MXPA00000862A - Method for preventing and treating hearing loss using a neurturin protein product - Google Patents

Abstract

The present invention relates generally to methods for preventing and/or treating injury or degeneration of cochlear hair cells and spiral ganglion neurons by administering a neurturin neurotrophic factor protein product. The invention relates more specifically to methods for treating sensorineural hearing loss.

Description

METHOD FOR PREVENTING AND TREATING HEARING LOSS USING A PRODUCT OF NEURTURINE PROTEIN BACKGROUND OF THE INVENTION The present invention relates in general to methods for preventing and / or treating lesions or degeneration of the sensory cells of the inner ear, such as hair cells and auditory neurons, by administration of a protein product neurotrophic factor. The present invention relates specifically to methods for preventing and / or treating hearing loss caused by a variety of causes. Neurotrophic factors are natural proteins, found in the nervous system or in non-nervous tissues under the nervous system, which function to promote the survival and maintenance of the phenotypic differentiation of certain nerves and / or populations of gual cells (Male et al. al., Ann. Rev. Neuroscience, 1: 327, 1979; Thoenen et al., Science, 229: 238, 1985). Due to this physiological function, it has been found that certain neurotrophic factors are useful in the treatment of the degeneration of certain nerve cells and the loss of differentiated function as a result of damage to nerves. Nervous damage is caused by conditions that compromise survival REF .: 32264 and / or the appropriate function of one or more types of nerve cells, including: (1) physical injury, which causes the degeneration of axonal processes (which, in turn, causes the death of the cell nervous) and / or nerve cell bodies near the site of injury, (2) temporary or permanent cessation of blood flow (ischemia) to parts of the nervous system, such as in a heart attack, (3) intentional or accidental exposure to neurotoxins, such as chemotherapeutic agents for cancer and AIDS, cisplatin and dideoxycytidine, respectively, (4) chronic metabolic diseases, such as diabetes or renal dysfunction or (5) neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease and Amyotrophic Lateral Sclerosis, which are the result of the degeneration of specific neuronal populations. In order for a particular neurotrophic factor to be potentially useful in the treatment of nerve damage, the class or classes of damaged nerve cells must respond to the factor. It has been established that not all neuron populations respond or are equally affected by all neurotrophic factors. The first neurotrophic factor to be identified was nerve growth factor (NGF). NGF is the first member of a defined family of trophic factors, termed neurotroids, which currently include brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), NT-4/5 and NT- 6 (Thoenen, Trends, Neurosci., 14: 165-170, 1991; Snider, Cell, 77: 627-638, 1994; Bothwell, Ann. Rev. Neurosci., 18: 223-253, 1995). These neurotrophins are known to act through the family of trk tyrosine kinase receptors, ie, trkA, trkB, trkC and the low affinity receptor p75 (Snider, Cell, 77: 627-638, 1994; Bothwell, Ann. Neuroscí., 18: 223-253, 1995; Chao et al., TINS 18: 321-326, 1995). The cell line-derived neurotrophic factor (GDNF) is a protein that is identified and purified using assays based on its efficacy to promote survival and stimulation of the transmitting phenotype of mesencephalic dopaminergic neurons in vi tro (Lin et al., Science, 260: 1130-1132, 1993). GDNF is a homodimer coupled with glycosylated disulfide bridges that has some structural homology with the protein superfamily of transforming growth factor-beta (TGF-β) (Lin et al., Science, 260: 1130-1132, 1993; Krieglstein et al., EMBO J., 14: 736-742, 1995; Poulsen et al., Neuron, 13: 1245-1252, 1994). GDNF mRNA has been detected in muscle and in Schwann cells in the peripheral nervous system (Henderson et al., Science, 266: 1062-1064, 1994, Trupp et al. , J. Cell Biol., 130: 137-148, 1995) and in type I astrocytes in the central nervous system (Schaar et al., Exp. Neurol., 124: 368-371, 1993). In vivo treatment with exogenous GDNF stimulates the dopaminergic phenotype of black substance neurons and restores functional deficiencies induced by axotomy or by inert neurotoxins in animal models of Parkinson's disease (Hudson et al., Brain Res. Bull ., 36: 425-432, 1995; Beck et al. , Nature, 373: 339-341, 1995; To ac et al. , Nature, 373: 335-339; nineteen ninety five; Hoffer et al. , Neurosci. Lett., 182: 107-111, 1994). Although originally thought to be relatively specific for dopaminergic neurons, at least in vi tro, evidence is beginning to emerge indicating that GDNF may have a greater spectrum of neurotrophic targets, in addition to mesencephalic dopaminergic neurons and somatic motor neurons (Yan and Matheson, Nature, 373: 341-344, 1995; Oppenheim et al., Nature, 373: 344-346, 1995; Mathenson et al., Soc. Neurosci. Abstr, 21, 544, 1995; Trupp et al., J. Cell Biol., 130: 137-148, 1995). In particular, GDNF has been found to have neurotrophic efficacy on brain stem neurons and cholinergic motor neurons of the spinal cord, both in vivo and in vi tro (Oppenheim et al., Nature, 373: 344-346, 1995; et al., Neuroreport, 6: 113-118, 1994; Yan et al., Nature, 373: 341-344, 1995; Henderson et al., Science, 266: 1062-1064, 1994), on retinal neurons such as photoreceptors and cells of the retinal ganglion and on sensory neurons of the dorsal root ganglion. The neuroepithelial hair cells in the organ of Corti of the inner ear translate sound into neuronal activity, which is transmitted along the cochlear division of the eighth cranial nerve. This nerve consists of two fibers of three types of neurons (Spoendlin, HH, in: Friedmann, I. Ballantyne, J., eds., Ultrastructural Atlas of the Inner Ear, London, Butterworth, pp. 133-164, 1984): 1) afferent neurons, which lie in the spiral ganglion and connect the cochlea to the brainstem; 2) efferent olivocochlear neurons, which originate in the upper olivic complex and 3) autonomous adrenergic neurons, which originate in the cervical sympathetic trunk and innervate the cochlea. In humans, there are approximately 30,000 afferent cochlear neurons, with myelinated axons, each consisting of approximately 50 lamellae of 4-6 μm in diameter. This histological structure forms the basis of uniform conduction velocity, which is an important functional characteristic. Throughout the length of the auditory nerve, there is a trophic arrangement of afferent fibers, with "basal" fibers wrapped around the "aplícal" fibers placed in the center, in a manner similar to a twisted cord. Spoendlin (Spoendlin, HH, in: Nauton, RF, Fernadex, C. eds, Evoked Electrical Activity in the Auditory Nervous System, London, Academic Press, pp. 21-39, 1978) identified two types of afferent neurons in the spiral ganglion. , based on morphological differences: type I cells (95%) are bipolar and have myelinated cell bodies and axons that project to the inner hair cells. Type II cells (5%) are monopolar with unmyelinated axons and project to the outer hair cells of the organ of Corti. Each inner hair cell is innervated by approximately 20 fibers, each of which synapses with only one cell. In contrast, each outer hair cell is innervated by approximately six fibers and each fiber branches to contact about 10 cells. Within the cochlea, the fibers are divided into: 1) an internal spiral group, which arises mainly in an ipsilateral manner and synapses with the afferent neurons towards the internal hair cells and 2) a larger external radial group, which arises mainly contralaterally and synapse directly with the outer hair cells. There is a minimum threshold at a frequency, the characteristic frequency or better frequency, but the threshold rises sharply at frequencies above and below this level (Pickles, JO, in: Introduction to the Physiology of Hearing. Press, pp. 71-106, 1982). Therefore, simple auditory nerve fibers appear to behave as bandpass filters. The basilar membrane vibrates preferentially at different frequencies, at different distances along its length and the frequency selectivity of each cochlear nerve fiber is similar to that of the internal hair cell to which the fiber is connected. Thus, each cochlear nerve fiber exhibits a curve that covers a different range of frequencies from its neighboring fiber (Evans, EF, in: Beagley HA ed., Auditory research: The Scientific and Technological Basis, New York, Oxford University Press, 1979). Through this mechanism, complex sounds are broken into component frequencies (frequency resolution) through the filters of the inner ear. Hearing loss of a sufficient degree to interfere with social and work-related communications is among the most common chronic nervous impairments in the population of the United States. Based on dfrom health interviews (Vital and Health Statistics, Series 10, No. 176, Washington, DC (DHHS Publication No. (PHS) 90-1504), it is estimated that approximately 4% of people under 45 years of age of age and approximately 29% of those over 65 years of age suffer from a disabling loss of hearing.It has been estimated that more than 28 million Americans suffer from hearing impairments and that an amount as large as 2 million in this group are profoundly deaf (report of the National Strategic Plan working group, Bethesda, Md .: National Institute of Health, 1989) The prevalence of hearing loss increases dramatically with age Approximately 1 in every 1,000 infants has a sufficient hearing loss severe, as to prevent him from developing a spoken language without help (Gentile, A. et al., Characteristics of persons with impaired hearing, United States, 1962-1963, Series 10, No. 35, Washington, DC .: Government Printing Office, 1967 (DHHS, publication No. (PHS) 1000) (Human communication and its disorders: an overview, Bethesda, Md .: National Institutes of Health, 1970). More than 360 people out of 1,000, over 75 years of age, have a disabling hearing loss (Vital and Health Statistics, Series 10, No. 176, Washington, DC (DHHS, Publication No. (PHS) 90-1504). It has been estimated that the cost of losses due to lack of productivity, special education and medical treatments, can exceed 30 billion dollars per year for hearing, speech and language disorders. (1990 annual report of the National Office of Deafness and Other Communication Disorders Washington, D.C .: Government Printing Office, 1991. (DHHDS, publication No. (NIH) 91-3189)). The main common causes of profound deafness in children are genetic disorders and meningitis, constituting approximately 13 percent and 9 percent of the total, respectively (Hotchkiss, D., Demographic aspects of hearing impatience: questions and answers, 2nd ed., Washington, D.C.: Gallaudet University Press, 1989). In about 50 percent of cases of childhood deafness, the cause is unknown, but it is probably due to genetic causes or predisposition (Nance WE, Sweeney A. Otolaringol, Clin North Am 1975, 8: 1948). ). Damages anywhere along the auditory path, from the external auditory canal to the central nervous system, can cause hearing loss. The auditory apparatus can be subdivided into the outer ear and middle ear, inner ear and auditory nerve, and central auditory pathways. The auditory information in humans is transduced from a mechanical signal to an electrical impulse driven neurally by the action of approximately 15,000 neuroepithelial cells (hair cells) and 30,000 first order neurons (spiral ganglion cells) in the inner ear. All the central fibers of the neurons of the spiral ganglion form synapses in the cochlear nucleus of the pontine brain stem. The number of neurons involved in the ear increases drastically from the cochlea to the auditory brain stem and the auditory cortex. All auditory information is transduced by only 15,000 hair cells, of which the so-called internal hair cells, which number 3,500, are critically important, since they form synapses with approximately 90 percent of the 30,000 primary auditory neurons. Thus, damage to a relatively small number of cells in the auditory periphery can cause substantial hearing loss. Hence, much of the causes of sensorineural loss can be attributed to injuries in the inner ear (Nadol, J.B., New England Journal of Medicine, 1993, 329: 1092-1102). The hearing loss can be at the level of conductivity, sensorineural and central level. Conductive hearing loss is caused by injuries that involve the outer or middle ear, resulting in the destruction of the normal pathway of sound amplification by the tympanic membrane and the ossicles to the fluids of the inner ear. Sensorineural hearing loss is caused by lesions of the cochlea or the auditory division of the eighth cranial nerve. Central hearing loss is due to injuries to the central auditory pathways. These consist of the cochlear complex and the dorsal olivárico nucleus, inferior coliculos, medial geniculate bodies, auditory cortex in the temporal lobes and the interconnection of afferent and efferent fiber sections (Adams, RD and Maurice, V., Eds. In: Principies of Neurology , 1989, McGraw-Hill Information Services Company, pp 226-246). As previously mentioned, at least 50 percent of cases of profound deafness in children have genetic causes (Brown, K.S., Med.Clin.North AM., 1969; 53: 741-72). If one takes into account the probability that the genetic predisposition is a major causal factor of presbycusis hearing loss, or. is related to age, which affects a third of the population over 75 years old (Nador, JB, in: Beasley DS, Davis GA, Aging: Communications Processes and Disorders, New York: Grunt &Stratton, 1981: 63-85), genetic and hereditary factors are probably the most common cause of hearing loss. Genetic abnormalities are much more commonly expressed as sensorineural hearing loss rather than as conductive hearing loss. Genetically determined sensorineural hearing loss is clearly a major cause, if not the major cause, of sensorineural loss, particularly in children (Nance WE, Sweeney A., Otolaryngol, Clin. North Am, 1975; 8: 19-48). Among the most common forms of sensorineural loss syndromes are Waardenburg syndrome, Alport syndrome and Usher syndrome. A variety of commonly used drugs have ototoxic properties. The best known are aminoglycoside antibiotics (Lerner, SA, et al., Eds Aminoglycoside ototoxicity, Boston: Little, Brown, 1981, Smith, CR et al., N. Engl. J. Med. 1980; 302: 1106- 9), loop diuretics (Bosher, SK, Acta Otolaryngol. (Stockholm) 1980; 90: 4-54), salicylates (Myers, EN, et al., N. Engl. J. Med. 1965; 273 : 587-90) and antineoplastic agents such as cisplatin (Strauss, M. et al., Laryngoscope 1983; 143: 1263-5). Ototoxicity has also been described during oral or parenteral administration of erythromycin (Kroboth, PD et al., Arch. Intern Med. 1983; 1: 169-79; Achweitzer, VG, Olson, N. Arch. Otolaryngol., 1984; 110: 258-60). Many ototoxic substances cause hearing loss by damaging the cochlea, particularly the auditory hair cells and the vascular tape, which is a specialized epithelial organ within the inner ear responsible for the hemostasis of fluids and electrolytes (Nadol, JB, New England J Med., 1993, 329: 1092-1102). Secondary neural degeneration can occur many years after an ototoxic event that affects hair cells. There is evidence that some ototoxic substances can be selectively concentrated within the inner ear, resulting in a progressive sensorineural loss despite the discontinuation of systemic administration (Federspil, P. et al., J. Infect. Dis 1976, supplement 134 : S200-S205). Trauma due to acoustic overstimulation is another major cause of deafness. There is an individual susceptibility to noise traumas. Clinically important sensorineural hearing loss can occur in some people exposed to high intensity noise, even below levels approved by the Occupational Safety and Health Agency (Osguthorpe, JD, ed. Washington, DC: American Academy of Otolaryngology-Head and neck Surgery Foundation, 1988). Demyelination processes, such as multiple sclerosis, can cause sensorineural hearing loss (Noffsinger, D. et al., Acta Otolaryngol Suppl (Stockholm) 1972; 303: 1-63). More recently, a form of sensorineural loss mediated by the immune system has been recognized (McCabe, B.F., Ann Otol Rhinol Laryngol, 1979; 88: 585-9). Hearing loss is usually bilateral, rapidly progressive (measured in weeks and months) and may or may not be associated with vestibular symptoms. A variety of tumors, both primary and etastatic, can produce either conductive hearing loss, or a sensorineural hearing loss, by invading the inner ear or the auditory nerve (Houck, JR, et al., Otolaryngol Head Neck Surg, 1992).; 106: 92-7). A variety of degenerative disorders of unknown cause can cause sensorineural hearing loss. Meniere's syndrome (Nadol, J.B., ed. Meniere's disease: pathogenesis, pathophysiology, diagnosis, and treatment.Amsterdam: Kugler &Ghedini, 1989), characterized by a fluctuating sensorineural loss. episodic vertigo and tinnitus, appear to be caused by a disorder of fluid hemostasis within the inner ear, although the pathogenesis is still unknown. Sudden idiopathic sensorineural hearing loss (Wilson, WR, et al., Arch Otolaryngol, 1980; 106: 772-6), which causes moderate to severe sensorineural deafness, can be due to several causes, including inner ear ischemia and viral labirhinitis. . Presbycusis, which is the hearing loss associated with age, affects more than a third of people over 75 years of age. The most common histopathology that correlates with presbycusis is the loss of neuroepithelial cells (hair), neurons and vascular bands of the peripheral auditory system (Schuknecht HF, Pathology of the Ear, Cambridge, Mass .: Harvard University Press, 1974: 415-420): Presbycusis is best understood as the result of the cumulative effects of various influences harmful during life, including trauma from noise, ototoxicity and genetically influenced degeneration. It has been shown that certain neurotrophic factors regulate neuronal differentiation and survival during development (Korsching, SJ, Neurosci., 13: 2739-2748, 1993) and protect neurons from injury and toxins in adulthood (Hefti, Neurosci ., 6: 2155-2162, 1986; Apfel et al., Ann Neurol, 29: 87-89, 1991; Hy an et al. , Nature 350: 230-233, 1991; Knusel et al. , J. Neurosci. 12: 4391-4402, 1992; Yan et al. , Nature, 360: 753-755, 1992; Koliatsos et al. , Neuron, 10: 359-367, 1993). In situ hybridization studies indicate that the mRNA of the neurotrophin receptors TrkB and TrkC is expressed by developing cochleovestibular ganglia (Ylikoski et al., Hear, Res. 65: 69-78, 1983; Schecterson et al., Hearing Res., 73 : 92-100, 1994) and that the BDNF and NT-3 mRNAs are found in the inner ear, including the organ of Corti (Pirvola et al., Proc. Nati. Acad. Sci., USA, 89: 9915- 9919, 1992; Schecterson et al., Hearing Res., 73: 92-100, 1994; Wheeler et al., Hearing Res., 73: 46-56, 1994). The physiological function of BDNF and NT-3 in the development of the vestibular and auditory system was investigated in mice carrying a deletion of the BDNF and / or NT-3 gene (Ernfors et al., Neuron, 14: 1153-1164, nineteen ninety five). In the cochlea, the BDNF mutant animals lost the spiral neurons type 2, causing the absence of innervation to the outer hair cells. The mutant animals of NT-3 showed a shortage of afferent fibers and the loss of 87% of the spiral neurons, presumably corresponding to the type 1 neurons, which innervate the inner hair cells. The mutant double animals presented a hearing loss, lacking all the vestibular and spiral neurons. The requirement of the TrkB and TrkC receptors for the survival of specific neuronal populations and for the maintenance of white innervation in the peripheral sensory system of the inner ear was demonstrated by studies of mice carrying a mutation in the catalytic domain of tyrosine kinase. of these genes (Schimmang et al., Develop ent, 121: 3381-3391, 1995). Gao et al. , (J. Neurosci., 15: 5079-5087, 1995) demonstrated the survival-promoting potency of NT-4/5, BDNF and NT-3 for spiral ganglion neurons, in postnatal rats, in dissociated cultures and that NT-4/5 protected these neurons from the neurotoxic effects of the cancer drug, cisplatin. Likewise, it has been shown that BDNF and NT-3 support the survival of auditory neurons in adult rats, in dissociated cultures (Lefebvre et al., Neuroreport, 5: 865-868, 1994). There have been no previous reports of the use of neurturin in the treatment of hearing loss. Because hearing damage is a serious condition, the identification of any agent and method of treatment that can protect auditory neurons and hair cells from damage would be of great benefit. BRIEF DESCRIPTION OF THE INVENTION The present invention provides methods for the treatment of sensorineural hearing loss, comprising administering to a subject having an injury to the inner ear, a therapeutically effective amount of a product of the neurturin neurotrophic factor protein. For example, hearing loss can be associated with lesions or degeneration of neuroepithelial hair cells (cochlear hair cells) or neurons of the spiral ganglion in the inner ear. The present invention is based on the findings that hair cells respond to neurturin resisting the toxic effects of ototoxins, such as cisplatin and neomycin, and that auditory neurons also respond to neurturin by resisting the toxic effects of a variety of ototoxins, such as for example cisplatin, neomycin and sodium salicylate. Therefore, a therapeutically effective amount of the neurturin protein product can be administered to promote the protection, survival or regeneration of hair cells and spiral ganglion neurons. It has also been discovered that lesions or alterations of the vestibular apparatus can also be treated by administration to a subject suffering from such lesions or alterations., of a therapeutically effective amount of a product of the neurturin protein. Such injuries can cause dizziness, vertigo or loss or balance. It is contemplated that such products of the neurturin protein will preferably include a neurturin protein such as that described by the amino acid sequence presented in the figures, as well as variants and derivatives thereof. It is also contemplated that such products of the neurturin protein will include [Met-] neurturin. In accordance with the present invention, the product of the neurturin protein can be administered parenterally, at a dose ranging from about 1 μg / kg / day to about 100 mg / kg / day, typically at a dose of about 0.1 to 25 mg / kg / day and usually at a dose of about 5 mg / kg / day to about 20 mg / kg / day. It is also contemplated that, depending on the needs of individual patients and the route of administration, the product of the neurturin protein may be administered at a lower frequency, for example weekly or several times a week, instead of daily. It is further contemplated that the product of the neurturin protein can be administered directly in the middle ear or inner ear. Those skilled in the art will note that by such administration, a smaller amount of the neurturin protein product can be used, for example, a direct dose in the middle ear or inner ear in the range of about 1 μg / ear to about 1 mg / ear, in a single injection or multiple injections. Alternatively, if administered topically or orally, a comparatively higher dose may be used. It is further contemplated that the product of the neurturin protein may be administered in combination or in conjunction with an effective amount of a second therapeutic agent, such as GDNF, BDNF and NT-3. The present invention also provides the use of a product of the neurturin protein in the manufacture of a medicament or pharmaceutical composition for the treatment of lesions or degeneration of hair cells and auditory neurons, for a variety of causes of sensorineural hearing loss. Such pharmaceutical compositions include product formulations of neurturin protein for topical, oral or in the middle and inner ear administration, or in combination with cochlear implants. Likewise, those skilled in the art will also observe that the administration process can be achieved by cell therapy and gene therapy, as will be described below. For example, in gene therapy, the cells have been modified to produce and secrete the product of the neurturin protein. The cells can be modified ex vivo or in vivo. Numerous additional aspects and advantages of the present invention will be apparent to those skilled in the art upon considering the following detailed description of the invention, which describes the presently preferred embodiments thereof. BRIEF DESCRIPTION OF THE FIGURES Numerous features and advantages of the present invention will be apparent upon review of the Figures, wherein: Figure 1 illustrates an amino acid sequence (SEQ ID No. 1) of the neurotrophic factor neurturin human. Figure 2 illustrates an amino acid sequence (SEQ ID No. 2) of murine neurturin neurotrophic factor. Figure 3 illustrates a nucleic acid sequence (SEQ ID No. 3) encoding a neurturin neurotrophic factor analogue. Figure 4 illustrates an amino acid sequence (SEQ ID Nos. 3 or 4) of a human neurotrophic factor neurturin analogue. Figure 5 illustrates an amino acid sequence (SEQ ID No. 5) of the human pre-pro-neurturin neurotrophic factor. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for preventing and / or treating sensorineural hearing loss by administering a therapeutically effective amount of a product of the neurotrophic factor protein neurturin. In accordance with one aspect of the present invention, methods are provided for the treatment of damaged hair cells and auditory neurons, by administering a therapeutically effective amount of the neurturin protein product by means of a pharmaceutical composition, the implant of cells that they express neurturin, or neurturin gene therapy. The present invention can be practiced using a product of the biologically active neurturin protein, including the proteins represented by the amino acid sequences presented in Figures 1, 2, 4 and 5 (SEQ ID Nos. 1, 2, 3, 4 and 5), including variants and derivatives thereof. In addition to oral, parenteral or topical administration of the neurturin protein product, administration by cell therapy and gene therapy methods is also contemplated. The present invention is based on the initial findings that neurturin protects hair cells from ototoxin-induced cell death in explant cultures of rat cochlea and dissociated neurons of the spiral ganglion of adult rats in culture. It is contemplated that administration of a neurturin protein product will protect hair cells and spiral ganglion neurons from traumatic damage (such as trauma from noise and acute or chronic treatments of cisplatin and aminoglycoside antibiotics) or from damage due to lack of neurotrophic factors caused by the interruption of the transport of the factors from the axon to the body of the cell. Such treatment is expected to allow hair cells and / or auditory neurons to tolerate intermittent aggression by traumas or ototoxins and to decrease the progressive degeneration of auditory neurons and hair cells which is responsible for hearing loss in pathological disorders such as presbycusis (hearing loss related to age), hereditary sensorineural degeneration and posidiopathic hearing losses, and preserve the functional integrity of the inner ear. It will also support the auditory neurons to have a better and more prolonged performance of cochlear implants. In accordance with the present invention, the product of the neurturin protein can be administered in the middle ear and at doses ranging from 1 μg / kg / day to about 100 mg / kg / day, typically at a dose of approximately 0.1 mg / kg. / day at approximately 25 mg / kg / day and usually at a dose of approximately 5 mg / kg / day to approximately 20 mg / kg / day. A product of the neurturin protein can be administered directly to the inner ear in cases where the invasion of the inner ear is already programmed, such as in a cochlear implant procedure or inner ear surgeries. In such cases, a smaller amount of the neurturin protein product will be administered, for example, from about 1 μg / ear to about 1 mg / ear in a single injection or in multiple injections. In situations where chronic administration of the protein product is necessary, a delivery device such as an Alzet minipump can be attached to a cannula, whose tip will be inserted in the inner ear or in the middle ear, for a continuous administration of the protein product. Alternatively, a product of the neurturin protein can be administered in the form of eardrops, which will penetrate the tympanic membrane of the blister. It is further contemplated that a product of the neurturin protein may be administered together with an effective amount of a second therapeutic agent, for the treatment of the degeneration of auditory neurons, for example GDNF, BDNF and NT-3, as well as other neurotrophic factors. or drugs used in the treatment of various pathologies of the inner ear. A variety of pharmaceutical formulations and different administration techniques are described in greater detail below. As used herein, the term "neurturin protein product" includes the natural, synthetic or recombinant neurturin neurotrophin factor, biologically active neurturin variants (including insertion, substitution and deon variants) and chemically modified derivatives thereof. . Also included are neurturin proteins that are substantially homologous to the human neurturin protein, having the amino acid sequence set forth in Figures 1 and 4 (SEQ ID Nos. 1, 3 and 4). In addition, the chemically modified derivatives of these various proteins are included in the present invention. Products of the neurturin protein can also exist in the form of homodimers or heterodimers, in their biologically active form. The term "biologically active", as used herein, means that the product of the neurturin protein demonstrates similar neurotrophic properties, but not necessarily all of the same properties, and not necessarily to the same degree, as the neurturin that has the sequence of amino acids established in the Figures, but have at least the same activity of promoting the protection, survival or regeneration of hair cells and neurons of the spiral ganglion. The selection of the particular neurotrophic properties of interest depends on the use for which the product of the neurturin protein is being administered. The term "substantially homogeneous", as used herein, means that it has a degree of homology with the neurturin protein having the amino acid sequence set forth in Figures 1, 2, 4 and 5 (SEQ ID Nos. 1 , 2, 3, 4 and 5), which preferably exceeds 70%, more preferably exceeds 80% and even more preferably exceeds 90 or 95%. For example, the degree of homology between murine and human protein is about 91% and it is contemplated that neurturin proteins from mammals will have a similarly high degree of homology. The percentage of homology or percent identity is calculated as the percentage of amino acid residues found in the smaller of the two sequences that are aligned with identical amino acid residues in the sequence being compared, when four different amino acid residues can be introduced. 100 amino acids in length to aid alignment (as set forth in Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p.124, National Biochemical Research Foundation, Washington, DC (1992), the description of which is incorporated in present as reference). Any product of the neurturin protein that can be isolated by virtue of its cross-reaction with antibodies directed against neurturin of Figure 1 or 2 (SEQ ID No. 1 or 2) or whose genes can be isolated is also included as a substantial homolog. through hybridization with the gene or with segments of the neurturin coding gene of Figure 1 or 2 (SEQ ID No. 1 or 2). The products of the neurturin protein according to the present invention can be isolated or generated by various means. Exemplary methods for the production of neurturin protein products useful in the present invention, are substantially similar to GDNF production methods as described in US Patent Application Serial No. 08 / 182,183 filed May 23, 1994 and its related applications; PCT Application No. PCT / US 92/07888, filed September 17, 1992, published as WO 93/06116 (Lin et al., Syntex-Synergen Neuroscience Joint Venture); European Patent Application No. 92921922.7, published as EP 610 254; and U.S. Patent Application Serial No. 08 / 535,681, filed September 28, 1995 ("Truncated Glial Cell-Line Derived Neurotrophic Factor"), the disclosures of which are incorporated herein by reference. The products of the neurturin protein can be synthesized chemically or recombinantly by means known to those skilled in the art, see for example Kotzbauer et al. , Nature, 384: 467-470, 1996. Products of the neurturin protein are preferably produced by recombinant techniques, because such methods are capable of achieving comparatively higher amounts of protein, with a higher purity. The forms of the neurturin protein products include glycosylated and non-glycosylated forms of the protein and include, but are not limited to, the product of the protein expressed in bacteria, mammalian cell systems or insect systems. In general, recombinant techniques involve isolating the genes responsible for encoding neurturin, cloning the genes into suitable vectors and / or cell types, modifying the genes if necessary to encode a desired variant, and expressing the genes in order to produce the neurturin protein product. Alternatively, a nucleotide sequence coding for the product of the desired neurturin protein can be chemically synthesized. It is contemplated that a product of the neurturin protein can be expressed using nucleotide sequences that vary in the use of codons due to degenerations of the genetic code or allelic variations or alterations made, to facilitate the production of the protein product by the selected cells. Kotzbauer et al. , Nature, 384: 467-470, describes the identification of a murine cDNA and an amino acid sequence, and a human cDNA and an amino acid sequence for the neurturin protein. International Publication WO 93/06116 describes a variety of factors, host cells and growth conditions in culture, for the expression of the GDNF protein product, which can also be used for the expression of the neurturin protein product. Additional vectors suitable for the expression of the neurturin protein product in E. coli are described in European Patent Application No. EP 0 423 980 ("stern Cell Factor"), published on April 24, 1991, the description of which is incorporated herein as a reference. The molecular weight of purified neurturin indicates that the protein is a dimer with disulfide bonds in its biologically active form. The material isolated after expression in a bacterial system is essentially biologically inactive and exists as a monomer. A reconformation is necessary to produce the dimer with biologically active disulfide bonds. Suitable processes for the reconformation and naturalization of neurturin expressed in bacterial systems are substantially similar to those described in International Publication WO 93/06116. The standard in vi tro assays for the determination of neurturin activity are also substantially similar to those used to determine the activity of GDNF, as described in International Publication WO 93/06116 and in the copending US Patent Application. and co-owner Serial No. 08 / 535,681 filed September 28, 1995 and which are incorporated herein by reference. A. Neurturin variants The term "neurturin variants", as used herein, includes polypeptides in which one or more amino acids have been deleted from ("deletion variants"), inserted into ("variants by addition"). ) or replaced by ("variants by substitution"), residues within the amino acid sequence of neurturin of Figures 1, 2, 4 and 5. Such variants are prepared by introducing appropriate nucleotide changes into the DNA encoding the polypeptide or by chemical synthesis within the desired polypeptide. Those skilled in the art will recognize that numerous combinations of deletions, insertions and substitutions can be made, as long as the final molecule possesses a biological activity of neurturin. An exemplary substitution variant is illustrated in Figure 4. Mutagenesis techniques for the replacement, insertion or deletion of one or more selected amino acid residues are known to those skilled in the art (eg, US Patent No. 4,518,584, The description of which is incorporated herein by reference There are two main variables in the construction of variants: the location of the mutation site and the nature of the mutation When designing neurturin variants, the selection of the mutation site and the nature of the mutation mutation may depend on the characteristics of neurturin to be modified.The mutation sites can be modified individually or in series, eg, (1) substituting first with conserved amino acid selections and then with more radical selections, depending on the results achieved, (2) eliminating the white amino acid residue or (3) inserting amino acid residues ad lying to the localized site. Changes conserved in the form of 1 to 20 amino acids are preferred. Once the amino acid sequence of the product of the desired neurturin protein is determined, the nucleic acid sequence to be used in the expression of the protein is easily determined. N-terminal and C-terminal deletion variants can also be generated by proteolytic enzymes. For neurturin deletion variants, deletions generally vary from about 1 to 30 residues, typically from about 1 to 10 residues and typically from about 1 to 5 contiguous residues. The N-terminal and C-terminal and internal deletions within the sequence are included. Deletions can be made in regions of low homology with other members of the TGF-β superfamily, to modify the activity of neurturin. Deletions in areas of substantial homology to other sequences of the TGF-β superfamily will be more likely to modify the biological activity of neurturin more significantly. The number of consecutive deletions will be selected such that the tertiary structure of the product of the neurturin protein in the affected domain, e.g., cross-links with cysteine, is retained. Non-limiting examples of deletion variants include the truncated neurturin protein products lacking one to seven N-terminal amino acids or variants lacking the C-terminal residue, or combinations thereof. In a basic embodiment, the truncated neurturin proteins can be represented by the following amino acid sequences, wherein the amino acid residue numbering scheme of Figure 1 is used to facilitate comparison with the human neurturin protein: X- [Cys8 -Cys101] -And where [Cys -Cys] represents the amino acid sequence from Cys to Cys, as illustrated in Figure 1 (SEQ ID No. 1); Y represents zero or the amino acid residues of the terminal carboxyl terminus one or more, for example -Val- 102; and X represents zero, a methionine residue or one or more amino acid residues of the amino terminal end, for example: P RP ARP GARP LGARP RLGARP ARLGARP As used herein, the term "product of the truncated neurturin protein" includes truncated, synthetic or recombinant neurturin proteins, biologically active, truncated neurturin proteins produced from mature neurturin, biologically active truncated neurturin variants (including insertion, substitution and deletion variants) and chemically derived modified thereof. Also included are truncated neurturin proteins that are substantially homologous to the human neurturin protein having the amino acid sequence set forth in Figure 1 (SEQ ID No. 1). For neurturin addition variants, additions of amino acid sequences typically include N-terminal and / or C-terminal fusions that vary in length from one residue to polypeptides containing one hundred or more residues, as well as internal additions within the sequence of single or multiple amino acid residues. The internal additions can generally vary from about 1 to 10 residues, typically from about 1 to 5 residues and usually from about 1 to 3 amino acid residues. Examples of N-terminal addition variants include neurturin with an N-terminal methionyl residue (eg, an artifact of direct expression of neurturin in bacterial recombinant cell culture), which is designated as [Met ~] neurturin and Fusion of a heterologous N-terminal signal sequence to the N-terminal end of neurturin, to facilitate the secretion of. Neurturin matures in recombinant host cells. Such signal sequences will usually be obtained from and, therefore will be homologous to, the intended host cell species. Additions may also include amino acid sequences derived from the sequence of other neurotrophic factors, for example, from 1 to 35 N-terminal amino acid residues of human or rat GDNF proteins. A preferred neurturin protein product to be used in accordance with the present invention is recombinant human [Met-] neurturin. In neurturin substitution variants, at least one amino acid residue of the amino acid sequence of human or murine neurturin has been removed and a different residue has been inserted in its place. Such substitution variants include allelic variants, which are characterized by changes in the nucleotide sequence of natural origin in the population of the species, which may or may not result in an amino acid change. An example of a substitution variant is illustrated in Figure 4 (SEQ ID Nos. 3 6 4). Specific mutations of the amino acid sequence of neurturin may include modifications to a glycosylation site (e.g., serine, threonine or asparagine). Absence of glycosylation or only partial glycosylation is the result of an amino acid substitution or deletion at any site of glycosylation recognition linked to asparagine or at any site of the molecule that is modified by the addition of an O-linked carbohydrate. . An asparagine-linked glycosylation recognition site comprises a tripeptide sequence that is specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are Asn-Xaa-Thr or Asn-Xaa-Ser, wherein Xaa can be any amino acid other than Pro. A variety of substitutions or deletions of amino acids in one or both of the first or third amino acid positions of a site of glycosylation recognition (and / or deletion of the amino acid in the second position) results in a non-glycosylation in the modified tripeptide sequence. Thus, the expression of altered nucleotide sequences produces variants that are not glycosylated at that site. Alternatively, the amino acid sequence of neurturin can be modified by adding glycosylation sites. A method for identifying amino acid residues or regions of neurturin for mutagenesis is called "alanine scanning utagénesis" as described by Cunningham and Wells (Science, 244: 1081-1085, 1989). In this method, a residue of amino acid or a group of white residues is identified (eg, charged residues such as Arg, Asp, His, Lys and Glu) and is replaced by a neutral or negatively charged amino acid (preferably alanine or polyalanine) to affect the interaction of the amino acids that surround the aqueous environment inside or outside the cell. Those domains that demonstrate functional sensitivity to substitutions are subsequently refined by introducing additional or alternative residues at the substitution sites. Thus, the target site is determined to introduce a variation of amino acid sequence, mutagenesis is carried out by alanine scanning or randomization in the target codon or in the region of the corresponding DNA sequence and the neurturin variants expressed are select for the optimal combination of desired activity and degree of activity. The sites of greatest interest for substitutional mutagenesis include sites where the amino acids found in the neurturin proteins of various species are substantially different in terms of side chain block, charge and / or hydrophobicity. Other sites of interest are those in which particular residues of neurturin-like proteins, obtained from several species, are identical. Such positions are usually important for the biological activity of a protein. Initially, these sites are replaced in a relatively conservative manner. Such conservative substitutions are shown in Table 1, under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes are introduced (example substitutions) and / or additions or deletions can be made, and the resulting products are selected with respect to their activity. TABLE 1 Amino Acid Substitutes TABLE 1 Substitutions of Amino Acids Residue Substitutions Preferred Original Substitutions of Example Ala (A) Val Val; Leu; lie Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Lys; Arg Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met; Ala Phe; norleucine Leu (L) lie norleucine; lie; Val; Met; To; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; He Phe (F) Leu Leu; Val; He; Wing Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu lie; Leu; Met; norleucine Conservative modifications to the amino acid sequence (and corresponding modifications to coding nucleic acid sequences) are expected to produce neurturin protein products that have functional and chemical characteristics similar to those of natural neurturin. In contrast, substantial modifications in the functional and / or chemical characteristics of neurturin protein products can be achieved by selecting substitutions that differ significantly in their effect of maintaining (a) the structure of the polypeptide framework in the area of substitution, for example, in sheet form or helical conformation, (b) the charge or hydrophobicity of the molecule in the target site, or (c) the block of side chains. The residues of natural origin are divided into groups based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, lie; 2) neutral hydrophilic: Cys, Ser, Thr; 3) Acids: Asp, Glu; 4) Basic: Asn, Gln, His, Lys, Arg; 5) residues that influence the chain orientation: Gly, Pro; and 6) aromatics: Trp, Tyr, Phe. Non-conservative substitutions may include changing a member of one of these classes for another. Such substituted residues can be introduced into regions of the neurturin protein that are homologous to other proteins of the TGF-β superfamily, including GDNF, or to non-homologous regions of the molecule. B. Neurturin Derivatives The chemically modified derivatives of neurturin protein products can also be prepared by those skilled in the art given the descriptions herein. The most suitable chemical portions for derivation include water-soluble polymers. A water-soluble polymer is desirable because the protein to which it binds does not precipitate in an aqueous environment, such as a physiological environment. Preferably, the polymer will be pharmaceutically acceptable for the preparation of a therapeutic product or composition. Those skilled in the art will be able to select the desired polymer, based on considerations such as whether the polymer / protein conjugate will be used therapeutically and, if so, the desired dose, time in circulation, resistance to proteolysis and other considerations. . Suitable water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers) and dextran or poly- (n-vinylpyrrolidone) -polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol) polyvinyl alcohol and mixtures thereof. Propionaldehyde d polyethylene glycol may have advantages in manufacturing, due to its stability in water. The polymer can have any molecular weight and can be branched or not. For polyethylene glycol, the preferred molecular weight ranges from about 2 to about 100 kDa for easy handling and manufacturing (The term "about" indicates that in polyethylene glycol preparations, some molecules will weigh more, others less, than the established molecular weight) . Other sizes may be used, depending on the desired therapeutic profile (eg, the duration of the desired sustained release, the effects, if any, on biological activity, ease of handling, the degree or lack of antigenicity and other known effects of polyethylene glycols on a protein or therapeutic variant The number of polymer molecules thus bound may vary and those skilled in the art will be able to foresee the effect on the function, a monoderivation may be performed or a biderivation, triderivation may be performed, tetraderivation or some combination of derivations, with the same or different chemical portions (e.g., polymers such as different weights of polyethylene glycols). The ratio of polymer molecules to protein (or peptide), as well as their concentrations in the reaction mixture. In general, the optimal ratio (in terms of reaction efficiency in which there is no excess of unreacted protein or polymer) will be determined by factors such as the desired degree of derivation (eg, mono-derivation, biderivation, triderivation, etc.), the molecular weight of the selected polymer, if the polymer is branched or unbranched and the reaction conditions. The propylene glycol molecules (or other chemical moieties) must be bound to the protein taking into account the effects exerted on the functional or antigenic domains of the protein. There are a number of joining methods available to those skilled in the art.

