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WO1997019693A1 - Procede de traitement des neuropathies sensorielles utilisant un produit proteique du facteur neurotrophique derive d'une lignee de cellules gliales - Google Patents

Procede de traitement des neuropathies sensorielles utilisant un produit proteique du facteur neurotrophique derive d'une lignee de cellules gliales Download PDF

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Publication number
WO1997019693A1
WO1997019693A1 PCT/US1996/018729 US9618729W WO9719693A1 WO 1997019693 A1 WO1997019693 A1 WO 1997019693A1 US 9618729 W US9618729 W US 9618729W WO 9719693 A1 WO9719693 A1 WO 9719693A1
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Prior art keywords
gdnf
protein product
gdnf protein
use according
pharmaceutical composition
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PCT/US1996/018729
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English (en)
Inventor
Qial Yan
Christine Matheson
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Amgen Inc.
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Application filed by Amgen Inc. filed Critical Amgen Inc.
Priority to AU11219/97A priority Critical patent/AU1121997A/en
Publication of WO1997019693A1 publication Critical patent/WO1997019693A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3

Definitions

  • the present invention relates generally to methods for treating sensory neuropathy, by administering glial cell line-derived neurotrophic factor (GDNF) protein product.
  • GDNF glial cell line-derived neurotrophic factor
  • Neurotrophic factors are natural proteins, found in the nervous system or in non-nerve tissues innervated by the nervous system, that function to promote the survival and maintain the phenotypic differentiation of certain nerve and/or glial cell populations (Varon et al., Ann. Rev. Neuroscience, 1 :327, 1979; Thoenen et al., Science, 229:238, 1985). Because of this physiological role, neurotrophic factors are useful in treating the degeneration of such nerve cells and the loss of differentiated function that results from nerve damage. Nerve damage is caused by conditions that compromise the survival and/or proper function of one or more types of nerve cells, including: (1) physical injury, which causes the degeneration of the axonal processes (which in turn causes nerve cell death) and/or nerve eel!
  • neurotoxins such as the cancer and AIDS chemotherapeutic agents cisplatinum 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 Amyorrophic Lateral Sclerosis, which result from the degeneration of specific neuronal populations.
  • the class or classes of damaged nerve cells must be responsive to the factor. It has been established that all neuron populations are not responsive to or equally affected by all neurotrophic factors.
  • NGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • NT-4/5 neurotrophin-6
  • NT-6 Thienen, Trends. Neurosci., 14: 165-170, 1991; Snider, Cell, 77:627-638, 1994; Bot ell, Ann. Rev. Neurosci., 18:223-253, 1995).
  • neurotrophins are known to act via the family of trk tyrosine kinase receptors, i.e., trkA, trkB, trkC, and the low affinity p75 receptor (Snider, Cell, 77:627-638, 1994; Bothwell, Ann. Rev. Neurosci., 18:223-253, 1995; Chao et al., TINS 18:321-326, 1995).
  • NGF is the classic molecule for sensory neurons (Theonen and Barde, Physiol. Rev., 60:1284-335, 1980) and has been demonstrated recently to be effective in the prevention of taxol-induced small fiber neuropathy (Apfel et al., Ann.
  • Glial cell line-derived neurotrophic factor is a recently discovered protein identified and purified using assays based upon its efficacy in promoting the survival and stimulating the transmitter phenotype of mesencephalic dopaminergic neurons in vitro (Lin et al, Science, 260:1130- 1132, 1993).
  • GDNF is a glycosylated disulfide-bonded homodimer that has some structural homology to the transforming growth factor-beta (TGF- ⁇ ) super family of proteins (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).
  • TGF- ⁇ transforming growth factor-beta
  • GDNF neurotrophic efficacy on brainstem and spinal cord cholinergic motor neurons, both in vivo and in vitro (Oppenheim et al., Nature, 373:344-346, 1995; Zurn et al., Neuroreport, 6:113-118, 1994; Yan et al., Nature, 373: 341-344, 1995; Henderson et al., Science, 266:1062-1064, 1994).
  • Sensory neuropathy is a relatively common neurological condition which can occur spontaneously or as a secondary complication of trauma or other diseases such as diabetes mellitus, HIV infection, and cancer chemotherapy. About 66% of diabetes mellitus patients develop sensory neuropathy, and about 1% of HIV-infected patients develop sensory neuropathy every year. For cancer patients, the dose of chemotherapeutic agent used is often limited by the development of toxic neuropathies. Sensory neuropathy can result in neuropathic pain, loss of external sensation, or the loss of protective sensation such as nociceptive, vibratory and proprioceptive perception.
  • Two major somatic sensory pathways serve both exteroception (perception of environmental influences, including pressure, touch, temperature and pain) and conscious proprioception (including vestibular system perception, joint position sense and muscle sensation).
  • Sensory neurons innervating the head reside in cranial ganglia, and they directly project to the sensory nuclei in the brain stem. All primary sensory afferents of the rest of the body have their cell bodies in the dorsal root ganglia.
  • the two different sensory pathways are anatomically separated and have different synapse patterns. The first pathway relays sensations of pain, temperature and crude touch and begins as free nerve endings of small myeiinated and unmyelinated fibers.
  • These small fibers of the first pathway enter the spinal cord via the dorsal root ganglia, lateral to the large myeiinated fibers of the second pathway, and then ascend or descend for one or two segments before synapsing in the dorsal horn and thereafter ascending to the brain.
  • the second pathway consists predominantly of larger myeiinated fibers and relays sensations of light touch, position sense and tactile localization.
  • These large fibers also enter the spinal cord via the dorsal root ganglia, and lie in a position medial to the smaller fibers of the first pathway. Most disorders of the peripheral somatosensory system occur in the axon itself or in the myelin sheath encasing the axon.
