Compositions and Methods for Preventing and Treating Hearing Loss

Abstract

In one aspect, the use of a compound as an active agent for treating and preventing hearing impairment is disclosed, as well as methods of using a composition for treating and / or preventing hearing impairment or disorder. In zebrafish and mice, (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and derivatives exhibited excellent protective effects against antibiotic-induced hearing loss. In one aspect, (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and derivatives can be used as a therapy for treating and / or preventing hearing loss. This abstract is intended as a scanning tool for retrieval purposes in a specific field and is not intended to limit the present invention.

Description

[0001] Cross - Reference to Related Applications: This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 078,571, filed on September 15, 2020 (which is hereby incorporated herein by reference in its entirety), under 35 USC§119(e) and 35 U.S.C.§111(a).

[0002] Statement Regarding Federally Sponsored Research or Development: This invention was made with government support under NIH grants R01DC015010, R01DC015444, R43DC019065, Navy / ONR grant N00014 - 18 - 1 - 2507, and Army / MRMC grant W81XWH - 18 - 1 - 0442. The government has certain rights in this invention. Background of the Invention

[0003] (1) Field of the Invention.

[0004] The present invention relates to the therapeutic use of active agents, such as for the treatment, inhibition, and / or prevention of hearing loss.

[0005] (2) Background Art, including information disclosed under 37 CFR 1.97 and 37 CFR 1.98.

[0006] More than 35 million Americans suffer from hearing impairment. In mammals, hair cell damage is permanent. Antibiotic - induced hearing loss (AIHL) is a permanent side effect that often requires medical care. The rate of hearing loss in newborns admitted to neonatal intensive care units is ten times that of non - admitted newborns and is often associated with the administration of antibiotics. The spiral cochlea in 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 received by receptors on auditory nerve fibers, which send electrical impulses to the brain. Inner ear cells lining the cochlea can be damaged by the use of antibiotics, resulting in a condition called sensorineural hearing impairment.

[0007] Currently, there are no clinically proven drugs for the treatment of hearing impairment (sensorineural and neural) or tinnitus related to the inner ear. Thus, there is a great need for drugs that can be used to prevent, reduce, or eliminate hearing impairment (or tinnitus). For individuals with severe sensorineural hearing impairment, the most common remedy is a hearing aid, which functions to amplify sound. Hearing aids are non - invasive and can improve an individual's hearing. 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 sound 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 antibiotic - induced hearing loss. Summary of the Invention

[0008] The present invention provides a method for preventing or treating hearing loss, the method comprising the step of administering to an animal or a human in need thereof an effective amount of a pharmaceutical composition comprising a therapeutic active agent, wherein the therapeutic active agent comprises: piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and its derivatives.

[0009] The subject matter of the present 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, and the active agent comprises: piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and its derivatives.

[0010] The subject matter of the present invention also includes a kit made of the following: an active agent, wherein the active agent comprises: piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and its derivatives or a pharmaceutically acceptable salt 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 impairment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings incorporated in and forming a part of this specification illustrate several aspects and, together with the description, are used to explain the principles of the present invention.

[0012] This patent or application file contains at least one color drawing. Copies of this patent or patent application publication with one or more color drawings will be provided by the Patent Office upon request and payment of the necessary fees.

[0013] Figure 1A is a diagram showing (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one (piperlongumine) having a structure represented by the shown formula. Figure 1B is a diagram showing the formula of a derivative of piperlongumine.

[0014] Figure 2 Shows a synthetic scheme for preparing the derivatives of the present invention.

[0015] Figure 3 Shows a synthetic scheme for preparing the derivatives of the present invention.

[0016] Figure 4A and Figure 4B Shows a synthetic scheme for preparing the derivatives of the present invention.

[0017] Figures 5A - 5C showed that piperlongumine protected zebrafish neuromasts from neomycin-induced hair cell death ( Figure 5A and Figure 5B ) and piperlongumine protected zebrafish neuromasts from gentamicin-induced hair cell death ( Figure 5C ).

[0018] Figure 6 showed that piperlongumine derivatives protected zebrafish neuromasts from excitotoxic injury. 5 dpf Tg(Brn3c:mGFP) larvae were used for the experiment. Zebrafish were pretreated with kainic acid (300 μM) for one hour to mimic excitotoxic injury induced by noise exposure and then post-treated with piperlongumine-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 piperlongumine-25 alone (+). The animals were then fixed and immunostained for GFP. Quantification was performed by evaluating three neuromasts at the same anatomical location in each animal (n = 5). Data were plotted as mean + SD. Statistical tests were performed using one-way ANOVA and Dunnett's post hoc test (P < 0.0001 compared to KA).

