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

Be used to protect, the recombinant tissue protectiveness cell factor and the code nucleic acid thereof of recovery and enhancement effect cell, tissue and organ

The application requires in the U.S. Provisional Patent Application the 60/392nd of application on July 1st, 2002, No. 455 and with the U.S. Provisional Patent Application the 60/393rd of on July 3rd, 2002 application, No. 423 right of priority, the full content of described patented claim all is attached to herein by reference.

1. technical field

The present invention relates to be used to protect, keep, strengthen or recover the function of mammal effector cell and relevant cell, tissue and organ or the mutain recombinant tissue protectiveness cell factor with one or more aminoacid replacement of vigor, relate to the Pharmaceutical composition that comprises described cell factor.This comprises that protection such as excitable tissues such as neuron and heart tissue avoid neurotoxin, hypoxemia and other unfavorable stimulation, and comprises enhancing excitable tissue function, for example is used to promote learning and memory.The invention still further relates to and use mutain recombinant tissue protectiveness cell factor to be used for transport molecule or to promote that molecule passes through the composition of the transhipment of endothelial barrier by transcytosis.

2. background technology

For many years, people only know that the physiological action of erythropoietin(EPO) is the erythrocytic generation of control.In recent years, some evidences show, erythropoietin(EPO) is as the cell factor superfamily member, have by with erythropoietin receptor (interact other important physiological function of mediation of erythropoietin(EPO)-R).These effects comprise that it is vessel retraction/vasodilation, superactivation blood platelet and to the influence of middle metabolism that mitosis generation, calcium current are gone into the adjusting of smooth muscle cell and neurocyte, vasoactive effect.It is believed that erythropoietin(EPO) is provided for the compensatory reaction that improves hypoxic cell micro-environment and regulate the apoptotic cell death that is caused by metabolic stress.Though study definite, the intracranial injection erythropoietin(EPO) can neuroprotective unit exempt from hypoxic neuron injury,, the encephalic administration is used, especially is unpractical to normal individual for treatment, also is unacceptable method of administration.In addition, in the past the anaemia patient was given the existing conclusion of research of erythropoietin(EPO), promptly the administered peripherally erythropoietin(EPO) can not be transported to brain (Marti etc., 1997, Kidney Int.51:416-8; Juul etc., 1999, Pediatr.Res.46:543-547; Buemi etc., 2000, Nephrol.Dial.Transplant.15:422-433).

Described the various modified forms that have at the erythropoietin(EPO) of the activity of improving described molecule erythropoiesis activity, for example had the amino acid whose modified forms of change, be described in United States Patent (USP) 5 at its c-terminus, 457,089 and United States Patent (USP) 4,835,260; Have the erythropoietin(EPO) isotype of the sialic acid residues of different numbers in each molecule, for example be described in United States Patent (USP) 5,856,298; Polypeptide is described in United States Patent (USP) 4,703,008; Activator is described in United States Patent (USP) 5,767,078; The peptide that combines with erythropoietin receptor is described in United States Patent (USP) 5,773, and 569 and 5,830,851; And small molecule mimetics, be described in United States Patent (USP) 5,835,382.

The present invention relates to recombinant tissue protectiveness cell factor in position and exsomatize protect, keep, the purposes in enhancing or recovery Effects cell and relevant cell, tissue and the organ, and in order to protect and to strengthen the purpose of the effector cell that leaves vascular system and relevant cell, tissue and organ and send recombinant tissue protectiveness cell factor and pass through endothelial cell barrier or carry the purposes that correlation molecule passes through endothelial cell barrier.

3. summary of the invention

In one aspect, the present invention relates to various forms of recombinant tissue protectiveness cell factors are used for protecting, keep, strengthen or recover the Pharmaceutical composition of the function of mammal effector cell and relevant cell, tissue and organ or vigor in preparation purposes.One concrete aspect, described mammal effector cell and relevant cell, tissue or organ are because endothelial cell barrier and away from vascular system closely.Another concrete aspect, described cell, tissue, organ or other body partly are separated portions in the mammalian body, for example the expection part that is used to transplant.As limiting examples, effector cell or tissue can be neuronal cell, retina cell, myocyte, core cell, pneumonocyte, liver cell, nephrocyte, small intestine cells, adrenal cortical cell, adrenal medullary cell, capillary endothelial cell, testicular cell, gonad cell, pancreatic cell, osteocyte, Skin Cell or endometrial cell or tissue.In addition, effector cell's limiting examples comprises photosensory cell (rod cell and awl cell), gangliocyte, Beale's ganglion cells, horizontal cell, amakrine, Miller (M ü eller) cell, Purkinje cell, the cardiac muscle cell, pacemaker cells, sinus node cells, sinus node cells, the hole nodal cell, the conjunctive tissue cell, the atrioventricular node cell, the atrioventircular bundle cell, liver cell, astrocyte, Kupffer cell, mesangial cell, renal epithelial cell, the tubulose enterocyte, goblet cell, enteraden cell (crypt), enteroendocrine cell, messangial cell, pencil cell (fasciculate), desmacyte, chromaffin cell, pericyte, interstitial cell, trophocyte, spermatid, ripe follicle cell (Graffian follicles), original follicular cells, pancreas islet, A cells, beta cell, γ-cell, the F-cell, osteogenic cell, osteoclast, Gegenbaur's cell, endometrial stromal cell, endometrial cell, stem cell and endothelial cell.These examples of effector cell only are illustrative.In one aspect, described effector cell or its relevant cell, tissue or organ be neither excitable cell, tissue or organ, dominant excitable cell or the tissue of also not comprising.In a specific embodiment, mammalian cell, tissue or the organ that has used above-mentioned recombinant tissue protectiveness cell factor is the cell of having spent or being about to spend a period of time under the condition of at least a vigor that is unfavorable for described cell, tissue or organ.In a specific embodiment, used mammalian cell, tissue or the organ of above-mentioned recombinant tissue protectiveness cell factor to express the EPO acceptor.Described condition comprises original position hypoxemia or metabolic dysfunction or the original position toxin exposure that traumatic original position hypoxemia or metabolic dysfunction, operation are induced; The latter may be relevant with chemotherapy or radiotherapy.In one embodiment, described adverse condition is the result who for example is used for some operating cardiopulmonary bypass (heart-lung machine).

Recombinant tissue protectiveness cell factor of the present invention can be used for the therapeutic treatment or the prophylactic treatment of following human diseases: central nervous system (CNS) disease or peripheral nervous disease and illness in eye, angiocardiopathy, heart and lung diseases, breathing problem, ephrosis, urethral disease and genital diseases, enterogastric diseases and cryptorrhea and the metabolic disorder that mainly have neurology symptom or psychiatric symptom.

The present invention also relates to comprise and give mammal, the Pharmaceutical composition of human specific above-mentioned recombinant tissue protectiveness cell factor preferably.Described Pharmaceutical composition can be mixed with in oral, the nose or parenteral usefulness, perhaps to be used to keep the perfusion liquid form of isolated cells, tissue or organ vitality.

The recombinant tissue protectiveness cell factor that is used for above-mentioned purpose can be the erythropoietin(EPO) of mutain or genetic modification, just has the erythropoietin(EPO) of a modification at least at the natural molecule amino acid backbone." variant protein " or " mutain " is meant the albumen that comprises the mutating acid sequence and comprises because of amino acid deletions, replacement or the two and be different from the polypeptide of natural erythropoietin(EPO) amino acid sequence." native sequences " is meant wild type or identical amino acid sequence or the nucleotide sequence of native form with gene or albumen.In addition; in one embodiment; recombinant tissue protectiveness cell factor of the present invention has cell protection activity; but also have the one or more effects of erythropoietin(EPO), promptly increase the generation and the procoagulant activity of hematocrit (red blood cell generation), vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, increase blood platelet marrow.In another embodiment; recombinant tissue protectiveness cell factor of the present invention has cell protection activity; but do not have the one or more effects of erythropoietin(EPO), promptly increase the generation and the procoagulant activity of hematocrit (red blood cell generation), vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, increase blood platelet marrow.Preferred cytoprotective recombinant tissue protectiveness cell factor of the present invention lacks erythropoietin(EPO) at least one effect to marrow; More preferably recombinant tissue protectiveness cell factor lacks the erythropoiesis activity; Most preferably recombinant tissue protectiveness cell factor lacks erythropoietin(EPO) all effects to marrow.

