromast HCs, to reduce the chance of losing some HCs after treatment, because as previously noted, incubation with the compound would affect GFP expression, making it more difficult to detect under a fluorescence microscope. Identify otic neuromasts, median neuromasts, and tegmental neuromasts, and manually count HCs at SO3 (supraorbital neuromasts) and O1-2 (ear neuromasts) using a Zeiss AxioSkop 2 fluorescence microscope with a 40x oil objective. Control animals were treated with DMSO (-) or Neo or GM (+) alone. The animals were then fixed and immunostained for GFP and otoferlin. Three neuromasts were assessed at the same anatomical position in each animal (n=5) for quantification. Data were plotted as mean + SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett's post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001 compared with Neo or GM, respectively).

[0050] Now refer to Figure 6 , piperlongumine-25 protects neuromast hair cells from excitotoxic damage induced by kainic acid (KA). To screen piperlongumine derivatives that protect against noise-induced hearing loss, we used a zebrafish model that mimics excitotoxic damage. Tg(brn3c:GFP) larvae at five days post fertilization (dpf) were incubated with 300μM KA for 1 hour, followed by incubation with 0.001μM, 0.1μM, 10μM, and 100μM piperlongumine-25 for 2 hours. DMSO and KA were used as controls. In addition, Tg(brn3c:GFP) larvae at five days post fertilization (dpf) were incubated with piperlongumine-25 alone for 2 hours to confirm that it did not cause any systemic toxicity by itself.

[0051] Now refer to Figure 7(E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one protects mouse embryonic fibroblasts (MEFs) from aminoglycoside antibiotics. MEFs were incubated with neomycin (100 mM) or gentamicin (8 mM) in the presence or absence of 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, and 500 nM of (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one (0.1 nM - 5 mM) for 15 hours. Cell viability was assessed using the MTT (3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability kit test (Thermo Fisher Scientific). Data were plotted as mean + SD. Statistical testing was performed with one-way ANOVA and Dunnett’s post-test (*P < 0.05, **P < 0.01 compared to Neo or GM, respectively). 100% protection against neomycin was observed for 0.1 nM to 10 nM of (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and 100% protection against gentamicin was observed for 0.1 nM to 1 nM of (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one.

[0052] Reference is now made to Figure 8A , Capsaicin protects mice from aminoglycoside toxicity in vivo. 6-7 week old C57BL / 6 (Cdh23 corrected), male and female were mixed throughout the experiment. The procedure was approved by the IACUC committee of Clayton University. Mice were treated with capsaicin (40 mg / kg b.w., for 17 days, IP) in the presence or absence of kanamycin treatment (700 mg / kg b.w., twice a day for 14 days, s.q.). The protective effect of capsaicin against hearing loss was assessed by measuring ABR threshold shift in the mouse ears.

[0053] Before ABR assessment, animals were anesthetized with a mixture of ketamine / xylazine. Subcutaneous needle electrodes were inserted into the auricle (inverting), the top of the skull (non-inverting), and the base of the tail (ground). BioSigRZ software and RZ6 multi-I / O processor system (Tucker-Davis Technology, Florida) were used to generate a short pure tone (Tone burst) of 5ms duration with a 0.5ms cosine square envelope (envelope) delivered at a rate of 21 stimulations per second with alternating polarity. Via the loudspeaker (MF1, TDT, FL) placed 10cm before the animal's auricle, stimulation was presented as an open field. The responses induced (20x) were expanded, bandpass filtered (300-3,000Hz) and the mean value of 512 responses of 10ms duration was recorded. The stimulus intensity was reduced in 5dB increments, from 100dB SPL to 0dB SPL. Thresholds at 4, 5.6, 8, 16, 22.6, 32, 45.2, and 64 kHz were identified by visual inspection of the stacked waveforms as the lowest level where reproducible responses could be identified. Before each start, the stimulus presentation speaker (MF1) was calibrated using a 1 / 4" microphone (PCB-378C10; Piezotronics, NY) (also placed 10 cm in front of the speaker). Similar experiments have been previously described in Rai V. et al., Sci Rep. 2020 Sep 16;10(1):15167.

[0054] Saline was administered to the aged control group of mice. A significant difference in threshold was observed at 22.6 kHz (p = 0.0408, two-way ANOVA with Holm Sidak multiple comparisons). Data are presented as mean ± SD, n = 3-4 / group. Figure 8B Kanamycin alone and in combination with (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one showed no systemic toxicity in C57BL / 6 mice (Cdh23 corrected) during a 17-day exposure period.

