AU727037B2 - Methods of treating disorders of non-visual sensory epithelia - Google Patents

Description

WO 97/17983 PCT/US96/18031 METHODS OF TREATING DISORDERS OF NON-VISUAL SENSORY EPITHELIA GOVERNMENT SUPPORT This invention was made with the support of federal grants from the U.S. Government.

The Government has certain rights in the invention.

FIELD OF THE INVENTION 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.

BACKGROUND OF THE INVENTION 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. 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.

I. Anatomy and function of sensory epithelia A. Hearing and the Sense of Balance 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 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. 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. These hair cells are similar to cochlear hair cells, and transmit electrical signals to the brain via neurons of the vestibular ganglion. These two systems share a common embryological origin, and are physiologically similar.

WO 97/17983 PCT/US96/18031 1. Anatomy and function of the cochlear-vestibular epithelia The Cochlear Epithelium 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. 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.

These 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. At their basal surfaces, 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 WO 97/17983 PCT/US96/18031 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. In summary, perception of tone is a function of which hair cells are stimulated, and 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. As with cochlear hair cells, the hair cells of the vestibular system form synapses with fibers from the vestibular ganglion to transmit information to the central nervous system.

Cochlear Innervation and the Primary Auditory Pathway The circuitry of the auditory pathway, the path by which hair cell responses are transmitted through the brain, 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 VIIIth 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 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. It is at the cortical level that higher order auditory processing occurs sound recognition, language extraction, etc.).

WO 97/17983 PCT/US96/18031 Two properties of auditory projections are of particular interest. First, 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. Second, 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. At the cortical level, auditory neurons display specificities for increasingly complex temporal patterns of frequency modulation.

The VHlIth cranial nerve also contains axons originating from the vestibular system. As with the auditory 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).

B. The senses of smell and taste Both the sense of smell and a large part of what is considered the sense of taste are mediated by the olfactory system. In the case of smell, airborne volatile molecules (odorants) are inhaled through the nose and sensed by a discreet patch of sensory epithelium (the olfactory epithelium) that resides in the rearmost region of the nasal passages upon bony ridges known as turbinates. Though the fundamental flavors identified as sweet, sour, salty and bitter are detected by sensory cells of the tongue, the bulk of gustatory experience is the product of flavor molecules volatilizing in the oral cavity and interacting with the olfactory epithelium via passages connecting the oral and nasal cavities. Thus, a normal sense of smell and taste is dependent upon the normal development and function of the olfactory epithelium and its connections to the central nervous system.

1. Anatomy of the olfactory system 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 propria WO 97/17983 PCT/US96/18031 and contains primarily connective and vascular tissues. Separating the lamina propria 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 propria 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. In addition there are 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.

III. Peptide Growth Factors The development and physiology of multicellular organisms requires multiple modes of intercellular communication. Such communication may be systemic, as in the case of hormones delivered via the bloodstream, or can be highly localized. In the latter case, two modes are commonly recognized: synaptic signaling from neurons; and paracrine signaling from adjacent or nearby cells (Alberts et al., Molecular Biology of the Cell, 2nd ed. Garland Publishing, New York, NY, 1989). A function of such signaling is to coordinate cell survival, proliferation, differentiation, and/or metabolic activity. The molecules that serve as transmitted signals vary in their chemical composition; one group of molecules are proteins, the peptide growth factors. Peptide growth factors act upon cells by binding to cell surface receptors. These receptors are coupled to intracellular signal transduction pathways that give rise to the above described activities when activated by growth factor binding.

WO 97/17983 PCT/US96/18031 A. Peptide Growth Factors in the cochlear-vestibular epithelia 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, Ylikowki, Palgi, J., Lethonen, E. Arumae, U. and Saarma, PNAS USA 89: 9915-9919, 1992) and the receptors trkB and trkC (Enfors, Merlio, and Persson, H. Eur. J. Neurosci.

4:1140-1158, 1992); culture studies demonstrating that BDNF and NT3 support survival and neurite extension of developing cochlear vestibular ganglia neurons (Avila, M. Varela- Nieto, Romero, Mato, J. Giraldez, Van De Water, T. R. and Represa, J.

Dev. Biol. 159:266-275, 1993), and; analysis of mice deficient in BDNF (Emfors, Lee, Kucera, J. and Jaenisch, R. Cell 77:503-512, 1994; Jones, K. Farinas, I., Backus, C. and Reichardt, L. F. Cell 76:989-999, 1994) and NT3 (Erfors, Lee, K. F.

and Jaenisch, R. Nature 368:147-150, 1994; Farinas, Jones, K. Backus, Wang, X. Y. and Reichardt, L. F. Nature 369:658-661, 1995) showed deficits in innervation and hair cell morphology.

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. Thus, 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, Merlio, and Persson, H. Eur. J. Neurosci. 4:1140-1158, 1992).

In concert with the known distribution of neurotrophins and trk receptors, the results from gene knockout experiments have led to the conclusion that BDNF is the major survival factor for vestibular ganglia neurons and NT3 for spinal ganglion neurons (Erfors, Lee, Kucera, J. and Jaenisch, R. Cell 77:503-512, 1994; Emfors, Lee, K. F. and Jaenisch, R. 1994; Enfors, Van De Water, Loring, and Jaenishce, R. Neuron 14:1153-1164, 1995).

A variety of culture systems have been used to study the activities of neurotrophins on cells of the inner ear and their associated ganglia. In these studies BDNF has been shown to promote survival and neurite outgrowth of vestibular and spinal ganglia neurons (Pirvola, U., Ylikowki, Palgi, Lethonen, E. Arumae, U. and Saarma, M. PNAS USA 89:9915- WO 97/17983 PCT/US96/18031 9919, 1992; Avila, M. Varela-Nieto, Romero, Mato, J. Giraldez, Van De Water, T. R. and Represa, J. Dev. Biol. 159:266-275, 1993).

More recently, 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. Stewart, R. R. and Gao, J. Neurobiology 28:3, 330-3401, 1995).

