Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-05-27
2001-07-31
Wilson, James O. (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S047000, C424S001730, C424S009350, C424S009351, C424S009600, C424S070100, C435S006120
Reexamination Certificate
active
06268351
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of auditory research. Specifically, the invention pertains to methods which promote the regeneration of auditory hair cells following loss due to trauma or disease.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application by numerals in parenthesis in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Hair cells are sensory cells that transduce motion into neural signals. In the cochlea, they are used to detect sound waves in the environment and convert them into auditory signals that can be processed in the brain. Loss of receptor hair cells in the cochlea accounts for a significant proportion of hearing impairment in the population (1). This loss can occur as a consequence of viral or bacterial insult, aging, and damage from intense sound or aminoglycoside antibiotics. The generation of replacement hair cells following damage by sound or drugs has been clearly demonstrated in birds (2-4), the chick being the best-studied model for auditory hair cell regeneration (5). New hair cells arise as progeny from an otherwise non-dividing supporting cell population induced to proliferate by the damage (6-12). Functional recovery of hearing accompanies this cellular recovery process (13,14). The signals and pathways responsible for regenerative proliferation are presently unknown. However, evidence in the art indicates that such a regenerative response also occurs in mammals.
SUMMARY OF THE INVENTION
The present invention provides a novel system for the regeneration of auditory receptor hair cells. The generation of replacement hair cells following damage by sound or drugs has been clearly demonstrated in birds where the hair cells arise from a normally non-dividing supporting cell population induced to proliferate by the damage. The present invention demonstrates that agents which increase cAMP levels induce cell proliferation in explanted but otherwise undamaged receptor epithelium of cochleas, and that protein kinase A (PKA) inhibitors block this proliferative response. Furthermore, the proliferative response which follows in vitro gentamicin damage is also significantly blocked by PKA inhibitors. Accordingly, biochemical manipulation of the cAMP pathway is now possible. Thus, in one embodiment of the invention, a research tool is provided for the elucidation of the molecular events involved in the restoration of auditory receptor hair cells. In a preferred embodiment, stimulation of proliferation is achieved via the administration of agents which activate the cAMP pathway, such as stimulators of adenylate cyclase (i.e., forskolin). Forskolin may be used in a range of about 1-200 &mgr;M. Narrower ranges of forskolin concentration are also contemplated in practicing the present invention, i.e, between about 10 &mgr;M-100 &mgr;M, or between about 75 &mgr;M-150 &mgr;M. Alternatively, cAMP levels may be augmented by direct delivery of cAMP or analogues thereof (i.e., 8-Br-cAMP). Proliferation of auditory receptor cells may be assessed by co-administration and detection of agents that are incorporated into replicating DNA (i.e., Bromo-deoxyuridine).
In another embodiment, the explant cochlea cultures are exposed to ototoxic agents. Damage to these cultures results in a proliferative response. Following damage, this regenerative response may be abrogated by agents that inhibit the cAMP mediated signal transduction pathway, such as PKA inhibitors. The culture systems of the present invention therefore provide a valuable research tool for dissecting the signal transduction pathway responsible for the restoration of hearing loss.
In yet another embodiment of the invention, a method for treating a patient suffering from hearing loss is provided. The term “patient” as used herein may be a human or an animal subject. The method comprises delivering to a patient's auditory receptor epithelium, a cAMP activating agent in a suitable pharmaceutical carrier in an effective amount to activate the signal transduction pathway responsible for regeneration of hair cells in the cochlea epithelium. In an alternative embodiment of the invention, the method is used to assess the regeneration of hair receptor cells in the bird. In this embodiment, an agent for detecting DNA replication is co-administered to the damaged auditory epithelium with the cAMP pathway activating agent. Following stimulation of a proliferative response, the animal is sacrificed and the level of proliferation assessed.
The following definitions are provided to facilitate the understanding of the subject matter of the present invention:
Receptor hair cells are cells which lie in the upper part of the epithelium with their stereocilia embedded in the tectorial membrane. Hair cells are surrounded by supporting cells. The transduction of the mechanical energy of sound to a coded neural signal is effected by hair cells. Receptor hair cells are located in the sensory epithelia of both the auditory and vestibular portions of the inner ear.
The vestibular system is composed of the utricle, the saccule and the three ampullae of the semicircular canals. Impairments in the vestibular sensory epithelia often cause impaired balance.
The auditory receptor epithelia is located within the cochlea and is referred to the organ of corti in mammals and basilar papilla in avian species. Damage to the sensory epithelium in the cochlea can lead to impaired hearing.
The methods of the invention provide a novel approach for stimulating the regeneration of auditory receptor epithelium. The methods of the present invention may be beneficially used to elucidate the molecular mechanisms relevant in the restoration of hearing in subjects whose hearing has been damaged due to trauma or disease.
REFERENCES:
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patent: 5527772 (1996-06-01), Horlick
patent: 5529769 (1996-06-01), Cho et al.
patent: 5837681 (1998-11-01), Magal
patent: 5840690 (1998-11-01), Horlick
patent: 6066618 (2000-05-01), Horlick
patent: 6136785 (2000-10-01), Corwin et al.
Douglas A. Cotanche, et al. Hair cell regeneration in the bird cochlea following noise damage or ototoxic drug damage.Anatomy and Embryology. 1994 189: 1-18.
Hiroshi Yamashita, et al. Induction of cell proliferation in mammalian inner-ear sensory epithelia by transforming growth factor &agr; and epidermal growth factor.Proc. Natl. Acad. Sci. USA. Apr. 1995 92: 3152-3155.
J. Lisa Zheng, et al. Induction of Cell Proliferation by Fibroblast and Insulin-Like Growth Factors in Pure Rat Inner Ear Epithelial Cell Cultures.The Journal of Neuroscience. Jan. 1, 1997 17(1): 216-226.
Jeffrey T. Corwin, et al. Regeneration of Sensory Hair Cells After Acoustic Trauma.Science. Jun. 1998 240: 1772-1774.
Jennifer S. Stone, et al. Identification of the Timing of S Phase and the Patterns of Cell Proliferation During Hair Cell Regeneration in the Chick Cochlea.The Journal Of Comparative Neurology. 1994 341:50-67.
Paul R. Lambert, M.D. Inner Ear Hair Cell Regeneration in a Mammal: Identification of a Triggering Factor.Laryngoscope. Jun. 1994 104: 701-718.
Navarathan Dhasakumar S.
Oberholtzer J. Carl
Dann Dorfman Herrell and Skillman
The Trustees of the University of Pennsylvania
Wilson James O.
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