[0055] Reference is now made to Figure 9, a series of experiments were performed to confirm that (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(lH)-one and its derivatives act by inhibiting NF-kB activity. A zebrafish reporter line expressing GFP under the NF-kB promoter was used. In individual experiments, 5 dpf animals were incubated with vehicle (E3 water) for 1 hour or with 10 ng / mL TNFa for 30 minutes to induce the NF-kB pathway. Next, zebrafish were incubated with vehicle (DMSO 0.1%) for 2 hours or with one of (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(lH)-one or its derivatives at 1 nM and 100 nM. Zebrafish neuromasts were fixed and immunostained for GFP (green) and otoferlin (red). The fluorescence intensity of GFP, used as an indicator of NF-kB activation, was quantified using ImageJ and expressed as arbitrary fluorescence units / neuromast. TNFa (an activator of NF-kB) was used as a positive control. Zebrafish incubated with 100 nM of (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(lH)-one or 1 nM of piperlongumine-25 (PG25 (SHJ-25)) showed a decrease in NF-kB pathway activation compared to fish exposed to KA or TNFa alone. 100 nM of piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(lH)-one or 1 nM of piperlongumine-25 (PG25 (SHJ-25)) showed a decrease in NF-kB pathway activation compared to fish exposed to KA or TNFa alone.

[0056] Zebrafish were incubated with vehicle alone (control), KA 300 mM (KA) or TNFa (10 ng / mL) (TNFa) or a combination thereof with one of piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(lH)-one or its derivatives at a concentration of 1 nM or 100 nM for 2 hours. Animals were fixed and immunostained for GFP (green) and otoferlin (red). GFP fluorescence intensity was quantified using ImageJ and expressed as mean + / - SEM. Statistical analysis: one-way ANOVA. (*p,0.05, **p<0.01, ****p<0.0001 compared to the corresponding ototoxin alone). 100 nM of piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(lH)-one or 1 nM of piperlongumine-25 PG25 showed a decrease in NF-kB pathway activation compared to fish exposed to KA or TNFa alone.

[0057] Now refer to Figure 10 and 11 , compared with piperlongum amide against kainic acid (KA)-induced hair cell death, the tested piperlongum amide derivatives, PG3 (SHJ-3), PG 18 (SHJ-18), PG25 (SHJ-25), PG 53 (SHJ-53) and PG54 (SHJ-54) showed better performance.

[0058] Figure 10 The excitotoxicity of piperlongumamide derivatives was tested in a zebrafish model. Fish at 5 dpf were incubated with KA 300 μM for 1 hour, followed by incubation with one of the piperlongumamide derivatives ranging from 1 nM to 100 μM for two hours. The animals were fixed and immunostained for the hair cell marker, otoferlin. Neuromast hair cells were counted under a fluorescence microscope. At least 3 neuromasts in the lateral line of each fish were examined, for a total of 6 fish per treatment. Results are expressed as mean + / - SD. Control, KA only, PG only, PG + KA, derivatives that did not protect against excitotoxicity, derivatives that performed better than PG. ( Figure 10 Figure 1 shows the legend).

[0059] Figure 11 PG3, PG18, PG25, PG53, and PG54 were shown to outperform piperlongum amide against excitotoxicity in a zebrafish model. 5dpf fish were incubated with KA 300 μM for 1 hour, followed by an additional 2 hours with different concentrations of one of the PG derivatives. The animals were fixed and immunostained for otoferlin. Neuromast hair cells were quantified in at least 3 rostral neuromasts per fish, with a total of 6 fish per treatment. Results are expressed as mean + / - SD. Statistical analysis: One-way ANOVA *P<0.05, **P<0.01, P<0.001 compared to KA alone. These five piperlongum amide derivatives performed better than piperlongum amide at at least one dose.

[0060] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0061] While the invention has been described with reference to details of the illustrated embodiments, these details are not intended to limit the scope of the invention as defined in the appended claims.The embodiments of the invention in which an exclusive property or privilege is claimed are defined in the claims.

Claims

1. A method for preventing or treating hearing loss, comprising: administering to an animal or human in need thereof an effective amount of a pharmaceutical composition containing a therapeutically active agent, wherein The therapeutically active agent is selected from the group consisting of piperlongamide or a derivative of piperlongamide.

2. The method of claim 1 comprising protecting inner ear cells from death caused by antibiotics.

3. The method of claim 1, wherein the antibiotic is an aminoglycoside.

4. The method of claim 1 comprising protecting inner ear cells from noise-induced death.

5. The method of claim 1, comprising protecting inner ear cells from cell death caused by cisplatin treatment.

6. A composition for preventing or treating hearing loss by protecting inner ear cells from death, wherein said composition is an effective amount of an active agent, wherein the active agent is selected from the group consisting of: Piper longum amide; a derivative of piper longum amide; or a pharmaceutically acceptable salt thereof.

7. The composition of claim 6, which is used to protect the inner ear cells Protection from antibiotic-induced death.

8. The composition of claim 6, which is used to protect the inner ear cells Protection from noise-induced death.

9. The composition of claim 6, which is used to protect the inner ear cells Protection from cisplatin-induced mortality.

10. The composition of claim 6, wherein the derivative of piperlongumamide (PG3(SHJ-3)) is composed of the following formula:

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