Tissue protective cytokines for the protection, restoration, and enhancement of responsive cells, tissues and organs

Abstract

Methods and compositions are provided for protecting or enhancing a 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 a tissue protective cytokine.

What is claimed is:  
     
         1 . A tissue protective cytokine comprised of a chemically modified erythropoietin lacking at least one activity selected from the group consisting of increasing hematocrit, vasoconstriciton, hyperactivating platelets, pro-coagulant activities and increasing production of thrombocytes, the cytokine having at least one responsive cellular protective activity selected from the group consisting of protecting, maintaining, enhancing or restoring the function or viability of responsive mammalian cells and their associated cells, tissues and organs.  
     
     
         2 . The tissue protective cytokine of  claim 1  wherein the tissue protective cytokine is capable of traversing an endothelial cell barrier.  
     
     
         3 . The tissue protective cytokine of  claim 2  wherein the endothelial cell barrier comprises the blood-brain barrier, the blood-eye barrier, the blood testes barrier, the blood-ovary barrier, and the blood-uterus barrier.  
     
     
         4 . The tissue protective cytokine of  claim 1  wherein the responsive mammalian cells comprise neuronal, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, capillary, endothelial, testes, ovary, endometrial, or stem cells.  
     
     
         5 . The tissue protective cytokine of  claim 4  wherein the responsive mammalian cells further comprise photoreceptor, ganglion, bipolar, horizontal, amacrine, Muieller, myocardium, pace maker, sinoatrial node, sinoatrial node, sinus node, atrioventricular node, bundle of His, hepatocyte, stellate, Kupffer, mesangial, goblet, intestinal gland, enteral endocrine, glomerulosa, fasciculate, reticularis, chromaffin, pericyte, Leydig, Sertoli, sperm, Graffian follicles, primordial follicles, endometrial stroma, and endometrial cells.  
     
     
         6 . The tissue protective cytokine of  claim 1  wherein the chemically modified erythropoietin is 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 having at least one or more oxidized carbohydrates;  
 v. An erythropoietin having at least one or more oxidized carbohydrates and is 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 glutamic acid residue;  
 x. An erythropoietin having at a modified tryptophan residue;  
 xi. An erythropoietin having at least one amino acid group removed;  
 xii. An erythropoietin having at least one opening of at least one of the cystine linkages in the erythropoietin molecule; and  
 xiii. A truncated erythropoietin.  
 
     
     
         7 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is asialoerythropoietin.  
     
     
         8 . The tissue protective cytokine of  claim 7  wherein said asialoerythropoietin is human asialoerythropoietin.  
     
     
         9 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is an erythropoietin with no N-linked carbohydrates.  
     
     
         10 . The tissue protective cytokines of  claim 6  wherein said tissue protective cytokine is an erythropoietin with no O-linked carbohydrates.  
     
     
         11 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is a erythropoietin treated with at least one glycosidase.  
     
     
         12 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is periodate-oxidized erythropoietin.  
     
     
         13 . The tissue protective cytokine of  claim 12  wherein said periodate-oxidized erythropoietin is chemically reduced with sodium cyanoborohydride.  
     
     
         14 . The tissue protective cytokine of  claim 4  wherein said tissue protective cytokine comprises an erythropoietin having a R-glyoxal moiety on the one or more arginine residues, wherein R is aryl or alkyl moiety.  
     
     
         15 . The tissue protective cytokine of  claim 14  wherein said erythropoietin is phenylglyoxal-erythropoietin.  
     
     
         16 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is an erythropoietin in which an arginine residue is modified by reaction with a vicinal diketone selected from the group consisting of 2,3-butanedione and cyclohexanedione.  
     
     
         17 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is an erythropoietin in which an arginine residue is reacted with 3-deoxyglucosone.  
     
     
         18 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is an erythropoietin molecule having at least one biotinylated lysine or N-terminal amino group.  
     
     
         19 . The tissue protective cytokine of  claim 18  wherein said erythropoietin molecule is biotinylated erythropoietin.  
     
     
         20 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is a glucitolyl lysine erythropoietin or fructosyl lysine erythropoietin.  
     
     
         21 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is an erythropoietin having at least one carbamylated lysine residue.  
     
