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    • 专利摘要:
    The present invention relates generally to methods of treating damage or degeneration of retinal ganglion cells by administering glial cell line-derived neurotrophic factor (GDNF). In particular, the present invention relates to a method of treating optic nerve damage or degeneration associated with glaucoma.
    公开号:KR19990071541A
    申请号:KR1019980703815
    申请日:1996-11-22
    公开日:1999-09-27
    发明作者:진-클로드 루이스
    申请人:스티븐 엠. 오드레；암젠 인코포레이티드；
    IPC主号:
    专利说明:

    Treatment method of retinal ganglion cell injury using glial cell line-derived neurotrophic factor (HDNF) protein product
    Background of the Invention
    The present invention generally relates to methods of treating damage or degeneration of retinal ganglion cells by administering a glial cell line-derived neurotrophic factor (GDNF) protein product. The present invention relates in particular to methods of treating glaucoma or other diseases / conditions that lead to retinal ganglion cell degeneration.
    Neurotrophic factors are natural proteins found in the nervous system or in non-neuronal tissues stimulated by the nervous system and serve to aid and survive phenotypic differentiation of specific neuronal and / or glial cell populations (Varon et al. Ann. Rev. Neuroscience , 1: 327, 1979; Thoenen et al. Science, 229: 238, 1985). Due to this physiological function, neurotrophic factors are useful for treating the loss of differentiation function due to neuronal degeneration and nerve damage. Nerve damage is caused by conditions that jeopardize the survival and / or intrinsic function of one or more types of neurons, including: (1) physical damage, which is the degeneration of axons (in turn of neurons Causes death) and / or denaturation of neuronal cell bodies near the site of injury, (2) temporary or permanent disruption of blood circulation to parts of the nervous system (such as ischemic), (3) cancer and AIDS, respectively Parkinson's disease, caused by intentional and accidental exposure to neurotoxins such as cisplatinum and dideoxycytidine, (4) chronic metabolic diseases such as diabetes or renal dysfunction, or (5) degeneration of certain neuronal populations, Neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. In order for a particular neurotrophic factor to be potentially useful in treating neuronal damage, a class or classes of damaged nerve cells must react with this factor. It has been demonstrated that not all neuronal populations respond or are equally affected by all neurotrophic factors.
    The first neurotrophic factor identified is nerve growth factor (hereinafter referred to as "NGF"). NGF is called neurotrophin as the first member of defined trophic factors and is generally brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4 / 5 and NT -6 (see Thoenen, Trends. Neurosci., 14: 165-170, 1991; Snider, Cell, 77: 627-638, 1994; Bothwell, Ann. Rev. Neurosci., 18: 223-253, 1995). ). This neurotrophin is known to act through trk tyrosine kinase receptors, namely trkA, trkB, trkC, and low-affinity p75 receptors (Snider, Cell, 77: 627-638, 1994; Bothwell, Ann. Rev. Neurosci., 18: 223-253, 1995; see TINS 18: 321-326, 1995 by Chao et al.).
    Glial cell line-derived neurotrophic factor (GDNF) is a recently discovered protein that has been identified and purified by in vitro assays based on the efficacy of stimulating phenotypes of midbrain dopaminergic neuronal transporters and promoting survival (Lin et al. Science, 260: 1130-1132, 1993). GDNF is a glycosylated disulfide-binding homodimer with some structural homology with the transforming growth factor-beta (TGF-β) superfamily in proteins (Lin et al. Science, 260: 1130-1132, 1993; Krieglstein Et al. EMBO J., 14: 736-742, 1995; Neuron, Poulsen et al., 13: 1245-1252, 1994). GDNF mRNA has been detected in muscle and Schwann cells of the peripheral nervous system (see Henderson et al. Science, 266: 1062-1064, 1994; J. Cell Biol., Trupp et al., 130: 137-148, 1995). It was detected in glial glial cells (see Schaar et al. Exp. Neurol., 124: 368-371, 1993). Treatment with exogenous GDNF in vivo stimulates the dopaminergic phenotype of melanoma neurons and restores the functional deficiency induced by axon cleavage or dopamine neurotoxicity in Parkinson's disease animal models (Hudson et al. 36: 425-432, 1995; See Beck et al., 373: 339-341, 1995; Tomac et al. Nature, 373: 335-339, 1995; Hoffer et al. Neurosci. Lett., 182: 107-111, 1994 ). Initially considered to be relatively specific for dopaminergic neurons, evidence is beginning to appear, at least in vitro, to show that GDNF may have a broader spectrum of neurotrophic targets in addition to midbrain dopamine agonist and somatic motor neurons. Yan and Matheson, Nature 373: 341-344, 1995; Nature of Oppenheim et al., 373: 344-346, 1995; Matheson et al., Soc. Neurosci.Abstr., 21, 544, 1995; Trupp et al. J. Cell Biol. , 130: 137-148, 1995). In particular, GDNF has been shown to have neurotrophic effects on brainstem and spinal cholinergic motor neurons both in vivo and in vitro (Nov., et al., Nature, 373: 344-346, 1995; Neuroreport, Zurn et al., 6: 113). 118, 1994; Yan et al. Nature, 373: 341-344, 1995; Henderson et al. Science, 266: 1062-1064, 1994).
    Generally related to the present invention is WO 93/06116, published on April 1, 1993, which discloses that GDNF is useful for the treatment of neurological damage, including damage associated with Parkinson's disease (syn. Neuroscience Joint Venture). Mol. Schmidt-Kastner et al. Also reported that GDNF mRNA could be detected and upregulated after pyrocarpin-induced seizures. Brain Res., 26: 325-330, 1994; Under culture conditions, basal forebrain astrocytes expressed moderate levels of GDNF mRNA, but GDNF did not alter basal forebrain ChAT activity. Neurol., 124: 368-371, 1993 and Schaar et al. Neurol., 130: 387-393, 1994; And currently pending US patent application Ser. No. 08 / 535,682, filed Sep. 28, 1995, which reports that GDNF is useful for treating damage or degeneration of basal forebrain cholinergic neurons. There is no previous description of how GDNF promotes survival or regeneration of damaged retinal ganglion cells.
    Reticulum ganglion cells play an important role in visual perception, which manifests in several stages. First, differentiated neurons, called photoreceptors, located on the outer membrane of the retina turn light into electrical signals, which are then combined and transmitted by intervening neurons to the retinal ganglion cells in the retina's inner membrane. Send this information to the area. The retinal ganglion cell axons are concentrated to form the optic nerve, which protrudes not only in the nucleus of the brain stem, but also in the lateral epithelium and the brain's upper extremity.
    Damage to retinal ganglion cells is the primary damage seen in glaucoma, the third leading cause of blindness. Glaucoma is a term used for a group of diseases characterized by visual neuropathy, including progressive loss of retinal ganglion cells. This damage to retinal ganglion cells is characterized by axonal transport defects in the optic nerve head and histopathological abnormalities of axons, and is typically associated with the appearance of perforated optic heads. Optic neural degeneration may also result from other conditions of the optic disc, such as papillary edema, papilloma (a type of optic neuritis) or ischemia due to elevated intracranial pressure.
    In most glaucoma, optic nerve damage is due to elevated intraocular pressure. The main types of glaucoma associated with elevated intraocular pressure are open angle glaucoma, right angle glaucoma, and secondary glaucoma. In some intestines, a similar reflex of neurons occurs with normal intraocular pressure. In all cases, elevated intraocular pressure usually results in greater nerve damage. Glaucoma is usually treated by medically or surgically attempting to lower intraocular pressure.
    Chronic open angle glaucoma is the most common form and occurs in about 5% of the US and European adult population. In this type of glaucoma, the waterproof reabsorption in the eye is impaired, causing the intraocular pressure to rise above the maximum normal value of 21 mmHg, and the axons are gradually destroyed and support the tissue in the optic disc. Such glaucoma usually shows no signs until it has fully developed. Early vision loss is seen in the peripheral field. Diagnosis is made by measuring intraocular pressure, examining the optic disc, and examining the patient's field of vision. First, it is treated by topical administration of parasympathetic nerve excitability (phylocarpine and carbacol), beta-adrenergic blocker (timorrole) and sympathetic nerve excitability (epinephrine), which act to lower intraocular pressure. When these medications, individually or in combination, can no longer prevent progressive damage, they are prescribed secondarily as parasympathetic nerve excitability (ecothioate) and carbonic anhydrase inhibitors (acetazolamide and metazolamide). If medication fails, surgical treatment may be performed to open the outflow passage. Some open angle glaucoma is congenital due to lack of specific anatomical structures of the eye. In right angle glaucoma, the aqueous outflow is mechanically obstructed by the shallow front. The intraocular pressure remains steady until the pupil block (which prevents the flow of waterproofing through the pupil) intersects the angled reabsorption surface and then rises rapidly, often above 50 mmHg. Such glaucoma is generally monocular, characterized by redness of the eye, ocular pain, and associated with pupil fixation and dilatation, decreased vision, sweating and nausea. Acute seizures require first aid and are prescribed parenterally with acetazolamide, orally with glycerol or with mannitol, and with topical administration of pilocarpine and timolol. If the acute attack calms down, surgery may solve the anatomical problem.
    Secondary glaucoma is caused by other eye diseases. For example, glaucoma progresses due to swelling of the lens, angiogenesis of each structure (formation of new blood vessels), chronic inflammation, or severe trauma to each structure. Secondary glaucoma caused by the lens is treated with a surgical operation to remove the lens. Most other secondary glaucoma are treated in the same way as primary open angle glaucoma. Angiogenic glaucoma is difficult to treat but can be improved with laser photocoagulation.
    There remains a need for methods and therapeutic compositions that are useful for treating retinal ganglion cell damage associated with conditions such as glaucoma. Such methods and therapeutic compositions will be able to fully protect retinal ganglion cells and optic nerves from progressive damage and promote the survival or regeneration of damaged neurons without serious side effects.