[0019] Figure 7 showed that piperlongumine protected mouse embryonic fibroblasts (MEFs) from aminoglycoside antibiotics damage. MEFs were incubated with neomycin (100 μM) or gentamicin (8 μM) in the presence or absence of piperlongumine (0.1 nM - 5 μM) for 15 hours. Cell viability was assessed using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability kit (Thermo Fisher). Data were plotted as mean + SD. Statistical tests were performed using one-way ANOVA and Dunnett's post hoc test (*P < 0.05, **P < 0.01 compared to Neo or GM, respectively).

[0020] Figure 8A and Figure 8BIt shows that in mice exposed to antibiotics, (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one retains normal hair cell function (A). C57Bl6 (Cdh23 corrected) (6 - 7 weeks old) were exposed to vehicle + kanamycin (Veh+Kan) (700 mg / kg b.w. twice a day for 14 days) (shown as circles with four breaks); 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 b.w. once a day for 17 days) (shown as circles without breaks). Age-matched controls (age, shown as circles with three breaks) were shown. Auditory brainstem response (ABR) thresholds at frequencies (4 kHz, 5.6 kHz, 8 kHz, 16 kHz, 22.6 kHz, 32 kHz, 45.2 kHz, and 64 kHz) were recorded before and after the treatment protocol. Compared to animals treated with Kan alone, we observed significant protection when animals were co-treated with PLM at 22.6 kHz. Statistical analysis was performed using repeated measures analysis of variance (ANOVA) between groups. B shows that piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one is non-toxic in C57Bl6 mice over a 17-day treatment protocol with and without kanamycin exposure.

[0021] Figure 9 shows that in zebrafish, piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one and its derivatives reduce NF-kB activity.

[0022] Figure 10 It shows that the excitotoxicity of piperlongumine derivatives was tested in a zebrafish model.

[0023] Figure 11 It shows that in a zebrafish model, piperlongumine derivatives PG3, PG18, PG25, PG53, and PG54 exhibit better excitotoxicity than piperlongumine. Detailed Description

[0024] The present invention can be more readily understood by reference to the following detailed description of the invention and the examples included therein. Before the compounds, compositions, articles, systems, devices, and / or methods of the invention are disclosed and described, it is to be understood that, unless otherwise specified, they are not limited to particular synthetic methods or, unless otherwise specified, to particular reagents, and thus may of course vary. It is also to 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 or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are now described.

[0025] While aspects of the present invention may be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and those skilled in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly specified, no aspect of any method or aspect set forth herein is ever intended to be construed as requiring that its steps be performed in a particular order. Accordingly, when a method claim does not specifically recite steps in the claim or the specification as being limited to a particular order, no order is ever to be inferred in any respect. This holds for any possible non-explicitly construed basis, including logical issues regarding step or operational flow arrangement, simple meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0026] 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 a pharmaceutical composition, which is formulated according to the intended method of administration. The compounds of the present invention are defined as a therapeutic active agent in a treatment regimen or procedure, which is intended to prevent hearing loss caused by noise or aging by protecting inner ear cells from death, and to prevent hearing loss caused by chemotherapy or antibiotic-induced hearing loss. A therapeutic agent means a chemical substance for treating or alleviating a disease condition or ailment.

[0027] 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 a pharmaceutical composition, which is formulated according to the intended method of administration. The compounds of the present invention are defined as a therapeutic active agent in a treatment regimen or procedure, which is intended to prevent hearing loss caused by noise or aging by protecting inner ear cells from death, and to prevent hearing loss caused by chemotherapy or antibiotic-induced hearing loss. A therapeutic agent means a chemical substance for treating or alleviating a disease condition or ailment.

