CN1688883B - Recombinant tissue protective cytokines and encoding nucleic acids thereof for protection, restoration, and enhancement of responsive cells, tissues, and organs - Google Patents

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 an erythropoietin receptor activity modulator, such as an recombinant tissue protective cytokine.

Description

Recombinant tissue protective cytokines and nucleic acids encoding them for the protection, restoration and enhancement of effector cells, tissues and organs

This application claims priority to U.S. Provisional Patent Application No. 60/392,455, filed July 1, 2002, and U.S. Provisional Patent Application No. 60/393,423, filed July 3, 2002, all of which The contents are hereby incorporated by reference in their entirety. the

1. technical field

The present invention relates to a mutant protein recombinant tissue protective cytokine with one or more amino acid substitutions for protecting, maintaining, enhancing or restoring the function or viability of mammalian effector cells and related cells, tissues and organs, and relates to a recombinant tissue protective cytokine comprising said Pharmaceutical composition of cytokines. This includes protection of excitable tissues such as neurons and cardiac tissue from neurotoxins, hypoxia and other adverse stimuli, and enhancement of excitable tissue function, for example to facilitate learning and memory. The present invention also relates to compositions using muteins to recombine tissue protective cytokines for the transport of molecules or to facilitate the transport of molecules across the endothelial barrier by transcytosis. the

2. Background technique

For many years, the only known physiological role of erythropoietin was to control the production of red blood cells. In recent years, some evidence has shown that erythropoietin, as a member of the cytokine superfamily, has other important physiological functions mediated by interacting with the erythropoietin receptor (erythropoietin-R). These effects include mitogenesis, regulation of calcium influx into smooth muscle cells and nerve cells, vasoactive effects ie vasoconstriction/vasodilation, hyperactivation of platelets and effects on intermediary metabolism. Erythropoietin is believed to provide a compensatory response for improving the hypoxic cellular microenvironment and regulating apoptotic cell death caused by metabolic stress. Although studies have established that intracranial injection of erythropoietin protects neurons from hypoxic neuronal injury, intracranial administration is impractical for therapeutic applications, especially in normal individuals, and Unacceptable route of administration. Furthermore, previous studies of erythropoietin administration in anemic patients have concluded that peripherally administered erythropoietin does not transport to the brain (Marti et al., 1997, Kidney Int. 51:416-8; Juul et al., 1999, Pediatr. Res. 46: 543-547; Buemi et al., 2000, Nephrol. Dial. Transplant. 15: 422-433). the

Various modified forms of erythropoietin having activity directed at improving the erythropoietic activity of the molecule have been described, for example with altered amino acids at its carboxyl terminus, as described in U.S. Patent 5,457,089 and U.S. Patent 4,835,260; each molecule Erythropoietin isoforms with different numbers of sialic acid residues, such as described in US Patent 5,856,298; polypeptides, described in US Patent 4,703,008; agonists, described in US Patent 5,767,078; peptides that bind to the erythropoietin receptor , described in US Patents 5,773,569 and 5,830,851; and small molecule mimetics, described in US Patent 5,835,382. the

The present invention relates to the use of recombinant tissue protective cytokines for the protection, maintenance, enhancement or restoration of effector cells and associated cells, tissues and organs in situ and ex vivo, and for the protection and enhancement of effector cells and associated cells exiting the vasculature, Use for delivering recombinant tissue protective cytokines across the endothelial cell barrier or carrying related molecules across the endothelial cell barrier for the purpose of tissues and organs. the

3. Contents of the invention

In one aspect, the present invention relates to various forms of recombinant tissue protective cytokines in the preparation of pharmaceutical compositions for protecting, maintaining, enhancing or restoring the function or vitality of mammalian effector cells and related cells, tissues and organs the use of. In a specific aspect, the mammalian effector cells and their associated cells, tissues or organs are kept away from the vasculature due to the tight endothelial cell barrier. In another specific aspect, the cell, tissue, organ or other body part is a part isolated from a mammal, such as a part intended for transplantation. As non-limiting examples, the effector cells or tissues may be neuronal cells, retinal cells, myocytes, cardiac cells, lung cells, liver cells, kidney cells, intestinal cells, adrenal cortical cells, adrenal medulla cells, capillary endothelial cells , testicular cells, ovarian cells, pancreatic cells, bone cells, skin cells, or endometrial cells or tissues. Furthermore, non-limiting examples of effector cells include photoreceptor cells (rods and cones), ganglion cells, bipolar cells, horizontal cells, amacrine cells, Müeller cells, Purkinje cells, cardiomyocytes , pacemaker cells, sinoatrial node cells, sinoatrial node cells, sinus node cells, junctional histiocytes, atrioventricular node cells, atrioventricular bundle cells, hepatocytes, astrocytes, hepatic macrophages, mesangial cells, kidney Epithelial cells, tubular enterocytes, goblet cells, intestinal gland cells (crypt), enteroendocrine cells, glomerular cells, fasciculate cells, reticulocytes, chromaffin cells, adventitial cells, mesenchymal cells, trophoblasts cells, sperm cells, mature follicles (Graffian follicles), primitive follicles, islets, α-cells, β-cells, γ-cells, F-cells, osteoprogenitors, osteoclasts, osteoblasts, uterus Endometrial stromal cells, endometrial cells, stem cells and endothelial cells. These examples of effector cells are illustrative only. In one aspect, the effector cells or their associated cells, tissues or organs are neither excitable cells, tissues or organs nor predominately comprise excitable cells or tissues. In a specific embodiment, the mammalian cell, tissue or organ to which the above-mentioned recombinant tissue protective cytokine has been used has survived or is about to survive at least one condition that is not conducive to the viability of said cell, tissue or organ. cells over time. In a specific embodiment, the mammalian cell, tissue or organ to which the recombinant tissue protective cytokine described above is used expresses the EPO receptor. Such conditions include traumatic in situ hypoxia or metabolic dysfunction, surgically induced in situ hypoxia or metabolic dysfunction, or in situ toxin exposure; the latter may be associated with chemotherapy or radiotherapy. In one embodiment, the adverse condition is, for example, the result of cardiopulmonary bypass (heart-lung machine) as used in certain surgical procedures. the

The recombinant tissue protective cytokines of the present invention can be used in the therapeutic treatment or prophylactic treatment of the following human diseases: central nervous system (CNS) diseases or peripheral nervous system diseases mainly with neurological symptoms or psychiatric symptoms, as well as eye diseases, heart diseases, etc. Vascular disease, cardiopulmonary disease, respiratory disease, kidney disease, urinary tract disease and reproductive tract disease, gastrointestinal disease and endocrine and metabolic abnormalities. the

The present invention also relates to pharmaceutical compositions comprising specific recombinant tissue protective cytokines as described above for administration to mammals, preferably humans. The pharmaceutical composition may be formulated for oral, intranasal or parenteral administration, or in the form of a perfusate for maintaining the viability of isolated cells, tissues or organs. the

The recombinant tissue protective cytokine used for the above purpose can be a mutant protein or a genetically modified erythropoietin, that is, an erythropoietin with at least one modification in the amino acid backbone of the natural molecule. "Variant protein" or "mutant protein" refers to a protein comprising a mutated amino acid sequence and includes polypeptides that differ from the amino acid sequence of native erythropoietin by amino acid deletions, substitutions, or both. "Native sequence" refers to an amino acid sequence or nucleic acid sequence identical to the wild-type or native form of a gene or protein. Furthermore, in one embodiment, the recombinant tissue protective cytokines of the invention have cytoprotective activity but also have one or more of the effects of erythropoietin on the bone marrow, i.e. increased hematocrit (erythropoiesis), vasoactive effects (vasoconstriction/vasodilation), hyperactivation of platelets, increased production of thrombus cells and procoagulant activity. In another embodiment, the recombinant tissue protective cytokines of the invention have cytoprotective activity but do not have one or more of the effects of erythropoietin on the bone marrow, i.e. increasing hematocrit (erythropoiesis), vasoactive effects ( vasoconstriction/vasodilation), hyperactivation of platelets, increased production of thrombus cells and procoagulant activity. Preferably, the cytoprotective recombinant tissue protective cytokine of the invention lacks at least one effect of erythropoietin on the bone marrow; more preferably the recombinant tissue protective cytokine lacks erythropoietic activity; most preferably the recombinant tissue protective cytokine lacks the effect of erythropoietin on All effects of bone marrow. the

As a non-limiting example, one or more amino acids may be altered or deleted from or added to the native erythropoietin molecule. In a preferred embodiment, the recombinant tissue protective cytokine has one or more modifications in one or more of the following regions: VLQRY (amino acids 11-15 of natural human erythropoietin; SEQ ID NO: 1) and /or TKVNFYAW (amino acids 44-51 of natural human erythropoietin; SEQ ID NO: 2) and/or SGLRSLTTL (amino acids 100-108 of natural human erythropoietin; SEQ ID NO: 3) and/or SNFLRG ( Amino acids 146-151 of native human erythropoietin; SEQ ID NO: 4). At amino acids 7, 20, 21, 29, 33, 38, 42, 59, 63, 67, 70, 83, 96, 126, 142, 143, 152, 153, 155, 156 and 161 of SEQ ID NO: 10 Other mutations may also be provided. These other mutations may be alone or in addition to at least one mutation of at least one of the aforementioned regions. In certain embodiments, one or more amino acid changes in TKVNFYAW (amino acids 44-51 of natural human erythropoietin; SEQ ID NO: 2) produce partial-function (i.e., have lower erythrocytes than rhu-EPO production activity) modified erythropoietin molecules. In other embodiments, one or more amino acid changes in SGLRSLTTL (amino acids 100-108 of natural human erythropoietin; SEQ ID NO: 3) result in partial function (i.e., lower erythropoietic activity than rhu-EPO) ) recombinant tissue protective cytokine. The above-mentioned recombinant tissue protective cytokines exhibit tissue protective activity or cytoprotective activity. With regard to erythropoietic activity, the recombinant tissue protective cytokines described above lack erythropoietic activity or exhibit a decrease in one or more erythropoietic activities. Examples of erythropoietic activity include increased hematocrit, vasoconstriction, hyperactivation of platelets, procoagulant activity, and increased production of thrombin cells. Erythropoietic activity can be measured using standard techniques in the art. For example, hematocrit can be determined using the UT-7 cell assay described in Section 6.17, or using the technique described in Physicians' Desk Reference (Medical Economics Company, Inc., Montvale, NJ, 2000.), which is incorporated by reference in its entirety to this article. In particular, pages 519-525 and 2125-2131 disclose methods for determining hematocrit levels and disclose different hematocrit ranges that can be used as targets to avoid toxicity. For example, in patients with chronic renal failure, the PDR recommends administering erythropoietin to achieve a non-toxic target hematocrit in patients ranging from 30% to 36% (see e.g. PDR, p. 523, pp. 1, 11 columns 17-96 and p. 2129, columns 1, 11, 11.8-93, and the accompanying tables in columns 2 and 3). The PDR notes that toxicity in the form of polycythemia (a condition characterized by an abnormal increase in the number of circulating red blood cells) can be avoided by carefully monitoring the hematocrit and adjusting the dose of EPO if the hematocrit approaches the upper end of the target range (36% for this patient population) or when there is an increase of more than 4 points in any 2-week period, stop erythropoietin until the hematocrit returns to the recommended target range (30%-36% for this patient population ; see PDR, page 523, column 1, and page 2129, column 1, under "Dose Adjustment"). In contrast, for cancer patients receiving chemotherapy, the PDR recommends dose adjustment at different hematocrit levels, ie if the hematocrit exceeds 40% (see page 2129, column 2, under "Dose Adjustment"). In one embodiment, the recombinant tissue protective cytokine has one or more erythropoietic activities, but at levels insufficient to cause side effects, ie, effects that outweigh the therapeutic benefit of the cytoprotective activity of the recombinant tissue protective cytokine. In one embodiment, recombinant tissue protective cytokines having one or more erythropoietic activities are also useful in the methods of the invention, if the level of erythropoietic activity can be determined. In embodiments where the recombinant tissue protective cytokine has one or more erythropoietic activities, the erythropoietic activity can be assayed and the amount and/or regimen of the cytokine administered can be adjusted to ensure that the Recombinant tissue protective cytokines are nontoxic. In embodiments where the recombinant tissue protective cytokine has one or more erythropoietic activities, the erythropoietic activity can be assayed and the amount and/or dosing regimen of the cytokine can be adjusted to ensure that the recombinant Tissue protective cytokines have low toxicity. In one embodiment, the recombinant tissue protective cytokine exhibits a decrease in one or more erythropoietic activities by about 1%, 2%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. the

The present invention provides recombinant tissue protective cytokines lacking at least one activity selected from the group consisting of increasing hematocrit, vasoconstriction, hyperactivating platelets, procoagulant activity and increasing production of thrombin cells. The cytokines comprise at least one effector cell protective activity selected from the group consisting of protecting, maintaining, enhancing or restoring the function or viability of mammalian effector cells, tissues or organs. the

In one embodiment of the present invention, the recombinant tissue protective cytokine is comprised between positions 11-15 of SEQ ID NO: 10 [SEQ ID NO: 1], between positions 44-51 of SEQ ID NO: 10 [SEQ ID NO: 2], between positions 100-108 of SEQ ID NO: 10 [SEQ ID NO: 3] or between positions 146-151 of SEQ ID NO: 10 [SEQ ID NO: 4] or multiple altered amino acid residues.

In another embodiment, the recombinant tissue protective cytokine comprises altered amino acid residues at one or more of the following positions of SEQ ID NO: 10: 7, 20, 21, 29, 33, 38, 42, 59 , 63, 67, 70, 83, 96, 126, 142, 143, 152, 153, 155, 156, or 161. the

In yet another embodiment, the recombinant tissue protective cytokine comprises the amino acid sequence of SEQ ID NO: 10 with one or more of the following changes (each changed sequence has been assigned a separate sequence identification number): SEQ ID NO: 10 ID NO: the alanine of 10 residue 6 (SEQ ID NO: 15); the alanine of SEQ ID NO: 10 residue 7 (SEQ ID NO: 16); the serine of SEQ ID NO: 10 residue 7 ( SEQ ID NO: 17); SEQ ED NO: 10 isoleucine at residue 10 (SEQ ID NO: 18); SEQ ID NO: 10 serine at residue 11 (SEQ ID NO: 19); SEQ ID NO: The alanine of 10 residue 12 (SEQ ID NO:20); The alanine of SEQ ID NO:10 residue 13 (SEQ ID NO:21); The alanine of SEQ ID NO:10 residue 14 (SEQ ID NO: ID NO: 22); SEQ ID NO: glutamic acid of 10 residue 14 (SEQ ID NO: 23); SEQ ID NO: glutamine of 10 residue 14 (SEQ ID NO: 24); SEQ ID NO: 10 residue Alanine at base 15 (SEQ ID NO: 25); SEQ ID NO: phenylalanine at residue 15 at 10 (SEQ ID NO: 26); SEQ ID NO: isoleucine at residue 15 at 10 (SEQ ID NO: 27); SEQ ID NO: glutamic acid (SEQ ID NO: 28) of 10 residues 20; alanine (SEQ ID NO: 29) of SEQ ID NO: 10 residues 20; SEQ ID NO: 10 residues Alanine at base 21 (SEQ ID NO:30); Lysine at residue 24 of SEQ ID NO:10 (SEQ ID NO:31); Serine at residue 29 of SEQ ID NO:10 (SEQ ID NO:32) ; SEQ ID NO: tyrosine (SEQ ID NO: 33) of 10 residue 29; Asparagine (SEQ ID NO: 34) of SEQ ID NO: 10 residue 30; Su of SEQ ID NO: 10 residue 32 amino acid (SEQ ID NO: 35); SEQ ID NO: the serine (SEQ ID NO: 36) of 10 residue 33; the tyrosine (SEQ ID NO: 37) of SEQ ID NO: 10 residue 33; SEQ ID NO: 10 residue 38

Lysine (SEQ ID NO: 38); SEQ ID NO: Lysine (SEQ ID NO: 39) of 10 residue 83; Asparagine (SEQ ID NO: 40) of SEQ ID NO: 10 residue 42 ; SEQ ID NO: the alanine of 10 residue 42 (SEQ ID NO: 41); the alanine of SEQ ID NO: 10 residue 43 (SEQ BD NO: 42); the iso of SEQ ID NO: 10 residue 44 Leucine (SEQ ID NO: 43); Aspartic acid at residue 45 of SEQ ID NO: 10 (SEQ ID NO: 44); Alanine at residue 45 of SEQ ID NO: 10 (SEQ ID NO: 45); SEQ ID NO: the alanine of 10 residue 46 (SEQ ID NO: 46); the alanine of SEQ ID NO: 10 residue 47 (SEQ ID NO: 47); the alanine of SEQ ID NO: 10 residue 48 Isoleucine (SEQ ID NO: 48); Alanine at residue 48 of SEQ ID NO: 10 (SEQ ID NO: 49); Alanine at residue 49 of SEQ ID NO: 10 (SEQ ID NO: 50 ); SEQ ID NO: the serine of 10 residue 49 (SEQ ID NO: 51); the phenylalanine of SEQ ID NO: 10 residue 51 (SEQ ID NO: 52); the asparagus of SEQ ID NO: 10 residue 51 Amide (SEQ ID NO:53); SEQ ID NO:Alanine at residue 52 of SEQ ID NO:10 (SEQ ID NO:54); Asparagine at residue 59 of SEQ ID NO:10 (SEQ ID NO:55); SEQ ID NO:10 NO: threonine (SEQ ID NO: 56) of 10 residue 62; Serine (SEQ ID NO: 57) of SEQ ID NO: 10 residue 67; Alanine (SEQ ID NO: 10 residue 70) of SEQ ID NO: 10 NO: 58); SEQ ID NO: 10 arginine at residue 96 (SEQ ID NO: 59); SEQ ID NO: 10 alanine at residue 97 (SEQ ID NO: 60); SEQ ID NO: 10 residues Arginine at 100 (SEQ ID NO:61); Glutamic acid at residue 10 of SEQ ID NO:100 (SEQ ID NO:62); Alanine at residue 10 at 100 (SEQ ID NO:63) ); SEQ ID NO:10 residue 100 of threonine (SEQ ID NO:64); SEQ ID NO:10 residue 101 of alanine (SEQ ID NO:65); SEQ ID NO:10 residue 101 of Isoleucine (SEQ ID NO: 66 ); SEQ ID NO: the alanine of 10 residue 102 (SEQ ID NO: 67); the alanine of SEQ ID NO: 10 residue 103 (SEQ ID NO: 68); the valley of SEQ ID NO: 10 residue 103 amino acid (SEQ ID NO: 69); SEQ ID NO: alanine (SEQ ID NO: 70) of 10 residue 104; isoleucine (SEQ ID NO: 71) of SEQ ID NO: 10 residue 104 ; SEQ ID NO:10 residue 105 alanine (SEQ ID NO:72); SEQ ID NO:10 residue 106 alanine (SEQ ID NO:73); SEQ ID NO:10 residue 106 Isoleucine (SEQ ID NO: 74); Alanine (SEQ ID NO: 75) of SEQ ID NO: 10 residue 107; Leucine (SEQ ID NO: 76) of SEQ ID NO: 10 residue 107 ); SEQ ID NO: lysine at 10 residue 108 (SEQ ID NO: 77); SEQ ID NO: alanine at 10 residue 108 (SEQ ID NO: 78); SEQ ID NO: 10 residue 108 Serine (SEQ ID NO: 79); SEQ ID NO: alanine (SEQ ID NO: 80) of 10 residue 116; alanine (SEQ ID NO: 81) of SEQ ID NO: 10 residue 126; SEQ ID NO: IDNO: Alanine at 10 residue 132 (SEQ ID NO: 82); SEQ ID NO: Alanine at 10 residue 133 (SEQ ID NO: 83); SEQ ED NO: Alanine at 10 residue 134 (SEQ ID NO: 84); SEQ ID NO: alanine at 10 residue 140 (SEQ ID NO: 85); SEQ ID NO: isoleucine at 10 residue 142 (SEQ ID NO: 86); SEQ ID NO : the alanine of 10 residue 143 (SEQ ID NO:87); SEQ ID NO: the alanine of 10 residue 146 (SEQ ID NO:88); the lysine of SEQ ID NO:10 residue 147 (SEQ ID NO: 89); SEQ ID NO: alanine at 10 residue 147 (SEQ ID NO: 90); SEQ ID NO: tyrosine at 10 residue 148 (SEQ ID NO: 91); SEQ ID NO: 10 Alanine at residue 148 (SEQ ID NO:92); Alanine at residue 149 of SEQ ID NO:10 (SEQ ID NO:93); Alanine at residue 150 of SEQ ID NO:10 (SEQ ID NO: 94); SEQ ID NO: the glutamic acid of 10 residue 150 (SEQ ID NO: 95); the alanine of SEQ ID NO: 10 residue 151 (SEQ ID NO: 96); SEQ ID NO: 10 residue 152 Alanine (SEQ ID NO: 97); SEQ ID NO: Tryptophan (SEQ ID NO: 98) of 10 residue 152; Alanine (SEQ ID NO: 99) of SEQ ID NO: 10 residue 153 ; SEQ ID NO: the alanine of 10 residue 154 (SEQ ID NO: 100); the alanine of SEQ ID NO: 10 residue 155 (SEQ ID NO: 101); the alanine of SEQ ID NO: 10 residue 158 Acid (SEQ ID NO: 102); Serine (SEQ ID NO: 103) of SEQ ID NO: 10 residue 160; Alanine (SEQ ID NO: 104) of SEQ ID NO: 10 residue 161; or SEQ ID NO : Alanine of 10 residue 162 (SEQ ID NO: 105). In one embodiment, the recombinant tissue protective cytokine comprises the amino acid sequence of SEQ ID NO: 10 with one or more amino acid substitutions among SEQ ID NOs: 15-105 and 119. the

In yet another embodiment, the recombinant tissue protective cytokine comprises the amino acid sequence of SEQ ID NO: 10 with a deletion at amino acid residues 44-49 of SEQ ID NO: 10. the

In yet another embodiment, the recombinant tissue protective cytokine comprises the amino acid sequence of SEQ ID NO: 10 with at least one of the following changes (each changed sequence has been assigned an independent sequence identification number): i) SEQ ID NO: 10 ID NO: Aspartic acid at residue 45 of 10 and glutamic acid at residue 100 (SEQ ID NO: 106); ii) SEQ ID NO: Asparagine at residue 30 of 10, threonine at residue 32 (SEQ ID NO: 107); iii) SEQ ID NO: 10 aspartic acid at residue 45, glutamic acid at residue 150 (SEQ ID NO: 108); iv) SEQ ID NO: 10 glutamate at residue 103 Serine (SEQ ID NO: 109) of amino acid and residue 108; v) alanine of SEQ ID NO: 10 residue 140 and alanine of residue 52 (SEQ ID NO: 110); vi) SEQ ID NO: NO: the alanine of 10 residue 140, the alanine of residue 52, the alanine of residue 45 (SEQ ID NO: 111); vii) the alanine of SEQ ID NO: 10 residue 97 and residue Alanine at base 152 (SEQ ID NO: 112); iix) Alanine at residue 97 of SEQ ID NO: 10, alanine at residue 152, alanine at residue 45 (SEQ ID NO: 113 ); ix) SEQ ID NO: 10 alanine at residue 97, alanine at residue 152, alanine at residue 45, and alanine at residue 52 (SEQ ID NO: 114); x) SEQ ID NO: 10 alanine at residue 97, alanine at residue 152, alanine at residue 45, alanine at residue 52, and alanine at residue 140 (SEQ ID NO: 115 ); xi) SEQ ID NO: 10 alanine at residue 97, alanine at residue 152, alanine at residue 45, alanine at residue 52, alanine at residue 140, residue Alanine at base 154, lysine at residue 24, lysine at residue 38, lysine at residue 83, lysine at residue 24, and alanine at residue 15 (SEQ ID NO : 116); xii) the lysine of SEQ ID NO: 10 residue 24, the lysine of residue 38 and the lysine of residue 83 (SEQ ID NO: 117); or xiv) SEQ ID NO: 10 residue Lysine at residue 24 and alanine at residue 15 (SEQ ID NO: 118). In one embodiment, the recombinant tissue protective cytokine comprises the amino acid sequence of SEQ ID NO: 10 with at least one substitution of the following amino acid residues in SEQ ID NOs: 106-118. the

One embodiment of the present invention relates to the above-mentioned recombinant tissue protective cytokine further comprising chemical modification of one or more amino acids. In another embodiment, said chemical modification comprises altering the charge of said recombinant tissue protective cytokine. In yet another embodiment, a positive or negative charge is chemically added to an amino acid residue, wherein the charged amino acid residue is modified to an uncharged residue. the

In addition, such recombinant tissue protective cytokines described above may be further modified to have chemical modifications of one or more amino acids, such as described in the following co-pending application: PCT application serial number filed on December 28, 2001 No. PCT/US01/49479, U.S. Patent Application Serial No. 09/753,132, filed December 29, 2000, and U.S. Patent Application Attorney Docket No. KW00-009C02-US, filed July 3, 2002, of which Each is hereby incorporated by reference in its entirety. These further chemical modifications can be used to enhance the tissue protective activity of the recombinant tissue protective cytokine or to inhibit any effect of the recombinant tissue protective cytokine on the bone marrow. In another embodiment, an additional chemical modification is provided to restore the solubility of the molecule, which may have been reduced by the genetic modification described above, such as by chemically adding a positive or negative charge to the molecule. Charge, if a charged amino acid residue is modified to an uncharged residue. the

As non-limiting examples, recombinant tissue protective cytokines of the present invention include human erythropoietin mutein S100E (SEQ ID NO: 5), human erythropoietin mutein K45D (SEQ ID NO: 6) and any non-erythropoietic Recombinant tissue protective cytokines that are still cytoprotective or erythropoietin that can benefit effector cells, tissues or organs are described in Elliott et al., 1997, Blood 89:493-502; Boissel et al., Journal of Biological Chemistry , Vol. 268, No. 21, pp. 15983-15993 (1993); Wen et al., Journal of Biological Chemistry, Vol. 269, No. 36, pp. 22839-22846 (1994); and Syed et al., Nature, pp. 395, pp. 511-516 (1998), which is hereby incorporated by reference in its entirety. The present invention relates to methods of use of any of the aforementioned recombinant tissue protective cytokines for the protection, restoration and enhancement of effector cells, tissues and organs. the

Other recombinant tissue protective cytokines of the invention include erythropoietin as described above comprising at least one genetically altered amino acid with at least one additional modification, which may be another modification of at least one additional amino acid of the erythropoietin molecule, or is a modification of at least one sugar of the erythropoietin molecule. The genetically altered amino acids may be unique or these amino acids may be further modified. Of course, recombinant tissue protective cytokine molecules for purposes herein may have many modifications compared to native erythropoietin molecules, such as modifications of the amino acid portion of the molecule, modifications of the sugar portion of the molecule or at least a second modification of the amino acid portion of the molecule and at least one modification of the sugar portion of the molecule. The recombinant tissue protective cytokine molecule retains its ability to protect, maintain, enhance or restore the function or viability of mammalian effector cells, but compared to the natural molecule, the recombinant tissue protective cytokine has other properties, ideals and Required features may not be present. In a preferred embodiment, the recombinant tissue protective cytokine is non-erythropoietic. the

In another embodiment, the recombinant tissue protective cytokine may be modified by fucosylation to alter the glycosylation pattern of the glycoprotein. the

The above-mentioned recombinant tissue protective cytokine according to one embodiment of the present invention is human erythropoietin mutein. In another embodiment of the present invention, the recombinant tissue protective cytokine is human phenylglyoxal erythropoietin mutein. In another embodiment of the present invention, the recombinant tissue protective cytokine is human asialoerythropoietin mutein. the

In one embodiment, the recombinant tissue protective cytokine as described above comprises at least one effector cell protective activity selected from the group consisting of protecting, maintaining, enhancing or restoring mammalian effector cell, tissue or organ function or viability. In such embodiments, mammalian effector cells include neuronal cells, myocytes, cardiac cells, lung cells, hepatic cells, renal cells, intestinal cells, adrenal cortical cells, adrenal medullary cells, capillary cells, endothelial cells, Testicular cells, ovarian cells, endometrial cells or stem cells. In other embodiments, the cells include photoreceptor cells, ganglion cells, bipolar cells, horizontal cells, amacrine cells, Müller cells, cardiomyocytes, pacemaker cells, sinus node cells, sinus node cells, atrial Ventricular node cells, atrioventricular bundle cells, hepatocytes, astrocytes, hepatic macrophages, mesangial cells, goblet cells, intestinal gland cells, enteroendocrine cells, glomerular cells, bundle cells, reticulocytes, phagocytes Chromocytes, adventitial cells, stromal cells, trophoblasts, sperm cells, mature follicular cells, primitive follicular cells, endometrial stromal cells, or endometrial cells. the

According to another aspect of the invention, the recombinant tissue protective cytokine as described above is capable of crossing the endothelial cell barrier. In a related embodiment, the endothelial cell barrier includes the blood-brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier, blood-fetal barrier, blood-heart barrier, blood-kidney barrier, and blood- Uterine barrier. the

In another embodiment of the present invention, the above-mentioned recombinant tissue protective cytokines are further modified. In one embodiment, the recombinant tissue protective cytokine is selected from: i) a cytokine with a reduced number of sialic acid or asialic acid moieties; ii) a reduced number of N-linked or O-linked sugars or no N-linked Cytokines with sugar-linked or O-linked sugars; iii) cytokines with at least reduced sugar content by treating the native cytokine with at least one glycosidase; iv) cytokines with at least one or more oxidized sugars; v) with At least one or more cytokines that oxidize sugars and are chemically reduced; vi) cytokines with at least one or more modified arginine residues; vii) cytokines with at least one or more modified lysine residues or cytokines A cytokine modified with one N-terminal amino group of the molecule; viii) a cytokine with at least one modified tyrosine residue; ix) a cytokine with at least one modified aspartic acid or glutamic acid residue; x) a cytokine with a Cytokines with modified tryptophan residues; xi) cytokines with at least one amino acid group removed; xii) cytokines with at least one open cystine bond within the cytokine molecule; xiii) Truncated cytokines; xiv) cytokines linked to at least one polyethylene glycol molecule; xv) cytokines linked to at least one fatty acid; xvi) having non-mammalian glycosyls due to recombinant cytokine expression in non-mammalian cells and xvi) cytokines having at least one histidine-tagged amino acid to facilitate purification. the

In one embodiment, the recombinant tissue protective cytokines of the invention have a reduced number or no sialic acid moieties. In a preferred embodiment, the recombinant tissue protective cytokine is the asialo form of erythropoietin (ie without the sialic acid moiety), most preferably human asialoerythropoietin. In another embodiment, the recombinant tissue protective cytokine has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 sialic acid moieties. The number of available positions for sialylation may be altered by one or more altered or modified amino acids present in the recombinant tissue protective cytokine. Accordingly, the invention includes embodiments wherein the recombinant tissue protective cytokine is either hyposialylated or hypersialylated. In a preferred aspect, the erythropoietin mutein has more than the 14 sialic acid moieties found on native erythropoietin. the

In one embodiment, the recombinant tissue protective cytokine is N-linked sugar-free erythropoietin. In another embodiment, the recombinant tissue protective cytokine is erythropoietin with a reduced number of N-linked sugars. In one embodiment, the recombinant tissue protective cytokine is O-linked sugar-free erythropoietin. In another embodiment, the recombinant tissue protective cytokine is erythropoietin with a reduced number of O-linked sugars. the

In another embodiment, the recombinant tissue protective cytokine can be modified by fucosylation to alter the glycosylation pattern on the glycoprotein. the

In yet another embodiment, the recombinant tissue protective cytokine is treated with at least one glycosidase. In another embodiment, the recombinant tissue protective cytokine has at least a reduced sugar content as a result of treatment of the recombinant tissue protective cytokine with at least one glycosidase. the

In yet another embodiment, the carbohydrate moiety of the recombinant tissue protective cytokine has at least a non-mammalian glycosylation pattern as a result of expression of recombinant erythropoietin in non-mammalian cells. In a preferred embodiment, the recombinant tissue protective cytokine is expressed in insect cells, plant cells, bacterial cells or yeast cells. the

In yet another embodiment, the recombinant tissue protective cytokine also has at least one or more oxidized sugars that can also be chemically reduced. In a preferred embodiment, the recombinant tissue protective cytokine is periodate oxidized erythropoietin. In certain embodiments, the periodate-oxidized erythropoietin is preferably chemically reduced by sodium cyanoborohydride. the

In yet another embodiment, the recombinant tissue protective cytokine for use above has at least one or more modified arginine residues. In one embodiment, the recombinant tissue protective cytokine comprises an R-glyoxal moiety at one or more arginine residues, wherein R is an aryl or alkyl moiety. In yet another embodiment, the recombinant tissue protective cytokine is phenylglyoxal-erythropoietin. In yet another embodiment, the recombinant tissue protective cytokine is an erythrocyte wherein the arginine residue has been modified by diketone reaction with, for example, but not limited to, 2,3-butanedione and cyclohexanedione Progenin. In yet another embodiment, the recombinant tissue protective cytokine is erythropoietin wherein the arginine residue is reacted with 3-deoxyglucosone. the

In yet another embodiment, the recombinant tissue protective cytokine comprises at least one or more modified lysine residues or an N-terminal amino modification of the erythropoietin molecule, said modification being derived from a lysine residue Or the N-terminal amino group reacts with an amino modification reagent. Modified lysine residues can also be reduced chemically. In a preferred embodiment, the recombinant tissue protective cytokine is biotinylated or carbamylated or acylated (eg acetylated) via one or more lysine groups. In another preferred embodiment, lysine is reacted with an aldehyde or a reducing sugar to form an imine which, upon reduction by sodium cyanoborohydride, forms an N-alkylated lysine such as glucosyllysine acid or, in the case of reducing sugars, by Amadori or Heyns rearrangement to form α-deoxy-α-amino sugars (eg α-deoxy-α-fructosyllysine). In another preferred embodiment, the lysine group is carbamylated, for example by reaction with cyanate ion, by reacting with an alkyl-isocyanate, aryl-isocyanate or arylisocyanate, respectively Thiocyanates are reacted to be alkyl-carbamylated, aryl-carbamylated or aryl-thiocarbamylated, or can be reacted, for example, by reaction with acetic anhydride, succinic anhydride or phthalic anhydride It is acylated by active alkyl carboxylic acid or aryl carboxylic acid derivatives. At least one lysine group can also be modified with a trinitrophenyl group by reaction with trinitrobenzenesulfonic acid, or preferably a salt thereof. In another embodiment, lysine residues may be modified by reaction with glyoxal derivatives, such as with glyoxal, methylglyoxal or 3-deoxyglucosone, to form the corresponding α-carboxyalkyl derivatives. In a related embodiment, the carbamylated cytokine comprises α-N-carbamyl erythropoietin; N-ε-carbamyl erythropoietin; α-N-carbamyl, N-ε -Carbamyl erythropoietin; α-N-carbamoylasialo erythropoietin; N-ε-carbamoylasialo erythropoietin; α-N-carbamoyl, N-ε-carbamoyl erythropoietin Acylasialoerythropoietin; α-N-carbamoyl, N-ε-carbamoylasialoerythropoietin; α-N-carbamoyllowsialoerythropoietin; N-ε-carbamoyl acyl-low sialyl erythropoietin; and α-N-carbamoyl, N-ε-carbamoyl-low sialyl erythropoietin. In yet another embodiment, the recombinant tissue protective cytokine comprises at least one acylated lysine residue. In yet another embodiment, the recombinant tissue protective cytokine comprises at least one acylated lysine residue. In yet another embodiment, the recombinant tissue protective cytokine comprises at least one acylated lysine residue. In a related embodiment, the acetylated cytokine comprises α-N-acetyl erythropoietin; N-ε-acetyl erythropoietin; α-N-acetyl, N-ε-acetyl erythropoietin; α- N-acetylasialoerythropoietin; N-ε-acetylasialoerythropoietin; α-N-acetyl, N-ε-acetylasialoerythropoietin; α-N-acetylhyposialoerythropoietin N-ε-acetyl low sialo erythropoietin; α-N-acetyl, N-ε-acetyl low sialo erythropoietin; α-N-acetyl high sialo erythropoietin; N-ε-acetyl Homosialyl erythropoietin; alpha-N-acetyl and N-ε-acetyl homosialyl erythropoietin. the