    Summary of the Invention
    The present invention provides a method for treating damage or degeneration of retinal ganglion cells by administering a therapeutically effective amount of a glial cell line-derived neurotrophic factor (GDNF) protein product. According to one specific aspect of the present invention, a method of treating glaucoma is provided by administering a therapeutically effective amount of a GDNF protein product. In another aspect, there is provided a method for treating optic nerve damage or degeneration due to conditions detrimental to the optic head, such as glaucoma, papilloma edema, papilloma and ischemia. Administration of a therapeutically effective amount of GDNF protein product promotes the survival and regeneration of optic nerve retinal ganglion cell axons. Such GDNF protein products will include variants and derivatives thereof as well as the GDNF protein shown in the amino acid sequence set forth in SEQ ID NO: 1. The present invention finds that retinal ganglion cells selectively absorb GDNF protein products and transport them in reverse, and that GDNF promotes the survival of retinal ganglion cells in vitro, and that GDNF is damaged in vivo in retinal ganglion cells, ie, damaged neurons in glaucoma. It is based on the discovery that it promotes the survival of major groups of people.
    The GDNF protein product according to the present invention can range from about 1 μg / kg / day to about 100 mg / kg / day, typically from about 0.1 mg / kg / day to about 25 mg / kg / day, usually about 5 mg / kg Parenteral administration may be in dosages ranging from / day to about 20 mg / kg / day. It is also believed that depending on the requirements and route of administration of each patient, the GDNF protein product may be administered at lower frequency, such as weekly or several times per week, rather than daily. GDNF protein products may also be administered by intraocular administration. Those skilled in the art will appreciate that a smaller amount of GDNF protein product can be intraocularly administered as above, for example, one injection of an intraocular dose of about 1 μg / eye to about 1 mg / eye. Alternatively, the injection may be divided into several times. It is also contemplated that GDNF protein products will be administered to glaucoma in conjunction with or in combination with an effective amount of other therapeutic agents.
    The present invention also provides the use of a GDNF protein product in the manufacture of a medicament or pharmaceutical composition for treating damage or degeneration of retinal ganglion cells, including glaucoma treatment. Such pharmaceutical compositions include topical, oral or parenteral GDNF protein product formulations. Those skilled in the art will also recognize that such a course of administration may be carried out by means of cell therapy and gene therapy, as described further below. Many additional aspects and advantages of the present 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 of the invention.
    The present invention provides a method for treating damage or degeneration of retinal ganglion cells by administering a therapeutically effective amount of a glial cell line-derived neurotrophic factor (GDNF) protein product. According to one aspect of the invention, a method of treating retinal ganglion cell damage due to glaucoma is provided by administering a therapeutically effective amount of a GDNF protein product in the form of a pharmaceutical composition, transplantation of GDNF-expressing cells, or GDNF gene therapy. The present invention can be practiced with all biologically active GDNF protein products and variants and derivatives thereof, including the GDNF protein represented by the amino acid sequence shown in SEQ ID NO: 1. In addition to oral, parenteral or topical delivery of GDNF protein products, administration via cell therapy and gene therapy can also be considered.
    The present invention is based on the initial discovery that GDNF promotes the survival of retinal ganglion cells in the medium, and that retinal ganglion cells selectively absorb and reverse transport GDNF in a receptor-specific manner. GDNF is known to play a role in the development, survival and maintenance of these neurons. The present invention is also based on the discovery that GDNF protein products enhance the in vivo survival of damaged retinal ganglion cells, which constitute a major population of neurons damaged by glaucoma. Administration of exogenous GDNF protein products should be able to protect retinal ganglion cells from damage or trauma resulting from a deficiency of neurotrophic factors caused by disrupted factor transport of cellular axons. These treatments are expected to make the retinal ganglion cells resistant to intermittent attacks caused by intraocular pressure, vascular malnutrition or other conditions that cause glaucoma or other optic nerve diseases, and to maintain optic nerve function.
    The GDNF protein product according to the present invention can range from about 1 μg / kg / day to about 100 mg / kg / day, typically from about 0.1 mg / kg / day to about 25 mg / kg / day, usually about 5 mg / kg It may be administered parenterally at a dosage of / day to about 20 mg / kg / day. GDNF protein products can be administered directly to the intraocular. In such cases, a smaller amount of GDNF protein product will be administered, for example from about 1 μg / eye to about 1 mg / eye for one injection or divided into several injections. GDNF may also be administered in the subretinal space between the photoreceptor layer and the pigment epithelial layer of the retina. It is believed that GDNF protein may be administered with an effective amount of other therapeutic agents for the treatment of glaucoma. Such other therapeutic agents include, for example, parasympathetic nerve excitability (phylocarpine and carbacol), beta-adrenergic blockers (timolol), sympathetic nerve excitability (epinephrine), secondary parasympathetic nerve excitability (ecothioate) and carbonic acid. Dehydratase inhibitors (acetazolamide and metazolamide). The invention also provides the use of a GDNF protein product in the manufacture of a medicament for the treatment of damage or degeneration of retinal ganglion cells, including the treatment of glaucoma. Various pharmaceutical formulations and transport techniques are described in detail below.
    As used herein, the term “GDNF protein product” refers to a purified natural, synthetic or recombinant glial cell line-derived neurotrophic factor, biologically active GDNF variants (including insertions, substitutions, deletion variants) and derivatives thereof chemically modified. It includes. Also included are GDNF substantially homologous to human GDNF having the amino acid sequence shown in SEQ ID NO: 1. GDNF protein products will exist as homodimers or heterodimers in their biologically active form.
    As used herein, the term "biological activity" means that the GDNF protein product exhibits neurotrophic properties similar to GDNF having the amino acid sequence shown in SEQ ID NO: 1, but need not necessarily have the same properties or to the same extent. The particular neurotrophic properties of interest are selected depending on the use of the GDNF protein product being administered.
    As used herein, the term “substantially homologous” means that there is some homology to the GDNF having the amino acid sequence shown in SEQ ID NO: 1, preferably at least 70%, most preferably at least 80%, and 90 Much more preferable is% or 95%. For example, the degree of homology between rats and human proteins is about 93%, and it is believed that the preferred mammalian GDNF will similarly have high homology. The percent homology described herein is calculated as the percentage of amino acid residues found in the smaller of the two sequences to be in line with the same amino acids of the sequences being compared, where 100 amino acids in length to aid in such alignment. Four gaps can be introduced in (Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, DC (1972), the contents of which are disclosed herein. Citations). Substantially homologous also includes any GDNF protein product that can be isolated by cross-reacting with an antibody against GDNF of SEQ ID NO: 1, or a gene that can be isolated by hybridizing to a gene or gene fragment encoding GDNF of SEQ ID NO: 1 do.
    Any means known to those skilled in the art can separate or produce the GDNF protein product according to the invention. Typical methods for producing GDNF protein products useful in the present invention include US Application No. 08 / 182,183, filed May 23, 1994, and its patent application; PCT Application No. PCT / US92 / 07888, filed Sep. 17, 1992, published as WO 93/06116 to Syntex-Synergen Neuroscience Joint Venture of Lin et al .; European Patent Application No. 92921022.7, published as EP 610 254; And co-owned, co-pending US Application No. 08 / 535,681 filed on Sep. 28, 1995 ("fragmented glial cell line-derived neurotrophic factor"), the disclosures of which are incorporated herein by reference. Cited by literature.
    Naturally occurring GDNF protein products can be isolated from mammalian neuronal cell preparations or from mammalian cell lines that secrete or express GDNF. For example, the separation of GDNF from serum-free growth media of B49 glioma cells is described in WO 93/06116. GDNF protein products may also be chemically synthesized by any means known to those skilled in the art. It is desirable to produce GDNF protein products by recombinant technology because relatively higher amounts of protein can be obtained with higher purity. Forms of recombinant GDNF protein products include glycosylated and aglycosylated forms of this protein and proteins expressed in bacterial, mammalian or insect cell systems.
    In general, recombinant techniques isolate genes encoding GDNF, clone these genes into appropriate vectors and cell types, modify the genes to encode desired variants if necessary, and express these genes to produce GDNF protein products. Includes sikim. Alternatively, nucleotide sequences encoding the desired GDNF protein products may be chemically synthesized. The synonym of genetic code and allelic variants is believed to allow the use of different nucleotide sequences to express GDNF protein products. WO 93/06116 describes the isolation and sequencing of cDNA clones of the rat GDNF gene and the isolation, sequencing and expression of genomic DNA clones of the human GDNF gene. WO 93/06116 also describes vectors, host cells, and culture growth conditions for expressing GDNF protein products. Other vectors suitable for expressing the GDNF protein product in E. coli are described in published European Patent Application No. EP 0 423 980 (“Hepatocyte Factor”), published April 24, 1991, which is incorporated herein by reference. Is incorporated by reference herein. The DNA sequence of the gene encoding the mature human GDNF and amino acid sequence of this GDNF is shown in Figure 19 (SEQ ID NO: 5) of WO 93/06116. FIG. 19 does not show the entire coding sequence for the pre-pro portion of GDNF, but the initial 50 amino acids of human pre-pro GDNF are shown in FIG. 22 (SEQ ID NO: 8) of WO 93/06116.
    Naturally occurring GDNF is a disulfide bound dimer as its biologically active form. Substances isolated after expression in the bacterial system are inherently biologically inert and exist as monomers. Refolding is required to produce biologically active disulfide bound dimers. Procedures for regenerating and naturalizing GDNF expressed in bacterial systems are described in WO 93/06116. Standard in vitro assays for measuring GDNF activity are described in WO 93/06116 and co-owned co-pending US Application No. 08 / 535,681 filed September 28, 1995, which is herein incorporated by reference. Cited as a reference.
    A. GDNF Variants
    As used herein, the term “GDNF variant” refers to the deletion of an amino acid at a residue in an amino acid sequence of a naturally occurring GDNF (“deleted variant”), inserting an amino acid within this residue (“additional variant”), or an amino acid instead of this residue. Polypeptides substituted with (“substituent variants”). Such variants are prepared by introducing appropriate nucleotide changes into the DNA encoding the polypeptide or by chemical synthesis of the desired polypeptide in vitro. Those skilled in the art will recognize that with a combination of many deletions, insertions and substitutions, the final molecule can be prepared to have GDNF biological activity.
