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WO1997017983A1 - Procedes de traitement de troubles dans des epitheliums sensoriels non visuels - Google Patents

Procedes de traitement de troubles dans des epitheliums sensoriels non visuels Download PDF

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Publication number
WO1997017983A1
WO1997017983A1 PCT/US1996/018031 US9618031W WO9717983A1 WO 1997017983 A1 WO1997017983 A1 WO 1997017983A1 US 9618031 W US9618031 W US 9618031W WO 9717983 A1 WO9717983 A1 WO 9717983A1
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Prior art keywords
visual sensory
sensory epithelial
cells
epithelial cells
cell
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PCT/US1996/018031
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English (en)
Inventor
Mark A. Marchionni
Nagesh K. Mahanthappa
Gerald Schwarting
Jeffrey Corwin
Original Assignee
Cambridge Neuroscience, Inc.
University Of Virginia Patent Foundation
E.K. Shriver Center For Mental Retardation
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Application filed by Cambridge Neuroscience, Inc., University Of Virginia Patent Foundation, E.K. Shriver Center For Mental Retardation filed Critical Cambridge Neuroscience, Inc.
Priority to EP96940360A priority Critical patent/EP0866716A4/fr
Priority to AU77258/96A priority patent/AU727037B2/en
Priority to CA 2237400 priority patent/CA2237400A1/fr
Priority to JP9518966A priority patent/JP2000505781A/ja
Publication of WO1997017983A1 publication Critical patent/WO1997017983A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors

Definitions

  • This invention relates to methods of affecting function of non-visual sensory epithelial cells, including, but not limited to, cells of the vestibular, cochlear and olfactory epithelia.
  • the invention relates to prophylactic or affirmative treatment of diseases and disorders of non-visual epithelia and associated tissues of the sensory organs by administering polypeptides found in vertebrate species, which polypeptides are growth, differentiation and survival factors for several cell types.
  • Polypeptides found in vertebrate species, which polypeptides are growth, differentiation and survival factors for several cell types.
  • Normal function of sensory epithelia cells including survival, proliferation, differentiation, and maintenance is dependent upon the controlled expression of a variety of peptide growth factors. Some of these factors can be produced by neuronal cells and by other cells of the sensory epithelia, which provide a signal to regulate sensory epithelial cell function.
  • the senses of hearing and balance depend upon the normal development and activity of tissues that comprise the auditory and vestibular pathways, respectively.
  • the auditory system consists of the outer ear (the external ear and tympanic membrane), the middle ear (location of the middle ear bones: malleus, incus, and stapes) and the cochlea of the inner ear (see Figure 1).
  • the sensory cells that respond to sound, the hair cells reside within the cochlea and transform sound information into electrical impulses that are transmitted to the brain via neurons of the spiral ganglion.
  • the vestibule and semicircular canals Immediately adjacent to the cochlea are the vestibule and semicircular canals. Within these structures are hair cells that respond to acceleration and thus give rise to the sense of balance and head position.
  • the cochlea of mammals is a spiral structure containing two fluid-filled tubes, the scala vestibuli and the scala tympani (see Figure 2).
  • These parallel tubes wrap around a central bony pillar (the modiolus) two and a half times, and connect with each other at the apex (via the helicotrema) of the cornucopia-shaped complex.
  • the modiolus When sound impinges upon the eardrum it sets the bones of the middle ear vibrating, and they in turn vibrate the membranous oval window. Since the scala vestibuli begins at the oval window, fluid within the tubes is set into motion and the scalae vestibuli and tympani vibrate in unison. The vibrations dissipate at the terminus of the scala tympani, the membranous round window.
  • a third tube, the cochlear duct, is closed at both ends and is sandwiched between the above-described tubes.
  • the floor of the cochlear duct is called the basilar membrane, and on this membrane sits the organ of Corti.
  • the organ of Corti constitutes the primary sensory receptor surface of the auditory system; one row of inner hair cells and three to four rows of outer hair cells run the entire length of this epithelium.
  • the hair cells are surrounded by support cells known as pillar cells, Deiter's cells, Henson's cells and phalangeal cells, and these are attached to the cartilaginous basilar membrane.
  • the hair cells are mechanosensitive cells that bear cilia ("hairs") on their apical surfaces.
  • cilia contact an overlying membrane such that vibrations moving through the scalae cause the cilia to move back and forth.
  • the back and forth motion causes the opening and closing of apical ion channels, and thus cochlear hair cells respond to sound by electrical depolarization and hyperpolarization.
  • hair cells form chemical synapses with neural fibers originating from the spiral ganglion, and these changes in polarization are communicated to the central nervous system by release of a neurotransmitter.
  • the three tubular structures within the cochlea are narrowest towards the oval and round windows, and broaden towards the apex.
  • This structure confers a critical mechanical property on the system; when sound sets the fluid within the scalae vestibuli and tympani in motion, a standing wave is produced such that low frequency sounds set the widest part of the organ of Corti in motion, and high frequency sounds maximally stimulate the narrower portion of the organ of Corti.
  • the various regions of the organ of Corti are preferentially stimulated by specific frequencies of sound, thus in humans a continuous "tonotopic" field (ranging in responsiveness from 20 Hz to 20,000 Hz) is mapped over the length of the epithelium.
  • Perceived loudness is proportional to the amplitude of the sonic vibrations.
  • perception of tone is a function of which hair cells are stimulated
  • perception of loudness is a function of how strongly the given hair cells are stimulated.
  • the vestibular system works in a similar fashion, but rather than making use of a topographic continuum to encode information, vestibular hair cells are segregated into five distinct substructures that respond to distinct stimuli.
  • the hair cells of the vestibular system are embedded in gelatinous material such that those within the vestibule respond to linear/translational motion, and those within the semicircular canals respond to rotational/angular motion.
  • the extracellular gel encasing hair cells within the vestibule contain calcium carbonate crystals (otoliths) that directly stimulate the hair cells by falling on them.
  • otoliths calcium carbonate crystals
  • the hair cells of the vestibular system form synapses with fibers from the vestibular ganglion to transmit information to the central nervous system.
  • the circuitry of the auditory pathway is quite complex. An overview of this circuitry follows. Neurons of the spiral ganglion are bipolar, extending one process to synapse with hair cells and another process that splits and synapses with two cochlear nuclei (ventral and dorsal) within the ipsilateral brainstem. Hair cell signals therefore pass through two synapses to reach neurons within the brain.
  • the projection from cochlea to cochlear nuclei is known as the Vlllth cranial nerve. (It is worth noting that an efferent, inhibitory pathway synapses primarily onto outer hair cells, and that these axons are of olivary origin. These are thought to play a feedback role in modulation of hair cell responsiveness.)
  • Neurons of the cochlear nuclei project contralaterally to the inferior colliculus (IC), a region responsible for auditory reflexes such as the startle response. Some axons projecting to the IC send branches to other brain nuclei including the contralateral nucleus of the lateral lemniscus (NLL). Some neurons of the NLL project to the ipsilateral IC, while the ipsilateral and contralateral IC communicate directly through the commissure of the IC. Thus at this level of the auditory system, connections cross heavily and information from both ears is available to both sides of the brain. IC neurons send non-crossing projections to the medial geniculate nucleus of the thalamus, which then projects to the auditory cortex.
  • IC inferior colliculus
  • auditory projections throughout the nervous system maintain tonotopic alignment. In other words, if one were to measure electrical responses to various pure tones in nuclei of the primary auditory pathway, one would find response properties within individual nuclei distributed along an axis corresponding to the axis of tonotopy found in the organ of Corti.
  • the "tuning curves" of single auditory neurons sharpen as one ascends the auditory pathway. This is to say that auditory hair cells respond to a increasingly broad range of frequencies as sound amplitude increases, but higher order auditory neurons exhibit increasingly specific frequency responses.