  • Mononeuropathies are generally caused by local disease (such as compression entrapment or other trauma, ischemia, tumor or granulomatous infiltration, amyloid deposition, leprosy, etc.).
  • Vascular mononeuritis result from vascular disorders, such as polyarteritis.
  • Symmetric generalized polyneuropathies result from immunologic or metabolic disorders (for example, demyelination or inflammatory neuropathies, diabetes, toxin or drug-induced injury, and metabolic diseases such as uremia or nutritional deficiency syndromes).
  • Acute inflammatory sensory polyneuropathy involves an inflammatory response that is usually associated with a preceding infection, typically one occurring several weeks earlier.
  • the diabetic neuropathies include isolated mononeuropathies and symmetrical polyneuropathies, which generally manifest with distal sensory loss.
  • the cause of diabetic neuropathies is unknown but a metabolic basis has been suggested.
  • Uremic polyneuropathy can be associated with chronic renal insufficiency of any type; the cause is unknown but is presumed to be related to toxins or other metabolites normally excreted by the kidneys.
  • Hypothyroidism is associated with mononeuropathy and symmetrical polyneuropathy. Serum monoclonal gammopathies are also associated with polyneuropathy.
  • Cancer-associated mononeuropathies can be caused by direct compression or infiltration of nerves by tumors; polyneuropathy can be caused by multiple myeloma.
  • Other vascular-related neuropathies (caused by vasculitis or ischemia) are associated with various conditions such as polyarteritis nodosa, rheumatoid arthritis, systemic lupus erythematosus, hypersensitivity angiitis, allergic granulomatosis, Sjogren's syndrome, Wegener's granulomatosis and cranial arteritis.
  • Infectious and granulomatous neuropathies include those associated with Lyme disease, HIV infection, herpes zoster infection and leprosy.
  • the hereditary sensory neuropathies, types I, II and III, are a group of slowly progressive disorders probably caused by inbom errors of metabolism.
  • Toxic neuropathies are caused by pharmaceutical or chemical agents, such as chloramphenicol, dapsone, disulfiram, dichloracetate, ethionamide, gold, glutethimide, hydralazine, isoniazid, lithium, metronidazole-misonidazole, nitrofurantoin, nitrous oxide, platinum, cis-platinum, pyridoxine, sodium cyanate, thalidomide, vincristine, acrylamide, arsenic, buckthorn toxin, carbon disulfide, cyanide, dimethylaminoproprionitrile, dichlorophenoxyacetic acid, diptheria toxin, ethylene oxide, hexacarbons (n-hexane, methyl n-butyl ketone), lead, Lucel-7, methyl bromide, organophosphates, thallium, and trichlorethylene.
  • pharmaceutical or chemical agents such as chloramphenicol, dapsone
  • Neuropathy is also associated with neuropathy. Many neuropathies are treated with corticosteroids, but there are currently no specific treatments for sensory neuropathies. Other growth factors such as nerve growth factor (NGF), neurotrophin-3 (NT-3), and insulin growth factor-I (IGF-I) are reportedly in clinical trials for treatment of peripheral neuropathies. These agents, however, may cause undesirable side effects and toxicity.
  • NGF nerve growth factor
  • NT-3 neurotrophin-3
  • IGF-I insulin growth factor-I
  • the present invention provides methods for treating sensory neuropathy caused by injury to, insults to, or degeneration of, sensory neurons, by administering a therapeutically effective amount of glial cell line-derived neurotrophic factor (GDNF) protein product.
  • the sensory neuropathy may be a secondary complication of a non-neurological condition, such as trauma. It is contemplated that such GDNF protein products would include a GDNF protein such as that depicted by the amino acid sequence set forth in SEQ ID NO: 1, as well as variants and derivatives thereof.
  • the invention is based on the finding that sensory neurons selectively take up and retrogradely transport GDNF protein product, and the novel discovery that GDNF enhances the survival of injured sensory neurons.
  • the GDNF protein product may be administered parenterally at a dose ranging from about 1 ⁇ g/kg/day to about 100 mg/kg/day, typically at a dose ranging from about 1 mg/kg/day to about 25 mg/kg/day, and usually at a dose of about 5 mg/kg day to 20 mg/kg/day.
  • GDNF protein product is preferably administered directly subcutaneously or intramuscularly close to or at the site of injury or degeneration; in such cases, a smaller dose of GDNF protein product, from about 1 ⁇ g/kg to 1 mg/kg, will be administered, for example, 1 ⁇ g/kg to 1 mg/kg administered at intervals ranging from weekly to daily . It is further contemplated that the GDNF protein product be administered in combination or conjunction with an effective amount of a second therapeutic agent for the treatment of sensory neuropathy.
  • the invention also provides for the use of GDNF protein product in the manufacture of a medicament or pharmaceutical composition for the treatment of sensory neuropathy.
  • Such pharmaceutical composition include topical, oral or parenteral GDNF protein product formulations.
  • the administration process can be accomplished via cell therapy and gene therapy means, as further described below. Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof.
  • the present invention provides methods for treating sensory neuropathy caused by injury to, insults to, or degeneration of, sensory neurons, by administering a therapeutically effective amount of glial cell line-derived neurotrophic factor (GDNF) protein product by means of a pharmaceutical composition, the implantation of GDNF-expressing cells, or GDNF gene therapy.
  • GDNF glial cell line-derived neurotrophic factor
  • the invention may be practiced using any biologically active GDNF protein product, including a GDNF protein represented by the amino acid sequence set forth in SEQ ID NO: l, including va ⁇ ants and derivatives thereof.
  • administration via cell therapy and gene therapy procedures is contemplated.