    Mutagenesis techniques for substitution, deletion or deletion of one or more selected amino acid residues are well known to those skilled in the art (see, eg, US Pat. No. 4,518,584, the disclosure of which is incorporated herein by reference). Quoted). There are two main variables in the construction of the variant: the location of the mutation site and the characteristics of the mutation. In designing GDNF variants, the choice of mutation site and the characteristics of that mutation will depend on the GDNF characteristic (s) to be modified. Sites for mutation can be mutated individually or continuously, e.g. (1) by first replacing with conservative amino acid selections and then further by radical selection depending on the results to be achieved, and (2) by target amino acid residues. Or by inserting an amino acid residue adjacent to this sited position (3). Conservative substitutions of 1 to 20 amino acids are preferred. Once the amino acid sequence of the desired GDNF protein product is determined, the nucleic acid sequence to be used for expression of that protein is readily determined. N-terminal and C-terminal deletion variants can also be generated using proteolytic enzymes.
    In GDNF deletion variants, the deletion is usually about 1 to 30 residues, more generally about 1 to 10 residues, and typically about 1 to 5 contiguous residues. N-terminal, C-terminal and internal sequences can be deleted. To modify the activity of GDNF, one may delete low homology sites with other TGF-β super families. Deletion within sites substantially homologous to other TGF-β phases and the sequence will significantly further modify GDNF biological activity. The number of consequent deletions will be chosen to preserve the tertiary structure of the GDNF protein product in the deletion domain, eg, cysteine-crosslinked structure. Non-limiting examples of deletion variants include truncated GDNF protein products that lack 1 to 40 N-terminal amino acids of GDNF, variants that lack the C-terminal residues of GDNF, or combinations thereof. Co-owned, co-pending US Application No. 08 / 535,681, filed September 28, 1995, which is incorporated by reference in its entirety.
    For GDNF addition variants, amino acid sequence additions typically include N- and / or C-terminal fusions across polypeptides that include one to 100 or more residues in length, as well as internal sequences of single or multiple amino acid residues. Addition. Usually internal additions can range from about 1 to 10, more generally about 1 to 5 residues, and usually about 1 to 3 amino acid residues. Examples of N-terminal addition variants include GDNF (an artificial product of direct expression of GDNF in bacterial recombinant cell culture) and recombinant host cells carrying an N-terminal methionyl residue, termed [Met- 1 ] GDNF. Fusions in which heterologous N-terminal signal sequences are fused to the N-terminus of GDNF to promote secretion of GDNF are included. Such signal sequences will usually be obtained from the intended host cell species and thus will be homologous to that host cell species. The adduct may also include amino acid sequences derived from sequences of other neurotrophic factors. Preferred GDNF protein products for use in the present invention are recombinant human [Met- 1 ] GDNF.
    GDNF substitutional variants are those wherein at least one amino acid residue in the GDNF amino acid sequence is removed and another residue is inserted therein. Such substitutional variants include allelic variants, which are characterized by naturally occurring nucleotide sequence changes in the population of the species, which in turn may or may not result in amino acid changes. Examples of substitutional variants are described in co-owned, co-pending US Application No. 08 / 535,681, filed Sep. 28, 1995, incorporated herein by reference (see, eg, SEQ ID NO: 50). .
    Specific mutations in the GDNF amino acid sequence will be associated with modifications to the glycosylation site (eg, serine, threonine or asparagine). Non-glycosylation or only partial glycosylation is caused by the substitution or deletion of an amino acid at any asparagine-linked glycosylation recognition site or any site of the molecule modified by addition of a 0-linked carbohydrate. Asparagine-binding glycosylation recognition sites include tripeptide sequences that are specifically recognized by appropriate cellular glycosylation enzymes. This tripeptide sequence is either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro. Various amino acid substitutions or deletions (and / or amino acid deletions at the second position) at one or both of the first or third amino acid positions of the glycosylation recognition site result in non-conformity in the modified tripeptide sequence. Glycosylation results. Thus, expression of the appropriate modified nucleotide sequence produces variants in which the site is not glycosylated. Alternatively, glycosylation sites may be added to modify the GDNF amino acid sequence.
    One method of identifying GDNF amino acid residues or mutagenesis sites is "alanine scanning mutagenesis" as described in Cunningham and Wells (see Science, 244: 1081-1085, 1989). In this way, amino acid residues or groups in the target residues can be identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) to affect the interaction of these amino acids with the surrounding aqueous environment, either inside or outside the cell. Is substituted with a neutral or negatively charged amino acid (alanine or polyalanine is most preferred). The domains that exhibit functional sensitivity to substitution are then purified by introducing additional or replacement residues at the substitution sites. Thus, a target site for introducing amino acid sequence mutations is determined to perform alanine injection or random mutagenesis on the corresponding target codon or site in the DNA sequence, followed by expression of the GDNF expressed for the optimal combination with the desired activity and activity grade. Variants are selected.
    The sites of interest in substitution mutagenesis include sites where amino acids found in various species of GDNF proteins differ substantially in side chain size, charge and / or hydrophobicity. Other sites of interest are those with the same specific residues of GDNF-like proteins obtained from various species. Initially, these sites are replaced in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the category of preferred substitutions. If such substitutions result in a change in biological activity, then a wider variety of substitutions (exemplary substitutions) are introduced and / or other additions or deletions result in screening the resulting product for activity.
    Amino acid substitutions Original residue Preferred Substitution Exemplary Substitution Ala (A) Val Val; Leu; Ile 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) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Leu Leu; Val; Ile; Ala 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 Ile; Leu; Met; Phe; Ala; Norleucine
    Conservative modifications to amino acid sequences (and corresponding modifications to coding nucleic acid sequences) are expected to result in GDNF protein products having properties similar to the functional and chemical properties of native GDNF. In contrast, by selecting substitutions with significantly differing effects in maintaining the following, substantial modifications of the functional and / or chemical properties of the GDNF protein product can be carried out: (a) the polypeptide backbone structure of the substitution region, eg a sheet or Helical conformation, (b) charge or hydrophobicity of the molecule at the target site, or (c) side chain size. Based on general side chain properties, naturally occurring residues are divided into the following groups:
    1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
    2) neutral hydrophilic: Cys, Ser, Thr;
    3) acidic: Asp, Glu;
    4) basic: Asn, Gln, His, Lys, Arg;
    5) residues that influence chain orientation: Gly, Pro; And
    6) Directionality: Trp, Tyr, Phe.
    Non-conservative substitutions may involve the exchange of one of these classes with another. The residues thus substituted can be introduced into GDNF protein sites homologous to other TGF-β phases and proteins, or into non-homologous sites of this molecule.
    B. GDNF Derivatives
    Those skilled in the art given herein can prepare chemically modified derivatives or GDNF variants of GDNF. Most suitable chemical components for derivatization include water soluble polymers. Water-soluble polymers are preferred because the protein binding thereto is not precipitated in an aqueous environment, such as a physiological environment. Preferably, the polymer will be pharmaceutically acceptable in the manufacture of the therapeutic product or composition. Those skilled in the art will be able to select the desired polymer based on whether the polymer protein conjugate will be used therapeutically and, if so, on the desired dosage, cycle time, resistance to proteolysis and other considerations. will be. Effectiveness of induction by administering the derivative in a preferred form (ie, by osmotic pump or more preferably by injection or infusion, or in a more formulated form for oral, pulmonary or other delivery routes) and determining its effectiveness can confirm.
    Suitable water soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, 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 -vinyl pyrrolidone) polyethylene glycols, proppropylene glycol homopolymers, Prolipropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof, including but not limited to. Polyethylene glycol propionaldehyde is advantageous in manufacturing as it is stable in water.
    The polymer may have any molecular weight and may or may not have one. For polyethylene glycols, the preferred molecular weight is in the range of about 2 kDa to about 100 kDa, which is easy to handle and prepare (the term "about" indicates that some molecules in the polyethylene glycol formulation will be greater or less than the molecular weight mentioned). ). According to the desired therapeutic profile (e.g., the desired sustained release duration, effect, biological activity, ease of handling, degree or lack of antigenicity and other known effects of polyethylene glycol on the therapeutic protein or variant, if present) Other sizes may be used.
    The number of polymer molecules attached as described above will vary, and those skilled in the art will be able to ascertain the functional effect. Those skilled in the art can synthesize mono-derived or provide derivatization of di-, tri-, tetra- or some combination with the same or different chemical components (eg, polymers such as polyethylene glycol with different weights). Can be. The ratio of polymer molecules to protein (or peptide) molecules will vary because of their concentration in the reaction mixture. In general, the optimum ratio (efficiency of the reaction) depends on factors such as the degree of derivatization desired (eg mono-, di-, tri-, etc.), the molecular weight of the selected polymer, whether or not the polymer is present and the reaction conditions. In this regard, ie no excess unreacted protein or polymer is present).
    Polyethylene glycol molecules (or other chemical components) should be attached to account for the effect on the functional or antigenic domains of the protein. There are several methods of attachment available to those skilled in the art. See, for example, EP 0 401 384 (PEG bonds to G-CSF), the disclosure of which is incorporated herein by reference, see Malik et al. See also Hematol., 20: 1028-1035, 1992, which reports on polyethylene glycolation of GM-CSF with tresyl chloride. For example, polyethylene glycol can be covalently linked through amino acid residues by a reactor such as a free amino group or a free carboxyl group. Reactors are groups that can be bound to activated polyethylene glycol molecules. Amino acid residues having free amino groups may include lysine residues and N-terminal amino acid residues. Amino acid residues having free carboxyl groups may include aspartic acid residues, glutamic acid residues and C-terminal amino acid residues. A sulfhydryl group may also be used as the reactor for attaching the polyethylene glycol molecule (s). For therapeutic purposes, attachment at amino groups such as attachment at the N-terminus or lysine group is preferred. If receptor binding is required, attachment at residues important for receptor binding should be avoided.