Specific growth factors that stimulate hair cell regeneration have not been identified.

B. Peptide Growth Factors in the olfactory system In a serum-free culture system, Mahanthappa and Schwarting demonstrated that epidermal growth factor (EGF) promotes basal cell division and transforming growth factor Ps (TGF-Ps) promote neurogenesis (Mahanthappa, et al. Neuron 10:293-305, 1993).

Whether EGF plays the role of basal cell mitogen in vivo, is not known. Furthermore, it remains to be determined whether EGF or another member of the EGF superfamily of peptide growth factors (Massague, et al. Ann Rev Biochem 62:515-541, 1993) is actually responsible for basal cell proliferation. Within the embryonic mouse olfactory epithelium, the most differentiated zones show high levels of TGF-02 signal by in situ hybridization (Millan, et al. Develop 111:131-144, 1991). Thus, the finding that TGF-p2 promotes neurogenesis in the cultured olfactory epithelium and is more potent than TGF-01 is entirely consistent with known patterns of localization in vivo (Mahanthappa, et al. Neuron 10:293-305, 1993).

Furthermore, the finding that antibody-neutralization of TGF-p2 completely blocks baseline levels of olfactory neurogenesis in vitro supports the hypothesis that TGF-p2 is in fact the endogeneously produced neurogenic factor in the OE (Mahanthappa, et al. Neuron 10:293- 305, 1993).

Though neurotrophins are expressed in the olfactory bulb (Guthrie, et al. J Comp Neurol 313:95-102, 1991), a potential source of trophic support, neither nerve growth factor nor brain-derived growth factor promote survival of olfactory neurons in vitro beyond one week (Mahanthappa, et al. Neuron 10:293-305, 1993). In a culture system established by Pixley (Pixley Neuron &:1191-1204, 1992), cultured epithelium demonstrated a tremendous degree of neurogenesis when cultured upon a monolayer of astrocytes. Furthermore, coculture with astrocytes induced the expression of olfactory marker protein (OMP), a maturation marker. Though Pixley has shown that astrocyte-conditioned medium is insufficient to promote OMP expression, specific astrocyte factors responsible for the modulation of neurogenesis, maturation, and survival were not identified.

WO 97/17983 PCT/US96/18031 IV. Pharmaceutical need for treating disorders of the ear A. Hearing Loss More than twenty-eight million Americans experience hearing loss. Two million lack sufficient hearing to understand speech and three-hundred and fifty thousand are unable to hear anything. Twenty percent of Americans with hearing problems are of school age.

There are two major forms of hearing loss. 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.

Much progress has been made in treating these forms of hearing loss primarily through surgical intervention and the creation of biocompatible prostheses. Such forms of hearing loss, however, constitute the minority.

Eighty percent of patients with significant hearing loss, approximately seventeen million Americans, suffer from sensorineural hearing loss. This loss of auditory sensitivity is due to damage to hair cells and/or lesions of neurons in the primary auditory pathway. The primary cause of sensorineural hearing loss is hair cell death. Hair cell death can have a variety of causes: loud sound, aminoglycoside antibiotic treatment, and aging among them. Hair cell loss has long been considered permanent in mammals, thus in addition to sophisticated electronic hearing aids, an increasingly pursued therapeutic approach to sensorineural hearing loss is the use of cochlear implants. Such implants are placed within the cochlea and serve to directly stimulate the VIIIth nerve in response to sound. The degree to which such devices succeed in improving patient quality of life is highly variable. Recent studies, however, suggest that hair cell loss may not be permanent and thus may herald therapies that promote full functional recovery through promotion of hair cell regeneration.

A summary of these and other relevant statistics follow: More than 28 million Americans have a hearing loss; 80 percent of those affected have irreversible and permanent hearing damage.

More than 1/3 of the U.S. population has a significant hearing impairment by age Approximately 2 million people are profoundly deaf.

One of every 1,000 infants is born totally deaf.

WO 97/17983 PCT/US96/18031 At least 1 million children are deaf or have a communication disorder.

One of every 22 infants has hearing problems.

Genetic factors are known to cause over 50 percent of all cases of severe childhood deafness.

The average age of diagnosis of hearing loss is close to age 3.

Sensorineural damage (damage to the hair cells and cochlea caused by genetics or exposure to noise) is the largest, single form of hearing loss affecting 17 million Americans.

At least 15 percent of the U.S. population is affected by tinnitus.

Persons over age fifty are twice as likely to have tinnitus.

Otitis media accounted for 10 million visits to doctors' offices in 1975.

Presbycusis affects 1/3 of the U.S. population over age 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).

B. Balance disorders Over 90 million Americans, age 17 and older, have experienced a dizziness or balance problem. In the United States, there are an estimated 97,000 cases of Meniere's disease each year.

The above information was obtained from the National Strategic Research Plan published by the NIDCD, NIH, April 1989.

WO 97/17983 PCTIUS96/18031 C. Therapeutic need for olfaction 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.

Keeping in mind that the primary olfactory neurons of the olfactory epithelium also undergo mitotic renewal, it is reasonable to expect that anti-mitotic chemotherapeutic agents would interfere with both the senses of taste and smell.

SUMMARY OF THE INVENTION In general, the present invention provides methods for promoting the function of nonvisual 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. Thus, the above factors, regulatory compounds that induce synthesis of these factors, and small molecules which mimic the effect of these factors by binding to the receptors on non-visual sensory epithelial tissues or by stimulating through other means the second messenger systems activated by the ligand-receptor complex are all extremely useful as prophylactic and affirmative therapies for non-visual tissue diseases and related disorders of the sense organs.

WO 97/17983 PCT/US96/18031 The survival of non-visual epithelial cells as used herein 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.

Methods for treatment of diseases or disorders using the polypeptides or other compounds described herein are also part of the invention. Examples of 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. Thus, 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 p185erbB2 (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.