     
         22 . The tissue protective cytokine of  claim 21  wherein said carbamylated erythropoietin is comprised of alpha-N-carbamoylerythropoietin; N-epsilon-carbamoylerythropoietin; alpha-N-carbamoyl, N-epsilon-carbamoylerythropoietin; alpha-N-carbamoylasialoerythropoietin; N-epsilon-carbamoylasialoerythropoietin; alpha-N-carbamoyl, N-epsilon-carbamoylasialoerythropoietin; alpha-N-carbamoylhyposialoerythropoietin; N-epsilon-carbamoylhyposialoerythropoietin; and alpha-N-carbamoyl, N-epsilon-carbamoylhyposialoerythropoietin.  
     
     
         23 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is an erythropoietin in which at least one lysine residue is acylated.  
     
     
         24 . The tissue protective cytokine of  claim 23  wherein a lysine residue of said erythropoietin is acetylated.  
     
     
         25 . The tissue protective cytokine of  claim 24  where said acetylated erythropoietin is comprised of alpha-N-acetylerythropoietin; N-epsilon-acetylerythropoietin; alpha-N-acetyl, N-epsilon-acetylerythropoietin; alpha-N-acetylasialoerythropoietin; N-epsilon-acetylasialoerythropoietin; alpha-N-acetyl, N-epsilon-acetylasialoerythropoietin; alpha-N-acetylhyposialoerythropoietin; N-epsilon-acetylhyposialoerythropoietin; and alpha-N-acetyl, N-epsilon-acetylhyposialoerythropoietin.  
     
     
         26 . The tissue protective cytokine of  claim 23  wherein a lysine residue of said erythropoietin is succinylated.  
     
     
         27 . The tissue protective cytokine of  claim 26  where said succinylated erythropoietin is comprised of alpha-N-succinylerythropoietin; N-epsilon-succinylerythropoietin; alpha-N-succinyl, N-epsilon-succinylerythropoietin; alpha-N-succinylasialoerythropoietin; N-epsilon-succinylasialoerythropoietin; alpha-N-succinyl, N-epsilon-succinylasialoerythropoietin; alpha-N-succinylhyposialoerythropoietin; N-epsilon-succinylhyposialoerythropoietin; and alpha-N-succinyl, N-epsilon-succinylhyposialoerythropoietin.  
     
     
         28 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is an erythropoietin with at least one lysine residue modified by 2, 4, 6 trintrobenzenesulfonate sodium or another salt thereof.  
     
     
         29 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is an erythropoietin in which at least one tyrosine residue is nitrated or iodinated.  
     
     
         30 . The tissue protective cytokine of  claim 6  wherein said tissue protective cytokine is an erythropoietin in which an aspartic acid or glutamic acid residue is reacted with a carbodiimide followed by reaction with an amine.  
     
     
         31 . The tissue protective cytokine of  claim 30  wherein said amine is glycinamide.  
     
     
         32 . A pharmaceutical composition comprising an therapeutically effective amount of a tissue protective cytokine comprised of a chemically modified erythropoietin that lacks at least one activity selected from the group consisting of increasing hematocrit, vasoconstriction, hyperactivating platelets, pro-coagulant activity and increasing production of thrombocytes, and wherein the tissue protective cytokine is effective in protecting, maintaining, enhancing or restoring the function or viability of responsive mammalian cells and their associated cells, tissues and organs.  
     
     
         33 . A pharmaceutical composition of  claim 32  wherein said chemically modified erythropoietin is 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 having at least one or more oxidized carbohydrates;  
 v) an erythropoietin having at least one or more oxidized carbohydrates which is 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; and  
 xiii) a truncated erythropoietin.  
 
     
     
         34 . The pharmaceutical composition of  claim 33  wherein said erythropoietin is asialoerythropoietin or phenylglyoxal-erythropoietin.  
     
     
         35 . A method for protecting, maintaining or enhancing the viability of a cell, tissue or organ isolated from a mammalian body comprising exposing said cell, tissue or organ to a pharmaceutical composition comprising a tissue protective cytokine comprised of a chemically modified erythropoietin that lacks at least one activity selected from the group consisting of increasing hematocrit, vasoconstriction, hyperactivating platelets, pro-coagulant activity and increasing production of thrombocytes.  
     
     
         36 . The method of  claim 35  wherein said chemically modified erythropoietin is 
 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 having at least one or more oxidized carbohydrates;  
 v) an erythropoietin having at least one or more oxidized carbohydrates and is 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; or  
 xiii) a truncated erythropoietin.  
 