[0028] Referring now to FIG. 1, piperlonguminine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one is shown. Piperlonguminine is an alkaloid extracted from Piper longum Linn, and exhibits anti-atherosclerotic, anti-anxiety, anti-diabetic, anti-depressant, antibacterial, anti-platelet aggregation, anti-anxiety and anti-inflammatory properties. Piperlonguminine prevents the production of tumor necrosis factor-α and interleukin-6. It is also known as piplartin, 5,6-dihydro-1-[(2E)-1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propen-1-yl]-2(1H)-pyridinone, Piplartine (Piplartine), 5,6-dihydro-1-(1-oxo-3-[3,4,5-trimethoxyphenyl]-trans-2-propenyl)-2[1H]-pyridinone.

[0029] A variety of derivatives of piperlonguminine also show efficacy as a therapy for treating and / or preventing hearing loss. The general structure of the derivatives within the scope of the present invention is shown. More particularly, acryloyl derivatives are considered. The acryloyl group is in the enone form having the structure H2C=CH-C(=O)-; it is an acyl group derived from acrylic acid.

[0030] The derivatives of piperlonguminine are formed by the following structure:

[0031] The wavy bond represents E and Z isomers.

[0032] N = 1, 2, 3, 4, 5, 6

[0033] R = H, alkyl, hydroxy, mercapto, halogen, acid, ester, amide, amine, substituted alkyl chain, substituted cyclic alkyl ring, heterocycle, piperidine, cyclohexanol, heteroaromatic ring, substituted heteroaromatic ring, cycloaliphatic ring, heterocycloaliphatic ring. On the other hand, the derivatives of piperlonguminine are formed by the following structure:

[0034]

[0035] The wavy bond represents E and Z isomers.

[0036] N = 0, 1, 2, 3, 4

[0037] R = H, alkyl, alkoxy, hydroxy, mercapto, halogen, acid, ester, amide, amine, substituted alkyl chain, substituted cyclic alkyl ring, heterocycle, piperidine, cyclohexanol, heteroaromatic ring, substituted heteroaromatic ring, cycloaliphatic ring, heterocycloaliphatic ring.

[0038] As used herein, the term "alkyl" is a branched or unbranched saturated hydrocarbon group having from 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. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. As described herein, for example, the alkyl group can be substituted with one or more groups including but not limited to alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfonyloxy, or thiol. A "lower alkyl" group is an alkyl group containing from one to six (e.g., one to four) carbon atoms. The term alkyl group can also be a 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. A cyclic compound (or ring compound) is a term for a compound in the chemical field in which one or more series of atoms are connected to form a ring. The size of the ring can vary from three to many atoms and includes examples where all atoms are carbon (i.e., a carbocyclic ring), no atoms are carbon (inorganic cyclic compound), or both carbon and non-carbon atoms are present (heterocyclic compound).

[0039] The basic structure of the derivative of piperlongumineamide is shown in Figure 1B and Table 1 is:[[]]END]]

[0040]

[0041] R1 = 2-furyl, 2-phenylthio, phenyl 3-methoxyphenyl, 3-trifluoromethylphenyl, 4-trifluoromethylphenyl, 4-chloro, 4-nitro, 2,2-difluoro-1,3-benzodioxole, 2-naphthyl; and

[0042] R2 = N-linked β-, γ-, δ-, or ε-lactam.

[0043] Table 1

[0044]

[0045]

[0046]

[0047]

[0048] can be through Figures 2 - 4A and Figure 4BThe compounds described in [reference] were used to synthesize the piperlongumine derivatives shown in Table 1. The piperlongumine derivatives were derived from the following reaction scheme:

[0049] Now referring to Figure 3 , the semisynthesis 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 enhance the otoprotective and biopharmaceutical properties of PG.

[0050] The synthesized analogs focused on para-substituted aromatic compounds to expand the SAR on the aromatic ring. We proposed two main aromatic series, i) mono- and polysubstituted benzene rings and ii) unsubstituted or substituted heteroarenes.

[0051] In Scheme 1, briefly, commercially available 3-aryl-α,β-unsaturated carboxylic acids were converted to acid chlorides with oxalyl chloride. Various lactams were activated with n-butyllithium and reacted with the acid. The 3-aryl-α,β-unsaturated carboxylic acids were converted to acid chlorides with oxalyl chloride. Various lactams were activated with n-butyllithium and reacted with the acid chloride to produce the final product. R1 = 3-methoxy, 3-trifluoromethyl, 4-trifluoromethyl, 4-cyano, 4-nitro, 4-chloro, 3,4,5-trimethoxy, 2,2-difluoroacetal ( Figure 2 ).