As limiting examples, can change or lack or on it, add one or more amino acid to one or more amino acid of natural erythropoietin molecule.In a preferred embodiment, described recombinant tissue protectiveness cell factor has one or more modifications: VLQRY (the amino acid/11 1-15 of natural human erythropoietin(EPO) one or more with inferior segment; SEQ ID NO:1) and/or TKVNFYAW (the amino acid 44-51 of natural human erythropoietin(EPO); SEQ ID NO:2) and/or SGLRSLTTL (the amino acid/11 00-108 of natural human erythropoietin(EPO); SEQ ID NO:3) and/or SNFLRG (the amino acid/11 46-151 of natural human erythropoietin(EPO); SEQ ID NO:4).On the amino acid 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, also can provide other sudden change.These other sudden changes can be independent, perhaps also have these other sudden changes except at least one sudden change at least one above-mentioned district.In certain embodiments, at one or more amino acid whose variation (the amino acid 44-51 of natural human erythropoietin(EPO) of TKVNFYAW; SEQ ID NO:2) produces modification erythropoietin molecule with partial function (promptly having the erythropoiesis activity lower) than rhu-EPO.In other embodiments, SGLRSLTTL (the amino acid/11 00-108 of natural human erythropoietin(EPO); SEQ ID NO:3) one or more amino acid whose variation produces has the recombinant tissue protectiveness cell factor of partial function (promptly having the erythropoiesis activity lower than rhu-EPO).Above-mentioned recombinant tissue protectiveness cell factor shows organization protection's activity or cell protection activity.As for the erythropoiesis activity, it is active or show reduction in one or more erythropoiesis activity that above-mentioned recombinant tissue protectiveness cell factor lacks erythropoiesis.The example of erythropoiesis activity comprises the generation that increases hematocrit, vessel retraction, superactivation blood platelet, procoagulant activity and increase blood platelet.Can use the measured by standard techniques erythropoiesis activity of this area.For example, UT-7 raji cell assay Raji or the employing that can adopt 6.17 trifles to describe are described in Physicians ' Desk Reference (MedicalEconomics Company, Inc., Montvale, NJ, 2000.) the technical measurement hematocrit, described document all is attached to herein by reference.Specifically, the 519-525 page or leaf discloses with the 2125-2131 page or leaf and has been used to measure the method for hematocrit levels and discloses can be used as target to avoid the different hematocrit scopes of toxicity.For example, in suffering from the patient of chronic renal failure, PDR recommend to give erythropoietin(EPO) with the hematocrit scope from 30% to 36% that reaches nontoxic target in the patient (for example referring to PDR, the 523rd page, 1st, 11 hurdles, 17-96 and the 2129th page, the 1st, 11 hurdles, 11.8-93, and subordinate list in the 2nd hurdle and the 3rd hurdle).PDR notices, can avoid the toxicity of polycythemia (increasing to the illness of feature with the circulation RBC number unusually) form by the following method: carefully monitor hematocrit and adjust the dosage of EPO, reach at 4 when above if hematocrit value increases near the height of target zone limit (is 36% for this patient colony) or in any 2 time-of-weeks, stop erythropoietin(EPO) and return to the target zone of suggestion up to hematocrit value (for this patient colony is 30%-36%; Referring to PDR, the 523rd page, the 1st hurdle, and the 2129th page, the 1st hurdle, at " Dose Adjustment " down).By contrast, for the cancer patient of chemotherapy, the PDR suggestion is adjusted dosage in different hematocrit levels, if promptly hematocrit surpasses at 40% o'clock (referring to the 2129th page, the 2nd hurdle, at " Dose Adjustment " down).In one embodiment, described recombinant tissue protectiveness cell factor has one or more erythropoiesis activity, promptly surpasses the effect of treatment benefit of the cell protection activity of recombinant tissue protectiveness cell factor but its level is not enough to cause spinoff.In one embodiment, the recombinant tissue protectiveness cell factor with one or more erythropoiesis activity also can be used for method of the present invention, if the erythropoiesis activity level can be measured.Have in the embodiment of one or more erythropoiesis activity in recombinant tissue protectiveness cell factor; it is active and can regulate the dosage and/or the dosage regimen of described cell factor to measure described erythropoiesis, to guarantee described recombinant tissue protectiveness cell factor avirulence.Have in the embodiment of one or more erythropoiesis activity in recombinant tissue protectiveness cell factor, can measure described erythropoiesis active and dosage and/or dosage regimen that can regulate described cell factor and have hypotoxicity to guarantee described recombinant tissue protectiveness cell factor.In one embodiment; Epo compares with reorganization, and described recombinant tissue protectiveness cell factor shows the active reduction 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% of one or more erythropoiesis.

The invention provides and lack at least a recombinant tissue protectiveness cell factor that is selected from following activity: increase the generation of hematocrit, vessel retraction, superactivation blood platelet, procoagulant activity and increase blood platelet.Described cell factor comprises and at least aly is selected from protection, keeps, strengthens or recovers the function of mammal effector cell, tissue or organ or the effector cell of vigor protects activity.

In one embodiment of the invention, described recombinant tissue protectiveness cell factor is included in the amino acid residue of one or more changes of [SEQ ID NO:4] between the position 146-151 of [SEQ ID NO:3] between the position 100-108 of [SEQ ID NO:2], SEQ ID NO:10 between the position 44-51 of [SEQ ID NO:1], SEQ ID NO:10 between the position 11-15 of SEQ ID NO:10 or SEQ ID NO:10.

In another embodiment, the amino acid residue of described recombinant tissue protectiveness cell factor one or more changes with upper/lower positions of being included in SEQID NO:10: 7,20,21,29,33,38,42,59,63,67,70,83,96,126,142,143,152,153,155,156 or 161.

In another embodiment, described recombinant tissue protectiveness cell factor comprises the amino acid sequence (sequence of each change has all been specified an independently sequence identification number) of the SEQ ID NO:10 with one or more following variations: the alanine of SEQ ID NO: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); The isoleucine of SEQ ED NO:10 residue 10 (SEQ ID NO:18); The serine of SEQ ID NO:10 residue 11 (SEQ ID NO:19); The alanine of SEQ ID NO: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:22); The glutamic acid (SEQID NO:23) of SEQ ID NO:10 residue 14; The glutamine of SEQ ID NO:10 residue 14 (SEQ ID NO:24); The alanine of SEQID NO:10 residue 15 (SEQ ID NO:25); The phenylalanine of SEQ ID NO:10 residue 15 (SEQ ID NO:26); The isoleucine (SEQID NO:27) of SEQ ID NO:10 residue 15; The glutamic acid of SEQ ID NO:10 residue 20 (SEQ ID NO:28); The alanine of SEQ IDNO:10 residue 20 (SEQ ID NO:29); The alanine of SEQ ID NO:10 residue 21 (SEQ ID NO:30); The lysine (SEQ IDNO:31) of SEQ ID NO:10 residue 24; The serine of SEQ ID NO:10 residue 29 (SEQ ID NO:32); The tyrosine of SEQ IDNO:10 residue 29 (SEQ ID NO:33); The asparagine of SEQ ID NO:10 residue 30 (SEQ ID NO:34); The threonine (SEQ IDNO:35) of SEQ ID NO:10 residue 32; The serine of SEQ ID NO:10 residue 33 (SEQ ID NO:36); The tyrosine of SEQ IDNO:10 residue 33 (SEQ ID NO:37); SEQ ID NO:10 residue 38