In yet another embodiment, the recombinant tissue protective cytokine has a succinylated lysine residue. In a related embodiment, the succinylated cytokine comprises α-N-succinyl erythropoietin; N-ε-succinyl erythropoietin; α-N-succinyl, N-ε-succinyl erythropoietin α-N-succinoylasialoerythropoietin; N-ε-succinoylasialoerythropoietin; α-N-succinyl, N-ε-succinoylasialoerythropoietin; - N-succinyl hyposialyl erythropoietin; N-ε-succinyl hyposialyl erythropoietin; alpha-N-succinyl, N-ε-succinyl hyposialyl erythropoietin; N-ε-succinylhomosialyl erythropoietin; and N-ε-succinyl homosialyl erythropoietin. the

In one embodiment, at least one tyrosine residue of the recombinant tissue protective cytokine may be modified, for example, by nitration or iodination within an aromatic ring position by an electrophile. In a related embodiment, the aforementioned recombinant tissue protective cytokine comprises at least one lysine residue modified with sodium 2,4,6-trinitrobenzenesulfonate or another salt thereof. the

In another embodiment, the recombinant tissue protective cytokine comprises at least one nitrated and/or iodinated tyrosine residue. the

In another embodiment, the recombinant tissue protective cytokine comprises an aspartic acid residue and/or a glutamic acid residue reacted with a carbodiimide followed by an amine. In a related embodiment, the amine is glycinamide. the

In one embodiment, at least one tryptophan residue of the recombinant tissue protective cytokine is modified, eg, by reaction with n-bromosuccinimide or n-chlorosuccinimide. the

In another embodiment, recombinant tissue protective cytokines are provided with at least one erythropoietin amino group removed, eg, by reaction with ninhydrin followed by reduction of the resulting carbonyl group by reaction with borohydride. the

In yet another embodiment, reformation of the disulfide bond is prevented by reaction with a reducing agent such as dithiothreitol, followed by reaction of the subsequently generated sulfhydryl group with iodoacetamide, iodoacetic acid, or another electrophile, A recombinant tissue protective cytokine is provided having at least one opening of a cystine bond in said molecule. the

In yet another embodiment, the recombinant tissue protective cytokine is subjected to limited chemical proteolysis targeting specific residues (eg, cleavage after a tryptophan residue). The resulting recombinant tissue protective cytokine fragments are included herein. the

As mentioned above, the recombinant tissue protective cytokines used for the purposes herein may optionally have at least one of the above-mentioned chemical modifications, but may also have more than one, in addition to the genetically altered amino acids. Exemplary recombinant tissue protective cytokines having one modification in the sugar portion of the molecule and one modification in the amino acid portion of the molecule, recombinant tissue protective cytokines that are biotinylated, acylated ( Such as acetylated) or carbamylated asialoerythropoietin. Recombinant tissue protective cytokines can also be modified by the addition of fatty acid chains. In another embodiment, the recombinant tissue protective cytokine is PEGylated by the addition of polyethylene glycol (PEG), resulting in a PEGylated tissue protective cytokine. the

According to one aspect of the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the recombinant tissue protective cytokine described above. In one embodiment, the nucleic acid molecule of described separation comprises the nucleotide sequence of SEQ ID NO:208 vector construct nucleotide residue 5461-6041, the core of SEQ ID NO:209 nucleotide residue 5461-6041 Nucleotide sequence, the nucleotide sequence of SEQ ID NO: 210 nucleotide residues 5461-6041, the nucleotide sequence of SEQ ID NO: 211 nucleotide residues 5461-6041 or SEQ ID NO: 212 nucleotide residues The nucleotide sequence of bases 5461-6041. the

In one embodiment of the invention there is provided an isolated nucleic acid molecule comprising a nucleotide sequence (i.e. cDNA, nucleotide sequence with or without intervening introns) encoding the The aforementioned recombinant erythropoietin or a polypeptide consisting of the aforementioned recombinant erythropoietin, provided that the nucleic acid molecule does not encode a recombinant tissue protective cytokine comprising one or more of the following amino acid substitutions: I6A, C7A, K20A, P42A, D43A, K45D, K45A, F48A, Y49A, K52A, K49A, S100E, R103A, K116A, T132A, I133A, K140A, N147K, N147A, R150A, R150E, G151A, K152A, K154A, G158A, C161A, or R162A. In a related embodiment, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising a recombinant tissue protective cytokine as described above, with the proviso that the nucleic acid molecule does not encode a recombinant tissue comprising any of the following combinations of substitutions Protective cytokines: N24K/N38K/N83K or A30N/H32T. In one embodiment, the nucleotide sequence encoding the recombinant tissue protective cytokine is synthesized using codons preferred for optimal expression in a particular host cell. The preferred codons may be optimized for expression in one of plant cells, bacterial cells, yeast cells, mammalian cells, fungal cells or insect cells. the

The invention also provides vectors comprising said nucleic acid molecules. The present invention also provides expression vectors comprising said nucleic acid molecule and at least one regulatory region operably linked to said nucleic acid molecule. In one embodiment, the vector is a pCiNeo vector. In another embodiment, the present invention provides cells comprising said expression vector. In yet another embodiment, a genetically engineered cell comprising said nucleic acid molecule is provided. the

In another embodiment, the present invention also includes compositions, including pharmaceutical compositions, comprising one or more of the recombinant tissue protective cytokines described above. the

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the above-mentioned recombinant tissue protective cytokine lacking at least one erythropoietic activity selected from the group consisting of increasing hematocrit, vasoconstriction, hyperactivating platelets, stimulating Coagulation activity and increased production of thrombin cells. According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the above-mentioned recombinant tissue protective cytokine, but said cytokine does not lack at least one erythropoietic activity selected from the group consisting of: increasing hematocrit, vasoconstricting , hyperactivated platelets, procoagulant activity and increased production of thrombus cells. The cytokine comprises at least one effector cell protective activity selected from the group consisting of protecting, maintaining, enhancing or restoring mammalian effector cell, tissue or organ function or viability. The recombinant tissue protective cytokine of the pharmaceutical composition may comprise the amino acid sequence of SEQ ID NO: 10 with one of the following changes, namely substitutions (each change or combination of listed changes has been assigned a separate sequence identification number) : i) SEQ ID NO: 10 aspartic acid at residue 45 and glutamic acid at residue 100 (SEQ ID NO: 106); ii) SEQ ID NO: 10 asparagine at residue 30, residue 32 Threonine (SEQ ID NO: 107); iii) Aspartic acid at residue 45 of SEQ ID NO: 10, glutamic acid at residue 150 (SEQ ID NO: 108); iv) SEQ ID NO: 10 Glutamic acid at residue 103 and serine at residue 108 (SEQ ID NO: 109); v) alanine at residue 140 of SEQ ID NO: 10 and alanine at residue 52 (SEQ ID NO: 110) ; vi) alanine of SEQ ID NO: 10 residue 140, alanine of residue 52, alanine of residue 45 (SEQ ID NO: 111); vii) SEQ ID NO: 10 of residue 97 Alanine and alanine of residue 152 (SEQ ID NO: 112); iix) SEQ ID NO: 10 alanine of residue 97, alanine of residue 152, alanine of residue 45 (SEQ ID NO: ID NO: 113); ix) the alanine of SEQ ID NO: 10 residue 97, the alanine of residue 152, the alanine of residue 45 and the alanine of residue 52 (SEQ ID NO: 114 ); x) SEQ ID NO: 10 alanine of residue 97, alanine of residue 152, alanine of residue 45, alanine of residue 52 and alanine of residue 140 (SEQ ID NO: 115); xi) SEQ ID NO: 10 alanine at residue 97, alanine at residue 152, alanine at residue 45, alanine at residue 52, alanine at residue 140 acid, alanine at residue 154, lysine at residue 24, lysine at residue 38, lysine at residue 83, lysine at residue 24, and alanine at residue 15 ( SEQ ID NO: 116); xii) SEQ ID NO: 10 lysine at residue 24, lysine at residue 38 and lysine at residue 83 (SEQ ID NO: 117); or xiv) SEQ ID NO: 10 Lysine at residue 24 and Alanine at residue 15 (SEQ ID NO: 118). the

According to another aspect of the present invention, there is provided a pharmaceutical composition for protecting, maintaining, enhancing or restoring the function or vitality of mammalian effector cells and related cells, tissues and organs, said composition comprising a therapeutically effective amount of Recombinant tissue protective cytokines containing at least one substitution of the following amino acid residues (each change or combination of listed changes has been assigned a separate sequence identification number): Tryptophan at residue 152 of SEQ ID NO: 10 (SEQ ID NO: 98); SEQ ID NO: the alanine of 10 residue 14 and the alanine of residue 15 (SEQ ID NO: 119); The alanine of SEQ ID NO: 10 residue 6 (SEQ ID NO: 15 ); SEQ ID NO: the alanine of 10 residue 7 (SEQ ID NO: 16); the alanine of SEQ ID NO: 10 residue 43 (SEQ ID NO: 42); the alanine of SEQ ID NO: 10 residue 42 Alanine (SEQ ID NO:41); Alanine at residue 48 of SEQ ID NO:10 (SEQ ID NO:49); Alanine at residue 49 of SEQ ID NO:10 (SEQ ID NO:50) ; SEQ ID NO:10 residue 32 of threonine (SEQ ID NO:35); SEQ ID NO:10 residue 133 of alanine (SEQ ID NO:83); SEQ ID NO:10 residue 134 of Alanine (SEQ ID NO:84); Alanine of SEQ ID NO:10 residue 147 (SEQ ID NO:90); Alanine of SEQ ID NO:10 residue 148 (SEQ ID NO:92) ; SEQ ID NO: the alanine of 10 residue 150 (SEQ ID NO: 94); the alanine of SEQ ID NO: 10 residue 151 (SEQ ID NO: 96); the alanine of SEQ ID NO: 10 residue 158 Alanine (SEQ ID NO: 102); Alanine at residue 161 of SEQ ID NO: 10 (SEQ ID NO: 104); or Alanine at residue 162 of SEQ ID NO: 10 (SEQ ID NO: 105) . the

In one embodiment, the pharmaceutical composition described above is formulated for oral, intranasal or parenteral administration. In another embodiment, the pharmaceutical composition is formulated as a perfusate. the

In certain embodiments, the pharmaceutical composition of the present invention for protecting, maintaining, enhancing or restoring the function or viability of mammalian effector cells and associated cells, tissues and organs comprises a therapeutically effective amount of a recombinant tissue protective cytokine, The cytokine comprises at least one substitution of an amino acid residue of the amino acid sequence of native human erythropoietin. the

In other embodiments, the pharmaceutical composition of the present invention for protecting, maintaining, enhancing or restoring the function or vitality of mammalian effector cells and related cells, tissues and organs comprises a therapeutically effective amount of a recombinant tissue protective cytokine, so Said cytokines comprise cytoprotective activity which may lack one or more erythropoietic activities or effects such as: increasing hematocrit, vasoactive effects (vasoconstriction/vasodilation), hyperactivating platelets, procoagulant activity and increasing coagulation Cell production. the

In other embodiments, the pharmaceutical composition of the present invention for protecting, maintaining, enhancing or restoring the function or vitality of mammalian effector cells and related cells, tissues and organs comprises a therapeutically effective amount of a recombinant tissue protective cytokine, so Said cytokines comprise cytoprotective activities which may also have one or more erythropoietic activities or effects such as: increasing hematocrit, vasoactive effects (vasoconstriction/vasodilation), hyperactivating platelets, procoagulant activity and increasing The production of blood clotting cells. the

According to one aspect of the present invention, there is provided a method for protecting, maintaining or enhancing the viability of cells, tissues or organs isolated from a mammal, said method comprising contacting said cells, tissues or organs with a compound comprising the absence of at least one Pharmaceutical composition of recombinant tissue protective cytokines of erythropoietin with erythropoietic activity selected from the group consisting of increased hematocrit, vasoactive effect (vasoconstriction/vasodilation), hyperactivated platelets, procoagulant activity and increased coagulation Cell production. In certain embodiments, the protection does not affect the bone marrow. the

The present invention also provides a method for protecting, maintaining or enhancing the viability of cells, tissues or organs isolated from a mammal, said method comprising contacting said cells, tissues or organs with a Pharmaceutical composition of recombinant tissue protective cytokines with erythropoietic activity: increases hematocrit, vasoactive effect (vasoconstriction/vasodilation), hyperactivation of platelets, procoagulant activity and increased production of thrombin cells. the

The present invention also provides the above-mentioned recombinant tissue protective cytokine lacking at least one erythropoietic activity selected from the group consisting of in the preparation of a pharmaceutical composition for preventing and preventing mammalian tissue damage and restoring and regenerating mammalian tissue and tissue function Uses for: Increase of hematocrit, vasoactive effect (vasoconstriction/vasodilation), hyperactivation of platelets, procoagulant activity and increased production of thrombus cells. In one embodiment, the injury is caused by epilepsy, multiple sclerosis, stroke, hypotension, cardiac arrest, ischemia, myocardial infarction, inflammation, age-related cognitive decline, radiation injury, cerebral palsy , neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, subacute necrotizing encephalomyelopathy (Leigh disease), AIDS dementia, memory loss, amyotrophic lateral cord Cirrhosis, alcoholism, mood disorders, anxiety disorders, ADHD, autism, transmissible spongiform encephalopathy (Creutzfeld-Jakob disease), brain or spinal cord trauma, cerebral or spinal cord ischemia, cardiopulmonary Shunt surgery, chronic heart failure, macular degeneration, diabetic neuropathy, diabetic retinopathy, glaucoma, retinal ischemia, or retinal trauma. the

According to another aspect of the present invention, there is provided a method for promoting the transcytosis of molecules across the endothelial cell barrier in a mammal, said method comprising administering to said mammal the above-mentioned recombinant Compositions of said molecules associated with tissue protective cytokines: increase hematocrit, increase blood pressure, hyperactivate platelets and increase thrombin cell production. In one embodiment, the association is a labile covalent bond, a stable covalent bond, or a non-covalent association with the binding site of the molecule. According to another aspect of the present invention, there is provided a method for promoting the transcytosis of molecules across the endothelial cell barrier in a mammal, the method comprising administering to the mammal a recombinant tissue protective agent comprising the above-mentioned recombinant tissue having an activity selected from the group consisting of: Composition of said molecules associated with cytokines: increase hematocrit, increase blood pressure, hyperactivate platelets and increase thrombin cell production. In one embodiment, the association is a labile covalent bond, a stable covalent bond, or a non-covalent association with the binding site of the molecule. In another embodiment, the endothelial cell barrier is selected from the group consisting of blood-brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier, blood-heart barrier, blood-kidney barrier and blood-fetal barrier. In yet another embodiment, the molecule is a receptor agonist or antagonist hormone, neurotrophic factor, antimicrobial drug, antiviral drug, radiopharmaceutical, antisense oligonucleotide, antibody, immunosuppressant, dye, markers or anticancer drugs.

According to another aspect of the present invention, there is provided a composition for transporting molecules across the endothelial cell barrier by transcytosis, said composition comprising a recombinant tissue protective cytokine as described above lacking at least one erythropoietic activity selected from The molecules associated: increased hematocrit, vasoactive effects (vasoconstriction/vasodilation), hyperactivation of platelets, procoagulant activity and increased generation of thrombin cells. According to another aspect of the present invention, there is provided a composition for transporting molecules across the endothelial cell barrier by transcytosis, said composition comprising a recombinant tissue protective cytokine as described above having at least one erythropoietic activity selected from The molecules associated: increased hematocrit, vasoactive effects (vasoconstriction/vasodilation), hyperactivation of platelets, procoagulant activity and increased generation of thrombin cells. In one embodiment, the association is a labile covalent bond, a stable covalent bond, or a non-covalent association with the binding site of the molecule. In another embodiment, the molecule is a receptor agonist or antagonist hormone, neurotrophic factor, antimicrobial drug, radiopharmaceutical, antisense oligonucleotide, antibody, immunosuppressant, dye, marker, or anti- cancer drugs. the

The present invention also provides the use of a recombinant tissue protective cytokine-associated molecule as described above lacking at least one erythropoietic activity selected from the group consisting of: Increases hematocrit, vasoactive effects (vasoconstriction/vasodilation), hyperactivation of platelets, procoagulant activity and increased production of thrombus cells. In one embodiment, the association is a labile covalent bond, a stable covalent bond, or a non-covalent association with the binding site of the molecule. In another embodiment, the molecule is a receptor agonist or antagonist hormone, neurotrophic factor, antimicrobial drug, radiopharmaceutical, antisense oligonucleotide, antibody, immunosuppressant, dye or marker, or anti- cancer drugs. the

Accordingly, the present invention relates to the cytoprotective use of any recombinant tissue protective cytokine having at least one amino acid alteration on the native erythropoietin counterpart, wherein said recombinant tissue protective cytokine has cytoprotective activity as described herein. Said cytoprotective activity includes, but is not limited to, neuroprotective activity. The present invention also relates to the use of any of the aforementioned recombinant tissue protective cytokines in the treatment of effector cells, tissues or organs, in particular in the treatment of disorders or diseases involving said effector cells, tissues or organs. In one such embodiment, the recombinant tissue protective cytokine has at least one erythropoietic activity selected from the group consisting of increasing hematocrit, vasoactive effects (vasoconstriction/vasodilation), hyperactivating platelets, procoagulant Activates and increases the production of blood clotting cells. The recombinant tissue protective cytokines of the present invention preferably maintain the three-dimensional conformation of native erythropoietin. The recombinant tissue protective cytokine may or may not have erythropoietic activity. the

In one embodiment of the invention, the recombinant tissue protective cytokine is produced as a recombinant protein with an N-terminal fused His tag (6xHis residues). In certain embodiments, additional amino acid sequences may be added as spacer sequences. In a specific embodiment, the histidine-tagged recombinant tissue protective cytokine muteins of the present invention include but are not limited to K45D-6xHis and S100E-6xHis. the

In another aspect of the present invention, any of the above-mentioned recombinant tissue protective cytokines can be used in preparation for ex vivo treatment of cells, tissues and organs to protect, maintain, enhance or restore mammalian effector cells and their related cells, tissues and organs. Pharmaceutical compositions for organ function or vitality purposes. The ex vivo treatment is used, for example, for the preservation of cells, tissues or organs for transplantation, whether autologous or xenotransplantation. The cells, tissue or organ may be immersed in a solution comprising erythropoietin mutein or recombinant tissue protective cytokine, or the perfusate may be instilled into the organ through the vasculature or otherwise, in which Cellular function is maintained during periods when a cell, tissue or organ is not integrated with the recipient's or donor's vasculature. Perfusates can be administered to donors, as well as to harvested organs and recipients, prior to organ harvesting. In addition, any of the above uses of recombinant tissue protective cytokines may be used when cells, tissues or organs are separated from the vasculature of an individual and therefore must exist ex vivo for a period of time, the term isolated means confining or immobilizing a cell, tissue, organ or organism Part of the vasculature, or access to the vascular system of cells, tissues, organs or body parts, such as may be performed during surgery, especially cardiopulmonary bypass surgery; shunting the vasculature of cells, tissues, organs or body parts; from breastfeeding Removal of cells, tissues, organs or body parts from animals, either prior to xenotransplantation or before or during autologous transplantation; or during traumatic amputation of cells, tissues, organs or body parts. Accordingly, this aspect of the invention relates to in situ and ex vivo perfusion with erythropoietin muteins. The recombinant tissue protective cytokine can be provided ex vivo in a cell, tissue or organ preservation solution. For either aspect, exposure can be by means of continuous infusion, pulse infusion, infusion, immersion, injection or catheterization. the

In yet another aspect, the invention relates to methods for protecting, maintaining, enhancing or restoring the viability of mammalian cells, tissues, organs or body parts (including effector cells or tissues), wherein said cells, tissues, organs or body parts isolated from mammals. The method comprises exposing isolated mammalian cells, tissues, organs or body parts to at least an amount of erythropoietin mutein or recombinant tissue protective cytokine for a period of time effective to protect, maintain, enhance or restore the aforementioned vigor. In a non-limiting example, to isolate means to confine or clamp the vasculature of, or access to, a cell, tissue, organ, or body part, such as may be used during surgery, especially in cardiopulmonary Shunt surgery; shunting the vasculature of cells, tissues, organs, or body parts; removing cells, tissues, organs, or body parts from mammals, either prior to xenotransplantation or before or during autologous transplantation; or may be performed in Performed during traumatic amputation of cells, tissues, organs, or body parts. Accordingly, this aspect of the invention involves in situ and ex vivo perfusion with erythropoietin muteins or recombinant tissue protective cytokines. The recombinant tissue protective cytokine can be provided ex vivo in a cell, tissue or organ preservation solution. For either aspect, exposure can be by means of continuous infusion, pulse infusion, infusion, immersion, injection or catheterization. the

As non-limiting examples, the above ex vivo effector cells or tissues may be or may include neuronal cells, retinal cells, myocytes, cardiac cells, lung cells, liver cells, kidney cells, intestinal cells, adrenal cortical cells, adrenal medulla cells, capillary endothelial cells, testicular cells, ovarian cells, pancreatic cells, bone cells, bone marrow cells, skin cells, umbilical cord blood cells, or endometrial cells or tissues. These examples of effector cells are illustrative only. the

All of the above methods and uses are preferably applied to humans, but may be applied to any mammal such as, but not limited to, companion animals, domesticated animals, livestock and zoo animals. The routes of administration of the above-mentioned pharmaceutical compositions include oral, intravenous, intranasal, topical, intraluminal, inhalation or parenteral administration, the latter including intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, mucosal Administer subcutaneously or intradermally. For ex vivo applications, the perfusate or immersion fluid is preferred. This includes perfusing isolated sections of the vasculature in situ. the

In yet another aspect of the present invention, any of the above-mentioned recombinant tissue protective cytokines is used for the preparation of a pharmaceutical composition for restoring the function of dysfunctional cells, tissues or organs. Give after attack. As a non-limiting example, in animals with a previous brain injury, administration of a pharmaceutical composition comprising a recombinant tissue protective cytokine restores cognitive function even long after the initial trauma has subsided (e.g., 1 day, 3 days, 5 days, 1 week, 1 month or longer) when administered. The present invention includes pharmaceutical compositions for treating (ie, alleviating or reversing the symptoms or effects) and preventing (ie, delaying onset, inhibiting or halting) subsequent damage to cells and tissues resulting from the cascade of reactions caused by the initial trauma. Recombinant tissue protective cytokines for use in such applications include any of the specific recombinant tissue protective cytokines described above. Any form of recombinant tissue protective cytokine that benefits effector cells is encompassed within this aspect of the invention. the

In yet another embodiment, the present invention provides a method for the above-mentioned recombinant tissue protective cytokines for restoring dysfunctional cells, tissues or organs, the method being administered after the onset of a disease or condition causing dysfunction. As a non-limiting example, in animals with a previous brain injury, administration of a pharmaceutical composition comprising a recombinant tissue protective cytokine restores cognitive function even long after the trauma has subsided (e.g., 3 days, 5 days, 1 week, 1 month or longer) when administered. Recombinant tissue protective cytokines and further modifications of said cytokines are as described above. Any form of recombinant tissue protective cytokine that benefits effector cells is encompassed within this aspect of the invention. the

In yet another aspect of the present invention, there is provided a method for promoting transcytosis of a molecule across the endothelial cell barrier in a mammal by administering to the mammal an erythropoietin mutein or a recombinant tissue protective cytokine as described above. The composition of the molecule. the

The association between the molecule to be transported and the recombinant tissue protective cytokine can be, for example, a labile covalent bond, a stable covalent bond, or a non-covalent association with the binding site of the molecule. Recombinant tissue protective cytokines and proteins to be transported can be expressed as fusion polypeptides. The endothelial cell barrier can be blood-brain barrier, blood-heart barrier, blood-kidney barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier and blood-fetal barrier. Suitable molecules to be delivered by the methods of the invention include hormones such as growth hormone, antibiotics and anticancer drugs. the

Another aspect of the present invention provides a composition for promoting transcytosis of a molecule across an endothelial cell barrier in a mammal, said composition comprising said molecule in association with a recombinant tissue protective cytokine as described above. the

In another aspect of the present invention, any of the above-mentioned recombinant tissue protective cytokines is used for the preparation of a pharmaceutical composition for promoting the transcytosis of molecules across the endothelial cell barrier in mammals, the composition comprising the above-mentioned recombinant tissue The molecules associated with protective cytokines. the

The association can be, for example, a labile covalent bond, a fusion polypeptide, a stable covalent bond, or a non-covalent association with the binding site of the molecule. The endothelial cell barrier can be blood-brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier and blood-fetal barrier. Suitable molecules to be delivered by the methods of the invention include hormones such as growth hormone, neurotrophic factors, antibiotics, antivirals, or antifungals such as those normally excluded by the brain and other barrier-protected organs, peptide radiopharmaceuticals, antisense drugs , antibodies against biologically active factors, pharmaceutical substances, dyes, markers and anticancer drugs. the

These and other aspects of the invention will be better understood with reference to the following figures and detailed description. the

4. Description of drawings

Figure 1 shows the distribution of erythropoietin receptors in normal human brain in thin sections stained with anti-erythropoietin antibody. the

Figure 2 is a higher magnification image of Figure 1. the

Figure 3 shows the ultramicroscopic distribution of the erythropoietin receptor using a gold-labeled secondary antibody. the

Figure 4, prepared in a manner similar to Figure 3, shows a high density of erythropoietin receptors on the luminal surface and reverse surface of human brain capillaries. the

Figure 5 depicts the transport of parenterally administered erythropoietin into the cerebrospinal fluid. the

Figures 6A and 6B show the results of an SK-N-SH neuroblastoma cell neuroprotection assay (against rotenone) with erythropoietin and recombinant tissue protective cytokines K45D and S100E. The Y-axis of the graph represents the absorbance readings, and the data are mean ± range of replicates. The graph in Figure 6A clearly shows that cell viability is maintained in K45D and S100E samples, demonstrating their cytoprotective effect. Figure 6B shows the plasmid map of hEPO-6xHis tag-PciNeo. the

Figure 7 compares the in vitro efficacy of erythropoietin and asialoerythropoietin on the viability of serum starved P19 cells. the

Figure 8 is another experiment comparing the in vitro efficacy of erythropoietin and asialoerythropoietin on the viability of serum starved P19 cells. the

Figure 9 shows the protective effects of erythropoietin and asialoerythropoietin in a rat model of focal cerebral ischemia. the

Figure 10 shows a dose response comparing the efficacy of human erythropoietin and human asialoerythropoietin in middle cerebral artery occlusion in an ischemic stroke model. the

Figure 11 shows the activity of iodinated erythropoietin in the P19 assay. the

Figure 12 shows the effect of biotinylated erythropoietin and asialo erythropoietin in the P19 assay. the

Figure 13 compares the in vitro efficacy of erythropoietin and phenylglyoxal-modified erythropoietin on the viability of serum starved P19 cells. the

Figure 14 shows the effect of tissue protective cytokines in the water intoxication assay. the

Figure 15 shows erythropoietin maintenance of heart function in preparation for transplantation. the

Figure 16 shows that erythropoietin protects cardiomyocytes from ischemic injury after transient vascular occlusion. the

Figures 17A, 17B, 17C and 17D depict the effect of erythropoietin treatment in a rat glaucoma model. the

Figure 18 shows the degree of protection of erythropoietin on retinal function in a rat glaucoma model. the

Figure 19 depicts the recovery of cognitive function induced by brain trauma by administration of erythropoietin starting 5 days after brain trauma. the

Figure 20 depicts the restoration of brain trauma induced cognitive function by administration of erythropoietin starting 30 days after brain trauma. the

Figure 21 depicts the efficacy of human asialoerythropoietin in the kainic acid model of brain toxicity. the

Figure 22 depicts the efficacy of tissue protective cytokines in a rat spinal cord injury model. the

Figure 23 shows the efficacy of tissue protective cytokines in the rabbit spinal cord injury model. the

Figures 24A, 24B, and 24C show coronal sections of cerebral cortex stained with hematoxylin and eosin. the

Figures 25A, 25B and 25C show coronal sections of the anterior cortex adjacent to the infarct stained with GFAP antibody. the

Figures 26A and 26B show coronal sections of cerebral cortex stained with OX-42 antibody. the

Figures 27A and 27B show coronal sections of cerebral cortex adjacent to the infarct area stained with OX-42 antibody. the

Figure 28 shows the anti-inflammatory effect of erythropoietin in the EAE model. the

Figure 29 compares the anti-inflammatory effects of dexamethasone and erythropoietin in the EAE model. the

Figures 30A and 30B show that erythropoietin inhibits inflammation associated with neuronal death. the

Figure 31 shows that adding human erythropoietin and recombinant tissue protective cytokines R130E and R150E to primary hippocampal neuron cell cultures prior to MNDA treatment effectively reduced cell death induced by NMDA. Cells treated with R103E (5 nM) showed significantly reduced cell death compared to vehicle control cells (p=0.01). Cells treated with R103E (5 nM) showed significantly reduced cell death compared to vehicle control cells (p=0.01). Cells treated with R150E (5 nM) showed an approximately 20% reduction in cell death compared to vehicle control cells (p=0.001). Statistics: ANOVA with Tukey's post hoc test. the

Figure 32 shows neuronal protection in P19 cells after serum depletion. The percentage of apoptotic cell death decreased for cells pretreated with Epo, EpoWT and recombinant tissue protective cytokine S100E. Cells treated with Epo showed about a 20% decrease in apoptotic cell death compared to untreated control cells. Cells treated with EpoWT and S100E both showed about 10% decrease in apoptotic cell death compared to untreated control cells. the

Figures 33A and 33B show the effect of pre-incubation with S100E in NGF-depleted differentiated PC12 cells in two independent experiments. Differentiated PC12 cells were pretreated with the indicated concentrations of S100E for 24 hours, Figure 33A (3pM), Figure 33B (0.00003pM-3pM). Viability was determined in the MTT assay. NGF (100 ng/ml) was used as a positive control, while medium without NGF (-NGF) was used as a negative control. Data shown in Figure 33 are positive control (+NGF) and % viability (n=8 in both experiments). Using one-way ANOVA with Bonferroni post hoc test, there was a statistically significant increase in the viability of S100E-treated cells compared to negative control cells (-NGF). ^*** p<0.001, ^* p<0.05. In terms of potency and efficacy, the effects observed with S100E were similar to those with Epo in this experimental system.