Currently, there are limited options for therapy for the promotion of inner ear cell function, including survival, proliferation, differentiation, growth and changes in gene activity and metabolic activity. Such a therapy would be useful for treatment of a variety of inner ear disorders resulting in loss of hearing and/or balance. Thus, treatments may be provided to slow or halt net cell loss or to increase the amount or quality of inner ear tissue present in the vertebrate.

WO 97/17983 PCT/US96/18031 Advantages 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.

By analogy to the diseases of the ear, 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. As 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.

BRIEF DESCRIPTION OF THE FIGURES 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.

-12- WO 97/17983 PCT/US96/18031 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.

DETAILED DESCRIPTION OF THE INVENTION It is intended that all references cited shall be incorporated herein by reference.

General 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.

Definition of key terms The term administration as used herein 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.

The term affectine as used herein refers to the induction of a quantitative change in the response of a target cell, as a result of an interaction with a neuregulin.

The term basal cell as used herein refers to epithelial cells that reside in immediate apposition to the extracellular matrix rich, basement membrane. Such cells can also be support cells.

-13- WO 97/17983 PCT/US96/18031 The term differentiation as used herein 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.

The term disorder as used herein 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.

The term erbB ptor as used herein refers to erbB2, erbB3 and erbB4 (also HER-2, HER-3 and HER-4 of human) existing as monomeric, homodimeric and heterodimeric erbB2/erbB3) cell surface receptor tyrosine kinases that bind and/or are activated by one or more neuregulins.

The term function as used herein 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.

The term hair cell as used herein 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 sound waves) causes signaling by the hair cells.

The term mammal as used herein describes a member of the Class Mammalia (Subphylum Vertebrata).

The term mitosis as used herein 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 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, The term MWM-i as used herein 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. Patent Application SN 08/249,322 which is now issued as U.S. Patent No. 5,716,930. The PCT application, which claims priority from USSN 08/249,322 was published on 7 December 1995, as PCT publication WO 95/32724.; WO 92/20798; EPO 0 505 148 Al; Marchionni, et al., Nature 362 313, 1993; Benveniste, et al., PNAS 82:3930, 1985,. Kimura, et al., Nature 34.8..25 7, 1990; Davis and Stroobant, J£ Cell. Biol. I110: 13 53, 1990; Wen, et al., Cell 69.'5 59, 1992; Yarden and Ullrich, Ann. Rev. Biochem.57:443, 1988; Holmes, et al., Science 256: 1205.

1992; Dobashi, et al., Proc. Natl. Acad. Sci. 88:8582, 199 1; Lupu, et al., Proc. Natl. Acad. Sci.

89:2287, 1992; Peles and Yarden, BioEssaysl 5..815, 1993, Mudge, Current Biology 2:361, 1993, all hereby incorporated by reference.

The term .neurlogiadsodr as described herein refers to a disorder of the nervous sytThe term _non-visual sensor epiheiu as used herein 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 epithelia] layer of the skin. Not included are the sensory epithelium of the visual system, the retina.

The term olfactory neuron as used herein 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.

The term su cl as used herein 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.

The term survv as used herein refers to any process where a cell avoids death, The term 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 WO 97/17983 PCT/US96/18031 indicates a decrease in the rate of cell death by at least 10%, more preferably by at least 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.

The term sustentacular cell as used herein refers to support cells of the olfactory epithelium that reside in the luminal most portion of the epithelium. The cells contribute to the nasal mucosa and abut the chemosensory cilia of the olfactory neurons.

The term therapeutically effective amount as used herein 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.

The term treating as used herein may refer to a procedure medical procedure) designed to exert a beneficial effect on a disorder. Treating as used herein means any administration of a substance described herein for the purpose of increasing inner ear cell function. Most preferably, the treating is for the purpose of reducing or diminishing the symptoms or progression of a disease or disorder of inner ear cells. Treating or treatment, as used herein, 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.

The term vertebrate as used herein 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.

Few factors have been identified that play a role in hair cell production. A candidate factor for a role in hair cell development and regeneration is TGFz, and another is retinoic acid.

WO 97/17983 PCT/US96/18031 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. Very recently it has been reported that 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.

Neurogenesis and Cell Survival in the olfactory system.

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 2:55-83, 1977). This ability to regenerate is necessary, because olfactory neurons are directly exposed to airborn 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.

A variety of studies have demonstrated that the basal cells give rise to new olfactory neurons. Early studies in mice (summarized by (Graziadei, et al. Handbook of Sensory Physiology 9:55-83, 1977) found that soon after injection of 3 H-thymidine, label was observed in basal cells, and subsequently in maturing neurons. Horizontal basal cells take up very little label while globose basal cells take up high levels of 3 H-thymidine soon after injection (Monti-Graziadei, et al. J Neurocytol 8:197-213, 1979; Miragall, et al. Brain Res 239:245-250, 1982). This finding has been confirmed in studies using bromodeoxyuridine (Yamagishi, et al. Arch Histol Cytol 52 Suppll:375-381, 1989; Suzuki, et al. Cell Tissue Res 266:239-245, 1991), and has been interpreted to mean that horizontal basal cells are not precursors of olfactory neurons. Longer term labeling studies, however, reveal a more complex pattern of cell division (Mackay-Sim, et al. J Neurosci 11:979-984, 1991): cells lying within one nuclear diameter of the basal lamina displayed a biphasic pattern of labeling.

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).

-17- WO 97/17983 PCT/US96/18031 Neuregulins A recently described family of growth factors, the neuregulins (reviewed by Mudge, Curr. Biol. 3:361, 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 p185erbB2 have been purified, cloned and expressed (Benveniste et al. PNAS, 82:3930, 1985; Kimura et al., Nature 348:257, 1990; Davis and Stroobant, J. Cell Biol. 110:1353, 1990; Wen et al., Cell 69:559, 1992; Yarden and Ullrich, Ann. Rev.

Biochem. 57:443, 1988; Dobashi et al., Proc. Natl. Acad. Sci. 88:8582, 1991; Lupu et al., Proc. Natl. Acad. Sci. 89:2287, 1992; Wen et al., Mol. Cell. Biol. 14:1909, 1994).