  • auditory neurons display specificities for increasingly complex temporal patterns of frequency modulation.
  • the Vlllth cranial nerve also contains axons originating from the vestibular system.
  • the neurons of the vestibular ganglion send one process to the hair cells, and one process that projects to the cerebellum and four vestibular nuclei in the ipsilateral brainstem.
  • the vestibular nuclei project broadly throughout the brain stem (where vestibular information is integrated with oculomotor information, etc.) and into the spinal cord (where vestibular information contributes to the modulation of posture and muscle tone).
  • the architecture of the olfactory epithelium is that of a relatively simple sheet in which various cell types occupy discrete strata (reviewed in (Graziadei, et al. Handbook of Sensory Physiology 9:55-83, 1977).
  • Figure 3 shows a schematic diagram of the olfactory epithelium.
  • the deepest layer, that which abuts the bony turbinates, is referred to as the lamina intestinal and contains primarily connective and vascular tissues. Separating the lamina intestinal from the actual sensory epithelium, or neuroepithelium, is the basal lamina.
  • the basal lamina appears to be a typical, acellular, basement membrane comprised of many extracellular matrix proteins such as collagens, laminins, and fibronectin.
  • the neuroepithelium is populated by multiple cell types that are restricted in their laminar position. Immediately adjacent to the basal lamina are the basal cells; those abutting the basal lamina are known as horizontal basal cells, and those sitting somewhat less tightly upon the horizontal basal cells are known as globose basal cells. Between the basal cell layer and the most superficial layer of the olfactory epithelium lie the cell bodies of the primary olfactory neurons. The olfactory neurons are the actual chemosensory cells that detect odorants and transmit odor information to the central nervous system.
  • This function depends on two extensions from each neuron cell body: a single dendrite that extends towards the nasal cavity where it is capped by cilia that bear odorant receptor proteins (see below); and a single axon that extends in the opposite direction through the basal cells and basal lamina and via the lamina basement connects directly to neurons of the olfactory bulb in the central nervous system.
  • the binding of specific odorants by specific odorant receptors causes the neuron to depolarize and thus signal the olfactory bulb though an action potential that travels the olfactory axon.
  • "Support" cells of unknown function, called sustentacular cells fill in around the olfactory dendrites and constitute the bulk of the exposed epithelial surface.
  • the cells that make up Bowman's glands - mucus secreting glands that are periodically distributed across the breadth of the olfactory epithelium and that span the depth of the neuroepithelium.
  • NT3 and BDNF Three independent lines of evidence provide strong support for the role of the neurotrophic factors NT3 and BDNF in the development of the inner ear.
  • the studies include: in situ hybridization to map the factor NT3 and BDNF (Pirvola, U., Ylikowki, J., Palgi, J., Lethonen, E. Arumae, U. and Saarma, M., PNAS USA 89; 9915-9919, 1992) and the receptors trkB and trkC (Enfors, P., Merlio, J.-P., and Persson, H. Eur. J. Neurosci.
  • Principal sites of NT3 expression include both support cells and hair cells of developing cochlear sensory epithelium and basal cells of the vestibular epithelium.
  • the expression of BDNF is localized to sensory cells (hair cells), but is not found in the support cells of the sensory epithelium.
  • the distributions of these two neurotrophins are overlapping, but not identical.
  • Neurons of the spinal ganglia and the vestibular ganglia which innervate the sensory epithelia of the cochlear and the three epithelium of the vestibular system, respectively, have been shown to express trkB and trkC receptors (Enfors, P., Merlio, J.-P., and Persson, H. Eur. J. Neurosci. 4: 1 140-1 158, 1992).
  • BDNF is the major survival factor for vestibular ganglia neurons and NT3 for spinal ganglion neurons
  • BDNF, NT3 and NT4/5 have been shown to promote survival of cultured vestibular ganglion neurons and to protect them from cell death induced by cytotoxins (Zheng, J. L., Stewart, R. R. and Gao, W.-Q. J. Neurobiology 28:3, 330-3401, 1995).
  • EGF epidermal growth factor
  • TGF- ⁇ s transforming growth factor ⁇ s
  • TGF- ⁇ 2 promotes neurogenesis in the cultured olfactory epithelium and is more potent than TGF- ⁇ l is entirely consistent with known patterns of localization in vivo (Mahanthappa, et al. Neuron 10:293-305, 1993).
  • TGF- ⁇ 2 completely blocks baseline levels of olfactory neurogenesis in vitro supports the hypothesis that TGF- ⁇ 2 is in fact the endogeneously produced neurogenic factor in the OE (Mahanthappa, et al. Neuron 10:293- 305, 1993).
  • Conductive hearing loss may be brought about by blockade of the ear canal, lesions of the ear drum, or lesions of the middle ear bones.
  • At least 15 percent of the U.S. population is affected by tinnitus.
  • Presbycusis affects 1/3 of the U.S. population over age 65.
  • Meniere's syndrome causes bilateral hearing loss in 5 to 20 percent of cases.
  • the estimated cost of care per year for persons with hearing impairment is $56 billion (based on $2,000 per patient annual costs for special education, speech therapy, hearing aids, physician and specialist fees, and other expenses).
  • the sense of smell is a primary mediator of appetite.
  • Patients undergoing chemotherapy for cancer treatment are often treated with high doses of anti-mitotic agents, and among the many side effects of such agents is the loss of appetite.
  • This loss of appetite is most often attributed to the severe disruption of the gastrointestinal tract since these tissues undergo continual mitotic renewal, and anti-mitotic agents interfere with their normal functioning.
  • anti-mitotic chemotherapeutic agents would interfere with both the senses of taste and smell.
  • the present invention provides methods for promoting the function of non- visual epithelial cells using neuregulins.
  • a novel aspect of the invention involves the use of neuregulins as growth factors to promote function of inner ear cells. Treating of the inner ear cells to provide these effects may be achieved by contacting inner ear cells with a polypeptide described herein.
  • the invention teaches how to stimulate mitogenesis of inner ear cells using neuregulins and therefore, treatments may be provided to slow or halt net cell loss or to increase the amount or quality of inner ear tissue present in a vertebrate.
  • the invention also teaches how to promote survival of olfactory neurons using neuregulins and therefore, treatments may be provided to slow or halt net cell loss or to increase the amount or quality of olfactory tissue present in the vertebrate.
  • Neuregulins are a family of protein factors heretofore described as glial growth factors, acetylcholine receptor inducing activity (ARIA), heregulins, neu differentiation factor, which are encoded by one gene.
  • a variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show binding to and activation of erbB2 (neu) and closely related receptors erbB3 and erbB4.
  • the invention provides methods for using all of the known products of the neuregulin gene, as well as other not yet discovered splicing variants of the neuregulin gene.
  • non-visual epithelial cells refers to the prevention of loss of non-visual sensory epithelial cells by necrosis or apoptosis or the prevention of other mechanisms of cell loss. Survival, as used herein, indicates a decrease in the rate of cell death of at least 10%, more preferably by at least 50%, and most preferably by at least 100% relative to an untreated control. The rate of survival may be measured by counting cells stainable with a dye specific for dead cells (such as propidium iodide) in culture.
  • a dye specific for dead cells such as propidium iodide
  • inner ear tissue disorders that may be treated, include inner ear diseases and disorders resulting from sensorineural pathologies, such as loss of hearing and/or balance, which may also be treated using the methods of the invention.
  • These disorders of the inner ear include, but are not necessarily limited to: sensorineural hearing loss caused by ototoxic antibiotics, excessive noise, viral infection or autoimmune disease; tinnitus; and, Meniere's syndrome.
  • administration of neuregulin in a therapeutically effective amount can provide a treatment for disorders of the ear, which otherwise left untreated would result in the loss of hearing and/or balance.
  • the methods of the invention make use of the fact that the various neuregulin proteins are encoded by the same gene.