Figure 34A, Figure 34B and Figure 34C show the effect of pre-incubation with Epo in NGF-depleted differentiated PC12 cells. Differentiated PC12 cells were pretreated with Epo, S100E or carbamylated Epo (30pM-30nM) for 24 hours. The chemically modified Epo molecule AA24496 was 10,000-fold less active than EPO in the UT-7 cell assay. Viability was determined in the MIT assay. NGF (100 ng/ml) was used as a positive control, while NGF-free medium (-NGF) was used as a negative control. the

Figure 35 shows the concentration-response curves of Epo, K45D and S100E in UT-7 cells. Different concentrations of Epo, EpoWT, K45D and S100E were added to UT-7 cells. In the WST-1 assay, viability was measured after 48 hours. Data are means ± SD of duplicate determinations in each of three different experiments. The curve is a nonlinear regression curve fit. the

Figure 36 shows the dose response curves of Epo, R103E and R150E in UT-7 cells. Different concentrations of Epo, EpoWT, R103E and R150E were added to UT-7 cells. In the WST-1 assay, viability was measured after 48 hours. Data are means ± SD of duplicate determinations in each of three different experiments. The curve is a nonlinear regression curve fit. the

Figure 37 is a graph demonstrating locomotor levels in rats recovering from spinal cord injury over a 42-day period. As can be seen from the figure, rats administered S100E recovered more readily from injury and demonstrated better overall recovery than control rats and rats administered methylprednisolone. the

Figure 38 shows the ratio of the latency of the injured eye to the latency of the normal eye for different treatment regimens. Rats treated with EPO showed a latency of 1.2, which was better than rats treated with saline. For each of the four recombinant tissue protective cytokines, R103E, R150E and S100E produced latencies equal to or better than EPO, indicating statistically better results than EPO. the

5. Specific implementation

The present invention relates to a mutant protein recombinant tissue protective cytokine. In particular, the present invention provides compositions comprising an isolated nucleic acid molecule encoding a recombinant tissue protective cytokine mutein, as well as isolated and/or recombinant cells and vectors comprising said nucleic acid molecule. The present invention also includes isolated polypeptides of recombinant tissue protective cytokines lacking at least one mutein of erythropoietic activity selected from the group consisting of increasing hematocrit, vasoactive effects (vasoconstriction/vasodilation), hyperactivating platelets, promoting Coagulation activity and increased production of thrombus cells, said cytokine having at least one effector cell protective activity selected from protecting, maintaining, enhancing or restoring mammalian effector cell, tissue or organ function or viability. The present invention also includes methods of using the recombinant tissue protective cytokine muteins of the present invention to protect, maintain or enhance the viability of cells, tissues or organs isolated from mammals, and the use of the muteins in the treatment and treatment of diseases and disorders. Use in prevention. the

"Effector cells" refer to mammalian cells whose function or viability can be maintained, promoted, enhanced, regenerated or otherwise benefited by exposure to erythropoietin. Non-limiting examples of such cells include neuronal cells, retinal cells, myocytes, cardiac cells, lung cells, liver cells, kidney cells, intestinal cells, adrenal cortical cells, adrenal medullary cells, capillary endothelial cells, testicular cells , ovarian cells, pancreatic cells, bone cells, skin cells and endometrial cells. Specifically, effector cells would include, but are not limited to, neuronal cells; Purkinje cells; retinal cells: photoreceptors (rods and cones), ganglion cells, bipolar cells, horizontal cells, amacrine cells, and rice Leukocytes; myocytes; heart cells: cardiomyocytes, pacemaker cells, sinoatrial node cells, sinus node cells, and combined tissue cells (atrioventricular node and atrioventricular bundle); lung cells; hepatocytes: hepatocytes, astrocytes and hepatic macrophages; renal cells: mesangial cells, renal epithelial cells, and tubular enterocytes; small intestinal cells: goblet cells, intestinal gland cells (cryts), and enteroendocrine cells; adrenal cortical cells: glomerular cells, fascicular cells and reticular cells; adrenal medullary cells: chromaffin cells; capillary cells: adventitial cells; testicular cells: Leydig cells, trophoblasts and sperm cells and their primitive cells; ovarian cells: mature follicular cells and primitive follicles cells; pancreatic cells: islets, α-cells, β-cells, γ-cells, and F-cells; bone cells: osteoprogenitors, osteoclasts, and osteoblasts; skin cells; endometrial cells: endometrial stroma cells and endometrial cells; and stem cells and endothelial cells present in the organs listed above. In addition, the effector cells and the benefits provided by the recombinant tissue protective cytokines can be extended to provide indirect protection or enhancement to other cells that are not direct effectors, or to tissues or organs containing the non-effector cells. These other cells or tissues or organs that indirectly benefit from the enhancement of effector cells present as part of the cell, tissue or organ are "associated" cells, tissues and organs. Thus, the benefit of the recombinant tissue protective cytokines described herein may be provided by the presence of a small number or proportion of effector cells in a tissue or organ, such as excitable or neuronal tissue present in said tissue, or Leydig cells in the testes that produce testosterone. In one aspect, the effector cells or their associated cells, tissues or organs are neither excitable cells, tissues or organs nor predominately comprise excitable cells or tissues. the

The method of the present invention provides local or systemic protection or enhancement of cells, tissues and organs in a mammal under various normal and adverse conditions, or provides protection or enhancement of cells, tissues that are intended to be transplanted (relocation) into another mammal. or organ protection. Additionally, recovery or regeneration of dysfunction is also provided. As noted above, the ability of erythropoietin muteins or recombinant tissue protective cytokines to cross the tight endothelial cell barrier and exert positive effects on effector cells (as well as other cell types) exiting the vasculature offers prophylactic as well as therapeutic potential for various Potential for conditions and diseases that would otherwise cause significant cellular and tissue damage in animals, including humans, but also enabling hitherto untried surgical procedures where risks outweigh benefits success. The duration and extent of deliberate disadvantages for ultimate benefit, such as high dose chemotherapy and radiotherapy, prolonged ex vivo graft survival and prolonged surgically induced ischemic periods, can be overcome by the advantages of the present invention. However, the present invention is not limited thereto, and also includes, as an aspect, methods or compositions wherein target effector cells leave the vasculature due to endothelial cell barriers and endothelial tight junctions. The present invention generally relates to any effector cells and associated cells, tissues and organs that may benefit from exposure to recombinant tissue protective cytokines. In addition, cellular, tissue or organ dysfunction can be restored or regenerated following acute adverse events such as trauma by exposure to recombinant tissue protective cytokines. the

Thus, the present invention generally relates to the use of recombinant tissue protective cytokines for the preparation of pharmaceutical compositions for the above purposes, wherein cellular functions are maintained, promoted, enhanced, regenerated or otherwise benefited. The invention also relates to methods of maintaining, enhancing, promoting or regenerating cellular function by administering to a mammal an effective amount of a recombinant tissue protective cytokine as described herein. The invention also relates to methods of maintaining, promoting, enhancing or regenerating cellular function ex vivo by contacting cells, tissues or organs with recombinant tissue protective cytokines. The invention also relates to perfusion compositions comprising recombinant tissue protective cytokines for organ or tissue preservation. the

The various methods of the present invention employ a pharmaceutical composition comprising at least an effective amount of a recombinant tissue protective cytokine for a particular route and duration of exposure such that there is no effect on or from a mammal. The effector cells exert a positive effect or benefit. When the target cell, tissue or organ intended to be treated requires recombinant tissue protective cytokines to cross the endothelial cell barrier, the pharmaceutical composition includes the recombinant tissue at a concentration capable of exerting its desired effect on the effector cells after crossing the endothelial cell barrier Protective cytokines. Molecules herein that are capable of interacting with the erythropoietin receptor and modulating cytoprotective activity in said cells are useful in the present invention. the

5.1. Nucleic acid of the present invention

Recombinant tissue protective cytokines comprising nucleic acid molecules of the invention include nucleic acids encoding tissue protective cytokines comprising erythropoietin muteins lacking or exhibiting reduced at least one erythropoietic activity selected from the group consisting of increased Hematocrit, vasoactive effects (vasoconstriction/vasodilation), hyperactivated platelets, procoagulant activity and increased production of thrombin cells, said cytokines having at least one selected from the group consisting of protecting, maintaining, enhancing or restoring mammalian effector cells , effector cytoprotective activity of tissue or organ function or viability. The tissue protective cytokine comprising the nucleic acid molecule of the present invention comprises a nucleic acid encoding an erythropoietin mutein having the above activity, said nucleic acid being contained between positions 11-15 of SEQ ID NO: 10 [SEQ ID NO: 1], Between positions 44-51 of SEQ ID NO: 10 [SEQ ID NO: 2], between positions 100-108 of SEQ ID NO: 3] or between positions 146-151 of SEQ ID NO 10 [ One or more altered amino acid residues of SEQ ID NO: 4]. The tissue protective cytokine comprising the nucleic acid molecule of the present invention comprises a nucleic acid encoding an erythropoietin mutein having the above activity, said nucleic acid comprising altered amino acid residues at one or more positions in the following SEQ ID NO: 10: 7 , 20, 21, 29, 33, 38, 42, 59, 63, 67, 70, 83, 96, 126, 142, 143, 152, 153, 155, 156, or 161. A tissue protective cytokine comprising a nucleic acid molecule of the present invention comprises a nucleic acid encoding an erythropoietin mutein having the activity described above, said nucleic acid comprising the amino acid sequence of SEQ ID NO: 10 having one or more of the following changes: SEQ ID NO: Alanine at 10 residue 6, SEQ ID NO: Alanine at 10 residue 7, Serine at SEQ ID NO: 10 residue 7, Isoleucine at 10 residue 10, SEQ ID NO : Serine of 10 Residue 11, SEQ ID NO: Alanine of 10 Residue 12, SEQ ID NO: Alanine of 10 Residue 13, SEQ ID NO: Alanine of 10 Residue 14, SEQ ID NO : Glutamic acid at residue 14 of 10, glutamine at residue 14 of SEQ ID NO: 10 residue 14, alanine at residue 15 of SEQ ID NO: 10, phenylalanine at residue 15 of SEQ ID NO: 10, SEQ ID NO: 10 residue 15 isoleucine, SEQ ID NO: 10 residue 20 glutamic acid, SEQ ID NO: 10 residue 20 alanine, SEQ ID NO: 10 residue 21 alanine amino acid, lysine of SEQ ID NO:10 residue 24, serine of SEQ ID NO:10 residue 29, tyrosine of SEQ ID NO:10 residue 29, day of SEQ ID NO:10 residue 30 Paragine, the threonine of SEQ ID NO:10 residue 32, the serine of SEQ ID NO:10 residue 33, the tyrosine of SEQ ID NO:10 residue 33, the lysine of SEQ ID NO:10 residue 38 amino acid, lysine of SEQ ID NO:10 residue 83, asparagine of SEQ ID NO:10 residue 42, alanine of SEQ ID NO:10 residue 42, SEQ ID NO:10 residue 43 Alanine of SEQ ID NO: 10 residue 44, Aspartic acid of SEQ ID NO: 10 residue 45, Alanine of SEQ ID NO: 10 residue 45, SEQ ID NO: Alanine at residue 46 of 10, Alanine at residue 47 of SEQ ID NO:10, Isoleucine at residue 48 of SEQ ID NO:10, Alanine at residue 48 of SEQ ID NO:10 , alanine of SEQ ID NO: 10 residue 49, serine of SEQ ID NO: 10 residue 49, phenylalanine of SEQ ID NO: 10 residue 51, asparagine of SEQ ID NO: 10 residue 51, SEQ ID NO: 10 alanine at residue 52, SEQ ID NO: 10 asparagine at 59 residue, SEQ ID NO: 10 threonine at 10 residue 62, SEQ ID NO: 10 serine at 67 residue, SEQ ID NO : 10 alanine at residue 70, SEQ ID NO: 10 arginine at 96 residue, SEQ ID NO: 10 alanine at 97 residue, SEQ ID NO: 10 arginine at 100 residue, SEQ ID NO : glutamic acid at 10 residue 100, SEQ ID NO: alanine at 10 residue 100, threonine at 10 residue 100, SEQ ID NO: alanine at 10 residue 101, SEQ ID NO : Isoleucine at 10 residue 101, SEQ ID NO: Alanine at 10 residue 102, Alanine at SEQ ID NO: 10 residue 103, Glutamic acid at 10 residue 103, SEQ ID NO:10 alanine at residue 104, SEQ ID NO:10 isoleucine at residue 104, SEQ ID NO:10 alanine at residue 105, SEQ ID NO:10 alanine at residue 106 amino acid, isoleucine of SEQ ID NO:10 residue 106, alanine of SEQ ID NO:10 residue 107, leucine of SEQ ID NO:10 residue 107, residue 10 of SEQ ID NO:10 Lysine at 108, Alanine at residue 108 of SEQ ID NO:10, Serine at residue 108 of SEQ ID NO:10, Alanine at residue 116 of SEQ ID NO:10, Residue 10 of SEQ ID NO:10 Alanine at 126, Alanine at residue 132 of SEQ ID NO:10, Alanine at residue 133 of SEQ ID NO:10, Alanine at residue 134 of SEQ ID NO:10, SEQ ID NO:10 Alanine at residue 140, Isoleucine at residue 142 of SEQ ID NO: 10, Alanine at residue 143 of SEQ ID NO: 10, Alanine at residue 146 of SEQ ID NO: 10, SEQ ID NO: 10 lysine at residue 147, SEQ ID NO: 10 alanine at 10 residue 147, SEQ ID NO: 10 tyrosine at 10 residue 148, SEQ ID NO: 10 alanine at 10 residue 148, SEQ ID NO: Alanine at residue 149 of 10, Alanine at residue 150 of SEQ ID NO:10, Glutamic acid at residue 150 of SEQ ID NO:Alanine at residue 151 of SEQ ID NO:10, Alanine at residue 151, SEQ ID NO: Alanine of 10 residue 152, Tryptophan of SEQ ID NO: 10 residue 152, Alanine of SEQ ID NO: 10 residue 153, Alanine of SEQ ID NO: 10 residue 154, SEQ ID NO: 10 alanine at residue 155, SEQ ID NO: alanine at 10 residue 158, SEQ ID NO: serine at 10 residue 160, SEQ ID NO : alanine at residue 161 of 10 or alanine at residue 162 of SEQ ID NO: 10. the

Nucleic acid molecules of the present invention also include codes that have at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, The nucleotide sequence of the recombinant erythropoietin mutein having 80%, 85%, 90%, 95%, 98% or higher amino acid sequence identity. To determine the % identity of two amino acid sequences or two nucleic acids encoding erythropoietin muteins, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment with a second amino acid sequence or nucleic acid sequence, the sequence can be found at A gap is introduced into the sequence of the first amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The % identity between two sequences is a function of the number of identical positions shared by the sequences (ie % identity = number of identical overlapping positions/total number of overlapping positions x 100%). In one embodiment, the two sequences are equal in length. the

The nucleic acid molecule of the present invention also includes a nucleotide sequence encoding a recombinant erythropoietin mutein, wherein the nucleic acid sequence encoding erythropoietin altered by one or more substitutions, deletions or the above-mentioned modifications comprises at least the same sequence as that of SEQ ID NO: 7 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity. The nucleic acid molecules of the present invention also include nucleotide sequences encoding recombinant erythropoietin muteins, wherein the erythropoietin-encoding nucleic acid sequence altered by one or more substitutions, deletions, or modifications described above is a nucleic acid encoding a non-human erythropoietin . the

The determination of % identity between two sequences can also be accomplished using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm for comparing two sequences is the Karlin and Altschul, 1990, Proc. Revised from .Acad.Sci.USA 90:5873-5877. The algorithm is incorporated into the NBLAST and XBLAST programs described in Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. Alignments incorporating gaps for comparison purposes can be obtained using Gapped BLAST as described by Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform iterative searches to determine intermolecular distance relationships (Altschul et al., 1997, supra). When using the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used (see http://www.nobi.nlm.nih.gov). Another non-limiting example of a preferred mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. The algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for amino acid sequence comparison, the PAM120 weight residue table can be used, the gap length penalty is 12, and the gap penalty is 4. the

The % identity of two sequences can be determined using techniques analogous to those described above, allowing or disallowing gaps. When calculating % identity, usually only exact matches are counted. the

The nucleic acid molecule of the present invention also includes: (a) a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid molecule encoding the above-mentioned erythropoietin mutein or recombinant tissue protective cytokine of the present invention, such as a filter-binding DNA Hybridization in 6x sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by one or more washes in 0.2xSSC/0.1% SDS at approximately 50-65°C, or (b) under high stringency conditions For example, filter-bound nucleic acids are hybridized at 45°C in 6xSSC, followed by one or more washes at about 68°C in 0.1xSSC/0.2% SDS, or under other hybridization conditions apparent to those skilled in the art (see, e.g., Ausubel F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & sons, Inc., New York, pp. 6.3.1-6.3.6 and 2.10.3). Preferably, the nucleic acid molecule encoding an erythropoietin mutein that hybridizes under the conditions of (a) and (b) above is a complementary nucleic acid molecule comprising a nucleic acid molecule encoding an erythropoietin mutein. In a preferred embodiment, the nucleic acid molecules hybridized under the above conditions (a) and (b) encode protein products, for example, functionally equivalent to erythropoietin mutant eggs that have one or more of the above-mentioned erythropoietin activities protein product. The nucleic acids of the invention are preferably human nucleic acids. the

The nucleic acid molecule of the present invention also includes the above-mentioned nucleotide sequence hybridized with the above-mentioned erythropoietin mutein or recombinant tissue protective cytokine, and the above-mentioned erythropoietin mutein or recombinant tissue protective cytokine also lacks at least one selected from The following erythropoietic activities or exhibit reduced said activities: increased hematocrit, vasoactive effects (vasoconstriction/vasodilation), hyperactivated platelets, procoagulant activity and increased production of thrombus cells, said cytokines or muteins comprising at least one effector cell protective activity selected from protecting, maintaining, enhancing or restoring mammalian effector cell, tissue or organ function or viability. The decrease can be a slight decrease or almost lack of an erythropoietic activity. The reduction can be determined by standard techniques known in the art (Gruber et al., 2002, J. Biol Chem. 277(81):27581-27584; Page et al., 1996, Cytokine 8(1):66-69; Park et al. , 1997, Mol.Cells 7(6): 699-704; Wolf et al., 1997, Thromb Haemost 78: 1505-1509; and Dale et al., 2002, Nature 415: 175-179. The UT-7 cell assay is described in subsection 6.17, is a non-limiting example of a technique for determining reduced or diminished erythropoietic activity.

The nucleic acid molecules of the present invention also include complementary nucleic acids to the aforementioned nucleic acids. the

Erythropoietin mutein nucleic acid molecular fragment refers to the above-mentioned erythropoietin mutein nucleic acid sequence, and its length can be at least 10, 12, 15, 20, 30, 40, 50, 60, 70 , 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1050 or more consecutive nucleotides. Alternatively, the fragment may comprise a sequence encoding at least 10, 20, 30, 40, 50, 60, 70, 80 or more contiguous amino acid residues of an erythropoietin mutein. In one embodiment, the erythropoietin mutein nucleic acid molecule encodes a gene product that exhibits at least one corresponding biological activity of the erythropoietin mutein. The erythropoietin mutein nucleic acid molecular fragment may also refer to the portion of the erythropoietin mutein coding region encoding the erythropoietin mutein domain or encoding the mature erythropoietin mutein. the

Erythropoietin derived from other organisms can be used to produce the erythropoietin muteins of the present invention. As for cloning of erythropoietin muteins or recombinant tissue protective cytokine nucleic acids and variants of homologs and orthologs from other species, the isolated erythropoietin nucleic acid sequences disclosed herein can be labeled and used To screen a cDNA library constructed from mRNA obtained from suitable cells or tissues derived from the organism of interest. When the cDNA library is derived from a different type of organism than the source of the marker sequence, the hybridization conditions used are generally of low stringency, and cDNA libraries can be routinely assayed based on, for example, the relative relationship of the target and reference organisms. the

Alternatively, using appropriate stringency conditions, the marker fragments can also be used to screen genomic libraries derived from the organism of interest. Conditions of appropriate stringency are well known to those of skill in the art, as described above, and are expected to vary depending on the particular organism from which the library and marker sequences are derived. For guidance on such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989-1999, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y, both of which are hereby incorporated by reference in their entirety. the

)，可以进行PCR。聚合酶链式反应(PCR)通常用于获得目标基因或基因片段。使用邻接编码可读框的核苷酸序列的PCR引物，可以产生例如编码任何所需长度的重组组织保护性细胞因子的核苷酸序列。或者，可以在合适位点用限制性内切核酸酶切割重组组织保护性细胞因子基因序列(如果有这样的位点的话)，释放编码重组组织保护性细胞因子基因的DNA片段。如果没有常规限制位点，可以通过定点诱变和/或本领域已知的DNA扩增方法，在合适位置产生所述位点(参见例如Shankarappa等，1992，PCR Method增刊1：277-278)。然后分离编码重组组织保护性细胞因子的DNA片段，并连接到合适的表达载体上，小心操作以确保合适的翻译阅读框。 In a preferred embodiment, for the preparation of recombinant tissue protective cytokines, amplification by polymerase chain reaction (PCR) using primers designed from the known sequences of related or homologous recombinant tissue protective cytokines , to amplify DNA from genomic or cDNA (ie, SEQ ID NO: 7). PCR is used to amplify desired sequences from DNA clones or genomic or cDNA libraries followed by selection. For example, by using a thermal cycler and Taq polymerase (

), PCR can be performed. Polymerase chain reaction (PCR) is commonly used to obtain target genes or gene fragments. Using PCR primers adjacent to the nucleotide sequence encoding the open reading frame, one can generate, for example, a nucleotide sequence encoding a recombinant tissue protective cytokine of any desired length. Alternatively, the recombinant tissue protective cytokine gene sequence (if such site exists) can be cleaved with restriction endonuclease at the appropriate site, releasing the DNA fragment encoding the recombinant tissue protective cytokine gene. If conventional restriction sites are not available, the sites can be generated at suitable positions by site-directed mutagenesis and/or DNA amplification methods known in the art (see e.g. Shankarappa et al., 1992, PCR Method Suppl. 1:277-278) . The DNA fragment encoding the recombinant tissue protective cytokine is then isolated and ligated into an appropriate expression vector, taking care to ensure the proper reading frame for translation.

Individual nucleotides in the DNA sequence may be modified using any mutagenesis technique known in the art for amino acid substitutions in the expressed peptide sequence, or for the creation/deletion of restriction sites for further manipulation. Such techniques include, but are not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551), oligonucleotide-directed mutagenesis (Smith, 1985, Ann. Rev. Genet. 19:423-463; Hill et al., 1987, Methods Enzymol.155:558-568) and PCR-based overlap extension as described in Section 6.3 (Ho et al., 1989, Gene 77:51-59), PCR-based megaprimers Mutagenesis (Sarkar et al., 1990, Biotechniques 8:404-407) and the like. Modifications can be confirmed, for example, by double-stranded dideoxynucleotide DNA sequencing. the

The invention also includes nucleic acid molecules, preferably DNA molecules, which are complementary to the nucleotide sequences of the above paragraphs. the

In certain embodiments, a nucleic acid molecule of the invention is a portion of a nucleic acid molecule that includes a nucleic acid sequence that contains or encodes a heterologous (eg, vector, expression vector, or fusion protein) sequence. the

5.2. Recombinant tissue protective cytokines of the present invention

The recombinant tissue protective cytokines of the present invention include erythropoietin muteins that retain part or all of their erythropoietic activity. Erythropoietin is a glycoprotein hormone with a molecular weight of about 34kDa in the human body. Its mature protein contains 165 amino acids, and sugar residues account for about 40% of the molecular weight. Forms of recombinant tissue protective cytokines useful in the practice of the invention include at least one amino acid alteration of naturally occurring, synthetic and recombinant forms of the following human and other mammalian erythropoietin-related molecules: erythropoietin, asialoerythrocyte Epoietin, deglycosylated erythropoietin, erythropoietin analogs, erythropoietin mimetics, erythropoietin fragments, hybrid erythropoietin molecules, erythropoietin receptor binding molecules, erythropoietin agonists, renal erythrocytes Poietin, brain erythropoietin, its oligomers and multimers and its congeners. Such equivalent recombinant tissue protective cytokines include mutant erythropoietins, which may contain substitutions, deletions including internal deletions, additions including additions producing melt proteins, or within and/or near the amino acid sequence Amino acid residues are conservatively substituted, but they produce "silent" changes in that they produce functionally equivalent erythropoietin muteins or recombinant tissue protective cytokines. In a preferred embodiment, the recombinant tissue protective cytokine is non-erythropoietic, ie lacks or exhibits reduced erythropoietic activity. Conservative amino acid substitutions may be made on the basis of similar polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathicity of the residues involved. Examples of nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids Includes glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged Charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, non-conservative amino acid changes and larger insertions and deletions can be used to generate recombinant tissue protective cytokines with altered function. Such mutants can be used to alter the properties of erythropoietin in a desired manner. For example, in one embodiment, the erythropoietin used in the practice of the present invention may be a recombinant tissue protective cytokine with one or more amino acid changes in the following four functional domains of erythropoietin that affect receptor binding: VLQRY( SEQ ID NO: 1) and/or TKVNFYAW (SEQ ID NO: 2) and/or SGLRSLTTL (SEQ ID NO: 3) and/or SNFLRG (SEQ ID NO: 4). In another embodiment, erythropoietins containing mutations in regions surrounding the molecule that affect the kinetics or receptor binding properties of the molecule may be used. Which alteration or position in the domain will affect binding can be determined by standard methods. For example, domains can be altered by paired alanine mutations (ala scanning mutagenesis) and the binding kinetics of the mutants assayed to determine the effect on receptor binding (Bernat et al., 2003, PNAS 100:952-957; Wells et al., 1989, Science 244:1081-1085). the

The terms "recombinant tissue protective cytokine" and "recombinant tissue protective cytokine" may be used interchangeably or in combination to include the recombinant tissue protective cytokine of the present invention and further modifications thereof, such as the recombinant tissue protective cytokine Deglycosylation, desialylation and other chemical modifications of partially glycosylated forms or amino acids. Non-limiting examples of such variants are described in Tsuda et al., 1990, Eur. J. Biochem. 188:405-411, which is incorporated herein by reference. Cytokines are highly flexible, and in the case of human growth hormone, flexibility is known to be required for activation (Wells et al., 1989, Science 244:1081-1085). Therefore, mutations that stabilize the three-dimensional structure of the cytokine, preventing normal activation of the erythropoietin receptor, are included in the present invention. In addition, various host systems are available for the expression and production of recombinant tissue protective cytokines, including but not limited to bacterial cell systems, yeast cell systems, insect cell systems, plant cell systems, and mammalian cells including human cells cell system. For example, recombinant erythropoietin produced by bacteria without glycosylation, desialylation, or partial glycosylation can be used to produce non-glycosylated forms of recombinant tissue protective cytokines, or can be further glycosylated by methods known in the art. The technology includes, but is not limited to, the technology of regulating protein glycosylation with fucosylation disclosed in the following documents: US Patent Application Nos. US 2003/0040037 A1 and US 2003/0003529. Alternatively, recombinant tissue protective cytokines can be produced in other systems capable of glycosylation of expressed proteins, such as plant cells and human cells. the

As noted above, the invention encompasses any and all molecules that are modulators of erythropoietin receptor activity that exert a positive effect on effector cells, regardless of the molecule's structural relationship to erythropoietin. the

In addition, recombinant tissue protective cytokines can be modified to tailor their activity to specific tissues. Several non-limiting strategies can be employed to achieve this desired tissue specificity, including modifications that shorten the circulating half-life and thus reduce the time during which recombinant tissue protective cytokines interact with erythroid precursors, or the modification of erythropoietin muteins Or the modification of the primary structure of recombinant tissue protective cytokine molecules. One approach to shortening the circulatory half-life is to remove or modify the glycosylated portion of erythropoietin, which has three N-linked sugars and one O-linked sugar. Said variants of the glycosylated recombinant tissue protective cytokines can be produced in various ways. For example, the techniques for modifying the primary structure of erythropoietin to produce the tissue protective cytokines of the present invention are numerous, including the substitution of one or more specific amino acids, i. Amino acid mutations and/or chemical modifications of one or more amino acids, or addition of other structures that interfere with the interaction of erythropoietin with its receptor. Such forms using recombinant tissue protective cytokines are included in the present invention. The sialic acid can be removed by special sialidase according to the chemical bonding between the sialic acid at the end of the sugar chain and the sugar chain. Alternatively, glycosylated structures can be removed in a different manner, using other enzymes that cleave specific bonds. In a preferred embodiment, the non-erythropoietic recombinant tissue protective cytokine of the invention has a half-life that is about 90% shorter than native erythropoietin. the

However, some of these recombinant tissue protective cytokine molecules will mimic the actions of erythropoietin itself in other tissues or organs. For example, a 17-mer containing the amino acid sequence 31-47 of native erythropoietin is inactive in erythropoiesis, but is fully active in neurons in vitro (Campana & O'Brien, 1998: Int.J.Mol.Med .1:235-41). the

In addition, derivatized recombinant tissue protective cytokine molecules as desired for use as described herein can be produced by guanidination, amidation, carbamylation, trinitrosulphenylation, acetylation or succinylation Acylation such as acylation, nitration, or removal of arginine, aspartic acid, glutamic acid, lysine, tyrosine, tryptophan, or cysteine in other methods such as limited proteolysis, removal of amino groups modification of acid residues or carboxyl groups, and/or mutated substitutions of arginine, lysine, tyrosine, tryptophan, or cysteine residues by molecular biology techniques to produce specific organ and tissue Erythropoietin muteins or recombinant tissue protective cytokines (e.g. Satake et al; 1990, Biochim. Biophys. Acta 1038: 125-9; fully incorporated by reference) that maintain appropriate levels (but not for other cells such as erythrocytes) incorporated into this article). A non-limiting example is the modification of erythropoietin arginine residues by reaction with glyoxal such as phenylglyoxal (according to Takahashi, 1977, J. Biochem. 81:395-402). plan). As will be seen below, the recombinant tissue protective cytokine molecule fully retains the neurotrophic effects of erythropoietin. Such recombinant tissue protective cytokine molecules are all included in the various uses and compositions described herein. In addition, these chemical modifications can be further used to enhance the protective effect of recombinant tissue protective cytokines or to neutralize any changes in molecular charge caused by amino acid mutations in native erythropoietin. Said modification is described in co-pending application serial number PCT/US01/49479 filed December 28, 2001; serial number 09/753,132 filed December 29, 2000 and attorney company filed July 3, 2002 Docket No. KW00-009C02-US, all of said patent applications are hereby incorporated by reference in their entirety. the

Provided herein are synthetic and recombinant molecules, such as brain and renal erythropoietin, recombinant mammalian forms of erythropoietin, and their natural isoforms, tumor-derived isoforms, and recombinant isoforms, such as recombinant Expressed molecules and molecules produced by homologous recombination. In addition, the invention includes molecules comprising peptides that bind the erythropoietin receptor, as well as recombinant constructs or other molecules that possess some or all of the structural and/or biological properties of erythropoietin, including fragments of erythropoietin or fragments thereof and polymer. The invention includes erythropoietin muteins or other recombinant tissue protective cytokines with increased or decreased glycosylation sites. As noted above, the terms "erythropoietin" and "mimetic" and other terms are used interchangeably herein to refer to effector cell protective and enhancing molecules associated with erythropoietin and molecules capable of crossing the endothelial cell barrier. In addition, the present invention also includes molecules produced by transgenic animals. It should be noted that the erythropoietin molecules included herein are not necessarily similar to erythropoietin in structure or otherwise, except to interact with the erythropoietin receptor or to modulate erythropoietin receptor activity or to activate erythropoietin as described herein Generin beyond its ability to activate signaling cascades. the

By way of non-limiting example, forms of recombinant tissue protective cytokines useful in the practice of the invention include recombinant tissue protective cytokines, such as those with altered amino acids at their carboxyl termini, as described in US Patent 5,457,089 and US Patent 4,835,260; Asialoerythropoietin and erythropoietin isoforms with varying numbers of sialic acid residues per molecule, such as described in US Patent 5,856,298; polypeptides, described in US Patent 4,703,008; agonists, described in US Patent 5,767,078 ; peptides that bind to the erythropoietin receptor, described in U.S. Patents 5,773,569 and 5,830,851; and small molecule mimetics, described in U.S. Patent 5,835,382; and erythropoietin analogs, described in WO 9505465, WO 9718318, and WO 9818926. All of the above cited documents are hereby incorporated by reference in the sense that their disclosures pertain to various alternative forms or methods of preparation of the recombinant tissue protective cytokine forms for use in the present invention. the

Erythropoietin is commercially available, for example, from Ortho Biotech Inc., Raritan, NJ, under the trademark PROCRIT, and from Amgen, Inc., Thousand Oaks, CA, under the trademark EPOGEN. the

The activity (in units) of erythropoietin (EPO) and erythropoietin-like molecules is usually defined in terms of their potency to stimulate red blood cell production in rodent models (also served as the source of the International Standard for erythropoietin). Typically one unit (U) of erythropoietin (MW ~30,000-34,000) is about 8 ng of protein (1 mg of protein is about 125,000 U). However, certain tissue protective cytokines of the invention are defined in terms of erythropoietic activity because the effect on erythropoiesis is concomitant with the desired activity herein and is not necessarily a detectable property of the recombinant tissue protective cytokines of the invention activity is inappropriate. Therefore, the activity unit of erythropoietin or erythropoietin-related molecules used herein is defined as the amount of protein required to cause the same activity in the nerve cell system or other effector cell system as that caused by the WHO international standard erythropoietin in the same system. Units of non-erythropoietic recombinant tissue protective cytokines or related molecules will be readily determined by the skilled artisan following the guidance herein. the

Recombinant tissue protective cytokine muteins include, but are not limited to, proteins and polypeptides encoded by the erythropoietin nucleic acid sequence described in Section 6.3. The present invention includes muteins that are functionally equivalent to the erythropoietin gene product described in Section 6.3. The erythropoietin gene product may contain one or more deletions, additions or substitutions of erythropoietin amino acid residues within the amino acid sequence encoded by the erythropoietin nucleic acid sequence, but which produce silent changes, thus resulting in functionally equivalent erythropoietin gene product. Amino acid substitutions may be made on the basis of similar polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathicity of the residues involved. the

The recombinant tissue protective cytokine muteins of the invention may be produced by mutagenesis, eg, discrete point mutations or truncations. The recombinant tissue protective cytokine muteins of the invention retain the cytoprotective biological activity of the native form, but may lack one or more erythropoietic activities of the native form of the protein. Thus, specific biological effects can be elicited by adding muteins of limited function. the

Recombinant tissue protective cytokine muteins may be structurally modified (for example, altering the phosphorylation pattern of the mutein) for purposes such as enhancing efficacy, stability, or post-translational modification. Said modified recombinant tissue protective cytokine mutein may be considered a recombinant tissue protective cytokine mutein when designed to retain at least one cytoprotective activity of the native form of said protein, or to generate a specific antagonist thereof functional equivalent of . Such modified recombinant tissue protective cytokine muteins can be produced, for example, by amino acid substitutions, deletions or additions. the

For example, it is reasonable to believe that substituting isoleucine or valine for leucine, glutamic acid for aspartic acid, serine for threonine, or a structurally related amino acid for a similar amino acid (i.e., the same mutations and/or isoelectric mutations) will not have a significant impact on the biological activity of the resulting molecule. the

Whether changes in the amino acid sequence of a recombinant tissue-protective cytokine mutein produce a functional homolog or a non-functional homolog (i.e., lacking one or more activities of the non-mutated cytokine) can be assessed by evaluating the variant mutant protein to resemble wild-type The ability of a cytokine to produce an effect in a cell, or to competitively inhibit that effect, is readily determined. Recombinant tissue protective cytokine muteins in which more than one substitution has occurred can be readily detected in the same manner. the

Muteins of the invention exhibiting altered function can be identified by screening combinatorial libraries of mutants, eg, truncation mutants of recombinant tissue protective cytokines of the invention that possess desired activities or lack such activities. In one embodiment, a variegated library of variants is generated and encoded by a variegated gene library by combinatorial mutagenesis at the nucleic acid level. Mottled libraries of variants can be generated, for example, by enzymatic ligation of synthetic oligonucleotide mixtures to nucleic acid sequences, such that degenerate sequences of underlying protein sequences can be expressed as distinct polypeptides, or, alternatively, as a larger set of fusions. proteins (e.g. for phage display). Libraries of potential variants of the recombinant tissue protective cytokines of the invention can be generated from degenerate oligonucleotide sequences using various methods. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198: 1056; Ike et al., 1983, Nucleic Acids Res. 11:477). the

In addition, the fragment library of the recombinant tissue protective cytokine coding sequence of the present invention can be used to generate a mottled population of the recombinant tissue protective cytokine for screening and subsequent mutein selection. For example, a library of coding sequence fragments can be generated by treating double-stranded PCR fragments of the coding sequence of interest with nucleases, denaturing the double-stranded DNA, and annealing the DNA from different The nicked product forms double-stranded DNA that can include sense/antisense pairs, the single-stranded portion is removed from the de novo duplex by treatment with S1 nuclease, and the resulting library of fragments is ligated into an expression vector. Through this method, expression libraries encoding N-terminal and internal fragments of target recombinant tissue-protective cytokine muteins of different sizes can be obtained. the

Several techniques are known in the art for screening combinatorial library gene products resulting from point mutations or truncations and for screening cDNA libraries for gene products with selected properties. The most widely used technique for high-throughput analysis suitable for screening large gene libraries generally involves cloning the gene library into replicable expression vectors, transforming suitable cells with the resulting vector library, and testing for the desired activity therein to facilitate isolation of the encoded The combined gene is expressed under the conditions of the gene vector whose product can be detected. The technique of recursive ensemble mutagenesis (REM), which increases the frequency of functional mutants in the library, can be used in conjunction with screening assays to identify muteins of the recombinant tissue protective cytokines of the invention (Arkin and Yourvan USA 89:7811-7815; Delgrave et al., 1993, Protein Engineering 6(3):327-331). the

An isolated nucleic acid molecule encoding a mutein can be produced by introducing one or more nucleotide substitutions, additions or deletions into the erythropoietin nucleotide sequence to introduce one or more amino acid substitutions, additions or deletions into the encoded of recombinant tissue protective cytokines. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Briefly, PCR primers were designed that deleted the trinucleotide codon for the amino acid to be altered and substituted with the trinucleotide codon for the amino acid to be included. The primers are used for PCR amplification of DNA encoding target recombinant tissue protective cytokines. This fragment is then isolated and inserted into a full-length cDNA encoding the tissue-protective cytokine of interest for recombinant expression. The resulting recombinant tissue protective cytokine includes the amino acid substitutions. the

Conservative or non-conservative amino acid substitutions may be made at one or more amino acid residues. Conservative and non-conservative substitutions can be made. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Generally, the encoded amino acids can be divided into 4 families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polarities = glycine, asparagine , glutamine, cysteine, serine, threonine, tyrosine. In a similar manner, the amino acid repertoire can be divided into (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine, (3) aliphatic = glycine , alanine, valine, leucine, isoleucine, serine, threonine, while serine and threonine are optionally divided into aliphatic-hydroxyl groups; (4) aromatic = phenylalanine , tyrosine, tryptophan; (5) amides = asparagine, glutamine; and (6) sulfur = cysteine and methionine (see e.g. Biochemistry, 4th edition, L. Stryer Ed., WH Freeman and Co., 1995). the

Alternatively, mutations can be randomly introduced into all or part of the coding sequence of a recombinant tissue protective cytokine, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the recombinant tissue protective cytokine can be assayed. the

In addition to the erythropoietin modifications described above for use herein, the following discussion details various recombinant tissue protective cytokines of the invention. As described above by Elliott et al., Boissel et al., and Wen et al., the following erythropoietin muteins are useful for the purposes described herein and can be provided in pharmaceutical compositions for use in the methods herein. In the mutein nomenclature adopted herein, the altered amino acid is firstly represented by the single-letter codon of the natural amino acid, followed by its position on the erythropoietin molecule, and finally by the single-letter codon of the substituted amino acid. For example, "human erythropoietin S100E" or "recombinant tissue protective cytokine 100E" refers to a human erythropoietin molecule in which the serine of amino acid 100 is changed to glutamic acid. Muteins useful in the practice of the invention include, but are not limited to, human erythropoietin with at least one of the following amino acid changes:

I6A, C7A, C7S,

R10I, V11S, L12A, E13A, R14A, R14E, R14Q, Y15A, Y15F, Y15I,

K20E, K20A,

E21A,

N24K, C29S, C29Y, A30N, H32T,

C33S, C33Y, N38K, N83K,

P42N,

P42A, D43A, T44I, K45D, K45A, V46A, N47A, F48I, F48A, Y49A, Y49S, 44-49 deletions,

W51F, W51N, K52A,

Q59N,

E62T,

L67S,

L70A,

D96R, K97A

S100R, S100E, S100A, S100T, G101A, G101I, L102A, R103A, R103E, S104A, S104I,

L105A, T106A, T106I, T107A, T107L, L108K, L108A, L108S,

K116A,

S126A,

T132A,

I133A, T134A,

K140A,

F142I,

R143A,

S146A, N147K, N147A, F148Y, P148A, L149A, R150A, R150E, G151A,

K152A, K152W,

L153A,

K154A,

L155A, G158A,

A160S, C161A, or R162A. the

In preferred embodiments, the erythropoietin muteins or recombinant tissue protective cytokines of the invention comprise one or more of the above substitutions. In other embodiments, the erythropoietin mutein or another recombinant tissue protective cytokine of the invention comprises more than one substitution or a combination thereof. the

In an alternative embodiment, the recombinant tissue protective cytokines, pharmaceutical compositions, uses and methods of treatment of the present invention comprise one or more of the above substitutions, provided they do not comprise one or more of the following substitutions: I6A, C7A, K20A, P42A, D43A, K45D, K45A, F48A, Y49A, K52A, K49A, S100E, R103A, K116A, T132A, I133A, K140A, N147K, N147A, R150A, R150E, G151A, K152A, K154A, CG162A. In a related embodiment of the invention, the recombinant tissue protective cytokines, pharmaceutical compositions, uses and methods of treatment of the invention comprise one or more of the above substitutions, provided they do not comprise any combination of the following substitutions: N24K /N38K/N83K or A30N/H32T. the

In certain embodiments, more than one amino acid change can be combined to generate a mutein. Examples of such combinations include, but are not limited to: K45D/S100E, A30N/H32T, K45D/R150E, R103E/L108S, K140A/K52A, K140A/K52A/K45A, K97A/K152A, K97A/K152A/K45A, K97A/K152A/ K45A/K52A, K97A/K152A/K45A/K52A/K140A, K97A/K152A/K45A/K52A/K140A/K154A, N24K/N38K/N83K, and N24K/Y15A. In certain embodiments, the recombinant tissue protective cytokine muteins of the invention do not comprise one or more of the above multiple substitutions. In certain embodiments, a pharmaceutical composition of the invention comprising a recombinant tissue protective cytokine mutein of the invention does not comprise one or more of the above multiple substitutions. In certain embodiments, the uses and methods of treatment of the invention using the recombinant tissue protective cytokine muteins of the invention do not include one or more of the above multiple substitutions. the

Certain modifications or combinations of modifications may affect the flexibility of the erythropoietin mutein to effect binding to a receptor, such as the erythropoietin receptor or a second receptor to which erythropoietin or the erythropoietin mutein binds. Examples of such modifications or combinations thereof for use in the compositions and methods of the invention include, but are not limited to, K152W, R14A/Y15A, I6A, C7A, D43A, P42A, F48A, Y49A, T132A, I133A, T134A, N147A, F148A, R150A , G151A, G158A, C161A and R162A. These corresponding mutations are known to be deleterious in human growth hormone (Wells et al.). In certain embodiments, the recombinant tissue protective cytokine muteins of the invention do not comprise one or more of the above substitutions. In certain embodiments, a pharmaceutical composition of the invention comprising a recombinant tissue protective cytokine mutein of the invention does not include one or more of the above substitutions. In certain embodiments, the uses and methods of treatment of the invention using the recombinant tissue protective cytokine muteins of the invention do not comprise one or more of the above substitutions. the

测序级唾液酸酶A^TM(N-乙酰神经氨酸糖基水解酶，EC 3.2.1.18)用于从复杂糖和糖蛋白例如红细胞生成素中切割所有非还原端唾液酸残基。它也会切割支化唾液酸(连接内部残基)。唾液酸酶A是从产脲节杆菌(Arthrobacter ureafaciens)的克隆中分离出来的。 In addition to one of the amino acid modifications described above, the recombinant tissue protective cytokines of the present invention may also lack a sialic acid moiety, referred to as asialoerythropoietin muteins. The asialoerythropoietin mutein of the present invention is preferably human asialoerythropoietin. In alternative embodiments, the recombinant tissue protective cytokines of the present invention may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or 13 sialic acid residues. They can be prepared by desialylation of recombinant tissue protective cytokines with a sialidase such as that described on the package of the manufacturer of Sialidase A from ProZyme Inc., San Leandro, California. usually,

Sequencing grade Sialidase A ^™ (N-acetylneuraminic acid glycosyl hydrolase, EC 3.2.1.18) was used to cleave all non-reducing sialic acid residues from complex sugars and glycoproteins such as erythropoietin. It also cleaves branched sialic acids (linking internal residues). Sialidase A was isolated from a clone of Arthrobacter ureafaciens.