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 69:559, 1992) and heregulins (Holmes et al., Science 256:1205, 1992), which were purified as ligands of the pl85erbB2 (also known as neu or HER2) receptor tyrosine kinase, also are splicing variants of the neuregulin gene. The acetylcholine receptor inducing activity (ARIA) 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 (Ig) 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 256:1205, 1992 Wen et al., Cell 69:559, 1992; Mearr and Birchmeier, PNAS 91:1064, 1994). Sites of gene expression have been localized in the brain and spinal chord and in other tissues. (Orr-Urteger et al., PNAS 20:1867, 1993; Falls et al., Cell 72:801, 1993; Marchionni et al., Nature 362:313, 1993; Mearr and Birchmeier, PNAS 91:1064, 1994 Chen et al., J. Comp. Neurol. 349; 389, 1994; Corfas et al., Neuron 14:103, 1995). Specifically in the inner ear neurepithelium, expression of neuregulin transcripts has been detected at embryonic day 18 in rat (Mearr and Birchmeier, PNAS 21:1064, 1994).

WO 97/17983 PCT/US96/18031 Although a large number of 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 celladhesion or juxtacrine mechanisms (reviewed by Massagu6 and Pandiella, Ann. Rev.

Biochem. 62:515, 1993). Alternatively, 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 (St6ckli 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. Based on studies demonstrating phosphorylation on tyrosine residues or cross-linking experiments, neuregulins are candidate ligands for the receptor tyrosine kinases p185erbB 2 (or HER-2 in human), pl85erbB3 (HER-3 in human), pl 8 5 erbB4 (or HER-4 in human) or related members of the EGFR gene family. Collectively, these receptors can be referred to as erbB receptors.

Though the precise ligand-receptor relationship of each neuregulin protein with each member of the EGFR family is yet to be clarified, several lines of evidence suggest that binding of ligands is mediated by either erbB3 and erbB4 but signaling occurs through either erbB2, erbB4 and heterodimers of the various subunits Carraway and Cantley, Cell 78:5, 1994). These receptors are known to be present on Schwann cells and muscle cells (Jo et al., Nature 373: 158, 1995), and other neuregulin targets, such as cell lines derived from various tumor tissues, such as breast and gastric epithelia. 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.

-19- WO 97/17983 PCT/US96/18031 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 heregulin BI (Levi et al, J Neurosci. 15:1329, 1995). On human muscle culture, 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 72:801, 1993) and heregulin Bl (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. 13:2118, 1993). Glial growth factor (GGF), and more specifically rhGGF2, can restrict neural crest stem cells to differentiate into glial cells in vitro (Shah et al., Cell 77:349, 1994). Activities of neuregulin on inner ear cells have not been described. In summary, there are examples of 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 rise 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).

Further evidence to support the fact a single gene encodes the various neuregulins derives from nucleotide sequence comparisons. Holmes et al., (Science 256:1205, 1992) demonstrate the purification of a 45-kilodalton human protein (Heregulin-ca) which specifically interacts with the receptor protein p185erbB2. Peles et al., (Cell 69:559, 1992) describe a complementary DNA isolated from rat cells encoding a protein call "neu differentiation factor" (NDF). The translation product of the NDF cDNA has pl85erbB2 binding activity. Several other groups have reported the purification of proteins of various molecular weights with erbB receptor binding activity. These groups include the following: Lupu et al., Proc. Natl. Acad. Sci. USA 89:2287, 1992; Yarden and Peles, Biochemistry 30:3543, 1991; Lupu et al., Science 249:1552, 1990; Dobashi et al., Biochem. Biophys.

Res. Comm. 179:1536, 1991; and Huang et al., J. Biol. Chem. 257:11508, 1992.

We have found that proteins that bind pl85erbB2 and related receptors pl85erbB3 and p185erbB4) affect inner ear cell mitogenesis (Examples 1 and Further, the presence of 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. We also have found that neuregulins affect survival of olfactory cells (Example 5) and that neuregulins transcripts are detected in cells of the olfactory epithelium (Example 6).

These 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 69:559,1992; Lupu et al., Proc. Natl. Acad. Sci. USA 89:2287, 1992; Yarden and Peles, Biochemistry 30:3543, 1991; Lupu et al., Science 249:1552, 1990; Dobashi et al, Biochem. Biophys. Res. Comm. 179:1536, 1991; Huang et al., J Biol. Chem. 257:11508- 11512, 1992; Marchionni et al, Nature 362:313, 1993; and in U.S. Patent Application Serial No.07/931,041, filed August 17, 1992 (US Patent No. 5,594,114) which corresponds to W094/04560 published 3 March 1994, and U.S. Patent No. 5,530,109 issued on June 25, 1996, all of which are incorporated herein by reference.

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 of Neuregulin Effects on Non-visual Sensory Epithelial Cells Several 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). General references on the culture of neural cells and tissues are Methods in Neurosciences, Vol. 2 (Ed. by P. M. Conn. Academic Press, San Diego, CA, 1990) and Culturing Nerve Cells (Ed. by G. Banker and K. Goslin, MIT Press, Cambridge, MA, 1991). General references of immunocytochemistry are Antibodies: A Laboratory Manual Harlow and D. Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988), and -21- WO 97/17983 PCT/US96/18031 Immunocytochemistry II (Ed. by A. C. Cuello, John Wiley and Sons, New York, NY, 1993).

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 Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing non-visual sensory epithelial cells. In addition, any of the media described in Ham and Wallace, Meth. Enz. 58:44, 1979; Barnes and Sato, Anal. Biochem. 102:255, 1980; U.S. Pat. Nos. 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), nucleosides (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.

The use of 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.

In Vitro Method I 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 10,000 cells/cm 2 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.