  • a variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show binding to pl85 er ⁇ B2 (or related receptors erbB3 and erbB4) and activation of the same. Products of this gene are used to show inner ear cell mitogenic activity (see Examples 1 and 2, below).
  • This invention provides a use for all of the known products of the neuregulin gene (described herein and in the references listed above), which have the stated activities as promoting inner ear cell function. Most preferably, recombinant human GGF2 (rhGGF2) is used in these methods.
  • the invention also relates to the use of other, not yet naturally isolated, splicing variants of the neuregulin gene.
  • the splicing variants are fully described in Goodearl et al., US Patent No. 5,530,109, issued June 25, 1996 (formerly USSN 08/036,555, filed March 24, 1993), incorporated herein by reference.
  • inventions of the present invention include the development of new therapeutic approaches to injury or diseases of the ear, more specifically degenerative diseases of the cochlear- vestibular epithelia, based on the promotion of inner ear cell function through the use of neuregulins.
  • Loss of inner ear cells is a common feature of degenerative ear diseases, and there are no available treatments, including growth factors. The factor can be formulated for injection and administered to patients that suffer from degenerative disorders, which lead to loss of hearing and/or balance. Thus, this approach to therapy can halt or slow the progressive loss of hearing and/or balance, which ensues in various ear diseases.
  • additional advantages of the invention include treatments for any type of non- visual sensory epithelia, such as diseases of olfactory, gustatory and other non-visual sensory epithelia.
  • Agents that can promote olfactory neuron survival are likely to restore, at least in part, normal chemosensation and thereby facilitate recovery of normal appetite. This restoration of normal appetite is nontrivial, as the inability of patients undergoing chemotherapy to maintain reasonable levels of nutrition often complicates recovery.
  • neuregulins in the form of rhGGF2 can promote olfactory neuron survival, it may be an important adjunct therapy for such patients.
  • Anosmia the lack of a sense of smell, is also known to be a source of depression in patients who are otherwise normal. Again, for the reason that it promotes olfactory neuron survival, neuregulins may prove as useful adjunct therapeutics in the context of rhinal and/or psychiatric dysfunction.
  • Figure 1 is a diagram of the gross anatomy of the ear.
  • Figure 2 is a diagram of the cochlea in cross-sectional view that depicts the organ of Corti, which contains the sensory hair cells.
  • Figure 3 is a schematic diagram of the olfactory epithelium.
  • Figure 4 shows the mitogenic response (% BrdU-labelled) of dissociated cochlear chick support cells in culture treated with various doses of rhGGF2.
  • Figure 5 shows the mitogenic response (number of BrdU-labelled support cells per epithelium) of cultured sensory epithelia (isolated from the rat cuticle—vesticular system) treated with rhGGF2.
  • Figure 6 shows neuregulin transcripts detected by in situ hybridization in the spiral ganglia of a day 18 mouse embryo.
  • Figure 7 shows neuregulin transcripts detected by in situ hybridization in the vestibular epithelium of a day 21 rat embryo.
  • Figure 8 shows that rhGGF2 promotes olfactory neuron survival in vitro; figure 8A represents 2 days in vitro and figure 8B represents 4 days in vitro.
  • Figure 9 shows neuregulin transcripts detected by in situ hybridization in the olfactory neuron layer of postnatal rat olfactory epithelium.
  • the invention pertains to methods of promoting function of non-visual sensory epithelial cells.
  • the function is affected by the administration of a neuregulin to a vertebrate where the neuregulin interacts with a non-visual sensory epithelial cell to promote one or more aspects of non-visual sensory epithelial cell function, including proliferation, differentiation, growth, survival, migration, changes in the pattern of gene expression and secretion, and metabolic change of the non- visual sensory epithelial cell.
  • administration refers to the act of delivering a substance, including but not limited to the following routes: parenteral, intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intranasal, intrathecal, aerosol or oral.
  • affecting refers to the induction of a quantitative change in the response of a target cell, as a result of an interaction with a neuregulin.
  • basal cell refers to epithelial cells that reside in immediate apposition to the extracellular matrix rich, basement membrane. Such cells can also be support cells.
  • differentiation refers to a morphological and or chemical change that results in the generation of a different cell type or state of specialization.
  • the differentiation of cells as used herein refers to the induction of a cellular developmental program which specifies one or more components of a cell type. The therapeutic usefulness of differentiation can be seen, in increases in quantity of any component of a cell type in diseased tissue by at least 10% or more, more preferably by 50% or more, and most preferably by more than 100% relative to the equivalent tissue in a similarly treated control animal.
  • disorder refers to a disturbance of function and/or structure of a living organism, resulting from an external source, a genetic predisposition, a physical or chemical trauma, or a combination of the above, including but not limited to any mammalian disease.
  • erbB receptor refers to erbB2, erbB3 and erbB4 (also HER-2,
  • HER-3 and HER-4 of human existing as monomeric, homodimeric and heterodimeric (e.g., erbB2/erbB3) cell surface receptor tyrosine kinases that bind and/or are activated by one or more neuregulins.
  • function refers to any activity or response of a cell. These include but are not limited to proliferation, differentiation, growth, survival, changes in the pattern of gene expression and secretion, and metabolic changes.
  • hair cell refers to the sensory cells of the cochlear and vestibular epithelia. Such cells possess stereocilia on their apical surfaces, and the bending of the stereocilia in response to mechanical stimuli (e.g. sound waves) causes signaling by the hair cells.
  • mechanical stimuli e.g. sound waves
  • mammal as used herein describes a member of the Class Mammalia (Subphylum Vertebrata).
  • mitosis refers to the division of a cell where each daughter nucleus receives identical complements of the numbers of chromosomes characteristic of the somatic cells of the species. Mitosis as used herein refers to any cell division which results in the production of new cells in the patient. More specifically, a useful therapeutic is defined in vitro as an increase in mitotic index relative to untreated cells of 50%, more preferably 100%, and most preferably 300%, when the cells are exposed to labeling agent for a time equivalent to two doubling times.
  • the mitotic index is the fraction of cells in the culture which have labeled nuclei when grown in the presence of a tracer which only incorporates during S phase (i.e., BrdU) and the doubling time is defined as the average time required for the number of cells in the culture to increase by a factor of two.
  • neuregulin refers to the glial growth factors, the heregulins, neu differentiation factor, acetylcholine receptor inducing activity, and erbB2, 3 and 4 binding proteins.
  • a more complete definition of neuregulins can be found in the specification herein and in the following materials: U.S. Patent No. 5,237,056; U.S.
  • neurological disorder refers to a disorder of the nervous system.
  • non-visual sensory epithelium refers to composite of cells and extracellular matrix that comprise the sheet-like layer of tissue in sensory organs that include sensory cells such as hair cells and olfactory neurons. Examples include, but are not limited to the cochlear epithelium, the three vestibular epithelia, the olfactory epithelium and the epithelial layer of the skin. Not included are the sensory epithelium of the visual system, the retina.
  • olfactory neuron refers to the primary sensory neurons of the olfactory epithelium. These cells project chemosensory cilia into nasal mucosa, and the cilia bind odorant molecules. Odorant binding results in olfactory neuron signaling.
  • support cell refers to cells residing within epithelia that are not sensory in nature, and that may play supporting functions for the maintenance and regeneration of the epithelium. Such cells may provide trophic support for sensory cells, and may give rise to new sensory cells in the regenerative situation.
  • survival refers to any process where a cell avoids death.
  • survival as used herein also refers to the prevention of cell loss as evidenced by necrosis or apoptosis or the prevention of other mechanisms of cell loss.
  • Survival as used herein indicates a decrease in the rate of cell death by at least 10%, more preferably by at least 50%, and most preferably by at least 100% relative to an untreated control.
  • the rate of survival may be measured by counting cells stainable with a dye specific for dead cells (such as propidium iodide) in culture.
