Protection, restoration, and enhancement of erythropoietin-responsive cells, tissues and organs

Abstract

Methods and compositions are provided for protecting or enhancing an erythropoietin-responsive cell, tissue, organ or body part function or viability in vivo, in situ or ex vivo in mammals, including human beings, by systemic or local administration of an erythropoietin receptor activity modulator, such as an erythropoietin or a modified erythropoietin.

1 . Use of an erythropoietin selected from the group consisting of
 i) an erythropoietin having at least no sialic acid moieties;   ii) an erythropoietin having at least no N-linked or no O-linked carbohydrates;   iii) an erythropoietin having at least a reduced carbohydrate content by virtue of treatment of native erythropoietin with at least one glycosidase;   iv) an erythropoietin with a carbohydrate portion of the erythropoietin molecule having at least a non-mammalian glycosylation pattern by virtue of the expression of a recombinant erythropoietin in non-mammalian cells;   v) an erythropoietin has at least one or more oxidized carbohydrates which also may be chemically reduced;   vi) an erythropoietin having at least one or more modified arginine residues;   vii) an erythropoietin having at least one or more modified lysine residues or a modification of the N-terminal amino group of the erythropoietin molecule   viii) an erythropoietin having at least a modified tyrosine residue;   ix) an erythropoietin having at least a modified aspartic acid or a glutamic acid residue;   x) an erythropoietin having at least a modified tryptophan residue;   xi) an erythropoietin having at least one amino group removed;   xii) an erythropoietin having at least an opening of at least one of the cystine linkages in the erythropoietin molecule;   xiii) an erythropoietin is provided having at least one substitution of at least one amino acid; and   xiv) a truncated erythropoietin;   for the preparation of a pharmaceutical composition for protecting, maintaining, enhancing or restoring the function or viability of erythropoietin-responsive mammalian cells and their associated cells, tissues and organs.   
   
   
       2 .- 57 . (canceled) 
   
   
       58 . A composition comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto, wherein said glycans comprise, on average, a) between 1 and 2 Lewis-X structures per EPO molecule, wherein, when an EPO molecule comprises 2 Lewis-X structures, said 2 Lewis-X structures are located on either the same or different glycans, and b) at least 6 terminal sialic acid moieties per EPO molecule, said terminal sialic acid moieties located on either the same or different glycans. 
   
   
       59 . The composition of  claim 58 , wherein said glycans comprise, on average, a) 1-2 Lewis-X structure per EPO molecule, and b) at least 7 sialic acid moieties per EPO molecule. 
   
   
       60 . The composition of  claim 58 , wherein said glycans comprise, on average, a) 1-2 Lewis-X structure per EPO molecule, and b) at least 8 sialic acid moieties per EPO molecule. 
   
   
       61 . The composition of  claim 58 , wherein said glycans comprise, on average, a) between 1 and 2 Lewis-X structures per EPO molecule, and b) between 8 and 10 sialic acid moieties per EPO molecule. 
   
   
       62 . A composition comprising one or more isoforms of an erythropoietin (EPO) comprising glycans linked thereto, wherein said glycans comprise, on average, a) at least 0.5 and no more than 2 Lewis-X structure per EPO molecule, wherein, when an EPO molecule comprises 2 Lewis-X structures, said 2 Lewis-X structures are located on either the same or different glycans linked to said EPO molecule, and b) at least 10 terminal sialic acid moieties per EPO molecule, wherein at least a portion of said terminal sialic acid moieties are located on different glycans. 
   
   
       63 . The composition of  claim 62 , wherein said glycans comprise, on average, a) 0.5-2 Lewis-X structure per EPO molecule, and b) at least 11 sialic acid moieties per EPO molecule. 
   
   
       64 . The composition of  claim 62 , wherein said glycans comprise, on average, a) between 0.5 and 1 Lewis-X structures per EPO molecule, and b) between 11 and 13 sialic acid moieties per EPO molecule. 
   
   
       65 . The composition of  claim 58 , wherein said erythropoietin is human erythropoietin. 
   
   
       66 . The composition of  claim 61 , wherein said erythropoietin is human erythropoietin. 
   
   
       67 . The composition of  claim 62 , wherein said erythropoietin is human erythropoietin. 
   