  • the present invention is based on the finding that GDNF protein product is selectively taken up and retrogradely transported to the cell body of sensory neurons through a receptor-mediated mechanism. It was then discovered that GDNF protein product serves as a survival factor for sensory neurons as demonstrated in the neonatal axotomy-induced cell death paradigm. After injection into the hindlimb of neonatal rats or the sciatic nerve of adult rats, GDNF protein product was retrogradely transported to sensory neurons in the lumbar dorsal root ganglia in a receptor-mediated fashion.
  • GDNF protein product will be advantageous in treating patients suffering from sensory neuropathy which can occur spontaneously or as a secondary complication of trauma, infection, inflammatory response, or other diseases or conditions, such as diabetes mellitus, immunologic or metabolic disorders and cancer, as described above.
  • GDNF protein product therapy may be advantageous in treating the hereditary sensory neuropathies, nutritional deficiency syndromes and toxic neuropathies, as described above.
  • GDNF protein product therapy may be advantageous in treating vascular-related neuropathies (caused by vasculiris or ischemia) associated with various conditions such as polyarteritis nodosa, rheumatoid arthritis, systemic lupus erythematosus, hypersensitivity angiitis, allergic granulomatosis, Sjogren's syndrome, Wegener's granulomatosis and cranial arteritis.
  • vascular-related neuropathies caused by vasculiris or ischemia
  • various conditions such as polyarteritis nodosa, rheumatoid arthritis, systemic lupus erythematosus, hypersensitivity angiitis, allergic granulomatosis, Sjogren's syndrome, Wegener's granulomatosis and cranial arteritis.
  • the GDNF protein product may be administered parenterally at a dose ranging from about 1 ⁇ g/kg/day to about 100 mg/kg/day, typically at a dose of about 1 mg/kg/day to about 25 mg/kg/day, and usually at a dose of about 5 mg/kg/day and 20 mg/kg/day.
  • GDNF protein product is preferably administered directly subcutaneously or intramuscularly close to the site of injury or degeneration. It is also contemplated that GDNF protein product may be administered subcutaneously or intramuscularly at a dose of about 1 ⁇ g/kg/day to 1 mg/kg/day.
  • GDNF protein product may be administered subcutaneously or intramuscularly at a dose of 1 ⁇ g/kg to 10 mg/kg once per week or several times per week.
  • the GDNF protein product also may be administered in combination or conjunction with an effective amount of a second therapeutic agent for the treatment of sensory neuropathy, such as NGF, NT-3 and IGF-I.
  • the invention also provides for the use of GDNF protein product in the preparation of a medicament for the treatment of sensory neuropathy.
  • a variety of pharmaceutical formulations and different delivery techniques are described in further detail below.
  • GDNF protein product includes purified natural, synthetic or recombinant glial cell line-derived neurotrophic factor, biologically active GDNF variants (including insertion, substitution and deletion variants), and chemically modified derivatives thereof. Also included are GDNFs that are substantially homologous to the human GDNF having the amino acid sequence set forth in SEQ ID NO:l. GDNF protein products may exist as homodimers or heterodimers in their biologically active form.
  • biologically active means that the GDNF protein product demonstrates similar neurotrophic properties, but not necessarily all of the same properties, and not necessarily to the same degree, as the GDNF having the amino acid sequence set forth in SEQ LD NO:l. The selection of the particular neurotrophic properties of interest depends upon the use for which the GDNF protein product is being administered.
  • substantially homologous as used herein means having a degree of homology to the GDNF having the amino acid sequence set forth in SEQ LD NO: 1 that is preferably in excess of 70%, most preferably in excess of 80%, and even more preferably in excess of 90% or 95%.
  • the degree of homology between the rat and human protein is about 93%, and it is contemplated that preferred mammalian GDNF will have a similarly high degree of homology.
  • the percentage of homology as described herein is calculated as the percentage of amino acid residues found in the smaller of the two sequences which align with identical amino acid residues in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to assist in that alignment (as set forth by Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), the disclosure of which is hereby inco ⁇ orated by reference).
  • any GDNF protein product which may be isolated by virtue of cross-reactivity with antibodies to the GDNF of SEQ LD NO:l or whose genes may be isolated through hybridization with the gene or with segments of the gene encoding the GDNF of SEQ ID NO: 1.
  • the GDNF protein products according to this invention may be isolated or generated by any means known to those skilled in the art. Exemplary methods for producing GDNF protein products useful in the present invention are described in U.S. Patent Application Serial No. 08/182,183 filed May 23, 1994 and its parent applications; PCT Application No. PCT/US92/07888 filed September 17, 1992, published as WO 93/06116 (Lin et al., Syntex-Synergen Neuroscience Joint Venture); European Patent Application No. 92921022.7, published as EP 610 254; and co-owned, co-pending 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 hereby incorporated by reference.
  • Naturally-occurring GDNF protein products may be isolated from mammalian neuronal cell preparations, or from a mammalian cell line secreting or expressing GDNF.
  • GDNF protein products may also be chemically synthesized by any means known to those skilled in the art.
  • GDNF protein products are preferably produced via recombinant techniques because they are capable of achieving comparatively higher amounts of protein at greater purity.
  • Recombinant GDNF protein product forms include glycosylated and non-glycosylated forms of the protein, and protein expressed in bacterial, mammalian or insect cell systems.
  • recombinant techniques involve isolating the genes responsible for coding GDNF, cloning the gene in suitable vectors and cell types, modifying the gene if necessary to encode a desired variant, and expressing the gene in order to produce the GDNF protein product.
  • a nucleotide sequence encoding the desired GDNF protein product may be chemically synthesized. It is contemplated that GDNF protein product may be expressed using nucleotide sequences which differ in codon usage due to the degeneracies of the genetic code or allelic variations.