Lysine (SEQ ID NO:38); The lysine (SEQ IDNO:39) of SEQ ID NO:10 residue 83; The asparagine of SEQ ID NO:10 residue 42 (SEQ ID NO:40); The alanine of SEQ IDNO:10 residue 42 (SEQ ID NO:41); The alanine of SEQ ID NO:10 residue 43 (SEQ BD NO:42); The isoleucine (SEQID NO:43) of SEQ ID NO:10 residue 44; The aspartic acid of SEQ ID NO:10 residue 45 (SEQ ID NO:44); The alanine of SEQID NO:10 residue 45 (SEQ ID NO:45); The alanine of SEQ ID NO:10 residue 46 (SEQ ID NO:46); The alanine (SEQ IDNO:47) of SEQ ID NO:10 residue 47; The isoleucine (SEQ ID NO:48) of SEQ ID NO:10 residue residue 48; The alanine of SEQ ID NO:10 residue 48 (SEQ ID NO:49); The alanine of SEQ ID NO:10 residue 49 (SEQ ID NO:50); The serine (SEQID NO:51) of SEQ ID NO:10 residue 49; The phenylalanine of SEQ ID NO:10 residue 51 (SEQ ID NO:52); The asparagine of SEQID NO:10 residue 51 (SEQ ID NO:53); The alanine of SEQ ID NO:10 residue 52 (SEQ ID NO:54); The asparagine (SEQID NO:55) of SEQ ID NO:10 residue 59; The threonine of SEQ ID NO:10 residue 62 (SEQ ID NO:56); The serine of SEQ IDNO:10 residue 67 (SEQ ID NO:57); The alanine of SEQ ID NO:10 residue 70 (SEQ ID NO:58); The arginine (SEQ IDNO:59) of SEQ ID NO:10 residue 96; The alanine of SEQ ID NO:10 residue 97 (SEQ ID NO:60); The arginine of SEQ IDNO:10 residue 100 (SEQ ID NO:61); The glutamic acid of SEQ ID NO:10 residue 100 (SEQ ID NO:62); The alanine (SEQ IDNO:63) of SEQ ID NO:10 residue 100; The threonine of SEQ ID NO:10 residue 100 (SEQ ID NO:64); The alanine of SEQ IDNO:10 residue 101 (SEQ ID NO:65); The isoleucine of SEQ ID NO:10 residue 101 (SEQ ID NO:66); The alanine (SEQID NO:67) of SEQ ID NO:10 residue 102; The alanine of SEQ ID NO:10 residue 103 (SEQ ID NO:68); The glutamic acid of SEQID NO:10 residue 103 (SEQ ID NO:69); The alanine of SEQ ID NO:10 residue 104 (SEQ ID NO:70); The isoleucine of SEQ ID NO:10 residue 104 (SEQ ID NO:71); The alanine of SEQ ID NO:10 residue 105 (SEQ ID NO:72); The alanine of SEQ ID NO:10 residue 106 (SEQ ID NO:73); The isoleucine of SEQ ID NO:10 residue 106 (SEQ ID NO:74); The alanine of SEQ ID NO:10 residue 107 (SEQ ID NO:75); The leucine of SEQ ID NO:10 residue 107 (SEQ ID NO:76); The lysine of SEQ ID NO:10 residue 108 (SEQ ID NO:77); The alanine of SEQ ID NO:10 residue 108 (SEQ ID NO:78); The serine of SEQ ID NO:10 residue 108 (SEQ ID NO:79); The alanine (SEQ IDNO:80) of SEQ ID NO:10 residue 116; The alanine of SEQ ID NO:10 residue 126 (SEQ ID NO:81); The alanine of SEQ IDNO:10 residue 132 (SEQ ID NO:82); The alanine of SEQ ID NO:10 residue 133 (SEQ ID NO:83); The alanine (SEQ IDNO:84) of SEQ ED NO:10 residue 134; The alanine of SEQ ID NO:10 residue 140 (SEQ ID NO:85); The isoleucine of SEQ IDNO:10 residue 142 (SEQ ID NO:86); The alanine of SEQ ID NO:10 residue 143 (SEQ ID NO:87); The alanine (SEQ IDNO:88) of SEQ ID NO:10 residue 146; The lysine of SEQ ID NO:10 residue 147 (SEQ ID NO:89); The alanine of SEQ IDNO:10 residue 147 (SEQ ID NO:90); The tyrosine of SEQ ID NO:10 residue 148 (SEQ ID NO:91); The alanine (SEQ IDNO:92) of SEQ ID NO:10 residue 148; The alanine of SEQ ID NO:10 residue 149 (SEQ ID NO:93); The alanine of SEQ IDNO:10 residue 150 (SEQ ID NO:94); The glutamic acid of SEQ ID NO:10 residue 150 (SEQ ID NO:95); The alanine (SEQ IDNO:96) of SEQ ID NO:10 residue 151; The alanine of SEQ ID NO:10 residue 152 (SEQ ID NO:97); The tryptophane of SEQ IDNO:10 residue 152 (SEQ ID NO:98); The alanine of SEQ ID NO:10 residue 153 (SEQ ID NO:99); The alanine (SEQ IDNO:100) of SEQ ID NO:10 residue 154; The alanine of SEQ ID NO:10 residue 155 (SEQ ID NO:101); The alanine of SEQ IDNO:10 residue 158 (SEQ ID NO:102); The serine of SEQ ID NO:10 residue 160 (SEQ ID NO:103); The alanine (SEQ IDNO:104) of SEQ ID NO:10 residue 161; Or the alanine of SEQ ID NO:10 residue 162 (SEQ ID NO:105).In one embodiment, described recombinant tissue protectiveness cell factor comprises the amino acid sequence of the SEQ ID NO:10 with one or more aminoacid replacement in SEQ ID NO:15-105 and 119.

In another embodiment, described recombinant tissue protectiveness cell factor is included in the amino acid sequence that has the SEQ ID NO:10 of disappearance on the SEQID NO:10 amino acid residue 44-49.

In a further embodiment, described recombinant tissue protectiveness cell factor comprises the amino acid sequence (sequence of each change has all been specified an independently sequence identification number) with at least one following SEQ ID NO:10 that changes: the i) glutamic acid of the aspartic acid of SEQ ID NO:10 residue 45 and residue 100 (SEQ ID NO:106); The ii) threonine of the asparagine of SEQ ID NO:10 residue 30, residue 32 (SEQ ID NO:107); The iii) glutamic acid of the aspartic acid of SEQ ID NO:10 residue 45, residue 150 (SEQ ID NO:108); The iv) serine of the glutamic acid of SEQ IDNO:10 residue 103 and residue 108 (SEQ ID NO:109); The v) alanine of the alanine of SEQ ID NO:10 residue 140 and residue 52 (SEQ ID NO:110); The vi) alanine of the alanine of the alanine of SEQ ID NO:10 residue 140, residue 52, residue 45 (SEQ ID NO:111); The vii) alanine of the alanine of SEQ ID NO:10 residue 97 and residue 152 (SEQ ID NO:112); Iix) alanine of the alanine of the alanine of SEQ ID NO:10 residue 97, residue 152, residue 45 (SEQ ID NO:113); Ix) alanine of the alanine of the alanine of the alanine of SEQ ID NO:10 residue 97, residue 152, residue 45 and residue 52 (SEQ ID NO:114); X) alanine of the alanine of the alanine of the alanine of the alanine of SEQ ID NO:10 residue 97, residue 152, residue 45, residue 52 and residue 140 (SEQ ID NO:115); Xi) alanine of the lysine of the lysine of the lysine of the lysine of the alanine of the alanine of the alanine of the alanine of the alanine of the alanine of SEQ ID NO:10 residue 97, residue 152, residue 45, residue 52, residue 140, residue 154, residue 24, residue 38, residue 83, residue 24 and residue 15 (SEQ ID NO:116); Xii) lysine of the lysine of the lysine of SEQ ID NO:10 residue 24, residue 38 and residue 83 (SEQ ID NO:117); Or xiv) alanine of the lysine of SEQ IDNO:10 residue 24 and residue 15 (SEQ ID NO:118).In one embodiment, described recombinant tissue protectiveness cell factor comprises the amino acid sequence with SEQID NO:10 that at least one following amino acid residue replaces among the SEQ ID NO:106-118.