Non-limiting examples of glycoprotein sialylation can be found in US Patent Application No. US2003/0040037, which discloses methods for sialylation with mammalian or bacterial sialyltransferases. Another non-limiting example of a sialylation method and alteration of the sialic acid pattern on a glycoprotein can be found in US Patent Application Nos. US 2002/0160460A1 and US 6,399,336 B1. This application discloses methods for in vitro sialylation of recombinant glycoproteins wherein a sialic acid donor moiety is bound to a glycoprotein having a galactose or N-acetylgalactosamine acceptor moiety. In this way, the sialyltransferase linking the acceptor and donor attaches sialic acid to the sugar. the

The recombinant tissue protective cytokines of the invention may have at least a reduced number of N-linked sugars. To remove N-linked sugars, recombinant tissue protective cytokines can be treated with hydrazine as described, for example, in Hermentin et al., 1996, Glycobiology 6(2): 217-30. As noted above, erythropoietin has 3 N-linked saccharide moieties; the invention includes erythropoietin with 2, 1, or no N-linked saccharide. the

The recombinant tissue protective cytokine of the invention may have at least a reduced sugar content as a result of treating the recombinant tissue protective cytokine with at least one glycosidase. For example, the method described by Chen and Evangelista, 1998, Electrophoresis 19(15):2639-44 can be used. In addition, O-linked sugars can be removed as described in Hokke et al., 1995, Eur. J Biochem. 228(3): 981-1008. the

As a result of expressing the recombinant erythropoietin mutein in non-mammalian cells, the carbohydrate portion of the recombinant tissue protective cytokine molecule may have at least a non-mammalian glycosylation pattern. The recombinant tissue protective cytokines of the present invention are preferably expressed in insect cells or plant cells. As a non-limiting example, recombinant tissue protective cytokines can be expressed in insect cells using the baculovirus expression system according to Quelle et al., 1989, Blood 74(2):652-657. Another method is described in US Patent 5,637,477. the

Expression in plant systems can be carried out according to the method of Matsumoto et al., 1993, Biosci. Biotech. Biochem. 57(8): 1249-1252. Alternatively, expression in bacteria will produce a non-glycosylated form of the recombinant tissue protective cytokine. These are merely exemplary methods of production of recombinant tissue protective cytokines useful in the present invention and are by no means limiting. the

A non-limiting example of modification of the glycosylation pattern is the use of fucosylation, as disclosed in US Patent Application No. US2003/0040037 Al and US Patent Application No. US2003/0003529 Al. Disclosed therein is a method for modifying the glycosylation pattern of a glycopeptide by contacting a reaction mixture having a fucose donor moiety with a glycopeptide having an acceptor moiety of a fucosyltransferase to modify the glycopeptide's sugar Base mode. Methods of using recombinant glycopeptides to modify glycosylation patterns are also disclosed. the

The recombinant tissue protective cytokines of the present invention can have at least one or more oxidized sugars that can also be chemically reduced. For example, the recombinant tissue protective cytokine can be periodate oxidized erythropoietin mutein; periodate oxidized erythropoietin mutein can also be treated with borohydride salts such as sodium borohydride or sodium cyanoborohydride. chemical reduction. Periodate oxidation of erythropoietin muteins can be performed, for example, by the method described by Linsley et al., 1994, Anal. Biochem. 219(2):207-17. Chemical reduction followed by periodate oxidation can be carried out according to the method of Tonelli and Meints, 1978, J. Supramol. Struct. 8(1): 67-78. the

It should be noted that some of the above and below amino acid modifications to native erythropoietin are not possible because the specific target amino acids for chemical modification have been altered in the native molecule to form the recombinant tissue protective cytokines of the present invention . Altered amino acids may, of course, undergo chemical modification by themselves, and the invention includes all such molecules. Those skilled in the art will readily determine the amino acid residues available and modifications thereto for the recombinant tissue protective cytokines of the invention. the

The recombinant tissue protective cytokines used in the above uses may have at least one or more modified arginine residues. For example, a recombinant tissue protective cytokine can comprise an R-glyoxal moiety at one or more arginine residues, where R is aryl, heteroaryl, lower alkyl, lower alkoxy, or cycloalkyl , or α-deoxy sugar alcohol group. The term lower "alkyl" as used herein means a straight or branched chain saturated aliphatic hydrocarbon group preferably containing 1 to 6 carbon atoms. Representative of such groups are methyl, ethyl, isopropyl, isobutyl, butyl, pentyl, hexyl, and the like. The term "alkoxy" refers to a lower alkyl group as described above attached through an oxygen to the rest of the molecule. Examples of alkoxy include methoxy, ethoxy, propoxy, isopropoxy and the like. The term "cycloalkyl" refers to cycloalkyl groups of 3 up to about 8 carbons including, for example, cyclopropyl, cyclobutyl, cyclohexyl, and the like. The term aryl refers to phenyl and naphthyl. The term heteroaryl refers to a heterocyclic group containing 4-10 ring atoms and 1-3 heteroatoms selected from oxygen, nitrogen and sulfur. Examples include, but are not limited to, isoxazolyl, phenylisoxazolyl, furyl, pyrimidinyl, quinolinyl, tetrahydroquinolinyl, pyridyl, imidazolyl, pyrrolidinyl, 1,2,4-tris Azolyl, thiazolyl, thienyl, etc. The R group may be substituted, eg 2,3,4-trihydroxybutyl for 3-deoxyglucosone. Typical examples of R-glyoxal compounds are glyoxal, methylglyoxal, 3-deoxyglucosone and phenylglyoxal. Preferred R-glyoxal compounds are methylglyoxal or phenylglyoxal. Exemplary methods for such modifications using phenylglyoxal can be found in Werber et al., 1975, Isr. J. Med. Sci. 11(11):1169-70. the

In another embodiment, preferably in about 50 millimolar borate buffer, at pH 8-9, by reaction with diketones such as 2,3-butanedione or cyclohexanedione, Modify at least one arginine residue. The latter modified method with 2,3-butanedione can be carried out according to the method of Riordan, 1973, Biochemistry 12 (20): 3915-3923; and the method with cyclohexanedione can be carried out according to Patthy et al., 1975, J.Biol .Chem 250(2):565-9 method. the

The recombinant tissue protective cytokines of the present invention may comprise at least one or more modified lysine residues or an N-terminal amino modification of the erythropoietin molecule, for example from the reaction of a lysine residue with an amino modifier The resulting modification. In another embodiment, lysine residues can be modified by reaction with glyoxal (eg, with glyoxal, methylglyoxal, or 3-deoxyglucosone) to form the corresponding α- Carboxyalkyl Derivatives. Examples of reaction with glyoxal to form carboxymethyllysine can be found in Glomb and Monnier, 1995, J. Biol. Chem. 270(17): 10017-26, or with methylglyoxal to form (1 Examples of -carboxyethyl)lysine can be found in Degenhardt et al., 1998, Cell. Mol. Biol. (Noisy-le-grand) 44(7): 1139-45. Modified lysine residues can also be further chemically reduced. For example, recombinant tissue protective cytokines can be biotinylated via a lysine group in which D-biotinyl-ε-aminocaproic acid-N-hydroxysuccinimide ester is reacted with erythropoietin, followed by Literature Description Removal of unreacted biotin by gel filtration on a Centricon 10 column: Wojchowski and Caslake, 1989, Blood 74(3):952-8. In this paper, the authors use 3 different methods of biotinylation of erythropoietin, each of which can be used to prepare erythropoietin for the use herein. Biotin can be added to (1) sialic acid moieties (2) carboxyl or (3) amino groups. the

In another preferred embodiment, the lysine can be reacted with an aldehyde or a reducing sugar to form an imine, which can be reduced by sodium cyanoborohydride to form an N-alkylated lysine residue such as glucose Alcohol lysine, or in the case of reducing sugars can be rearranged by Amadori or Heyns to form α-deoxy-α-amino sugars such as α-deoxy-α-fructosyllysine in the erythropoietin molecule residues are stable. As an example, fructosyllysine-modified proteins can be prepared by incubation with 0.5M glucose in sodium phosphate buffer (pH 7.4) for 60 days, as described in Makita et al., 1992, J.Biol.Chem.267: 5133-5138. In another example, a lysine group can be carbamylated, for example by reaction with a cyanate ion, or with an alkyl-isocyanate or aryl-isocyanate, an alkyl-isothiocyanate Alkyl-carbamylation or aryl-carbamylation or alkyl-thiocarbamylation or aryl-thiocarbamylation by reaction of salt or aryl-isothiocyanate, Alternatively it may be acylated by reactive alkyl or aryl carboxylic acid derivatives, for example by reaction with acetic or succinic anhydride or phthalic anhydride. Examples are the modification of lysine groups with 4-sulfophenylisothiocyanate or acetic anhydride, both described in Gao et al., 1994, Proc Natl AcadSci USA 91(25):12027-30. The lysine group can also be modified with a trinitrophenyl group by reaction with trinitrobenzenesulfonic acid, or preferably a salt thereof.

At least one tyrosine residue of the recombinant tissue protective cytokine may be modified, for example, by nitration or iodination within an aromatic ring position by an electrophile. As a non-limiting example, erythropoietin can be reacted with tetranitromethane (Nestler et al., 1985, J. Biol. Chem. 260(12):7316-21; or iodized as described in Example 4.

At least one aspartic acid residue or one glutamic acid residue of the recombinant tissue protective cytokine can be modified, for example, by reaction with carbodiimide followed by reaction with an amine such as, but not limited to, glycinamide. the

In another example, a tryptophan residue of a recombinant tissue protective cytokine can be obtained, for example, by reacting with n-bromosuccinimide or n-chlorosuccinimide and then passing it through, for example, as described in Josse et al., Chem Biol Interact 1999. Modified by the method of May 14;119-120. the

In yet another example, recombinant tissue protective cytokines can be prepared by removal of at least one amino group by reaction with ninhydrin followed by reduction of the subsequent carbonyl group by reaction with borohydride. the

In yet another example, there is provided a recombinant tissue protective cytokine having at least one opening of at least one cysteine bond within the erythropoietin molecule by reacting with a reducing agent such as dithiothreitol , followed by reaction of the subsequently generated thiol with iodoacetamide, iodoacetic acid, or another electrophile to prevent reformation of the disulfide bond. As mentioned above, disulphide bonds can be eliminated by either or a combination of two methods, i.e. altering the cysteine molecules involved in the actual cross-linking, or at least one of the erythropoietin muteins which renders the erythropoietin mutein unable to form at least one native Other amino acid residues of disulfide bonds present in the molecule. the

Recombinant tissue protective cytokines can be produced by limited chemical proteolysis of erythropoietin targeting specific residues (eg, cleavage after a tryptophan residue). The resulting recombinant tissue protective cytokine fragments are included herein. the

As noted above, recombinant tissue protective cytokines for purposes herein may have at least one of the above modifications, but may also have more than one of the above modifications. As an example of a recombinant tissue protective cytokine having one modification in the sugar portion of its molecule and one modification in its amino portion, the recombinant tissue protective cytokine may be asialoerythropoietin and its lysine at position 45 The residue becomes aspartic acid. the

Accordingly, various recombinant tissue protective cytokine molecules and pharmaceutical compositions containing them are included for the uses described herein. As described above, the erythropoietin molecules include, but are not limited to, muteins that are asialoerythropoietin, N-deglycosylated erythropoietin, O-deglycosylated erythropoietin, Erythropoietin with reduced sugar content, Erythropoietin with altered glycosylation pattern, Erythropoietin with oxidized and then reduced sugars, Erythropoietin modified with arylglyoxal, Erythropoietin modified with alkylglyoxal Erythropoietin, 2,3-butanedione modified erythropoietin, cyclohexanedione modified erythropoietin, biotinylated erythropoietin, N-alkylated lysyl erythropoietin, glucosyl lysine Acidic erythropoietin, α-deoxy-α-fructosyllysine-erythropoietin, carbamylated erythropoietin, acetylated erythropoietin, succinylated erythropoietin, α-carboxyalkyl erythropoietin Erythropoietin, nitroerythropoietin, iodinated erythropoietin, are some representative but non-limiting examples named according to the definition of the present invention. Preference is given to the above-mentioned modified forms based on human erythropoietin. the

In addition, the present invention includes the aforementioned recombinant tissue protective cytokines and pharmaceutical compositions comprising said compounds. As non-limiting examples, the recombinant tissue protective cytokines include periodate oxidized erythropoietin muteins, glucosyllysine erythropoietin muteins, fructosyllysine erythropoietin muteins, 3-Deoxyglucosone erythropoietin mutein and carbamylated asialo erythropoietin mutein. the

5.3. Expression system

Various host expression vector systems can be used to produce recombinant tissue protective cytokines including the erythropoietin mutein molecules of the invention. The host expression system represents a vector by which the recombinant tissue protective cytokine of interest can be produced and thereafter purified, and also represents a cell which, when transformed or transfected with an appropriate nucleotide coding sequence The modified erythropoietin gene product is expressed in situ upon staining. These include, but are not limited to, bacterial, insect, plant, mammalian host systems including humans, such as, but not limited to, insects infected with recombinant viral expression vectors (e.g., baculoviruses) containing sequences encoding recombinant tissue-protective cytokine products Cellular systems; plant cell systems infected with recombinant viral expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing recombinant tissue protective cytokine coding sequences, or with recombinant tissue protective cytokine coding sequences A plant cell system transformed with a recombinant plasmid expression vector (such as a Ti plasmid) of the sequence; or includes a promoter (such as a metallothionein promoter) from a mammalian cell genome or a promoter from a mammalian virus (such as an adenovirus late promoter) ; vaccinia virus 7.5K promoter) in mammalian cell systems including human cell systems (eg HT1080, COS, CHO, BHK, 293, 3T3). the

As used herein, an expression construct refers to a recombinant tissue protective cytokine-encoding nucleotide sequence operably linked to one or more regulatory regions that permit expression of the recombinant tissue protective cytokine in a suitable host cell. "Operably linked" refers to a connection in which the regulatory region and the recombinant tissue protective cytokine polypeptide sequence to be expressed are combined and positioned in a manner that allows transcription, and ultimately translation, of the recombinant tissue protective cytokine sequence. A variety of expression vectors can be used to express recombinant tissue protective cytokines, including but not limited to plasmids, cosmids, phages, phagemids or modified viruses. Examples include phages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene). Typically, the expression vector comprises a functional origin of replication for replication of the vector in a suitable host cell, one or more restriction endonuclease sites for insertion of a recombinant tissue protective cytokine gene sequence, and a or multiple selection marks. the

In a preferred embodiment, the pCI-neo vector is used to anneal oligonucleotides to the original human EPO cDNA clone to introduce the mutations described above. The pCI-neo vector contains the neomycin phosphotransferase gene, which is a selectable marker for use in mammalian cells. The pCI-neo vector can be used for transient or stable expression by selecting transfected cells with the antibiotic G-418. (Brondyk, 1995, New Mammalian Expression Vector with a selectable marker: pCI-neo. Promega Notes 51, 10-14). the

For expression of recombinant tissue protective cytokines in mammalian host cells, various regulatory regions are available, such as SV40 early and late promoters, cytomegalovirus (CMV) immediate early promoter, and Rous sarcoma virus long terminal repeat (RSV-LTR) promoter. Inducible promoters that can be used in mammalian cells include, but are not limited to, promoters linked to the metallothionein II gene, the mouse mammary tumor virus glucocorticoid-responsive long terminal repeat (MMTV-LTR), and the alpha-interferon gene ( Williams et al., 1989, Cancer Res. 49:2735-42; Taylor et al., 1990, Mol. Cell. Biol. 10:165-75). the

Expression efficacy of recombinant tissue protective cytokines in host cells can be enhanced by including appropriate transcriptional enhancer elements in the expression vector, such as those found in SV40 virus, hepatitis B virus, cytomegalovirus, immunoglobulin Enhancer elements in genes, metallothionein, alpha-actin (see Bittner et al., 1987, Methods in Enzymol. 153:516-544; Gorman, 1990, Curr. Op. in Biotechnol. 1:36-47). the

Expression vectors may also contain sequences that allow the vector to be maintained and replicated in more than one type of host cell, or to integrate the vector into the host chromosome. Such sequences may include, but are not limited to, origins of replication, autonomously replicating sequences (ARS), centromeric DNA, and telomeric DNA. Shuttle vectors that replicate and are maintained in at least two types of host cells may also be advantageously used. the

In addition, the expression vector may contain a selectable or selectable marker gene for the primary isolation or identification of host cells containing the DNA encoding the recombinant tissue protective cytokine. For long-term, high-yield production of recombinant tissue protective cytokines, stable expression in mammalian, plant, bacterial or fungal cells can be used. Various selection systems are available for mammalian cells, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski and Szybalski, 1962 USA 48:2026) and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be used in tk-cells, hgprt-cells or aprt-cells, respectively. Additionally, antimetabolite resistance can be used as a basis for selection based on dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981, Proc.Natl.Acad.Sci.USA78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc.Natl.Acad.Sci.USA78: 2072); neomycin phosphotransferase (neo), which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygromycin phosphotransferase (hyg), which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Other selectable markers such as, but not limited to, histidinol and Zeocin ^(TM) may also be used.

In order to insert the recombinant tissue protective cytokine coding sequence into the cloning site of the vector, a DNA sequence with regulatory functions such as a promoter must be linked to the coding sequence. To do this, linkers providing suitable compatible restriction sites can be ligated to the ends of cDNA or synthetic DNA encoding recombinant tissue protective cytokines by techniques well known in the art (Wu et al., 1987, Methods Enzymol. 152 : 343-349). After cleavage with restriction enzymes, modifications can be made before ligation, by reverse digestion or blunt ends of single-stranded DNA to generate blunt ends. Alternatively, the desired restriction enzyme site can be introduced into the DNA fragment by amplifying the DNA by PCR using primers containing the desired restriction enzyme site. the

An expression construct comprising a recombinant tissue protective cytokine coding sequence operably linked to a regulatory region can be directly introduced into a suitable host cell for the expression and production of the recombinant tissue protective cytokine of the present invention without further cloning (see eg US Patent No. 5,580,859). Expression constructs may also contain DNA sequences that facilitate integration of the coding sequence into the host cell genome, eg, by homologous recombination. In this case, it is not necessary to use an expression vector containing an origin of replication suitable for a suitable host cell in order to replicate and express the recombinant tissue protective cytokine in the host cell. the

Expression constructs containing cloned recombinant tissue protective cytokine coding sequences can be introduced into mammalian host cells by various techniques known in the art, including but not limited to calcium phosphate-mediated transfection (Wigler et al. , 1977, Cell 11: 223-232), liposome-mediated transfection (Schaefer-Ridder et al., 1982, Science 215: 166-168), electroporation (Wolff et al., 1987, Proc.Natl.Acad.Sci 84:3344) and microinjection (Cappechi, 1980, Cell 22:479-488). the

In addition, host cell strains can be selected that modulate the expression of the inserted sequences, or modify and process the gene product in the specific manner desired. Such modification (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Different host cells have characteristic mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed foreign protein. For this, eukaryotic host cells for the correct processing of primary transcripts, glycosylation and phosphorylation of the cellular machinery of the gene product can be used. Mammalian host cells, including human host cells, include, but are not limited to, HT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38. the

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered to stably express gene products of recombinant tissue protective cytokine-related molecules. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA and selectable markers controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After the introduction of exogenous DNA, the engineered cells can be grown in a nourishing medium for 1-2 days, and then transferred to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows the cell to stably integrate the plasmid into its chromosome, allowing it to grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing recombinant tissue protective cytokine gene products. The engineered cell lines are particularly useful in screening and evaluating compounds that affect the endogenous activity of recombinant tissue protective cytokine gene products. the

Any of the cloning and expression vectors described can be synthesized and assembled from known DNA sequences by techniques well known in the art. Regulatory regions and enhancer elements can be of various origins, both natural and synthetic. Certain vectors and host cells are commercially available. Non-limiting examples of useful vectors are described in Appendix 5 of Current Protocols in Molecular Biology, 1988, edited by Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, which are incorporated herein by reference; and companies such as Clontech Laboratories, Stratagene Inc. and Invitrogen , Inc and other suppliers' product catalogs. the

Alternatively, various viral-based expression systems are also available for recombinant expression of tissue protective cytokines in mammalian cells. Vectors using DNA viral backbones are derived from simian virus 40 (SV40) (Hamer et al., 1979, Cell 17:725), adenovirus (Van Doren et al., 1984, Mol. Cell Biol. 4:1653), adeno-associated virus (McLaughlin et al., 1988, J. Virol. 62: 1963) and bovine papilloma virus (Zinn et al., 1982, Proc. Natl. Acad. Sci. 79: 4897). In cases where an adenovirus is used as an expression vector, the donor DNA sequence may be linked to an adenovirus transcriptional/translational control region, such as the late promoter and tripartite leader sequence. The chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a heterologous product in an infected host (see, for example, Logan and Shenk, 1984, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659). the

Alternatively, the vaccinia virus 7.5K promoter can be used (see, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79:7415-7419; Mackett et al., 1984, J. Virol. , 1982, Proc. Natl. Acad. Sci. U.S.A. 79:4927-4931). In cases where human host cells are used, vectors based on the Epstein-Barr virus (EBV) origin (OriP) and EBV nuclear antigen 1 (EBNA-1; trans-acting replication factor) can be used. The vectors can be used in a variety of human host cells, such as EBO-pCD (Spickofsky et al., 1990, DNA Prot. Eng. Tech. 2:14-18), pDR2 and αDR2 (available from Clontech Laboratories). the

Expression of recombinant tissue protective cytokines can be performed by retrovirus-based expression systems. Unlike transfection, retroviruses can efficiently infect and transfer genes to a wide variety of cell types, including, for example, primary hematopoietic cells. In retroviruses such as Moloney murine leukemia virus, most viral gene sequences can be removed and replaced by recombinant tissue-protective cytokine-encoding sequences, which in turn supply their lost viral functions. The host range of retroviral vector infection can also be manipulated by the choice of envelope of the packaging vector. the

For example, a retroviral vector may contain a 5' long terminal repeat (LTR), a 3' LTR, a packaging signal, a bacterial origin of replication, and a selectable marker. The recombinant tissue protective cytokine DNA is inserted at a position between the 5'LTR and 3'LTR such that transcription from the 5'LTR promoter can transcribe the cloned DNA. The 5'LTR comprises a promoter, which in turn includes, but is not limited to, the LTR promoter, the R region, the U5 region, and the primer binding site. The nucleotide sequences of these LTR elements are well known in the art. Heterologous promoters and multidrug selectable markers are also included in the expression vectors for selection of infected cells (see McLauchlin et al., 1990, Prog. Nucleic Acids Res. and Molec. Biol. 38:91-135; Morgenstern et al., 1990 , Nucleic Acids Res.18: 3587-3596; Choulika et al., 1996, J. Virol70: 1792-1798; Boesen et al., 1994, Biotherapy 6: 291-302; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141 and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3: 110-114). the

In one embodiment of the invention, recombinant tissue protective cytokines deficient in sialic acid residues or completely lacking in sialic acid residues can be produced by mammalian cells, including human cells. The cells can be engineered to reduce or lack the enzymes that add sialic acid, namely β-galactoside-α-2,3-sialyltransferase (α-2,3-sialyltransferase@) and β - Galactoside-α-2,6-sialyltransferase (α-2,6-sialyltransferase@) activity. In one embodiment, mammalian cells are used in which one or both of the alpha-2,3-sialyltransferase gene and/or the alpha-2,6-sialyltransferase gene are deleted. Such deletions can be constructed using gene knockout techniques well known in the art. In another embodiment, dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CHO) cells are used as host cells for the production of recombinant tissue protective cytokines. CHO cells do not express alpha-2,6-sialyltransferase and therefore do not add sialic acid to N-linked oligosaccharides linked at 2,6 linkages to glycoproteins produced by these cells. As a result, recombinant proteins produced by CHO cells lack sialic acid linked to galactose in a 2,6 linkage (Sasaki et al., (1987; Takeuchi et al., supra; Mutsaers et al., Eur. J. Biochem. 156, 651 (1986); Takeuchi et al., J. Chromotgr.400, 207 (1987). In one embodiment, in order to produce host cells for the production of asialoerythropoietin, the α-2,3-sialic acid encoded in CHO cells is transferred Enzyme gene deletion. The CHO cells that have deleted α-2,3-sialyltransferase completely lack sialyltransferase activity, and the result can be used for recombinant expression and production of asialoerythropoietin mutant protein. 

In another embodiment, asialoglycoproteins can be produced by interfering with the transport of sialic acid to the Golgi apparatus, e.g. Eckhardt et al., 1998, J. Biol. Chem. 273:20189-95). Using methods well known to those skilled in the art (such as Oelmann et al., 2001, J.Biol.Chem.276:26291-300), the mutagenesis of the nucleotide sugar CMP-sialic acid transporter can be completed to produce Chinese hamster ovary cell mutant. These cells are unable to add sialic acid residues to glycoproteins such as recombinant tissue protective cytokines and so can only produce asialoerythropoietin muteins. the

Transfected mammalian cells that produce erythropoietin muteins also produce cytoplasmic sialidase that, if leaked into the medium, efficiently degrades sialic acid erythropoietin muteins (e.g. Gramer et al., 1995 Biotechnology 13:692 -698). Cell lines can be transfected, mutated, or otherwise produced to constitutively produce sialidase using methods well known to those skilled in the art (eg, information provided by Ferrari et al., 1994, Glycobiology 4:367-373). In this manner, the asialoerythropoietin mutein can be produced during the production of the asialoerythropoietin mutein. the

Recombinant cells can be cultured under standard conditions of temperature, culture time, optical density, and medium composition. Alternatively, modified culture conditions and media can be used to increase production of recombinant tissue protective cytokines. For example, recombinant cells can be grown under conditions that promote the expression of inducible recombinant tissue protective cytokines. Any technique known in the art can be used to establish optimal conditions for recombinant tissue protective cytokine production. Cell lysates or extracts containing recombinant tissue protective cytokines can be used for further purification to isolate recombinant tissue protective cytokines. the

To facilitate the purification of recombinant tissue protective cytokines, the marker amino acid sequence is a hexahistidine peptide, such as that provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311) and others, many of which are is commercially available. For example, routinely purified hexahistidine for fusion proteins is provided as described by Gentz et al., 1989, PNAS 86:821. Other peptide tags used for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the "flag" tag . Any purification method known in the art can be used (see, e.g., International Patent Publication No. WO 93/21232; EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Patent 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al., 1991, J Immunol 146:2446-2452). the

5.4. Determination of tissue protective properties of recombinant tissue protective cytokines

After preparing recombinant tissue protective cytokines and, in certain embodiments, further chemically modifying the tissue protective cytokines of the invention, one of ordinary skill in the art can use well-known assays to test the tissue protective properties of the cytokines and their effect on Lack of myeloid effect. the

For example, non-erythropoietic effects of recombinant tissue protective cytokines can be tested by using the TF-1 assay. In this assay, TF-1 cells were cultured for 1 day in complete RPMI medium supplemented with 5 ng/ml GM-CSF and 10% FCS in a 37 °C _CO2 incubator. The cells were then washed in starvation medium (5% FCS, without GM-CSF) and suspended in starvation medium (5% FCS, without GM-CSF) at a density of 10 ⁶ cells/ml for 16 hours. Prepare a 96-well plate by: (1) adding 100 μl sterile water to peripheral wells to maintain humidity; (2) adding medium (10% FCS, cell-free or GM-CSF) to 5 wells; and (3) ) Inoculate the culture medium containing 10% FCS and the recombinant tissue protective cytokine in the remaining wells with 25,000 cells/well (5 wells for each cytokine to be tested). If the cells proliferate, the recombinant tissue protective cytokines can be erythropoietic. The in vivo effects of the compounds can then be tested in an in vivo assay that monitors the increase in hematocrit induced by recombinant tissue protective cytokines. Negative results - no proliferation of cells in the TF-1 in vitro assay and/or no increase in hematocrit in the in vivo assay - indicate that the recombinant tissue protective cytokine is non-erythropoietic.