    In particular, technicians may want proteins that are chemically modified at the N-terminus. Those skilled in the art will appreciate that when using polyethylene glycol as an example of the composition, various polyethylene glycol molecules (by molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mixture, the type of polyethylene glycolation reaction to be performed and The selected N-terminus can be selected from the method of obtaining the polyethylene glycolated protein. A method of obtaining an N-terminally polyethylene glycolated agent (i.e., separating this component from other monopolyethylene glycolylated components if necessary) is based on the polyethylene glycolated protein molecular population from the N-terminal polyethylene glycol. It may be to purify the ized material. Selective N-terminal chemical modifications can be performed by reductive alkylation utilizing the differential reactivity of other types of primary amino groups (N-terminal to lysine) available for derivatization of certain proteins. Under appropriate reaction conditions, the carbonyl group-containing polymer obtains selective derivatization of the protein substantially at the N-terminus. For example, technicians may selectively polymerize N-terminal polyethylene glycols by performing a reaction at a pH that allows for a pKa difference between the e-amino group of the lysine residue and the a-amino group of the N-terminal residue of the protein. Can be. By such selective derivatization, the attachment of the water soluble polymer to the protein is regulated as follows: the binding to the polymer occurs preferentially at the N-terminus of the protein and no significant modification of other reactors such as lysine side chain amino groups occurs. . When using reductive alkylation, the water soluble polymer may be of the type described above and should have a single reactive aldehyde that binds to the protein. Polyethylene glycol propionaldehyde, including a single reactive aldehyde, may also be used.
    The present invention relates to the use of derivatives or variants thereof in which prokaryotic-expressing GDNFs are bound to at least one polyethylene glycol molecule, as well as the use of GDNF or variants thereof to which one or more polyethylene glycol molecules are attached by acyl or alkyl bonds. .
    Polyethylene glycolation can be carried out with any of the polyethylene glycolation reactions known in the art. See, eg, Focus on Growth Factors, 3 (2): 4-10, 1992; EP 0 154 316, which is incorporated by reference herein; EP 0 401 384; And other publications cited herein relating to polyethylene glycolation. Polyethylene glycolation can be carried out via acylation or alkylation with reactive polyethylene glycol molecules (or structure-like reactive water soluble polymers).
    Polyethylene glycolation by acylation usually involves reacting an active ester derivative of polyethylene glycol with a GDNF protein or variant. Any known reactive PEG molecule, or the reactive PEG molecule found thereafter, can also be used to polyethylene glycolate GDNF proteins or variants. Preferred activated PEG esters are PEG esterified to N-hydroxysuccinimide. As used herein, "acylation" is intended to include, without limitation, the following types of linkages between water soluble polymers, such as PEG, and therapeutic proteins: amides, carbamates, urethanes, and the like. See Bioconjugate Chem., 5: 133-140, 1994. The reaction conditions may be selected from any of the conditions known in the polyethylene glycolation art or those found thereafter, but conditions of temperature, solvent and pH that inactivate the GDNF or variant to be modified should be avoided.
    Polyethylene glycolation by acylation will generally produce poly- polyethylene glycolated GDNF proteins or variants. Preferably, the linkage bond will be an amide. It is also preferred that the resulting product is only substantially mono-, di- or tri- polyethylene glycolated (eg> 95%). However, some species may be formed which are polyethylene glycolated to a higher degree, depending on the specific reaction conditions used. If desired, more purified polyethylene glycolated species can be separated from the mixture, and in particular by standard purification techniques, including techniques including dialysis, salting out, ultrafiltration, ion exchange chromatography, gel filtration chromatography and electrophoresis. The reacted species can be separated.
    Polyethylene glycolation by alkylation usually involves reacting a terminal aldehyde derivative of PEG with a GDNF protein or variant in the presence of a reducing agent. Polyethylene glycolation by alkylation can also result in poly- polyethylene glycolated GDNF proteins or variants. In addition, the technicians can manipulate the reaction conditions such that polyethylene glycolation is substantially only beneficial at the a-amino group at the N-terminus of the GDNF protein or variant (ie, mono-polyethylene glycolated protein). In either case of monopolyethylene glycolation or polypolyethylene glycolation, it is preferred to add PEG groups to the protein by -CH 2 -NH- groups. In particular with respect to the —CH 2 — group, this type of bond is referred to herein as an “alkyl” bond.
    Derivatization with reductive alkylation resulting in monopolyethylene glycolated products utilizes the differential reactivity of other types of primary amino groups (N-terminal versus lysine) useful for derivatization. This reaction is carried out at a pH at which a pKa difference between the e-amino group of the lysine residue and the a-amino group at the N-terminal residue of the protein is available. Such selective derivatization regulates the attachment of water-soluble polymers, including reactors, such as aldehydes, to proteins: binding to the polymer occurs preferentially at the N-terminus of the protein, with the significance of other reactors such as lysine side chain amino groups. No deformation occurs. In one important aspect, the present invention is directed to substantially the monopolymer / GDNF protein (or variant) conjugate molecule, meaning that the GDNF protein or variant is attached to the polymer molecule only at a single location (ie> 95%). It relates to the use of a homogeneous formulation. More particularly, if polyethylene glycol is used, the present invention also includes the use of polyethylene glycolated GDNF proteins or variants with the potential antigenic linking groups and having polyethylene glycol molecules directly attached to the GDNF protein or variant.
    Thus, it is anticipated that GDNF protein products for use in accordance with the present invention may include polyethylene glycolated GDNF proteins or variants, wherein the PEG group (s) are attached by acyl or alkyl groups. As noted above, such products may be mono- polyethylene glycolated or poly- polyethylene glycolated (eg containing 2-6, preferably 2-5 PEG groups). Although it is usually thought that a PEG group can be attached to a protein at the a-or e-amino group of an amino acid, it is also believed that any amino group attached to the protein can be attached to the PEG group, which amino group is sufficiently reactive to attach to the PEG group under suitable reaction conditions.
    The polymer molecules used in both the acylation and alkylation methods can be selected from the above water-soluble polymers. The polymer chosen to have a single reactor, such as an active ester of acylation or an aldehyde of alkylation, should be modified so that the degree of polymerization can be controlled as provided in this process. Typical reactive PEG aldehydes are water stable polyethylene glycol propion aldehydes or mono C1-C10 alkoxy or aryloxy derivatives thereof (see US Pat. No. 5,252,714). This polymer may or may not be present. In the acylation reaction, the selected polymer (s) should have a single reactive ester group. For the present reductive alkylation, the selected polymer (s) must have a single reactive aldehyde group. Generally, water soluble polymers will not be selected from naturally occurring glycosyl residues because they are usually made more suitable by mammalian recombinant expression systems. This polymer may have any molecular weight and may or may not have one.
    Particularly preferred water soluble polymers for use herein are polyethylene glycols. As used herein, polyethylene glycol is meant to include any of the forms of PEG used to induce other proteins, such as mono- (C1-C10) alkoxy- or aryloxy-polyethylene glycol.
    In general, chemical derivatization can be carried out under all suitable conditions used to react biologically active substances with activated polymer molecules. Methods for preparing polyethylene glycolated GDNF proteins or variants usually include the following steps: (a) GDNF protein or variant is prepared from polyethylene glycol (eg PEG) under conditions such that the protein is attached to one or more Reaction ester or aldehyde derivative) and (b) obtaining the reaction product (s). In general, the optimal reaction conditions for the acylation reaction will be determined from case to case based on known parameters and the desired result. For example, the greater the ratio of PEG to protein, the higher the percentage of poly- polyethylene glycolated products.
    Reductive alkylation resulting in a substantially homogeneous population of mono-polymer / GDNF protein (or variant) conjugate molecules will generally comprise the following steps: (a) under reducing alkylation conditions, i.e., the amino terminus of the GDNF protein or variant Reacting the GDNF protein or variant with a reactive PEG molecule at a pH suitable for selectively modifying the a-amino group in the group; And (b) obtaining the reaction product (s).
    For mono-polymer / GDNF protein (or variant) conjugate molecules, which are a substantially homogeneous population, reductive alkylation reaction conditions are conditions under which the water-soluble polymer component can be selectively attached to the N-terminus of the GDNF protein or variant. Such reaction conditions usually give a pKa difference between the lysine amino group and the a-amino group at the N-terminus (pKa is the pH at which 50% of the amino groups are protonated and 50% are not). This pH also affects the ratio of polymer to protein to be used. In general, lower pHs will require more excess polymer for the protein (ie, the less reactive N-terminal a-amino groups, the more polymer will be needed to achieve optimal conditions). If the pH is higher, the polymer: protein ratio does not have to be as high (ie, fewer polymer molecules are needed because more reactors are used). The pH for the present invention will usually be in the range 3-9, preferably in the range 3-6.
    Another important consideration is the molecular weight of the polymer. In general, the higher the molecular weight of the polymer, the less polymer molecules can be attached to the protein. Similarly, pruning of the polymer should be taken into account when optimizing these variables. In general, the higher the molecular weight (or the more), the larger the polymer to protein ratio. Usually, the preferred average molecular weight for the polyethylene glycolation reaction as intended herein is about 2 kDa to about 100 kDa. Preferred average molecular weights are from about 5 kDa to about 50 kDa, more preferably from about 12 kDa to about 25 kDa. The ratio of water soluble polymer to GDNF protein or variant will usually range from 1: 1 to 100: 1, preferably 1: 1 to 20: 1 (for polyethylene glycolylation) and 1: 1: 5: 1 ( Monopolyethylene glycolation).