  • sustentacular cell refers to support cells of the olfactory epithelium that reside in die luminal most portion of the epithelium. The cells contribute to the nasal mucosa and abut the chemosensory cilia of the olfactory neurons.
  • therapeutically effective amount refers to that amount which will produce a desirable result upon administration and which will vary depending upon a number of issues, including the dosage to be administered, and the route of administration.
  • treating may refer to a procedure (e.g. medical procedure) designed to exert a beneficial effect on a disorder.
  • Treating means any administration of a substance described herein for the pu ⁇ ose of increasing inner ear cell function. Most preferably, the treating is for the pu ⁇ ose of reducing or diminishing the symptoms or progression of a disease or disorder of inner ear cells.
  • Treating or treatment also means the administration of a substance to increase or alter the cells in healthy individuals. The treating may be brought about by the contacting of the cells which are sensitive or responsive to the neuregulins described herein with an effective amount of the neuregulin.
  • vertebrate refers to an animal that is a member of the Subphylum Vertebrata (Phylum Chordata).
  • Hair cell regeneration an approach to therapy
  • the first evidence for regeneration of mammalian hair cells came from studies of vestibular hair cell regeneration in guinea pigs treated with an ototoxic antibiotic. An accompanying in vitro study demonstrated the production of hair cells with an immature phenotype. In both cases, however, any hair cell regeneration might have been unexpected. Support cell division was observed to take place prior to production of new hair cells, but it is evident that certain factors that promote appropriate regeneration are either absent or at low levels in these systems or are inhibited.
  • a candidate factor for a role in hair cell development and regeneration is TGF ⁇ , and another is retinoic acid.
  • Retinoic acid has been detected in embryonic and early postnatal mouse cochlea. When placed in culture with exogenous retinoic acid, embryonic mouse cochleas develop supernumerary hair cells in the absence of cell division. This finding suggests that retinoic acid acts as a regulator of differentiation rather than acting as a mitogen for support/precursor cells.
  • retinoic acid stimulates regeneration of antibiotic-treated postnatal rat cochleas in vitro, and that this regeneration is blocked by anti-mitotic agents. In light of the previous retinoic acid result, it appears likely that an endogenous mitogen stimulates division of a hair cell precursor and that retinoic acid stimulates hair cell differentiation.
  • a unique aspect of the olfactory epithelium is that primary olfactory neurons constitute the only population of neurons in all vertebrates that are known to be able to regenerate throughout the lifetime of the animal (Graziadei, et al. Handbook of Sensory Physiology 9:55-83, 1977). This ability to regenerate is necessary, because olfactory neurons are directly exposed to airbom pollutants and pathogens, and thus are vulnerable to irreversible damage and death. The regeneration process requires both the production of new olfactory neurons and appropriate targeting of the new axons to the olfactory bulb.
  • the model proposed to account for the observed pattern of labeling posits a rapid phase of 2-3 successive cell divisions that give rise to migrating immature neurons, and a superimposed slower rate of basal cell division.
  • the fast initial divisions thus correspond to the activity of neurogenic globose cells, while the slower divisions correspond to the horizontal cell production of globose cells (Mackay-Sim, et al. J Neurosci 11:979-984, 1991).
  • neuregulins A recently described family of growth factors, the neuregulins (reviewed by Mudge, Curr. Biol. 1:361 , 1993; Peles and Yarden, Bioessays 15:815. 1993), are synthesized by neurons (Marchionni et al. Nature 362:313. 1993) and by mesenchymal cells from several parenchymal organs (Mearr and Birchmeier, PNAS 91 : 1064, 1994). The neuregulins and related factors that bind pl85 erD B2 jj ave been purified, cloned and expressed (Benveniste et al.
  • Recombinant neuregulins have been shown to be mitogenic for peripheral glia (Marchionni et al., Nature 362:313. 1993) and have been shown to influence the formation of the neuromuscular junction (Falls et al., Cell 72:801 , 1993; Jo et al., Nature 373: 158, 1995; Chu et al., Cell 14: 329, 1995).
  • the neuregulin gene consists of at least thirteen exons.
  • the neuregulin transcripts are alternatively spliced and these encode many distinct peptide growth factors, which are referred to as the neuregulins (Marchionni et al., Nature 362:313. 1993).
  • DNA sequence comparisons revealed that neu differentiation factor (NDF) (Wen et al., Cell 62:559, 1992) and heregulins (Holmes et al., Science 256: 1205. 1992), which were purified as ligands of the pl 85 er °B2 ( a j so known as neu or HER2) receptor tyrosine kinase, also are splicing variants of the neuregulin gene.
  • the acetylcholine receptor inducing activity also is a product of the neuregulin gene (Falls et al., Cell 72:801, 1993). Common structural features of the neuregulins are the presence of a single immunoglobulin-like (lg) fold and a single epidermal growth factor-like (EGF) domain.
  • the sites of neuregulin gene expression have been characterized by use of nucleic acid probes to analyze RNA samples by a variety of methods, such as Northern blotting, RNase protection, or in situ hybridization. Transcripts have been detected in the nervous system and in a variety of other tissues (Holmes et al., Science 2 ⁇ 6: 1205, 1992 Wen et al., Cell 69:559, 1992; Mearr and Birchmeier, PNAS 21: 1064, 1994). Sites of gene expression have been localized in the brain and spinal chord and in other tissues.
  • neuregulins may be produced by alternative splicing, they can be broadly sorted into the putative membrane-bound and the soluble isoforms.
  • the former contains a putative trans-membrane domain and may be presented at the cell surface.
  • Membrane-anchored peptide growth factors may mediate cell-cell interactions through cell- adhesion or juxtacrine mechanisms (reviewed by Massague and Pandiella, Ann. Rev. Biochem. 62:515, 1993).
  • the putative membrane-bound isoforms may be cleaved from the cell surface and function as soluble proteins (Wen et al., Cell 69:559, 1992; Falls et al., Cell 72:801 , 1993).
  • the soluble neuregulin isoforms contain sequence corresponding to the extracellular domains of the putative membrane-bound isoforms, but terminate before the transmembrane domain. These neuregulin isoforms may be secreted, and hence could affect cells at a distance; or they may be present in the cytoplasm, but could be released upon cellular injury. In the latter case, neuregulins may function as injury factors, as has been postulated for the ciliary neurotrophic factor (St ⁇ ckli et al., Nature 342:920. 1989). Any one of these modes of action of the neuregulins may occur in the cochlear- vestibular epithelia.
  • Cellular targets of peptide growth factors are those which bear receptors for the factor(s) and those that are shown to respond in a bioassay either in vitro or in vivo.
  • neuregulins are candidate ligands for the receptor tyrosine kinases pl85 erD B2 ( or HER-2 in human), p l 85 erbB3 (HER-3 in human), pl85 erbB 4 (or HER-4 in human) or related members of the EGFR gene family. Collectively, these receptors can be referred to as erbB receptors.
  • HER-4 gene Sites of expression of the HER-4 gene have been localized by in situ hybridization to several regions of the brain, including: hippocampus, dentate gyrus, neo cortex, medial habenula, reticular nucleus of the thalamus, and the amygdala (Lai and Lemke, Neuron 6:691 , 1991).
  • the distribution of the HER-4 receptor has not been studied by methods that allow detection of the protein or the activated receptor tyrosine kinase in vivo or in cultures of primary cells.
  • the expression pattern of erbB2, erbB3 and erbB4 in the cochlear-vestibular epithelia has not been described.
  • Neuregulins have been shown to have a variety of biological activities depending on the cell type being studied.
  • Several neuregulins including native bovine GGFI, II and III and recombinant human GGF2 (rhGGF2) are mitogenic for Schwann cells (Marchionni et al , Nature 362 313, 1993), as is hereguhn Bl (Levi et al, J Neurosci 15_: 1329, 1995)
  • rhGGF2 has a potent trophic effect on myotubes (Sklar et al., U.S. Pat. Applic. # 08/059, 022).