See, for example, European Patent EP 0 401 384, the description of which is incorporated herein by reference (coupling of PEG to G-CSF), see also Malik et al. , Exp. He atol., 20: 1028-1035, 1992 (report of pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol can be covalently linked through amino acid residues by a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule can be attached. The amino acid residues that have a free amino group can include lysine residues and the N-terminal amino acid residue. Those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups can also be used as a reactive group to bind to the polyethylene glycol molecule (s). For therapeutic purposes, binding to an amino group, such as the N-terminus or a lysine group, is preferred. If it is desired that the molecule binds to its receptor, binding in residues important for receptor binding should be avoided. A chemically modified protein at the N-terminus can be specifically desired. Using the propylene glycol as an illustration of the compositions herein, one can select from a variety of polyethylene glycol molecules (by molecular weight, branches, etc.), the proportion of polyethylene glycol molecules relative to the protein (or peptide) molecules. in the reaction mixture, the type of pegylation reaction to be performed and the method for obtaining the pegylated protein at the selected N-terminal end. The method for obtaining the pegylated preparation at the N-terminus (ie, separating this portion from other non-pegylated portions, if necessary) can be purification of the pegylated material at the N-terminal position from a population of protein molecules. pegilada. Selective N-terminal chemical modification can be achieved by a reductive alkylation that exploits the differential reactivity of the different types of primary amino groups (lysine versus the N-terminus) available for derivation in a particular protein. Under the appropriate reaction conditions, a substantially selective derivation of the protein at the N-terminus with a carbonyl group containing the polymer is achieved. For example, the protein can be selectively pegilated at the N-terminal end when performing the reaction at a pH that allows taking advantage of the pKa differences between the e-amino group of the lysine residues and that of the a-amino group of the residue N-terminal protein. By such selective derivation, the binding of a water-soluble polymer to a protein is controlled: the conjugation with the polymer is carried out predominantly at the N-terminus of the protein without significantly modifying the other reactive groups, such as the amino groups of the protein. the side chain of lysine. When using reductive alkylation, the water-soluble polymer can be of the type described above and must have only one reactive aldehyde to couple with the protein. Polyethylene glycol propionaldehyde, which contains only one reactive aldehyde, can be used. The present invention contemplates the use of derivatives that are neurturin or variants thereof expressed in prokaryotes, linked to at least one molecule of polyethylene glycol, as well as the use of neurturin or variants thereof bound to one or more polyethylene glycol molecules through of an acyl or alkyl bond. The pegylation can be carried out by any pegylation reaction known in the art. See for example: Focus on Growth Factors, 3 (2): 4-10, 1992; EP 0 154 316, the disclosure of which is incorporated herein by reference; European Patent EP 0 401 384; and the other publications cited herein with reference to pegylation. PEGylation can be carried out by an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer). PEGylation by acylation generally includes reacting an active ester derivative of polyethylene glycol, with neurturin protein or a variant. Any known or subsequently discovered reactive PEG molecule can be used to carry out the pegylation of the neurturin or variant protein. A preferred activated PEG ester is PEG esterified with N-hydroxysuccinimide. As used herein, the term "acylation" includes, without limitation, the following types of linkages between the therapeutic protein and a water-soluble polymer such as PEG: amide, carbamate, urethane, and the like. See Bioconjugate Chem., 5: 133-140, 1994. The reaction conditions can be selected from any of those known in the pegylation art or between those subsequently developed, but temperature, solvent and pH conditions that inactivate the neurturin or variant to be modified. PEGylation by acylation will generally result in a neurturin protein or polypeglylated variant. Preferably, the linking link will be an amide. Also preferably, the resulting product will be substantially only (e.g., >95%) monopegylated, dipegylated or tripegylated. However, some species with higher degrees of pegylation can be formed, in amounts that depend on the specific reaction conditions used. If desired, more purified pegylated species can be separated from the mixture, particularly unreacted species, by standard purification techniques including, among others, dialysis, salination, ultrafiltration, ion exchange chromatography, gel filtration chromatography and electrophoresis. . PEGylation by alkylation generally includes reacting a terminal aldehyde derivative of PEG with a neurturin or variant protein, in the presence of a reducing agent. Pegylation by alkylation can also produce neurturin protein or polypeglylated variant. In addition, the reaction conditions can be manipulated to favor pegylation substantially only in the a-amino group of the N-ternimal end of the neurturin or variant protein (i.e., a monopegylated protein). In the case of monopegilation or polypegilation, the PEG groups are preferably bound to the protein through a -CH2-NH- group. With particular reference to the group -CH2-, this type of bond is referred to herein as an "alkyl" bond. The derivation by reductive alkylation to produce a monopegylated product exploits the differential reactivity of the different types of primary amino groups (lysine verses N-terminal) available for derivation. The reaction is carried out at a pH that allows taking advantage of the pKa differences between the e-amino groups of the lysine residues and that of the a-amino group of the N-terminal residue of the protein. By such selective derivation, the binding of a water-soluble polymer containing a reactive group such as an aldehyde to a protein is controlled: the conjugation with the polymer is carried out predominantly at the N-terminal end of the protein without significantly modifying the proteins. other reactive groups, such as the amino groups of the lysine side chain. In an important aspect, the present invention contemplates the use of a substantially homogeneous preparation of monopolymer / neurturin protein (or variant) conjugated molecules (which means a neurturin or variant protein to which a polymer molecule has been bound, only substantially (ie, > 95%) in a single location). More specifically, if polyethylene glycol is used, the present invention also encompasses the use of the pegylated neurturin protein or variant possibly lacking antigenic binding groups and having the polyethylene glycol molecule directly coupled to the neurturin or variant protein. Thus, it is contemplated that the products of the neurturin protein to be used in accordance with the present invention may include the pegylated neurturin protein or variants thereof, wherein the PEG group or groups are attached through acyl groups or I rent. As described above, such products can be monopegylated or polypelylated (e.g., containing 2-6 and preferably 2-5 PEG groups). PEG groups are usually linked to the protein in the a-amino or e-amino groups of amino acids, but it is also contemplated that PEG groups can be attached to any amino group attached to the protein that is sufficiently reactive to bind to a PEG group, under suitable reaction conditions. The polymer molecules used in both acylation and alkylation can be selected from water-soluble polymers such as those described above. The selected polymer must be modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, such that the degree of polymerization can be controlled as provided in the present methods. An example reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or C1-C10 alkoxy or aryloxy derivatives thereof (see, U.S. Patent No. 5,252,714). The polymer may be branched or unbranched. For acylation reactions, the selected polymers must have a single reactive ester group. For the reductive alkylation herein, the polymer or polymers selected must have a single reactive aldehyde group. In general, the water-soluble polymer will not be selected from naturally occurring glucosyl residues, since these are usually prepared more conveniently with recombinant expression systems in mammals. The polymer can be of any molecular weight and can be branched or not. A preferred water-soluble polymer for use herein is polyethylene glycol. As used herein, polyethylene glycol encompasses any form of PEG that has been used to derive other proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. In general, chemical derivatization can be performed under any suitable condition used to react a biologically active substance with an activated polymer molecule. Methods for preparing a product of the pegylated neurturin protein, will generally comprise the steps of: (a) reacting a product of the neurturin protein with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions in which the protein binds one or more PEG groups and (b) obtain the product or products of the reaction. In general, the optimal reaction conditions for the acylation reactions will be determined, as the case may be, based on the known parameters and the desired result. For example, the larger the PEG / protein ratio, the higher the percentage of polypeglylated product. Reductive alkylation to produce a substantially homogeneous population of a monopolymer conjugate / neurturin protein product, will generally comprise the steps of: (a) reacting a product of the neurturin protein with a reactive PEG molecule, under alkylation conditions reductive, at a suitable pH to allow selective modification of the a-amino group at the amino terminus of the neurturin protein product; and (b) obtain the product or products of the reaction. For a substantially homogeneous population of monopolymer / neurturin protein product conjugate molecules, the conditions of the reductive alkylation reaction are such as to permit selective binding of the water-soluble polymer portion to the N-terminus of the neurturin protein product. . Such reaction conditions usually provide pKa differences between the amino groups of the lysine and the a-amino group of the N-terminal end (the pKa being the pH at which 50% of the amino groups are protonated and 50% they are not) . The pH also affects the ratio of the polymer to the protein to be used. In general, if the pH is lower, a greater excess of polymer with respect to protein will be desired (i.e., the less reactive the N-terminal a-amino group, the more polymer will be needed to achieve optimum conditions). If the pH is higher, the polymer / protein ratio will not be as great (i.e., more reactive groups are available, so fewer polymer molecules are needed). For the purposes of the present invention, the pH will generally fall within the range of 3 to 9, preferably 3 to 6. Another important consideration is the molecular weight of the polymer. In general, the larger the molecular weight of the polymer, the less polymer molecules will bind to the protein. Similarly, the branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or more branches), the higher the polymer / protein ratio. In general, for the pegylation reactions contemplated herein, the preferred average molecular weight is from about 2 kDa to about 100 kDa. The preferred average molecular weight is from about 5 kDa to about 50 kDa, particularly from about 12 to about 25 kDa is particularly preferred. The ratio of the water-soluble polymer to the product of the neurturin protein will generally be in the range of 1: 1 to 100: 1, preferably (for polypegilation) from 1: 1 to 20: 1 and (for monopegylation) of 1: 1 to 5: 1. Using the above-indicated conditions, the reductive alkylation will reduce the selective binding of the polymer to any product of the neurturin protein having an α-amino group at the amino terminus and will provide a substantially homogeneous preparation of the monopolymer conjugate / neurturin protein product. The term "monopolymer / neurturin protein product conjugate", as used herein, means a composition comprised of a single polymer molecule bound to a molecule of a product of the neurturin protein. The monopolymer / neurturin protein product conjugate will preferably have a polymer molecule located at the N-terminal end, but not at the amino side groups of lysine. The preferred preparation will be greater than 90% monopolymer conjugate / neurturin protein product and more preferably greater than 95% monopolymer conjugate / neurturin protein product, where the rest of the observable molecules remain unreacted (ie, protein lacking the polymer portion).