    Reductive monocyclization using the conditions described above will result in the selective attachment of the polymer to any GDNF protein or variant having an a-amino group at the amino terminus, and the substantially homogeneous preparation of the monopolymer / GDNF protein (or variant) conjugate Will be provided. As used herein, the term “monopolymer / GDNF protein (or variant) conjugate” refers to a composition consisting of a single polymer molecule attached to a GDNF protein or GDNF variant protein molecule. Preferably the monopolymer / GDNF protein (or variant) conjugate will have a polymer molecule located at the N-terminus rather than the lysine amino side chain group. The formulation will preferably contain at least 90% monopolymer / GDNF protein (or variant) conjugates, and at least 95% of monopolymer / GDNF protein (or variant) conjugates will be more preferred, with the remaining observable molecules reacting. (Ie, a protein lacking a polymer component).
    In the present reductive alkylation, the reducing agent should be stable in aqueous solution and preferably capable of reducing only the Schiff base formed in the initial process of reductive alkylation. Preferred reducing agents can be selected from sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane and pyridine borane. Particularly preferred reducing agents are sodium cyanoborohydride. Other reaction parameters such as solvent, reaction time, temperature and the like and the means for purifying the product may be determined on a case-by-case basis based on published information relating to the derivatization of the protein with a water soluble polymer (see publication cited herein).
    C. Pharmaceutical Compositions of GDNF Protein Products
    Pharmaceutical compositions of GDNF protein products generally comprise a therapeutically effective amount of a GDNF protein product together with one or more pharmaceutical formally acceptable pharmaceutical substances. Suitable formulation materials include, but are not limited to, antioxidants, preservatives, colorants, flavors, excipients, emulsifiers, suspensions, solvents, fillers, swelling agents, buffers, carriers, diluents, prostheses, and / or pharmaceutical adjuvants. Does not. For example, a suitable excipient may be injectable solution, physiological saline or artificial CSF and will be supplemented with other materials common to compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are also typical excipients.
    The main solvent of the excipient will in reality be either aqueous or non-aqueous. In addition, excipients will include other pharmaceutically acceptable excipients to modify or maintain pH, osmolality, viscosity, clarity, color, sterilization, stability, rate of degradation or flavor of the formulation. Similarly, excipients will include other pharmaceutically acceptable excipients to modify or maintain the free rate of the GDNF protein product, or to promote the absorption or penetration of the GDNF protein product across the membrane of the eye. Such excipients are substances commonly and commonly used to formulate dosages for parenteral administration in unit or plural dosage forms.
    Once the therapeutic composition is formulated, it will be stored in sterile vials as a solution, suspension, gel, emulsion, solid or dehydrated powder or lyophilized powder. Such formulations will be stored in an immediate use form or in a form that needs to be reconstituted prior to administration, such as lyophilized.
    The optimal pharmaceutical formulation will be determined by those skilled in the art depending on considerations such as route of administration and preferred dosage. See, eg, Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) p 1435-1712, Remington, which is incorporated herein by reference. Such formulations will affect the physical state, stability, in vivo release and in vivo purification of the present GDNF proteins, variants and derivatives.
    Other effective dosage forms are also contemplated, such as slowly liberal parenteral formulations, inhaled formulations or orally active formulations. For example, in a continuously released formulation, the GDNF protein product may be inserted or incorporated into the particulate formulation of a polymer compound (such as polyacetic acid, polyglycolic acid, etc.) or liposomes. Hyaluronic acid may also be used, which will have the effect of enhancing the duration of blood circulation. Pharmaceutical compositions of GDNF protein products will also be formulated for parenteral administration, for example by intraocular infusion or injection, and will also include slowly liberated or sustained circulating formulations. Such parenterally administered therapeutic compositions are typically in the form of a parenterally acceptable aqueous solution containing the GDNF protein product in a pharmaceutically acceptable excipient that is pyrogenic. One of the preferred excipients is sterile distilled water.
    It is also contemplated that certain formulations containing GDNF protein products will be administered orally. The GDNF protein product administered in this manner may be encapsulated and formulated with or without the carriers commonly used in the preparation of solid dosage forms. Capsules will be prepared to release the active portion of the formulation in the gastrointestinal tract when the bioavailability is at maximum and pre-systemic degradation is minimal. Additional excipients will be included to facilitate uptake of the GDNF protein product. Diluents, spices, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.
    Formulation forms of topical ophthalmic preparations, including ophthalmic solutions, suspensions and ointments are known to those skilled in the art [Pharmamological Sciences, 18th Edition, Chapter 86, p 1581-1592, Mack Publishing Company, 1990] See literature. Other methods of administration are also available, including anterior infusion (which can be administered directly to the anterior or vitreous cavity), subconjunctival infusion, and post-ocular implantation, and methods and means for producing suitable eye drops for such administration methods are also known. have.
    As used herein, "inner eye" refers to the eye surface and (outer) space between the eye and the eyelid. The eyelid palate, the cyst, the surface of the conjunctiva, and the surface of the cornea belong to the external eye region. This site is external to all inner tissues and does not need to be reached by this infiltration process. Examples of the extraocular system include inserts and “locally” dropping drops, gels or ointments that are used to transport therapeutic material to these sites. Intraocular instruments are generally easy to remove, even in patients.
    The following patent document describes an extraocular system used to administer a drug to the extraocular site. Higuchi et al. Describe biodegradable ocular inserts containing drugs in US Pat. Nos. 3,981,303, 3,986,510 and 3,995,635. This insert can be made in other forms to preserve the extraocular space between the eye and the eyelid, and the ocular sac. Several common biocompatible polymers are also described that are suitable for use in the fabrication of this device. These polymers include zinc alginate, poly (lactic acid), poly (vinyl alcohol), poly (anhydrides) and poly (glycolic acid). The patent document also describes a membrane coating apparatus that reduces penetration into the drug and a hollow chamber that accommodates the drug formulation.
    Theeuwes describes a micropore reservoir used to regulate drug transport in US Pat. No. 4,217,898. These instruments are put into and out of the eye bag. Important among the polymer systems are poly (vinylchloride) -co-poly (vinyl acetate) copolymers. Kaufman, in US Pat. Nos. 4,865,846 and 4,882,150, describes an ophthalmic transport system comprising at least one biodegradable substance or ointment carrier to the conjunctival sac. Also described are crosslinked collagen suitable as polymer and transport systems, such as poly (lactide), poly (glycolide), poly (vinyl alcohol).
    In the use of the GDNF protein products described for the treatment of retinal diseases or damage, the topical ophthalmic formulations also preferably include drugs that aid in the penetration or transport of the therapeutic drug into the eye. Such drugs are known in the art. For example, US Pat. No. 5,221,696 to Ke et al. Describes the use of materials to enhance penetration of ocular agents through the cornea.
    The intraocular system is a system suitably used for all tissue components within, between, or around the tissue layer of the eye itself. These locations include subconjunctival (below the mucosa adjacent to the eye), orbital (behind the eye) and anterior (in the chamber of the eye). In contrast to the extraocular system, it is necessary to reach these sites by an infiltration process consisting of injection or transplantation.
    The following patent document describes a guide mechanism. Wong, US Pat. No. 4,853,224, describes a drug encapsulated in microcapsules that is introduced into the eye chamber. Polymers used in these systems are polyesters and polyethers. Lee's US Pat. No. 4,863,457 describes a biodegradable instrument that is surgically implanted to allow continuous release of a therapeutic drug. The instrument is designed to be implanted under the conjunctiva. US Patent No. 4,188,373 to Krezancaki describes a pharmaceutical excipient that gels at human body temperature. This excipient is an aqueous suspension of the drug and synthetic derivatives derived from rubber or cellulose. Haslam et al., In US Pat. Nos. 4,474,751 and 4,474,752, describe polymer-drug systems that are liquid at room temperature and gel at body temperature. Suitable polymers for use in the present system include polyoxyethylene and polyoxypropylene. Davis et al. Describe a biodegradable injectable drug transport polymer that prolongs drug release in US 5,384,333. Such drug compositions are formulated with pharmaceutically active drugs against biodegradable polymer matrices, wherein the polymer matrix is solid when the temperature is between 20 ° and 37 ° C. and fluid at 38 ° to 52 ° C. Drug transport polymers are not limited to the transport of solid or liquid drug formulations. For example, the polymer can be used as a substrate to ensure that the drug containing the centrosome, liposome or other particle-binding drug remains stable at the injection site.
    Particularly suitable excipients for intraocular injection are sterile distilled water and formulate the GDNF protein product in appropriately stored sterile isotonic solution. However, other ophthalmic agents include formulating a GDNF protein product with a drug such as an injectable centroid or liposome so that the protein is released slowly or continuously, which protein is transported by accumulation injection. Other suitable means for introducing the GDNF protein product into the intraocular includes implantable drug transport mechanisms.
    The ocular therapeutic formulations, in particular topical formulations, of the present invention may contain other ingredients such as eye preservatives, tonics, cosolvents, wetting agents, combinations, buffers, antimicrobials, antioxidants and surfactants as are well known in the art. Will contain. For example, suitable tension enhancers include alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol, and the like. It is desirable to add sufficient tension enhancer such that the formulation to be injected into the eye is hypotonic or substantially isotonic. Suitable preservatives include, but are not limited to benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and the like. Hydrogen peroxide can also be used as a preservative. Suitable cosolvents include, but are not limited to, glycerin, propylene glycol and polyethylene glycol. Suitable co-agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agents include, but are not limited to, sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapol, and the like. The buffer may be a conventional buffer such as borate, citrate, phosphate, bicarbonate or tris-HCl.
    Formulation ingredients are present in acceptable concentrations at the intraocular or intraocular administration site. For example, a buffer is used to maintain the composition at a physiological pH or slightly lower pH, generally at a pH in the range of about 5 to about 8.
    In order to maximize local contact and promote absorption, the additional formulation component may include a substance that allows the therapeutic agent administered intraocularly to stay longer in the eye. Suitable materials include polymers or gel forming materials that increase the viscosity of the ophthalmic agent. Particularly suitable materials for glass-rate regulators which continuously release the ophthalmic liquid formulation are chitosan (see US 5,422,116 to Jen et al.). Suitability of the formulation of the invention for controlling the release of ophthalmic agents (eg, sustained transport and extension thereof) can be determined by various methods known in the art. See, eg, Journal of Controlled Release, 6: 367-373, 1987 and variations thereof.