One embodiment of the invention relate to above-mentioned recombinant tissue protectiveness cell factor, and described cell factor also comprises one or more amino acid whose chemical modifications.In another embodiment, described chemical modification comprises the electric charge that changes described recombinant tissue protectiveness cell factor.In another embodiment, positive charge or negative charge are added on the amino acid residue through chemical method, wherein charged amino acid residue is modified to uncharged residue.

In addition; above-mentioned recombinant tissue protectiveness cell factor like this can also further be modified; make it have one or more amino acid whose chemical modifications; for example be described in following copending application: the PCT application Ser. No PCT/US01/49479 of application on Dec 28 calendar year 2001; the U.S. Patent application serial number 09/753 of application on Dec 29th, 2000; 132; with the reel number KW00-009C02-US of U.S. Patent application agency of application on July 3rd, 2002, each in these applications all is attached to herein by reference.These further chemical modifications can be used for strengthening organization protection's activity of recombinant tissue protectiveness cell factor or suppress any effect of recombinant tissue protectiveness cell factor to marrow.In another embodiment, provide extra chemical modification to recover the solubleness of described molecule, and this solubleness may be lowered because of above-mentioned genetic modification, described genetic modification is such as adding positive charge or negative charge through chemical method on described molecule, if charged amino acid residue is modified to uncharged residue.

As limiting examples, recombinant tissue protectiveness cell factor of the present invention comprise erythropoietin mutain S100E (SEQ ID NO:5), erythropoietin mutain K45D (SEQ ID NO:6) and any non-erythropoietic, be still the erythropoietin(EPO) that cytoprotective recombinant tissue protectiveness cell factor maybe can be of value to effector cell, tissue or organ, they are described in Elliott etc., 1997, Blood 89:493-502; Boissel etc., Journal of Biological Chemistry, the 268th volume, the 21st phase, 15983-15993 page or leaf (1993); Wen etc., Journal of Biological Chemistry, the 269th volume, the 36th phase, 22839-22846 page or leaf (1994); With Syed etc., Nature, the 395th volume, 511-516 page or leaf (1998), described document all is attached to herein by reference.The present invention relates to any above-mentioned recombinant tissue protectiveness cell factor be used to protect, the using method of recovery and enhancement effect cell, tissue and organ.

Other recombinant tissue protectiveness cell factor of the present invention comprises the amino acid whose above-mentioned erythropoietin(EPO) that contains at least one hereditary change with at least one extra modification; another that described extra modification can be at least one additional amino acid of erythropoietin molecule modified, or the modification of at least one sugar of erythropoietin molecule.The amino acid of described hereditary change can be unique or these amino acid are further modified.Certainly; the recombinant tissue protectiveness cell factor molecule that is used for this paper purpose is compared with natural erythropoietin molecule; can have many modifications, modify and at least one modification partly of described molecular saccharides at least the second of a plurality of modifications of the sugar moieties of a plurality of modifications of the amino acid moiety of all molecules as described, described molecule or the amino acid moiety of described molecule.Described recombinant tissue protectiveness cell factor molecule keeps it and protects, keeps, strengthens or recover mammal effector cell's the function or the ability of vigor; but compare with natural molecule, recombinant tissue protectiveness cell factor and above-mentioned other irrelevant characteristic, the characteristic of wanting can not exist.In a preferred embodiment, described recombinant tissue protectiveness cell factor right and wrong are erythropoietic.

In another embodiment, described recombinant tissue protectiveness cell factor can be modified by fucosylation, to change the glycosylation pattern of glycoprotein.

The above-mentioned recombinant tissue protectiveness cell factor that one embodiment of the invention relate to is the erythropoietin mutain.In another embodiment of the invention, described recombinant tissue protectiveness cell factor is people's phenyl glyoxal erythropoietin(EPO) mutain.In another embodiment of the invention, described recombinant tissue protectiveness cell factor is that the people takes off sialic acid erythropoietin(EPO) mutain.

In one embodiment, aforesaid recombinant tissue protectiveness cell factor comprises the effector cell who at least aly is selected from protection, keeps, strengthens or recovers mammal effector cell, tissue or organ dysfunction or vigor and protects activity.In such embodiments, the mammal effector cell comprises neuronal cell, myocyte, core cell, pneumonocyte, liver cell, nephrocyte, small intestine cells, adrenal cortical cell, adrenal medullary cell, capillary cell, endothelial cell, testicular cell, gonad cell, endometrial cell or stem cell.In other embodiments, described cell comprises photosensory cell, gangliocyte, Beale's ganglion cells, horizontal cell, amakrine, muller cell, the cardiac muscle cell, pacemaker cells, sinus node cells, the hole nodal cell, the atrioventricular node cell, the atrioventircular bundle cell, liver cell, astrocyte, Kupffer cell, mesangial cell, goblet cell, the enteraden cell, enteroendocrine cell, messangial cell, the pencil cell, desmacyte, chromaffin cell, pericyte, interstitial cell, trophocyte, spermatid, the ripe follicle cell, original follicular cells, endometrial stromal cell or endometrial cell.

According to a further aspect in the invention, aforesaid recombinant tissue protectiveness cell factor can be passed through endothelial cell barrier.In a related embodiment, described endothelial cell barrier comprises blood brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier, blood-tire barrier, blood-heart barrier, blood-kidney barrier and blood-uterus barrier.

In another embodiment of the invention, above-mentioned recombinant tissue protectiveness cell factor is further modified.In one embodiment, described recombinant tissue protectiveness cell factor is selected from: i) have the sialic acid of decreased number or the cell factor of asialo part; Ii) have the N connection sugar or the O connection sugar of decreased number or do not have N connection sugar or the cell factor of O connection sugar; Iii) has the cell factor that reduces sugared content at least by handling the n cell factor with at least a glycosidase; The cell factor that iv) has at least one or a plurality of oxosugars; V) have at least one or a plurality of oxosugar and by the cell factor of electronation; The cell factor that vi) has at least one or a plurality of modification arginine residues; An amido modified cell factor of N end that vii) has at least one or a plurality of modification lysine residue or cell factor molecule; Viii) has the cell factor that at least one modifies tyrosine residue; Ix) has the cell factor that at least one modifies aspartic acid or glutaminic acid residue; X) has a cell factor of modifying trp residue; Xi) has a removed cell factor of amino acid group at least; Xii) cell factor that has at least one to open at intramolecular at least one cystine linkage of described cell factor; Xiii) cell factor of brachymemma; Xiv) connect the cell factor of at least one peg molecule; Xv) connect the cell factor of at least one fatty acid; Xvi) in the nonmammalian cell, express and have the cell factor of nonmammalian glycosylation pattern owing to recombinant cytokine; And xvi) has the amino acid of at least one histidine mark so that the cell factor of purifying.

