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Assay for compounds that protect against sensory hair cell death and compounds identified by sameRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, In Vivo Diagnosis Or In Vivo Testing, Testing Efficacy Or Toxicity Of A Compound Or Composition (e.g., Drug, Vaccine, Etc.)Assay for compounds that protect against sensory hair cell death and compounds identified by same description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060188445, Assay for compounds that protect against sensory hair cell death and compounds identified by same. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0002] Aminoglycosides are clinically used drugs that cause dose-dependent sensorineural hearing loss (Smith et al., New Engl J Med, (1977) 296:349-53) and are known to kill hair cells in the mammalian inner ear (Theopold, Acta Otolaryngol (1977) 84:57-64). In the U.S, over 2,000,000 people receive treatment with aminoglycosides per year. The clinical efficacy of these drugs in treating resistant bacterial infections and their low cost globally account for their continued use and need. Cisplatin, a chemotherapeutic agent, is also used for its benefit to life despite its toxic effects on the hair cells of the inner ear. High frequency hearing loss (>8 kHZ) has been reported to be as high as 90% in children undergoing cisplatin therapy (Allen, et al., Otolaryngol Head Neck Surg (1998) 118:584-588). The incidence of vestibulotoxic effects of such drugs on patient populations has been less well studied. Estimates range between 3% and 6% with continued reports in the literature of patients with aminoglycoside induced vestibulotoxicity (Dhanireddy et al., Arch Otolarngol Head Neck Surg (2005) 131:46-48). Other clinically important and commonly used drugs also have documented ototoxic effects, including loop diuretics (Greenberg, Am J Med Sci, (2000) 319:10-24), antimalarial sesquiterpene lactone endoperoxides (i.e., artemesinins) (Toovey and Jamieson, Trans R Soc Trop Med Hyg (2004) 98:261-7), antimalarial quinines (Claessen, et al., Trop Med Int Health, (1998) 3:482-9), salicylates (Matz, Ann Otol Rhinol Laryngol Suppl (1990) 148:39-41), and interferon polypeptides (Formann, et al., Am J Gastroenterol (2004) 99:873-77). [0003] Zebrafish are an advantageous animal model for studying hair cell development and function (see, Grant, et al., Neuron (2005) 45:69-80). U.S. Pat. No. 6,656,449 discloses general methods of screening unknown agents in zebrafish for cell death activity, but does not describe preferential labeling of any particular tissue or cell type in a live zebrafish. Idziorek, et al., disclose that the cell impermeant nuclear dye YOPRO-1 (Molecular Probes, Eugene, Oreg.) does not interfere with cell viability of human immune cells, but does not disclose labeling zebrafish or hair cells. Harris, et al., disclose exposing zebrafish lateral hair cells labeled with the fluorescent vital dye DASPEI to neomycin and identifying regenerating hair cells (J Assoc Res Otolaryngol (2003) 4:219-34). Harris, et al., assert to have provided a preparation for studying and identifying genes that influence vertebrate hair cell death. None of the foregoing references disclose using zebrafish for screening for compounds that inhibit or prevent ototoxicity induced by one or more noxious stimuli. Accordingly, there remains a need for the identification of compounds that can counteract sensory hair cell loss. The present invention fulfills this and other needs. BRIEF SUMMARY OF THE INVENTION [0004] The present invention provides a methods for identifying compounds that decrease, inhibit or prevent sensory hair cell damage or death induced by one or more noxious stimuli, the methods comprising: [0005] a) preferentially labeling the lateral line hair cells of zebrafish in comparison to other cells, wherein the labeling is substantially non-toxic to the zebrafish hair cells, wherein said label is detectably distinct in a live cell in comparison to a dying cell or a dead cell; [0006] b) contacting the zebrafish with a test compound suspected of decreasing, inhibiting or preventing sensory hair cell damage or death; [0007] c) contacting the zebrafish with one or more noxious stimuli, each known to cause sensory hair cell damage or death; and [0008] d) detecting the label, wherein a compound that decreases, inhibits or prevents sensory hair cell damage or death is identified when the number of live lateral line hair cells is greater in zebrafish contacted with the test compound in comparison to a control zebrafish not contacted by the test compound. [0009] In certain embodiments, the methods include the step of washing away label unassociated with the lateral line hair cells of a zebrafish before contacting a zebrafish hair cell with a test compound. [0010] In certain embodiments, the noxious stimulus comprises one or more drugs known to cause sensory hair cell death. In certain embodiments the noxious stimulus is a sound pressure level (decibel level) known to cause sensory hair cell damage or death. In certain embodiments the noxious stimulus is age. [0011] The methods also include simultaneously screening a plurality of test compounds potentially capable of decreasing, inhibiting or preventing sensory hair cell damage or death induced by one or more noxious stimuli under high throughput conditions. Accordingly, the invention further provides high throughput methods of screening comprising: [0012] a) labeling the lateral line hair cells of members of a plurality of zebrafish, wherein said labeling preferentially labels lateral line hair cells in comparison to other cells of the zebrafish, wherein said labeling is substantially non-toxic to the zebrafish hair cells, and wherein said label is detectably distinct in a live cell in comparison to a dying cell or a dead cell; [0013] b) contacting each member of the plurality of zebrafish with one member of a plurality of test compounds suspected of decreasing, inhibiting or preventing sensory