    Other eye drops will still contain an effective amount of GDNF protein product in admixture with excipients that are acceptable for non-toxic eye treatment, which are suitable for tablet manufacture. By dissolving the tablet in sterile water or other suitable excipient, the ophthalmic solution may be prepared in unit dosage form. Suitable excipients include inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; Or binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc.
    Administration / Transport of GDNF Protein Products
    GDNF protein products can be administered parenterally via subcutaneous, intramuscular, intravenous, intrapulmonary, transdermal, intradermal or intracerebral routes. To treat the condition of the eye, the GDNF protein product may be administered intraocularly or intraocularly by topical application, insertion, injection, transplantation, cell therapy or gene therapy. For example, a slowly released insert containing neurotrophic factors embedded in a biodegradable polymer matrix can transport the GDNF protein product. The GDNF protein product will be administered extracellularly in chemically modified or packaged form to allow it to cross the blood brain barrier, or in combination with one or more drugs that can help penetrate the GDNF protein product across the barrier. Similarly, the GDNF protein product will be administered intraocularly or in or out with one or more drugs that can aid in the penetration or transport of the GDNF protein product across the membrane of the eye. The frequency of administration will depend on the pharmacodynamic parameters and route of administration of the GDNF protein product being formulated.
    The specific dosage will be calculated taking into account weight, body surface area or organ size. More accurate calculations need to be made to determine the appropriate dosage for treatment with each of the formulations mentioned above are routinely made by those skilled in the art, especially in view of the dosage information and assays described herein. Belongs to the scope of work being done. Appropriate dosages will be determined using predetermined assays to determine the dosage used in conjunction with specific dosage-response data. It will be apparent to those skilled in the art that the dosage used in the intraocular dosage form is very small compared to the amount used for systemic injection or oral administration.
    The final dosing regimen associated with the treatment of the condition is determined by the attending physician, taking into account several factors that alter drug action: age, condition, weight, patient's sex and usual food, severity of infection, time of administration and other clinical factors. Will be decided by. As the study progresses, more information will be poured out on appropriate dosage levels to treat various diseases and conditions.
    Continuous administration or continuous transport of GDNF is believed to be desirable for a given treatment. Continuous administration can be via mechanical methods such as using infusion pumps, while other modes of administration are expected to be continuous or nearly continuous. For example, chemical induction or encapsulation results in a sustained free form of the protein with a continuous presence effect in an amount that can be determined based on the determined dosage regimen. Thus, GDNF protein products include proteins formulated to induce or otherwise formulate such continuous administration to be effective.
    GDNF protein product cell therapies, such as intraocular transplantation of cells producing GDNF protein products, are also contemplated. This embodiment relates to transplanting a cell into a patient capable of synthesizing and secreting a biologically active form of GDNF protein product. This GDNF protein product-producing cell is a natural production cell of GDNF protein product (similar to B49 glioblastoma cells) or transduced with a gene encoding a desired GDNF protein product in a vector suitable for promoting its expression and secretion. It will be a recombinant cell with the ability to produce a GDNF protein product that is increased by conversion. In order to minimize the immune response that can occur in patients to which GDNF protein products of foreign species are administered, natural cells producing GDNF protein products are of human origin and preferably produce human GDNF protein products. Transplanted cells will encapsulate to prevent infiltration around the tissue. Human or non-human animal cells will be implanted into the patient into a biocompatible, semipermeable polymeric inclusion body or membrane that releases GDNF protein products but prevents other factors harmful from surrounding tissue or cell destruction by the patient's immune system. For example, an implant would attach to the sclera to produce the GDNF protein product and release it directly into the vitreous fluid.
    It is also contemplated that the patient's own cells will also be transformed ex vivo to produce the GDNF protein product and will be directly transplanted without encapsulation. For example, retinal neurons will be harvested, transformed with the appropriate vector to culture the cells and then transplanted back into the retina of the patient to produce and release the desired GDNF protein or GDNF protein variant.
    In vivo GDNF protein product gene therapy can also be contemplated by inserting the coding gene for the GDNF protein product into the target retinal cells via local injection of nucleic acid constructs or other suitable transport vectors [Hefti, J. Neurobiol., 25: 1418. -1435, 1994]. For example, nucleic acid sequences encoding GDNF protein products will be contained in adeno-binding viral vectors or adenovirus vectors for transport to retinal cells. Alternative viral vectors include, but are not limited to, retroviruses, Herpes simplex virus, papillomavirus vectors. Suitable physical transitions in vivo or ex vivo are liposome-mediated transitions, direct injection (as-is), receptor-mediated transitions (ligand-DNA complexes), electroporation, calcium phosphate precipitation or particulate bombardment (gene injectors). It can be achieved by
    The methodology for membrane encapsulation of living cells is familiar to those skilled in the art, and the preparation of encapsulated cells and transplantation into patients will be done without unnecessary experimental work. 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. Encapsulated viable cell lines are described in PCT Application WO91 / 10425 to Aebischer et al., Incorporated herein by reference. PCT Application WO91 / 10470 to Aebischer et al., Incorporated herein by reference, and Exper. Neurol., 113: 322-329, 1991, Exe. Neurol., 111: 269-275, 1991; See also Tresco et al., ASAIO, 38: 17-23, 1992. Various formulation techniques of sustained- or controlled-transportation methods such as liposome carriers, biodegradable particles or beads and cumulative injections are also known to those skilled in the art.
    The GDNF protein product formulation form described herein will apply to humans as well as animals, and the term "patient" is not to be interpreted in a limited form. When applied to animals, the dosage range will be as described above.
    Other aspects and advantages of the invention will be appreciated with reference to the following examples. Example 1 describes the effect of administration of GDNF protein products on retinal ganglion cell tissue culture systems. Example 2 uses radiolabeled GDNF protein products to experiment with a population of neurons capable of binding, internalizing and transporting GDNF protein products in a siphon-mediated manner. Example 3 describes the effect of administration of GDNF protein products on retinal ganglion cell injury models. Retinal ganglion cell damage studies have shown that GDNF protein products have neurotrophic activity against this neuronal population that was previously unknown to be GDNF-reactive.
    Example 1
    GDNF protein products may be used for in vitro retinal ganglion cells.
    Helps survive and develop
    matter
    The material used in the following examples was obtained as follows.
    Cell culture medium
    Dulbecco's modified Eagle's medium of high glucose (DMEM; # 11965-092), F12 medium of ham (F12; # 11765-021), Ribowitz L15 medium without sodium bicarbonate (# 41300-039); B27 medium supplement (# 17504-010), penicillin / streptomycin (# 15070-014), L-glutamine (# 25030-016), Dulbecco's phosphate buffered saline (D-PBS; # 14190-052), with calcium Hank's balanced salt solution containing magnesium salt (HBSS; # 24020-026), N -2-hydroxyethylpiperazine-N '-2 ethanesulfonic acid (HEPES; # 15630-015), mouse laminin (# 23017- 015), bovine serum albumin and fraction V (# 110-1817) were all obtained from Gibco / BRL, Grand Island, NY. Equine heat-inactive serum was obtained from high clones in Logan, Utah. Poly-L-ornithine hydrobromide (P-3655), bovine insulin (I-5500), human transferrin (T-2252), putrescine (P-6608), progesterone (P-6149) and selenic acid Sodium (S-9133) was purchased from Sigma Chemical Company, St. Louis, Missouri. Papain, deoxyribonuclease I (DNA aze) and egg albumin (papaine separation system) were purchased from Waterington Biochemicals, Freehold, NJ. Falcon sterile 96-well microplates (# 3072), tissue culture plastic articles, and polypropylene centrifuge tubes were purchased from Becton-Dickinson, Oxnard, CA. Nitex 20 μm nylon mesh (# 460) was purchased from Tetko, Elmford, NY. 4 "anatomical tweezers and 4" anatomical scissors were purchased from Robots Surgical, Washington, DC, USA.
    Antibodies and Related Reagents
    Mouse / rat monoclonal antibodies against mouse Thy-1, 2 were obtained from Schöllinger-Manheim (Indianapolis, Indiana). Rabbit polyclonal anti-rat and anti-mouse IgG antibodies, biotinylated horse anti-mouse IgG and peroxidase-conjugated avidin / biotin complexes (ABC Elite; Kit PK-6100) are based in Burlingame, California, USA. It was purchased from Vector Laboratories. 3 ', 3'-diaminobenzidine was purchased from Capel Laboratoris, West Chester, Pennsylvania. Superblock blocking buffer in PBS was purchased from Pierce, Rockford, Illinois, USA. Triton X-100 (X100), Nonidet P-40 (N6507) and hydrogen peroxide (30%, v / v; H1009) were purchased from Sigma. All other reagents were purchased from Sigma Chemical Company, St. Louis, MO, unless otherwise specified.
    Way
    Badge manufacturing
    Basal medium was prepared from a 1: 1 mixture of DMEM and F12 medium and supplemented with B27 medium supplement added with 50-fold concentrated stock solution. B27 medium supplements include biotin, L-carnitine, corticosterone, ethanolamine, D (+)-galactose, reduced glutathione, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, selenium, T3 (triodo- 1-tyronine, DL-alpha-tocopherol; vitamin E), DL-alpha-tocopherol acetate, bovine serum albumin, catalase, insulin, superoxide dismutase and transferrin. L-glutamine was added at a final concentration of about 2 mM, penicillin at about 100 IU / l and streptomycin at about 100 mg / l. Equine heat-inert serum is added to a final concentration of about 2.5%, D-glucose has a final concentration of about 5 g / l, HEPES buffer about 20 mM, bovine insulin about 2.5 mg / ml, human transferrin It was added to about 0.1 mg / ml. After mixing, the pH was adjusted to about 7.3 and the medium was kept at 4 ° C. In order to minimize deformation during the experiment, the medium was freshly prepared immediately before use. Plastic pipettes and vessels were used throughout to minimize protein adsorption.