For the present reductive alkylation, the reducing agent must be a stable solution in an aqueous medium and preferably must be able to reduce only the Schiff base formed in the initial reductive alkylation process. Preferred reducing agents can be selected from the group consisting of sodium borohydride, sodium cyanoborohydride, dimethylaminoborane, tri-ethylaminoborane and pyridinoborane. A particularly preferred reducing agent is sodium cyanoborohydride. Other reaction parameters, such as solvent, reaction time, temperatures, etc. and the means of purification of the products, can be determined as the case may be, based on the published information regarding the derivation of proteins with water-soluble polymers (see the publications cited herein). C. Neurturin Protein Product Pharmaceutical Compositions Neurturin protein product pharmaceutical compositions typically include a therapeutically effective amount of a neurturin protein product, mixed with one or more pharmaceutically and physiologically acceptable formulation materials, which are selected by its suitability according to the mode of administration. Suitable formulation materials include, but are not limited to, antioxidants, preservatives, colorants, flavors and diluents, emulsifying agents, suspending agents, solvents, fillers, bulk agents, pH regulating solutions, delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants. For example, a suitable vehicle can be injectable water, physiological saline or artificial perilymph, possibly supplemented with other materials common in the compositions for parenteral administration. Neutral regulatory saline or saline mixed with serum albumin are additional examples of vehicles. The primary solvent in a carrier can be aqueous or non-aqueous in nature. In addition, the carrier may contain other pharmaceutically acceptable excipients to modify or maintain the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution or aroma of the formulation. Similarly, the carrier may still contain other pharmaceutically acceptable excipients to modify or maintain the rate of product release from the neurturin protein or to promote the absorption or penetration of the product of the neurturin protein through the tympanic membrane. Such excipients are those substances normally used to formulate medicaments for administration in the middle ear, either in single dose units or in the form of multiple doses. Once the therapeutic composition has been formulated, it can be stored in sterile flasks in the form of a solution, suspension, gel, emulsion, solid or dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form, or in a form for example lyophilized, which requires their reconstitution before administration. The optimal pharmaceutical formulations will be determined by those skilled in the art depending on considerations such as the route of administration and the desired dose. See for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA (18042), pages 1435-1712, the disclosure of which is incorporated herein by reference.) Such formulations can influence physical state, the stability, the in vivo release rate and the in vivo clearance rate of the neurturin proteins of the present, variants and derivatives thereof, Other effective forms of administration, such as slow release formulations in the middle ear, mists Inhaled or orally active formulations are also included For example, in a sustained release formulation, the product of the neurturin protein can be attached or incorporated into particulate preparations of polymeric compounds (such as polylactic acid, polyglycolic acid, etc.) or liposomes. Hyaluronic acid can also be used and this can have the effect of promoting sustained duration in the blood circulation. Suitable biodegradable sustained release matrices include gelatin and polymers of, for example, lactic acid or collagen, including modified collagen such as atelocollagen, methylated collagen or succinylated collagen. See, for example, European Patent Application Publication No. EP 412 554 A2, published on February 13, 1991. Other suitable sustained-release matrices include copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, polymers. (2-hydroxyethyl methacrylate), ethylene vinyl acetate, poly-D- (-) - 3-hydroxybutyric acid, other polyesters, hyaluronic acid or liposomes. The controlled release matrix can be prepared by mixing a GDNF solution or a gel with the biodegradable matrix vehicle, followed by a concentration and dehydration of the mixture. It is contemplated that a controlled release composition can be prepared in which the protein is dispersed in preformed porous polymer microparticles. See PCT Publication Application No. WO 93/15722, published on August 19, 1993. The microparticles can be prepared from any suitable polymeric material, such as polyesters, polyamides, polyanhydrides or polyacrylates and, preferably, is a biodegradable polymer such as polylactic acid, polyglycolic acid, a copolymer of lactic acid and glycolic acid or polyacid l, 3-bis- (p-carboxyphenoxy) -propancosebasic acid} . The microparticles, which are generally 50 to 400 microns in diameter and are permeable by a network of pores ranging from 0.01 to 1 microns, are loaded with the protein by balancing them in a suspension or protein solution. Vacuum or pressure can be applied to facilitate the migration of the drug into the microparticles. The microparticles can be dried in air, under vacuum, by controlled evaporation, by flow of an inert gas, by freezing or by other techniques and then further processed to obtain the desired compositions for injection or implant. The neurturin protein product pharmaceutical composition can also be formulated for administration in the middle ear, e.g., by infusion into the tympanic membrane or injection, and can also include slow release or sustained circulation formulations. Such therapeutic compositions administered in the middle ear, typically are in the form of an aqueous solution acceptable to the middle ear, free of pyrogens, which comprises the product of the neurturin protein in a pharmaceutically acceptable vehicle. A preferred vehicle is sterile distilled water. It is also contemplated that certain formulations containing the product of the neurturin protein can be administered orally. A product of the neurturin protein which is administered in this manner can be formulated in the form of an elixir, tablet, capsule or gel and can be formulated with or without those vehicles customary in the preparation of solid dosage forms. The capsule can be designed to release the active portion of the formulation at the point of the gastrointestinal tract where the bioavailability is maximal and presystemic degradation is minimal. Additional excipients may be included to facilitate absorption of the neurturin protein product. Also use diluents, flavorings, waxes to lower the melting point, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders. The formulation of topical preparations for otic administration, including solutions, suspensions and ointments for the middle ear, is well known to those skilled in the art (see Remington's Pharmaceutical Sciences, 18th Edition, Chapter 86, pages 1581-1592, Mack Publishing Company, 1990). Other means of administration are available, including injections to the middle ear and methods and means for the production of preparations suitable for the middle ear are also known such modes of administration. As used herein, the term "middle ear" refers to the space between the tympanic membrane and the middle ear. This location is external to all the tissue of the inner ear and an invasive procedure may not be required to access this region, if a formulation is developed for neurturin to penetrate through the tympanic membrane. Alternatively, the material can be introduced into the middle ear by injection through the tympanic membrane or, if repeated administrations are necessary, a perforation can be made in the tympanic membrane. Examples of such systems include inserts and drops applied "topically", gels or ointments that can be used to distribute the therapeutic material in these regions. An opening in the tympanic membrane is a very common procedure that is performed in the office, in cases such as middle ear infections (usually in children). The opening closes spontaneously after a few days. In the presently described use of the neurturin protein product in the treatment of diseases or injuries of the inner ear, it is also advantageous that a formulation of topical application includes an agent to promote the penetration or transport of the therapeutic agent towards the middle ear and the inner ear. Such agents are known in the art. For example, Ke et al. , US Patent No. 5,221,696 describe the use of materials to increase the penetration of ophthalmic preparations through the cornea. The internal ear systems are those systems that are suitable for use in any tissue compartment within, between or around the tissue layers of the inner ear, such as the cochlea and the vestibular organ. These locations include the different structures of the cochlea such as the vascular tape, the Reissner's membrane, the organ of Corti, the spiral ligament and the cochlear neurons. An invasive procedure may not be required to access these structures, since it has been shown that proteins penetrate the round window membrane in the perillinfa of the inner ear. • A particularly suitable vehicle for introducing neurturin into the inner ear by penetration through the round window membrane is artificial perilymph. This solution consists of 10.00 mM D-glucose, 1.5 mM CaCl, 1.5 mM MgCl in a 1.0% solution of Dulbecco's phosphate buffer, in deionized water, at 280-300 mOsm and pH 7.2. Another preparation may include the product formulation of the neurturin protein with an agent such as injectable microspheres or liposomes in the middle ear, which provides for slow or sustained release of the protein, which then can be administered as an injected reservoir. Other suitable mechanisms for introducing neurturin protein product into the inner ear include implantable drug delivery devices or devices containing neurturin protein product and a cochlear implant with a tunnel through it, so that neurturin can be supplied continuously through the tunnel to the inner ear. The preparations for ear treatment of the present invention, particularly topical preparations, may include other components, for example conservatives acceptable for the middle ear, tonicity regulating agents, cosolvents, co-plete agents, pH regulating agents, antimicrobials, antioxidants and surfactants, which are well known in the art.

For example, tonicity improving agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol and the like. Advantageously, a sufficient amount of the tonicity enhancing agent is added so that the formulation to be introduced into the ear is compatible with the osmolality of the endolymph and the perilymph. Such suitable preservatives include, but are not limited to, benzalkonium chloride, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine, sodium benzoate, sorbic acid, and the like. You can also use hydrogen peroxide as a preservative. Suitable cosolvents include, but are not limited to, alcohols, glycerin, glycerol, propylene glycol and polyethylene glycol. The above-described complex agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin. Suitable antioxidants include sodium bisulfite and ascorbic acid. Suitable surfactants or wetting agents are, for example, sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal and the like. The pH regulating agents can be conventional regulating agents such as acetate, borate, citrate, phosphate, dicarbonate or tris-HCl. Other stabilizing agents can be used, including proteins such as serum albumin, gelatin or immunoglobulins; amino acids such as glycine, glutamate, aspartate, arginine, lysine or cysteine; and monosaccharides and disaccharides such as glucose, mannose or dextrin. The components of the formulation are present in a concentration that is acceptable for the administration site in the middle ear or inner ear. For example, pH regulating solutions are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of about 5 to about 8. Additional formulation components may include materials that provide a residence prolonged therapeutic agent administered in the middle ear, to maximize topical contact and promote absorption through the round window membrane. Suitable materials include polymers or gel-forming materials that provide an increase in viscosity of the preparation in the middle ear. The suitability of the formulations of the present invention for the controlled release (e.g., sustained and prolonged release) of an agent for the treatment of the inner ear can be determined by various methods known in the art. Another otic preparation may include an effective amount of the neurturin protein product mixed with non-toxic excipients, acceptable for the treatment of the middle ear, which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water or other suitable vehicle, solutions for the treatment of the middle ear can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium or bicarbonate carbonate, lactose or calcium phosphate, or binding agents such as starch, gelatin or acacia gum. Administration / Product Distribution of Neurturin Protein Neurturin protein product can be administered parenterally subcutaneously, intramuscularly, intravenously, intraarterially, intranasally, intrapulmonaryly, intraperitoneally, infraocularly, transcranially, intravitreally, subretinally, intrathecally or intracerebrally. In appropriate circumstances, intralesional administration may be indicated, e.g., in the irrigation fluid used to wash injured areas or implants in injured areas, with an adequate matrix. Alternatively, the product of the neurturin protein can be administered orally or in specific areas of the gastrointestinal tract or rectally, transdermally or topically.

For the treatment of disorders of the inner ear, the product of the neurturin protein can be administered in the middle ear (or directly in the inner ear, especially in cases where the invasive procedure is already programmed), through the topical application of inserts, injections, implants, cell therapy or gene therapy. For example, slow release implants containing the neurotrophic factor embedded in a biodegradable polymer matrix can deliver the product of the neurturin protein. A product of the neurturin protein can be administered extracerebrally in a form that has been chemically modified or packaged to pass through the blood-brain barrier, or it can be administered in connection with one or more agents capable of promoting the penetration of the product from the brain. Neurturin protein through said barrier. Similarly, the product of the neurturin protein can be administered in the middle ear or inner ear, or it can be administered over the tympanic membrane in connection with one or more agents capable of promoting penetration or transport of the product from the body. Neurturin protein through the ear membranes. The frequency of dosing will depend on the pharmacokinetic parameters of the neurturin protein product as formulated and the route of administration.