  • WO93/06116 describes the isolation and sequencing of a cDNA clone of the rat GDNF gene, and the isolation, sequencing and expression of a genomic DNA clone of the human GDNF gene.
  • WO93/061 16 also describes vectors, host cells, and culture growth conditions for the expression of GDNF protein product. Additional vectors suitable for the expression of GDNF protein product in E. coli are disclosed in published European Patent Application No.
  • Naturally-occurring GDNF is a disulfide-bonded dimer in its biologically active form.
  • the material isolated after expression in a bacterial system is essentially biologically inactive, and exists as a monomer. Refolding is necessary to produce the biologically active disulfide-bonded dimer. Processes for the refolding and naturation of the GDNF expressed in bacterial systems are described in WO93/06116. Standard in vitro assays for the determination of GDNF activity are described in WO93/06116 and in co- owned, co-pending U.S. Patent Application Serial No. 08/535,681 filed September 28, 1995, and are hereby inco ⁇ orated by reference.
  • GDNF variants includes polypeptides in which amino acids have been deleted from (“deletion variants"), inserted into (“addition variants”), or substituted for (“substitution variants”), residues within the amino acid sequence of naturally-occurring GDNF. Such variants are prepared by introducing appropriate nucleotide changes into the DNA encoding the polypeptide or by in vitro chemical synthesis of the desired polypeptide. It will be appreciated by those skilled in the art that many combinations of deletions, insertions, and substitutions can be made provided that the final molecule possesses GDNF biological activity.
  • Mutagenesis techniques for the replacement, insertion or deletion of one or more selected amino acid residues are well known to one skilled in the art (e.g., U.S. Patent Number 4,518,584, the disclosure of which is hereby inco ⁇ orated by reference.)
  • the selection of the mutation site and nature of the mutation will depend on the GDNF characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target amino acid residue, or (3) inserting amino acid residues adjacent to the located site.
  • deletions generally range from about 1 to
  • N-terminal, C-terminal and internal intrasequence deletions are contemplated. Deletions may be introduced into regions of low homology with other TGF- ⁇ superfamily members to modify the activity of GDNF. Deletions in areas of substantial homology with other TGF- ⁇ superfamily sequences will be more likely to modify the GDNF biological activity more significantly. The number of consecutive deletions will be selected so as to preserve the tertiary structure of the GDNF protein product in the affected domain, e.g., cysteine crosslinking.
  • Non-limiting examples of deletion variants include truncated GDNF protein products lacking from one to forty N-terminal amino acids of GDNF, or variants lacking the C-terminal residue of GDNF, or combinations thereof, as described in co-owned, co ⁇ pending U.S. Patent Application Serial No. 08/535,681 filed September 28, 1995, which is hereby inco ⁇ orated by reference.
  • amino acid sequence additions typically include N-and/or C-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as internal intrasequence additions of single or multiple amino acid residues.
  • N-terminal addition variants include GDNF with an N-terminal methionyl residue (an artifact of the direct expression of GDNF in bacterial recombinant cell culture), which is designated [Mer ⁇ jGDNF, and fusion of a heterologous N-terminal signal sequence to the N-terminus of GDNF to facilitate the secretion of mature GDNF from recombinant host cells.
  • signal sequences generally will be obtained from, and thus be homologous to, the intended host cell species.
  • Additions may also include amino acid sequences derived from the sequence of other neurotrophic factors.
  • a preferred GDNF protein product for use according to the present invention is the recombinant human [Met _ 1 ]GDNF.
  • GDNF substitution variants have at least one amino acid residue of the GDNF amino acid sequence removed and a different residue inserted in its place.
  • substitution variants include allelic variants, which are characterized by naturally-occurring nucleotide sequence changes in the species population that may or may not result in an amino acid change. Examples of substitution variants (see, e.g., SEQ ID NO: 50) are disclosed in co-owned, co- pending U.S. Patent Application Serial No. 08/535,681 filed September 28, 1995, and are hereby inco ⁇ orated by reference.
  • GDNF amino acid sequence may involve modifications to a glycosylation site (e.g., serine, threonine, or asparagine).
  • a glycosylation site e.g., serine, threonine, or asparagine.
  • the absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at any asparagine-linked glycosylation recognition site or at any site of the molecule that is modified by addition of an O-linked carbohydrate.
  • An asparagine-linked glycosylation recognition site comprises a tripeptide sequence which is specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro.
  • a variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and or amino acid deletion at the second position) result in non-glycosylation at the modified tripeptide sequence.
  • the expression of appropriate altered nucleotide sequences produces variants which are not glycosylated at that site.
  • the GDNF amino acid sequence may be modified to add glycosylation sites.
  • alanine scanning mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (Science, 244:1081-1085, 1989).
  • an amino acid residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains demonstrating functional sensitivity to the substitutions then are refined by introducing additional or alternate residues at the sites of substitution.
  • the target site for introducing an amino acid sequence variation is determined, alanine scanning or random mutagenesis is conducted on the corresponding target codon or region of the DNA sequence, and the expressed GDNF variants are screened 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 GDNF proteins from various species are substantially different in terms of side-chain bulk, charge, and/or hydrophobicity.
  • Other sites of interest are those in which particular residues of GDNF-like proteins, obtained from various species, are identical. Such positions are generally important for the biological activity of a protein. Initially, these sites are substituted 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 (exemplary substitutions) are introduced, and/or other additions or deletions may be made, and the resulting products screened for activity.
  • Non-conservative substitutions may involve the exchange of a member of one of these classes for another. Such substituted residues may be introduced into regions of the GDNF protein that are homologous with other TGF- ⁇ superfamily proteins, or into the non-homologous regions of the molecule.
  • GDNF Derivatives Chemically modified derivatives of GDNF or GDNF variants may be prepared by one of skill in the art given the disclosures herein.