-22- WO 97/17983 PCT/US96/18031 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-11-dUTP using terminal deoxynucleotidyl transferase (TUNEL) according to the protocol described in Gavrieli et al., J. Cell Biol. 119: 493-501, 1992. The labeled DNA strands are detected using standard kits available from commercial vendors Genius kit from Boehringer Mannheim). Further, 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.

In Vitro Method III The release into the culture medium of the cytosolic enzyme lactate dehydrogenase (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. 271:1080, 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. Briefly, 80 ul samples of the culture medium are mixed with an equal volume of the substrate solution containing (in mg/1) INT, 334; phenazine methosulfate, 86; nicotinamide adenine dinucleotide, 862; L-(+)-lactate, 4900 (lithium salt); and 0.1% Triton X-100 in 0.2 M Tris buffer, pH 8.2. In the assay, LDH activity is directly proportional to the rate of appearance of the resulting INT formazan (absorbance max. at 492 nm). The product is monitored quantitatively in a microplate reader (UVmax, Molecular Devices, Menlo Park, CA) as the change in absorbance at 490 nm over a 2 minute interval.

In Vitro Method IV The proliferative function of neuregulins on non-visual sensory epithelial cells can be assayed by incorporation of 12 5 I-Urd, 3 H-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 incorporation of DNA synthesis precursors (Brockes et al., Brain Res. 165:105, 1979; Davis and Stroobant, J. Cell Biol. 110: 1353, 1990) can be adapted to non-visual sensory epithelial cells by one of normal skill in the art of cell culture.

-23- WO 97/17983 PCT/US96/18031 In Vitro Method V 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.

In Vitro Method VI Several peptide 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 nonvisual 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 epithelia. 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 epithelia 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.

In Vivo Assays of Neuregulin Effects on non-visual sensory epithelial cells.

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.

-24- WO 97/17983 PCT/US96/18031 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.

Acoustic overstimulation for 48 hours at 1.5 kHz leads to severe damage to hair cells in the cochlea (Corwin and Cotanche, Science 240: 1772-1774, 1988). The damage to the cochlea and the recovery process can be monitored by several techniques, including microscopy and tests for hearing function or impairment. Regions of the epithelia located at a point approximately 30% of the distance along the epithelium from the proximal end in a cochlea (that is fixed immediately after 48 hours of acoustic overstimulation at 1.5 kHz) can be seen to have hair cell stereocilia bundles missing in the area damaged by the sound. Normal stereocilia bundles surround the lesion. 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.

Thus, 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.

In Vivo Method II 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 WO 97/17983 PCT/US96/18031 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 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.

In Vivo Method III Bulbectomy (based on M. Schwartz Levey, D.M. Chikaraishi, and J.S. Kauer, J. Neurosci.

11:3556-3564 1991): 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 370 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. By 3 days, the olfactory epithelium on the experimental side starts to show structural changes accompanied by neuronal degeneration.

By 7 days post-lesion, newly formed epithelial cells can begin to be detected. By 14 days post-lesion, a nearly normal olfactory epithelium has been regenerated.

One could inject rhGGF2 systemically and/or apply it topically, at various times and doses and look for either neuroprotection and/or the acceleration of regeneration.

In Vivo Method IV 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.

One could inject rhGGF2 systemically and/or apply it topically, at various times and doses and look for either neuroprotection and/or the acceleration of regeneration.

-26- WO 97/17983 PCT/US96/18031 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. A variety of 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. Thus, 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. 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 invention includes the use of the above named family of proteins neuregulins) as extracted from natural sources (tissues or cell lines) or as prepared by recombinant means.

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 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 WO 97/17983 PCT/US96/18031 occurring neuregulin polypeptides for the purpose 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 incorporated 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.

Use of Neuregulins 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 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 -28dosage form. In using the factors of the invention, 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 be used to deliver neuregulin directly to the perilymphatic fluid compartments of the inner ear via a cannula. There is also the option of delivering the factor using ethylene-vinyl acetate copolymer implants or by delivery to the cerebral spinal fluid by intrathecal injection.

Thus, the 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 (US Patent No. 5,681,568) which corresponds to W096/05857 published 29 February 1996, hereby incorporated by reference).

The 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.

Methods well-known in the art for making formulations are to be found in, for example, "Remington's Pharmaceutical Sciences." 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 polyoxyethylenepolyoxypropylene copolymers may be used to control the release of the present factors.

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.

S-29- WO 97/17983 PCT/US96/18031 The present factors can be used as the sole active agents, or can be used in combination with other active ingredients, other growth factors which could facilitate neuronal survival in neurological diseases, or peptidase or protease inhibitors.

The 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.

In general terms, 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 jig/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 the 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 GGF2HBS5, GGF2BPP3) and, when present alone GGF2 OR rhGGF2), to indicate recombinant human protein encoded by plasmids isolated with peptide sequence data derived from the GGF-II protein as produced in insect cells from the plasmid HBS5). Recombinant human GGF from the GGFHBS5 clone is called GGF2, rhGGF2 and GGF2HBS5 polypeptide.

Methods for treatment of diseases or disorders using nucleic acid constructs encoding neuregulins or neuregulin producer cells are also part of the invention.

Delivery of DNA to a cell or tissue that will take up the DNA, express the DNA and produce neuregulin as shown by Wolff et al., (Science 247:1465, 1990) and Ascadi et al., (Nature 352:815, 1991) is an aspect of the invention. Genetic modification of cultured cells (or their precursors) such as fibroblasts (as shown by Wolff et al. Proc. Nat'lAcad. Sci. USA 86:1575, 1988) or such as those derived from the nervous system (as shown by Weiss et al.

International Patent Application number PCT/US94/01053; publication number WO 94/16718) to induce the production of neuregulin from the cultured cells is another aspect of this 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.

WO 97/17983 PCT/US96/18031 As will be seen from Examples 1 and 2, below, the present factors exhibit mitogenic activity on inner ear cells. In addition to stimulating support cell division, 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.

Also, the present factors exhibit survival activity on olfactory cells. The general statements of invention above in relation to formulations and/or medicaments and their manufacture should clearly be construed to include appropriate products and uses.