As an alternative to the TF-1 assay described above, one skilled in the art may employ other erythropoiesis assays known in the art, including but not limited to UT-7 cell assays such as those described in the Examples section below. the

The tissue protective properties of recombinant tissue protective cytokines can be examined using the P-19 in vitro assay or the in vivo mouse water intoxication assay, both of which are outlined in more detail below. Alternative assays include, but are not limited to, other assays outlined in the Examples below, such as PC-12 and hypocampal slice assays. The above assays are examples only, and other suitable assays to determine the tissue protective and/or myeloid effects of recombinant tissue protective cytokines are known to those of ordinary skill in the art and contemplated by the present invention. the

5.5. The pharmaceutical composition of the present invention

In the practice of one aspect of the invention, the above-described pharmaceutical composition comprising a recombinant tissue protective cytokine may be administered to a mammal by any route that provides sufficient levels of the recombinant tissue protective cytokine in the vasculature to allow transport Crosses the endothelial cell barrier and has beneficial effects on effector cells. Similar results were obtained when used to perfuse tissues or organs. In cases where the erythropoietin mutein is used for ex vivo perfusion, the recombinant tissue protective cytokine may be any form of the erythropoietin mutein, such as the recombinant tissue protective cytokine described above. In cases where the cells or tissues are nonvascularized and/or administered by soaking the cells or tissues with the compositions of the invention, the pharmaceutical compositions provide effective amounts of recombinant tissue protective cytokines that are beneficial to the effector cells. Endothelial barriers across which recombinant tissue protective cytokines are transported include tight junctions, perforated junctions, fenestrated junctions and any other type of endothelial barrier present in mammals. A preferred barrier is tight junctions of endothelial cells, but the invention is not limited thereto. the

The above-mentioned recombinant tissue protective cytokines are generally used in the therapeutic treatment or prophylactic treatment of the following human diseases: diseases of the central nervous system or peripheral nervous system mainly with neurological or psychiatric symptoms and diseases of the eye, cardiovascular diseases, cardiopulmonary diseases , respiratory diseases, kidney diseases, urinary tract diseases and reproductive tract diseases, gastrointestinal diseases and endocrine and metabolic abnormalities. In particular, the conditions and diseases include hypoxic conditions that adversely affect excitable tissues, such as central nervous system tissues such as brain, heart, retina/eye, peripheral nervous system tissues or Excitable tissue in cardiac tissue or retinal tissue. Accordingly, the present invention is useful in the treatment or prevention of damage to excitable tissues resulting from hypoxic conditions in a variety of conditions and conditions. Non-limiting examples of such disorders and conditions are provided in the table below. the

In protected examples of neuronal histopathological conditions treatable in accordance with the invention, said pathological conditions include conditions resulting from decreased oxygenation of neuronal tissues. Any condition in which reduced oxygen availability to neuronal tissue results in stress, injury and ultimately neuronal cell death can be treated by the methods of the present invention. These conditions, commonly referred to as hypoxia and/or ischemia, are caused by or include, but are not limited to, stroke, vascular occlusion, antepartum or postpartum hypoxia, suffocation, choking, Near drowning, carbon monoxide poisoning, smoke inhalation, trauma including surgery and radiation, asphyxia, seizures, hypoglycemia, chronic obstructive pulmonary disease, emphysema, adult respiratory distress syndrome, hypotensive shock , septic shock, anaphylactic shock, insulin shock, sickle cell crisis, cardiac arrest, rhythm disorders, nitrogen anesthesia, and neurological deficits caused by cardiopulmonary bypass surgery. the

In one embodiment, for example, particular compositions of recombinant tissue protective cytokines can be administered to prevent injury or tissue damage caused by the risk of injury or tissue damage during surgical procedures such as tumor resection or aneurysm resection. Other pathological conditions caused by or resulting from hypoglycemia that may be treated by the methods described herein include insulin excess (also known as iatrogenic hyperinsulinemia), insulinoma, growth hormone deficiency, adrenal insufficiency, drug overdose, and certain some tumors. the

Other pathological conditions resulting from damage to excitable neuronal tissue include epilepsy, such as epilepsy, convulsions or chronic epilepsy. Other treatable conditions and diseases include, but are not limited to, for example, stroke, multiple sclerosis, hypotension, cardiac arrest, Alzheimer's disease, Parkinson's disease, cerebral palsy, brain or spinal cord trauma, AIDS dementia, age-related cognitive function memory loss, amyotrophic lateral sclerosis, epilepsy, alcoholism, retinal ischemia, optic nerve damage and neuronal loss due to glaucoma. the

Particular compositions and methods of the invention are useful for treating inflammation, such as physically or chemically induced, caused by a disorder or various traumas. The trauma may include vasculitis, chronic bronchitis, pancreatitis, osteomyelitis, rheumatoid arthritis, glomerulonephritis, optic neuritis, temporal arteritis, encephalitis, meningitis, transverse myelitis, cutaneous Myositis, polymyositis, necrotizing fasciitis, hepatitis, and necrotizing enterocolitis. the

There is evidence that activated astrocytes can exert cytotoxic effects on neurons by producing neurotoxins. Glial cells release nitric oxide, reactive oxygen species, and cytokines in response to cerebral ischemia (see Becker, K.J. 2001. Targeting the central nervous system inflammatory response in ischemic stroke (Targeting the central nervous system in ischemic stroke Inflammatory response). Curr Opinion Neurol 14:349-353 and Mattson, M.P., Culmsee, C. and Yu, Z.F. 2000. Apoptotic and Antiapoptotic mechanisms in stroke. Cell Tissue Res 301: 173 -187.). Studies have further shown that glial activation and subsequent production of inflammatory cytokines depend on primary neuronal injury in models of neurodegeneration (see Viviani, B., Corsini, E., Galli, C.L., Padovani, A. , Ciusani, E. and Marinovich, M. 2000. Dying neural cell activate glia through the release of a protease product (dying neural cell activates glia by releasing protease product). Glia 32:84-90 and Rabuffetti, M. , Scioratti, C., Tarozzo, G., Clementi, E., Manfredi, A.A. and Beltramo, M. 2000. Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone includes long lasting neuroprotection incerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines Reduction of inflammatory cytokines, long-term sustained neuroprotection). J Neurosci. 20: 4398-4404). Inflammation and glial activation are common to different forms of neurodegenerative diseases including cerebral ischemia, brain trauma, and experimental allergic encephalomyelitis, where erythropoietin exerts cytoprotective effects. Inhibition of cytokine production by erythropoietin may at least partially mediate its protective effects. However, unlike "classic" anti-inflammatory cytokines such as IL-10 and IL-13, which directly inhibit tumor necrosis factor production, erythropoietin appears to be active only in the presence of neuronal death. the

While not wishing to be bound by any particular theory, it appears that this anti-inflammatory activity can be hypothetically explained by several non-limiting theories. First, because erythropoietin prevents apoptosis, inflammatory events triggered by apoptosis will be prevented. In addition, erythropoietin can prevent dying neurons from releasing molecular signals that can stimulate glial cells or can act directly on glial cells to reduce their response to these products. Another possibility is that erythropoietin targets members of the inflammatory cascade (eg caspase 1, reactive oxygen or nitrogen intermediates) in closer proximity to trigger apoptosis and inflammation. the

Furthermore, erythropoietin appears to provide anti-inflammatory protection without the rebound effect commonly associated with other anti-inflammatory compounds such as dexamethasone. Also, while not wishing to be bound by any particular theory, this appears to be due to the effect of erythropoietin on multi-target neurotoxins such as nitric oxide (NO). Although activated astrocytes and microglia produce neurotoxic amounts of NO in response to various traumas, NO has many uses in the body, including the regulation of essential physiological functions. Thus, although the use of anti-inflammatory drugs can alleviate inflammation by inhibiting NO or other neurotoxins, if the anti-inflammatory drugs have too long half-lives, they can also interfere with these chemicals in the repair of trauma-induced damage that lead to inflammation. It is hypothesized that the recombinant tissue protective cytokines of the present invention can alleviate inflammation without interfering with the repair capacity of neurotoxins such as NO. the

Particular compositions and methods of the invention are useful for treating disorders and damage to retinal tissue. Such diseases include, but are not limited to, retinal ischemia, macular degeneration, retinal detachment, retinitis pigmentosa, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, hypotension, and diabetic retinopathy. the

In another embodiment, the methods and principles of the present invention can be used to protect or treat damage caused by radiation injury to excitable tissues. A further use of the method of the invention is for the treatment of neurotoxin poisoning such as mussel poisoning by domoic acid, lathyrus poisoning and Guam's disease, amyotrophic lateral sclerosis and Parkinson's disease. the

As noted above, the present invention also relates to methods of enhancing the function of excitable tissues in mammals by peripherally administering the recombinant tissue protective cytokines described above. Various diseases and conditions are amenable to treatment by this method, and this method can be used to enhance cognitive function in the absence of any condition or disease. These uses of the invention are described in more detail below and include enhancements in learning and training in humans and non-human mammals. the

Disorders and diseases involving the central nervous system that may be treated by the methods of this aspect of the invention include, but are not limited to, mood disorders, anxiety disorders, depression, autism, ADHD, and cognitive dysfunction. These conditions benefit from enhanced neuronal function. Other disorders treatable in accordance with the present invention include, for example, sleep disruption, sleep apnea, and travel-related disorders; subarachnoid and aneurysmal hemorrhage, hypotensive shock, concussion injury, septic shock, anaphylactic shock, and various Sequelae of encephalitis and meningitis eg cerebritides such as lupus. Other uses include prophylaxis or protection against neurotoxin poisoning such as mussel domoic acid poisoning, lathyrus poisoning and Guam's disease, amyotrophic lateral sclerosis, Parkinson's disease; embolic injury or ischemic injury postoperative treatment of; whole brain irradiation; sickle cell crisis; and convulsions. the

Other types of disorders that may be treated by the methods of the present invention include inherited or acquired mitochondrial dysfunction, which is the cause of various neurological diseases typically characterized by neuronal damage and death. For example, subacute necrotizing encephalomyelopathy (subacute necrotizing encephalopathy) is characterized by progressive visual loss and encephalopathy due to neuronal loss and myopathy. In these cases, defective mitochondrial metabolism fails to supply high enough energetic substrates to power excitable cellular metabolism. Modulators of erythropoietin receptor activity optimize disrupted function in various mitochondrial diseases. As mentioned above, hypoxic conditions negatively affect excitable tissues. The excitable tissue includes, but is not limited to, central nervous system tissue, peripheral nervous system tissue, and cardiac tissue. In addition to the conditions described above, the methods of the present invention are useful in the treatment of inhalational poisoning such as inhalation of carbon monoxide and smoke, severe asthma, adult respiratory distress syndrome, suffocation and near drowning. Other conditions that produce hypoxic conditions or otherwise induce damage to excitable tissues include hypoglycemia or insulin-producing tumors (insulinomas) that can occur with inappropriate insulin administration. the

A variety of neuropsychological disorders believed to arise from damage to excitable tissues can be treated by the methods of the present invention. Chronic disorders in which neuronal damage is involved and which are treated with the present invention include disorders related to the central nervous system and/or peripheral nervous system, including age-related cognitive decline and Alzheimer's disease, chronic epilepsy, Alzheimer's disease, Parkinson's disease, dementia, Memory loss, amyotrophic lateral sclerosis, multiple sclerosis, tuberous sclerosis, Wilson disease, cerebral palsy and progressive supranuclear palsy, Guam disease, Lewy body dementia, prion diseases such as Spongiform encephalopathies such as transmissible spongiform encephalopathy, Huntington's disease, myotonic dystrophy, Freidrich ataxia and other ataxias, and Tourette's syndrome ( Gilles de la Tourettle's syndrome), epilepsy such as epilepsy and chronic epilepsy, stroke, brain trauma or spinal cord trauma, AIDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic dysfunction such as hypertension and sleep disturbance, And including but not limited to the following neuropsychiatric disorders (neuropsychiatric disorders): schizophrenia, schizoaffective disorder, attention deficit hyperactivity disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, Psychoactive substance use disorder, anxiety disorders, panic disorder, and unipolar and bipolar disorders. Other neuropsychiatric disorders and neurodegenerative diseases include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM), the most recent edition of which is hereby incorporated by reference in its entirety. . the

In another embodiment, recombinant chimeric toxin molecules comprising recombinant tissue protective cytokines are useful for therapeutic administration of toxins for the treatment of proliferative diseases such as cancer, or viral diseases such as subacute sclerosing panencephalitis. the

The following table lists additional exemplary, non-limiting indications that are various conditions and diseases that may be treated by the recombinant tissue protective cytokines described above. the

As noted above, these diseases, disorders or conditions are merely illustrative of the extent to which the recombinant tissue protective cytokines of the invention provide benefits. Thus, the present invention generally provides therapeutic or prophylactic treatment of the consequences of mechanical trauma or human disease. Preferably for the therapeutic or prophylactic treatment of diseases, disorders or conditions of the CNS and/or peripheral nervous system. Provides therapeutic treatment or preventive treatment for diseases, disorders or conditions with psychiatric problems. Provides therapeutic or prophylactic treatment for diseases, disorders, or conditions including, but not limited to: eye disease, cardiovascular disease, cardiopulmonary disease, respiratory disease, kidney disease, urinary tract disease, reproductive tract disease, gastrointestinal disease, endocrine disorders and metabolic abnormalities. the

In one embodiment, pharmaceutical compositions of recombinant tissue protective cytokines can be administered systemically to protect or enhance target cells, tissues or organs. The administration can be parenteral, by inhalation or transmucosally, eg orally, nasally, rectally, intravaginally, sublingually, submucosally or transdermally. Administration is preferably parenteral, such as by intravenous or intraperitoneal injection, and also includes, but is not limited to, intraarterial, intramuscular, intradermal and subcutaneous administration. the

For other routes of administration such as by use of perfusates, injection into organs or other local routes of administration, pharmaceutical compositions producing similar levels of recombinant tissue protective cytokines as described above would be provided. A preferred level is from about 0.01 pM to 30 nM. the

The pharmaceutical composition of the present invention may comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government, or listed in the US Pharmacopoeia or other recognized foreign pharmacopoeia, for use in animals, especially humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. The pharmaceutical carrier can be a sterile liquid, such as saline solution and oils including petroleum, animal, vegetable or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solution is preferred as the carrier when the pharmaceutical composition is administered intravenously. Saline solutions and solutions in water dextrose and glycerol can also be employed as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, dextrose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skim milk, Glycerin, propylene glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. The compounds of the present invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and salts formed with free carboxyl groups, such as those derived from sodium hydroxide, potassium hydroxide, hydrogen Salts of ammonium oxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions may contain a therapeutically effective amount of the compound, preferably in pure form, together with an appropriate amount of carrier so as to provide a form suitable for administration to a patient. The formulation should suit the mode of administration. the

Pharmaceutical compositions suitable for oral administration may be in the form of capsules or tablets; powders or granules; solutions, syrups or suspensions (in water or non-aqueous liquids); edible foams or whipped preparations ( whip); or emulsion. Tablets or hard gelatine capsules may contain lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatine capsules may contain vegetable oils, waxes, fats, semi-solid or liquid polyols and the like. Solutions and syrups may contain water, polyols and sugars. the

Active agents intended for oral administration may be coated with or mixed with a material which delays disintegration and/or absorption of the active agent in the gastrointestinal tract (for example, glyceryl monostearate or glyceryl distearate may be used). esters). Thus, a sustained release of the active agent can be achieved for several hours and, if necessary, the active agent can be protected from degradation in the stomach. Pharmaceutical compositions for oral administration can be formulated so that the active agent is released at a specific site of the gastrointestinal tract as a result of a specific pH or enzymatic conditions. the

Pharmaceutical compositions adapted for transdermal administration may be in the form of discrete patches intended to remain in intimate contact with the epidermis of the recipient for an extended period of time. Pharmaceutical compositions adapted for topical administration may be in the form of ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eye or other external tissues, topical ointments or creams are preferred. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. In these compositions, the active ingredient may be dissolved or suspended in a suitable carrier such as an aqueous solvent. Pharmaceutical compositions suitable for topical administration in the oral cavity include lozenges, pastilles and mouthwashes. the

Pharmaceutical compositions suitable for nasal and pulmonary administration may contain solid carriers such as powders (preferably in the particle size range of 20-500 microns). The powder can be administered by inhalation (ie, by bringing the container of powder close to the nose and inhaling it quickly into the nasal cavity). Alternatively, compositions suitable for nasal administration may comprise a liquid carrier such as nasal spray or nasal drops. Alternatively, direct inhalation into the lungs can be achieved by deep inhalation or through an oral device leading into the pharynx. These compositions may contain aqueous or oily solutions of the active ingredient. Compositions for administration by inhalation may be presented in specially suitable devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which may be manufactured so as to provide predetermined doses of the active ingredient. In a preferred embodiment, the pharmaceutical composition of the present invention is administered directly to the nasal cavity or lungs through the nasal cavity or pharynx. the

Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations. the

Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the composition substantially isotonic with the blood of the intended recipient formulations or suspensions. Other ingredients that may be present in the composition include, for example, water, alcohols, polyols, glycerin and vegetable oils. Compositions suitable for parenteral administration may be presented in single-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of sterile Liquid carriers such as sterile saline solutions for injection are sufficient. Reconstituted injection solutions and suspensions can be prepared from sterile powders, granules and tablets. In one embodiment, autoinjectors containing recombinant tissue protective cytokine injections are available for emergency use in ambulances, emergency rooms, and battlefield situations, and even for self-administration at home, especially when trauma may occur severe cutting such as in the case of careless use of a lawn mower. By administering recombinant tissue-protective cytokines at multiple sites at the amputated site as soon as possible (even before medical personnel arrive on the scene, or before the amputated toe arrives in the emergency room), when the amputated foot or toe is replanted, its The likelihood of cell and tissue survival increases. the

In a preferred embodiment, the composition is formulated according to conventional methods into a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the components are presented singly or mixed in unit dosage form, eg, a lyophilized powder or a water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the composition is administered by infusion, it may be formulated in an infusion bottle filled with sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile saline can be provided so that the components can be mixed prior to administration. the

Suppositories usually contain the active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient. the

Perfusate compositions may be provided for soaking an organ for transplantation, perfusing in situ, or administering a vessel of an organ donor prior to organ harvesting. The pharmaceutical composition may comprise recombinant tissue protective cytokines or forms of recombinant tissue protective cytokines that are not suitable for acute or chronic, local or systemic administration to an individual, but the recombinant tissue protective cytokines or recombinant tissue protective The form of sexual cytokines can be administered to cadavers, organ soaks, organ perfusates, or In situ perfusate plays the expected role in this paper. the

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of a pharmaceutical composition of the invention. The container optionally bears a notice in the form prescribed by a governmental agency requiring the manufacture, use or sale of pharmaceuticals or biological products, the notice reflecting approval for the manufacture, use or sale of pharmaceuticals for human use. the

In another embodiment, recombinant tissue protective cytokines can be administered, for example, in a controlled release system. For example, the polypeptides may be administered by intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In one embodiment, a pump (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, the compound can be administered in a carrier, especially a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); WO91/04014; US Patent No. 4,704,355; Lopez-Berestein, supra, pp. 317-327; see generally above). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds), CRC Press: Boca Raton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball ( Ed.), Wiley: New York (1984); Ranger and Peppas, J.Macromol.Sci.Rev.Macromol.Chem.23:61, 1953; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). the

In yet another embodiment, the controlled release system can be placed in close proximity to the therapeutic target, i.e. the target cell, tissue or organ, thus requiring only a fraction of the amount administered systemically (see, e.g., Goodson in Medical Applications of Controlled Release, pp. 2, pp. 115-138, cf. supra, 1984). A review of other controlled release systems can be found in Langer (1990, Science 249: 1527-1533). the

In another embodiment, appropriately formulated recombinant tissue protective cytokines may be administered nasally, orally, rectally, vaginally or sublingually. the

In a specific embodiment, it is desirable to administer the recombinant tissue protective cytokine composition of the present invention locally to the site in need of treatment; this may be, for example but not limited to, by intraoperative local infusion, topical application such as with a post-operative wound dressing It is done using, by injection, by catheter, by suppository, or by implant, which is porous, non-porous, or gelatin-like material, including membranes or fibers such as silicone rubber membranes and the like. the

Selection of a preferred effective dose will be readily determined by the skilled artisan based on several factors known to those of ordinary skill in the art. Said factors include the specific form of the recombinant tissue protective cytokine and its pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which have been established in the routine development program usually used to obtain statutory approval of pharmaceutical compounds. Other factors in considering dosage include the condition or disease being treated or the desired benefit in a normal individual, the patient's body weight, the route of administration (whether acute or chronic), co-administered drugs, and effects on the efficacy of given drugs. other well-known factors. The precise dosage will therefore be determined according to the judgment of the physician and the circumstances of each patient, for example according to the condition and immune status of each patient and according to standard clinical techniques. the

In another aspect of the present invention, there is provided a perfusate or perfusion solution for perfusion and preservation of transplanted organs, said perfusate comprising recombinant tissue protective cytokines in an effective amount to protect effector cells and related cells, tissues or organs. Transplantation includes, but is not limited to, allogeneic transplantation, in which an organ (including cells, tissues, or other body parts) is harvested from a donor and transplanted into a different recipient; Removal of one part and replacement of another, including ex vivo surgery in which an organ is removed, removed ex vivo, repaired, or otherwise manipulated, such as tumor removal, and then replaced. In one embodiment, the perfusate is a University of Wisconsin (UW) solution (U.S. Patent No. 4,798,824) containing about 1 U/ml to about 25 U/ml erythropoietin, 5% hydroxyethyl starch ( Molecular weight of about 200,000 to about 300,000 and substantially free of ethylene glycol, 2-chloroethanol, sodium chloride, and acetone); 25 mM KH ₂ PO ₄ ; 3 mM glutathione; 5 mM adenosine; 10 mM glucose; 10 mM HEPES Buffer; 5mM magnesium gluconate; 1.5mM CaCl ₂ ; 105mM sodium gluconate; 200,000 units of penicillin; 40 units of insulin; 16 mg of dexamethasone; 12 mg of phenol red; l. The solution is used to maintain cadaveric kidneys and pancreas prior to transplantation. Using the solution, the storage period can be extended beyond the limit of 30 hours recommended for cadaveric kidney preservation. This particular perfusate is only intended to illustrate that bulk solutions containing effective amounts of recombinant tissue protective cytokines are suitable for current use. In another embodiment, the perfusate contains about 0.01 pg/ml to about 400 ng/ml recombinant tissue protective cytokine, or about 40 ng/ml to about 300 ng/ml recombinant tissue protective cytokine. As noted above, any form of recombinant tissue protective cytokine may be used in this aspect of the invention.

While the preferred recipient of recombinant tissue protective cytokines for purposes herein is human, the methods herein are equally applicable to other mammals, especially domesticated, farm, companion and zoo animals. However, the invention is not limited thereto and the benefits are applicable to any mammal. the

5.6. Therapeutic and preventive uses of recombinant tissue protective cytokines

As described in Example 1 below, erythropoietin receptors exist in human brain capillary endothelium, indicating that the targets of the recombinant tissue protective cytokines of the present invention are present in the human brain, and that the targets of these recombinant tissue protective cytokines of the present invention are Animal research can be directly transferred to human treatment or prevention. the

In another aspect of the present invention, methods and compositions are provided for enhancing the viability of cells, tissues or organs not separated from the vasculature by the endothelial cell barrier, i.e. by directly contacting said cells, tissues or organs comprising recombinant tissue A pharmaceutical composition of a protective cytokine, or a pharmaceutical composition comprising a recombinant tissue protective cytokine is administered to or contacts the vasculature of said cell, tissue or organ. The enhanced activity of effector cells in the treated tissue or organ is responsible for the exertion of positive effects. the

As noted above, the present invention is based in part on the discovery that erythropoietin molecules can be transported from the luminal surface of endothelial cells to the basement membrane surface of capillaries with endothelial cell tight junction organs including, for example, brain, retina and testicles. Thus, effector cells that cross the barrier are targets susceptible to the beneficial effects of recombinant tissue protective cytokines, as are other cell types or tissues or organs that contain effector cells and depend in whole or in part on effector cells, are also targets of the methods of the invention. While not wishing to be bound by any particular theory, following transcytosis of recombinant tissue protective cytokines, recombinant tissue protective cytokines may interact with erythropoietin receptors on effector cells such as neuronal cells, Retinal cells, muscle cells, heart cells, lung cells, liver cells, kidney cells, small intestinal cells, adrenal cortex cells, adrenal medulla cells, capillary endothelial cells, testicular cells, ovarian cells, pancreatic cells, bone cells, skin cells or endometrial cells, and receptor binding can initiate a signal transduction cascade leading to the activation of gene expression programs in said effector cells or tissues, resulting in protection of said cells or tissues or organs from, for example, toxins, chemotherapeutics, radiotherapy, Hypoxia and other damage. Accordingly, methods for protecting tissue containing effector cells from injury or hypoxic stress and enhancing the function of said tissue are described in detail below. As noted above, the methods of the invention are equally applicable to humans and other animals. the

In the practice of one embodiment of the invention, a mammalian patient undergoes systemic chemotherapy for the treatment of cancer, including radiation therapy, often with side effects of nerve damage, lung damage, heart damage, ovarian damage, or testicular damage. The pharmaceutical composition comprising the above-mentioned recombinant tissue-protective cytokines is administered before and simultaneously with chemotherapy and/or radiotherapy to protect various tissues and organs from damage caused by chemotherapy drugs, for example, to protect the testis. Treatment is continued until circulating levels of the chemotherapeutic agent fall below a level that is potentially dangerous to the mammalian organism. the

In the practice of another embodiment of the present invention, various organs are planned to be harvested from victims of car accidents for transplantation to multiple recipients, some of which require long distances and prolonged transport. Prior to organ harvesting, the victim is infused with a pharmaceutical composition comprising a recombinant tissue protective cytokine as described herein. Harvested organs for shipping are perfused with a perfusate containing recombinant tissue protective cytokines as described herein and stored in an infusion solution containing recombinant tissue protective cytokines. Certain organs are continuously perfused with a pulsatile perfusion device with a perfusate containing a recombinant tissue protective cytokine according to the invention. Organ function is minimally attenuated during transport and transplantation and organ reperfusion in situ. the

In another embodiment of the invention, surgery to repair a heart valve requires temporary cardioplegia and arterial occlusion. Before the operation, the patients were infused with 4 μg of recombinant tissue protective cytokines per kilogram of body weight. The treatment prevents hypoxic ischemic cell injury, especially after reperfusion. the

In another embodiment of the present invention, the recombinant tissue protective cytokines of the present invention may be used during any surgical procedure such as cardiopulmonary bypass. In one embodiment, before, during and/or after shunt surgery, a pharmaceutical composition comprising the recombinant tissue protective cytokines described above is administered to protect the function of the brain, heart and other organs. the

Wherein the recombinant tissue protective cytokines of the invention are used for ex vivo application or treatment of effector cells such as neuronal tissue, retinal tissue, heart cells, lung cells, liver cells, kidney cells, intestinal cells, adrenal cortical cells, adrenal medullary cells , capillary endothelial cells, testicular cells, ovarian cells, or endometrial cells or tissues, the present invention provides a pharmaceutical composition in dosage unit form suitable for protecting or enhancing effector cells leaving the vascular system, Tissues or organs, said composition comprising a dose range of about 0.01 pg-5 mg, 1 pg-5 mg, 500 pg-5 mg, 1 ng-5 mg, 500 ng-5 mg, 1 μg-5 mg, 500 μg-5 mg or 1 mg-5 mg per dosage unit Recombinant tissue protective cytokine, and a pharmaceutically acceptable carrier. In a preferred embodiment, the amount of the recombinant tissue protective cytokine is in the range of about 1 ng-5 mg. In a preferred embodiment, the recombinant tissue protective cytokines of the above compositions are non-erythropoietic. the

In another aspect of the present invention, it was found that administration of EPO to animals subjected to brain trauma restores cognitive function. The recombinant tissue protective cytokine of the present invention is expected to have the same cytoprotective effect as EPO. After a delay of either 5 or 30 days, administration of EPO still restored function compared to sham-treated animals, suggesting the ability of EPO to regenerate or restore brain activity. Accordingly, the present invention also relates to the use of recombinant tissue protective cytokines in preparation for the treatment of brain trauma (including treatment long after injury, such as 3 days, 5 days, 1 week, 1 month or longer) and other cognitive dysfunction. use in pharmaceutical compositions. The invention also relates to methods for treating cognitive dysfunction following injury by administering an effective amount of a recombinant tissue protective cytokine. Any recombinant tissue protective cytokine as described herein may be used in this aspect of the invention. the

Furthermore, this restorative aspect of the invention relates to the use of any of the recombinant tissue protective cytokines herein for the manufacture of a pharmaceutical composition for the restoration of cell, tissue or organ dysfunction wherein Treatment begins immediately after the injury and long afterward. Furthermore, treatment with the recombinant tissue protective cytokines of the invention can span the course of the disease or disorder in the acute phase as well as the chronic phase. the

In the case where the recombinant tissue protective cytokine of the present invention has erythropoietic activity, in a preferred embodiment, each administration of the recombinant tissue protective cytokine can be between about 0.01 pg and about 100 μg/kg body weight , preferably about 1-50 μg/kg body weight, most preferably about 5-30 μg/kg body weight for systemic administration. The effective dose should be sufficient to achieve a serum level of the recombinant tissue protective cytokine in serum of greater than about 10,000 mU/ml, 15,000 mU/ml, or 20,000 mU/ml following administration of the recombinant tissue protective cytokine. Said serum levels may be achieved about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours after administration. Said administration can be repeated if necessary. For example, administration may be repeated daily, or at appropriate intervals, such as every 1-12 weeks, but preferably every 1-3 weeks, for as long as clinically necessary. In one embodiment, an effective amount of a recombinant tissue protective cytokine and a pharmaceutically acceptable carrier may be packaged in a single dose vial or other container. In another embodiment, the recombinant tissue protective cytokines used for the purposes herein are non-erythropoietic, ie, are capable of exerting the activities described herein without causing an increase in hemoglobin concentration or hematocrit. In cases where long-term delivery of the methods of the invention is contemplated, non-erythropoietic forms of recombinant tissue protective cytokines are preferred. In another embodiment, the recombinant tissue protective cytokine is administered at a dose greater than that required to maximally stimulate erythropoiesis. As mentioned above, the recombinant tissue protective cytokines of the present invention do not necessarily have erythropoietic activity, so the above doses expressed in units are only exemplary for recombinant tissue protective cytokines; The molar equivalent of the dose of any recombinant tissue protective cytokine. the

The present invention also relates to a method for promoting the transport of molecules across the endothelial cell barrier in a mammal by administering a composition comprising a specific molecule associated with a recombinant tissue protective cytokine as described above. As mentioned above, tight junctions between endothelial cells in certain organs of the body create a barrier to the entry of certain molecules. For the treatment of various conditions within barrier organs, means to facilitate the passage of pharmaceutical agents are needed. The recombinant tissue protective cytokines of the present invention are used as carriers for delivery of other molecules across the blood-brain barrier or other similar barriers. A composition comprising a molecule desired to cross the barrier and a recombinant tissue protective cytokine is prepared and administered peripherally, resulting in transcytosis of the composition across the barrier. The association between the molecule to be transported across the barrier and the recombinant tissue protective cytokine may be a labile covalent bond, in which case the molecule is removed from the association with the recombinant tissue protective cytokine after crossing the barrier. He was released. The complex can be administered if the association between the molecule and the recombinant tissue protective cytokine is maintained or does not affect the desired pharmacological activity of the molecule. the

The skilled artisan is aware of various means for associating molecules with the recombinant tissue protective cytokines of the invention and the other drugs mentioned above, ie by covalent, non-covalent and other means. Furthermore, evaluation of the efficacy of the compounds can be readily performed in experimental systems. Association of molecules with tissue protective cytokines can be achieved by various means including destabilization, covalent binding, cross-linking, and the like. A biotin/avidin interaction can be used. As noted above, hybrid molecules, eg, domains comprising a molecule having the desired pharmacological activity and domains that result in modulation of erythropoietin receptor activity, can be prepared recombinantly or synthetically. the

A molecule can be conjugated to a recombinant tissue protective cytokine via a multifunctional molecule, ie a multifunctional crosslinker. As used herein, the term "multifunctional molecule" includes molecules having functional groups capable of reacting more than once in succession, such as formaldehyde, as well as molecules having more than one reactive group. The term "reactive group" as used herein refers to a functional group on a cross-linking agent that reacts with a molecule such as a peptide, protein, sugar, nucleic acid, especially a hormone, antibiotic or anticancer drug to be delivered across the endothelial cell barrier to Functional groups that form crosslinkers and covalent bonds between the molecules. The term "functional group" retains its standard meaning in organic chemistry. Said multifunctional molecules that can be used are preferably biocompatible linkers, ie they are non-carcinogenic, non-toxic and substantially non-immunogenic in vivo. Multifunctional crosslinkers, such as those known in the art and described herein, can be readily tested in animal models to determine their biocompatibility. The multifunctional molecule is preferably bifunctional. As used herein, the term "bifunctional molecule" refers to a molecule having two reactive groups. The bifunctional molecule can be a heterobifunctional molecule or a homobifunctional molecule. Heterobifunctional crosslinkers allow vector conjugation. It is particularly preferred that the multifunctional molecule has sufficient solubility in water, since the cross-linking reaction takes place in aqueous solution, e.g. buffered aqueous solution at pH 6-8, and the resulting conjugate remains water soluble for more efficient biodistribution. Typically, multifunctional molecules are covalently bonded to amino or thiol functional groups. However, multifunctional molecules reactive with other functional groups such as carboxylic acid or hydroxyl groups are also included in the present invention. the

Homobifunctional molecules have at least two identical reactive functional groups. Reactive functional groups on homobifunctional molecules include, for example, aldehyde groups and active ester groups. Homobifunctional molecules having an aldehyde group include, for example, glutaraldehyde and subaraldehyde. The use of glutaraldehyde as a crosslinking agent is disclosed in Poznansky et al., Science 223, 1304-1306 (1984). Homobifunctional molecules having at least two active ester units include esters of dicarboxylic acids and N-hydroxysuccinimide. Some examples of the N-succinimidyl esters include disuccinimidyl disuccinimidyl suberate and dithio-bis-(succinimidyl propionate) and its soluble Bis-sulfonic acids and bis-sulfonates such as their sodium and potassium salts. These homobifunctional reagents are available from various commercial sources (Pierce, Rockford, Illinois). the

Heterobifunctional molecules have at least two different reactive groups. The reactive group reacts with different functional groups such as the erythropoietin mutein and the molecule. The two different functional groups that react with reactive groups on heterobifunctional crosslinkers are usually amino groups, such as the epsilon amino group of lysine; sulfhydryl groups, such as the sulfhydryl group of cysteine; a carboxylic acid; or a hydroxyl group, such as that on serine. Of course, the recombinant tissue protective cytokines of the invention may lack specific amino acid residues that would facilitate cross-linking of native erythropoietin, but those skilled in the art know that the portion of residues available in the muteins of the invention and thus the cross-linking couplet. the

In addition, the various recombinant tissue protective cytokine molecules of the present invention may not have groups suitable for reacting with a certain cross-linking agent; however, those skilled in the art will fully understand that according to the available groups to select crosslinkers. the

When the reactive group of the heterobifunctional molecule forms a covalent bond with an amino group, the covalent bond is usually an amido bond or an imide bond. A reactive group forming a covalent bond with an amino group can be, for example, an activated carboxylate, halocarbonyl or ester group. The halocarbonyl is preferably chlorocarbonyl. The ester group is preferably an active ester group such as N-hydroxy-succinimide ester. the

Typically, the other functional group can be a thiol, a group that can be converted into a thiol, or a group that can form a covalent bond with a thiol. Typically, the covalent bond is a thioether bond or a disulfide bond. A reactive group that forms a covalent bond with a sulfhydryl group can be, for example, a double bond that reacts with a sulfhydryl group or an activated disulfide. Reactive groups containing double bonds capable of reacting with sulfhydryl groups can be eg maleimide and other groups such as acrylonitrile. Active disulfide bonds such as 2-pyridinedithiol or 5,5'-dithio-bis-(2-nitrobenzoic acid) groups. Some examples of heterobifunctional reagents containing reactive disulfide bonds include N-succinimidyl 3-(2-pyridine-dithio)propionate (Carlsson et al., 1978, Biochem J., 173:723-737 ), S-4-Succinimidyloxycarbonyl-α-methylbenzyl thiosulfate and 4-Succinimidyloxycarbonyl-α-methyl-(2-pyridyldithio) toluene. N-succinimidyl 3-(2-pyridinedithio)propionate is preferred. Some examples of heterobifunctional reagents containing reactive groups with double bonds that react with sulfhydryl groups include succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and succinimidyl Imido-maleimide benzoate. the

Other heterobifunctional molecules include succinimidyl 3-(maleimide)propionate, sulfosuccinimidyl 4-(p-maleimide-phenyl)butyrate, thio Succinimidyl 4-(N-maleimidomethyl-cyclohexane)-1-carboxylate, Maleimidobenzoyl-N-hydroxy-succinimidyl ester. The sodium sulfonate salt of succinimidyl m-maleimide benzoate is preferred. Many of the aforementioned heterobifunctional reagents and their sulfonates are available from Pierce Chemical Co., Rockford, Illinois USA. the

A skilled artisan can readily determine the need for such conjugation to be reversible or unstable. The conjugates can be tested in vitro for recombinant tissue protective cytokine and desired pharmacological activity. If the conjugate retains both properties, its suitability can be tested in vivo. If the conjugated molecule needs to be separated by activity from the recombinant tissue protective cytokine, labile bonding or reversible association with the recombinant tissue protective cytokine is preferred. The labile properties can also be tested using standard in vitro methods prior to in vivo testing. the

Additional information on how to prepare and use them and other polyfunctional reagents can be obtained from the following publications or other information available in the art:

1.Carlsson, J. et al., 1978, Biochem.J.173:723-737.