    GDNF Protein Product Solution
    Purified human recombinant GDNF protein product was prepared as a 1 mg / ml solution in D-PBS (phosphate buffered saline prepared with distilled water) containing 5% bovine serum albumin. The solution was aliquoted and stored at -85 ° C. Serial dilutions were prepared on 96-well microplates. 10 μl of a 10 times concentrated GDNF protein product solution was added to the cell medium containing the culture medium (90 μl). Control medium was added D-PBS with 5% albumin (10 μl). One hour after seeding the cells, GNDF protein product treatment was started and, if necessary, repeated every other day.
    Badge underlayer
    To aid in optimal binding of spiral ganglion cells to the lower layer and neurite side branches, the surface of the microtiter plate (lower medium) was modified by subsequent coating with poly-L-ornithine followed by laminin according to the following method. The plate surface was completely covered with 0.1 mg / ml polyornithine sterile solution in 0.1 M boric acid (pH 8.4) at room temperature for at least 1 hour, followed by sterile washing with Super-Q water. The wash solution was extracted and then 1 μg / ml mouse laminin solution in PBS was added and incubated at 37 ° C. for 2 hours. This procedure was performed just before using the plate to ensure the reproducibility of the results.
    Preparation of Retinal Ganglion Cell Medium in Rats
    Rats 7 days old (obtained from Jackson Laboratories, Harbor, Maine, USA) were killed and killed, and eyes were sterilely transferred to L15 medium without sodium bicarbonate. Up to 24 eyes were treated per experiment. The eyes were cut in half to remove the lens and grass paper. The neural retina was carefully removed, the pigmented epithelium was excised, cut into small pieces (about 1 mm 2 or less) and placed in ice-cold D-PBS. The cells were collected and then transferred to 10 ml of separation medium (120 units of papain and 2000 units of DNA aze in HBSS). The cells were incubated for 45 minutes at about 37 ° C. on a rotating platform shaker at about 200 rpm. Cells were dispersed by grinding through a fire-paste pipette and distributed in 20 μm nylon mesh nets to remove unseparated tissue and centrifuged at 200 × g for 5 minutes using an IEC clinical centrifuge. The resulting cell pellet was resuspended in HBSS containing egg albumin and about 500 units of DNAase, placed on top of 4% egg albumin solution (in HBSS) and centrifuged at 500 × g for about 10 minutes. The final pellet was resuspended to a concentration of 10 7 cells / ml in DMEM / F12 supplemented with 20% fetal bovine serum, and retinal ganglion cells were purified by immunopanning according to the method described above (J. Neurosci et al., Lehwalder et al.). Res., 24: 329-337, 1989).
    At panning, 35-mm tissue culture plastic dishes were pre-coated with anti-mouse IgG antibody (1: 100 dilution with PBS) for 2 hours at room temperature, washed three times with PBS containing 1% fetal bovine serum and then Thy- Incubated with mouse monoclonal antibodies for 1, 2 (1: 100 dilution). After washing the dish with DMEM / F12, about 1 ml of retinal cell suspension was incubated in a culture dish pretreated for 1 hour at room temperature. Unattached cells were removed by repeated washing with culture medium until only tightly attached cells remained. Bound cells were collected with a rubber polyman and resuspended in complete culture medium (as described above). The cell concentration was adjusted to about 11,000 cells / ml and the cell suspensions were inoculated at 90 μl at a density of about 1,000 cells / 6-mm well aliquots in 96-well microplates precoated with polyornithine and laminin medium. The attachment of the cells occurred quickly, and the plate efficiency was about 50%.
    Immunohistochemistry of Retinal Ganglion Cells
    To identify retinal ganglion cells in rats, indirect immune peroxidation as described in Louis et al. (J. Pharmacol. Exp. Therap., 262: 1274-1283, 1992; Science, 259: 689-692, 1993). The enzyme method was used with a slight modification as follows. Retinal ganglion cell medium was fixed with 4% paraformaldehyde in D-PBS, pH 7.4 for about 30 minutes at room temperature and washed three times with D-PBS (200 μl per 6-mm well). Fixed medium was incubated in superblock blocking buffer in PBS containing 1% Nonidet P-40. Monoclonal anti-Thy-1 antibodies (purchased from Schöllinger-Manheim) were added to 1: 100-1: 400 dilutions in the same buffer and the medium was incubated for 1 hour at 37 ° C. on a rotary shaker. . After three washes with D-PBS, retinal ganglion cell-binding antibodies using equine anti-mouse biotinylated IgG (Vectasterin kit purchased from Vector Laboratories, Burlingame, Calif.) At approximately 1: 500 dilution. Was detected. The secondary antibody was incubated with the cells at 37 ° C. for about 1 hour and then incubated at 37 ° C. for about 45 minutes. The secondary antibody was labeled with an avidin-biotin peroxidase complex diluted 1: 500 and the cells were incubated at 37 ° C. for about 45 minutes. After washing three more times with D-PBS, label in 0.1 M Tris-HCl, pH 7.4 solution containing 0.04% 3 ′, 3′-diaminobenzidine- (HCl) 4 , 0.06% NiCl 2 and 0.02% hydrogen peroxide Cell medium was reacted for 5-20 minutes.
    Determination of Retinal Ganglion Cell Survival Rate
    After several hours of culture (24 hours, 3 days and 6 days), the rat retinal ganglion cell medium was fixed and treated and immunostained for Thy-1, 2 as above. The medium was then observed at 200 × magnification with bright-light optics. The number of all Thy-1, 2-positive neurons present in 6-mm wells was counted. Viable retinal ganglion cells are characterized by regular shaped cell bodies ranging in size from 30-40 μm and having approximately three long neuritic processes. The number of retinal ganglion cells showing signs of degeneration, such as having an irregular vesicular periplasmic nucleus or collapsed neurites, was not counted (but most degenerative retinal ganglion cells were isolated from the media underlayer). Cell number is shown as one of the relative fold changes for cell / 6-mm wells or control cell density.
    result
    Purified rat retinal ganglion cell medium was used to explain the effect of GDNF protein products on survival and morphological maturation. Retinal ganglion cells were purified from the retina of 7 days old rats by immunopanning against plastic surfaces coated with anti-Thy-1, 2 antibodies. In the rat retina, the Thy-1 antigen is known to be located in retinal ganglion cells (see Leifer et al. Exp. Neurol., 113: 386-390, 1991). Pure results of retinal ganglion cell medium at a density of about 1,000 per 6-mm well in DMEM / F12 supplemented with B27 medium supplement, 2.5% horse's heat-inactive serum, D-glucose, HEPES, insulin and transferrin Pure retinal ganglion cell medium was made by inoculating the populations with polyornithine-laminin-coated microplates. Retinal ganglion cells were identified by the presence of Thy-1, 2 immunoreactivity. In this way, a relatively low amount of retinal ganglion cells (about 5,000 cells / retina) was obtained but the purity was high (about 90% of Thy-1, 2-positive cells).
    Rat retinal ganglion cell medium was evaluated for the effect of administration of GDNF protein product on survival and morphological maturation. Retinal ganglion cell medium was treated with human recombinant GDNF protein product (3-fold passage dilution over 10 ng / ml-1 pg / ml). Medium was fixed in vitro after 24 hours, 3 days and 6 days, 4% paraformaldehyde immobilized and then immunostained for Thy-1, 2. For medium not treated with GDNF protein product, only about 32 ± 4% (n = 6) of the inoculated retinal ganglion cells survived in the medium after 24 hours. About 11 ± 3% (n = 6) of the initially retinal ganglion cells were present after 3 days, and only 3 ± 2% (n = 3) were found in the medium after 6 days. Treatment of the medium with E. coli-expressing recombinant human GDNF protein product resulted in an approximately 2.5-fold increase in the number of retinal ganglion cells that could survive in the medium after 24 hours (80 ± 6, n = 6). After 3 days in the presence of the GDNF protein product, about 50 ± 5% (n = 5) of the original retinal ganglion cell numbers survived (4.5 fold increase compared to control), and after about 6 days, about 39 ± 4% (n = 3) was able to survive (9.6-fold increase compared to control). After 3 days in vitro, the effect of GDNF protein product on retinal ganglion cells was about 30 pg / ml with ED50 of about 300 pg / ml.
    In addition to promoting the survival of retinal ganglion cells, GDNF protein products stimulated its morphological development. Treatment with GDNF protein products increases the cell body size and length and branching of neuritic processes of retinal ganglion cells. After 3 days in vitro, many retinal ganglion cells in GDNF treated media had neurites of 500-1000 μm in length, whereas the longest neurites found in control medium were 300 μm in length or less.
    Example 2
    Reverse Transport of GDNF Protein Products in Retinal Ganglion Cells
    In this experiment, 125 I-radiolabeled GDNF protein products were injected into the brains of adult rats to identify neuronal populations that could bind, internalize, and transport GDNF back in a receptor-mediated manner. The GDNF protein product tested was recombinant human [Met- 1 ] GDNF and was expressed and produced in E. coli as generally described in Examples 6B and 6C of WO93 / 06116. Purified [Met- 1 ] GDNF was iodinated using the lactoperoxidase technique and G-25 Sephadex Quick Spin Column (G) as described in Yan et al., J. Neurobiol., 24: 1555-77, 1993. 25 Sephadex quick spin columns) were used to separate from glass 125 I. 43 mg / ml of ketamine hydrochloride, 8.6 mg / ml of xylazine and 1.43 mg / ml of acepromazine cocktail were anesthetized mature Sprag-Doleley rats at a dose of 0.7 ml / kg body weight. 1 the group consisting of 6 rats for, PBS / BSA in the 111-fold excess of unlabeled [Met -1] 125 μl of 5 carrying the GDNF I - [Met -1] GDNF ( total of 4 - 5 × 10 6 cpm of radiation) or 5 μl of 125 I-[Met- 1 ] GDNF were injected stereotactically into the right lateral ventricle with a 5 μl Hamilton syringe. Group 2, consisting of six rats, was injected in the middle of the right striatum with 1 μl of 125 I- [Met- 1 ] GDNF with or without 111-fold unlabeled [Met- 1 ] GDNF. Group 3, consisting of six rats, was injected into the gut with 1 μl of 125 I- [Met- 1 ] GDNF with or without 111-fold unlabeled [Met- 1 ] GDNF. In all cases of injection, the injection rate was 0.1 μl per 15 seconds.