  • the differentiation response to rhGGF2 also includes induction of acetylcholine receptors in cultured myotubes (Jo et al., Nature 373 158, 1995) This activity is associated with other forms of neuregulin, including ARIA (Falls et al , Cell 22:801 , 1993) and hereguhn B l (Chu et al , Neuron 14.329, 1995), as well as with rhGGF2 Further, ARIA has been shown to induce synthesis of voltage-gated sodium channels in chick skeletal muscle (Corfas and Fischbach, J Neurosci. 13:21 18, 1993).
  • G al growth factor GGF
  • rhGGF2 G al growth factor 2
  • rhGGF2 can restrict neural crest stem cells to differentiate into g al cells in vitro (Shah et al., Cell 27:349, 1994) Activities of neuregulin on inner ear cells have not been described.
  • neuregulin proteins affecting proliferation, survival and differentiation of target cells
  • a novel aspect of the present invention relates to the ability of neuregulins to affect function of inner ear cells and olfactory epithelial cells.
  • Neuregulins are the products of a gene which produce a number of variably-sized, differentially-spliced RNA transcripts that give nse to a series of proteins. These proteins are of different lengths and contain some common peptide sequences and some unique peptide sequences. The conclusion that these factors are encoded by a single gene is supported by the differentially-spliced RNA sequences which are recoverable from bovine posterior pituitary, human spinal chord and human breast cancer cells (MDA-MB-231). Further support for this conclusion derives from the size range of proteins which act as ligands for the erbB receptors (see below)
  • neuregulin transcripts (Example 3 and 4) in inner ear cells in vivo indicates that neuregulin has a role in inner ear cell function in vivo.
  • neuregulins affect survival of olfactory cells (Example 5) and that neuregulins transcripts are detected in cells of die olfactory epithelium (Example 6).
  • neuregulins may be identified using the protocols described herein and in Holmes et al., Science 256: 1205, 1992; Peles et al, Cell 69:205, 1992; Wen et al., Cell 62:559, 1992; Lupu et al., Proc. Natl. Acad. Sci. USA 82:2287, 1992; Yarden and Peles, Biochemistry 30:3543, 1991 ; Lupu et al., Science 249: 1552, 1990; Dobashi et al., Biochem. Biophys. Res. Comm. 122: 1536, 1991 ; Huang et al., J. Biol. Chem.
  • Compounds can be assayed for their usefulness in vitro using the methods provided in the description and examples below. Following the in vitro demonstration of the effect of neuregulins on inner ear cell function, the in vivo therapeutic benefit of the effect can be accomplished by the administration of neuregulins, neuregulin producing cells or DNA encoding neuregulins to a vertebrate requiring therapy.
  • in vitro assays are used to determine which neuregulin protein(s) promote non- visual sensory epithelial cell function and which non-visual sensory epithelial cell types are affected by contacting neuregulin protein. Described below are methods for detecting the ability of a neuregulin to promote function of a non-visual sensory epithelial cell. In vitro assays for determining neuregulin effects on non-visual sensory epithelial cell function depend on establishing non-visual sensory epithelial cell cultures. A general reference on cell and tissue culture is Cell and Tissue Culture: Laboratory Procedures (Ed. by A. Doyle, J. B. Griffiths, and D. G. Newell, John Wiley and Sons, New York, NY, 1994).
  • the non-visual sensory epithelial cell from a vertebrate used in this invention may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI- 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing non-visual sensory epithelial cells.
  • 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195 may be used as culture media for non-visual sensory epithelial cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucieosides (such as adenosine and thymidine), antibiotics (such as GentamycinTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, will be apparent to the ordinarily skilled artisan.
  • non-visual sensory epithelial cell cultures to demonstrate that neuregulin promotes non-visual sensory epithelial cell function is in accordance with the methods described in general terms above and further described below.
  • the survival function is assayed by methods that identify and count either viable cells or dead non-visual sensory epithelial cell cells following culture at low density (e.g., 10,000 cells/cm2) over a period from one to six days in the presence of varying amounts of neuregulin added to the culture medium. Included in these methods are specific stains for dead cells, such as propidium iodide, which enters the nucleus of dead cells and is detected by fluorescence microscopy. Alternatively, the counting of non-visual sensory epithelial cells adhering to the culture substratum over a six day period also can be used as an indicator of cell survival. In Vitro Method II
  • An alternative procedure to monitor non-visual sensory epithelial cell death utilizes labeling of nicked DNA strands, which are characteristic of cells undergoing apoptotic cell death, with digoxygenin-1 1-dUTP using terminal deoxynucleotidyl transferase (TUNEL) according to the protocol described in Gavrieli et al., J. Cell Biol. U_2: 493-501 , 1992.
  • the labeled DNA strands are detected using standard kits available from commercial vendors (e.g., Genius kit from Boehringer Mannheim).
  • a cell death detection ELISA system which is based on the DNA fragmentation that occurs in dying cells (Boehringer Mannheim catalog no. 1585 045) can be utilized to quantify cell death in accordance with the instructions provided by the commercial vendor.
  • LDH also can be used to quantify the extent of non-visual sensory epithelial cell death in vitro (Kirk et al, J. Pharmacol. Exper. Therapeut. 221: 1080, 1994). LDH levels are measured by an automated kinetic colorimetric assay in which oxidation of lactate to pyruvate is coupled to reduction of the tetrazolium dye, INT.
  • the proliferative function of neuregulins on non-visual sensory epithelial cells can be assayed by inco ⁇ oration of 125 ⁇ _ ⁇ j r d, 3 ⁇ -dT or BrdU into replicating DNA strands of dividing cells, or by cell counting.
  • the assays developed to measure the mitogenic activity of neuregulins on Schwann cells by inco ⁇ oration of DNA synthesis precursors can be adapted to non-visual sensory epithelial cells by one of normal skill in the art of cell culture.
  • the differentiation function of neuregulins on non-visual sensory epithelial cells can be assayed by employing analytical methods, such as staining of filamentous actin of hair cell stereocilia and cuticular plates by fluorescently conjugated phalloidin, immunostaining of in situ hybridization, which can detect and quantify marker proteins associated with the various cell types of non-visual sensory epithelia, such as myosin 7, osteospondin, hair cell antigens (HCA), calbindin, tectorins, and other antigens in hair cells and supporting cells of the cochlear-vestibular epithelia which are specifically bound by monoclonal antibodies.
  • analytical methods such as staining of filamentous actin of hair cell stereocilia and cuticular plates by fluorescently conjugated phalloidin, immunostaining of in situ hybridization, which can detect and quantify marker proteins associated with the various cell types of non-visual sensory epithelia, such as myosin 7, osteospondin, hair cell antigens (HCA), calbindin
  • RNA growth factors and their receptors have been identified in non-visual sensory epithelia, as described in the prior act. Methods utilized to detect those molecules and activities can be employed to demonstrate a differentiation function of neuregulin on non- visual sensory epithelial cells. For example, neuregulins can be shown to induce the synthesis of growth factors and/or their receptors expressed in the cochlear-vestibular epi elia. The analysis can be by in situ hybridization or other methods of quantitative RNA analysis, such as, but not limited to, reverse transcription-PCR, RNAse protection and Northern blotting. Alternatively, induced expression of growth factors or their receptors can be assayed by immunocytochemical staining or cell biological assays designed to measure growth factor activity.
  • the in vitro assays described above to identify neuregulins that have biological activity on non-visual sensory epithelial cells can be applied to dissociated cells, semi-dissociated cells, explants of whole non-visual sensory epimelia and parts thereof, such as preparations of the cochlear-vestibular epithelia or of the olfactory epithelia.
  • the cultures can be established and maintained using methods described above. In some cases, minor modifications or substitutions to the procedures described herein, which do not alter the reduction to practice of the invention, can be provided by one of ordinary skill in the art.