[0052] To synthesize piperlongumine derivatives with optimized aromatic groups i) mono- and polysubstituted benzene rings, and ii) unsubstituted or substituted heteroarenes, follow Scheme 2 ( Figure 2 ):

[0053]

[0054] Scheme 2. Synthesis of β-aryl-α,β-unsaturated carboxylic acids.

[0055] The synthesized β-aryl-α,β-unsaturated carboxylic acids were followed by the reactions of Scheme 1 ( Figure 2 ).

[0056] In Scheme 2, β-aryl-α,β-unsaturated carboxylic acids were synthesized according to Scheme 2. Briefly, aryl or heteroaryl aldehydes were reacted with methyl 2-bromoacetate under Wittig conditions. Saponification of the methyl ester produced β-aryl-α,β-unsaturated carboxylic acids. We plan to synthesize PG analogs with at least 50 - 75 unique aromatic groups.

[0057] To synthesize piperlongumine derivatives with optimized linker groups i) saturated amide linker, ii) reduced alkyl linker, iii) β-aryl-α,β-unsaturated sulfonamide, and iv) saturated sulfonamide, follow Scheme 3 ( Figure 4A ) and 4 (Figure 4B ):

[0058] After the piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one analogues are synthesized according to Scheme 1 ( Figure 2 ) and reduced according to Scheme 3, saturated amide linkers and alkyl linker groups are generated. According to Scheme 4 ( Figure 4B ), β-aryl-α,β-unsaturated sulfonamides will be synthesized. In Scheme 4, 2-bromomethylsulfonyl chloride can react with various lactams in the presence of n-butyllithium to produce sulfonamides.

[0059] It is revealed that the compounds can prevent hair cell apoptosis. Through the presented models and data, the compounds are identified as being able to counter hair cell loss in animals. The models reveal the properties necessary for otoprotective compounds, such as high efficacy against hair cell loss and relatively low toxicity. It is revealed that the compounds have high efficacy and high affinity in mouse and zebrafish models used to demonstrate protection against hair cell loss. The lateral line neuromasts of zebrafish are a valuable model for testing the protective effect of compounds against hearing loss in vivo because their HCs are considered homologous to those in the mammalian inner ear and are readily accessible to drugs in vivo. Teitz et al., J. Exp. Med. [Journal of Experimental Medicine] 2; 215(4):1187-1203 (2018). A mouse model involving embryonic fibroblast viability has been demonstrated to be able to effectively validate 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. [Journal of Experimental Medicine] 2; 215(4):1187-1203 (2018).

[0060] The compounds and compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, the pharmaceutical compositions can be formulated for topical or systemic administration, such as by instilling or injecting into the ear, insufflating (such as into the ear), intravenously, topically, or orally. The compounds can be synthesized by a variety of methods known in the art.

[0061] The nature of the pharmaceutical composition for administration depends on the mode of administration and can be readily 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 buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), 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, the administration can be parenteral, intravenous, subcutaneous, or oral. The regulatory compounds 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 can be found, for example, in Remington's Pharmaceutical Sciences, 18th edition, edited by Gennaro, Mack Publishing Co., Easton, PA 1990.

[0062] In various aspects, the disclosed pharmaceutical compositions include the disclosed compounds (including one or more pharmaceutically acceptable salts thereof) as active ingredients, a pharmaceutically acceptable carrier, 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 particular host and the nature and severity of the condition for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any method well known in the pharmaceutical art.

[0063] 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 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 can be, for example, solid, liquid, or gaseous.

[0064] The pharmaceutical composition of the present invention comprises 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 additional therapeutic agents or adjuvants. The compositions of the present invention include compositions 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 particular host and the nature and severity of the condition for which the active ingredient is being administered. The pharmaceutical compositions may conveniently be presented in unit dosage form and prepared by any methods well known in the pharmaceutical art.

[0065] The pharmaceutical composition of the present invention suitable for parenteral administration may be prepared as a solution or suspension of the active compound in water. Suitable surfactants, such as hydroxypropylcellulose, may be included. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof in oils. In addition, preservatives may be included to prevent the harmful growth of microorganisms.

[0066] The pharmaceutical composition of the present invention suitable for injectable use comprises a sterile aqueous solution or dispersion. In addition, the composition may be in the form of a sterile powder for the extemporaneous preparation of such sterile injectable solution or dispersion. In all cases, the final injectable form must be sterile and must be a fluid effective for easy injection.