The specific dose can be calculated in accordance with factors such as body weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dose for the treatment involving each of the aforementioned formulations is routinely done by those skilled in the art and is within the scope of the tasks performed routinely, especially in light of the Dosage information and the assays described herein. Appropriate dosages can be determined by using established assays to determine the doses used in conjunction with appropriate dose-response data. Those skilled in the art will note that the dosage used in formulations administered in the inner ear will be miniscule compared to that used in a systemic injection or by oral administration. The final dose regimen involved in a method for the treatment of the above-described disorders will be determined by the attending physician, taking into account several factors that modify the action of the drugs, eg, the age, the patient's condition , body weight, sex and diet, the severity of any infection, the time of administration and other clinical factors. As studies are conducted, additional information will emerge regarding the appropriate dose levels for the treatment of various diseases and disorders. It is contemplated that continuous administration or sustained distribution of a product of the neurturin protein may be advantageous for a given treatment. While continuous administration can be accomplished by mechanical means such as an infusion pump, it is contemplated that other modes of continuous or near continuous administration can be practiced. For example, chemical derivatization or encapsulation can result in sustained release forms of the protein that have the effect of a continuous presence, in predictable amounts, based on a given dosage regimen. Thus, the products of the neurturin protein include proteins derived or formulated in some other way to effect such continuous administration. Cell therapy with products of the neurturin protein, e.g., implants in the middle ear or inner ear of cells producing the neurturin protein product, is also contemplated. This modality would involve implanting patients with cells capable of synthesizing and secreting a form biologically active product of the neurturin protein. Such cells producing the neurturin protein product can be cells that are natural producers of the neurturin protein product or can be cells that have been modified to express the protein. Such modified cells include recombinant cells whose ability to produce a product of the neurturin protein has been increased by a transformation with a gene encoding the product of the desired neurturin protein., in a suitable vector to promote its expression and secretion. In order to minimize the potential for immunological reactions in patients given the neurturin protein product of a foreign species, it is preferred that the natural cells producing the neurturin protein product be of human origin and produce the product of the human neurturin protein. Similarly, it is preferred that the recombinant cells producing the neurturin protein product be transformed with an expression vector containing a gene encoding a product of the human neurturin protein. The implanted cells may be encapsulated to prevent infiltration of surrounding tissue. Human or non-human animals can be implanted in patients, in semi-permeable and biocompatible polymeric shells or membranes that allow the release of neurturin protein product, but that prevent the destruction of cells by the patient's immune system or by any other harmful factor of the tissue that surrounds them. Such implants, for example, can be attached to the round window membrane of the middle ear, to produce and release the product of the neurturin protein directly in the perilymph. The methodology of membrane encapsulation of living cells is familiar to those skilled in the art and the preparation of the encapsulated cells and their implantation in patients can be carried out without undue experimentation. See, for example, US Pat. Nos. 4,892,538; 5,011,472 and 5,106,627, each of which is specifically incorporated herein by reference. In PCT Publication WO 91/10425 of Aebischer et al. , which is incorporated herein by reference specifically, describes a system for encapsulating living cells. See also Publication WO 91/10470 of Aebischer et al. , Winn et al. , Exper. Neurol., 113: 322-329, 1991, Aebischer et al. , Exper. Neurol., 111: 269-275, 1991; Tresco et al. , ASAIO, 38: 17-23, 1992, each of which is specifically incorporated herein by reference. Techniques for formulating a variety of other sustained or controlled release mechanisms, such as liposome vehicles, bioerodible particles or injected beads and reservoirs, are also known to those skilled in the art. It is also contemplated that the patient's own cells can be transformed ex vivo to produce the product of the neurturin protein, which would be implanted directly without encapsulation. For example, cells of the organ of Corti can be excised, cultured and transformed with the appropriate vector and transplanted back into the patient's inner ear, where they would produce and release the desired neurturin protein or the desired neurturin protein variant. In vivo gene therapy is also included with a product of the neurturin protein, by introducing the gene coding for the neurturin protein product into target cells of the inner ear, by a local injection of a nucleic acid construct or other vector of appropriate administration (Hefti, J. Neurobiol., 25: 1418-1435, 1994). For example, a nucleic acid sequence encoding a product of the neurturin protein can be contained in an adeno-associated virus vector or an adenovirus vector, to be delivered to the cells of the inner ear. Alternative viral vectors include, but are not limited to, retroviruses, herpes simplex viruses and papillomavirus vectors. Physical transfer, either in vivo or ex vivo as appropriate, can also be achieved through a liposome-mediated transfer, a direct injection (naked DNA), a receptor-mediated transfer (ligand-DNA complex), electroporation, precipitation with calcium phosphate or bombardment with microparticles (gene gun). It should be noted that the product formulations of the neurturin protein described herein, can be used for veterinary applications as well as in human medicine and that the term "patient" should not be considered in any way as limiting. In the case of veterinary applications, the dose ranges will be the same as those specified above. Polynucleotides that Code for a Neurturin Protein Product The present invention also provides novel polynucleotides that encode products of the neurturin protein. When used as a hybridization probe or amplification primer, the nucleic acid sequence will be substantially free of other nucleic acid sequences. For use in expression of recombinant proteins, the nucleic acid sequence will generally be substantially free of nucleic acid sequences encoding other proteins, unless a fusion protein is intended. Based on the present description and using the universal codon table, a person skilled in the art will be able to easily determine all the nucleic acid sequences that code for the amino acid sequences of a product of the neurturin protein. It will also be appreciated by those skilled in the art that the novel polynucleotides encoding neurturin protein products include those amino acid sequences that encode variant proteins, whether naturally occurring or man-made. The recombinant expression techniques conducted in accordance with the descriptions presented below, they can be followed to produce these polynucleotides and express the various products of the neurturin protein. For example, by inserting a nucleic acid sequence encoding a protein into an appropriate vector, a person skilled in the art will readily be able to produce large quantities of the desired nucleotide sequence. Then, the sequences can be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding a product of the neurturin protein can be inserted into an expression vector. By introducing the expression vector into an appropriate host, the desired protein can be produced in large quantities. As will be described hereinafter, there are numerous host / vector systems available for the propagation of nucleic acid sequences and / or for the production of neurturin protein products. These include, but are not limited to, plasmids, viral vectors and insertional vectors, and prokaryotic and eukaryotic hosts. Those skilled in the art will be able to adapt a host / vector system that is capable of propagating or expressing heterologous DNA, to produce or express the sequences of the present invention. By means of such recombinant techniques, the proteins of the present invention are easily produced in commercial quantities. In addition, those skilled in the art will note that, in view of the present disclosure, the novel nucleic acid sequences include the degenerate nucleic acid sequences encoding the proteins specifically set forth in the Figures, variants of such proteins and acid sequences. nucleic acids with which they hybridize, preferably under stringent hybridization conditions, with complements of these nucleic acid sequences (see Maniatis et al., Molecular cloning (A Laboratory Manual); Cold Spring Harbor Laboratory, pages 387 to 389, 1982). An example of stringent hybridization conditions are the hybridization conditions in 3 x SSC at 62-67 ° C, followed by a wash in 0.1 x SSC 62-67 ° C for about one hour. Alternatively, an example of stringent hybridization conditions are the hybridization conditions in 45-55% for amide, 4 x SSC at 40-45 ° C. DNA sequences that hybridize to the sequences complementary to the neurturin protein under relaxed hybridization conditions and that encode a neurturin protein of the present invention are also included herein. Examples of such relaxed hybridization conditions are 4 x SSC at 45-55 ° C or hybridization with 30-40% of formamide at 40-45 ° C. The present invention also provides recombinant DNA constructs that involve the DNA vector together with the DNA sequence encoding a product of the neurturin protein. In such DNA constructs, the nucleic acid sequence encoding the protein (with or without signal peptides) is in operative association with a suitable regulatory or expression control sequence, capable of directing the replication and / or expression of the protein in the selected host. Recombinant Expression of a Neurturin Protein Product Preparation of polynucleotides that encode products of the neurturin protein. A nucleic acid sequence encoding a product of the neurturin protein can be readily obtained in a variety of ways, including, without limitation, chemical synthesis, selection of cDNA or genomic libraries, selection of expression libraries and / or amplification of CDNA by polymerase catalyzed chain reaction (PCR). These methods and other methods useful for the isolation of such nucleic acid sequences are described, for example, by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold spring Harbor, NY, 1989), by Ausubel et al. , eds. (Cursor Protocols in Molecular Biology, Current Protocols Press, 1994) and by Berger and Kimel (Methods in Enzymology: Guide to Molecular Cloning Techniques, vol.152, Academic Press, Inc., San Diego, CA, 1987). The chemical synthesis of a nucleic acid sequence encoding a product of the neurturin protein can also be carried out using methods known in the art, such as those described by Engels et al. (Angew, Chem. Intl. Ed., 28: 716-734, 1989). These methods include, inter alia, the phosphotriester, phosphoramidite and H-phosphonate methods of nucleic acid synthesis. The nucleic acid sequence encoding the protein will have a length of several hundred base pairs (bp) or nucleotides. Large nucleic acid sequences, for example greater than nucleotides in length, can be synthesized in the form of several fragments. Subsequently, the fragments can be ligated to form a nucleic acid sequence encoding the protein. A preferred method is the synthesis supported by polymers, using the standard phosphoramidite chemistry. Alternatively, a suitable nucleic acid sequence can be obtained by a selection in an appropriate cDNA library (ie, a library prepared from one or more tissue sources thought to express the protein) or a genomic library (a library prepared from total genomic DNA). The source of the cDNA library is typically a tissue of any species that is believed to express neurturin in reasonable amounts. The source of the genomic library can be any tissue or tissue from any mammal or other species, which is believed to carry a gene encoding neurturin or a homolog of neurturin. The library can be screened for the presence of neurturin cDNA / genes, using or more nucleic acid probes (oligonucleotides, CDNA or genomic DNA fragments having an acceptable level of homology with the cDNA or the neurturin gene or the neurturin homologue being cloned) wherein said probes will selectively hybridize with the cDNA or the. neurturin or neurturin homolog present in the library. The probes typically used for such library selection generally code for a small region of the neurturin DNA sequence from the same species or a similar species as the species from which the library was prepared. Alternatively, the probes may be degenerate, as described herein. The selection or library search is typically carried out by pairing oligonucleotide or cDNA probe chains with the clones of the library, under strict conditions that prevent non-specific binding, but that allow the binding of those clones that have a significant level of homology with the probe or the primer. Typical hybridization and stringency conditions depend in part on the size (i.e., number of nucleotides in length) of the cDNA or oligonucleotide probe and whether the probe is degenerate. The probability of obtaining a clone or clones in the design of the hybridization solution (i.e., if it is a cDNA or genomic library which is being selected) is also taken into account.; if it is a cDNA library, the probability that the cDNA of interest is present is greater). When DNA fragments (such as cDNAs) are used as probes, typical hybridization conditions include those set forth in Ausubel et al. , eds., supra. After hybridization, the stain containing the library is washed at a suitable stringency, depending on various factors such as the size of the probe, the expected homology of the probe with respect to the clone, the type of library being selected. , the number of clones being selected and the like. Examples of stringent washing solutions (which are generally of low ionic strength and are used at relatively high temperatures) are as follows. One such stringent wash is 0.015 M NaCl, 0.005 M sodium citrate and 0.1% Sodium Dodecyl Sulfate at 55-65 ° C. Another stringent regulatory solution is 1 mM Na2.EDTA, 40 mM NaHP04, pH 7.2 and 1% DSS, at about 40-50 ° C. Another stringent wash is 0.2 X SSC and 0.1% DSS at approximately 50-65 ° C. There are also exemplary protocols for stringent wash conditions, wherein oligonucleotide probes are used to select cDNA or genomic libraries. For example, the first protocol uses 6 X SSC with 0.05 percent sodium pyrophosphate, at a temperature between about 35 and 62 ° C, depending on the length of the probe. For example, probes of 14 bases were washed at 35-40 ° C, probes of 17 bases at 45-50 ° C, probes of 20 bases at 52-57 ° C and probes of 23 bases at 57-63 ° C. The temperature can be increased 2-3 ° C when the nonspecific background bond appears to be high. A second protocol with tetramethylammonium chloride (CTMA) is used for washing. One such stringent wash solution is 3 M CTMA, 50 mM Tris-HCl, pH 8.0 and 0.2% DSS. Another suitable method for obtaining a nucleic acid sequence encoding a product of the neurturin protein is the polymerase chain reaction (PCR). In this method, poly (A) + RNA or total RNA is extracted from a tissue that expresses neurturin. Next, the cDNA is prepared from the RNA using the reverse transcriptase enzyme. Two primers, typically complementary to two separate regions of the neurturin cDNA (oligonucleotides) are added to the cDNA together with a polymerase such as Taq polymerase and the polymerase amplifies the cDNA region between the two primers. When the selection method for the preparation of the nucleic acid sequence coding for the product of the desired neurturin protein requires the use of oligonucleotide primers or probes (eg, PC, selection in cDNA or genomic library), the oligonucleotide sequences selected as probes or primers must be of a suitable length and sufficiently unambiguous to minimize the amount of non-specific binding that will occur during library selection or PC amplification. The actual sequence of the probes or primers is usually based on conserved or highly homologous sequences or regions of the same gene or of a similar gene of another organism. Optionally, the probes or primers can be partially or totally degenerate (ie, contain a mixture of probes / primers, all coding for the same amino acid sequence, but using different codons to do it.) An alternative to the preparation of degenerate probes is to place an inosine in some or all of the positions of those codons that vary by species.The oligonucleotide probes or primers can be prepared by chemical DNA synthesis methods, as described above.The products of the neurturin protein based on these sequences Nucleic acid, as well as mutant sequences or variants thereof, are also contemplated within the scope of the present invention As described above, a mutant or variant sequence is a sequence containing one or more substitutions, deletions and / or insertions of nucleotides compared to the wild-type sequence and which results in the expression of variations of the amino acid sequence compared to the wild-type amino acid sequence. In some cases there may be variants or mutants of neurturin amino acids of natural origin, due to the existence of natural allelic variation. - Neurturin protein products based on such mutants or variants of natural occurrence, also fall within the scope of the present invention. The preparation of synthetic mutant sequences is also known in the art and is described, for example, in Wells et al. (Gene, 34: 315, 1985) and in Sambrook et al. , supra. Vectors The cDNA or genomic DNA encoding a product of the neurturin protein is inserted into a vector for cloning (amplification of DNA) or for expression. In commerce, suitable vectors are available or the vector can be specifically constructed). The selection or construction of the appropriate vector will depend on: 1) whether it is to be used for DNA amplification or for DNA expression, 2) the size of the DNA to be inserted in the vector and 3) the host cell (eg, mammalian cells, insects, yeasts, fungi, plants or bacteria) that will be transformed with the vector. Each vector contains several components depending on its function (DNA amplification or DNA expression) and its compatibility with the intended host cell. The vector components usually include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selection genes or markers, enhancer elements, promoters, a terminator sequence of transcription and the like. These components or regulatory elements of the expression can be obtained from natural sources or can be synthesized by known methods. The vectors of the present invention include a nucleic acid sequence encoding a product of the neurturin protein of interest, operatively linked to one or more of the following expression control or regulatory sequences capable of directing, controlling or affecting any another way the expression of the protein, by the host cell. Signal Sequence The signal sequence may be a component of the vector or may be part of the product DNA of the neurturin protein that is inserted into the vector. The neurturin DNA codes for a signal sequence at the amino terminus of the protein that is broken during the post-translational processing of the protein, to form the mature protein. Within the scope of the present invention polynucleotides of products of the neurturin protein with the native signal sequence and other pre-pro-sequences are included, as well as polynucleotides in which the negative signal sequence has been deleted and replaced by a sequence of heterologous signal. The selected heterologous signal sequence must be one that is recognized and processed, i.e., broken or degraded by a signal peptidase, in the host cell. For prokaryotic host cells that do not recognize and process the native neurturin signal sequence, the signal sequence is replaced by a prokaryotic signal sequence that is selected, for example, from the group consisting of alkaline phosphatase, penicillinase or enterotoxin II leaders. thermostable. For yeast secretion, the native neurturin signal sequence can be replaced by yeast invertase, alpha factor or acid phosphatase. In expression in mammalian cells, the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable. Origin of Replication Expression and cloning of vectors generally includes a nucleic acid sequence that makes it possible for the vector to replicate in one or more of the selected host cells. In vector cloning, this sequence is typically one that makes it possible for the vector to replicate independently of the chromosomal DNA of the host and includes origins of replication or autonomously replicating sequences. Such sequences are known for a variety of bacteria, yeasts and viruses. The origin of plasmid pBR322 replication is suitable for most Gram negative bacteria and diverse origins (e.g., SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. In general, the origin of replication component is not necessary for expression vectors in mammals (for example, the SV40 origin is often used only because it contains the early promoter). Selection gene The expression and cloning of vectors typically contains a selection gene. This gene encodes a "marker" protein necessary for the survival or growth of transformed host cells when grown in a selective culture medium. Host cells that were not tansformed with the vector will not contain the selection gene and, therefore, will not survive in the culture medium. Typical selection genes encode for proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline; (b) complement auxotrophic deficiencies or (c) supply critical nutrients that are not available in the culture medium. Other selection genes can be used to amplify the gene to be expressed. Amplification is the process in which the genes that have the greatest demand for the production of a protein critical for growth are reiterated in tandem with the chromosomes of successive generations of recombinant cells. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. Transforming mammalian cells are placed under a selective pressure, under which only the transformants are adapted to survive by virtue of the marker present in the vector. Selective pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the culture medium is successively changed, thereby causing the amplification of both the selection gene and the DNA encoding a product of the neurturin protein. As a result, increasing amounts of the neurturin protein product are synthesized from the amplified DNA. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate, which is a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is used is the Chinese hamster ovary cell line deficient in DHFR activity (see for example, Urlaub and Chasin, Proc. Nati. Acad. Sci., EUA 77 (7): 4216-4220 (1980)). Then, the transformed cells are exposed to increasing levels of methotrexate. This causes the synthesis of multiple copies of the DHFR gene and, concomitantly, multiple copies of another DNA present in the expression vector, such as the DNA encoding the neurturin protein. Promoter The expression and cloning vectors of the present invention will typically contain a promoter which is recognized by the host organism and is operably linked to the nucleic acid sequence encoding the product of the neurturin protein. The promoters are untranslated sequences located upstream (5 ') of the start codon of a structural gene (generally within about 100 to 1000 bp) that controls the transcription and translation of a particular nucleic acid sequence. The promoters are conventionally grouped into two classes, inducible promoters and constitutive promoters. Inducible promoters initiate increasing levels of transcription of the DNA under their control in response to some changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature. A large number of promoters, recognized by a variety of potential host cells, are known. These promoters are operably linked to the DNA encoding neurturin by removing the source DNA promoter by digestion with restriction enzymes and inserting the desired promoter sequence into the vector. The native neurturin promoter sequence can be used to direct the amplification and / or expression of neurturin DNA. Nevertheless, a heterologous promoter is preferred if it allows for greater transcription and higher yields of the expressed protein, compared to the native promoter and if it is compatible with the host cell system that has been selected for use. Promoters suitable for use with prokaryotic host cells include the beta-lactamase and lactose promoter systems; alkaline phosphatase, a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their nucleotide sequences have been published, which makes it possible for those skilled in the art to link them to the desired DNA sequences, using linkers or adapters as needed to supply any required restriction site. Promoter sequences suitable for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Promoters suitable for use with mammalian host cells are well known and include those obtained from the genomes of viruses such as polyoma virus, avian varicella virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, bovine papilloma virus, avian sarcoma, cytomegalovirus, a retrovirus, hepatitis B virus and more preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include promoters from heterologous mammals, e.g., heat shock promoters and the actin promoter. A promoter currently used in the production of proteins in CHO cells is SRa. See Takebe et al. , Mol. Cell. Biol., 8 (1): 466-472 (1988). Enhancer Element An enhancer sequence can be inserted into the vector to increase the transcription of a DNA sequence encoding a protein of the present invention, by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, which act on the promoter to increase its transcription. The intensifiers are of relatively independent orientation and position. They have been found towards 5 'and towards 3' of the transcription unit. Several intensifying sequences available for mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein and insulin) are known. However, an intensifier from a virus will typically be used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector in a 5 'or 3' position of the neurturin DNA, it is typically located at a site about 5 'of the promoter. Transcription Termination Expression vectors used in eukaryotic host cells (yeast cells, fungi, insects, plants, animals, humans or nucleated cells of other multicellular organisms) will also contain the sequences necessary for the termination of transcription and to stabilize the transcription. MRNA. Such sequences are commonly available from the 5 'regions and occasionally from the 3' untranslated regions of eukaryotic DNA or cDNA. These regions contain segments of nucleotides transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the protein. The construction of suitable vectors containing one or more of the above-listed components together with the sequence coding for the product of the desired neurturin protein is achieved by standard ligation techniques. The isolated plasmids or DNA fragments are broken, sized and re-ligated in the desired order, to generate the plasmids required. To confirm that the correct sequences have been constructed, the ligated mixtures can be used to transform E. coli and the successful transformants can be selected by known techniques, such as ampicillin or tetracycline resistance, as described above. Subsequently, plasmids of the transformants are prepared, analyzed by digestion with restriction endonucleases and / or sequenced to confirm the presence of the desired construct. Vectors that provide transient expression of the DNA encoding a product of the neurturin protein in mammalian cells can also be used. In general, transient expression involves the use of an expression vector that is capable of efficiently replicating in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of the desired protein encoded by the expression vector. Transient expression systems comprising a suitable expression vector in a host cell, allow convenient positive identification of proteins encoded by cloned DNA, as well as the rapid selection of such proteins for their desired biological or physiological properties. Thus, transient expression systems are particularly useful for identifying variants of the protein. Selection and Transformation of Host Cells Host cells (eg, bacterial, mammalian, insect, yeast or plant cells) transformed with the nucleic acid sequences to be used in the expression of a recombinant neurturin protein are also provided by the present invention . The transformed host cell is cultured under the appropriate conditions that allow the expression of the nucleic acid sequence. The selection of suitable host cells and methods for transformation, cultivation, amplification, selection and production of the product and purification thereof are known in the art. See for example, Gething and Sambrook, Nature 293: 620-625 (1981), or alternatively, Kaufman et al., Mol. Cell. Biol., 5 (7): 1750-1759 (1985) or Howley et al. , U.S. Patent No. 4,419,446. The transformed host cell is cultured in a suitable medium and then, optionally, the expressed factor is recovered, isolated and purified from the culture medium (or from the cell, if expressed intracellularly) by appropriate methods known to those skilled in the art. matter. Suitable host cells for the cloning or expression of the present vectors are prokaryotic cells, yeasts or higher eukaryotic cells, as described above. Prokaryotic host cells include, but are not limited to, eubacteria such as Gram-positive or Gram-negative microorganisms, for example E. coli, bacilli such as B. subtilis, pseudomonas species such as P. aeruginosa, Salmonella typhimurium or Srratia marcescens.

Alternatively, methods of in vitro cloning, e.g., PCR or other nucleic acid poimerase reactions are suitable.