  • the chemical moieties most suitable for derivatization include water soluble polymers.
  • a water soluble polymer is desirable because the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer will be pharmaceutically acceptable for the preparation of a therapeutic product or composition.
  • One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations.
  • the effectiveness of the derivatization may be ascertained by administering the derivative, in the desired form (i.e., by osmotic pump, or, more preferably, by injection or infusion, or, further formulated for oral, pulmonary or other delivery routes), and determining its effectiveness.
  • Suitable water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly- l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight ranges from about 2 kDa to about 100 kDa for ease in handling and manufacturing (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight).
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of polyethylene glycol on a therapeutic protein or variant).
  • polymer molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function.
  • One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical moieties (e.g., polymers, such as different weights of polyethylene glycols).
  • the proportion of polymer molecules to protein (or peptide) molecules will vary, as will their concentrations in the reaction mixture.
  • the optimum ratio in terms of efficiency of reaction in that there is no excess unreacted protein or polymer
  • the desired degree of derivatization e.g., mono-, di-, tri-, etc.
  • the molecular weight of the polymer selected whether the polymer is branched or unbranched, and the reaction conditions.
  • polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
  • attachment methods available to those skilled in the art. See for example, EP 0 4 1 384, the disclosure of which is hereby inco ⁇ orated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematoi, 20:1028-1035, 1992 (reporting pegylation of GM-CSF using tresyl chloride).
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may 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 may also be used as a reactive group for attaching the polyethylene glycol molecule(s).
  • attachment at an amino group, such as attachment at the N-terminus or lysine group is preferred. Attachment at residues impo ⁇ ant for receptor binding should be avoided if receptor binding is desired.
  • polyethylene glycol as an illustration of the present compositions, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective N-terminal chemical modification may be accomplished by reductive alkyiation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein.
  • substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • one may selectively N-terminally pegylate the protein by performing the reaction at a pH which allows one to take advantage of the pKa differences between the e-amino group of the lysine residues and that of the a- amino group of the N-terminal residue of the protein.
  • the water soluble polymer may be of the type described above, and should have a single reactive aldehyde for coupling to the protein.
  • Polyethylene glycol propionaldehyde, containing a single reactive aldehyde, may be used.
  • the present invention contemplates the use of derivatives which are prokaryote-expressed GDNF, or variants thereof, linked to at least one polyethylene glycol molecule, as well as use of GDNF, or variants thereof, attached to one or more polyethylene glycol molecules via an acyl or alkyl linkage.
  • Pegylation may be carried out by any of the pegylation reactions 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 hereby inco ⁇ orated by reference; EP O 401 384; and the other publications cited herein that relate to pegylation.
  • the pegylation may be carried out via an aeylation reaction or an alkyiation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
  • Pegylation by aeylation generally involves reacting an active ester derivative of polyethylene glycol with the GDNF protein or variant.
  • any known or subsequently discovered reactive PEG molecule may be used to carry out the pegylation of GDNF protein or variant.
  • a prefened activated PEG ester is PEG esterified to N-hydroxysuccinimide.
  • "aeylation” is contemplated to include 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.
  • Reaction conditions may be selected from any of those known in the pegylation art or those subsequently developed, but should avoid conditions of temperature, solvent, and pH that would inactivate the GDNF or variant to be modified.
  • Pegylation by aeylation will generally result in a poly-pegylated GDNF protein or variant.
  • the connecting linkage will be an amide.
  • the resulting product will be substantially only (e.g., > 95%) mono-, di- or tri-pegylated. Some species with higher degrees of pegylation, however, may be formed in amounts depending on the specific reaction conditions used. If desired, more purified pegylated species may be separated from the mixture, particularly unreacted species, by standard purification techniques, including, among others, dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel filtration chromatography and electrophoresis.
  • Pegylation by alkyiation generally involves reacting a terminal aldehyde derivative of PEG with the GDNF protein or variant in the presence of a reducing agent. Pegylation by alkyiation can also result in poly-pegylated GDNF protein or variant. In addition, one can manipulate the reaction conditions to favor pegylation substantially only at the a-amino group of the N-terminus of the GDNF protein or variant (i.e., a mono-pegylated protein). In either case of monopegylation or polypegylation, the PEG groups are preferably attached to the protein via a -CH2-NH- group.
  • this type of linkage is refened to herein as an "alkyl” linkage.
  • Derivatization via reductive alkyiation to produce a monopegylated product exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization.
  • the reaction is performed at a pH which allows one to take 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.
  • the present invention contemplates use of a substantially homogeneous preparation of monopolymer/GDNF protein (or variant) conjugate molecules (meaning GDNF protein or variant to which a polymer molecule has been attached substantially only (i.e., > 95%) in a single location). More specifically, if polyethylene glycol is used, the present invention also encompasses use of pegylated GDNF protein or variant lacking possibly antigenic linking groups, and having the polyethylene glycol molecule directly coupled to the GDNF protein or variant.
  • GDNF protein products to be used in accordance with the present invention may include pegylated GDNF protein or variants, wherein the PEG group(s) is (are) attached via acyl or alkyl groups.
  • PEG groups may be mono-pegylated or poly-pegylated (e.g., containing 2-6, and preferably 2-5, PEG groups).
  • the PEG groups are generally attached to the protein at the a- or e-amino groups of amino acids, but it is also contemplated that the PEG groups could be attached to any amino group attached to the protein, which is sufficiently reactive to become attached to a PEG group under suitable reaction conditions.
  • the polymer molecules used in both the aeylation and alkyiation approaches may be selected from among water soluble polymers as described above.
  • the polymer selected should be modified to have a single reactive group, such as an active ester for aeylation or an aldehyde for alkyiation, preferably, so that the degree of polymerization may be controlled as provided for in the present methods.