[0067] In various aspects, the compounds of the present invention may be used in combination with one or more other drugs in the form of a kit to prevent, control, improve hearing impairment, or reduce the risk of hearing impairment when it is already known that other drugs can damage hearing (such as antibiotics). Certain antibiotics, especially aminoglycoside antibiotics (such as gentamicin, streptomycin, and neomycin). The hearing-related side effects of these antibiotics are most common in people with kidney disease or those who already have ear or hearing problems.

[0068] Now refer to Figures 5A - 5C, (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one (piperlongumine) protects zebrafish lateral line neuromasts from aminoglycoside-induced hair cell loss in vivo. Zebrafish lateral line neuromast HC counting is a commonly used in vivo model for screening otoprotective agents. Zebrafish (Danio rerio) experimental larvae were obtained from adult fish pairs reared by standard methods approved by the Institutional Animal Care and Use Committee at Creighton University. The fish used were Tg(pou4f3:mGFP) that express membrane-bound GFP in HCs. The experimental fish were maintained at 28.5 °C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4, pH 7.2). The animals were cryoanesthetized after drug treatment and before fixation. The neuromasts SO3 and O1-2 examined are part of the cranial system and include the otic neuromasts, middle neuromasts, and opercular neuromasts. The zebrafish lateral line neuromasts are a valuable system for testing the protective effect of compounds against aminoglycoside toxicity in vivo because their HCs are thought to be homologous to those in the mammalian inner ear and are readily accessible to drugs.

[0069] For screening, 5-day post-fertilization (dpf) Tg(brn3c:GFP) larvae were pre-incubated with 10 nM, 10 nM, 1 μM, 10 μM, 100 μM, and 300 μM of (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one for 1 h, followed by co-incubation with 200 μM neomycin (Neo) for 30 min (as Figure 5A and Figure 5B shown), or co-incubation with 100 μM gentamicin (GM) for 1 h (as Figure 5C shown). DMSO, Neo alone, and GM alone were used as controls for their respective assays ( Figure 5A and Figure 5C ).

[0070] Subsequently, the animals were transferred to E3 water for 5 hours and fixed overnight in 4% paraformaldehyde (PFA). The neuromast HCs were immunolabeled with anti-harpegnathos (HCS-1, DSHB) and anti-GFP (NB100-1614, NovusBiologicals). These two markers were used to detect and count the neuromast HCs to reduce the chance of losing some HCs after treatment, because as previously noted, incubation with the compound affects GFP expression, making it more difficult to detect under a fluorescence microscope. The otic neuromasts, middle neuromasts, and tectal neuromasts were identified, and the HCs at SO3 (supraorbital line neuromast) and O1-2 (otic line neuromast) were manually counted using a Zeiss AxioSkop 2 fluorescence microscope with a 40× oil objective. Control animals were treated with DMSO (-) or Neo or GM alone (+). The animals were then fixed and immunostained for GFP and harpegnathos. Quantification was performed by evaluating three neuromasts at the same anatomical location in each animal (n = 5). The data were plotted as mean + SD. Statistical tests were performed using one-way ANOVA and Dunnett's post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001 compared with Neo or GM, respectively).

[0071] Now refer to Figure 6 , piperlongumine-25 protects neuromast hair cells from kainic acid (KA)-induced excitotoxic damage. To screen piperlongumine derivatives for protection against noise-induced hearing loss, we employed a zebrafish model mimicking excitotoxic damage. Five-day post-fertilization (dpf) Tg(brn3c:GFP) larvae were incubated with 300 μM KA for 1 hour, followed by incubation with 0.001 μM, 0.1 μM, 10 μM, and 100 μM piperlongumine-25 for 2 hours. DMSO and KA were used as controls. In addition, five-day post-fertilization (dpf) Tg(brn3c:GFP) larvae were incubated with piperlongumine-25 alone for 2 hours to confirm that it does not cause any systemic toxicity by itself.

[0072] 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 μM) or gentamicin (8 μM) for 15 h 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 μM). Cell viability was assessed using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability kit (Thermo Fisher Scientific). Data were plotted as mean + SD. Statistical tests were performed using one-way ANOVA and Dunnett's post hoc test (*P < 0.05, **P < 0.01 compared to Neo or GM, respectively). 100% protection against neomycin was observed with (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one from 0.1 nM to 10 nM and 100% protection against gentamicin with (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one from 0.1 nM to 1 nM.