2. Cumber, J.A. et al., 1985, Methods in Enzymology 112: 207-224.

3. Jue, R. et al., 1978, Biochem 17: 5399-5405.

4. Sun, T.T. et al., 1974, Biochem.13: 2334-2340.

5. Blattler, W.A. et al., 1985, Biochem.24: 1517-152.

6. Liu, F.T. et al., 1979, Biochem.18: 690-697.

7. Youle, R.J. and Neville, D.M.Jr, 1980, Proc.Natl.Acad.Sci.U.S.A.77:5483-5486.

8. Lerner, R.A. et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 3403-3407.

9. Jung, S.M. and Moroi, M., 1983, Biochem. Biophys. Acta 761: 162.

10. Caulfield, M.P. et al., 1984, Biochem.81: 7772-7776.

11. Staros, J.V., 1982, Biochem.21: 3950-3955.

12. Yoshitake, S. et al., 1979, Eur. J. Biochem. 101: 395-399.

13. Yoshitake, S. et al., 1982, J. Biochem.92: 1413-1424.

14. Pilch, P.F. and Czech, M.P., 1979, J.Biol.Chem.254:3375-3381.

15. Novick, D. et al., 1987, J. Biol. Chem. 262: 8483-8487.

16. Lomant, A.J. and Fairbanks, G., 1976, J. Mol. Biol. 104: 243-261.

17. Hamada, H. and Tsuruo, T., 1987, Anal. Biochem. 160: 483-488.

18. Hashida, S. et al., 1984, J. Applied Biochem. 6:56-63. the

Additionally, crosslinking methods are reviewed in Means and Feeney, 1990, Bioconjugate Chem. 1:2-12. the

The barriers crossed by the above methods and compositions of the present invention include, but are not limited to, blood-brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier, blood-heart barrier, blood-kidney barrier and blood-uterine barrier . the

Candidate molecules for transport across the endothelial cell barrier include, for example, hormones such as growth hormone, neurotrophic factors, antibiotics, antivirals, or antifungals normally excluded by the brain and other barrier-protected organs, peptide radiopharmaceuticals, Antibodies against bioactive factors and antiviral drugs, pharmaceutical substances and anticancer drugs. Non-limiting examples of such molecules include hormones such as growth hormone, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), Transforming growth factor beta 1 (TGF beta 1), transforming growth factor beta 2 (TGF beta 2), transforming growth factor beta 3 (TGF beta 3), interleukin 1, interleukin 2, interleukin 3 and interleukin 6, AZT, anti-tumor necrosis factor antibodies and immunosuppressive drugs such as cyclo sporins. In addition, dyes or labels can be attached to erythropoietin or a tissue protective cytokine of the invention to visualize cells, tissues or organs within the brain and other barrier organs for diagnostic purposes. As an example, markers used to visualize lesions in the brain can be linked to erythropoietin or tissue protective cytokines in order to determine the progression of Alzheimer's disease in a patient. the

The present invention also relates to a composition comprising a molecule to be transported across the tight junction barrier of endothelial cells by transcytosis and a recombinant tissue protective cytokine as described above. The present invention also relates to the use of a conjugate between a molecule and the aforementioned recombinant tissue protective cytokine for the preparation of a pharmaceutical composition for delivering said molecule across the aforementioned barrier. the

The invention may be better understood by reference to the following examples which are illustrative and non-limiting of the invention. The following examples are provided to more fully illustrate the preferred embodiments of the invention. However, they should in no way limit the broad scope of the invention. the

6. Example

6.1. Example 1: Distribution of erythropoietin receptor in human brain

Normal human brains excised during surgery (eg, to provide tumor-free margins during tumor resection) were immediately fixed in 5% acrolein in 0.1 M phosphate buffer (pH 7.4) for 3 hours. Cut into 40 µm thick sections using a vibratome. Immunohistochemical staining was performed with erythropoietin receptor antiserum (from Santa Cruz Biotechnology) at a dilution of 1:500 using free-floating sections and using the indirect antibody peroxidase-anti-peroxidase method. Tissue sections were pretreated with hydrogen peroxide to quench endogenous peroxidase activity (3% hydrogen peroxide in methanol for 30 minutes). Staining was also confirmed to be specific for the erythropoietin (EPO) receptor by primary antibody omission and by treatment of tissue controls with appropriate blocking peptides (obtained from Santa Cruz Biotech.). the

Figure 1 shows that human brain capillaries express very high levels of the EPO receptor as determined by immunohistochemistry with antibodies specific against the EPO receptor. This provides a mechanism by which EPO can penetrate the brain from the systemic circulation despite the blood-brain barrier. the

Figure 2 shows that EPO receptors are concentrated in and around capillaries forming the blood-brain barrier in the human brain. ``

In Figure 3 a similar protocol to Figures 1 and 2 was performed, except that 10 micron sections were sectioned in paraffin and embedded sections were fixed by immersion in 4% paraformaldehyde. Figure 3 shows the high density of EPO receptors on the luminal surface and reverse surface of human brain capillaries, which constitute the anatomical basis for the transport of EPO from the circulation to the brain. the

Figure 4 is obtained from a similar protocol to Figure 3, except that the tissue was sectioned with an ultramicrotome for microscopy and the secondary antibody was labeled with colloidal gold particles. The figure shows that EPO receptors are found on the endothelial surface of the human brain ( ^* ), within cytoplasmic vesicles (arrowheads) and on glial synaptic nubs (+), providing transport of EPO from the circulation into the brain anatomical basis.

6.2. Example 2: Erythropoietin crosses the blood-cerebrospinal fluid tight barrier

Sprague-Dawley adult male rats were anesthetized, and 5000 U/kg body weight of recombinant human erythropoietin was administered intraperitoneally. Cerebrospinal fluid samples were taken from Daci at 30-minute intervals up to 4 hours, and erythropoietin concentrations were determined using a sensitive and specific enzyme-linked immunoassay. As shown in Figure 5, the baseline erythropoietin concentration in CSF was 8 mU/ml. After a delay of several hours, measured erythropoietin levels in CSF began to rise and were significantly different from baseline concentrations by 2.5 hours and beyond (p<0.01 level). Peak levels of approximately 100 mU/ml are within the range known to exert protective effects in vitro (0.1-100 mU/ml). Peak time occurs at approximately 3.5 hours, which is significantly delayed (less than 1 hour) from peak serum levels. The results of this experiment demonstrate that significant levels of erythropoietin can be achieved by bolus parenteral administration of appropriate concentrations of erythropoietin across tight cell junctions. the

6.3. Example 3: Recombinant tissue protective cytokines

The following human erythropoietin constructs were prepared using the following procedure. The cDNA of human erythropoietin was cloned by PCR from human brain cDNA using a published human cDNA sequence (Accession No. NM_000799). Primers were introduced into the Xho I site at the 5' end and the Xba I site at the 3' end of the cDNA. The primer sequences are:

HEPO-5-Xho I 5'-AGCTCTCGAGGCGCGGAGATGGGGGTGCACGAATG-3'(SEQ.ID.8)

HEPO-3-Xba I 5'-ATGCTCTAGACACACCTGGTCATCTGTCCCCTGTCC-3'(SEQ.ID.9). 

The PCR product was cloned between the Xho I site and the Xba I site of the pCiNeo mammalian expression vector (Promega). The clone was sequenced and it was confirmed that the sequence matched that of NM_000799 except for one base. Base 418 (numbering from ATG) in the coding sequence is C instead of G, and Arg at amino acid 140 from the first methionine in the full-length EPO sequence is changed to Gly. However, this is a normal sequence change from the original sequence and is present in most forms of erythropoietin. the

The coding sequence of erythropoietin cDNA is as follows:

ATGGGGGTGCACGAATGTCCTGCCTGGCTGTGGCTTCTCCTGTCCCTGCTGTCGCTCCCTCTGGGCCTCCCAGTCCTGGGCGCCCCACCACGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGACGGGCTGTGCTGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAGAAGTCTGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTTGGTCAACTCTTCCCAGCCGTGGGAGCCCCTGCACTGCATGTGGATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTTCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGATGA(SEQ IDNO：7). 

The cDNA encodes the full-length amino acid sequence of erythropoietin as follows:

MGVHECPAWLWLLLSLLSLPLGLPVLGAPPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR(:

The first 27 amino acid residues of SEQ ID NO: 10 comprise a leader sequence. Unless prefixed with the term "full length", all amino acid positions in the erythropoietin sequence and mutations noted therein refer to positions in the mature protein.

By designing a new oligonucleotide, the 6xHis tag was introduced into the C-terminus of the human EPO protein, so that the following oligonucleotide was adopted, and the 6 histidines were connected to Asp 192 in the full-length sequence:

3'-6xhis-hEPO 5'-

GGTCTAGATCAATGGTGATGGTGATGATGGTCCCCTGTCCTGCAGGCC-3' (SEQ ID NO: 134)

Using HEPO-5-Xho I oligo and 6xHis-tagged oligo, the EPO cDNA was amplified by PCR and cloned between the Xho I site and the Xba I site of the pCiNeo mammalian expression vector. The insertions were then sequenced and the sequence confirmed. the

The mutations described in section 5.2 for the human EPO cDNA sequence with a C-terminal 6xHis tag were introduced by oligonucleotide site-directed mutagenesis as described in that section. Mutant clones were sequenced and mutations confirmed. the

Various methods of purifying the recombinant tissue protective cytokines of the invention can be used, including but not limited to the following protocols used in conjunction with histidine-tagged recombinantly expressed tissue protective cytokines of the invention. Recombinant cell (CHO-K1) supernatant (eg resin from (Ni-CAMHC RESIN: High Capacity Nickel Chelate Affinity Matrix, Sigma, Cat. No. N 3158)) was thoroughly resuspended and gently inverted. Then, 100 μl of the resin suspension (equivalent to 50 μl of packaging resin) was added to a microcentrifuge tube (size 1.7 ml). The mixture was centrifuged at 8,000 rpm for 5 minutes at 4°C to pellet the resin, and the supernatant was discarded. The microcentrifuge was a Megafuge 1.0R (Heraeus Instruments). The mixture was washed twice with 1 ml deionized water (0.2 μm filter) to remove ethanol. Resuspend the resin in 500 µl equilibration buffer (50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, 10 mM imidazole) and transfer the mixture to a 50 ml conical tube. The microcentrifuge tube was rinsed with 500 μl equilibration buffer and this amount was added to the mixture in the 50 ml conical tube. The mixture was centrifuged at 3,000 rpm for 5 minutes at 4°C to pellet the resin. The supernatant was decanted and discarded. Before binding, samples (CHO-KI supernatant) were centrifuged at 3,800 rpm for 5 minutes at 4°C. Cell supernatant was added to the resin. Loading buffer (50 mM sodium phosphate, pH 8.0, 3M NaCl, 100 mM imidazole) was added to 1X and mixed gently on a rotating platform at 4°C for 1 hour. An example of a platform used is Nutator (rotating platform) (Clay Adams Brand). The mixture was centrifuged at 3,000 rpm for 5 minutes at 4°C. The supernatant was removed and kept for SDS-PAGE analysis and ELISA (unbound). Resuspend the resin in 500 µl of wash buffer and transfer the mixture to a microcentrifuge tube. Rinse the 50 ml conical tube with 500 μl equilibration buffer and add this amount to the mixture in the microcentrifuge tube. The resin suspension was then mixed for 10 minutes at 4°C on a rotating platform. The suspension was centrifuged at 8,000 rpm at 4°C for 5 minutes (the first wash was reserved for ELISA). The resin was resuspended in 1 ml wash buffer, then the resin suspension was mixed for 10 minutes at 4°C on a rotating platform, and the resin was washed again. Discard the wash solution. Then, 75 μl of elution buffer (50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, 500 mM imidazole) was added. The resin was mixed for 10 minutes at 4°C on a rotating platform. The mixture was centrifuged at 8,000 rpm for 5 minutes at 4°C. Remove the supernatant and keep. Histidine-tagged protein is present in this fraction. To elute more protein, add 75 μl of elution buffer (50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, 500 mM imidazole). The resin was mixed for an additional 10 minutes at 4°C on a rotating platform. The mixture was centrifuged again at 8,000 rpm for 5 minutes at 4°C. Keep the eluted fractions as a pool or as separate fractions. the

Alternatively, isolate and purify histidine-tagged cytokines as follows. Conditioned medium was collected and filtered through a 0.45 μm filter. A 50 ml aliquot was then loaded onto a 5 ml HiTrap chelating column (Amersham biosciences) equilibrated with 20 mM sodium phosphate pH 7.4 and activated with 2.5 ml 100 mM _NiSO4 . The column was washed with 20 mM sodium phosphate pH 7.4 and eluted with a gradient of 0M-0.5M imidazole (dissolved in 20 mM sodium phosphate pH 7.4) over 25 column volumes. The flow rate was 5ml/min, and the fraction volume was 5ml.

Fractions were analyzed for recombinant tissue protective cytokines present by SDS-PAGE and EPO ELISA. Positive fractions were pooled and dialyzed against 10 mM Tris pH 7.0. The pool was loaded into 1 ml HiTrap Q HP (Amershambiosciences) equilibrated with 10 mM Tris pH 7.0. After washing with equilibration buffer, the sample was eluted with a NaCl gradient to 0.5 M over 10 column volumes at a flow rate of 1 ml/min. Im fractions were collected and analyzed by SDS-PAGE, EPO ELISA and Western blot using antibodies against the hexa-His tag (anti-His6, ROCHE). Fractions containing recombinant tissue protective cytokines were pooled and concentrated with a centricon with a cutoff size of 10 kDa (Amicon) in a final volume of 1-2 ml. the

The final pool of recombinant tissue protective cytokines was analyzed by SDS-PAGE (NuPage 4-12%; Invitrogen) and developed with NOVEX Colloidal Blue (Invitrogen) using the manufacturer's recommended protocol. The purity of the recombinant tissue protective cytokine can be judged according to the obtained gel. Only one band on each lane of the gel corresponds to glycosylated recombinant tissue protective cytokines, indicating that the isolated cytokines are of high purity. the

All plasmids were transfected into CHO-1 cells or COS-7 cells using lipofectamine. 48-72 hours after transfection, the culture medium of the cells was collected. This medium can be used directly or after purification for the detection of EPO by ELISA assays after erythropoiesis assays or neuronal assays. the

Mutations K45D, S100E, and A30N/H32T were introduced into the human EPO cDNA sequence by oligonucleotide-directed mutagenesis using the following oligonucleotides:

HEPO-S100E-Winding 5′-

CATGTGGATAAAGCCGTCGAGGGCCTTCGCAGCCTCACCACTCTG-3' (SEQ ID NO: 11)

HEPO-S100E-Lower Chain 5′-

CAGAGTGGTGAGGCTGCGAAGGCCCTCGACGGCTTTATCCACATG-3' (SEQ ID NO: 12)

HEPO-K45D-winding chain 5′-

GAGAATATCACTGTCCCAGACACCGACGTTAATTTCTATGCCTGG-3' (SEQ ID NO: 13)

HEPO-K45D-lower chain 5′-

CCAGGCATAGAAATTAACGTCGGTGTCTGGGACAGTGATATTCTC-3' (SEQ ID NO: 14)

HEPO-A30N/H32T-Winding 5′-

GAATATCACGACGGGCTGTAATGAAACCTGCAGCTTGAATGAG-3' (SEQ ID NO: 132)

HEPO-A30N/H32T-Lower chain 5′-

CTCATTCAAGCTGCAGGTTTCATTACAGCCCGTCGTGATATTC-3' (SEQ ID NO: 133)

For other erythropoietin muteins and recombinant tissue protective cytokines of the present invention, the oligonucleotide sequences used in the site-directed mutagenesis of oligonucleotides include:

For the R150E mutein:

R150E-F GTCTACTCCAATTTCCTCGAGGGAAAGCTGAAGCTG, (SEQ ID NO: 120)

R150E-R GCTTCAGCTTTTCCCTCGAGGAAATTGGAGTAGAC (SEQ ID NO: 121)

For the R103E mutein:

R103E-F CCGTCAGTGGCCTTGAGAGCCTCACCACTCTG, (SEQ ID NO: 122)

R103E-R CAGAGTGGTGAGGCTCTCAAGGCCACTGACGG (SEQ ID NO: 123)

For the R103E/L108S(103) combination mutein:

R103E-F CCGTCAGTGGCCTTGAGAGCCTCACCACTCTG (SEQ ID NO: 124)

R103E-R CAGAGTGGTGAGGCTCTCAAGGCCACTGACGG (SEQ ID NO: 125)

L108S(103)F CGCAGCCTCACCACTTCGCTTCGGGCTCTGG, (SEQ IDNO: 126)

L108S(103)R CCAGAGCCCGAAGCGAAGTGGTGAGGCTGCG (SEQ ID NO: 127)

For 44-49 missing:

d44-49F GAATATCACTGTCCCAGACGGTGGTGCCTGGAAGAGGATG, (SEQ ID NO: 128)

d44-49R CATCCTCTTCCAGGCACCACCGTCTGGGACAGTGATATTC (SEQ ID NO: 129)

For the K20A mutein:

K20A-F TACCTCTTGGAGGCCGCGGAGGCCGAGAATATC, (SEQ ID NO: 130)

K20A-R GATATTCTCGGCCTCCGCGGCCTCCAAGAGGTA (SEQ ID NO: 131)

For the K140A mutein:

K140A-F GCTGACACTTTCCGCGCACTTCTTCCGAGTCTACTC, (SEQ ID NO: 132)

K140A-R GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC (SEQ ID NO: 133)

For the K152A mutein:

K152A-F ATTTCCTCCGGGGAGCGCTGAAGCTGTACACAG, (SEQ ID NO: 134)

K152A-R CTGTGTACAGCTTCAGCGCTCCCCGGAGGAAAT (SEQ ID NO: 135)

For the K154A mutein:

K154A-F CTCCGGGGAAAGCTGGCGCTGTACACAGGGGA, (SEQ ID NO: 136)

K154A-R TCCCCTGTGTACAGCGCCAGCTTTTCCCCGGAG (SEQ ID NO: 137)

For the K45A mutein:

K45A-F ACTGTCCCAGACACCGCAGTTAATTTCTATGCCTG, (SEQ ID NO: 138)

K45A-R CAGGCATAGAAATTAACTGCGGTGTCTGGGACAGT (SEQ ID NO: 139)

For the K52A mutein:

K52A-F AGTTAATTTCTATGCCTGGGCGAGGATGGAGGTCG, (SEQ ID NO: 140)

K52A-R CGACCTCCATCCTCGCCCAGGCATAGAAATTAACT (SEQ ID NO: 141)

For the K97A mutein:

K97A-F TGCAGCTGCATGTGGATGCAGCCGTCAGTGGCC, (SEQ ID NO: 142)

K97A-R GGCCACTGACGGCTGCATCCACATGCAGCTGCA (SEQ ID NO: 143)

For the K116A mutein:

K116A-F CTCTGGGAGCCCAGGCGGAAGCCATCTCCCCT, (SEQ ID NO: 144)

K116A-R AGGGGAGATGGCTTCCGCCTGGGCTCCCAGAG (SEQ ID NO: 145)

For the K140A/K52A combination mutein:

K140A-F GCTGACACTTTCCGCGCACTTCTTCCGAGTCTACTC, (SEQ ID NO: 146)

K140A-R GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC (SEQ ID NO: 147)

K52A-F AGTTAATTTCTATGCCTGGGCGAGGATGGAGGTCG, (SEQ ID NO: 148)

K52A-R CGACCTCCATCCTCGCCCAGGCATAGAATTAACT (SEQ ID NO: 149)

For the K140A/K52A/K45A combination mutein:

K140A-F GCTGACACTTTCCGCGCACTTCTTCCGAGTCTACTC, (SEQ ID NO: 150)

K140A-R GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC (SEQ ID NO: 151)

K52A-F AGTTAATTTCTATGCCTGGGCGAGGATGGAGGTCG, (SEQ ID NO: 152)

K52A-R CGACCTCCATCCTCGCCCAGGCATAGAAATTAACT (SEQ ID NO: 153)

K45A-F ACTGTCCCAGACACCGCAGTTAATTTCTATGCCTG, (SEQ ID NO: 154)

K45A-R CAGGCATAGAAATTAACTGCGGTGTCTGGGACAGT (SEQ ID NO: 155)

For the K97A/K152A combination mutein:

K97A-FTGCAGCTGCATGTGGATGCAGCCGTCAGTGGCC, (SEQ ID NO: 156)

K97A-R GGCCACTGACGGCTGCATCCACATGCAGCTGCA (SEQ ID NO: 157)

K152A-F ATTTCCTCCGGGGAGCGCTGAAGCTGTACACAG, (SEQ ID NO: 158)

K152A-RCTGTGTACAGCTTTCAGCGCTCCCCGGAGGAAAT (SEQ ID NO: 159)

For the K97A/K152A/K45A combination mutein:

K97A-F TGCAGCTGCATGTGGATGCAGCCGTCAGTGGCC, (SEQ ID NO: 160)

K97A-R GGCCACTGACGGCTGCATCCACATGCAGCTGCA (SEQ ID NO: 161)

K152A-F ATTTCCTCCGGGGAGCGCTGAAGCTGTACACAG, (SEQ ID NO: 162)

K152A-R CTGTGTACAGCTTCAGCGCTCCCCGGAGGAAAT (SEQ ID NO: 163)

K45A-F ACTGTCCCAGACACCGCAGTTAATTTCTATGCCTG, (SEQ ID NO: 164)

K45A-R CAGGCATAGAAATTAACTGCGGTGTCTGGGACAGT (SEQ ID NO: 165)

For K97A/K152A/K45A/K52A combination muteins:

K97A-F TGCAGCTGCATGTGGATGCAGCCGTCAGTGGCC, (SEQ ID NO: 166)

K97A-R GGCCACTGACGGCTGCATCCACATGCAGCTGCA (SEQ ID NO: 167)

K152A-F ATTTCCTCCGGGGAGCGCTGAAGCTGTACACAG, (SEQ ID NO: 168)

K152A-R CTGTGTACAGCTTCAGCGCTCCCCGGAGGAAAT (SEQ ID NO: 169)

K45A-F ACTGTCCCAGACACCGCAGTTAATTTCTATGCCTG, (SEQ ID NO: 170)

K45A-R CAGGCATAGAAATTAACTGCGGTGTCTGGGACAGT (SEQ ID NO: 171)

K52A-F AGTTAATTTCTATGCCTGGGCGAGGATGGAGGTCG, (SEQ ID NO: 172)

K52A-R CGACCTCCATCCTCGCCCAGGCATAGAAATTAACT (SEQ ID NO: 173)

For K97A/K152A/K45A/K52A/K140A combination muteins:

K97A-F TGCAGCTGCATGTGGATGCAGCCGTCAGTGGCC, (SEQ ID NO: 174)

K97A-R GGCCACTGACGGCTGCATCCACATGCAGCTGCA (SEQ ID NO: 175)

K152A-F ATTTCCTCCGGGGAGCGCTGAAGCTGTACACAG, (SEQ ID NO: 176)

K152A-R CTGTGTACAGCTTCAGCGCTCCCCGGAGGAAAT (SEQ ID NO: 177)

K45A-F ACTGTCCCAGACACCGCAGTTAATTTCTATGCCTG, (SEQ ID NO: 178)

K45A-R CAGGCATAGAAATTAACTGCGGTGTCTGGGACAGT (SEQ ID NO: 179)

K52A-F AGTTAATTTCTATGCCTGGGCGAGGATGGAGGTCG, (SEQ ID NO: 180)

K52A-R CGACCTCCATCCTCGCCCAGGCATAGAAATTAACT (SEQ ID NO: 181)

K140A-F GCTGACACTTTCCGCGCACTTCTTCCGAGTCTACTC, (SEQ ID NO: 182)

K140A-R GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC (SEQ ID NO: 183)

For K97A/K152A/K45A/K52A/K140A/K154A combination muteins:

K97A-F TGCAGCTGCATGTGGATGCAGCCGTCAGTGGCC, (SEQ ID NO: 184)

K97A-R GGCCACTGACGGCTGCATCCACATGCAGCTGCA (SEQ ID NO: 185)

K152A-F ATTTCCTCCGGGGAGCGCTGAAGCTGTACACAG, (SEQ ID NO: 186)

K152A-R CTGTGTACAGCTTCAGCGCTCCCCGGAGGAAAT (SEQ ID NO: 187)

K45A-F ACTGTCCCAGACACCGCAGTTAATTTCTATGCCTG, (SEQ ID NO: 188)

K45A-R CAGGCATAGAAATTAACTGCGGTGTCTGGGACAGT (SEQ ID NO: 189)

K52A-F AGTTAATTTCTATGCCTGGGCGAGGATGGAGGTCG, (SEQ ID NO: 190)

52A-R CGACCTCCATCCTCGCCCAGGCATAGAAATTAACT (SEQ ID NO: 191)

K140A-F GCTGACACTTTCCGCGCACTTCTTCCGAGTCTACTC, (SEQ ID NO: 192)

K140A-R GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC (SEQ ID NO: 193)

K154A(152)F CTCCGGGGAGCGCTGGCGCTGTACACAGGGGA, (SEQ IDNO: 194)

154(152)R TCCCCTGTGTACAGCGCCAGCGCTCCCCGGAG (SEQ ID NO: 195)

For N24K/N38K/N83K combination muteins:

N24K-F CAAGGAGGCCGAGAAAATCACGACGGGCTGT, (SEQ ID NO: 196)

N24K-R ACAGCCCGTCGTGATTTTCTCGGCCTCCTTG (SEQ ID NO: 197)

N38K-F ACTGCAGCTTGAATGAGAAATCACTGTCCCAGACAC, (SEQ ID NO: 198)

N38K-R GTGTCTGGGACAGTGATTTTCTCATTCAAGCTGCAGT (SEQ ID NO: 199)

N83K-F AGGCCCTGTTGGTCAAATCTTCCCAGCCGTG, (SEQ ID NO: 200)

N83K-R CACGGCTGGGAAGATTTGACCAACAGGGCCT (SEQ ID NO: 201)

For the K152W mutant protein:

K152W-F ATTTCCTCCGGGGATGGCTGAAGCTGTGTACAG, (SEQ ID NO: 202)

K152W-R CTGTGTACAGCTTCAGCCATCCCCGGAGGAAAT (SEQ ID NO: 203)

For the R14A/Y15A combination mutein:

RY14AA-F AGCCGAGTCCTGGAGGCGGCCCTCTTGGAGGCCAA, (SEQ IDNO: 204)

RY14AA-RTTGGCCTCCAAGAGGGCCGCCTCCAGGACTCGGCT (SEQ ID NO: 205)

Y15A-F AGCCGAGTCCTGGAGAGGGCCCTCTTGGAGGCCAA (SEQ ID NO: 206)

Y15A-R TTGGCCTCCAAGAGGGCCCTCTCCAGGACTCGGCT (SEQ ID NO: 207)

The following are examples of constructed constructs: human EPO (hEPO)-6xHis tag-pCiNeo sequence (SEQ ID NO: 208); hEPO6xHis tag-A30N/H32T-pCiNeo (SEQ ID NO: 209); hEPO-6xHis tag-K45D- pCiNeo sequence (SEQ ID NO: 210); hEPO-6xHis tag-S100E-pCiNeo sequence (SEQ ID NO: 211); and hEPO-6xHis tag-K45D/S100E-pCiNeo sequence (SEQ ID NO: 212). The pCI-neo mammalian expression vector carries the human cytomegalovirus (CMV) immediate early enhancer/promoter region to drive constitutive expression of cloned DNA inserts in mammalian cells. the

These oligonucleotides were annealed to the original human erythropoietin cDNA clone in pCiNeo to introduce the mutations. Mutant clones were sequenced and mutations confirmed. All plasmids were transfected into CHO-1 cells or COS-7 cells using lipofectamine. 48-72 hours after transfection, the culture medium of the cells was collected. This medium can be used directly or after purification for the detection of erythropoietin by ELISA assays after erythropoiesis assays or neuronal assays. the

Then, K45D and S100E recombinant tissue protective cytokines were tested in neuronal assays. Specifically, an in vitro neuroprotection assay using SK-N-SH neuroblastoma cells was employed. SK-N-SH cells were seeded on 24-well plates at a density of 40,000 cells/well for 24 hours. Then the recombinant tissue protective cytokine was added at a concentration of 3 nM, and after another 24 hours (erythropoietin = commercially available preparation; EPO = erythropoietin and recombinant tissue protective cytokine expressed by CHO cells; pure vector = from Cell supernatant of CHO cells transfected with vector of Epo construct). After pre-incubation, cells were exposed to rotenone (5 [mu]M) for 4 hours, washed and allowed to recover for 24 hours. The presence of EPO variants was indicated in all these steps. At the end of the experiment, cell viability was quantified by incubating the cells with the tetrazolium dye WST-1 (according to manufacturer's instructions: Roche #1644807) for 2 hours, expressed as absorbance readings. the

Figures 6A and 6B show the results of an SK-N-SH neuroblastoma cell neuroprotection assay (against rotenone) with erythropoietin and recombinant tissue protective cytokines K45D and S100E. The Y-axis of the graph represents the absorbance readings, and the data are the mean ± the range of duplicate determinations. The graph in Figure 6A clearly shows that cell viability is maintained in K45D and S100E samples, demonstrating their cytoprotective effect. Figure 6B shows the plasmid map of hEPO-6xHis tag-PciNeo. the

6.4. Example 4: Tissue protective cytokines

Recombinant tissue protective cytokines desired for use as described herein may be further modified by desialylation, guanidinylation, carbamylation, amidation, trinitrosulphenylation, acetylation, succinylation, Nitration, or modification of arginine residues or carboxyl groups among other methods. Alternatively, such modifications to native erythropoietin or erythropoietin derivatives include, but are not limited to, desialylation, guanidinylation, carbamylation, amidation, tri- Nitrosulphenylated, acetylated, succinylated or nitrosated erythropoietin. Some examples of further modifications to recombinant tissue protective cytokines are described below. Those of ordinary skill in the art know that the following methods can also be used to chemically modify native erythropoietin or its derivatives prior to introducing mutations to produce recombinant tissue protective cytokines. the

6.4.1. Asialo recombinant tissue protective cytokines

Recombinant tissue protective cytokines can be asialized by the following exemplary methods. Sialidase (isolated from Streptococcacs sp 6646K.) was obtained from SEIKAGAKU AMERICA (No. 120050). Recombinant tissue protective cytokines were treated with sialidase (0.025 U/mg EPO) at 37° C. for 3 hours to desialolate them. The reaction mixture was desalted and concentrated using an Ultrafree centrifugal filter unit. The sample is then loaded onto the ion exchange column of the AKTAprime ^™ system. Proteins are eluted with the buffer of choice. The eluted fractions containing a large amount of protein were then used for IEF gel analysis. Fractions containing only the top two bands (migrated at pI ~ 8.5 and pI ~ 7.9 on IEF gels) were pooled, and the protein content of the pooled fractions was determined by adding 1/9 volume of 10x saline solution (1M NaCl, 0.2 M sodium citrate, 3mM citric acid). The sialic acid content was then determined. No significant sialic acid content was detected.

As shown in Figures 7-8, asialoerythropoietin was as effective as native erythropoietin in neuronal cells in vitro. In vitro experiments were performed with neural-like embryonal carcinoma cells (P19) that had undergone post-serum depletion apoptosis. 1-1000 ng/ml erythropoietin or modified erythropoietin was added to the culture 24 hours before serum was removed. The next day, the medium was removed, the cells were washed with a fresh serum-free medium, and a medium containing the test substance (serum-free) was added to the culture, followed by further culturing for 48 hours. To determine the number of viable cells, a tetrazolium reduction assay (CellTiter 96; Promega, Inc.) was performed. As shown in Figures 7-8, asialoerythropoietin was shown to be as effective in preventing cell death as erythropoietin itself. the

The persistence of neuroprotective activity in vivo was demonstrated using a rat focal ischemia model in which reversible damage to the middle cerebral artery region can be formed as previously described (Brines et al., 2000, Proc. Nat. Acad. Sci. U.S.A. 97:10526-31). Asialoerythropoietin or erythropoietin (5000 U/kgBW ip) or vehicle was administered at the onset of arterial occlusion in Sprague-Dawley adult male rats. After 24 hours, the animals were sacrificed to obtain the brains for research. Serial sections were made and stained with tetrazolium salts to identify viable regions in the brain. As shown in Figure 9, asialoerythropoietin was as effective as native erythropoietin in conferring neuroprotection, ie, reduced infarct volume, during 1 hour of ischemia. Figure 10 shows another focal ischemia model in which dose responses were compared with erythropoietin and asialoerythropoietin. At the lowest dose of 4 μg/kg, asialoerythropoietin provided protection whereas unmodified erythropoietin did not. The number n of rats in each group was greater than or equal to 4. the

Similar results are expected for the asialo recombinant tissue protective cytokines of the present invention. the

6.4.2. Carbamylation of recombinant tissue protective cytokines

Recombinant tissue protective cytokines can be used to prepare the corresponding carbamylated molecules according to the following method as described by Jin Zeng (1991). Lysine modification of metallothionein by carbamylation and guanidine reactions. Methods in Enzymology 205:433-437. Potassium cyanate was recrystallized. Prepare 1M borate buffer (pH 8.8). Mix the recombinant tissue protective cytokine solution with an equal volume of borate buffer. Add potassium cyanate directly into the reaction tube to a final concentration of 0.5M. The solution was mixed well and incubated at 37°C for 6-16 hours. The solution was then thoroughly dialyzed. The product was removed from the dialysis tube and collected into a new tube. Measure the volume and add 1/9 volume of 10X saline solution (1M NaCl, 0.2M sodium citrate, 3mM citric acid). Determine the protein content and calculate the product recovery. Products were analyzed by IEF gels and in vitro assays with TF-1 cells. the

6.4.3. Succinylated recombinant tissue protective cytokines

Recombinant tissue protective cytokines can be used to prepare the corresponding succinylated molecules following the procedure as described by Alcalde et al. (2001). Succinylation of cyclodextrin glycosyltransferase from Thermoanaerobacter sp. 501 uses starch as a donor to enhance its transferase activity. J. Biotechnology 86:71-80. Recombinant tissue protective cytokines (100 μg) in 0.5 M NaHCO ₃ (pH 8.0) were incubated with 15 molar excess of succinic anhydride at 15° C. for 1 hour. The reaction was terminated by dialysis against distilled water.

Dissolve 27 mg/ml succinic anhydride in dry acetone. Reactions were performed in microcentrifuge tubes in 10 mM sodium phosphate buffer (pH 8.0). Add the recombinant tissue protective cytokine protein and 50-fold molar amount of succinic anhydride into the test tube. Mix well and rotate the tube for 1 hour at 4°C. Reactions were terminated by dialysis against 10 mM sodium phosphate buffer using a dialysis cassette (Slide-A-Laze 7K, Pierce 66373). The product was removed from the dialysis cassette and collected into a new tube. Measure the volume and add 1/9 volume of 10X saline solution (1M NaCl, 0.2M sodium citrate, 3mM citric acid). Determine the protein content and calculate the product recovery. Products were analyzed by IEF gels and in vitro assays with TF-1 cells. the

6.4.4. Acetylated recombinant tissue protective cytokines

Recombinant tissue protective cytokines can be used to prepare the corresponding acetylated molecules according to the following method as described by Satake et al. (1990). Chemical modification of erythropoietin: increasing in vitro activity by guanidinization. Biochimica et Biophysica Acta. 1038:125-129. the

Reactions were performed in microcentrifuge tubes in 80 mM sodium phosphate buffer (pH 7.2). Add recombinant tissue protective cytokines and equimolar amounts of acetic anhydride. Mix well and incubate on ice for 1 hour. Reactions were terminated by dialysis against water using a dialysis cassette (Slide-A-Laze 7K, Pierce 66373). The product was removed from the dialysis cassette and collected into a new tube. After measuring the volume, add 1/9 volume of 10X saline solution (1M NaCl, 0.2 M sodium citrate, 3 mM citric acid). Determine the protein content and calculate the product recovery. Products were analyzed by IEF gels and in vitro assays with TF-1 cells. the

6.4.5. Carboxymethylated lysine of recombinant tissue protective cytokines

Recombinant tissue protective cytokines can be used to prepare corresponding N ^ε -(carboxymethyl)lysine (CML) modified molecules according to the following method as described by Akhtar et al. (1999), wherein one or Multiple lysyl residues are modified: Conformational study of N ^{ε -} (carboxymethyl)lysine adducts of recombinant a-crystallins . Current Eye Research, 18: 270-276.

Glyoxylic acid (200 mM) and _NaBH3CN (120 mM) in sodium phosphate buffer (50 mM, pH 7.5) were freshly prepared. In a microcentrifuge tube, add erythropoietin (dissolved in phosphate buffer). Calculate the lysine equivalents in solution (about 8 lysine residues/mol). Add 3 times more NaBH ₃ CN and 5 times more or 10 times more glyoxylic acid into the test tube. After each tube was vortex shaken, it was incubated at 37°C for 5 hours. Samples were dialyzed against phosphate buffer overnight at 4°C. After dialysis, the volume of each product was measured. Determine the protein concentration and calculate the product recovery. Products were analyzed by IEF gels and in vitro assays with TF-1 cells.