  • An exemplary reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono CI-CIO alkoxy or aryloxy derivatives thereof (see, U.S. Patent Number 5,252,714).
  • the polymer may be branched or unbranched.
  • the polymer(s) selected should have a single reactive ester group.
  • the polymer(s) selected should have a single reactive aldehyde group.
  • the water soluble polymer will not be selected from naturally-occurring glycosyl residues since these are usually made more conveniently by mammalian recombinant expression systems.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • a particularly prefened water-soluble polymer for use herein is polyethylene glycol.
  • polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono-(Cl-ClO) alkoxy- or aryloxy-polyethylene glycol.
  • chemical derivatization may be performed under any suitable condition used to react a biologically active substance with an activated polymer molecule.
  • Methods for preparing pegylated GDNF protein or variant will generally comprise the steps of (a) reacting a GDNF protein or variant with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the protein becomes attached to one or more PEG groups, and (b) obtaining the reaction product(s).
  • Reductive alkyiation to produce a substantially homogeneous population of mono-polymer/GDNF protein (or variant) conjugate molecule will generally comprise the steps of: (a) reacting a GDNF protein or variant with a reactive PEG molecule under reductive alkyiation conditions, at a pH suitable to permit selective modification of the a-amino group at the amino terminus of said GDNF protein or variant; and (b) obtaining the reaction product(s).
  • the reductive alkyiation reaction conditions are those which permit the selective attachment of the water soluble polymer moiety to the N-terrmnus of GDNF protein or va ⁇ ant
  • Such reaction condinons generally provide for pKa differences between the lysine amino groups and the a-amino group at the N-terminus (the pKa being the pH at which 50% of the amino groups are protonated and 50% are not)
  • the pH also affects the ratio of polymer to protein to be used In general, if the pH is lower, a larger excess of polymer to protein will be desired (I e., the less reacuve the N- terminal a-amino group, the more polymer needed to achieve optimal conditions). If the pH is higher, the polymer protein ratio need not be as large (i e., more reactive groups are available, so fewer polymer molecules are needed).
  • the pH will generally be used to be used in general, if the pH is lower, a larger excess of polymer to protein will be desired (I
  • the molecular weight of the polymer is the higher the molecular weight of the polymer, the fewer polymer molecules may be attached to the protein Similarly, branching of the polymer should be taken into account when optimizing these parameters Generally, the higher the molecular weight (or the more branches) the higher the polyme ⁇ protein ratio In general, for the pegylation reactions contemplated herein, the prefened average molecular weight is about 2 kDa to about 100 kDa.
  • the prefened average molecular weight is about 5 kDa to about 50 kDa, particularly preferably about 12 kDa to about 25 kDa
  • the ratio of water-soluble polymer to GDNF protein or va ⁇ ant will generally range from 1.1 to 100: 1, preferably (for polype gylation) 1 1 to 20 1 and (for monopegylation) 1 1 to 5.1 Using the conditions indicated above, reductive alkyiation will provide for selective attachment of the polymer to any GDNF protein or va ⁇ ant having an a-amino group at the amino terminus, and provide for a substantially homogenous preparation of monopolymer/GDNF protein (or va ⁇ ant) conjugate.
  • monopolymer/GDNF protein (or vanant) conjugate is used here to mean a composition compnsed of a single polymer molecule attached to a molecule of GDNF protein or GDNF va ⁇ ant protein
  • the monopolymer/GDNF protein (or vanant) conjugate preferably will have a polymer molecule located at the N-terrmnus, but not on lysine amino side groups.
  • the preparation will preferably be greater than 90% monopolymer/GDNF protein (or vanant) conjugate, and more preferably greater than 95% monopolymer/GDNF protein (or vanant) conjugate, with the remainder of observable molecules being unreacted (i.e., protein lacking the polymer moiety).
  • the reducing agent should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkyiation.
  • Prefened reducing agents may be selected from sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane and pyridine borane.
  • a particularly prefened reducing agent is sodium cyanoborohydride.
  • Other reaction parameters such as solvent, reaction times, temperatures, etc., and means of purification of products, can be determined case-by-case based on the published information relating to derivatization of proteins with water soluble polymers (see the publications cited herein).
  • GDNF protein product pharmaceutical compositions typically include a therapeutically effective amount of a GDNF protein product in admixture with one or more pharmaceutically and physiologically acceptable formulation materials.
  • suitable formulation materials include, but are not limited to, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants.
  • a suitable vehicle may be water for injection, physiological saline solution, or artificial CSF, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • the primary solvent in a vehicle may be either aqueous or non-aqueous in nature.
  • the vehicle may contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the vehicle may contain still other pharmaceutically-acceptable excipients for modifying or maintaining the rate of release of GDNF protein product, or for promoting the abso ⁇ tion or penetration of GDNF protein product across the blood-brain barrier.
  • excipients are tho.se substances usually and customarily employed to formulate dosages for parenteral administration in either unit dose or multi-dose form or for direct continuous or periodic infusion from an implanted pump.
  • the therapeutic composition Once the therapeutic composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder.
  • Such formulations may be stored either in a ready to use form or in a form, e.g., lyophilized, requiring reconstitution prior to administration.
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives.
  • the GDNF protein product pharmaceutical composition may be formulated for parenteral administration, e.g., by intracerebroventricular infusion or injection.
  • parenterally administered therapeutic compositions are typically in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the GDNF protein product in a pharmaceutically acceptable vehicle.
  • a pharmaceutically acceptable vehicle is physiological saline.
  • the GDNF protein product pharmaceutical composition also may include particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes to provide sustained release characteristics. Hylauronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • GDNF protein product which is administered in this fashion may be encapsulated and may be formulated with or without those carriers customarily used in the compounding of solid dosage forms.