[0073] Now refer to Figure 8A , Piperlongumine protects mice from aminoglycoside toxicity in vivo. C57BL / 6 (Cdh23 corrected) mice, 6 - 7 weeks old, were used with a mix of male and female throughout the experiment. The procedures were approved by the Creighton University IACUC committee. Mice were treated with piperlongumine (40 mg / kg b.w., for 17 days, IP) in the presence or absence of kanamycin treatment (700 mg / kg b.w., twice daily for 14 days, s.q.). The protective effect of piperlongumine against hearing loss was evaluated by measuring ABR threshold shift in the ears of mice.

[0074] Before ABR assessment, the animals were anesthetized with a mixture of ketamine / xylazine. Subcutaneous needle electrodes were inserted into the pinna (inverting), vertex (non-inverting), and base of the tail (ground). Using BioSigRZ software and an RZ6 multi-I / O processor system (Tucker-Davis Technologies, Florida), short tone bursts with a 5-ms duration, a 0.5-ms cosine-squared envelope, and an alternating polarity were delivered at a rate of 21 stimuli per second. The stimuli were presented as an open field via a speaker (MF1, TDT, FL) placed 10 cm in front of the animal's pinna. The evoked responses were amplified (20x), band-pass filtered (300 - 3,000 Hz), and the average of 512 responses with a 10-ms duration was recorded. The stimulus intensity was decreased in 5-dB increments from 100 dB SPL to 0 dB SPL. Thresholds at 4, 5.6, 8, 16, 22.6, 32, 45.2, and 64 kHz were identified by visually inspecting the stacked waveforms as the lowest level at which a reproducible response could be identified. Before each session, the stimulus presentation speaker (MF1) was calibrated with a 1 / 4” microphone (PCB-378C10; PCB Piezotronics, New York) (also placed 10 cm in front of the speaker). A similar experiment has been previously described in Rai V. et al., Sci Rep. September 16, 2020; 10(1):15167.

[0075] Saline was administered to the age-matched control mice. A significant difference in thresholds 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. Now refer Figure 8B , during the 17-day exposure period, in C57BL / 6 mice (Cdh23 corrected), neither kanamycin alone nor its combination with (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one showed systemic toxicity.

[0076] Now referring to Figure 9, a series of experiments were conducted to confirm that (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-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), KA 300 μM for 1 hour or with 10 ng / mL TNFa for 30 minutes to induce the NF-kB pathway. Next, the zebrafish were incubated with vehicle (DMSO 0.1%), or with 1 nM and 100 nM of (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one or one of its derivatives for 2 hours. The zebrafish neuromasts were fixed and immunostained for GFP (green) and otopetrin (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(1H)-one or 1 nM of piperlongumine-25 (PG25(SHJ-25)) showed reduced NF-kB pathway activation compared to fish exposed to KA or TNFa alone. Zebrafish incubated with 100 nM of piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one or 1 nM of piperlongumine-25 (PG25(SHJ-25)) showed reduced NF-kB pathway activation compared to fish exposed to KA or TNFa alone.

[0077] Zebrafish were incubated with vehicle alone (control), KA 300 μM (KA) or TNFa (10 ng / mL) (TNFa) or a combination thereof with 1 nM or 100 nM of piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one or one of its derivatives for 2 hours. The animals were fixed and immunostained for GFP (green) and otopetrin (red). The 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 respective ototoxin alone). Zebrafish incubated with 100 nM of piperlongumine (E)-1-(3-(3,4,5-trimethoxyphenyl)acryloyl)-S,6-dihydropyridin-2(1H)-one or 1 nM of piperlongumine-25 PG25 showed reduced NF-kB pathway activation compared to fish exposed to KA or TNFa alone.

[0078] Now referring to Figure 10 and Figure 11 ,the derivatives of piperlongumine tested, PG3 (SHJ-3), PG18 (SHJ-18), PG25 (SHJ-25), PG53 (SHJ-53) and PG54 (SHJ-54), showed better performance compared to piperlongumine against kainic acid (KA)-induced hair cell death.