A series of experiments follow which provide additional basis for the claims described herein. The following examples relating to the present invention should not be construed as specifically limiting the invention, or such variations of the invention, now known or later developed.

The examples illustrate our discovery that recombinant human GGF2 (rhGGF2) 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.

EXAMPLES

The following examples are designed to illustrate certain aspects of the present invention.

The examples are not intended to be comprehensive of all embodiments of the present invention, and should not be construed as limiting the claims presented herein.

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 Medium-199 with 20% Fetal Bovine Serum. After 96 hours, the serum containing media was removed and cells were maintained in serum free M-199 with N2 supplement for 24 hours.

M-199/N2 containing 0-200 ng/ml rhGGF2 was then added to the cells for 24 hours with BrdU added for the last 4 hours. Cells were then fixed and processed for BrdU immunohistochemistry using fluorescent double labeling techniques with BrdU labeled nuclei fluorescing red and unlabeled nuclei fluorescing green. Cell counts shown in Figure 4 are the fraction of labeled cells were counted randomly throughout the well in 600 x 600 gim squares.

-31- In the dissociated cell culture of the avian cochlea (data shown in Figure 4) 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 the percent labelled cells over the basal level.

Example 2: Neuregulin (rhGGF2) promotes mitogenesis of rat balance organ epithelia in vitro Sensory epithelia were isolated from rat utricle and plated onto fibronectin coated wells in DMEM/F12 with 10% Fetal Bovine Serum. After 8 days, medium was removed and fresh media added containing 12.5 to 100 ng/ml rhGGF2. After 72 hours, BrdU was added to the cultures for 48 hours. Cells were then fixed and processed for BrdU immunohistochemistry using fluorescent double labeling techniques with BrdU labeled nuclei fluorescing red and unlabeled nuclei fluorescing green. The data in Figure 5 show S. the number of BrdU labeled nuclei found in each culture. 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. For comparison, TGFa 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 Ten micron paraffin sections of the embryonic day X rat incubated with a single-stranded 35 S-labeled riboprobe (antisense strand) encoding the EGF-like domain through the cytoplasmic domain of the rat cDNA clone GGFRP3 (Marchionni et al., Nature 362:312, 1993), show (Figure 6) that neuregulin message is normally expressed in the developing spiral ganglion and thus that products of the neuregulin gene are normally expressed in a site where they may affect a hair cell epithelium. 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 (US Patent No.

6,087,323 which corresponds to W096/15812 published 30 May 1996: Use of neuregulins as modulators of cellular communication) and thus stimulate hair cell regeneration and promote recovery of hearing loss.

-32- WO 97/17983 PCT/US96/18031 Example 4: Epithelial cells of the vestibular system express high levels of neuregulin.

The vestibular epithelium (ve) is the hair cell epithelium of the vestibular system, and thus mediates the sense of balance. Using ten micron frozen sections incubated with a singlestranded 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), we show that (Fig. 7) neuregulin message is normally expressed in the embryonic day 21 rat vestibular system. Neuregulin transcripts are not detected in the nonsensory epithelium (nse).

Example 5: Recombinant human GGF2 promotes olfactory neuron survival in vitro.

All reagents were from Sigma unless otherwise noted. 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. To 250 ml of stock was added 200 ml water, 5 ml amino acid mix (AAM, see below), 0.5 ml Bvitamin/putrescine mix (BVP, see below), 2 ml 2.5 mg/ml insulin in 5 mM HCI, 0.5 ml mg/ml transferrin in PBS, 10 ml 5% bovine serum albumin (BSA) in PBS, 0.25 g glucose, ml 1.7 gg/ml sodium selenate, 5 ml 100 mM sodium pyruvate 20 itl ethanolamine, 0.7 g sodium bicarbonate, 5 ml of 10,000 U/ml penicillin-10 mg/ml streptomycin, and brought up to a final volume of 500 ml with 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 Gly (0.05 Pro (0.05 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 Recombinant human GGF2 was stored at -80 0 C, and diluted into NSFM at the indicated concentrations.

Cultures were produced by a simplification of the protocol of Calof and Lander (Calof, et al. J Cell Biol 115:779-794, 1991). Three to four day old rat pups were sacrificed individually, the head bisected with a sagittal cut, the nasal septum removed, and endoturbinates II-IV dissected out as a single unit. As turbinates were collected, they were placed in Hank's Balanced Salt Solution (HBSS) on ice. Turbinates collected bilaterally from 20-25 rats were then coarsely minced with iridectomy scissors, placed in a 15 ml centrifuge -33- WO 97/17983 PCT/US96/18031 tube with 10 ml of Ca++/Mg++-free HBSS (CMF-HBSS) (Gibco) containing 1 mg/ml trypsin (Type XIII) and 1 mg/ml collagenase (Type A, Boehringer Mannheim), and rotated end-overend at 37°. After 20 min, the tissue was triturated 5-10 times with a 5 ml tissue culture pipet, and returned to 370 for 25 min. All bony material and undigested tissue was allowed to settle out for approximately 2-3 min at 1 g, the supernatant transferred to a new 15 ml tube, and centrifuged at 100 g for 5 min in a benchtop centrifuge (IEC Centra-7) with the brake turned off. The pellet was resuspended with a flame-polished Pasteur pipet in 10 ml of CMF-HBSS containing 1 mg/ml trypsin inhibitor (Type II-0, from chicken egg white) and 0.5 mg/ml deoxyribonuclease I (DNase), and returned to the warm room for 20 min. 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. In order to reduce fibroblast number, the pellet was resuspended in 15 ml of NSFM, and the OE tissue preplated in 3, 60 mm polystyrene Petri dishes for 2-3 hr at 370 in a 5% CO 2 incubator.