     
     
         37 . Use of a tissue protective cytokine comprised of a chemically modified erythropoietin that lacks at least one activity selected from the group consisting of increasing hematocrit, vasoconstriction, hyperactivating platelets, pro-coagulant activity and increasing production of thrombocytes, for the preparation of a pharmaceutical composition for the protection against and prevention of a tissue injury as well as the restoration of and rejuvenation of tissue and tissue function in a mammal.  
     
     
         38 . The use of  claim 37  wherein the injury is caused by a seizure disorder, multiple sclerosis, stroke, hypotension, cardiac arrest, ischemia, myocardial infarction, inflammation, age-related loss of cognitive function, radiation damage, cerebral palsy, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, Leigh disease, AIDS dementia, memory loss, amyotrophic lateral sclerosis, alcoholism, mood disorder, anxiety disorder, attention deficit disorder, autism, Creutzfeld-Jakob disease, brain or spinal cord trauma or ischemia, heart-lung bypass, chronic heart failure, macular degeneration, diabetic neuropathy, diabetic retinopathy, glaucoma, retinal ischemia, or retinal trauma.  
     
     
         39 . The use of  claim 37  wherein said chemically modified erythropoietin is 
 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 having at least one or more oxidized carbohydrates;  
 v) an erythropoietin having at least one or more oxidized carbohydrates which is 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; or  
 xiii) a truncated erythropoietin.  
 
     
     
         40 . A method for facilitating the transcytosis of a molecule across an endothelial cell barrier in a mammal comprising administration to said mammal a composition comprising said molecule in association with a tissue protective cytokine comprised of a chemically modified erythropoietin lacking at least one activity selected from the group consisting of increasing hematocrit, increasing blood pressure, hyperactivating platelets, and increasing production of thrombocytes, the tissue protective cytokine 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 having at least one or more oxidized carbohydrates;    v) an erythropoietin having at least one or more oxidized carbohydrates which is 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; and    xiii) a truncated erythropoietin.    
     
     
         41 . The method of  claim 40  wherein said association is a labile covalent bond, a stable covalent bond, or a non-covalent association with a binding site for said molecule.  
     
     
         42 . The method of  claim 40  wherein said endothelial cell barrier is selected from the group consisting of the blood-brain barrier, the blood-eye barrier, the blood-testes barrier, the blood-ovary barrier and the blood-placenta barrier.  
     
     
         43 . The method of  claim 40  wherein said molecule is a receptor agonist or antagonist hormone, a neurotrophic factor, an antimicrobial agent, an antiviral agent, a radiopharmaceutical, an antisense oligonucleotide, an antibody, an immunosuppressant, a dye, a marker, or an anti-cancer drug.  
     
     
         44 . A composition for transporting a molecule via transcytosis across an endothelial cell barrier comprising said molecule in association with a tissue protective cytokine comprised of a chemically modified erythropoietin lacking at least one activity selected from the group consisting of increasing hematocrit, vasoconstricition, hyperactivating platelets, pro-coagulant activity and increasing production of thrombocytes, the chemically modified 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 having at least one or more oxidized carbohydrates;    v) an erythropoietin having at least one or more oxidized carbohydrates which is 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; and    xiii) a truncated erythropoietin.    
     
     
         45 . The composition of  claim 44  wherein said association is a labile covalent bond, a stable covalent bond, or a non-covalent association with a binding site for said molecule.  
     
     
         46 . The composition of  claim 44  wherein said molecule is a receptor agonist or antagonist hormone, a neurotrophic factor, an antimicrobial agent, a radiopharmaceutical, an antisense oligonucleotide, an antibody, an immunosuppressant, a dye, a marker, or an anti-cancer drug.  
     
     
         47 . Use of an tissue protective cytokine comprised of a chemically modified erythropoietin lacking at least one activity selected from the group consisting of increasing hematocrit, vasoconstriction, hyperactivating platelets, pro-coagulant activities and increasing production of thrombocytes, 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 having at least one or more oxidized;    v) an erythropoietin having at least one or more oxidized carbohydrates which is 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; and    xiii) a truncated erythropoietin associated with a molecule for the preparation of a pharmaceutical composition for transporting said molecule via transcytosis across an endothelial cell barrier.    
     
     
         48 . The use of  claim 47  wherein said association is a labile covalent bond, a stable covalent bond, or a non-covalent association with a binding site for said molecule.  
     
     
         49 . The use of  claim 47  wherein said molecule is a receptor agonist or antagonist hormone, a neurotrophic factor, an antimicrobial agent, a radiopharmaceutical, an antisense oligonucleotide, an antibody, an immunosuppressant, a dye, or a marker, or an anti-cancer drug.