In one embodiment, recombinant tissue protectiveness cell factor of the present invention has the sialic acid part of decreased number or does not have the sialic acid part.In a preferred embodiment, described recombinant tissue protectiveness cell factor is the sialic acid form of taking off (promptly not having the sialic acid part) of erythropoietin(EPO), and optimum is chosen and taken off the sialic acid erythropoietin(EPO).In another embodiment, described recombinant tissue protectiveness cell factor has 1,2,3,4,5,6,7,8,9,10,11,12 or 13 sialic acid parts.The number of sialylated available position can be changed by the one or more changes that exist in the recombinant tissue protectiveness cell factor or the amino acid of modification.Therefore, the present invention includes wherein said recombinant tissue protectiveness cell factor or by low sialylated (hyposialylated) or by the embodiment of high sialic acidization (hypersialylated).One preferred aspect, described erythropoietin(EPO) mutain has 14 the sialic acid parts that surpass that exist on the natural erythropoietin(EPO).

In one embodiment, described recombinant tissue protectiveness cell factor is the erythropoietin(EPO) that does not contain N connection sugar.In another embodiment, described recombinant tissue protectiveness cell factor is the erythropoietin(EPO) with N connection sugar of decreased number.In one embodiment, described recombinant tissue protectiveness cell factor is the erythropoietin(EPO) that does not contain O connection sugar.In another embodiment, described recombinant tissue protectiveness cell factor is the erythropoietin(EPO) with O connection sugar of decreased number.

In another embodiment, described recombinant tissue protectiveness cell factor can be modified by fucosylation, to change the glycosylation pattern on the glycoprotein.

In another embodiment, described recombinant tissue protectiveness cell factor is handled with at least a glycosidase.In another embodiment, described recombinant tissue protectiveness cell factor has the sugared content that reduces at least owing to handle recombinant tissue protectiveness cell factor with at least a glycosidase.

In another embodiment, the sugar moieties of described recombinant tissue protectiveness cell factor has nonmammalian glycosylation pattern at least because of recombinant erythropoietin express recombinant erythropoietin(EPO) in the nonmammalian cell.In preferred embodiments, described recombinant tissue protectiveness cell factor is expressed in insect cell, vegetable cell, bacterial cell or yeast cells.

In another embodiment, described recombinant tissue protectiveness cell factor also have at least one or a plurality of also can be by the oxosugar of electronation.In a preferred embodiment, described recombinant tissue protectiveness cell factor is the erythropoietin(EPO) of periodate oxidation.In certain embodiments, the erythropoietin(EPO) of periodate oxidation is preferably by the sodium cyanoborohydride electronation.

In another embodiment, the recombinant tissue protectiveness cell factor that is used for such use has at least one or a plurality of modification arginine residues.In one embodiment, described recombinant tissue protectiveness cell factor comprises R-glyoxal part on one or more arginine residues, and wherein R is aryl or moieties.In another embodiment, described recombinant tissue protectiveness cell factor is phenyl glyoxal-erythropoietin(EPO).In another embodiment, described recombinant tissue protectiveness cell factor be wherein arginine residues by with such as but not limited to 2, company's two reactive ketones of 3-diacetyl and cyclohexanedione and adorned erythropoietin(EPO).In another embodiment, described recombinant tissue protectiveness cell factor is the erythropoietin(EPO) of arginine residues and 3-deoxyglucosone reaction wherein.

In another embodiment; it is amido modified that described recombinant tissue protectiveness cell factor comprises a N end of at least one or a plurality of modification lysine residues or this erythropoietin molecule, described modification be by lysine residue or N end amino with amido modified reagent reacting.The lysine residue of modifying also can be by electronation.In a preferred embodiment, recombinant tissue protectiveness cell factor by one or more lysine groups by biotinylation or carbamylation or acidylate (for example acetylation).In another preferred embodiment, lysine and aldehyde or reducing sugar reaction, form imines, described imines by formed by sodium cyanoborohydride reduction N-alkylation lysine for example glucose alcohol radical lysine stablize, perhaps under the situation of reducing sugar, can reset formation α-deoxidation-alpha-amido sugar (for example α-deoxidation-α-fructosyl lysine) and stablize by Amadori or Heyns.In another preferred embodiment; the lysine group is for example by with cyanic acid ion reaction and by carbamylation; by respectively with alkyl-isocyanate, aryl-isocyanate or aryl isothiocyanic acid reactant salt by alkyl-carbamylation, aryl-carbamylation or aryl-thiocarbamoylization, perhaps can be for example by with acetic anhydride, succinic anhydride or phthalic anhydride by active alkyl carboxylic acid or arylcarboxylic acid derivative institute acidylate.At least one lysine group also can be by with trinitro-benzene-sulfonic acid or preferably and its reactant salt and being modified by trinitrophenyl.In another embodiment, lysine residue can form corresponding α-carboxyalkyl derivant by being modified with glyoxal derivative reaction (for example with glyoxal, methyl-glyoxal or the reaction of 3-deoxyglucosone).In a relevant embodiment, described carbamylation cell factor comprises α-N-carbamyl erythropoietin(EPO); N-ε-carbamyl erythropoietin(EPO); α-N-carbamyl, N-ε-carbamyl erythropoietin(EPO); α-N-carbamyl takes off the sialic acid erythropoietin(EPO); N-ε-carbamyl takes off the sialic acid erythropoietin(EPO); α-N-carbamyl, N-ε-carbamyl takes off the sialic acid erythropoietin(EPO); α-N-carbamyl, N-ε-carbamyl takes off the sialic acid erythropoietin(EPO); α-N-carbamyl hangs down the sialic acid erythropoietin(EPO); N-ε-carbamyl hangs down the sialic acid erythropoietin(EPO); And α-N-carbamyl, N-ε-carbamyl hangs down the sialic acid erythropoietin(EPO).In another embodiment, described recombinant tissue protectiveness cell factor comprises at least one acidylate lysine residue.In another embodiment, described recombinant tissue protectiveness cell factor comprises at least one acidylate lysine residue.In another embodiment, described recombinant tissue protectiveness cell factor comprises at least one acidylate lysine residue.In a relevant embodiment, described acetylation cell factor comprises α-N-acetyl erythropoietin(EPO); N-ε-acetyl erythropoietin(EPO); α-N-acetyl, N-ε-acetyl erythropoietin(EPO); α-N-acetyl takes off the sialic acid erythropoietin(EPO); N-ε-acetyl takes off the sialic acid erythropoietin(EPO); α-N-acetyl, N-ε-acetyl takes off the sialic acid erythropoietin(EPO); α-N-acetyl hangs down the sialic acid erythropoietin(EPO); N-ε-acetyl hangs down the sialic acid erythropoietin(EPO); α-N-acetyl, N-ε-acetyl hangs down the sialic acid erythropoietin(EPO); α-N-acetyl group high sialic acid erythropoietin(EPO); N-ε-acetyl high sialic acid erythropoietin(EPO); α-N-acetyl group and N-ε-acetyl high sialic acid erythropoietin(EPO).

In another embodiment, described recombinant tissue protectiveness cell factor has the lysine residue of a succinylation.In a relevant embodiment, described succinylation cell factor comprises α-N-succinyl erythropoietin(EPO); N-ε-succinyl erythropoietin(EPO); α-N-succinyl, N-ε-succinyl erythropoietin(EPO); α-N-succinyl takes off the sialic acid erythropoietin(EPO); N-ε-succinyl takes off the sialic acid erythropoietin(EPO); α-N-succinyl, N-ε-succinyl takes off the sialic acid erythropoietin(EPO); α-N-succinyl hangs down the sialic acid erythropoietin(EPO); N-ε-succinyl hangs down the sialic acid erythropoietin(EPO); α-N-succinyl, N-ε-succinyl hangs down the sialic acid erythropoietin(EPO); α-N-succinyl high sialic acid erythropoietin(EPO); N-ε-succinyl high sialic acid erythropoietin(EPO); With N-ε-succinyl high sialic acid erythropoietin(EPO).