Purified pieces of semi-dissociated OE do not adhere to Petri plastic, and were collected by repeating the very low speed centrifugation. Final plating in NSFM with the indicated factors resulted in cultures referred to as "semi-dissociated." Alternatively, the semidissociated pieces were collected after pre-plating, incubated in 2 ml of CMF-HBSS with 1 mg/ml trypsin and 1 mM EGTA for 10 min in a 370 water bath, and triturated to single cells with a flame-narrowed Pasteur pipet. To this was added 5 ml of CMF-HBSS with 2.5 mg/ml trypsin inhibitor and 1 mg/ml DNase, the cells incubated at 370 a further 10 min, and the pellet resuspended for final plating by centrifugation at 100 g for 10 min. Resultant cultures are referred to as "fully dissociated." On average, 7.5 x 105 viable cells (as judged by Trypan blue exclusion) were plated per 35 mm dish.

To prepare culture substrates, 22 mm 2 glass coverslips or regular glass microscope slides were washed serially for 30 min in 4M NaOH, 10 min in deionized water (3 times), 5 min in 100% ethanol, and allowed to dry completely under UV illumination in a laminar-air flow tissue culture hood. For the daily tracking of identified cells/clusters, microscope slides were inscribed with a grid using a diamond-tip scribe prior to cleaning. The cleaned glass was then incubated at 37°, overnight with a coating of 25 gg/ml fibronectin (from bovine plasma) in PBS: 100 l/coverslip, and 200 il/slide. It was found that rat OE derived cells do not adhere to laminin. For most cultures, the OE tissue derived from 20-25 rat pups was plated on coverslips, or 6-10 slides.

As can be seen in Figure 8, rhGGF2 promotes olfactory neuron survival in vitro. When assessed at 2 days in vitro, doses of 50 ng/ml or higher give a maximal increased in survival -34- WO 97/17983 PCTIUS96/18031 over control cultures (approximately 2.8- fold increase). By 4 days in vitro, a time at which very few neurons survive in serum-free medium, the presence of rhGGF2 at 100ng/ml gives rise to a approximately 10-fold increase in the number of surviving neurons.

Example 6: Olfactory neurons express high levels of neuregulin.

It has previously been reported that 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 91: 1064-1068, 1994). Using ten micron paraffin sections incubated with 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), we show (Fig.9) that neuregulin message is normally expressed in the 15 postnatal rat olfactory epithelium, and this message is expressed within the olfactory neuron layer (onl) and not in the sustentacular cell layer (scl).

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Claims (48)

1. A method of treating non-visual sensory epithelial cells of a mammal comprising administration to said mammal of a therapeutically effective amount of a polypeptide which binds an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4.

2. A method of claim 1, wherein said polypeptide which binds said erb B receptor comprises glial growth factor 2.

3. A method of claim 1, wherein said polypeptide which binds said erb B receptor comprises recombinant human glial growth factor 2.

4. A method of treating non-visual sensory epithelial cells of a mammal comprising administration to said mammal of a DNA sequence encoding a polypeptide which binds an erb B receptor, P wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4. A method of treating non-visual sensory epithelial cells of a mammal, said method comprising contacting said non-visual sensory epithelial cells with a therapeutically effective amount of a polypeptide which activates an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4.

6. A method for the prophylaxis of a pathophysiological condition of non-visual sensory epithelial cells in a mammal, wherein said condition involves a non- visual sensory epithelial cell type which is sensitive or responsive to a polypeptide which binds an erb B receptor, said method comprising administering a therapeutically effective amount of a polypeptide which activates an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4. A method for the treatment of a pathophysiological condition of non-visual sensory epithelial cells in a mammal, wherein said condition involves a non- visual sensory epithelial cell type which is sensitive or responsive to a polypeptide which binds an erb B receptor, said method comprising administering a therapeutically effective amount of a polypeptide which activates an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4.

8. A method for the treatment of a condition which involves non-visual sensory epithelial cell damage in a mammal, said method comprising contacting said non-visual sensory epithelial cells with an effective amount of a neuregulin polypeptide. ~i WO 97/17983 PCT/US96/18031

9. The method of claim 7, wherein said treatment of the pathophysiological condition of non-visual sensory epithelial cells results in decreasing the atrophy of said non-visual sensory epithelial cells.

10. The method of claim 7, wherein said treatment of the pathophysiological condition of non-visual sensory epithelial cells results in increasing the non-visual sensory epithelial cells present in said mammal.

11. The method of claim 7, wherein said treatment of the pathophysiological condition of non-visual sensory epithelial cells results in increasing the non-visual sensory epithelial cell survival in said mammal.

12. A method of claim 7, wherein said pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss.

13. A method of claim 7, wherein said pathophysiological condition of non-visual sensory epithelial cells is sensorineural loss of balance.

14. A method of claim 7, wherein said pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by ototoxic antibiotics. A method of claim 7, wherein said pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by excessive noise.

16. A method of claim 7, wherein said pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by viral infection.

17. A method of claim 7, wherein said pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by an autoimmune disease.

18. A method of claim 7, wherein said pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by tinnitus.

19. A method as claimed in claim 7, wherein said pathophysiological condition involves non- visual sensory epithelial cell damage. A method of claim 7, wherein said non-visual sensory epithelial cell type is an auditory hair cell. -37- WO 97/17983 PCT/US96/18031

21. A method of claim 7, wherein said non-visual sensory epithelial cell type is a vestibular hair cell.

22. A method of claim 7, wherein said non-visual sensory epithelial cell type is an auditory support cell.

23. A method of claim 7, wherein said non-visual sensory epithelial cell type is a vestibular support cell.

24. A method of claim 7, wherein said non-visual sensory epithelial cell type is a sustenactular cell. A method of claim 7, wherein said non-visual sensory epithelial cell type is a basal cell.