In addition to the prokaryotic host cells, eukaryotic microbes such as filamentous fungi or yeast may be suitable hosts for the expression of neurturin protein products. Saccharomyces cerevisiae or common baker's yeast, are the most commonly used lower eukaryotic host microorganisms, but a number of other genera, species and strains are well known and available. Suitable host cells for the expression of the products of the glycosylated neurturin protein are derived from multicellular organisms. Such host cells are capable of carrying out complex processes and glycosylation activities. In principle, any culture of higher eukaryotic cells can be used, regardless of whether. such a culture involves vertebrate or invertebrate cells, including plant cells and insects. The propagation of vertebrate cells in culture (tissue culture) is a well-known procedure. Examples of useful mammalian host cell lines include, but are not limited to, monkey kidney CV1 cell lines transformed by SV40 (COS-7), human embryonic kidney line (293 or 293 cells subcloned to grow in culture). suspension), lactating hamster kidney cells and Chinese hamster ovary cells. Other suitable mammalian cell lines include, but are not limited to, HeLa cells, murine L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK cells or HaK hamster cell lines. The bacterial cells are similarly useful as host cells suitable for the present invention. For example, the various strains of E. coli (e.g., HB101, DH5a, DH10 and MC1061) are host cells well known in the field of biotechnology. Several strains of Strptomyces spp may also be used. Currently preferred host cells for the production of neurturin proteins are bacterial cells (e.g., Escherichia coli) and mammalian cells (such as Chinese hamster ovary cells, COS cells, etc.). The host cells are transfected and preferably transformed with the expression or cloning vectors described above and cultured in a conventional nutrient medium. The medium can be modified, as appropriate, to induce promoters, select transformants or modify the genes encoding the desired sequences. Transfection and transformation are performed using standard techniques, which are known to those skilled in the art and which are selected as appropriate for the host cells involved. For example, for mammalian cells without cell wall, the calcium phosphate precipitation method can be used. Electroporation, microinjection and other known techniques can also be used. Culturing the Host Cells The transformed cells used to produce the proteins of the present invention are cultured in suitable media. The 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), pH-regulating solutions (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such gentamicin), trace elements (defined as inorganic compounds normally present at final concentrations of a micromolar range) and glucose or other energy source. Other supplements may also be included, at appropriate concentrations, as will be observed by those skilled in the art. Suitable culture conditions such as temperature, pH and the like, are also known to the technicians in the art for use with the selected host cells. It is also possible that the product of the neurturin protein is produced by homologous recombination or by recombinant production methods using control elements introduced into the cells that already contain DNA encoding neurturin or GDNF. Homologous recombination is a technique originally developed to target genes to introduce or correct mutations in transcriptionally active genes (Kucherlapati, Prog. In Nucí. Acid Res. And Mol. Biol. 36: 301 (1989)). The basic technique was developed as a method to introduce specific mutations in specific regions of the mammalian genome (Thomas et al., Cell., 44: 419-428, 1986; Thomas and Capecchi, Cell., 51: 503-512, 1987; Doetschman et al. , Proc. Nati Acad. Sci., 85: 8583-8587, 1988) or to correct specific mutations in defective genes (Doetschman et al., Nature, 330: 576-578, 1987). Exemplary homologous recombination techniques are described in U.S. Patent No. 5,272,071 (European Patent EP 91 90 3051, European Publication No. 505 500; PCT Publication / US 90/07642, International Publication No. WO 91/09955) whose description is incorporated herein by reference. Through homologous recombination, the DNA sequence to be inserted into the genome can be directed towards a specific region of the gene of interest, binding it to the target DNA. Target-directed DNA is a DNA that is complementary (homologous) to a region of genomic DNA. Small pieces of target DNA that are complementary to a specific region of the genome are brought into contact with the parent strand during the DNA replication process. It is a general property of DNA that has been inserted into a cell, hybridized and therefore recombined with other pieces of endogenous DNA through the shared homologous regions. If this complementary filament binds to an oligonucleotide containing a different mutation or sequence of DNA, it is also incorporated into the newly synthesized filament as a result of recombination. As a result of the correction tests function, it is possible that the new DNA sequence serves as a template. Thus, the transferred DNA is incorporated into the genome. If the sequence of a particular gene is known, such as the nucleic acid sequence of a product of the neurturin protein, the pre-pro-sequence or the expression control sequence, which is a DNA fragment complementary to a selected region of the gene can be synthesized or obtained in some other way, for example by appropriate restriction of the native DNA at specific recognition sites that bind to the region of interest. This fragment serves as a sequence targeted to the target at the time of insertion into the cell and will hybridize to its homologous region within the genome. If this hybridization occurs during DNA replication, this DNA fragment and any additional sequence attached to it will act as an Okazaki fragment and will be incorporated into the newly synthesized strand of DNA. In the present invention, attached to these target-directed DNA fragments are regions of DNA that can interact with the expression of a product of the neurturin protein. For example, a promoter / enhancer element, a suppressor or an exogenous transcription regulatory element is inserted into the genome of the host cell, in sufficient proximity and orientation to influence the transcription of the DNA encoding the product of the neurturin protein. The control element does not code for neurturin. it controls a portion of the DNA present in the genome of the host cell. Thus, expression of the protein can be achieved not by transfection of DNA encoding the neurturin protein product gene itself, but by the use of targeting DNA (which contains regions of homology with the endogenous gene of the neurturin gene). interest) coupled with regulatory DNA segments that provide the endogenous gene sequence with recognizable signals for the transcription of a product of the neurturin protein. In accordance with the present invention, homologous recombination methods can also be used to modify a cell containing a gene of the product of the transcriptionally silent neurturin protein, to produce a cell that expresses the product of the neurturin protein. Other aspects and advantages of the present invention will be understood by taking into account the following illustrative examples. Example 1 refers to the effects of administration of the neurturin protein product on hair cells, in an explant cochlea culture system. Example 3 refers to the effects of administration of the neurturin protein product on auditory neurons of the inner ear (spiral ganglion neurons), in a dissociated cell culture generated from the cochlea. EXAMPLES EXAMPLE 1 The product of the neurturin protein protects the cochlear hair cells against ototoxicity. MATERIALS The materials used for the following Example were obtained as follows. Corti Organ Dissection Solution: Dulbecco Phosphate Regulatory Saline Solution (PBS); lx, without calcium chloride, with magnesium chloride. Cat. # 14190-136, Gibco BRL), containing 1.5 g / 1 of D-Glucose (Dextrose, Cat. # 15023-021, Gibco BRL). Culture Medium Explantar for the Organ of Corti: 1. Eagle's Medium Modified by Dulbecco with High Glucose Content (DMEM, 1 X, with L-glutamine, without Sodium Pyruvate, Cat. # 11965-084, Gibco BRL). 2. D-Glucose 0.15 g / 100 ml (Dextrose, Cat. # 15023-021, Gibco BRL). 3. Supplement N-2 at 1% (100 X, Cat. # 17502-030, Gibco BRL). 4. 100 Units / ml of Penicillin G, potassium (Penicillin, Cat. # 21840-020, Gibco BRL). Methods Preparation of the Medium The DMEM medium was supplemented with 1% N-2 supplement and D-glucose was added to a final concentration of 1.5 g / 1. Penicillin was added at 100 units / ml. After mixing, the medium was filtered and maintained at 4 ° C. The medium was prepared just before use in order to minimize variations between experiments. Plastic pipettes and containers were used throughout the experiment to minimize protein adsorption. Neurturin Protein Product Solutions The recombinant human neurturin protein products were prepared in the form of 1 mg / ml solutions in D-PBS (phosphate buffer saline prepared with distilled water) containing 5% bovine serum albumin. The solutions were stored at -85 ° C in aliquots. Serial dilutions (0.1, 1, 10, 50, 100 ng / ml in normal culture medium) were prepared in 96-well microplates. 10 microliters of the concentrated neurturin protein product was added by a factor of ten to the explant culture medium of the organism of Corti containing ototoxins or not (control) (90 μl). The control cultures received normal medium (10 μl). Neurturin protein product treatments were initiated on the day of inoculation. On the second day, the media was switched to media containing the ototoxins alone, together with neurturin or without both (control). Dissection Instruments and Culture Plates 1. 4"and 5" dissection forceps and 4"dissection scissors were used from Roboz Surgical, Washington, DC, USA 2. Sterile 96-well Falcon microplates were used. of flat bottom, Cat. # 3072), plastic materials for tissue culture and polypropylene centrifuge tubes, from Becton-Dickinson, Lincoln Park, New Jersey, USA.

Ototoxins and Related Reagents 1. Neomycin solution (Cat. # N1142, Sigma, St. Louis, MO), was used at a final concentration of 0.6 mM (A fresh solution was prepared for each experiment, mixing 90 μl of neomycin at 1 mg / ml with 1410 μl of medium). 2. Cisplatin (Platinol-AQ, Cat. #NDC 0015-3220-22, Bristol-Myers Squibb Laboratories, Princeton, New Jersey, USA). It was used at a final concentration of 25 μg / ml (a fresh solution was prepared for each experiment, mixing 52.5 μl of cisplatin at 1 mg / ml, with 1447.5 μl of medium). 3. Triton X-100 (t-octylphenoxypolyethoxyethanol, Cat. # X-100, Sigma, St. Louis, MO). 4. Phalloidin (labeled FITC, Cat. # P-5282, Sigma, St. Louis, MO). 5. Vectashield (Mounting Medium, Cat. # H-1000, Vector, Burlingame, CA). Preparation of Explantar Cultivation of the Rat Corti Organ Explants were obtained from the Corti organ of Wistar P3-P4 rats. The rats were decapitated, the lower jaw was cut and the skin was removed. The temporal bone was collected in dissection solution, the otic capsule was exposed and the bone-cartilaginous cochlear capsule was separated from the temporal bone. Once the cochlea was released, it was transferred to another Petri dish with dissection solution for further dissection. Intact Corti organs were obtained using fine forceps to support the central nerve VIII and remove it, then the membrane of the vascular tape was carefully removed, starting from the apex or base. Subsequently the organ of Corti was transferred to a 35 mm Petri dish containing cold PBS supplemented with glucose and ready to be grown. Cochlear Explant Cultivation Procedure Cochlea explants were grown in 96-well microplates without coating. A single organ of Corti was placed in a well and kept floating in the middle. The explants were kept in normal medium for 24 hours (90 μl / well). The neurturin protein solution (10 μl) was added to the "treated" cultures and 10 μl of medium was added to the controls. After 24 hours of incubation, the media were changed and the explants were exposed to medium containing an ototoxin (90 μl), with neurturin protein solution (10 μl) or without it (control). The cultures were incubated for an additional 3 days. Then, the explants were fixed with 4% paraformaldehyde in 0.1 M D-PBS for 30 minutes at room temperature and processed for immunostaining. FITC-Faloidin Staining of Hairy Cells To identify and count the hair cells of the organ of Corti, a method of direct immunostaining was used to mark the actin naturally present within the boundaries of the stereocilia of the hair cells. The explants were washed three times with D-PBS (200 μl per well) and permeabilized with 1% Triton X-100 in D-PBS for 15 minutes at room temperature. After three washes with D-PBS, the explants were incubated with labeled FITC-phalloidin (1:60 stock, 50 μl / well) for 45 minutes at room temperature. The plates were covered with an aluminum foil since phalloidin is sensitive to light. After three more washes with C-PBS, the labeled explants were placed in a drop of glycerol on a microscope slide, covered with a coverslip and sealed with nail polish. The explants were observed under a Nikon Diaphot-300 inverted fluorescence microscope, using FITC filters and fluorescence optics. Determination of the Number of Hair Cells For each experimental point, 2 to 4 cochleae were used. In each cochlea, the number of hair cells was counted in 2-3 cuts of 175 mm in length each. Only sections of the middle part of the cochlea were analyzed. Each experiment was repeated several times. The number of hair cells in the control and in the cultures treated with cisplatin or with neomycin, was generated by analyzing 40 cochlea per point. RESULTS The hair cells in the floating explant cultures did not die during the experimental period of four days. So that, the number of cells stained with phalloidin presented at the end of the 4-day experimental period, in the absence of ototoxins and with the treatments, was 105.4 ± 6.9 (n = 28). The ototoxins added to the explants on the second day of culture, caused a significant loss of the number of hair cells found after 4 days in vi tro. Exposure to 35 μg / ml of cisplatin 24 hours after the start of the culture caused the loss of approximately 80 percent of the hair cells: only 20.8% ± 4.6 (n = 21) of the initial hair cell number survived (Table 1) and after exposure to 0.8 mM neomycin, only 5.9% ± 4.7 (n = 23) of the hair cells survived (Table 2). There was a marked difference in the morphology of the organs of Corti between these two treatments: while the treatment with neomycin caused the almost complete loss of the hair cells, those that remained were still organized in the typical structure of four rows (3 rows of outer hair cells and a row of internal hair cells). On the other hand, treatment with cisplatin caused a marked alteration of the four-row structure and the surviving cells were located at random. In cultures that received neurturin at the time the culture started (pretreatment), a significant number of hair cells survived during the 3 days of exposure to ototoxins (from day 2 to day 4). In cultures exposed to cisplatin, treatment with neurturin concentrations as low as 0.1 ng / ml, caused an increase in cell survival of 21% (untreated cultures) to 35%. Maximum protective activity was achieved with 1 ng / ml neurturin (50% survival) (Table 1). In cultures exposed to neomycin, neurturin at 0.1 ng / ml increased the number of hair cells from 6 to 22%; the maximum activity of neurturin (22% survival) was observed with 10 ng / ml of neurturin (Table 2). Treatment with neurturin retained the four-row morphology in cultures treated with neomycin, but did not prevent the alteration caused by cisplatin.

TABLE 1 Effect of Neurturin on Cochlear Hair Cells Exposed to Cisplatin Survival of hair cells (% with respect to untreated cells) Cisplatin only 20.8 ± 6.9 n = 28 (35 μg / ml) Cisplatin + Neurturin 35.5 + 9.1 n 0.1 ng / ml Cisplatin + Neurturin 50.0 ± 13.8 n = 5 1 ng / ml Cisplatin + Neurturin 37 ± 6.1 n = 7 10 ng / ml Cisplatin + Neurturin 40.8 ± 5.3 n = 4 50 ng / ml Cisplatin + Neurturin 46 ± 10.8 n = 10 100 ng / ml Neurturin was introduced to the explant cultures on the day of the inoculum. Cisplatin (35 μg / ml) was added 24 hours later and the cultures were incubated for an additional 3 days. The hair cells were stained with FITC-phalloidin. The number of hair cells in the middle portion of the cochlea was counted in 2-3 cuts of 175 μm each. The results were expressed as a percentage of hair cells present with respect to untreated cultures after 4 days in vi tro (105.4 + 6.9; n = 28) Each number is the average ± SD of n cochlea. TABLE 2 Effect of Neurturin on Cochlear Hair Cells Exposed to Neomycin Survival of hair cells (% with respect to untreated cells) Neomycin alone 5.9 ± 4.7 n = 23 (0.6 μg / ml) Neomycin + Neurturin 21.6 ± 3.1 n 0.1 ng / ml Neomycin + Neurturin 19.0 ± 3.3 n = 3 1 ng / ml Neomycin + Neurturin 21.6 ± 5.2 n 10 ng / ml Neomycin + Neurturin 17.4 ± 3.9 n = 3 50 ng / ml Neomycin + Neurturin 17.0 ± 1.3 n 100 ng / ml Neurturin was introduced to the explant cultures on the day of the inoculum. Neomycin (35 μg / ml) was added 24 hours later and the cultures were incubated for an additional 3 days. The hair cells were stained with FITC-phalloidin. The number of hair cells in the middle part of the cochlea was counted in 2-3 cuts of 175 μm each. The results are expressed as the percentage of hair cells present with respect to the untreated cultures after 4 days in vitro (105.4 ± 6.9, n = 28). Each number is the average ± SD of n cochlea. EXAMPLE 2 Recombinant Production of a Neurturin Protein Product in E. coli Exemplary neurturin protein products such as those described in the Figures were expressed as E. coli. Complementary, overlapping oligonucleotides comprising the coding nucleotide sequence (e.g., Figure 3) were synthesized in such a way that the codons used were optimized for expression in E. coli. The oligonucleotides were paired and used as templates for PCR procedures, as described in RCP Technology, Principles and Applications for DNA Amplification, Henry A. Erlich, ed., Stockton Press, NY, 1989- (Chapter 6, Using PCR to Engineer DNA) whose description is incorporated herein by reference. The product of the PCR reaction was the full-length neurturin gene. This DNA fragment was cloned into an expression vector for expression in E. coli. After verification of the DNA sequence, the expression plasmid was transformed into a host strain of E. coli.

EXAMPLE 3 The Product of Neurturin Protein Promotes Survival of Internal Ear Auditory Neurons (Spiral Ganglion Neurons) and Protects Against Ototoxins MATERIALS The materials used in the following Example can be obtained as follows. Medium Eagle Cell Culture Media Modified by Dulbecco with High Glucose Content (DMEM; # 11965-092), Ham's F12 medium (F12; # 11765-021), supplement of medium B27 (# 17504-010), penicillin / streptomycin (# 15070-014), L-glutamine (# 25030-016), Phosphate Regulatory Saline Solution Dulbecco (D-PBS; # 14190-052), murine laminin (# 23017-015), bovine serum albumin and fraction -V (- # 110-18-017), all are from GIBCO / BRL, Grand Island, NY. Heat inactivated horse serum is from HyClone, Logan, Utah. Poly-L-ornithine hydrobromide (P-3655), bovine insulin (1-5500), human transferrin (T-2252), putreccin (P-6024), progesterone (P-6149) and sodium selenite (S-9133) are from Sigma Chemical Company, Saint-Louis, MO.

Papain, deoxyribonuclease I (DNAase) and ovalbumin (papain dissociation system) come from Worthington Biochemicals, Freehold, NJ. Sterile 96-well Falcon microplates (# 3072), plastic materials for tissue culture and polypropylene centrifuge tubes, from Becton-Dickinson, Oxnard, CA. Nitex 20 μm nylon mesh (# 460) was used from Tetko, Elmsford, NY. Dissection forceps of 4"and dissection scissors of 4" were used from Roboz Surgical, Washington, D.C., USA. Antibodies and Related Reagents The rabbit polyclonal antibody against the Neuronal Specific Enolase (EEN) is Chemicon (# AB951), the serum of biotinylated rabbit IgG (# BA-1000) and the avidin / biotin complex conjugated with peroxidase ( ABC Elite; PD-6100 package) are. by Vector Laboratories, Burlingame, CA. The 3 ', 3' -diaminobenzidine is from Cappel Laboratories, West Chester, PA. The blocking solution of superblocks in PBS - (# 37515) is Pierce, Rockford, IL. Triton X-100 (X100), Nonidet P-40 (N6507) and hydrogen peroxide (30%, v / v; H1009) are from Sigma. All other reagents were obtained from Sigma Chemical Company (Saint-Louis, MO), unless otherwise specified. Ototoxins Cisplatin (Platinol-AQ, #NDC 0015-3220-22) is from Bristol-Myers Squibb, Princeton, NJ; Sodium salicylate is from J.T. Baker, Phillipsburg, NJ, (# 3872-01) and neomycin is from Sigma (# N1142).