  • the formulation may be designed to release the active portion of the pharmaceutical composition at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional excipients may be included to facilitate abso ⁇ tion of GDNF protein product. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed. D. Administration of GDNF Protein Product
  • the GDNF protein product may be administered topically or parenterally via a subcutaneous, intramuscular, intravenous, transpulmonary, transdermal, intrathecal, intracerebral or intraocular route.
  • a subcutaneous, intramuscular, intravenous, transpulmonary, transdermal, intrathecal, intracerebral or intraocular route For example, one route of administration is subcutaneously or intramuscularly close to the site of injury or degeneration, at a dose of about 1 ⁇ g/kg to 1 mg/kg delivered at intervals ranging from weekly to daily.
  • repeated daily or less frequent injections may be administered, or GDNF protein product may be infused continuously or periodically from a constant- or programmable-flow implanted pump. The frequency of dosing will depend on the pharmacokinetic parameters of the GDNF protein product as formulated, and the route of administration.
  • GDNF protein product may also be administered via a carrier means, such as a biodegradable material, containing GDNF protein product that may be surgically implanted at or near the cite of injury. It is also contemplated that such a carrier composition may be packed within a wound.
  • a carrier means such as a biodegradable material, containing GDNF protein product that may be surgically implanted at or near the cite of injury. It is also contemplated that such a carrier composition may be packed within a wound.
  • the specific dose is typically calculated according to body weight or body surface area. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them, especially in light of the dosage information and assays disclosed herein. Appropriate dosages may be ascertained through use of the established assays for determining dosages utilized in conjunction with appropriate dose-response data.
  • the final dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g., the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding the appropriate dosage levels for the treatment of various diseases and conditions.
  • GDNF continuous administration or sustained delivery of GDNF may be advantageous for a given treatment. While continuous administration may be accomplished via a mechanical means, such as with an infusion pump, it is contemplated that other modes of continuous or near continuous administration may be practiced. For example, chemical derivatization may result in sustained circulation or sustained release forms of the GDNF protein which have the effect of continuous presence in the blood stream, in predictable amounts, based on a determined dosage regimen. Thus, GDNF protein products include proteins derivatized to effectuate such continuous administration.
  • GDNF protein product cell therapy e.g., implantation of cells producing GDNF protein product
  • This embodiment would involve implanting into patients cells capable of synthesizing and secreting a biologically active form of GDNF protein product.
  • Such GDNF protein product-producing cells may be cells that are natural producers of GDNF protein product (analogous to B49 ghoblastoma cells) or may be recombinant ceils whose ability to produce GDNF protein product has been augmented by transformation with a gene encoding the desired GDNF protein product.
  • transformation may be accomplished by means of a vector suitable for delivering the gene as well as promoting its expression and secretion.
  • the natural cells producing GDNF protein product be of human origin and produce human GDNF protein product.
  • the recombinant cells producing GDNF protein product be transformed with an expression vector containing a gene encoding a human GDNF protein product.
  • Implanted cells may be encapsulated to avoid infiltration of sunounding tissue.
  • Human or non-human animal cells may be implanted in patients in biocompatible, semipermeable polymeric enclosures or membranes that allow release of GDNF protein product, but that prevent destruction of the cells by the patient's immune system or by other detrimental factors from the sunounding tissue.
  • the patient's own cells, transformed to produce GDNF protein product ex vivo could be implanted directly into the patient without such encapsulation.
  • GDNF protein product gene therapy in vivo is also envisioned, by introducing the gene coding for GDNF protein product into targeted cells via local injection of a nucleic acid construct or other appropriate delivery vectors.
  • a nucleic acid sequence encoding a GDNF protein product may be contained in an adeno-associated virus vector for delivery into the targeted cells.
  • Alternative viral vectors include, but are not limited to, retrovirus, adenovirus, he ⁇ es simplex virus and papilloma virus vectors.
  • Physical transfer may also be achieved by liposome-mediated transfer, direct injection (naked DNA), receptor-mediated transfer (ligand-DNA complex), eiectroporation, calcium phosphate precipitation or microparticle bombardment (gene gun).
  • the methodology for the membrane encapsulation of living cells is familiar to those of ordinary skill in the art, and the preparation of the encapsulated cells and their implantation in patients may be accomplished without undue experimentation. See, e.g., U.S. Patent Numbers 4,892,538, 5,011,472, and 5,106,627, each of which is specifically inco ⁇ orated herein by reference.
  • a system for encapsulating living cells is described in PCT
  • GDNF protein product formulations described herein may be used for veterinary as well as human applications and that the term "patient” should not be construed in a limiting manner. In the case of veterinary applications, the dosage ranges should be the same as specified above.
  • Example 1 addresses the use of radiolabelled GDNF protein product to show that neonatal and adult sensory neurons bind, internalize and retrogradely transport GDNF in a receptor-mediated fashion.
  • Example 2 addresses the effect of GDNF protein product administration in a rat sensory neuron injury model.
  • GDNF protein product ⁇ I-radiolabelled GDNF protein product was used to show that neonatal and adult sensory neurons bind, internalize and retrogradely transport GDNF protein product in a receptor-mediated fashion.
  • the GDNF protein product used in Examples 1 and 2 was recombinant human [Met'l] GDNF and was produced by expression in E. coli as generally described in Exampies 6B and 6C of WO93/06116.
  • the purified [Met ⁇ jGDNF was iodinated using the lactoperoxidase technique and separated from free * *"I using G-25 Sephadex quick spin columns as described in Yan et al., J.