  • Neuregulin activity on inner ear cells also can be shown through in vivo assays. Some in vivo assays represent animal models of loss of hearing, balance or smell. In Vivo Method I
  • Hair cell regeneration has been studied in vivo in a variety of vertebrate species.
  • a general model for the process as described in the avian cochlea is outlined below.
  • Damage to cochlear epithelia is brought on by acoustic trauma.
  • Ototoxic antibiotics can be used to damage both the cochlear and the vestibular epithelia.
  • Regeneration of chick sensory hair cells after acoustic trauma illustrates two key points: 1) the perturbations replicate the manner in which human hearing loss occurs, and; 2) regeneration of the damaged tissue leads to restoration of hearing function over a period of days to weeks. Since the lost neuroepithelial cells are part of a local neural circuitry the re-establishment of appropriate synaptic connections is quite feasible and has been demonstrated in this model and in other models of hair cell regeneration.
  • Regeneration that occurs in the cochlear sensory epithelium of chickens can be observed by utilizing scanning electron microscopy.
  • a chick cochlear epithelium fixed 10 days after acoustic trauma shows that nearly normal appearance has been restored.
  • Similar models are available in rat and guinea pig, where hair cell regeneration in vestibular end organs is much more limited.
  • the key to promoting mammalian hair cell regeneration would seem to lie in identifying and delivering the appropriate stimulus for proliferation of support cells and differentiation or repair of hair cells.
  • Neuregulins delivered to the damaged epithelial tissue can stimulate proliferation of support cells in the otic neuroepithelium (cochlear and vestibular) and thus promote the regeneration of hair cells.
  • neuregulin administered to the lesion site in an therapeutically effective amount can be shown to promote the process of hair cell regeneration leading to the recovery of inner ear function of hearing and balance.
  • Guinea pigs (3-6 weeks old) are given daily subcutaneous injections of gentamycin for ten consecutive days to traumatize both the vestibular and cochlear sensory epithelium (Forge et al. Science 259: 1616-1619, 1993). Following this treatment animals are fitted with osmotic micropumps to deliver either vehicle alone or rhGGF2 in vehicle directly to the perilymphatic fluid compartments of the inner ear via a cannula. Some groups of animals (10 animals/group) are sacrificed at 7-10 days to analyze the level of proliferation of support cells, while other groups are allowed to continue recovery for 28 to 60 days to assess hair cell regeneration. Labelling with bromodeoxyuridine, also delivered via osmotic micropumps, is used to monitor proliferation in whole mount and sectioned preparations, and hair cell regeneration can be assessed by microscopy.
  • mice Male mice, aged 3 months, are used for these experiments. Olfactory bulbectomy is performed on the right side of each animal under sterile conditions, the left side serving as a control. Under Avertin anesthesia (tribromoethanol and amyl alcohol, warmed to 37° C), the overlying skull is opened and the right olfactory bulb is removed by aspiration. A piece of Gelfoam is placed in the bulb site to prevent bleeding.
  • Avertin anesthesia tribromoethanol and amyl alcohol, warmed to 37° C
  • a piece of Gelfoam is placed in the bulb site to prevent bleeding.
  • rhGGF2 systemically and/or apply it topically, at various times and doses and look for either neuroprotection and/or the acceleration of regeneration.
  • Partial olfactory nerve lesion (based on M. Cagiano, J.S. Kauer, and D.D. Hunter, Neuron 13:339-352, 1994):
  • Rat pups, 2-5 days old, are anesthetized, and the experimental animals are lesioned at the level of the cranial extension of the olfactory nerve, by penetration through the cartilage with a blunt needle inserted just caudal to the cribriform plate. This resultant damage to the olfactory nerve causes degeneration of part of the olfactory epithelium.
  • rhGGF2 systemically and/or apply it topically, at various times and doses and look for either neuroprotection and/or the acceleration of regeneration.
  • Other compounds, in particular peptides, which specifically bind and/or activate erbB receptors also can be used according to the invention as effectors of non-visual sensory epithelial cell function.
  • a candidate compound can be routinely screened for erbB receptor binding, and if it binds, can then be screened for affecting inner ear cell function using the methods described herein.
  • the demonstration of biological activity of the neuregulins by promoting inner ear cell function in any of the animal models described above indicates efficacy in treating disorders of the ear.
  • inner ear diseases and related disorders are known that produce impaired hearing and/or balance and in some cases progress to total deafness.
  • These disorders of the ear include, but are not necessarily limited to: sensorineural hearing loss caused by ototoxic antibiotics, and by chemotherapeutic agents such as cisplatin which produces a peripheral neuropathy resulting in hearing loss, excessive noise, viral and bacterial infection or autoimmune disease; tinnitus; and, Meniere's syndrome. Meningitis often results in hearing loss.
  • administration of neuregulin in a therapeutically effective amount can provide a treatment for disorders of the ear, which otherwise left untreated would result in the loss of hearing and/or balance.
  • the invention includes use of any modifications or equivalents of the above polypeptide factors which do not exhibit a significantly reduced activity related to affecting inner ear cell function and more generally to non-visual sensory epithelial cell function.
  • modifications in which amino acid content or sequence is altered without substantially adversely affecting activity are included.
  • the statements of effect and use contained herein are therefore to be construed accordingly, with such uses and effects employing modified or equivalent factors being part of the invention.
  • the invention includes the use of the above named family of proteins (i.e. neuregulins) as extracted from natural sources (tissues or cell lines) or as prepared by recombinant means.
  • family of proteins i.e. neuregulins
  • the human peptide sequences described above represent a series of splicing variants which can be isolated as full length complementary DNAs (cDNAs) from natural sources
  • cDNA libraries prepared from the appropriate tissues can be assembled as DNA constructs with individual exons (e.g., derived as separate exons) by someone skilled in the art.
  • the invention includes methods for the use of any protein which is substantially homologous to coding segments which comprise neuregulin, as well as other naturally occurring neuregulin polypeptides for the pu ⁇ ose of promoting inner ear cell function. Also included are the use of: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid naturally occurring (for definitions of high and low stringency see Current Protocols in Molecular Biology, (1989) John Wiley & Sons, New York, NY, 6.3.1 - 6.3.6, hereby inco ⁇ orated by reference); and the use of polypeptides or proteins specifically bound by antisera to GGF polypeptides. The term also includes the use of chimeric polypeptides that include the neuregulin polypeptides.
  • a novel aspect of the invention involves the use of neuregulins as factors to promote non-visual sensory epithelial cell function. Treatment of the cells to achieve these effects may be achieved by contacting cells with a polypeptide described herein.
  • the methods of the invention make use of the fact that the neuregulin proteins are encoded by the same gene.
  • a variety of messenger RNA splicing variants (and their resultant proteins) are derived from this gene and many of these products show binding to erbB receptors and activation of the same.
  • This invention provides a use for all of the known products of the neuregulin gene (described herein and in the references listed above). Most preferably, recombinant human GGF2 (rhGGF2) is used in these methods.
  • the invention includes use of any modifications or equivalents of the above polypeptide factors which do not exhibit a significantly reduced activity. For example, modifications in which amino acid content or sequence is altered without substantially adversely affecting activity are included. The statements of effect and use contained herein are therefore to be construed accordingly, with such uses and effects employing modified or equivalent factors being part of the invention.
  • the human peptide sequences described above represent a series of splicing variants which can be isolated as full-length complementary DNAs (cDNAs) from natural sources (cDNA libraries prepared from the appropriate tissues) or can be assembled as DNA constructs with individual exons (e.g., derived as separate exons) by someone skilled in the art.
  • Another aspect of the invention is the use of a pharmaceutical or veterinary formulation comprising any factor, as defined above, formulated for pharmaceutical or veterinary use, respectively, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
  • a pharmaceutical or veterinary formulation comprising any factor, as defined above, formulated for pharmaceutical or veterinary use, respectively, optionally together with an acceptable diluent, carrier or excipient and/or in unit dosage form.