In one embodiment, at least one tyrosine residue of recombinant tissue protectiveness cell factor can be modified by for example nitrated or iodate in the aromatic ring position by electrophilic reagent.In a relevant embodiment, above-mentioned recombinant tissue protectiveness cell factor comprises at least one by 2,4, the lysine residue that 6-trinitrobenzene sulfonate or its another kind of salt are modified.

In another embodiment, described recombinant tissue protectiveness cell factor comprises at least one by the tyrosine residue of nitrated and/or iodate.

In another embodiment, described recombinant tissue protectiveness cell factor comprise one with carbodiimide reaction after again with the asparagicacid residue and/or the glutaminic acid residue of amine reaction.In a relevant embodiment, described amine is glycine amide.

In one embodiment, at least one trp residue of recombinant tissue protectiveness cell factor is for example by reacting with positive bromine succinimide or positive chloro-succinimide and being modified.

In another embodiment, for example by with ninhydrin reaction after again by with hydroborate reaction reduction gained carbonyl, provide at least one erythropoietin(EPO) amino removed recombinant tissue protectiveness cell factor.

In another embodiment; by with the reaction of reductive agent such as for example dithiothreitol (DTT); then form again to stop disulfide bond, be provided at the recombinant tissue protectiveness cell factor that cystine linkage has at least one to open in the described molecule by sulfydryl and iodoacetamide, iodoacetic acid or the reaction of another kind of electrophilic reagent that will produce subsequently.

In another embodiment, reorganization tissue protective cell factor is carried out the limited chemical proteolysis (for example behind trp residue, cutting) of the specific residue of target.Gained recombinant tissue protectiveness cell factor fragment is included in herein.

As mentioned above, the recombinant tissue protectiveness cell factor that is used for this paper purpose can be chosen wantonly and has at least one above-mentioned chemical modification except the amino acid of hereditary change, modifies more than one incessantly but also can have.The sugar moieties at its molecule as an example has one to be modified and has the recombinant tissue protectiveness cell factor of a modification at its amino acid moiety, and recombinant tissue protectiveness cell factor is to be taken off the sialic acid erythropoietin(EPO) at its lysine residue by biotinylation, acidylate (such as acetylation) or carbamylation.Recombinant tissue protectiveness cell factor also can be modified by adding fatty acid chain.In another embodiment, recombinant tissue protectiveness cell factor by the PEGization modification, produces PEGization tissue protective cell factor by adding polyglycol (PEG).

The isolated nucleic acid molecule of the nucleotide sequence of the polypeptide that comprising encodes contains above-mentioned recombinant tissue protectiveness cell factor is provided according to an aspect of the present invention.In one embodiment, described isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:208 vector construction body nucleotide residue 5461-6041, the nucleotide sequence of SEQ ID NO:209 nucleotide residue 5461-6041, the nucleotide sequence of SEQ ID NO:210 nucleotide residue 5461-6041, the nucleotide sequence of SEQ ID NO:211 nucleotide residue 5461-6041 or the nucleotide sequence of SEQ IDNO:212 nucleotide residue 5461-6041.

In one embodiment of the invention; it (is cDNA that a kind of nucleotide sequence that comprises is provided; interleave or do not interleave the nucleotide sequence of introne) isolated nucleic acid molecule; the described nucleotide sequence coded polypeptide that comprises above-mentioned recombinant erythropoietin or be made up of above-mentioned recombinant erythropoietin, condition are the recombinant tissue protectiveness cell factor that described nucleic acid molecules is not encoded and comprised one or more following aminoacid replacement: 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 relevant embodiment; a kind of nucleotide sequence isolated nucleic acid molecule of polypeptide that coding contains above-mentioned recombinant tissue protectiveness cell factor that comprises is provided, and condition is that described nucleic acid molecules is not encoded and contained the recombinant tissue protectiveness cell factor of any following replacement combination: N24K/N38K/N83K or A30N/H32T.In one embodiment, the nucleotide sequence of coding recombinant tissue protectiveness cell factor is to use the preferred codon that is easy to optimum expression in particular host cell and synthetic.Described preferred codon can be that expression is optimized in one of vegetable cell, bacterial cell, yeast cells, mammalian cell, fungal cell or insect cell.

The present invention also provides the carrier that comprises described nucleic acid molecules.The present invention also provides the expression vector that comprises the regulatory region that described nucleic acid molecules and at least one and described nucleic acid molecules effectively be connected.In one embodiment, described carrier is the pCiNeo carrier.In another embodiment, the invention provides the cell that comprises described expression vector.In another embodiment, provide the genetically engineered cell that comprises described nucleic acid molecules.

In another embodiment, the present invention also comprises composition, and described composition comprises pharmaceutical composition, and described Pharmaceutical composition comprises one or more above-mentioned recombinant tissue protectiveness cell factors.

According to a further aspect in the invention, provide lack at least a Pharmaceutical composition that is selected from the above-mentioned recombinant tissue protectiveness cell factor of following erythropoiesis activity a kind of comprising: to increase the generation of hematocrit, vessel retraction, superactivation blood platelet, procoagulant activity and increase blood platelet.According to a further aspect in the invention; provide a kind of Pharmaceutical composition that comprises above-mentioned recombinant tissue protectiveness cell factor, but described cell factor has no lack of at least a following erythropoiesis activity that is selected from: increase the generation of hematocrit, vessel retraction, superactivation blood platelet, procoagulant activity and increase blood platelet.Described cell factor comprises at least a mammal effector cell, tissue or the organ dysfunction that is selected from protection, keeps, strengthens or recover or the effector cell of vigor protects activity.The recombinant tissue protectiveness cell factor of described Pharmaceutical composition can comprise the amino acid sequence (an independently sequence identification number has all been specified in the combination of each variation or listed variation) with SEQ ID NO:10 that variation promptly replaces below: the i) glutamic acid of the aspartic acid of SEQ ID NO:10 residue 45 and residue 100 (SEQ ID NO:106); The ii) threonine of the asparagine of SEQ ID NO:10 residue 30, residue 32 (SEQ ID NO:107); The iii) glutamic acid of the aspartic acid of SEQ ID NO:10 residue 45, residue 150 (SEQ ID NO:108); The iv) serine of the glutamic acid of SEQ ID NO:10 residue 103 and residue 108 (SEQ ID NO:109); The v) alanine of the alanine of SEQ ID NO:10 residue 140 and residue 52 (SEQ ID NO:110); The vi) alanine of the alanine of the alanine of SEQ ID NO:10 residue 140, residue 52, residue 45 (SEQ ID NO:111); The vii) alanine of the alanine of SEQ ID NO:10 residue 97 and residue 152 (SEQ IDNO:112); Iix) alanine of the alanine of the alanine of SEQ ID NO:10 residue 97, residue 152, residue 45 (SEQ ID NO:113); Ix) alanine of the alanine of the alanine of the alanine of SEQ ID NO:10 residue 97, residue 152, residue 45 and residue 52 (SEQ ID NO:114); X) alanine of the alanine of the alanine of the alanine of the alanine of SEQ ID NO:10 residue 97, residue 152, residue 45, residue 52 and residue 140 (SEQ IDNO:115); Xi) alanine of the lysine of the lysine of the lysine of the lysine of the alanine of the alanine of the alanine of the alanine of the alanine of the alanine of SEQ ID NO:10 residue 97, residue 152, residue 45, residue 52, residue 140, residue 154, residue 24, residue 38, residue 83, residue 24 and residue 15 (SEQ ID NO:116); Xii) lysine of the lysine of the lysine of SEQ IDNO:10 residue 24, residue 38 and residue 83 (SEQ IDNO:117); Or xiv) alanine of the lysine of SEQ ID NO:10 residue 24 and residue 15 (SEQ ID NO:118).