  • the 125l-[Met ⁇ l]GDNF was retrogradely transported by L4 and L5 dorsal root ganglia ipsilateral to the injection side, as determined by direct measurement of transported radioactivity in the dissected L4 and L5 dorsal root ganglia. The specificity of this transport was demonstrated by the much higher accumulation of radioactivity in ipsilateral spinal cords.
  • Cell sizing histograms of autoradiogram s indicated that both large and small neurons take up GDNF protein product in neonatal rats. No silver grains above background were observed in the contralateral dorsal root ganglia.
  • Retrograde transport of a neurotrophic factors is significant because these factors initiate their effects by binding to cell surface receptor followed by uptake and retrograde transport to the cell body.
  • the specific retrograde transport of GDNF protein product by dorsal root ganglia neurons indicates that these neurons express a GDNF receptor and that GDNF may play a physiological role as a target-derived neurotrophic factor for these neurons.
  • GDNF Glial Cell Line- Derived Neurotrophic Factor
  • GDNF (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
  • Lys lie Leu Lys Asn Leu Ser Arg Asn Arg Arg Leu Val Ser Asp Lys 85 90 95

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Abstract

La présente invention se rapporte de manière générale à des procédés de traitement des neuropathies sensorielles qui consistent à administrer un facteur neurotrophique dérivé d'une lignée de cellules gliales (GDNF).
PCT/US1996/018729 1995-11-29 1996-11-22 Procede de traitement des neuropathies sensorielles utilisant un produit proteique du facteur neurotrophique derive d'une lignee de cellules gliales WO1997019693A1 (fr)

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WO1999006064A1 (fr) * 1997-07-30 1999-02-11 Amgen Inc. Procedes de prevention et de traitement des pertes d'audition a l'aide d'un produit proteique de neurturine
WO2000062798A2 (fr) * 1999-04-15 2000-10-26 St. Elizabeth's Medical Center, Inc. Facteurs de croissance angiogenique destines au traitement de neuropathie peripherique
EP1304119A2 (fr) * 1997-07-30 2003-04-23 Amgen Inc. Procédé de prévention et de traitement des pertes d'audition à l'aide d'un produit protéique de neurturine
US6677135B1 (en) 1996-05-08 2004-01-13 Biogen, Inc. Ret ligand (RetL) for stimulating neutral and renal growth
US7125856B1 (en) 1999-04-15 2006-10-24 St. Elizabeth's Medical Center Of Boston, Inc. Angiogenic growth factors for treatment of peripheral neuropathy
US8222378B2 (en) 1998-07-14 2012-07-17 Janssen Pharmaceutica N.V. Neurotrophic growth factor, enovin
US8722862B2 (en) 2004-08-19 2014-05-13 Biogen Idec Ma Inc. Refolding transforming growth factor beta family proteins
US9138461B2 (en) 2007-05-01 2015-09-22 Biogen Ma Inc. Compositions and methods for increasing vascularization
US10328125B2 (en) 2006-02-27 2019-06-25 Gloriana Therapeutics, Inc. Treatments for neurological disorders
US11912750B2 (en) 2016-11-10 2024-02-27 Keros Therapeutics, Inc. GDNF fusion polypeptides and methods of use thereof

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6677135B1 (en) 1996-05-08 2004-01-13 Biogen, Inc. Ret ligand (RetL) for stimulating neutral and renal growth
US6861509B1 (en) 1996-05-08 2005-03-01 Biogen, Inc. Antibodies to Ret and RetL3
US6274554B1 (en) 1997-07-30 2001-08-14 Amgen Inc. Method for preventing and treating hearing loss using a neurturin protein product
WO1999006064A1 (fr) * 1997-07-30 1999-02-11 Amgen Inc. Procedes de prevention et de traitement des pertes d'audition a l'aide d'un produit proteique de neurturine
EP1304119A2 (fr) * 1997-07-30 2003-04-23 Amgen Inc. Procédé de prévention et de traitement des pertes d'audition à l'aide d'un produit protéique de neurturine
EP1304119A3 (fr) * 1997-07-30 2004-01-21 Amgen Inc. Procédé de prévention et de traitement des pertes d'audition à l'aide d'un produit protéique de neurturine
US6043221A (en) * 1997-07-30 2000-03-28 Amgen Inc. Method for preventing and treating hearing loss using a neuturin protein product
US8222378B2 (en) 1998-07-14 2012-07-17 Janssen Pharmaceutica N.V. Neurotrophic growth factor, enovin
US8492336B2 (en) 1998-07-14 2013-07-23 Janssen Pharmaceutica N.V. Methods of treating neuropathic pain with an environ polypeptide
US7125856B1 (en) 1999-04-15 2006-10-24 St. Elizabeth's Medical Center Of Boston, Inc. Angiogenic growth factors for treatment of peripheral neuropathy
WO2000062798A3 (fr) * 1999-04-15 2001-01-04 St Elizabeth S Medical Ct Inc Facteurs de croissance angiogenique destines au traitement de neuropathie peripherique
WO2000062798A2 (fr) * 1999-04-15 2000-10-26 St. Elizabeth's Medical Center, Inc. Facteurs de croissance angiogenique destines au traitement de neuropathie peripherique
US8722862B2 (en) 2004-08-19 2014-05-13 Biogen Idec Ma Inc. Refolding transforming growth factor beta family proteins
US8969042B2 (en) 2004-08-19 2015-03-03 Biogen Idec Ma Inc. Refolding transforming growth factor beta family proteins
US10328125B2 (en) 2006-02-27 2019-06-25 Gloriana Therapeutics, Inc. Treatments for neurological disorders
US9138461B2 (en) 2007-05-01 2015-09-22 Biogen Ma Inc. Compositions and methods for increasing vascularization
US11912750B2 (en) 2016-11-10 2024-02-27 Keros Therapeutics, Inc. GDNF fusion polypeptides and methods of use thereof

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