6.4.6. Iodinated recombinant tissue protective cytokines

Recombinant tissue protective cytokines can be used to prepare the corresponding iodinated molecules as described below in the instructions for use in IODO-Gen pre-embedded iodinated tubes (Cat. No. 28601) provided by Pierce Chemical Company (Rockford, IL) . the

Prepare 0.1M NaI, the total reaction volume is 0.1ml/tube in IODO-Gen pre-embedded iodination tube (Pierce, 28601), and carry out iodination reaction in sodium phosphate buffer (40mM, pH 7.4). Protein substrates (recombinant tissue protective cytokines) were mixed with sodium phosphate buffer and transferred to IODO-Gen pre-embedded iodide tubes. Add NaI so that the final concentration is 1-2mM, and the NaI/protein molar ratio is 14-20. Mix well and incubate at room temperature for 15 minutes with gentle agitation. The reaction was terminated by removal of the reaction mixture into a tube containing 3.9 ml of sodium buffer (ie 40-fold dilution). The product was concentrated by passing through a pre-wetted Ultrafree centrifugal filter unit. Measure the volume of the concentrate and add 1/9 volume of 10X saline solution (1M NaCl, 0.2M sodium citrate, 3mM citric acid). Determine the protein concentration and then calculate the product recovery. Products were analyzed by IEF gels and in vitro assays with TF-1 cells. the

Alternatively, recombinant tissue protective cytokines can be iodinated as follows. Iodine beads (Pierce, Rockford, Il) were incubated for 5 min in 100 μl PBS (20 mM sodium phosphate, 0.15 M NaCl, pH 7.5) containing 1 mCi of free ^Na123I . 100 μg of recombinant tissue protective cytokines dissolved in 100 μl of PBS were then added to the mixture. After incubation at room temperature for 10 minutes, the reaction was terminated by removing 200 [mu]l of the solution from the reaction vessel (leaving the iodine beads). Excess iodine was then removed by gel filtration on a Centricon 10 column. As shown in Figure 11, iodine erythropoietin produced in this way was effective in protecting P19 cells after serum depletion. Those of ordinary skill in the art will recognize that iodination of the recombinant tissue protective cytokines of the invention is expected to yield similar results.

Yet another method for iodination of recombinant tissue protective cytokines is outlined below. 100 μg recombinant tissue protective cytokine dissolved in 100 μl PBS was added to 500 uCi N ¹²⁵ I and the mixture was mixed together in a microcentrifuge tube. Then 25 μl of chloramine T (2 mg/ml) was added and the mixture was incubated at room temperature for 1 min. Then 50 [mu]l Chloramine T stop buffer (2.4 mg/ml sodium metabisulfite, 10 mg/ml tyrosine, 10% glycerol, 0.1% xylene in PBS) was added. The iodinated tyrosine and iodinated recombinant tissue protective cytokines were then separated by gel filtration with a Centricon 10 column.

6.4.7. Biotinylated recombinant tissue protective cytokines

Recombinant tissue protective cytokines can be used to prepare the corresponding biotinylated molecules following the following procedure as described by Pierce Chemical Company (Rockford, IL) for EZ-LinkNHS-LC-Biotin (Cat. No. 21336). the

Just before the reaction, EZ-Link NHS-LC-Biotin (pierce, 21336) was dissolved in DMSO at a concentration of 2 mg/ml. Reactions were carried out in test tubes (17×100 mm) containing 50 mM sodium bicarbonate (pH 8.3) in a total volume of 1 ml. Add recombinant tissue protective cytokines and <10% EZ-Link NHS-LC-biotin, biotin/protein molar ratio is about 20. Mix well and keep on ice for 2 hours. The reaction product was desalted and concentrated using an Ultrafree centrifugal filter unit. Collect the product into a new tube. Measure the volume and add 1/9 volume of 10X saline solution (1 M NaCl, 0.2 M sodium citrate, 3 mM citric acid). The protein content was determined, and then the product recovery was calculated. Products were analyzed by IEF gels and in vitro assays with TF-1 cells. the

Methods for biotinylation of free amino groups of recombinant tissue protective cytokines are disclosed below. 0.2 mg D-biotinyl-ε-aminocaproic acid-N-hydroxysuccinimide ester (Boehringer Mannheim #1418165) was dissolved in 100 μl DMSO. The solution was mixed with 400 μl of PBS containing approximately 0.2 mg of recombinant tissue protective cytokine in a foil-covered tube. After incubation at room temperature for 4 hours, unreacted biotin was separated by gel filtration on a Centricon 10 column. As shown in Figure 12, the biotinylated erythropoietin protected p19 cells from depletion of serum. Those of ordinary skill in the art will recognize that biotinylation of the recombinant tissue protective cytokines of the invention is expected to yield similar results. the

Finally, in Wojchowski et al. "Biotinylated recombinant human erythropoietins: Bioactivity and Utility as a receptor ligand (biotinylated recombinant human erythropoietin: biological activity and use as a ligand for receptors)". Blood, 1989, 74(3): In 952-8, the authors used three different approaches to biotinylate erythropoietin. Biotin is added to (1) sialic acid moieties (2) carboxyl and (3) amino groups. The authors used a mouse splenocyte proliferation assay to demonstrate that (1) addition of biotin to the sialic acid moiety does not inactivate the biological activity of erythropoietin; (2) addition of biotin to the carboxyl group results in erythropoietin (3) Adding biotin to the amino group leads to complete biological inactivation of erythropoietin. These methods and modifications are fully included herein. Figure 12 shows the activity of biotinylated erythropoietin and asialoerythropoietin in a serum starvation P19 assay. One of ordinary skill in the art will recognize that iodination of recombinant tissue protective cytokines of the invention is expected to yield similar results, see Section 6.15. the

6.5. Example 5: Modification of recombinant tissue protective cytokines by other methods

6.5.1. Trinitrophenylation:

According to the method described by Plapp et al. ("Activity of bovine pancreatic deoxyribonuclease A with modified amino groups" 1971, J. Biol. Chem. 246, 939-845), the recombinant tissue Protective cytokines (100 μg) were modified with 2,4,6-trinitrobenzenesulfonate. the

6.5.2. Arginine modification

According to Riordan ("Functional arginyl residues in carboxypeptidase A. Modification with butanedione (functional arginyl residues in carboxypeptidase A. Modification with butanedione)." Riordan JF, Biochemistry 1973, 12 (20): 3915- 3923), recombinant tissue protective cytokines are modified with 2,3-butanedione. the

In another modification in which the amino acid residues of erythropoietin are modified, the arginine residues are modified with phenylglyoxal according to the method described by Takahashi (1977, J. Biochem. 81: 395-402), so that The reaction can be carried out at room temperature for a variable time range from 0.5 hours to 3 hours. The reaction was terminated by dialysis of the reaction mixture against water. The use of such modified forms of erythropoietin are all included in the present invention. Phenylglyoxal-modified erythropoietin was tested using the neural-like P19 cell assay described above. As shown in Figure 13, the chemically modified erythropoietin completely retained its neuroprotective effect. Similar results were obtained with similarly modified recombinant tissue protective cytokines of the present invention. the

According to Patthy et al. ("Identification of functional arginine residues inribonuclease A and lysozyme (identification of functional arginine residues of ribonuclease A and lysozyme)." Patthy, L, Smith EL, J.Biol.Chem 1975 250( 2): The method described in 565-9), the recombinant tissue protective cytokine is modified with cyclohexanone. the

According to Werber et al. ("Proceedings: Carboxypeptidase B: modification offfunctional arginyl residues (Proceedings: Carboxypeptidase B: Modification of functional arginyl residues)." Werber, MM, Sokolovsky M Isr J Med Sci1 975 11(11) : 1169-70), recombinant tissue protective cytokines were modified with phenylglyoxal. the

6.5.3. Tyrosine modification

According to Nestler et al. "Stimulation of rat ovarian cell steroidogenesis by high density lipoproteins modified with tetranitromethane." Nestler JE, Chacko GK, Strauss JF 3rd. J Biol Chem 1985 Jun 25;260(12):7316-21) as previously described, recombinant tissue protective cytokines (100 μg) were incubated with tetranitromethane. the

6.5.4. Glutamate (and Aspartate) Modifications

To modify the carboxyl group, follow Caraway et al. "Carboxyl group modification in chymotrypsin and chymotrypsinogen (carboxyl modification of chymotrypsin and chymotrypsinogen)." Carraway KL, Spoerl P, Koshland DE Jr.J Mol Biol 1969May 28; 42( 1): As described in 133-7, recombinant tissue protective cytokines (100 μg) were incubated with 0.02M EDC in 1M glycinamide at pH 4.5 for 60 minutes at room temperature. the

6.5.5. Tryptophan residue modification

According to the method described in Ali et al., J Biol Chem.1995 Mar 3; 270 (9): 4570-4, the recombinant tissue protective cytokine (100 μ g) was mixed with 20 μ M n-bromosuccinimide in 20 mM potassium phosphate buffer (pH 6.5) Insulate together at room temperature. The number of oxidized tryptophan residues was determined according to the method described by Korotchkina (Korotchkina, LG et al., Protein Expr Purif. 1995 Feb; 6(1):79-90). the

6.5.6. Removal of amino groups

In order to remove the amino groups of recombinant tissue-protective cytokines, according to Kokkini et al. -705), recombinant tissue protective cytokines (100 μg) were incubated with PBS (pH 7.4) containing 20 mM ninhydrin (Pierce Chemical, Rockford, Il) at 37° C. for 2 hours. Reduction of the resulting aldehyde is accomplished by reacting the product with sodium borohydride or lithium aluminum hydride. Specifically, erythropoietin (100 μg) was incubated with 0.1 M sodium borohydride in PBS for 30 minutes at room temperature. The reduction reaction was terminated by cooling the samples on ice for 10 minutes and dialyzed 3 times against PBS overnight. (Kokkini, G., Blood, Vol. 556, No. 4, 1980: 701-705). Reduction with lithium aluminum hydride was accomplished by incubating recombinant tissue protective cytokines (100 μg) with 0.1 M lithium aluminum hydride in PBS for 30 minutes at room temperature. The reduction reaction was terminated by cooling the samples on ice for 10 minutes and dialyzed 3 times against PBS overnight. the

6.5.7. Disulfide bond reduction and stabilization

Recombinant tissue protective cytokines (100μg) were incubated with 500mM DTT at 60°C for 15 minutes. A 20 mM aqueous solution of iodoacetamide was then added to the mixture and incubated for 25 minutes at room temperature in the dark. the

6.5.8. Limited proteolysis

Recombinant tissue protective cytokines can undergo limited chemical proteolysis targeting specific residues. Recombinant tissue protective cytokines were reacted with 2-(2-nitrophenylsulfinyl)-3-methyl-3'-bromoindolenine in a capped test tube under nitrogen pressure at room temperature in the dark Specific cleavage after tryptophan residues for 48 hours in 50-fold excess of 50% acetic acid. The reaction was terminated by quenching with tryptophan and desalting. the

As noted above, recombinant tissue protective cytokines can be modified, but multiple modifications of tissue protective cytokines as well as additional modifications can also be made without departing from the spirit of the invention. the

6.6. Example 6: Tissue protective cytokines have neuroprotective effects

According to Manley et al., 2000, Aquaporin-4 deletion in mice reduces brainedema after acute water intoxication and ischemic stroke (mice aquaporin-4 deletion reduces acute water intoxication and brain edema after ischemic stroke), Nat Med 2000Feb; 6( 2): The method described in 159-63, evaluating the neuroprotective effect of chemically modified erythropoietin in the water intoxication assay. C3H/HEN female mice were used. Mice were administered intraperitoneally with 20% of their body weight in water and 400 ng/kg bw DDAVP (desmopressin). Administration of mice with erythropoietin (A) or tissue protective cytokines: asialoerythropoietin (B), carbamylated asialoerythropoietin (C), succinylated asialoerythropoietin ( D), acetylated asialo erythropoietin (E), iodide asialo erythropoietin (F), carboxymethylated asialo erythropoietin (G), carbamylated erythropoietin (H ), acetylated erythropoietin (I), iodinated erythropoietin (J) or N ^ε -carboxymethyl erythropoietin (K). Mice were administered erythropoietin or chemically modified erythropoietin twice intraperitoneally at a dose of 100 micrograms/kg 24 hours before and at the time of water administration. A modified rating scale from Manley et al. was used to evaluate mice. The modified ratings are listed below:

1. Explore the cage/table

yes 0

No 1

2. Visual tracking target

yes 0

No 1

3. Beard movement

exists 0

does not exist 1

4. Leg-tail movement

Normal 0

stiff 1

Paralyzed 2

5. Painful withdrawal (toe pinch)

yes 0

No 1

6. Motor coordination

Normal 0

Exception 1

7. Stop at the table

yes 0

No 1

Possible total rating: 8

Mice were scored at the following time points: 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 120 minutes, 150 minutes and 180 minutes. Figure 14 shows the performance of mice receiving erythropoietin or chemically modified erythropoietin, as a percentage of neuron-deficient experiments in control mice. Figure 14 shows the protection of tissue protective cytokines in mice with neurological trauma caused by water intoxication. Recombinant tissue protective cytokines with similar chemical modifications are expected to give similar results. Statistical significance was also determined. Compared with the control, those dosage regimens with significant difference, p<0.05, are indicated by ^* , and those with highly significant difference, p<0.01, are indicated by ^** .

6.7. Example 7: Maintenance of heart function for transplantation

According to the protocol described by Delcayre et al., 1992, Amer. J. Physiol. 263: H1537-45, for Wistar male rats weighing 300-330 g, cytopoietin ( 5000U/kg body weight) or vehicle. Animals were sacrificed with pentobarbital (0.3 mL) and heparinized (0.2 mL) intravenously. The heart begins to let it equilibrate for 15 minutes. The left atrial sac was then inflated to a volume of 8 mm Hg end-diastolic pressure. Left ventricular pressure-volume curves were constructed by increasing balloon volume inflation to 0.02 ml. Zero volume is defined as the point where the left ventricular end-diastolic pressure is zero. Upon completion of the pressure volume curve, after checking coronary flow, the left ventricular sac was deflated to restore end-diastolic pressure to 8 mmHg and the control cycle was continued for 15 minutes. The heart was then stopped with 50 mL of Celsior+ molecules to rest at 4°C under a pressure of 60 cm _H2O . The hearts were then excised and stored at 4°C for 5 hours in plastic containers filled with the same solution surrounded by crushed ice.

After storage was complete, the hearts were transferred to the Langendorf apparatus. The balloon catheter was reinserted into the left ventricle and reinflated to the same preischemic volume. Hearts were reperfused for at least 2 hours at 37°C. The reperfusion pressure was set at 50 cm _H2O for 15 minutes of reflow, then returned to 100 cm _H2O for an additional 2 hours. Re-establish heartbeat (320 beats/min). Isometric measurements of systolic index and diastolic blood pressure were repeated three times at 25 minutes, 45 minutes, 60 minutes and 120 minutes of reperfusion. Pressure-volume curves were completed at this time point and coronary effluent was collected during 45 min of reperfusion to measure creatine kinase leakage. The two treatment groups were compared using an unpaired t-test, and linear regression of end-diastolic pressure data was used to design compliance curves. As shown in Figure 15, following treatment with erythropoietin, a marked improvement in left ventricular pressure variability occurred, as well as an improved volume-pressure curve, reduced left ventricular diastolic pressure, and reduced creatine kinase leakage. Similar results are expected to be obtained with treatment with the recombinant tissue protective cytokines of the present invention.

6.8. Example 8: Erythropoietin protects cardiomyocytes from ischemic injury

Adult male rats were administered recombinant human erythropoietin (5000 U/kg body weight) 24 hours before anesthesia and prepared for coronary artery occlusion. An additional dose of erythropoietin was given at the beginning of the procedure and the left main coronary artery was occluded for 30 minutes before release. The same dose of erythropoietin was given daily for 1 week after treatment. The heart function of the animals is then studied. As shown in Figure 16, animals that received a sham injection (saline) demonstrated a large increase in left end-diastolic pressure, indicative of an enlarged, stiff heart secondary to myocardial infarction. In contrast, animals receiving erythropoietin did not experience cardiac hypofunction compared to sham-operated controls (significant difference at the p<0.01 level). Similar results are expected to be obtained with treatment with the recombinant tissue protective cytokines of the present invention. the

6.9. Example 9: Protection of peripheral administration of erythropoietin on retinal ischemia

Retinal cells are very sensitive to ischemia, so after 30 minutes of ischemic stress, many cells die. Furthermore, subacute or chronic ischemia underlies the degenerative symptoms that accompany a number of common human diseases such as diabetes, glaucoma and macular degeneration. To date, there are no effective therapies to protect cells from ischemia. A tight endothelial barrier exists between the blood and retina, which excludes most macromolecules. To test whether peripheral administration of erythropoietin protects cells sensitive to ischemia, an acute, reversible rat model of glaucoma as described by Rosenbaum et al. (1997; Vis. Res. 37:3443-51) was used. Specifically, saline was injected into the anterior compartment of adult male rats to pressurize systemic arterial pressure and maintain it for 60 minutes. Twenty-four hours before the induction of ischemia, the animals were intraperitoneally administered with saline or 5000 U erythropoietin/kg body weight, and then continuously administered daily doses for 3 days. Electroretinography was performed on dark-adapted rats 1 week after treatment. Figures 17-18 illustrate that administration of erythropoietin is significantly more effective on the electroretinogram (ERG) (Fig. With good reservations. Figure 18 compares the electroretinogram a-wave and b-wave amplitudes of the erythropoietin-treated and saline-treated groups, showing that erythropoietin provides significant protection. Similar results can be obtained by treatment with the recombinant tissue protective cytokines of the present invention. the

6.10. Example 10: The Restoration Effect of Erythropoietin on Cognitive Function Decreased by Brain Injury

In a study demonstrating the effect of erythropoietin on the ability of mice to recover diminished cognitive function after brain trauma, female Balb/c mice were subjected to blunt trauma to the brain as described in Brines et al., PNAS 2000, 97; 10295-10672 5 days later, intraperitoneal administration of 5000 U/kg-bw erythropoietin per day was started. Twenty days after injury, animals were examined for cognitive function in the Morris water maze, 4 trials per day. Although both treated and untreated animals performed poorly in the test (swimming time was about 80 seconds instead of the possible 90 seconds), Figure 19 shows the Morris Water maze experiment, n=16 mice in each group. The first experiment was 1 week after EPO administration (12 days y after injury). Both groups of animals performed poorly (swimming time of about 80 seconds instead of the possible 90 seconds). Animals treated with erythropoietin fared better (negative values are better in this figure). 4 trials per day, averaged. Figure 19 shows. Restoration of cognitive function was observed even when initiation of erythropoietin treatment was delayed until 30 days after trauma (Fig. 20). In Fig. 20, n=7 mice per group were treated with 5000 U/kg EPO starting one month after injury, except on weekends. The average of experiments was also 4 experiments per day. Similar results are expected to be obtained with treatment with the recombinant tissue protective cytokines of the present invention. the

6.11. Example 11: Kainic acid model

In the kainic acid neurotoxicity model, according to the protocol described by Brines et al., Proc. Asialoerythropoietin at a dose of 5000 U/kg-bw was shown to be as effective as erythropoietin, as indicated by time to death (Fig. 21). Similar results are expected to be obtained with treatment with the tissue protective cytokines of the present invention. the

6.12. Example 12: Spinal cord injury model

6.12.1. Detection of erythropoietin and tissue protective cytokines in rat spinal cord compression

Wistar rats (female) weighing 180-300 g were used in this study. Animals were fasted and humanely restrained for 12 hours prior to surgery, and then anesthetized with intraperitoneal injection of thiopental (40 mg/kg-bw). After penetration into the skin (bupivacaine 0.25%), a total monolevel (T-3) laminectomy was performed through a 2 cm incision with the aid of a dissecting microscope. Traumatic spinal cord injury was induced by epidurally applying a temporary aneurysm clip to the spinal cord with a closure force of 0.6 Newtons (65 g) for 1 min. After the clips were removed, the skin incision was closed and the animal was allowed to fully recover from anesthesia and returned to its cage. The bladder was palpated at least 2 times per day, and rats were continuously monitored until voiding occurred spontaneously. the

40 animals were randomly divided into 5 groups. Control (I) animals (n=8) receiving normal saline (by iv injection) were sutured immediately after incision. Group (II; n=8) received rhEPO @ 16 μg/kg-bw iv, group (III) received the asialo-tissue protective cytokine (asialoerythropoietin) of the present invention @ 16 μg/kg-bw iv, group (IV) received asialo-tissue protective cytokine@30 μg/kg-bw iv, and group (V) received asialo-tissue-protective cytokine (asialoerythropoietin)@ of the present invention 30 μg/kg-bw; all given as a single bolus intravenous injection immediately after removal of the aneurysm clip. the

The motor function of the rats will be assessed by using the motor rating scale of Basso et al. On this scale, animals are scored on a scale from 0 (no hindlimb movement observed) to 21 (normal gait). Rats will be examined for functional deficits at 1 hour, 12 hours, 24 hours, 48 hours, 72 hours and 1 week after injury by the same examiner blinded to the treatment each animal received. the

Figure 22 is a graph demonstrating the level of motion recovered by rats within 30 days of spinal cord trauma. As shown, rats administered erythropoietin (Group II) or tissue protective cytokines (Group III-Group V) recovered readily from injury and demonstrated better overall recovery than control rats. Similar results are expected to be obtained with therapeutic treatment with the recombinant tissue protective cytokines of the present invention. the

In a second related study, animals were injected in the same manner. 40 animals were randomly divided into 3 groups. Control animals (n=8) receiving normal saline (by iv injection) were sutured immediately after incision. The second group (n=8) received methylprednisolone @ 30 micrograms/kg, 3 times a day, then 1 time in two weeks, methylprednisolone is a commonly used therapeutic drug for spinal cord injury; the third group received the drug of the present invention immediately after the injury Recombinant tissue protective cytokine S100E was administered at a dose of 10 μg/kg-bw, all given as a single bolus intravenous injection immediately after removal of the aneurysm clip. the

The motor function of the rats will be assessed by using the motor rating scale of Basso et al. On this scale, animals are scored on a scale from 0 (no hindlimb movement observed) to 21 (normal gait). Rats will be examined for functional deficits at 1 hour, 12 hours, 24 hours, 48 hours, 72 hours and 1 week after injury by the same examiner blinded to the treatment each animal received. the

Figure 37 is a graph demonstrating the level of motor recovery in rats within 42 days of spinal cord trauma. As shown, rats administered S100E recovered readily from injury and demonstrated better overall recovery than control rats and rats treated with methylprednisolone. the

6.12.2. Detection of erythropoietin and tissue protective cytokines in rabbit spinal cord ischemia

Thirty-six New Zealand White rabbits (8-12 months old, male) weighing 1.5-2.5 kg were used in this study. Animals were fasted for 12 hours and restrained humanely. Anesthesia was performed by inhalation of 3% halothane in 100% oxygen and maintained in 0.5-1.5% halothane in a mixture of 50% oxygen and 50% air. An intravenous catheter (22 gauge) was placed in the left ear vein. During surgery, Ringers Lactate was infused at a rate of 4 ml/kg body weight (bw) per hour. Cefazolin 10mg/kg-bw was given intravenously before the operation to prevent infection. The animal was placed in the right lateral position, the skin was prepared with povidone iodine, infiltrated with bupivacaine (0.25%) and a subflank skin incision was made at the 12th rib parallel to the spine. After incision of the skin and subcutaneous thoracolumbar fascia, the longissimus lumborum and lumboiliac costalis contract. The abdominal aorta was exposed and diverted under the left renal artery through the left retroperitoneal approach. A PE-60 tubing was looped around the aorta leaving the left renal artery with both ends threaded through large rubber tubing. The aorta was atraumatically occluded for 20 min by tensioning the PE tubing. High-dose intravenous heparin (400 IU) was administered before aortic occlusion. After 20 minutes of occlusion, the tubes and catheters were removed, the incision was closed, and the animals were monitored until complete recovery, after which they were serially evaluated for neurological function. the

36 animals were randomly divided into 6 groups. In the control group (I), animals (n=6) received normal saline intravenously immediately after release of the aortic occlusion. Group (II) received rhEPO @ 6.5 μg/kg-bw; Group (III) received a tissue protective cytokine (carbamylated erythropoietin) @ 6.5 μg/kg-bw; Group (IV) received another tissue Protective cytokine (asialoerythropoietin) @ 6.5 μg/kg-bw; Group (V) received the same tissue protective cytokine as Group (IV), but @ 20 μg/kg-bw; and Group (VI) Received another tissue protective cytokine (asialocarbamylated erythropoietin) @ 20 micrograms/kg-bw; all given intravenously immediately after reperfusion (n = for each group 6). the

Motor function was assessed by investigators blinded to treatment at 1 hour, 24 hours, and 48 hours after reperfusion according to the criteria of Drummond and Moore. Each animal was scored from 0 to 4 as follows: 0 = paraplegia with no motor function of the lower limbs; 1 = poor motor function of the lower limbs, only weak anti-gravity movements; 2 = good anti-gravity strength, but unable to step down the body Moderate lower extremity motor function with moving legs; 3 = good motor function, able to drag leg under body to jump, but not normal; 4 = normal motor function. The bladders were defecated manually twice daily in paraplegic animals. the

Figure 23 is a graph depicting motor function in recovered rabbits. The graph demonstrates that erythropoietin and the tissue protective cytokines of the present invention completely recovered rabbits from spinal cord injury even in a period of only 2 days. Similar results are expected to be obtained with therapeutic treatment with the recombinant tissue protective cytokines of the present invention. the

6.13. Example 13: Anti-inflammatory effect of erythropoietin

In vivo studies :

rat MCAO

Male Crl:CD(SD)BR rats weighing 250-280 g were obtained from Charles River, Calco, Italy. According to Brines, M.L., Ghezzi, P., Keenan, S., Agnello, D., de Lanerolle, N.C., Cerami, C., Itri, L. M., and Cerami, A. 2000. Erythropoietin crosses the blood-brain barrier to Protect against experimental brain injury (erythropoietin crosses the blood-brain barrier to prevent experimental brain injury). [In ProcessCitation] Proc Natl Acad Sci USA 97: 10526-10531 described the method of surgery on these rats. Briefly, rats were anesthetized with chloral hydrate (400 mg/kg-bw, i.p.), the carotid artery was exposed, the right carotid artery was occluded with two sutures and opened. The MCA is clearly visualized adjacent to and towards the right orbital foramen cauterized away from the nasal artery. To create a penumbra (border) around this fixed MCA lesion, the contralateral carotid artery was occluded for 1 hour before being released using fine forceps retraction. PBS or rhEPO (5,000 U/kg-bw, i.p.; previously shown to be protective in this model (1 )) was administered immediately after MCAO. When required, TNF and IL-6 were quantified in cerebral cortex homogenates as previously described (8). MCP-1 in the homogenate was determined using a commercially available ELISA kit (biosource, Camarillo, CA). the

Twenty-four hours after MCAO, rats were anesthetized as described above and perfused intracardiacly with 100 ml of saline followed by 250 ml of sodium phosphate buffered 4% paraformaldehyde solution. Brains were quickly dissected, fixed in sodium phosphate-buffered 4% paraformaldehyde solution for 2 h, transferred to 20% sucrose solution in PBS overnight, then in 30% sucrose solution until they were submerged, then in 2-methylbutane Freeze at -45°C in alkanes. Sectioning (30 μm) through the brain in a transverse plane on a cryostat (HM 500 OM, Microm) at -20°C and selecting every 5th section for staining for different antigens or hematoxylin-eosin Histochemistry. Following the protocol described by Houser et al. and the manufacturer's protocol, anti-glial fibrillary acid protein (GFAP) mouse monoclonal antibody (1:250, Boehringher Mannheim, Monza, Italy) and anti-cdl11b (MRC OX- 42) Mouse monoclonal antibody (1:50, Serotec, UK) treated free floating sections for immunoreactivity. All sections were dipped for light microscopy in saline on embedded slides, dehydrated by graded ethanol, fixed in xylene and coverslipped with DPX mounter (BDH, Poole, UK). Adjacent sections were stained with hematoxylin-eosin as described (10). the

Figure 24 shows a coronal section of the cerebral cortex stained with hematoxylin and eosin. Control rats (A), ischemic rats (B) were treated with PBS, ischemic rats (C) were treated with rhEPO (5,000U/kg-bw, i.p., administered immediately after MCAO). Section B shows a marked decrease in tissue staining consistent with inflammation, with loss of neuronal components compared to control (A). Systemic administration of rhEPO greatly reduced ischemic damage at the site of cell death or damage in focal areas (C). (Magnification 2.5x. Scale bar = 800μm.)

Figure 25 shows a coronal section of the anterior cortex adjacent to the infarct stained with GFAP antibody. Control rats (A), ischemic rats treated with PBS (B), ischemic rats treated with rhEPO (C). Activated astrocytes were manifested by their GFAP-positive processes (panel B). Staining intensity of activated astrocytes was significantly decreased both quantitatively and in representative rhEPO-treated animals (panel C). (Magnification 10x. Scale bar = 200μm.)

Figure 26 shows a coronal section of the cerebral cortex stained with OX-42 antibody. Ischemic rats were treated with PBS (A) and ischemic rats were treated with rhEPO (B). In the ischemic hemispheres, cellular staining in the tissue surrounding the infarct was particularly prominent in both treatment groups, but more dense and extensive in the saline-treated group. (Magnification 20x; scale bar = 100 μm). the

Figure 27 shows a coronal section of the cerebral cortex adjacent to the infarct area stained with OX-42 antibody. A much higher density of mononuclear inflammatory cells was observed in ischemic rats treated with PBS (A) compared to ischemic rats treated with rhEPO (B). Infiltrating leukocytes with a typical round shape will enlarge the infarct volume. (Magnification 10x; scale bar = 200 μm). the

Similar results are expected to be obtained with therapeutic treatment with the recombinant tissue protective cytokines of the present invention. the

Acute experimental allergic encephalomyelitis (EAE) in Lewis rats

Female Lewis rats aged 6-8 weeks were purchased from Charles River (Calco, Italy). Under slight ether anesthesia, rats were injected with an equal volume of complete Freund's adjuvant (Difco, Detroit, MI) with heat-inactivated Mycobacterium tuberculosis (M. CFA, Sigma) emulsified 50 μg of guinea pig MBP (Sigma, St. Louis, MO) in water (to a final volume of 100 μ), induced EAE in rats. Rats were examined blindly for EAE symptoms and scored as follows: 0, no disease; 1, tail flaccidity; 2, ataxia; 3, complete paralysis of the hind limbs with urinary incontinence. Starting from 3 days after immunization, the rats were intraperitoneally (i.p.) administered the specified dose of r-Hu-EPO (EPOetin alfa, Procrit, Ortho Biotech, Raritan, NJ) dissolved in PBS, once a day. Because clinical grade EPO contains human serum albumin, control animals are usually given PBS containing the same amount of human serum albumin. Daily administration of 5,000 U/kg-bw of EPO increased hematocrit by 30%. When needed, inject (s.c.) from days 3 to 18 with 1.3 mg/kg-bw of DEX disodium phosphate (Sigma) in PBS equivalent to 1 mg/kg-bw of dexamethasone (DEX) rat. When required, TNF and IL-6 were quantified in brain and spinal cord homogenates as previously described [Agnello, 2000 #10]. the

Figure 28 shows the protective effect of different doses of EPO on the clinical signs of EAE given from 3 days to 18 days after MBP immunization. EPO delayed disease onset and reduced disease severity in a dose-dependent manner, as summarized in Table 1. However, EPO does not delay the time to maximum severity. As shown in the table, EPO significantly lowered the mean cumulative score at doses of 2,500 and 5,000 U/kg-bw. the

In experiments in which EPO treatment was not continued and rats were monitored for up to 2 months after disease recovery, no relapse was observed, whereas DEX resulted in disease exacerbation after a delay in its administration (Fig. 29). Similar results are expected to be obtained with therapeutic treatment with the recombinant tissue protective cytokines of the present invention. the

In vitro studies :

。在微量板读数器上读取560nm吸光度。 Primary cultures of glial cells were prepared from 1-2 day old neonatal Sprague-Dawley rats. The brain hemispheres were detached from the meninges and disrupted mechanically. The cells were dispersed in a solution of 2.5% trypsin and 1% DNase, filtered through a 100 μm nylon mesh and seeded (140,000 cells/35 mm dish) in supplemented with 10% fetal bovine serum, 0.6% glucose, chloramphenicol (0.1 mg/ml ) and penicillin (100Ul/ml) on Eagle's minimal essential medium. Glial cultures were fed twice a week and incubated at 37 °C in a humidified incubator with 5% _CO2 . All experiments were performed on 2-3 week old glial cell cultures with 97% astrocytes and 3 % microglia. Establishment of neuronal cultures from 18-day-old rat fetal hippocampus. The brain was removed and peeled away from the meninges, and the hippocampus was isolated. The cells were dispersed by incubation in a 2.5% trypsin solution at 37°C for 15-20 minutes and the concentration was determined. The cell suspension was diluted in medium for glial cells and seeded onto polyornithine-embedded coverslips at a density of 160,000 cells per coverslip. One day after inoculation, coverslips were transferred to neuronal maintenance medium supplemented with 5 μM of cytarabine (supplemented with 5 μg/ml insulin, 100 μg/ml transferrin, 100 μg/ml putrescin, 30 nM Sodium selenite, 20nM progesterone and 100U/ml penicillin Dulbecco's modified Eagle's medium and Ham's nutrient mix F12) Petri dish. Flip the coverslip so that the hippocampal neurons face the glial monolayer. Paraffin dots glued to the coverslip support them on the glia, creating a slit that prevents the two cell types from contacting each other but allows soluble material to diffuse. These culture conditions allowed the growth of differentiated neuronal cultures with >98% homology as determined by immunochemical assays for microtubule-associated protein 2 and GFAP. Cells were then treated with 1 μM trimethyltin (TMT) for 24 h in the presence or absence of rhEPO (10 U (80 ng/ml), supernatants were used for TNF assays, and cell viability was assessed as described above. When needed , in the presence of LPS, with or without rhEPO, the glial cells were cultured for 24 hours, and the TNF in the culture supernatant was measured. Through 3-(4,5-dimethyl-thiazol-2-yl)-2 , 5-diphenyltetrabromide azole (MTT) assay, measure cell viability. Denizot, F. and Lang, R.1986.Rapid colorimetric assay for cell growth and survival.Modifications to the tetrazolium dye procedure reviving improved sensitivity and reliability.( Rapid colorimetric assay of cell growth and survival. Modification of the tetrazolium dye method for improved sensitivity and reliability). J Immunol Methods 89: 27 1-277. Briefly, MTT tetrazolium salt was dissolved in Serum-free medium to a final concentration of 0.75 mg/ml, and added to cells that had been treated at 37°C for 3 hours. The medium was then removed and extracted with 1N HCl:isopropanol (1:24) First

. Absorbance was read at 560 nm on a microplate reader.

Figure 30 shows that rhEPO prevents neuronal death-inducing TNF production in mixed neuron-glial cultures. Panel A: Percentage of neuronal cell death induced by TMT 1 μM with or without rhEPO (10 U/ml). Panel B: TNF release from glial cells exposed to TMT 1 μM in the presence (shaded bars) or absence (black bars) of neurons, with or without rhEPO (10 U/ml). Similar results are expected to be obtained with therapeutic treatment with the recombinant tissue protective cytokines of the present invention. the

6.14. Example 14: NMDA-induced cell death assay

Excitotoxicity can be defined as hyperactivation of glutamate receptors, such as N-methyl-D-aspartate (NMDA) receptors. NMDA receptors show increased activity in response to ischemic trauma or other trauma (Fauci et al., 1998, Harrison's Principles of Internal Medicine), (Nishizawa, 2001, Life Sci.69, 369-381), (White et al. , 2000, J. Neurol. Sci. 179, 1-33). Therefore, this assay serves as a model for evaluating compounds with effects on cell damage and death. the

Protocol for NMDA excitotoxicity in primary hippocampal neurons

Primary hippocampal neuronal cultures were prepared from neonatal mice (less than 24 hours) as described by Krohn et al. (1998). Briefly, hippocampi were sectioned in DMEM containing 0.02% BSA. Tissues were transferred to DMEM containing 0.1% papain and incubated at 37°C for 20 minutes. The medium containing papain was sucked off and the digestion was terminated by adding MEMII, and the hippocampal cells were dispersed by pipetting with a 1000 μl pipette. Tissue fragments were allowed to settle and supernatants containing single cells were transferred to MEMII containing 1% trypsin inhibitor (typeII-O) and 1% BSA. The pipetting step was repeated 3 times, and then single cells were centrifuged at 600 U/min for 10 min and resuspended in growth medium (MEMII, 20 mM D-glucose, 100 U/ml penicillin, 100 μg streptomycin, 2 mM L-glutamine , 10% Nu-serum (bovine), 2% B27 supplement, 26.2 mM NaHCO ₃ ). Cells from 10 hippocampi were used to seed 24-well plates. One day after seeding, cells were treated with cytidine-arabino-furanoside (1 [mu]M). On day 2, the medium was changed and cytidine-arabinose-furanoside (1 μM) was added.