According to a further aspect in the invention, a kind of Pharmaceutical composition that is used to protect, keep, strengthen or recover mammal effector cell and relevant cell, tissue and organ dysfunction or vigor is provided, and described composition comprises the recombinant tissue protectiveness cell factor (an independently sequence identification number has all been specified in the combination of each variation or listed variation) that at least one following amino acid residue replaces that contains of treatment effective dose: the tryptophane (SEQID NO:98) of SEQ ID NO:10 residue 152; The alanine (SEQID NO:119) of the alanine of SEQ ID NO:10 residue 14 and residue 15; The alanine of SEQ ID NO:10 residue 6 (SEQ ID NO:15); The alanine of SEQ IDNO: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 (SEQ ID NO:41); The alanine of SEQ ID NO:10 residue 48 (SEQ ID NO:49); The alanine of SEQ ID NO:10 residue 49 (SEQ ID NO:50); The threonine of SEQ ID NO:10 residue 32 (SEQ ID NO:35); The alanine of SEQ ID NO:10 residue 133 (SEQ ID NO:83); The alanine of SEQ ID NO:10 residue 134 (SEQ ID NO:84); The alanine of SEQ ID NO:10 residue 147 (SEQ ID NO:90); The alanine of SEQ ID NO:10 residue 148 (SEQ ID NO:92); The alanine of SEQ ID NO: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 (SEQ ID NO:102); The alanine of SEQ ID NO:10 residue 161 (SEQ ID NO:104); Or the alanine (SEQID NO:105) of SEQ ID NO:10 residue 162.

In one embodiment, above-mentioned Pharmaceutical composition be mixed with in oral, the nose or parenteral use.In another embodiment, described Pharmaceutical composition is mixed with perfusion liquid.

In certain embodiments; the Pharmaceutical composition of the present invention that is used to protect, keep, strengthen or recovers mammal effector cell and relevant cell, tissue and organ dysfunction or vigor comprises the recombinant tissue protectiveness cell factor for the treatment of effective dose, and described cell factor comprises at least one replacement of natural human erythropoietin(EPO) amino acid sequence amino acid residue.

In other embodiments; the Pharmaceutical composition of the present invention that is used to protect, keep, strengthen or recovers mammal effector cell and relevant cell, tissue and organ dysfunction or vigor comprises the recombinant tissue protectiveness cell factor for the treatment of effective dose, and described cell factor comprises can lack one or more for example following erythropoiesis activity or the cell protection activity of effect: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet.

In other embodiments; the Pharmaceutical composition of the present invention that is used to protect, keep, strengthen or recovers mammal effector cell and relevant cell, tissue and organ dysfunction or vigor comprises the recombinant tissue protectiveness cell factor for the treatment of effective dose, and described cell factor comprises also can have one or more for example following erythropoiesis activity or the cell protection activity of effect: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet.

According to an aspect of the present invention; be provided for protecting, keep or strengthen the method for the vigor of isolated cells, tissue or organ in the mammalian body, described method comprises the Pharmaceutical composition that described cell, tissue or organ contact is comprised contain the recombinant tissue protectiveness cell factor that lacks at least a erythropoietin(EPO) that is selected from following erythropoiesis activity: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet.In certain embodiments, described protective effect does not influence marrow.

The present invention also is provided for protecting, keep or strengthens the method for the vigor of isolated cells, tissue or organ in the mammalian body, and described method comprises that making described cell, tissue or organ contact comprise containing lacks at least a Pharmaceutical composition that is selected from the recombinant tissue protectiveness cell factor of following erythropoiesis activity: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet.

The present invention also provides and lacks at least a above-mentioned recombinant tissue protectiveness cell factor that is selected from following erythropoiesis activity and be used for preventing and prevent the mammalian tissues damage and recover and the purposes of the Pharmaceutical composition of regeneration mammalian tissues and function of organization in preparation: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet.In one embodiment, described damage is to be caused by following: epilepsy, multiple sclerosis, apoplexy, low blood pressure, asystole, ischaemic, miocardial infarction, inflammation, the age cognitive decrease of being correlated with, radiation damage, cerebral palsy, neurodegenerative disease, alzheimer's disease (Alzheimer ' s disease), Parkinson's (Parkinson ' s disease), SNE (Leigh disease), the acquired immune deficiency syndrome (AIDS) dementia, failure of memory, amyotrophic lateral sclerosis, alcoholism, mood disorder, anxiety disorder, scatterbrained hyperactivity disorder, autism, infectiousness spongiform encephalopathy (Creutzfeld-Jakob disease), cerebral trauma or spinal cord injuries receptor, cerebral ischemia or ischemia of spinal cord, cardiopulmonary bypass, chronic heart failure, macular degeneration, diabetic neuropathy, diabetic retinopathy, glaucoma, treat retinal ischemic or retina wound.

According to a further aspect in the invention; be provided for promoting the method that the molecule in the mammalian body passes through the transcytosis of endothelial cell barrier, described method comprises that giving described mammal comprises and the composition that lacks the described molecule that at least a above-mentioned recombinant tissue protectiveness cell factor that is selected from following activity associates: increase hematocrit, rising blood pressure, superactivation blood platelet and increase the generation of blood platelet.In one embodiment, described association be unsettled covalent bond, stable covalent bond or with the non-covalent association of described binding site molecule point.According to a further aspect in the invention; be provided for promoting the method that the molecule in the mammalian body passes through the transcytosis of endothelial cell barrier, described method comprises that giving described mammal comprises and the composition with described molecule that the above-mentioned recombinant tissue protectiveness cell factor that is selected from following activity associates: increase hematocrit, rising blood pressure, superactivation blood platelet and increase the generation of blood platelet.In one embodiment, described association be unsettled covalent bond, stable covalent bond or with the non-covalent association of described binding site molecule point.In another embodiment, described endothelial cell barrier is selected from blood brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier, blood-heart barrier, blood-kidney barrier and blood-tire barrier.In another embodiment, described molecule is receptor stimulating agent or antagonist hormone, neurotrophic factor, antimicrobial agents, antiviral agent, radiopharmaceutical, antisense oligonucleotides, antibody, immunodepressant, dyestuff, label or anticarcinogen.

According to a further aspect in the invention; be provided for passing through by the transcytosis transport molecule composition of endothelial cell barrier, described composition comprises and lacks the described molecule that at least a above-mentioned recombinant tissue protectiveness cell factor that is selected from following erythropoiesis activity is associated: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet.According to a further aspect in the invention; be provided for passing through by the transcytosis transport molecule composition of endothelial cell barrier, described composition comprises and has the described molecule that at least a above-mentioned recombinant tissue protectiveness cell factor that is selected from following erythropoiesis activity is associated: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet.In one embodiment, described association be unsettled covalent bond, stable covalent bond or with the non-covalent association of described binding site molecule point.In another embodiment, described molecule is receptor stimulating agent or antagonist hormone, neurotrophic factor, antimicrobial agents, radiopharmaceutical, antisense oligonucleotides, antibody, immunodepressant, dyestuff, label or anticarcinogen.

The present invention also provides and lacks molecule that at least a above-mentioned recombinant tissue protectiveness cell factor that is selected from following erythropoiesis activity associates and be used for passing through purposes aspect the Pharmaceutical composition of endothelial cell barrier by the transcytosis transport molecule in preparation: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet.In one embodiment, described association be unsettled covalent bond, stable covalent bond or with the non-covalent association of described binding site molecule point.In another embodiment, described molecule is receptor stimulating agent or antagonist hormone, neurotrophic factor, antimicrobial agents, radiopharmaceutical, antisense oligonucleotides, antibody, immunodepressant, dyestuff or label or anticarcinogen.