hair cell damage or death; [0014] c) contacting each member of the plurality of zebrafish with one or more noxious stimuli known to cause sensory hair cell damage or death; and [0015] d) detecting the label in each member of the plurality of zebrafish, wherein a compound that decreases, inhibits or prevent sensory hair cell damage or death is identified when the number of live lateral line hair cells is greater in zebrafish contacted with the test compound in comparison to a control zebrafish not contacted by the test compound. [0016] In addition, the invention provides several compounds identified by the methods of the invention to be protective against ototoxic drugs. These compounds include cepharanthine, amsacrine, drofenine, phenoxybenzamine, N,N-hexamethyleneamiloride, carvedilol and 9-amino-1,2,3,4-tetrahydroacridine. Also identified by the methods of the invention as protective against ototoxic drugs are compounds that contain a thiophene carboxamide moiety (Formula I or II) or a urea-thiophene-carboxamide moiety (Formula III or IV), and are structurally consistent with a drug-like profile, according to Lipinski's Rule of 5 criteria. These compounds include F5, H10, and Compounds A, B, C and D described herein. [0017] The invention further includes methods for decreasing, inhibiting or preventing ototoxicity induced by one or more noxious stimuli by administering a sufficient amount of a compound identified by the screening methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS [0018] FIGS. 1A and 1B illustrate labeling and neomycin-induced hair cell death of zebrafish lateral line hair cells. FIG. 1A depicts 5 days post fertilization (dpf) larvae hair cells labeled with FM 1-43 (red) to identify the cytoplasm of hair cells and Yo-Pro-1 (green) labeling the nucleus. After 1 hour exposure to 200 .mu.M, neomycin hair cells that are not protected undergo apoptotic death. Cytoplasmic (red) and nuclear fragments (green) are visible in FIG. 1B. [0019] FIG. 2 illustrates compounds F5 and H10, structurally related compounds identified using the present methods and which protect hair cells from the aminoglycoside neomycin. [0020] FIG. 3A illustrates how F5 is optimally protective against 200 .mu.m neomycin at 10 .mu.M. FIG. 3B shows that H10 is optimally protective against 200 .mu.M neomycin at 1 .mu.M. [0021] FIG. 4 illustrates how F5 protects lateral line hair cells against neomycin. [0022] FIG. 5 illustrates how F5 and H10 are protective through 24 hours post neomycin treatment. Hair cell regeneration in the lateral line prevents evaluation beyond this time. [0023] FIG. 6 illustrates how pretreatment with F5 is protective against cisplatin-induced lateral line hair cell death. [0024] FIG. 7 illustrates how compounds F5 and H10 do not prevent the rapid uptake of FMI-43. [0025] FIG. 8 illustrates additional compounds identified using the present screening methods. DETAILED DESCRIPTION OF THE INVENTION [0026] Methods of using a zebrafish model system to screen for small molecules capable of decreasing, inhibiting or preventing sensory hair cell damage or death are provided. Zebrafish are an advantageous animal model system for studying causes and prevention of hearing loss in comparison to mammalian animal model systems. The relative inaccessibility of hair cells in mammalian organisms limits their use as a high throughput model for identifying compounds that would prevent toxin mediated and other forms of hair cell death from occurring. The lateral line hair cells of zebrafish (Danio rerio) are structurally and functionally similar to mammalian sensory hair cells. The zebrafish is therefore an ideal model organism for in vivo high throughput screening to identify compounds that can prevent hair cell damage or death from occurring. [0027] The methods are exemplified by screening a combinatorial chemical library for compounds that counteract (i.e., decrease, prevent or inhibit) damage or death to the lateral line hair cells of zebrafish induced by one or more noxious stimuli (i.e., an ototoxic drug, a damaging sound pressure level, presbyacusis or age-related hearing loss). Using this approach, six structurally related small molecules have been identified as protective against the toxic effects of an exemplified aminoglycoside (neomycin) and an exemplified platinium coordination complex (cisplatin) on hair cells. The identified compounds: [0028] F5: 2-({[(4-chlorophenyl)amino]carbonyl}amino)-6-ethyl-4,5,6,7-tetrahydrothie- no[2,3-c]pyridine-3-carboxamide; [0029] H10: 2-{[({2,2,2-trichloro-1-[(4-methoxybenzoyl)amino]ethyl}amino)carbonothioy- l]amino}-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxamide; [0030] Compound A: 2-{{({2,2,2-trichloro-1-[(3-methylbenzoyl)amino]ethyl}amino)carbonothioyl- ]amino}-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxamide; [0031] Compound B: 2-({[(4-chlorophenyl)amino]carbonyl}amino)-4,5,6,7-tetrahydro-1-benzothio- phene-3-carboxamide; [0032] Compound C: 2-[(4-chloro-3-nitrobenzoyl)amino]-4,5,6,7-tetrahydro-1-benzothiophene-3-- carboxamide; and [0033] Compound D: 2-[(1-piperidinylacetyl)amino]-4,5,6,7-tetrahydro-1-benzothiophene-3-carb- oxamide each contain a thiophene carboxamide and are structurally consistent with a drug-like profile, according to Lipinski's Rule of 5 criteria. The methods find use in screening further chemical libraries for additional compounds and in refining identified lead compounds to optimize their protective efficacy. [0034] In addition, the high throughput screening method of the invention has been used to identify several compounds approved by the United States Food and Drug Administration (FDA) as protective against ototoxic drugs. These compounds include cepharanthine, amsacrine, drofenine, phenoxybenzamine, N,N-hexamethyleneamiloride, carvedilol and 9-amino-1,2,3,4-tetrahydroacridine. Definitions Continue reading about Assay for compounds that protect against sensory hair cell death and compounds identified by same... 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