26. A method of claim 7, wherein said non-visual sensory epithelial cell type is a Meissner's corpuscle.

27. A method of claim 7, wherein said non-visual sensory epithelial cell type is a Pacinian corpuscle.

28. A method for the prophylaxis or treatment of a pathophysiological condition of non- visual sensory epithelial cells in a vertebrate, wherein said condition involves a non- visual sensory epithelial cell type which is sensitive or responsive to a neuregulin polypeptide, said method comprising administering to said vertebrate a therapeutically effective amount of said neuregulin polypeptide. -38-

29. The use of a polypeptide which binds an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4 in a method of treating non-visual sensory epithelial cells of a mammal comprising administration to said mammal of a therapeutically amount of said polypeptide. The use of claim 29, wherein said polypeptide which binds said erb B receptor comprises glial growth factor 2.

31. The use of claim 29, wherein said polypeptide which binds said erb B receptor comprises recombinant human glial growth factor 2.

32. The use of a DNA sequence encoding a polypeptide which binds an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4 in a method of treating non-visual sensory S. epithelial cells of a mammal comprising administration to said mammal of said DNA sequence.

33. The use of a polypeptide which activates an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4 in a method of treating non-visual sensory epithelial cells of a mammal, said method comprising contacting said non-visual sensory epithelial cells with a therapeutically effective amount of said polypeptide.

34. The use of a polypeptide which activates an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb 0 B4, or erb B3/erb B4 in a method for the prophylaxis of a pathophysiological condition of non-visual sensory epithelial cells in a mammal, wherein said condition involves a non- visual sensory epithelial cell type which is sensitive or responsive to said polypeptide which binds an erb B receptor, said method comprising administering a therapeutically effective amount of said polypeptide to said mammal. i35. The use of a polypeptide which activates an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4 in a method for the treatment of a pathophysiological condition of non- visual sensory epithelial cells in a mammal, wherein said condition involves a non- visual sensory epithelial cell type which is sensitive or responsive to a polypeptide which binds an erb B receptor, said method comprising administering a therapeutically effective amount of said polypeptide to said mammal.

36. The use of neuregulin polypeptide in a method for the treatment of a condition which involves non-visual sensory epithelial cell damage in a mammal, said method comprising contacting said non-visual sensory epithelial cells with an effective amount of said polypeptide.

37. The use of claim 35 wherein said treatment of the pathophysiological condition of non-visual sensory epithelial cells results decreasing the atrophy of said non-visual sensory epithelial cells.

38. The use of claim 35 wherein said treatment of the pathophysiological condition of non-visual sensory epithelial cells results in increasing the non-visual sensory epithelial cells present in said mammal.

39. The use of claim 35 wherein said treatment of the pathophysiological condition of non-visual sensory epithelial cells results in increasing the non-visual sensory epithelial cell survival in said mammal.

40. The use of claim 35, wherein the pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss.

41. The use of claim 35, wherein the pathophysiological condition of non-visual sensory epithelial cells is sensorineural loss of balance.

42. The use of claim 35, wherein the pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by ototoxic antibiotics.

43. The use of claim 35, wherein the pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by excessive noise. The use of claim 35, wherein the pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by viral infection.

45. The use of claim 35, wherein the pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by an autoimmune disease.

46. The use of claim 35, wherein the pathophysiological condition of non-visual sensory epithelial cells is sensorineural hearing loss caused by tinnitus.

47. The use of claim 35, wherein said pathophysiological condition involves non-visual sensory epithelial cell damage.

48. The use of claim 35, wherein said non-visual sensory epithelial cell type is an auditory hair cell.

49. The use of claim 35, wherein said non-visual sensory epithelial cell type is a vestibular hair cell. 1WUS The use of claim 35, wherein said non-visual sensory epithelial cell type is an auditory support cell.

51. The use of claim 35, wherein said non-visual sensory epithelial cell type is a vestibular support cell.

52. The use of claim 35, wherein said non-visual sensory epithelial cell type is a sustenactular cell.

53. The use of claim 35, wherein said non-visual sensory epithelial cell type is a basal cell.

54. The use of claim 35, wherein said non-visual sensory epithelial cell type is a Meissner's corpuscle. The use of claim 35, wherein said non-visual sensory epithelial cell type is a Pacinian corpuscle. o

56. The use of a neuregulin polypeptide in a method for the prophylaxis or treatment of a physiological condition of non-visual sensory epithelial cells in a vertebrate, wherein said condition involves a non-visual sensory epithelial cell type which is sensitive or responsive to said neuregulin polypeptide, said method comprising administering to said vertebrate a therapeutically effective amount of said neuregulin polypeptide. S*57. A method of treating non-visual sensory epithelial cells of a mammal, said method comprising contacting said non-visual sensory epithelial cells with a therapeutically effective amount of a polypeptide which activates an erb B receptor, wherein said erb B receptor is erb S: B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4, said method being substantially as herein described with reference to S0;* the accompanying Examples.

58. A method for the treatment of a condition which involves non-visual sensory epithelial cell S damage in a mammal, said method comprising contacting said non-visual sensory epithelial cells with an effective amount of a neuregulin polypeptide, said method being substantially as herein described with reference to the accompanying Examples.

59. The use of a polypeptide which activates an erb B receptor, wherein said erb B receptor is erb B2, erb B3, erb B4, erb B2/erb B2, erb B3/erb B3, erb B4/erb B4, erb B2/erb B3, erb B2/erb B4, or erb B3/erb B4 in a method of treating non-visual sensory epithelial cells of a mammal, said method comprising contacting said non-visual sensory epithelial cells with a therapeutically effective amount of said polypeptide, said use being substantially as herein described with reference to the accompanying Examples. The use of neuregulin polypeptide in a method for the treatment of a condition which involves non-visual sensory epithelial cell damage in a mammal, said method comprising contacting said non-visual sensory epithelial cells with an effective amount of said polypeptide, said use being substantially as herein described with reference to the accompanying Examples. DATED this 2 7 th day of September 1999 CAMBRIDGE NEUROSCIENCE INC UNIVERSITY OF VIRGINIA PATENT FOUNDATION E. K. SHRIVER CENTER FOR MENTAL RETARDATION By their patent attorneys CALLINAN LAWRIE 0 0 0 e0 *0 00 *S 0 OS