METHODS Preparation of Media A basal medium was prepared in the form of a 1: 1 mixture of DMEM and F12 medium and supplemented with supplement B27, added in the form of a concentrated solution by a factor of 50. Supplement B27 consists of biotin, L -carnitine, corticosterone, ethanolamine, D (+) - galatose, reduced glutathione, linoleic acid, linolenic acid, progesterone, putrecine, retinyl acetate, selenium, T3 (triiodothyronitrone, DL-alpha-tocopherol, vitamin E) , DL-alpha-tocopherol acetate, bovine serum albumin, catalase, insulin, superoxide dismutase - and transferrin. L-glutamine was added to a final concentration of approximately 2 mM, penicillin to approximately 100 IU / 1 and streptomycin to approximately 100 mg / 1. Heat inactivated horse serum was added to a final concentration of approximately 2.5%, D-glucose was added to a final concentration of approximately 5 g / 1, HEPES buffer was added to a final concentration of approximately 20 M, insulin was added bovine at a concentration of approximately 2.5 mg / ml and human transferrin was added to a final concentration of approximately 0.1 mg / ml. After mixing, the pH was adjusted to approximately 7.3 and the medium was maintained at 4 ° C. The media were prepared fresh just before use, in order to minimize variations between experiments. Pipettes and plastic containers were used throughout the experiment to minimize protein adsorption. Product Solutions of Neurturin Protein Products of the purified recombinant neurturin protein (eg, Figures 1 and 3) were prepared as 1 mg / ml solutions in D-PBS (phosphate buffer saline prepared with distilled water) containing 5% of bovine serum albumin. The solution was stored at -85 ° C in aliquots. Serial dilutions were prepared in 96-well microplates. Ten microliters of concentrated neurturin protein product were added by a factor of ten to cell cultures containing culture medium (90 μl). The control cultures received D-PBS with 5 percent albumin (10 μl). Neurturin protein product treatments were added to the cultures one hour after the cells were inoculated or 24 hours later, alone or together with the ototoxins. Ototoxin preparations Neomycin alone was added from a stock solution (approximately 10 M) to 10 μl per well, to obtain a final concentration of approximately -4 M). The cisplatm was diluted with the culture medium of the stock solution (1 mg / ml) until a solution of 20 μg / ml was obtained and added at a rate of 10 μl per well, to obtain a final concentration of 2 μg / ml. Sodium salicylate was prepared from a powder to obtain a 1 M stock solution in PBS and then diluted in the culture medium to 100 mM, which produced a final concentration of 10 mM when added at a rate of 10 μL. / well to the crop. Crop Substrate In order to favor the optimal union of the cells of the spiral ganglion to the substrate and its growth, the surface of the microplates (the substrate of the culture) was modified by a sequential coating with poly-L-ornithine followed by laminin, in accordance with The following procedure. The surfaces of the plates were completely coated with a sterile 0.1 mg / ml solution of polyornithine in boric acid 0.1 M (pH 8.4) for at least one hour at room temperature, followed by a sterile wash with Super-Q water. Then, the water wash was aspirated and 10 μg / ml of murine laminin solution in PBS was added and incubated at 37 ° C for two hours. These procedures were performed just before using the plates, in order to ensure the reproducibility of the results.

Preparation of Rat Spiral Ganglion Cell Cultures Wistar rats were injected at three to four weeks of age (obtained at son Laboratories, Bar Harbor, Maine) with an overdose of the following solution: ketamine (100 mg / ml); xylazine (20 mg / ml) and acopromazine maleate (910 mg / ml) at 3: 3: 1 ratios. Then, the rats were sacrificed by decapitation and the temporal bone was dissected with the cochlea and transferred, sterilely, to PBS with 1.5 g / 1 of glucose on ice. A maximum of 30 cochlea per experiment was processed. The cochlea was opened and the organ of Corti was excised with the bone modiol in a sterile 50 ml tube containing 5 ml of dissociation medium (120 units of papain and 2000 units of DNAse in HBSS). The tissue was incubated for 30 minutes at about 37 ° C on a rotary platform shaker at approximately 200 rpm. The dissociation solution was replaced by fresh solution and the incubation was resumed for another 30 minutes. Subsequently, the cells were dispersed by trituration through Pasteur pipettes burnished on fire, screened through a Nitex nylon mesh of 40 μm to discard the undissociated tissue and centrifuged for 5 minutes at 200 xg using an IEC clinical centrifuge. . The resulting cell pellet was resuspended in HBSS containing ovalbumin and approximately 500 units of DNAse, placed on a 4% oval 1 -butanol solution (in HBSS) and centrifuged for approximately 6 minutes at 500 x g. The final pellet was resuspended in approximately 6 ml of culture medium and inoculated at 90 μl / well in the previously coated plates. Immunohistochemistry of Spiral Ganglion Cells Spiral ganglion neurons were identified by immunohistochemical staining for neuronal specific enolase (EEN). Cultures of spiral ganglion cells were fixed for approximately 10 minutes at room temperature with 8% paraformaldehyde in D-PBS, pH 7.4, added at 100 μl / well to the culture medium and then replaced by 100 μl of culture medium. four percent paraformaldehyde for an additional 10 minutes, followed by three washes with D-PBS (200 μl per 6 mm well). The fixed cultures were subsequently incubated in Superblock blocking solution in PBS, containing one percent Nonidet P-40 to increase the penetration of the antibody. The polyclonal rabbit anti-EEN (Chemicon) antibodies were then applied at a dilution of 1: 6000 in the same buffer and the cultures were incubated for two hours at 37 ° C on a rotary shaker. After three washes with D-PBS, antibodies bound to spiral ganglion cells were detected using a goat anti-biotinylated rabbit IgG serum (Vectastain kit from Vector Laboratories, Burlingame, CA) at a dilution of approximately 1: 300. The secondary antibody was incubated with the cells for approximately one hour at 37 ° C and then the cells were washed three times with D-PBS. Subsequently, the secondary antibody was labeled with an avidin-biotin-peroxidase complex diluted 1: 300 and the cells were incubated for approximately 60 minutes at 37 ° C. After three more washes with D-PBS, the labeled cell cultures were reacted for 5 minutes with a 0.1 M Tris-HCl solution, pH 7.4, containing .3 ', 3' -diaminobenzidine- (HC1) 4 0.04% , 0.06% NiCl2 and hydrogen peroxide 0. 02 percent. Determination of the Survival of the Spiral Ganglion Cells After a time in culture (24 hours, 3 days and 4 hours), the cultures of the spiral ganglion cells were fixed, processed and immunostained for EEN in the manner previously described. and the cultures were examined with bright light optics, with an increase of 200x. All the EEN-positive neurons present in the 6 mm wells were counted. The viable spiral ganglion cells are characterized because they have a round body with a size ranging from 15 to 40 μm and are carriers of neuritic processes. Spiral ganglion cells showing signs of degeneration, such as those with vacuolated pericaryas or irregular fragmented neutrites, are excluded from the account (however, most of the degenerating spiral ganglion cells were detached from the culture substrate). The number of cells was expressed as cells / well of 6 mm or as the change factor in relation to the density of control cells. RESULTS Cultures of rat spiral ganglion neurons can be used to demonstrate the effect of the neurturin protein product on survival and protection against ototoxins. Spiral ganglion cells came from rat cochleae three to four weeks old. Then, the dissociated cells were inoculated onto poly-nitrin-laminin-coated microplates at a density of about 1 cochlea per 2 wells, in DMEM / F12 supplemented with supplement B27, 2.5 percent heat-inactivated horse serum, D-glucose, HEPES , insulin and transferrin. The cultures will consist of a mixture of neurons and non-neuronal cells. Preferably, the only neurons present are spiral ganglion neurons and these can be enhanced by the presence of immunoreactivity against EEN. The effect of neurturin protein product administration on the survival and morphological maturation of rat spiral ganglion neurons in culture, as well as on their ability to resist the toxic effects of a known ototoxin, such as cisplatin, was evaluated. Cultures of spiral ganglion cells were treated 24 hours after being inoculated with a product of the recombinant human neurturin protein (varying from 50 ng / ml to 0.1 ng / ml) alone or in combination with cisplatin (35 μg / ml). Twenty-four hours after inoculation, it is expected that there will be no difference in the number of auditory neurons between the control cultures and those treated with neurturin at 1 ng / ml and 10 ng / ml). After an additional 3 days, treatment with neurturin at a concentration of 1 ng / ml was not expected to result in a significant increase in the number of neuronal cells. However, it is expected that there will be a marked trophic effect: neural bodies are larger and fibers longer and more elaborate than in control crops. In cultures treated with 10 ng / ml of neurturin, approximately 70% of neurons present after 24 hours are expected to survive, representing an average increase of 40% over control cultures. The trophic effect is expected to be even greater than in cultures treated with 1 ng / ml of neurturin. Neurturin is also expected to protect the neurons of the spiral ganglia from the toxicity caused by cisplatin. The exposure of the cultures to 5 μg / ml of cisplatin 24 hours after inoculation, can cause the loss of approximately 90% of the initial number (at 24 hours) of neurons after 4 days in culture. When neurturin is added together with cisplatin, the number of neurons found after 4 days is expected to be significantly higher. It is also anticipated that this protective effect of neurturin will be dose-dependent and that approximately 60% of neurons responding to neurturin (approximately 40% of the population of spiral ganglion neurons) is also protected against the toxicity caused by cisplatin. EXAMPLE 4 The Neurturin Protein Product Promotes In Vivo Survival of Cochlear Hair Cells The following Example describes the administration in the inner ear of the neurturin protein product to protect cochlear hair cells against ototoxicity in an animal model. The product of the neurturin protein is introduced into the inner ear through a cannula pushed towards the tympanic scale through a perforation made in the basal contortion of the cochlea. The cannula is connected to an Alzet minipump loaded with the neurturin protein product (50 ng / ml) at a release rate of 0.5 μl / hour for 14 days. Intramuscular injections of cisplatin were initiated two days after cannulation, either at a dose of 1 mg / ml per day for 15 days, or at a dose of 7.5 mg / kg two days at an interval of 5 days. The experiment concluded after 27 days. The hair cells were stained with FITC-phalloidin and their number was determined in the middle part of the cochlea (at least 20% of the middle part). The results are expressed as the percentage of hair cells lost for each individual guinea pig by ear treated with the neurturin protein product (right ear) and the untreated ear (left ear). MATERIALS The materials used in the following Example were obtained in the following manner. Mini-Pump Preparation Materials Vinyl Medical Intubation size V / 4, catalog number BB317-85, from Bolab Products, ((800) 331-7724). Fisher 5-ml plastic pipettes were used. Microlume polyimide intubations, Catalog No. 8004853 OG (Tampa, Florida, USA) were used. The MDX 4-4210 silicone medical product is from Dow Corning Corporation, Midland, MI. The flow moderator of the Alzet osmotic minipump and the osmotic minipump of Alzet, Catalog No. 2002, are from Alza Corp., Palo Alto, CA. Tape (TimeMed tape). Prosil-28, product No. 11975-0, from PCR Incorporated, Gainesville, Florida. The products of the purified neurturin protein were prepared as solutions at 50 ng / ml in D-PBS and 0.1% ASB. The sterile 0.1 methylene blue (Catalog No. M-9140) dissolved in PBS and the mineral oil (Catalog No. 400-5) are from Sigma. Mini Pump Preparation Procedure The vinyl intubation was cut into a section of approximately ten centimeters and placed in a miniature vise. A piece of microlume polyimide tube (7 mm) was placed on the end of the vinyl tube. Silicone was mixed by adding approximately 10 parts of base and one part of curing agent. A droplet was placed in the opening of the vinyl tube using a thin probe and the microlume tube was pushed inside the vinyl, leaving a 3.75 mm extension out of the vinyl tube. Using a drop of silicone in the probe, a small pellet was created around the microlume tube, 0.5 mm from the tip and left to dry overnight. The diameter of a 5 ml pipette was increased by applying three concentric layers of tape along the length of the pipette. A constant opening was left where the pipette remained uncoated. The tube V / 4 was wrapped around the pipette and the turns were adjusted so that both ends were loose and there was a continuous contact between all the turns. Two thin strips of tape were aligned with the edges of the pipette tape, to secure the helical in place. Two lines of supergoma were applied uniformly in the turns of the helical. After drying for a minimum of one hour, the loose ends were aligned approximately parallel to the pipette and secured in place with a strip of tape. A supergome drop was applied to secure the tube to the turns of the helical. It was allowed to dry overnight, the tape was removed and the helical was removed from the pipette. A flow moderator was inserted in one of the loose ends and secured with a drop of supergome. The helical was flooded with 1% Prosil-28 in water, rinsed well with water and then flooded with 70% ethanol. The ethanol was removed by means of a syringe or with air vacuum. The helicals were left in a vacuum desiccator for at least 30 minutes and kept overnight in a sealed, sealed desiccator, followed by gas sterilization. During the loading procedure, the helical device was kept as horizontal as possible to prevent the movement guided by the gravity of the neurturin protein, oil and dye product liquids. The formation of air bubbles in the pump or in the helicals was avoided. The bomb was submerged in sterile PBS and incubated overnight at 37 ° C. The loading of a pump with the methylene dye was carried out holding the pump in a vertical position. A syringe loaded with the dye was inserted and the dye was injected until the pump was flooded. It was avoided to inject any bubble of air in the pump. A small piece of sterile V / 4 tube was placed in the flow moderator. The neurturin protein product was loaded at a concentration of 50 ng / ml in PBS + 0.1% ASB, in a total volume of 230 μl, up to approximately 10 mm from the tip of the cannula, using a syringe connected to the V / 4 tube. For the vehicle control experiments, the same volume of PBS + 0.1% ASB was loaded into the pumps. The small piece of V / 4 tube was removed. Then mineral oil was loaded into the helical device with a syringe, so that an air space of 2 mm and 7 mm of mineral oil interposed between the liquid of the pump and the fluid line (infusion fluid) remained. A flow moderator was inserted completely into the pump. INSERTION OF THE PUMP IN THE INTERNAL EAR Materials: Adhesive adhesive for tissue-cyanoacrylate, is from Vetbond Tissue Adhesive, 3M Animal Care Products, St. Paul, MN. ESPE Durelon carboxylate cement, Catalog No. 03828, is from ESPE-Premier Sales Corp., Norristown, PA. Methyl methacrylate is from Lang Jet Acrylic, Lang Dental MFG, Co., Wheeling, IL. The dissection instruments are from Roboz Surgical. Xylazine, ketamine and buprenorphine were used. Lubricating Ophthalmic Ointment (AKWA Tears) is from Akorn Inc., Abita Springs, LA. The 2% xylocaine, Catalog No. NDC 0186-0160-01, is from ASTRA. Medical-grade silicone petroleum jelly, article No. 51,300, is from Unimed. Durelon Pulver carboxylate powder cement, Catalog No. De-8229, is from ESPE, Seefeld. The sulfate ointment (Bacitracin zinc-neomycin, Catalog No. 0168-0012-31) is from Fougera. Procedure: Albino guinea pigs (250-350 g) were anaesthetized with a mixture of xylazine 20 mg / kg, ketamine 40 mg / kg and buprenorphine 0.05 mg / kg. The area of the right ear was shaved caudally, starting approximately 2 cm from the apex, 4-5 cm posterior to the scapula and posauricularly. The shaved area was washed with betadine. Lubricant ophthalmic ointment was applied in both eyes. Xylocaine was injected subcutaneously into the tissue to be cut. Using an aseptic technique, a post-auricular incision was made. With a fine needle, a perforation was made in the ampulla to expose the middle ear cavity and visualize the cochlea. A small hole was drilled manually into the bony wall of the basal contortion, below the round window, using a fine needle. The tip of the cannula was inserted into the perforation until the drop of silicone was resting against the bone, which places the tip of the cannula approximately halfway on the scale of the tympanic canal. A drop of cyanoacrylate was placed in the perforation of the ampoule. Carboxylate cement was applied around the cannula on the cyanoacrylate. Once the cement hardened, the placement was confirmed and the rest of the perforation was covered with carboxylate cement over a layer of silicone petrolatum. A subcutaneous pocket was made between the scapulae to accommodate the pump, which was then inserted there. The subcutaneous pouch was rinsed once with 3 ml of a nitrofurazone solution dissolved in sterile PBS and then filled with 3 ml of sterile PBS plus 1% gentamicin to prevent infection. The incision was closed with wound clips and then powdered nitrofurazone was applied around the wound. STRENGTH Materials Cisplatin (Platinol-AQ), Catalog No. NDC 0015-3220-22, from Bristol-Myers Squibb Laboratories, Princeton, NJ. Procedure The cisplatin (intraperitoneal) injections started two days after the minipump implant.