  • conventional pharmaceutical or veterinary practice may be employed to provide suitable formulations or compositions.
  • a medicament is made by administering the polypeptide with a pharmaceutically effective carrier.
  • Neuregulins can be administered into the perilymphatic fluid compartments of the inner ear by injection of factor formulated in an appropriate vehicle for administration.
  • An osmotic mini-pump may also used to deliver neuregulin directly to the perilymphatic fluid compartments of the inner ear via a cannula.
  • formulations to be used as a part of the invention can be applied to parenteral administration, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intranasal, intrathecal, aerosol, transdermal and by other slow release devices (i.e., osmotic pump-driven devices; see also USSN 08/293,465, hereby inco ⁇ orated by reference).
  • parenteral administration for example, intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intranasal, intrathecal, aerosol, transdermal and by other slow release devices (i.e., osmotic pump-driven devices; see also USSN 08/293,465, hereby inco ⁇ orated by reference).
  • formulations of this invention may also be administered by the transplantation into the patient of host cells expressing the DNA encoding polypeptides which are effective for the methods of the invention or by the use of surgical implants which release the formulations of the invention.
  • Parenteral formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations maybe in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors.
  • polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes
  • biocompatible, biodegradable lactide polymer or polyoxyethylene-polyoxypropylene copolymers
  • Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or citric acid for vaginal administration.
  • the present factors can be used as the sole active agents, or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate neuronal survival in neurological diseases, or peptidase or protease inhibitors.
  • concentration of the present factors in the formulations of the invention will vary depending upon a number of issues, including the dosage to be administered, and the route of administration.
  • the factors of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration.
  • General dose ranges are from about 1 ⁇ g/kg to about lg/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day.
  • the preferred dosage to be administered is likely to depend upon the type and extent of progression of the pathophysiological condition being addressed, the overall health of the patient, the make up of tiie formulation, and the route of administration.
  • a further general aspect of the invention is the use of a factor of the invention in the manufacture of a medicament, preferably for the treatment of a disease or disorder.
  • the "GGF2" designation is used for all clones which were previously isolated with peptide sequence data derived from GGF-II protein (i.e., GGF2HBS5, GGF2BPP3) and, when present alone (i.e., GGF2 OR rhGGF2), to indicate recombinant human protein encoded by plasmids isolated with peptide sequence data derived from the GGF-II protein (i.e., as produced in insect cells from the plasmid HBSS).
  • Recombinant human GGF from the GGFHBS5 clone is called GGF2, rhGGF2 and GGF2HBS5 polypeptide.
  • nucleic acid constructs encoding neuregulins or neuregulin producer cells are also part of the invention.
  • the genetically modified neuregulin producer cells can be transplanted to a position near the inner ear cell type and elicit the responses described above.
  • the present factors exhibit mitogenic activity on inner ear cells.
  • other possible roles for neuregulin/rhGGF2 might include induction of hair cell regeneration, promotion of hair cell-neuron synaptogenesis, and maintenance of both hair cell survival and synaptic function.
  • the present factors exhibit survival activity on olfactory cells.
  • rhGGF2 recombinant human GGF2 confers effects on non-visual sensory epithelial cells. These activities indicate efficacy of GGF2 and other neuregulins in inducing wound repair and repair of other non-visual sensory epithelial tissue damage, and promoting regeneration and prophylactic effects on non-visual sensory epithelial tissue degeneration.
  • Example 1 Neuregulin (rhGGF2) promotes mitogenesis of chick hearing organ epithelia in vitro.
  • Dissociated chick cochlea supporting cells were plated onto fibronectin coated well in
  • the basal level of the support cell proliferation is 12.1 %, and therefore, the effect of rhGGF2 appears to be to produce roughly a doubling in tiie percent labelled cells over the basal level.
  • the mitogenic stimulation by rhGGF2 is roughly twenty-fold over the basal level at 50 ng/ml and nearly eight-fold over the basal level at 12.5 ng/ml.
  • TGFoc produces little more than a doubling of the basal rate of proliferation in cultures of mouse vestibular epithelia.
  • Example 3 Neurons of the spiral ganglion express high levels of neuregulin.
  • Neurons of the spiral ganglion innervate and make synapses with hair cells of the cochlear epithelium (ce) (see also Figure 2).
  • the survival of spiral ganglion neurons depends on the expression of trophic factors BDNF and NT3 from cells in the sensory epithelia.
  • Neuregulin growth factors expressed in the neurons of the spiral ganglia can elicit the expression of the neurotrophins NT3 and BDNF from cells in the sensory epithelia as part of a trophic support circuit (Gywnne et al., U.S. Patent Application# 08/341,018: Use of neuregulins as modulators of cellular communication) and thus stimulate hair cell regeneration and promote recovery of hearing loss.
  • Example 4 Epithelial cells of the vestibular system express high levels of neuregulin.
  • the vestibular epithelium is the hair cell epithelium of the vestibular system, and thus mediates the sense of balance.
  • a single- stranded digoxigenin-labeled riboprobe (antisense strand) encoding the EGF-like domain through the cytoplasmin domain of the rat cDNA clone GGFRP3 (Marchionni et al., Nature 362:312. 1993)
  • Fig. 7 neuregulin message is normally expressed in the embryonic day 21 rat vestibular system. Neuregulin transcripts are not detected in the non- sensory epithelium (nse).
  • Example 5 Recombinant human GGF2 promotes olfactory neuron survival in vitro.
  • the defined medium used for the culture of primary olfactory neurons, NSFM is a modification of NYSF-404 (Yabe, et al. In vitro 22:363-368, 1986): A 2x stock of basal medium was produced by dissolving the contents of 1 vial of MEM (Sigma Cat. #M-0643) and 1 vial of RPMI- 1640 (Sigma Cat. #R- 6504) (each meant for 1 liter) into 1 liter of tissue culture-grade water.
  • AAM was made by adding 0.15 g of each of the following, except where noted, to 100 ml of water, and stored at -20° in 5 ml aliquots: Arg, Asp, Gin (3 g), Gly (0.05 g), Pro (0.05 g), Ser, Thr, Val, and choline chloride (0.25 g).
  • BVP was made by dissolving 0.0125 g vitamin B-12, 0.025 g biotin, and 0.125 g putrescine into 10 ml water; this was diluted 1000-fold and stored as 0.5 ml aliquots at -20°.
  • Recombinant human GGF2 was stored at -80°C, and diluted into NSFM at the indicated concentrations.
  • HBSS Hank's Balanced Salt Solution
  • Turbinates collected bilaterally from 20-25 rats were then coarsely minced with iridectomy scissors, placed in a 15 ml centrifuge tube with 10 ml of Ca ++ /Mg ++ -free HBSS (CMF-HBSS) (Gibco) containing 1 mg/ml trypsin (Type XUI) and 1 mg/ml collagenase (Type A, Boehringer Mannheim), and rotated end-over- end at 37°. After 20 min, the tissue was triturated 5-10 times with a 5 ml tissue culture pipet, and returned to 37° for 25 min.
  • CMF-HBSS Ca ++ /Mg ++ -free HBSS
  • Type XUI trypsin
  • Type A Boehringer Mannheim
  • the tube was then placed in the centrifuged with the brake turned off, the power turned on for the amount of time necessary to reach 5 g, and immediately turned off; this spin lasted approximately 10 min and allowed the selective pelleting of semi-dissociated pieces of OE sheet.
  • the pellet was resuspended in 15 ml of NSFM, and the OE tissue pre- plated in 3, 60 mm polystyrene Petri dishes for 2-3 hr at 37° in a 5% CO2 incubator.
  • rhGGF2 promotes olfactory neuron survival in vitro.