    After 20-24 hour survival, animals were killed and perfused with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2. The brain was removed to allow cold defense. Rats injected with the upper mouth were killed, then their eyes were taken out and immersed-fixed with the same fixative. The radioactivity of the eyes was measured by scintillation counter. Observational sections of the frozen brain were cut into sliding microtomes 25 μm thick and 10 μm sections of the eyes were cut with cold bath. The sections were placed in a slide glass and lightly moistened with Kodak NTB-3 emulsion. Exposure time varied from 15 to 30 days. The slides were developed and counterstained with toluidine blue and observed under a microscope.
    After tissue parenchymal or cerebral ventricular (ICV) injection into the central nervous system (CNS), uptake and transport of 125 I-[Met -1 ] GDNF was observed in dopamine-functional neurons in the black matter and ventral canine region. Injection of excess unlabeled [Met- 1 ] GDNF was able to completely inhibit neuronal labeling by ICV or intraparenchymal injection of 125 I-[Met- 1 ] GDNF. These dopaminergic neurons biologically reactive to GDNF were expected to be able to express functional GDNF receptors with the ability to absorb and reverse transport. Thus, the specific labeling of these dopaminergic neurons, injected IC125 and tissue parenchyma with 125 I-[Met- 1 ] GDNF, is preferred as a positive control.
    In addition, low levels of neuronal markers were observed in the parenchyma and the central nucleus of the mastectomy following ICV injection. Labeled [Met- 1 ] GDNF uptake was inhibited by injecting an excess of unlabeled [Met- 1 ] GDNF together, suggesting that uptake occurs through a receptor-mediated mechanism. In the parenchymal parenchyma or after ICV injections, no markers were observed in the other mature or all other neuronal populations around the rod nucleus in the brains of whole mature rats.
    125 I-[Met -1 ] GDNF was injected into the upper gut in an attempt to find other neuronal populations in the eye, for example, that did not access the ventricle but could absorb and back transport GDNF. Radioactivity observed by direct counting was selectively accumulated in the ipsilateral region rather than the ipsilateral eye. This selectivity is probably due to the cross-protection of retinal ganglion cells against the ipsilateral superior. Accumulation of radioactivity can be inhibited by co-administration of excess unlabeled [Met- 1 ] GDNF. Autoradiograms of the cross sections of these eyes showed that radioactivity was specifically associated with retinal ganglion cells. As a result, 125 I-[Met -1 ] GDNF was shown to be selectively transported to ipsilateral retinal ganglion cells via ipsilateral receptor-mediated mechanisms.
    Example 3
    Retinal Ganglion Cells Through Administration of GDNF Protein Product
    Promotion of survival
    In this experiment, the effect of the GDNF protein product or excipient administered to the optic nerve axon cutting animal model was determined by Proc. Natl. Acad. Sci., 91: 1632-1636, 1994 were evaluated according to the method described in the literature. In this model, axon cutting of the optic nerve causes the death of retinal ganglion cells in mature rats. Retinal ganglion cells were pre-labeled by applying fluorogold-soaked gel foam to the surfaces of both the right and left upper globules of mature female Sprague-Doleley rats. One week after the addition of fluorogold (day 0), the right optic nerve was cut transversely 0.5 mm from the eye (n = 3-6 of each experiment). The left eye is an intrinsic contrast that represents the marker of retinal ganglion cells.
    As a negative control, animals were treated by intraocular injection of 1 μl of [Met- 1 ] GDNF (1 mg / ml in PBS) or cytochrome C into the right eye on days 0 or 7. Axon cutting and drug treatment effects were tested on day 7 or day 14 after optic nerve cutting, and are shown in Table 2.
    groupInjection dateExperiment day One07 2014 30, 714
    The eyes were immersed for 1 hour in 4% paraformaldehyde. The direction of the retina was indicated, and then the retina was dissected and the whole was placed on a slide glass and photographed under a fluorescence microscope. Retinal ganglion cells were counted in the photograph. Axons with or without cytochrome C (negative control) significantly reduced the number of labeled retinal ganglion cells on day 7, and further decreased on day 14. Treatment with [Met- 1 ] GDNF on day 0 completely rescued retinal ganglion cells on days 7 and 14. These results demonstrate that retinal ganglion cells are sensitive to GDNF and treated with GDNF protein products promote the survival of damaged ganglion cells, a major population of neurons damaged by glaucoma.
    In the practice of the present invention, it is contemplated that many variations and modifications may be made by those skilled in the art with reference to the preferred embodiments of the foregoing detailed description. As a result, what is most suitably limited to the scope of the present invention is those shown in the appended claims.
    Sequence list
    (1) General Information:
    (Ⅰ) Applicant: Yan, Qiao
    (Ii) Name of the invention: glial cell line-derived neurotrophic factor (GDNF)
    Retinal Ganglion Cells Using Protein Products
    Damage Treatment
    (Iii) Number of sequences: 1
    (Iii) communication address:
    (A) Recipient: Amgen Incorporated
    (B) Street: Dehavilland Drive 1840
    (C) Poetry: Thousand Oaks
    (D) State: California
    (E) Country name: United States
    (F) ZIP Code: 91320
    (Iii) computer-readable form:
    (A) Media Type: Floppy Disk
    (B) Computer: IBM PC compatible model
    (C) operating system: PC-DOS / MS-DOS
    (D) Software: PatentIn Release # 1.0, Version # 1.25
    (Iii) Current application data:
    (A) Application number:
    (B) Date of application:
    (C) classification:
    (Iv) Agent / Manager Information:
    (A) Statement: Curry, Daniel R.
    (B) Registration Number: 32, 727
    (C) Reference / Document Number: A-360
    (Iii) communication information:
    (A) Phone number: 805-447-8102
    (B) Fax: 805-499-8011
    (C) Telex:
    (2) Information about SEQ ID NO: 1
    (Iii) Sequence features:
    (A) Sequence length: 134 amino acid residues
    (B) Type of sequence: amino acid
    (D) topology: linear
    (Iii) Characteristic part:
    (A) Characteristic symbol: amino acid sequence deduced on mature human GDNF
    权利要求:
    Claims (10)
    [1" claim-type="Currently amended] Use of a glial cell line-derived neurotrophic factor (GDNF) protein product for the manufacture of a pharmaceutical composition for treating damage or degeneration of retinal ganglion cells.
    [2" claim-type="Currently amended] Use according to claim 1, characterized in that damage or degeneration of retinal ganglion cells is associated with glaucoma.
    [3" claim-type="Currently amended] Use according to claim 2, characterized in that the pharmaceutical composition further comprises another therapeutic agent for treating glaucoma.
    [4" claim-type="Currently amended] Use of glial cell line-derived neurotrophic factor protein products for the manufacture of pharmaceutical compositions for the treatment of damage or degeneration of retinal ganglion cell axons that make up the optic nerve.
    [5" claim-type="Currently amended] 5. Use according to claim 4, characterized in that the damage or degeneration of the optic nerve is associated with conditions detrimental to the optic head, selected from the group consisting of glaucoma, papilloma edema, papitis and ischemia.
    [6" claim-type="Currently amended] Use according to any one of claims 1 to 5, characterized in that the pharmaceutical composition comprises the GDNF amino acid sequence set forth in SEQ ID NO: 1 or variants or derivatives thereof.
    [7" claim-type="Currently amended] Use according to claim 6, characterized in that the pharmaceutical composition is [Met- 1 ] GDNF.
    [8" claim-type="Currently amended] 7. Use according to claim 6, wherein the derivative comprises a water soluble polymer.
    [9" claim-type="Currently amended] Use according to any one of claims 1 to 5, characterized in that the pharmaceutical composition is a continuously released pharmaceutical composition.
    [10" claim-type="Currently amended] Use according to any one of the preceding claims, characterized in that the composition comprises cells which have been mutated to produce and secrete GDNF protein products.
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    同族专利:
    公开号 | 公开日
    ES2148826T3|2000-10-16|
    US5641749A|1997-06-24|
    EP0866719A1|1998-09-30|
    IL124600D0|1998-12-06|
    CZ149998A3|1998-08-12|
    DE69609084D1|2000-08-03|
    WO1997019695A1|1997-06-05|
    BR9611748A|1999-06-01|
    DK0866719T3|2000-08-28|
    PT866719E|2000-11-30|
    NO982276L|1998-07-29|
    EP0866719B1|2000-06-28|
    CA2236158A1|1997-06-05|
    JP4066201B2|2008-03-26|
    MX9803968A|1998-09-30|
    HU0001078A2|2000-08-28|
    SK65898A3|1999-01-11|
    DE69609084T2|2000-11-16|
    AU1163197A|1997-06-19|
    AT194081T|2000-07-15|
    DK866719T3|
    AU696772B2|1998-09-17|
    CA2236158C|2002-07-16|
    GR3034194T3|2000-11-30|
    NO982276D0|1998-05-19|
    JP2000501393A|2000-02-08|
    CN1203531A|1998-12-30|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1995-11-29|Priority to US8/564458
    1995-11-29|Priority to US08/564,458
    1996-11-22|Application filed by 스티븐 엠. 오드레, 암젠 인코포레이티드
    1996-11-22|Priority to PCT/US1996/018851
    1999-09-27|Publication of KR19990071541A
    优先权:
    申请号 | 申请日 | 专利标题
    US8/564458|1995-11-29|
    US08/564,458|US5641749A|1995-11-29|1995-11-29|Method for treating retinal ganglion cell injury using glial cell line-derived neurothrophic factorprotein product|
    PCT/US1996/018851|WO1997019695A1|1995-11-29|1996-11-22|Method for treating retinal ganglion cell injury using glial cell line-derived neurotrophic factorprotein product|
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