   
       68 . The composition of  claim 64 , wherein said erythropoietin is human erythropoietin. 
   
   
       69 . The composition of  claim 58 , wherein said glycans comprise three N-linked glycans per EPO molecule with each N-linked glycan linked to a different asparagine residue. 
   
   
       70 . The composition of  claim 62 , wherein said glycans comprise three N-linked glycans per EPO molecule with each N-linked glycan linked to a different asparagine residue. 
   
   
       71 . The composition of  claim 58 , wherein said glycans comprise three N-linked glycans per EPO molecule and one O-linked glycan per EPO molecule, wherein said O-linked glycan is linked to a serine residue. 
   
   
       72 . The composition of  claim 62 , wherein said glycans comprise three N-linked glycans per EPO molecule and one O-linked glycan per EPO molecule, wherein said O-linked glycan is linked to a serine residue. 
   
   
       73 . The composition of  claim 58 , wherein at least one of said Lewis-X structures and at least one of said sialic acid moieties are located on the same glycan. 
   
   
       74 . The composition of  claim 58 , wherein at least one said Lewis-X structures and at least one of said sialic acid moieties are located on different glycans. 
   
   
       75 . The composition of  claim 58 , wherein at least one of said glycans per EPO molecule comprises an oligosaccharide having between 2 and 5 N-acetyl-glucosamine (GlcNAc) bearing branches. 
   
   
       76 . The composition of  claim 75 , further comprising at least one GlcNAc covalently linked to galactose. 
   
   
       77 . The composition of  claim 76 , wherein at least one galactose is covalently linked to a sialic acid via an alpha-2,3- or an alpha-2,6-linkage. 
   
   
       78 . The composition of  claim 76 , further comprising between 1 and 2 GlcNAc modified with galactose further modified with alpha-1,3-linked fucose so as to form said between 1 and 2 Lewis-X structures per EPO molecule. 
   
   
       79 . The composition of  claim 78 , further comprising a galactose of one of said Lewis-X structures modified with either alpha-2,3- or alpha-2,6-linked sialic acid to form a sialyl-Lewis-X structure. 
   
   
       80 . The composition of  claim 75 , wherein at least one GlcNAc is covalently linked to an N-acetyl-galactosamine (GalNAc). 
   
   
       81 . The composition of  claim 80 , wherein at least one GalNAc is covalently linked to a sialic acid via an alpha2,6-linkage. 
   
   
       82 . The composition of  claim 80 , further comprising between 1 and 2 GlcNAc covalently linked to GalNAc further covalently linked to alpha1,3-linked fucose so as to form said between 1 and 2 Lewis-X structures per EPO molecule. 
   
   
       83 . The composition of  claim 82 , further comprising a GalNAc of one of said Lewis-X structures covalently linked to a sialic acid via an alpha2,6-linkage to form a sialyl-Lewis-X structure. 
   
   
       84 . A method for the therapeutic treatment of a disorder selected from the group consisting of cerebral ischemia and myocardial infarction, said method comprising: administering to a human or animal subject suffering from or at risk of suffering from cerebral ischemia or myocardial infarction a composition comprising erythropoietin molecules, wherein said composition of erythropoietin molecules has an average number of Lewis-X structures on N-linked glycans per erythropoietin molecule of at least 2.7. 
   
   
       85 . A composition comprising a plurality of isolated erythropoietin molecules, wherein the plurality of erythropoietin molecules have an average number of Lewis-X structures on N-linked glycans per erythropoietin molecule of at least 2.7. 
   
   
       86 . The composition of  claim 85 , wherein the average number of Lewis-X structures on N-linked glycans per erythropoietin molecule is at least about 3.6. 
   
   
       87 . The composition of  claim 85 , wherein the average number of Lewis-X structures on N-linked glycans per erythropoietin molecule is at least about 4.1. 
   
   
       88 . The composition of  claim 85 , wherein the average number of Lewis-X structures on N-linked glycans per erythropoietin molecule is at least about 5.7. 
   
   
       89 . The composition of  claim 85 , further comprising a pharmaceutically acceptable diluent or carrier. 
   
   
       90 . The composition of  claim 85 , wherein the erythropoietin molecules are recombinant erythropoietin molecules.