  • doses of 50 ng/ml or higher give a maximal increased in survival over control cultures (approximately 2.8-fold increase).
  • rhGGF2 By 4 days in vitro, a time at which very few neurons survive in serum-free medium, the presence of rhGGF2 at 100 ng/ml gives rise to a approximately 10-fold increase in the number of surviving neurons.
  • Example 6 Olfactory neurons express high levels of neuregulin.
  • neuregulins are expressed in the olfactory epithelium of embryonic mice using in situ hybridization. This hybridization, however, was performed using 35 S-labeled probes and the resolution was such that the cell types expressing neuregulin could not be identified (Meyer, et al. Proc Natl Acad Sci USA 9k 1064-1068, 1994).

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Abstract

D'une manière générale, la présente invention concerne des procédés permettant de promouvoir la fonction de cellules de l'oreille interne en utilisant des neurégulines. Un aspect nouveau de l'invention concerne l'emploi de neurégulines comme facteurs de croissance, pour promouvoir la fonction de cellules épithéliales sensorielles non visuelles. Le traitement de ces cellules pour obtenir ces effets peut être effectué en mettant lesdites cellules en contact avec un polypeptide qui figure dans la description. Les traitements peuvent être appliqués pour ralentir ou arrêter la perte nette de cellules ou pour accroître la quantité ou la qualité du tissu épithélial sensoriel non visuel qui existe chez le vertébré considéré.
PCT/US1996/018031 1995-11-13 1996-11-12 Procedes de traitement de troubles dans des epitheliums sensoriels non visuels WO1997017983A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP96940360A EP0866716A4 (fr) 1995-11-13 1996-11-12 Procedes de traitement de troubles dans des epitheliums sensoriels non visuels
AU77258/96A AU727037B2 (en) 1995-11-13 1996-11-12 Methods of treating disorders of non-visual sensory epithelia
CA 2237400 CA2237400A1 (fr) 1995-11-13 1996-11-12 Procedes de traitement de troubles dans des epitheliums sensoriels non visuels
JP9518966A JP2000505781A (ja) 1995-11-13 1996-11-12 非視覚性感覚上皮の疾患の治療方法

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US654195P 1995-11-13 1995-11-13
US60/006,541 1995-11-13

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020889A1 (fr) * 1996-11-15 1998-05-22 Creative Biomolecules Inc Regeneration de tissus percepteurs sensoriels, induite par des peptides morphogenes
WO1999042088A3 (fr) * 1998-02-23 2000-03-02 Otogene Ag Procede pour le traitement de maladies ou de troubles de l'oreille interne
WO2000044397A3 (fr) * 1999-01-28 2001-01-11 Amgen Inc Anticorps monoclonal dirige contre l'epithelium utriculaire
US6838444B1 (en) 1999-06-01 2005-01-04 Baylor College Of Medicine Compositions and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis, and abnormal cell proliferation
US7053200B1 (en) 1999-06-01 2006-05-30 Baylor College Of Medicine Compositions and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis, and abnormal cell proliferation
US7741303B2 (en) 1998-02-23 2010-06-22 Sound Pharmaceuticals Incorporated Stimulation of cellular regeneration and differentiation in the inner ear
US9951351B2 (en) 2014-10-09 2018-04-24 Genvec, Inc. Adenoviral vector encoding human atonal homolog-1 (HATH1)

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US6087323A (en) * 1992-04-03 2000-07-11 Cambridge Neuroscience, Inc. Use of neuregulins as modulators of cellular communication

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Title
DAVIS J. G., ET AL.: "USE OF THE TELEOST SACCULE TO IDENTIFY GENES INVOLVED IN INNER EAR FUNCTION.", DNA AND CELL BIOLOGY, MARY ANN LIEBERT, NEW YORK, NY, US, vol. 14., no. 10., 1 January 1995 (1995-01-01), US, pages 833 - 839., XP000882867, ISSN: 1044-5498 *
MAHANTHAPPA N K SCHWARTING G A: "Peptide Growth Factor Control of Olfactory Neurogenesis and Neuron Surviva l in Vitro: Roles of EGF and TGF-betas", NEURON, CAMBRIDGE, MA, US, vol. 10, no. 2, 1 February 1993 (1993-02-01), US, pages 293 - 305, XP002955395, DOI: 10.1016/0896-6273(93)90319-M *
MAHANTHAPPA N, SCHWARTING G A: "THE ROLE OF NEUREGULINS IN OLFACTORY NEUROGENESIS", ABSTRACTS OF THE SOCIETY FOR NEUROSCIENCE, SOCIETY FOR NEUROSCIENCE, WASHINGTON, DC., US, vol. 221, 1 January 1995 (1995-01-01), US, pages 1512 + AN 595.04, XP001109117, ISSN: 0190-5295 *
MARCHIONNI M A: "NEU TACK ON NEUREGULIN", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 378, 23 November 1995 (1995-11-23), United Kingdom, pages 334/335, XP001092777, ISSN: 0028-0836, DOI: 10.1038/378334a0 *
See also references of EP0866716A4 *
SOCIETY FOR NEUROSCIENCE, WASHINGTON, DC.; 1 January 1996 (1996-01-01), GU R, MARCHIONNI M, CORWIN J T: "GLIAL GROWTH FACTOR ENCHANCES SUPPORTING CELL PROLIFERATION IN RODENT VESTIBULAR EPHITHELIA CULTURED IN ISOLATION", XP001052949 *
YAMASHITA H., OESTERLE E. C.: "INDUCTION OF CELL PROFILERATION IN MAMMALIAN INNER-EAR SENSORY EPITHELIA BY TRANSFORMING GROWTH FACTOR A AND EPIDERMAL GROWTH FACTOR.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 92., 1 January 1995 (1995-01-01), US, pages 3152 - 3155., XP000882869, ISSN: 0027-8424, DOI: 10.1073/pnas.92.8.3152 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998020889A1 (fr) * 1996-11-15 1998-05-22 Creative Biomolecules Inc Regeneration de tissus percepteurs sensoriels, induite par des peptides morphogenes
WO1998020890A1 (fr) * 1996-11-15 1998-05-22 Creative Biomolecules, Inc. Regeneration induite par peptide morphogene de tissus percepto-sensoriels
WO1999042088A3 (fr) * 1998-02-23 2000-03-02 Otogene Ag Procede pour le traitement de maladies ou de troubles de l'oreille interne
US7087581B1 (en) 1998-02-23 2006-08-08 Sound Pharmaceuticals Incorporated Method for the treatment of diseases or disorders of the inner ear
US7741303B2 (en) 1998-02-23 2010-06-22 Sound Pharmaceuticals Incorporated Stimulation of cellular regeneration and differentiation in the inner ear
WO2000044397A3 (fr) * 1999-01-28 2001-01-11 Amgen Inc Anticorps monoclonal dirige contre l'epithelium utriculaire
US6838444B1 (en) 1999-06-01 2005-01-04 Baylor College Of Medicine Compositions and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis, and abnormal cell proliferation
US7053200B1 (en) 1999-06-01 2006-05-30 Baylor College Of Medicine Compositions and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis, and abnormal cell proliferation
US7442688B2 (en) 1999-06-01 2008-10-28 Baylor College Of Medicine Composition and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis and abnormal cell proliferation
US7470673B2 (en) 1999-06-01 2008-12-30 Baylor College Of Medicine Composition and methods for the therapeutic use of an atonal-associated sequence for deafness, osteoarthritis and abnormal cell proliferation
US9951351B2 (en) 2014-10-09 2018-04-24 Genvec, Inc. Adenoviral vector encoding human atonal homolog-1 (HATH1)
US11279951B2 (en) 2014-10-09 2022-03-22 Genvec, Inc. Adenoviral vector encoding human atonal homolog-1 (HATH1)

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AU7725896A (en) 1997-06-05

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