Excitotoxicity Assay

12 day old cultures were pre-incubated with 5 nM of test compound (vehicle, R103E, R150E or EPO) for 24 hours. On day 13, the media was removed from the cells and maintained while the cultures were challenged with 300 μM NMDA for 5 min at room temperature. After excitotoxic damage, the preconditioned medium was returned to the cultures and after an additional 24 hr incubation damage was quantified by trypan blue exclusion. Approximately 300 neurons were counted in at least 4 separate wells for each condition and the experiment was repeated at least 2 times (Krohn, A.J., Preis, E. and Prehn, J.H.M. (1998) J. Neurosci. 18(20): 8186 -8197). the

Figure 3 1 shows that human erythropoietin and recombinant tissue protective cytokines R130E and R150E, when added to primary hippocampal neuron cell cultures before MNDA treatment, effectively reduced cell death induced by NMDA. Cells treated with R103E (5 nM) exhibited significantly reduced cell death compared to vehicle control cells (p=0.01). Cells treated with R103E (5 nM) showed significantly reduced cell death compared to vehicle control cells (p=0.01). Cells treated with R150E (5 nM) showed an approximately 20% reduction in cell death compared to vehicle control cells (p=0.001). Statistics: ANOVA with Tukey's post hoc test. the

6.15. Example 15: Neuronal protection in P19 cells after serum removal

To test the neuronal protective effect of the recombinant tissue protective cytokines of the present invention, serum-depleted P19 cells were used as a model. Clone P19S1801A1 was kindly provided by Dr. WHFischer. Cells were maintained in a humidified incubator supplemented with 2 mM L-glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate, and 10% fetal calf serum (FCS) in an atmosphere of ₇ % CO2 in a humidified incubator. ; all from Gibco, Paisley, Scotland, LTK) and Dulbecco's modified Eagles medium (DMEM) containing 1.2 g/L NaHCO ₃ , 10 mM Hepes buffer (Carlo Erba, Milano, Italy) (this is hereinafter referred to as complete medium )middle. Serum-free medium (N2) had the same composition as above, but lacked serum, and the following were added: 5 μg/ml insulin, 100 μg/ml transferrin, 20 nM progesterone, 100 μM putrescine, and ₃₀ nM _Na2SeO3 (all from Sigma). For death experiments, cells were dispersed with 10% trypsin preparation (Gibco), washed once with complete medium, washed twice with N2 medium, and plated, unless otherwise stated, in 25 ^cm2 tissue culture flasks (FalconBecton Dickinson, Lincoln Park, New Jersey) at a final concentration of 10 ⁴ cells/cm ² in 5 μl of serum-free medium. L-acetylcarnitine (100 μM) was used as a positive control, said substance conferring a protective reduction of the percentage of apoptotic nuclei up to 50% after 24 hours after deprivation of serum. After 24 hours of removal of serum, tap the flask to disperse the cells (without trypsin), centrifuge at 600 rpm (Shandon Southern, USA) for 10 minutes, inoculate on microscope slides and wash with Carnoy's solution (methanol:acetic acid, 3: 1) Fix for 10 minutes, stain with Hoechst 33258 (0.1 μr/ml PBS) at 37°C for 1 hour, wash with tap water for 15 minutes, air dry and install. Slides were observed with a fluorescence microscope (Zeiss, Germany) at an excitation wavelength of 365 nm. The percentage of apoptotic nuclei was determined by blind counting in a total of 100 cells in at least 5 assays.

P19 cells were pre-incubated with 3nM Epo or recombinant tissue protective cytokine S100E for 24 hours. Such treatment produced significant (p<0.001 ) protection against apoptosis triggered by serum depletion. Data are the average of 3 determinations in 1 experiment. The experiment was performed twice with similar results. the

Figure 32 shows neuronal protection in P19 cells after serum depletion. The percentage of apoptotic cell death decreased for cells pretreated with Epo, EpoWT and recombinant tissue protective cytokine S100E. Cells treated with EPO showed about a 20% decrease in apoptotic cell death compared to untreated control cells. Cells treated with EpoWT and S100E both showed about 10% decrease in apoptotic cell death compared to untreated control cells. the

6.16. Example 16: NGF removal in differentiated PC12 cells

To test the neuronal protective effect of the recombinant tissue protective cytokines of the present invention, NGF depleted differentiated PC12 cells were used as a model. The assay is a well established model of apoptosis. The PC12 rat cell line is derived from a phaeochromocytoma of the adrenal medulla and can differentiate into neuron-like cells in the presence of NGF (Masuda et al., 1993, J Biol Chem 268, 11208-11216). The PC 12 cell line is a neuroendocrine cell line that can differentiate to express a neuron-like phenotype in the presence of NGF (Vaudry et al., 2002, Science 296, 1648-1649). Once cells are fully differentiated, they become NGF-dependent, and when NGF is removed they lead to apoptosis. the

PC12 cells were incubated in Dulbecco's modified Dulbecco supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, 1% sodium pyruvate, and 1% penicillin-streptomycin (P/S) (Invitrogen, Carlsbad, USA). maintained in Eagle medium (DMEM). the

For the experiment, in the supplementation of 1% heat-inactivated horse serum, 1% sodium pyruvate, 1% P/S and 100ng/ml NGF (7S nerve growth factor, mouse submandibular gland, purchased from Calbiochem, catalog number 480354) Cells were differentiated at a density of 24,000 cells/well in collagen G-embedded 48-well plates in DMEM for 7 days, and the medium was changed every 2-3 days. On day 6, after replacing the medium with RPMI1640, 1% P/S to remove NGF from all cells, Epo mutants at amino acid 100 (=S100E) were added to the cells at the indicated concentrations for 24 hours. Then add S100E, when NGF (100ng/ml) is used as positive control (+NGF). After 24 hours, viability was determined by tetrazolium (MTT) reduction assay. the

Figures 33A and 33B show the effect of pre-incubation with S100E in NGF-depleted differentiated PC12 cells in two independent experiments. Differentiated PC12 cells were pretreated with the indicated concentrations of S100E for 24 hours, Figure 33A (3 pM) Figure 33B (0.00003 pM-3 pM). Viability was determined in the MTT assay. NGF (100 ng/ml) represents a positive control, while medium without NGF (-NGF) serves as a negative control. Data shown in Figure 33 are positive control (+NGF) and % viability (n=8 in both experiments). ^*** p<0.001, ^* p<0.05, using one-way ANOVA with Bonferroni post-hoc test, there was a statistically significant increase in the viability of S100E-treated cells compared to negative control cells (-NGF). In terms of potency and efficacy, the effects observed with S100E were similar to those observed with EPO in this experimental system.

Figure 34 shows the effect of pre-incubation with Epo in NGF-depleted differentiated PC12 cells. Differentiated PC12 cells were pretreated with Epo, S100E or carbamylated Epo (30 pM-30nM) for 24 hours. The chemically modified Epo molecule AA24496 was 10,000-fold less active than EPO in the UT-7 cell assay. Viability was determined in the MIT assay. NGF (100 ng/ml) was used as a positive control, while medium without NGF (-NGF) was used as a negative control. the

6.17. Example 17: EPO bioassay for UT-7 cell proliferation

UT-7 is a leukemic Epo-dependent cell line used to measure the erythroid effects of recombinant tissue protective cytokines such as K45D. UT-7 cells (Deutsche Sammlungvon Mikroorganismen und Zellkulturen (DSMZ), catalog number ACC 363) grew normally in the presence of 10% FBS and 5 ng/ml Epo. Proliferation/survival (=increased viability) effects of cells exposed to Epo are mediated through traditional peripheral Epo receptors. The proliferative effect was quantified and determined in relation to the ability of the Epo-variants to stimulate conventional Epo receptors. the

Method for UT-7 cell viability assay

The Epo-dependent human leukemia cell line UT7 was prepared, and the proliferative effect of the added Epo/recombinant tissue protective cytokine was used to measure its biological activity. On day 1 of the assay, cells were transferred to fresh complete RPMI1640 medium containing 10% serum and containing Epo (5 ng/ml) (10% donor calf serum, 4 mM L-glutamine, supplemented with 5 ng/ml ofrhuEPO )middle. Cells were grown in ^75cm2 flasks containing 20ml of culture. On day 2 of the assay, cells were transferred from the bottle to a 50 ml conical tube and centrifuged at 1,000 rpm for 5 min at room temperature. The old medium was discarded and the cells were washed twice with 10 ml starvation medium (3% donor calf serum, 4mM L-glutamine). Resuspend cells in starvation medium and pipette up and down to obtain a single cell suspension. To determine cell density, the resuspended cells were diluted with starvation medium to a density of 4 x ¹⁰⁵ cells/ml in a total volume of 10 ml of medium and seeded into 25 ^cm2 culture flasks. The mixture was incubated at 37 C for 4 h in a 5% _CO2 humidified incubator. During the last 1 h of incubation, prepare the 96-well plate. At the end of the 4 hour incubation, the cell culture was removed from the incubator and the cells were transferred from the flask to a 50 ml conical tube. Shake by hand to mix the contents and keep the cells in suspension. Add 50 ml starvation medium as a medium blank without cells. Five wells are control cells without reagent. The adjacent row of wells contains the lowest concentration of recombinant tissue protective cytokine. Each subsequent row of wells was sequentially added with a higher concentration. 100 [mu]l per well were seeded at 200,000 cells/ml in cell cultures incubated in medium containing 3% serum and without Epo in 96-well plates. The contents are mixed quickly and carefully with the orbital vibrating platform on top of the stir plate. The plate was incubated with different concentrations of Epo variants (from 0.2 pM-20 nM) in RPMI 1640 medium containing 3% serum in a humidified incubator with 5% _CO2 at 37 C for 48 hours. Four times a day, the 96-well plate was taken out of the incubator and placed in a laminar flow hood at room temperature. Measuring the formation of formazan by cellular metabolism of the tetrazolium dye WST1 The product (which correlates with cell viability and cell number) is immediately quantified for biological activity (photometric absorbance at 450 nm, subtracted from background absorbance at 620 nm).

result

UT7 cells grown on Epo-containing medium for 3 months showed stable and reliable growth. the

Viability-inducing K45D induced an Epo-dependent increase in viability of UT-7 cells in a dose-dependent manner with an EC ₅₀ of 294.0. In comparison, the _EC50 was 58.13 for Epo (Figure 35) and 608 for His-tagged Epo (EpoWT). At concentrations <50 nM (ie, within the measurable range), S100E did not increase the viability of Epo-dependent UT-7 cells (greater than 50%). Thus, K45D exhibited potency on the same order of magnitude as Epo, while S100E exhibited at least 1000-fold lower potency than Epo.

At concentrations up to 20 nM, R103E did not increase the survival of Epo-dependent UT-7 cells, ie, its potency was at least 4 orders of magnitude lower than that of Epo. R150E induces Epo-dependent survival of UT-7 cells in a dose-dependent manner with an EC ₅₀ of 20 nM. In contrast, for Epo (Epo#4) the _EC50 was 66.5 (Figure 36). Thus, R150E showed at least 3 orders of magnitude less potency than Epo.

Figure 35 shows the concentration-response curves of Epo, K45D and S100E in UT-7 cells. Different concentrations of Epo, EpoWT, K45D and S100E were added to UT-7 cells. In the WST-1 assay, viability was measured after 48 hours. Data are means ± SD of replicates each performed in three different experiments. The curve is a nonlinear regression curve fit. the

Figure 36 shows the dose response curves of Epo, R103E and R150E in UT-7 cells. Different concentrations of Epo, EpoWT, R103E and R150E were added to UT-7 cells. In the WST-1 assay, viability was measured after 48 hours. Data are means ± SD of replicates for each of three different experiments. The curve is a nonlinear regression curve fit. the

6.18. Example 18: Protection of Retinal Ischemia by Peripheral Administration of Recombinant Tissue Protective Cytokines

As mentioned in subsection 6.9, retinal cells are very sensitive to ischemia, so after 30 min of ischemic stress, many cells die. In this experiment, a rat model of reversible glaucoma as described by Rosenbaum et al. (1997; Vis. Res. 37:3443-51 ) was used. The effect of recombinant tissue protective cytokines on the ischemic stress response was examined. the

Following the example provided in subsection 6.9 for injecting saline into the anterior compartment of adult male rats, the injections were given to one eye of each rat. Upon reperfusion, that is, when the pressure in the anterior chamber is released, rats were administered intravenously with 10 μg/kg EPO of one of the four recombinant tissue-protective cytokines R103E, R150E, S100E, and S100e/K45D or saline. Electroretinography was performed on both the injured and normal eyes of each rat at 1 day, 3 days, 5 days and 6 days after injury. The latency of the injured eye of each rat was compared with that of the normal eye of the same rat. Data were recorded as the ratio of the latency of the injured eye to the latency of the normal eye, yielding a ratio when the injured eye had normal function. Two factors contribute to the outcome of the lesion: amplitude (the difference from peak to trough, shown in Figure 17, panel A, indicated by 'b' and latency), and the time it takes to reach the peak in response to the stimulus. the

Figure 38 shows the ratio of the latency of the injured eye to the latency of the normal eye for different treatment regimens. Rats treated with EPO showed a latency of 1.2, better than rats treated with saline. For each of the four recombinant tissue protective cytokines, R103E, R150E and S100E produced latencies equal to or better than EPO, indicating statistically better results than saline. the

The scope of the invention is not to be limited by the specific embodiments intended as individual illustrations of the various aspects of the invention and functionally equivalent methods and compositions are within the scope of the invention. Even from the foregoing description and drawings, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art. Such modifications are all within the scope of the appended claims. the

All references cited above are hereby incorporated by reference in their entirety for all purposes. the

Claims (80)

1. A pharmaceutical composition comprising a mutein recombinant tissue protective cytokine, wherein said cytokine comprises an amino acid sequence having one or more of the following amino acid substitutions in erythropoietin shown in SEQ ID NO: 10: I6A , C7A, C7S, R10I, V11S, L12A, E13A, R14A, R14E, R14Q, Y15A, Y15F, Y15I, K20A, K20E, E21A, N24K, C29S, C29Y, A30N, H32T, C33S, C33Y, N38K, P42N, P42A . 、G101I、L102A、R103A、R103E、S104A、S104I、L105A、T106A、T106I、T107A、T107L、L108K、L108A、L108S、K116A、S126A、T132A、I133A、T134A、K140A、F142I、R143A、S146A、N147K、N147A , F148A, F148Y, L149A, R150A, R150E, G151A, K152A, K152W, L153A, K154A, L155A, G158A, A160S, C161A, and/or R162A, wherein the amino acid positions are numbered according to the mature erythropoietin molecule; and (a) Absence or attenuation of at least one activity of said cytokine selected from the group consisting of increased hematocrit, vasoactive effects, hyperactivated platelets, procoagulant activity, and increased production of thrombin cells; and (b) said cytokine has at least an activity selected from the group consisting of: i. Preserving the function or viability of mammalian effector cells, tissues or organs; ii. maintaining the function or viability of mammalian effector cells, tissues or organs; iii. enhancing the function or viability of mammalian effector cells, tissues or organs; and iv. Restoring the function or viability of a mammalian effector cell, tissue or organ. 2. The pharmaceutical composition of claim 1, wherein said mutein recombinant tissue protective cytokine comprises at least one of the following mutations or combinations of mutations: R14Q, S 100E, R103E, R150E, K45D/S100E or K45D/R150E or R103E/ L108S. 3. The pharmaceutical composition of claim 1, wherein the mutein recombinant tissue protective cytokine comprises an altered amino acid residue at one or more amino acid positions selected from: positions 11, 12, 13, 14 , 15 is SEQ ID NO: 1, position 44, 45, 46, 47, 48, 49, 51 is SEQ ID NO: 2, position 100, 101, 102, 103, 104, 105, 106, 107, 108 is SEQ ID NO: 3, positions 146, 147, 148, 149, 150 and amino acid 151 are SEQ ID NO: 4. 4. The pharmaceutical composition of claim 1, wherein the mutein recombinant tissue protective cytokine comprises an altered amino acid residue at one or more amino acid positions selected from: 7, 20, 21, 29, 33 , 38, 42, 59, 67, 70, 83, 96, 126, 142, 143, 152, 153, 155 and 161. 5. A pharmaceutical composition comprising a mutein recombinant tissue protective cytokine, wherein said cytokine comprises the amino acid sequence of SEQ ID NO: 10 with the following changes: 1) One or more of the amino acid residue substitutions shown in SEQ ID NO: 15-105 or 119; 2) amino acid residues 44-49 are deleted, wherein the amino acid positions are numbered according to the mature erythropoietin molecule; or 3) At least one of the amino acid substitutions shown in SEQ ID NO: 106-118, and (a) at least one activity of said cytokine selected from the group consisting of lacking or weakening of: increasing hematocrit, vasoactive effect, hyperactivation platelets, procoagulant activity, and increased production of thrombin cells; and (b) the cytokine has at least one activity selected from the group consisting of: i. Preserving the function or viability of mammalian effector cells, tissues or organs; ii. maintaining the function or viability of mammalian effector cells, tissues or organs; iii. enhancing the function or viability of mammalian effector cells, tissues or organs; and iv. Restoring the function or viability of a mammalian effector cell, tissue or organ. 6. The pharmaceutical composition according to any one of claims 1-5, wherein the mutein recombinant tissue protective cytokine further comprises chemical modification of one or more amino acids. 7. The pharmaceutical composition of claim 6, wherein said chemical modification results in a change in charge. 8. The pharmaceutical composition of claim 7, wherein the charged amino acid residues are modified to uncharged residues. 9. The pharmaceutical composition of any one of claims 1-5, wherein the mammalian effector cells are selected from the group consisting of neuronal cells, muscle cells, heart cells, lung cells, liver cells, kidney cells, small intestinal cells, adrenal cortex cells, adrenal medulla cells, capillary cells, endothelial cells, testicular cells, ovarian cells, endometrial cells, and stem cells. 10. The pharmaceutical composition of any one of claims 1-5, wherein the mammalian effector cells are selected from the group consisting of photoreceptor cells, ganglion cells, bipolar cells, horizontal cells, amacrine cells, Müller cells, cardiac cells, pacemaker cells, sinoatrial node cells, sinus node cells, atrioventricular node cells, atrioventricular bundle cells, hepatocytes, astrocytes, hepatic macrophages, mesangial cells, goblet cells, intestinal gland cells, enteroendocrine cells, glomerular cells, fascicular cells, reticulocytes, chromaffin cells, adventitial cells, mesenchymal cells, trophoblast cells, sperm cells, mature follicular cells, primitive follicular cells, endometrial stromal cells, and uterine endometrial cells. 11. The pharmaceutical composition of any one of claims 1-5, wherein the mutein recombinant tissue protective cytokine is capable of crossing the endothelial cell barrier. 12. The pharmaceutical composition of claim 11, wherein said endothelial cell barrier is selected from the group consisting of blood-brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier and blood-uterine barrier. 13. The pharmaceutical composition of any one of claims 1-5, wherein the mutein recombinant tissue protective cytokine has at least one of the following modifications compared to the native erythropoietin molecule: i. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 sialic acid moieties; ii. Reduced number or absence of N-linked or O-linked sugars; iii. having reduced sugar content by treating the natural recombinant tissue protective cytokine with at least one glycosidase; iv. One or more oxidized sugars; v. One or more oxidized sugars, and the mutein recombinant tissue protective cytokine is also chemically reduced; vi. One or more modified arginine residues; vii. One or more modified lysine residues or N-terminal amino modification; viii. one or more modified tyrosine residues; ix. One or more modified aspartic acid or glutamic acid residues; x. One or more modified tryptophan residues; xi. One or more amino groups are removed; xii. At least one cystine bond is open; xiii. Truncated; xiv. Attaching one or more polyethylene glycol molecules; xv. linking one or more fatty acids; xvi. Non-mammalian glycosylation patterns; and xvi. One or more histidine-tagged amino acids. 14. The pharmaceutical composition of claim 13, wherein the recombinant tissue protective cytokine is asialoerythropoietin. 15. The pharmaceutical composition of claims 1-5, wherein the mutein recombinant tissue protective cytokine is hypersialylated. 16. The pharmaceutical composition of claim 13, wherein said mutein recombinant tissue protective cytokine has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 sialic acid moieties. 17. The pharmaceutical composition of claim 13, wherein the mutein recombinant tissue protective cytokine is N-linked sugar-free erythropoietin. 18. The pharmaceutical composition of claim 17, wherein the mutein recombinant tissue protective cytokine is erythropoietin without O-linked sugars. 19. The pharmaceutical composition of claim 13, wherein the mutein recombinant tissue protective cytokine is treated with at least one glycosidase by treating the native recombinant tissue protective cytokine with at least one glycosidase. 20. The pharmaceutical composition of claim 13, wherein said mutein recombinant tissue protective cytokine is periodate oxidized erythropoietin. 21. The pharmaceutical composition of claim 20, wherein said periodate oxidized erythropoietin is chemically reduced by sodium cyanoborohydride. 22. The pharmaceutical composition of claim 13, wherein the mutein recombinant tissue protective cytokine comprises an R-glyoxal moiety on one or more arginine residues, wherein R is an aryl or alkyl moiety . 23. The pharmaceutical composition of claim 22, wherein said mutein recombinant tissue protective cytokine is phenylglyoxal-erythropoietin. 24. The pharmaceutical composition of claim 13, wherein the mutein recombinant tissue protective cytokine is wherein the arginine residue is reacted with a diketone selected from 2,3-butanedione and cyclohexanedione And the modified erythropoietin. 25. The pharmaceutical composition of claim 13, wherein said mutein recombinant tissue protective cytokine is erythropoietin in which the arginine residue is reacted with 3-deoxyglucosone. 26. The pharmaceutical composition of claim 13, wherein the modified lysine or modified N-terminal amino group is at least one biotinylated lysine or biotinylated N-terminal amino group. 27. The pharmaceutical composition of claim 13, wherein the modified lysine is glucosyllysine or fructosyllysine. 28. The pharmaceutical composition of one of claims 1-5, wherein the mutein recombinant tissue protective cytokine comprises at least one carbamylated lysine residue. 29. The pharmaceutical composition of claim 28, wherein the mutein recombinant tissue protective cytokine is selected from the group consisting of: α-N-carbamyl erythropoietin; N-ε-carbamyl erythropoietin; α-N -Carbamyl, N-ε-carbamoyl erythropoietin; α-N-carbamoylasialo erythropoietin; N-ε-carbamoylasialo erythropoietin; Acyl, N-ε-carbamoylasialo erythropoietin; α-N-carbamoylhyposialo erythropoietin; N-ε-carbamoylhyposialo erythropoietin; and α-N-ammonia Formyl, N-ε-carbamoyl-low sialyl erythropoietin. 30. The pharmaceutical composition of claim 13, wherein said mutein recombinant tissue protective cytokine comprises at least one acylated lysine residue. 31. The pharmaceutical composition of claim 30, wherein said mutein recombinant tissue protective cytokine comprises at least one acetylated lysine residue. 32. The pharmaceutical composition of claim 31, wherein said mutein recombinant tissue protective cytokine is selected from the group consisting of: α-N-acetyl erythropoietin; N-ε-acetyl erythropoietin; α-N-acetyl, N -ε-acetylasialoerythropoietin; α-N-acetylasialoerythropoietin; N-ε-acetylasialoerythropoietin; α-N-acetyl, N-ε-acetylasialoerythropoietin; α-N-acetylhyposialyl erythropoietin; N-ε-acetylhyposialyl erythropoietin; and α-N-acetyl, N-ε-acetylhyposialyl erythropoietin. 33. The pharmaceutical composition of claim 30, wherein the lysine residues of the mutein recombinant tissue protective cytokine are succinylated. 34. The pharmaceutical composition of claim 33, wherein the mutein recombinant tissue protective cytokine is selected from the group consisting of: α-N-succinyl erythropoietin; N-ε-succinyl erythropoietin; α-N-succinyl erythropoietin; Acyl, N-ε-succinyl erythropoietin; α-N-succinylasialoerythropoietin; N-ε-succinylasialoerythropoietin; α-N-succinyl, N-ε-succino Acyl-asialo erythropoietin; α-N-succinyl hyposialo erythropoietin; N-ε-succinyl hyposialo erythropoietin; and α-N-succinyl, N-ε-succinyl hyposialo erythropoietin acid erythropoietin. 35. The pharmaceutical composition of claim 13, wherein the mutein recombinant tissue protective cytokine comprises at least one lysine modified by sodium 2,4,6-trinitrobenzenesulfonate or another salt thereof Residues. 36. The pharmaceutical composition of claim 13, wherein said modified tyrosine residues are nitrated and/or iodinated. 37. The pharmaceutical composition of claim 13, wherein the modified aspartic acid residue or glutamic acid residue is reacted with a carbodiimide followed by an amine. 38. The pharmaceutical composition of claim 37, wherein said amine is glycinamide. 39. The pharmaceutical composition of any one of claims 1-5, formulated for oral, intranasal or parenteral administration. 40. The pharmaceutical composition of any one of claims 1-5 formulated as a perfusate. 41. A mutein recombinant tissue protective cytokine, wherein said cytokine comprises at least one of the following amino acid substitutions: R150E, K45D/S100E or K45D/R150E or R103E/L108S, wherein said amino acid positions are according to the mature erythropoietin molecule numbering; and at least one activity of said cytokine selected from the group consisting of lack or attenuation of: increased hematocrit, vasoactive effect, hyperactivated platelets, procoagulant activity, and increased production of thrombus cells; said cytokine has at least one An effector cell protective activity selected from protecting, maintaining, enhancing or restoring mammalian effector cell, tissue or organ function or viability. 42. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising the mutein recombinant tissue protective cytokine of claim 41. 43. A vector comprising the nucleic acid molecule of claim 42. 44. An expression vector comprising the nucleic acid molecule of claim 42 and at least one regulatory region operably linked to said nucleic acid molecule. 45. The vector of claim 43 or 44, which is a pCI-neo vector. 46. A genetically engineered cell comprising the nucleic acid molecule of claim 42. 47. A cell comprising the expression vector of claim 43, 44 or 45. 48. A method for protecting, maintaining or enhancing the viability of cells, tissues or organs isolated from the body of a mammal, said method comprising contacting said cells, tissues or organs with any one of claims 1-5 medicinal composition. 49. The method of claim 48, wherein the mutein recombinant tissue protective cytokine is non-erythropoietic. 50. Use of a mutant protein recombinant tissue protective cytokine in the preparation of a pharmaceutical composition, wherein the cytokine comprises one or more of the following amino acid substitutions in erythropoietin shown in SEQ ID NO: 10 Sequence: I6A, C7A, C7S, R10I, V11S, L12A, E13A, R14A, R14E, R14Q, Y15A, Y15F, Y15I, K20A, K20E, E21A, N24K, C29S, C29Y, A30N, H32T, C33S, C33Y, N38K, P42N, P42A, D43A, T44I, K45A, K45D, V46A, N47A, F48A, F48I, Y49A, Y49S, W51F, W51N, K52A, Q59N, E62T, L67S, L70A, N83K, D96R, K97A, S100R, S100E, S100A S100T、G101A、G101I、L102A、R103A、R103E、S104A、S104I、L105A、T106A、T106I、T107A、T107L、L108K、L108A、L108S、K116A、S126A、T132A、I133A、T134A、K140A、F142I、R143A、S146A、 and (a) lack or attenuation of at least one activity of said cytokine selected from the group consisting of increased hematocrit, vasoactive effects, hyperactivated platelets, procoagulant activity and increased production of thrombin cells, and (b) said cells The factor has at least one activity selected from: i. Preserving the function or viability of mammalian effector cells, tissues or organs; ii. maintaining the function or viability of mammalian effector cells, tissues or organs; iii. enhancing the function or viability of mammalian effector cells, tissues or organs; and iv. Restoring the function or viability of a mammalian effector cell, tissue or organ, The pharmaceutical composition is used for: 1) preventing or preventing an injury, disease or condition affecting an effector cell, tissue or organ in a mammal; or 2) Enhancing, maintaining, restoring or regenerating the function or vitality of effector cells, tissues or organs in a mammal. 51. The use of claim 50, wherein the mutein recombinant tissue protective cytokine comprises at least one of the following mutations or combinations of mutations: R14Q, S100E, R103E, R150E, K45D/S100E or K45D/R150E or R103E/L108S. 52. The use of claim 50, wherein the altered protein recombinant tissue protective cytokine comprises an altered amino acid residue at one or more amino acid positions selected from: positions 11, 12, 13, 14, 15[ SEQ ID NO: 1], position 44, 45, 46, 47, 48, 49, 51 [SEQ ID NO: 2], position 100, 101, 102, 103, 104, 105, 106, 107, 108 [SEQ ID NO:3], positions 146, 147, 148, 149, 150 and amino acid 151 [SEQ ID NO:4]. 53. The use of claim 50, wherein the mutein recombinant tissue protective cytokine comprises altered amino acid residues at one or more of the following amino acid positions: 7, 20, 21, 29, 33, 38, 42, 59, 67, 70, 83, 96, 126, 142, 143, 152, 153, 155 and 161. 54. Use of a mutein recombinant tissue protective cytokine in the preparation of a pharmaceutical composition, wherein the cytokine comprises the amino acid sequence of SEQ ID NO: 10 with the following changes: (a) one or more of the amino acid residue substitutions shown in SEQ ID NO: 15-105 or 119; (b) amino acid residues 44-49 are deleted, wherein the amino acid positions are numbered according to the mature erythropoietin molecule; or (c) at least one of the amino acid substitutions shown in SEQ ID NO: 106-118, and (i) at least one activity of said cytokine selected from the group consisting of lacking or attenuating activity of: increasing hematocrit, vasoactive effect, hyperactivity Activation of platelets, procoagulant activity and increased production of thrombin cells; and (ii) said cytokine has at least one activity selected from the group consisting of: i. Preserving the function or viability of mammalian effector cells, tissues or organs; ii. maintaining the function or viability of mammalian effector cells, tissues or organs; iii. enhancing the function or viability of mammalian effector cells, tissues or organs; and iv. Restoring the function or viability of a mammalian effector cell, tissue or organ, The pharmaceutical composition is used for: 1) preventing or preventing an injury, disease or condition affecting an effector cell, tissue or organ in a mammal; or 2) Enhancing, maintaining, restoring or regenerating the function or vitality of effector cells, tissues or organs in a mammal. 55. The use of any one of claims 50-54, wherein the mammal suffers from: cognitive impairment, seizure disorder, chronic epilepsy, epilepsy, convulsions, nerve root compression, multiple sclerosis, stroke, low Blood pressure, cardiac arrest; central nervous system disease, disorder or condition; neuronal loss, ischemia, subarachnoid hemorrhage, aneurysm, aneurysmal bleeding, myocardial infarction, inflammation, age-related cognitive decline, radiation injury, Radiation therapy, whole brain irradiation, cerebral palsy, supranuclear palsy, progressive supranuclear palsy, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, Tourette's syndrome, subacute necrotizing cerebrospinal Acute febrile polyneuritis, dementia, AIDS dementia, senile dementia, Lewy body dementia, memory loss, amyotrophic lateral sclerosis, alcoholism, neuropsychiatric or neuropsychological disorders, mood disorders, Unipolar Disorder, Depression, Major Depressive Disorder, Dysthymic Disorder, Mania, Bipolar Disorder, Panic Disorder, Anxiety Disorder, Schizophrenia, Schizoaffective Disorder, OCD, Concentration Inattention, ADHD, autism, prion disease, transmissible spongiform encephalopathy, Friedreich's ataxia, Wilson's disease, trauma, concussion injury, brain trauma or spinal cord trauma, cerebral insufficiency Blood or spinal cord ischemia, cardiopulmonary bypass, neurological deficits caused by cardiopulmonary bypass, postoperative cognitive impairment, embolic injury, hypoxia, mitochondrial dysfunction, cardiac injury, chronic heart failure, ocular tissue damage, macula Degeneration, diabetic neuropathy, diabetic retinopathy, glaucoma, retinal ischemia, retinal trauma, retinitis pigmentosa, optic nerve damage, retinal detachment, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion , low blood pressure, conditions associated with low blood sugar, diabetes, nephrotic syndrome, acute kidney failure, or hepatitis. 56. The pharmaceutical composition of any one of claims 1-5, wherein the mutein recombinant tissue protective cytokine is alkylated. 57. The pharmaceutical composition of any one of claims 1-5, wherein the mutein recombinant tissue protective cytokine is carbamylated. 58. The pharmaceutical composition of any one of claims 1-5, wherein the mutein recombinant tissue protective cytokine is α-N-carbamoyl, N-ε-carbamoyl erythropoietin. 59. The composition of any one of claims 1-5 or 28, wherein the mutein recombinant tissue protective cytokine has at least one polyethylene glycol molecule linked thereto. 60. The mutein recombinant tissue protective cytokine of claim 41, wherein at least one lysine residue of said cytokine is carbamylated. 61. The mutein recombinant tissue protective cytokine of claim 41, wherein said cytokine is carbamylated. 62. The mutein recombinant tissue protective cytokine of claim 41, wherein said cytokine is alkylated. 63. The mutein recombinant tissue protective cytokine of claim 41, wherein said cytokine is asialoerythropoietin. 64. The mutein recombinant tissue protective cytokine of claim 41, wherein said cytokine is α-N-carbamoyl, N-ε-carbamoyl erythropoietin. 65. The mutein recombinant tissue protective cytokine of claim 41 or 60, wherein the cytokine has at least one molecule of polyethylene glycol linked thereto. 66. The method of claim 48, wherein at least one lysine residue of the mutein recombinant tissue protective cytokine is carbamylated. 67. The method of claim 48, wherein the mutein recombinant tissue protective cytokine is carbamylated. 68. The method of claim 48, wherein the mutein recombinant tissue protective cytokine is asialoerythropoietin or alkylated erythropoietin. 69. The method of claim 48, wherein said mutein recombinant tissue protective cytokine is alpha-N-carbamyl, N-epsilon-carbamyl erythropoietin. 70. The method of claim 48 or 66, wherein the mutein recombinant tissue protective cytokine has at least one polyethylene glycol molecule attached to it. 71. The use of any one of claims 50-54, wherein at least one lysine residue of the mutein recombinant tissue protective cytokine is carbamylated. 72. The use of any one of claims 50-54, wherein the mutein recombinant tissue protective cytokine is carbamylated. 73. The use of any one of claims 50-54, wherein the mutein recombinant tissue protective cytokine is asialoerythropoietin or alkylated erythropoietin. 74. The use of any one of claims 50-54, wherein the mutein recombinant tissue protective cytokine is α-N-carbamoyl, N-ε-carbamoyl erythropoietin. 75. The use of any one of claims 50-54, wherein the mutein recombinant tissue protective cytokine has at least one polyethylene glycol molecule linked thereto. 76. The use of any one of claims 50-54, wherein said pharmaceutical composition is formulated for administration after said injury, disease or condition has started. 77. The use of any one of claims 50-54, wherein the pharmaceutical composition is formulated for administration prior to the onset of the injury, disease or condition. 78. The use of claim 76 or 77, wherein the injury or condition is cardiopulmonary bypass, radiotherapy or chemotherapy. 79. The use of any one of claims 50-54, wherein said pharmaceutical composition is formulated for administration during the acute and/or chronic phase of said injury, disease or condition. 80. The use of any one of claims 50-54, wherein the mutein recombinant tissue protective cytokine is carbamylated and has at least one polyethylene glycol molecule attached thereto.

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