Two application paradigms were used: either two 7.5 mg / kg injections at 5-day intervals, or 1 mg / kg daily for 15 days. PERFUSION After four weeks, the guinea pigs underwent deep anesthesia with a mixture of xylazine and ketamine and they underwent a transcardial infusion of ice-cold PBS followed by 4% paraformaldehyde in ice-cold PBS. The temporal bones were removed and the cochlea was placed in 4% paraformaldehyde for postfixation overnight at 4 ° C. STAINING The methods of surface preparation and phalloidin staining were used to stain hair cells. The bone cochlea was opened with a fine needle or a # 11 blade. The vascular tape was removed using fine forceps. In a petri dish filled with PBS, the basement membrane was carefully dissected out of the modiolus, using fine needles. Care was taken to remove it intact. The phalloidin staining procedure is similar to that performed for in vitro explants, with the following changes: permeabilization was performed for 20-30 minutes and phalloidin was added for 90 minutes. Parts of the apex, medial contortion and basal were mounted on 60 x 22 glass slides. One drop of VECTASHIELD mounting medium was added and the samples were covered with 22 x 22 mm coverslips and sealed with nail polish, to prevent evaporation. DATA ANALYSIS Each cochlea was examined under a microscope with a set of FITC filters. Eight segments with the highest loss of hair cells from the middle part of the basement membrane were selected and photographed using an attached computer printer. Hairy cell counts were made manually using the photographs. In each animal, the loss of hair cells from the left ear (as control, i.e., without infusion of the neurturin protein product) was compared with the loss of hair cells from the right ear (infused with the product of the neurturin protein). RESULTS Cisplatin injections caused a significant loss of hair cells in the cochlea. This loss, in sections of the mean contortion analyzed in the left ears of guinea pigs injected with cisplatin at 1 mg / kg per day for 15 days, is expected to be 20 to 50%. Likewise, in guinea pigs injected with cisplatin at a dose of 7.5 mg / kg for two days, instead of 1 mg / kg daily, a loss of approximately 40% of the hair cells in the left ear was anticipated. The introduction of neurturin in the right inner ear of each guinea pig is expected to result in a significant reduction in hair cell loss. In animals implanted with a mini-pump filled with vehicle instead of the neurturin protein product, no differences are expected in the number of hair cells found in the left ear (untreated ear) and in the right ear (implanted ear). EXAMPLE 5 Neurturin Protein Product Injections for Promote In Vitro Cochlear Hairy Cell Survival The following Example describes the use of neurturin protein products to protect hairy cochlear cells against ototoxicity in an animal model, when applied to the middle ear. The neurturin protein product is introduced into the right middle ear by a single injection through the tympanic membrane, at a concentration of 1 mg / ml in PBS + 1% ASB in a volume of 125-135 μl. Intramuscular injections of cisplatin were initiated one day after the injection of the neurturin protein product at 7.5 mg / kg, twice, at a 5-day interval. The experiment was completed three days after the second injection of cisplatin. The hair cells were stained with FITC-phalloidin and their number was determined in the middle part of the cochlea (at least 20% of the part of the middle seizure). These results are expressed as the percentage of hair cell loss for each individual guinea pig per ear treated with the neurturin protein product (right ear) and the untreated ear (left ear). MATERIALS The materials used in this experiment are the same as those used in Example 4. Procedure: Albino guinea pigs (600 to 700 g of weight) were anesthetized with a mixture of xylazine 10 mg / kg, ketamine 40 mg / kg and buprenorphine 0.05 mg / kg. Under a surgical microscope, a perforation was made in the tympanic membrane of the right ear by inserting a 27-gauge needle into the membrane. In another location of the tympanic membrane, the product of the neurturin protein (at a concentration of 1 mg / ml in PBS + 1% ASB) was injected into the middle ear cavity, so that the entire cavity would be full (125- 135 μl). A few animals were injected only with vehicle (PBS + 0.1% ASB) instead of the neurturin protein product. The next day, an intramuscular injection of cisplatin was applied (7.5 mg / kg). Five days later, a second injection was applied at the same concentration. Three days later (8 days of total experimental period), the animals were sacrificed, the tissues were fixed and the cochleae were analyzed in the manner described in Example 4. RESULTS At eight days, the guinea pigs injected with cisplatin are expected to show a significant loss of hair cells in the cochlea. In the left ears, which did not receive the product of the neurturin protein, the loss of hair cells in the average convulsion of the cochlea is expected to be 35 to 50%. The injection of neurturin protein product into the right middle ear cavity, at 1 mg / ml, is expected to significantly reduce this loss: from approximately 16 to 30%. Guinea pigs who received vehicle injections in the right ear, instead of the neurturin protein product, are not expected to demonstrate a difference in the number of hair cells between the right (treated) ear and the left (untreated) ear. It is expected that those skilled in the art can devise numerous modifications and variations of the practice of the present invention by taking into account the above description of the presently preferred embodiments thereof. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANTS: Magal, Ella Delaney, John M. (ii) TITLE OF THE INVENTION: METHOD TO PREVENT AND TREAT HEARING LOSS USING A NEURTURINE PROTEIN PRODUCT (iii) SEQUENCE NUMBER: 5 (iv) POSTAL ADDRESS: (A) RECIPIENT: Amgen Inc. (B) STREET: One Amgen Center Drive (C) CITY: Thousand Oaks ( D) STATE: California (E) COUNTRY: USA (F) POSTAL CODE: 91320-1789 (v) COMPUTER LEGIBLE FORM (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reeléase # 1.0, Version # 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) NUMBER OF APPLICATION: US (B) DATE OF SUBMISSION: JUNE 29, 1998 ( C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 60/054184 (B) SUBMISSION DATE: 30-JULY-1997 (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Curry, Daniel R. (B) RECORD NUMBER: 32, 727 (C) REFERENCE NUMBER / CEDULA: A-444 (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 102 amino acids (B) TYPE: amino acids (C) TYPE OF CHAIN: simple '(D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1: Wing Arg Leu Gly Wing Arg Pro Cys Gly Leu Arg Glu Leu Glu Val Ara 1 5 10 15 Val Ser Glu Leu Gly Leu Gly Tyr Ala Ser Asp Glu Thr Val Leu ° he 20 25 30 Arg Tyr Cys Wing Gly Wing Cys Glu Wing Wing Wing Arg Val Tyr Asp Leu 35 40 5 Gly Leu Arg Arg Leu Arg Gln Arg Arg Arg Leu Arg Arg Glu Arg Val 50 55 60 Arg Ala-Gln Pro Cys Cys Arg Pro Thr Wing Tyr Glu Asp Glu Val Ser 65 70 75 80 Phe Leu Asp Ala His Ser Arg Tyr His Thr Val His Glu Leu Ser Wing 85 9o 95 Arg Glu Cys Ala Cys Val 100 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 100 amino acids (B) TYPE: amino acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein - (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Pro Gly Wing Arg Pro Cye Gly Leu Arg Glu Leu Glu Val Arg Val Ser 15 Glu Leu Gly Leu Gly Tyr Thr Ser Asp Glu Thr Val Leu Phe Are Tv 20 25 3Q * U? R Cys Ala Gly Ala Cys Glu Ala Ala lie Arg lie Tyr Asp Leu Glv L «_» 35 40 45 y ^ eu Arg Arg Leu Arg Arg Arg Arg Arg Val Arg Arg Glu Arg Ar R Ar 50 55 60 His Pro Cys Cys Arg Pro Thr Wing Tyr Glu Asp Glu Val Ser Phe Leu 65 70 75 80 Asp Val His Ser Arg Tyr His Thr Leu Gln Glu Leu Ser Wing Are Glu 85 90 55 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 312 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1.309 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 3: ATG GCA CGT CTG GGT GCT CGT CCG TGT GGT CTG CGT GAA CTG GAA GTT 48 Met Ala Arg Leu Gly Ala Arg Pro Cys Gly Leu Arg Glu Leu Glu val 1 5 10 15 CGT GTT CC "GAA CTG GGT CTG GGT TAC GCT TCC GAC GAA ACC GTT CTG 96 Arg Val Ser Glu Leu Gly Leu Gly Tyr Wing Ser Asp Glu Thr Val Leu 20 25 30 TC CGT TAC TGT GCA GGT GCT TGT GAA GCA GCT GCA CGT GTT TAC GAC 144 Phe Arg Tyr Cys Wing Gly Wing Cys Glu Wing Wing Wing Arg Val Tyr Asp 35 40 45 CTG GGT CTG CGT CGC CTG CGT CAG CGC CGT CGC CTG CGT CGC GAA CGT 192 Leu Gly Leu Arg Arg Leu Arg Gln Arg? Rg Arg Leu Arg Arg Glu Arg 50 55 60 GTT CGC GCA CAG CCG TGT TGC CGT CCG ACC GCA TAC GAA GAC GAA GTT 240 Val Arg Wing Glix Pro Cys Cys Arg Pro Thr Wing Tyr Glu Asp Glu Val 65 70 75 80 CC TTC CTG GAC GCT CAC CC CGT TAC CAC ACC GTT CAC GAA CTG TCC 288 Ser Phe Leu Asp Ala His Ser Arg Tyr His Thr Val His Glu Leu Ser 85 90 95 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 103 amino acids (B) TYPE: amino acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 4 Met Ala Arg Leu Gly Ala Arg Pro Cys Gly Leu Arg Glu Leu Glu Val 1 5 10 15 Arg Val Ser Glu Leu Gly Leu Gly Tyr Wing Ser Asp Glu Thr Val Leu 20 25 30 Phe Arg Tyr Cys Wing Gly Wing Cys Glu Wing Wing Wing Arg Val Tyr Asp 35 40 45 Leu Gly Leu Arg Arg Leu Arg Gln Arg Arg Arg Leu Arg Arg Glu Arg 50 55 60 Val Arg Ala Gln Pro Cys Cys Arg Pro Thr Ala Tyr Glu Asp Glu Val 65 70 75 80 Be Phe Leu Asp Ala His Ser Arg Tyr His Thr Val His Glu Leu Ser 85 90 95 Ala Arg His Cye Ala Cys Val 100 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 197 amino acids (B) TYPE: amino acids (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 5: Met Gln Arg Trp Lys Ala Ala Ala Ala Leu Ala Ser Val Leu Cys Ser Ser 1 5 10 15 Val Leu Ser He Trp Met Cys Arg Glu Gly Leu Leu Leu Ser His Arg 20 25 30 Leu Gly Pro Ala Leu Val Pro Leu His Arg Leu Pro Arg Thr Leu Asp 35 40 45 Wing Arg lie Wing Arg Leu Wing Gln Tyr Arg Ala Leu Leu Gln Gly Wing 50 55 60 Pro Asp Wing Met Glu Leu Arg Glu Leu Thr Pro Trp Wing Gly Arg Pro 65 70 75 80 Pro Gly Pro Arg Arg Arg Ala Gly Pro Arg Arg Arg Arg Ala Arg Ala 85 90 95 Arg Leu Gly Ala Arg Pro Cys Gly Leu Arg Glu Leu Glu Val Arg Val 100 105 110 Ser Glu Leu Gly Leu Gly Tyr Ala Ser Asp Glu Thr Val Leu Phe Arg 115 120 125 Tyr Cys Ala Gly Ala Cys Glu Ala Ala Ala Arg Val Tyr Asp Leu Gly 130 135 140 Leu Axg Arg Leu Arg Gln Arg Arg Arg Leu Arg Arg Glu Arg Val Arg A "1S0 155 160 Wing Gln Pro Cys Cys Arg Pro Thr Wing Tyr Glu Asp Glu Val Ser Phe 165 170 175 Leu Asp Ala His Ser Arg Tyr His Thr Val His Glu Leu Ser Ala Arg 180 185 l? 0 Glu Cys Ala Cys Val 195

Claims (40)

1. CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property: 1. Use of a neurturin protein product It will give the manufacture of a composition for the treatment of sensorineural hearing loss of a subject suffering from an inner ear injury.
2. 2. The use according to claim 1, characterized in that the hearing loss is associated with a lesion or degeneration of the neuroepithelial hair cells of the inner ear.
3. 3. The use according to claim 1, characterized in that the hearing loss is associated with a lesion or degeneration of spiral ganglion neurons.
4. 4. The use according to claim 1, characterized in that the product of the neurturin protein is the amino acid sequence established in Figure 1, 2, 4 or 5 (SEQ ID Nos. 1, 2, 3, 4 or 5) or a variant or derivative thereof.
5. 5. The use according to claim 4, characterized in that the product of the neurturin protein has the amino acid sequence in Figure 1 (SEQ ID No. 1).
6. 6. The use according to claim 4, characterized in that the product of the neurturin protein has the amino acid sequence set forth in Figure 4 (SEQ ID Nos. 3 and 4).
7. The use according to claim 4, characterized in that the product of the neurturin protein is [Met-] neurturin.
8. 8. The use according to claim 1, characterized in that the product of the neurturin protein is administered at a dose of about 1 μg / kg / day to about 100 mg / kg / day.
9. The use according to claim 1, characterized in that the product of the neurturin protein is administered by cell therapy or by means of gene therapy, wherein the cells have been modified to produce and secrete the product of the neurturin protein.
10. 10. The use according to claim 8, characterized in that the cells have been modified ex vivo.
11. 11. The use according to claim 8, characterized in that the cells have been modified in vivo.
12. 12. Use of a product of the neurturin protein for the manufacture of a composition for the treatment of injuries or alterations of the vestibular apparatus.
13. 13. The use according to claim 12, characterized in that the injury or alteration results in dizziness, vertigo or loss of balance.
14. The use according to claim 12, characterized in that the product of the neurturin protein is the amino acid sequence set forth in Figure 1, 2, 4 or 5 (SEQ ID Nos. 1, 2, 3, 4 or 5) or a variant or a derivative thereof.
15. 15. The USo according to claim 14, characterized in that the product of the neurturin protein has the amino acid sequence set forth in Figure 1 (SEQ ID No. 1).
16. 16. The use according to claim 14, characterized in that the product of the neurturin protein has the amino acid sequence set forth in Figure 4 (SEQ ID Nos. 3 6 4).
17. 17. The use according to claim 14, characterized in that the product of the neurturin protein is [Met] neurturin.
18. 18. The use according to claim 12, characterized in that the product of the neurturin protein is administered at a dose of about 1 μg / kg / day to about 100 mg / kg / day.
19. The use according to claim 12, characterized in that the product of the neurturin protein is administered by cell therapy or by gene therapy, wherein the cells have been modified to produce and secrete the product of the neurturin protein.
20. 20. The use according to claim 19, characterized in that the cells have been modified ex vivo.
21. 21. The use according to claim 19, characterized in that the cells have been modified in vivo.
22. 22. A neurturin protein product, characterized in that it comprises the amino acid sequence illustrated in Figure 4 (SEQ ID Nos. 3 or 4).
23. 23. The protein according to claim 22, characterized in that the amino acid sequence has a methionine residue at the amino-terminal end.
24. 24. The protein according to claim 22, characterized is modified by the union of one or more polymer portions.
25. 25. The protein according to claim 24, characterized in that the polymer portion is polyethylene glycol.
26. 26. A pharmaceutical composition characterized in that it comprises a neurotrophic protein according to claim 22 in combination with a pharmaceutically suitable carrier.
27. 27. A nucleic acid sequence encoding a product of the neurturin protein, characterized in that it comprises the amino acid sequence illustrated in Figure 4 (SEQ ID Nos. 3 or 4).
28. 28. The nucleic acid sequence according to claim 27, characterized in that it comprises the sequence illustrated in Figure 3 (SEQ ID No. 3).
29. 29. A vector characterized in that it comprises operably regulating elements linked to a nucleic acid sequence according to claim 27.
30. 30. A host cell transformed or transfected with the vector according to claim 29.
31. 31. The host cell according to claim 30, characterized in that it is selected from the group consisting of mammalian cells and bacterial cells.
32. 32. The host cell according to claim 30, characterized in that the cell is suitable for implantation in humans and wherein the cell expresses and secretes the product of the neurturin protein.
33. 33. The host cell according to claim 30, characterized in that the cell is transformed or transfected ex vivo.
34. 34. The host cell according to claim 30, characterized in that the cell is enclosed in a semipermeable membrane suitable for implantation in humans.
35. 35. A method for the production of a neurotrophic factor, characterized in that it comprises the steps of: (a) culturing a transformed or transfected host cell with a nucleic acid sequence encoding a neuratrophic factor comprising the amino acid sequence illustrated in Figure 4 (SEQ ID Nos. 3 or 4), under conditions suitable for the expression of the neurotrophic factor by the host cells; and (b) optionally, isolating the neurotrophic factor expressed by the host cells.
36. 36. The method according to claim 35, characterized in that the nucleic acid sequence comprises the sequence illustrated in Figure 3 (SEQ ID No. 3).
37. 37. The method according to claim 35, characterized in that it also comprises the step of reshaping the isolated neurotrophic factor.
38. 38. The method according to claim 35, characterized in that the host cell is a prokaryotic cell.
39. 39. The method according to claim 35, characterized in that the host cell is a eukaryotic cell.
40. 40. An article for the treatment of nerve damage, characterized in that it comprises: (a) a semipermeable membrane suitable for implantation; and (b) cells encapsulated within said membrane, wherein the cells secrete a neurotrophic factor comprising the amino acid sequence illustrated in Figure 4 (SEQ ID Nos. 3 or 4); wherein the membrane is permeable to the neurotrophic factor and impermeable to materials that damage cells.