[0079] Figure 10 Illustrated the excitotoxicity of piperlongumine derivatives tested in a zebrafish model. Fish at 5 dpf were incubated with 300 μM KA for 1 hour, followed by incubation with one of the piperlongumine derivatives at 1 nM to 100 μM for two hours. The animals were fixed and immunostained against the hair cell marker, otolithin. Neuromast hair cells were counted under a fluorescence microscope. At least 3 neuromasts of the head lateral line of each fish were examined, with 6 fish for each treatment. Results are expressed as mean + / - SD. Controls, KA only, PG only, PG+KA, derivatives without protective effect against excitotoxicity, derivatives showing better performance than PG. ( Figure 10 legend shown in

[0080] Figure 11 Illustrated that PG3, PG18, PG25, PG53 and PG54 showed better performance against excitotoxicity than piperlongumine in a zebrafish model. 5 dpf fish were incubated with 300 μM KA for 1 hour, followed by incubation with one of the derivatives of PG at different concentrations for another 2 hours. The animals were fixed and immunostained against otolithin. Neuromast hair cells were quantified in at least 3 rostral neuromasts of each fish, with 6 fish for each treatment. Results are expressed as mean + / - SD. Statistical analysis: compared to KA alone, one-way ANOVA *P<0.05, **P<0.01, P<0.001. These five piperlongumine derivatives showed better performance than piperlongumine at at least one dose.

[0081] All publications, patents, and patent applications mentioned in this specification are hereby 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.

[0082] While the invention has been described in detail with reference to the details of 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 claiming proprietary ownership or privilege are defined as follows:

Claims

1. Use of an effective amount of a pharmaceutical composition in the preparation of a medicament for preventing or treating hearing loss caused by excitotoxic injury in an animal or human in need thereof, wherein the pharmaceutical composition comprises a therapeutic active agent, wherein the therapeutic active agent is a derivative of piperlongumine, and wherein the derivative of piperlongumine is selected from the group consisting of: PG3 (SHJ-3), which is constituted by the following formula: ; PG18 (SHJ-18), which is constituted by the following formula: ; PG25 (SHJ-25), which is constituted by the following formula: ; PG53 (SHJ-53), which is constituted by the following formula (C16H15F2NO4): ; and PG54 (SHJ-54), which is constituted by the following formula (C15H13F2NO4): .

2. The use according to claim 1, wherein the use comprises protecting inner ear cells from death caused by antibiotics.

3. The use according to claim 2, wherein the antibiotic is an aminoglycoside.

4. The use according to claim 1, wherein the use comprises protecting inner ear cells from death caused by noise.

5. The use according to claim 1, wherein the use comprises protecting inner ear cells from death caused by cisplatin treatment.

6. The use according to claim 2, wherein the use comprises protecting inner ear cells from death caused by neomycin.

7. The use according to claim 2, wherein the use comprises protecting inner ear cells from death caused by kanamycin.

8. 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 is a derivative of piperlongumine, and wherein the derivative of piperlongumine is selected from the group consisting of: PG3 (SHJ-3), which has the following formula: ; PG18 (SHJ-18), which has the following formula: ; PG25 (SHJ-25), which has the following formula: ; PG53 (SHJ-53), which has the following formula (C16H15F2NO4): ; and PG54 (SHJ-54), which has the following formula (C15H13F2NO4): ; or a pharmaceutically acceptable salt thereof.

9. The composition according to claim 8, wherein the composition is for protecting the inner ear cells from death caused by antibiotics.

10. The composition according to claim 8, wherein the composition is for protecting the inner ear cells from death caused by noise.

11. The composition according to claim 8, wherein the composition is for protecting the inner ear cells from death caused by cisplatin treatment.

12. A kit, the kit comprising: an active agent, wherein the active agent is a derivative of piperlongumine, and the derivative of piperlongumine is selected from the group consisting of: PG3 (SHJ-3), which is constituted by the following formula: ; PG18 (SHJ-18), which is constituted by the following formula: ; PG25 (SHJ-25), which is constituted by the following formula: ; PG53 (SHJ-53), which is constituted by the following formula (C16H15F2NO4): ; and PG54 (SHJ-54), which is constituted by the following formula (C15H13F2NO4): ; or a pharmaceutically acceptable salt thereof; and one or more of the following: (A) at least one antibiotic; (B) at least one cancer drug; (C) instructions for preventing hearing impairment.

13. The kit according to claim 12, wherein the at least one antibiotic is an aminoglycoside.

14. The kit according to claim 12, wherein the at least one cancer drug is cisplatin.

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