Therefore; the present invention relates to have on natural erythropoietin(EPO) homologue the cytoprotection purposes of any recombinant tissue protectiveness cell factor of at least one amino acid change, wherein said recombinant tissue protectiveness cell factor has cell protection activity as described herein.Described cell protection activity includes but not limited to neuroprotective activity.The invention still further relates to any above-mentioned recombinant tissue protectiveness cell factor and comprise the illness of described effector cell, tissue or organ or the purposes in the disease in treatment effector cell, tissue or organ, particularly treatment.In such embodiment, described recombinant tissue protectiveness cell factor has at least a following erythropoiesis activity that is selected from: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet.Recombinant tissue protectiveness cell factor of the present invention preferably keeps the three-dimensional conformation of natural erythropoietin(EPO).Described recombinant tissue protectiveness cell factor can have or not have the erythropoiesis activity.

In one embodiment of the invention, described recombinant tissue protectiveness cell factor produces as the recombinant protein of the His mark (6xHis residue) with the fusion of N end.In certain embodiments, extra amino acid sequence can be used as the intervening sequence adding.In a specific embodiment, the recombinant tissue protectiveness cell factor mutain of histidine mark of the present invention includes but not limited to K45D-6xHis and S100E-6xHis.

In another aspect of the present invention, any above-mentioned recombinant tissue protectiveness cell factor all can be used for preparing and is used for ex vivo treatment cell, tissue and organ to reach protection, keep, strengthen or to recover the Pharmaceutical composition of mammal effector cell and relevant cell thereof, tissue and organ dysfunction or vigor purpose.Described ex vivo treatment is used for for example for the preservation of transplanting with cell, tissue or organ, no matter be autotransplatntation or heterograft.Described cell, tissue or organ can be immersed in the solution that comprises erythropoietin(EPO) mutain or recombinant tissue protectiveness cell factor; perhaps described perfusion liquid can be instilled in the described organ by vascular system or alternate manner, and between the vascular system integration period of acceptor or donor does not keep cell function at described cell, tissue or organ.Can before the organ results, give donor with perfusion liquid, and organ of gathering in the crops and acceptor.In addition, therefore when cell, tissue or organ separate with individual vascular system and must exsomatize when having a period of time, can use the such use of any recombinant tissue protectiveness cell factor, term separates the vascular system that is meant restriction or clamps down on cell, tissue, organ or body part, or lead to the vascular system of cell, tissue, organ or body part, for example can be in operation, especially in the cardiovascular shunt operation, carry out; The vascular system of shunting cell, tissue, organ or body part; In mammalian body, take out cell, tissue, organ or body part, these can before the heterograft or before the autotransplatntation or during carry out; Perhaps can in the traumatic amputation of cell, tissue, organ or body part, carry out.Therefore, this aspect of the present invention relates to the erythropoietin(EPO) mutain and carries out original position and ex vivo perfusion.Can exsomatize in cell, tissue or organ preservative fluid provides described recombinant tissue protectiveness cell factor.For either side, exposure can be undertaken by the mode of continous pouring, pulse perfusion, infusion, immersion, injection or cathterization.

Aspect another; the present invention relates to be used for protect, keep, strengthen or recover the method for the vigor of mammalian cell, tissue, organ or body part (comprising effector cell or tissue), wherein said cell, tissue, organ or body partly separate in mammalian body.Described method is included in the mammalian cell, tissue, organ or the body that make separation in a period of time at least and partly contacts a certain amount of erythropoietin(EPO) mutain or recombinant tissue protectiveness cell factor, protects, keeps, strengthens to reach effectively or recover above-mentioned vigor.In limiting examples, separate the vascular system that is meant restriction or clamps down on cell, tissue, organ or body part, or lead to the vascular system of cell, tissue, organ or body part, for example can be in operation, especially in the cardiovascular shunt operation, carry out; The vascular system of shunting cell, tissue, organ or body part; In mammalian body, take out cell, tissue, organ or body part, these can before the heterograft or before the autotransplatntation or during carry out; Perhaps can in the traumatic amputation of cell, tissue, organ or body part, carry out.Therefore, this aspect of the present invention relates to erythropoietin(EPO) mutain or recombinant tissue protectiveness cell factor and carries out original position and ex vivo perfusion.Can exsomatize in cell, tissue or organ preservative fluid provides described recombinant tissue protectiveness cell factor.For either side, exposure can be undertaken by the mode of continous pouring, pulse perfusion, infusion, immersion, injection or cathterization.

As limiting examples, above-mentioned stripped effector cell or tissue can be or can comprise neuronal cell, retina cell, myocyte, core cell, pneumonocyte, liver cell, nephrocyte, small intestine cells, adrenal cortical cell, adrenal medullary cell, capillary endothelial cell, testicular cell, gonad cell, pancreatic cell, osteocyte, bone marrow cell, Skin Cell, cord blood cell or endometrial cell or tissue.These examples of effector cell only are illustrative.

All said methods and purposes preferably are used for the mankind, but also can be used for any mammal, such as but not limited to companion animals, performing animal, domestic animal and zoo animal.That the method for administration of above-mentioned Pharmaceutical composition comprises is oral, in the intravenous, nose, in local, the tube chamber, suction or parenteral, the latter comprises in intravenous, intra-arterial, subcutaneous, intramuscular, the peritonaeum, mucous membrane down or intradermal administration.Use for exsomatizing, preferably perfusion liquid or soak solution.This comprises the separating part of situ perfusion vascular system.

Of the present invention aspect another; any above-mentioned recombinant tissue protectiveness cell factor is used to prepare the Pharmaceutical composition that has handicapped cell, tissue or organ dysfunction to use for recovering, and described composition gives after causing the outbreak of handicapped disease or illness.As limiting examples; formerly have in the animal of brain damage; the Pharmaceutical composition that comprises recombinant tissue protectiveness cell factor can recover cognitive function, even after initial wound has disappeared during (for example 1 day, 3 days, 5 days, 1 week, January or longer time) administration for a long time.The present invention includes and be used for the treatment of that (promptly alleviate or reverse described symptom or effect) and prevention (promptly postpone, suppress or stop) causing cascade reaction by initial wound and the Pharmaceutical composition of the follow-up infringement of pair cell and tissue.The recombinant tissue protectiveness cell factor that is used for described application comprises any concrete above-mentioned recombinant tissue protectiveness cell factor.Any form that can be of value to effector cell's recombinant tissue protectiveness cell factor all is included within this one side of the present invention.

In another embodiment, the invention provides the method that above-mentioned recombinant tissue protectiveness cell factor is used to recover to have handicapped cell, tissue or organ dysfunction, described method is to cause handicapped disease or illness outbreak back administration.As limiting examples; formerly have in the animal of brain damage; the Pharmaceutical composition that comprises recombinant tissue protectiveness cell factor can recover cognitive function, even after described wound has disappeared during (for example 3 days, 5 days, 1 week, January or longer time) administration for a long time.The described cell factor of recombinant tissue protectiveness cell factor and further modification thereof as mentioned above.Any form that can be of value to effector cell's recombinant tissue protectiveness cell factor all is included within this one side of the present invention.

In still another aspect of the invention; be provided for promoting the method that the molecule in the mammalian body passes through the transcytosis of endothelial cell barrier, i.e. the composition of the described molecule by giving the association of mammal and erythropoietin(EPO) mutain or above-mentioned recombinant tissue protectiveness cell factor.

Treat association between transport molecule and recombinant tissue protectiveness cell factor can be for example unsettled covalent bond, stable covalent bond or with the non-covalent association of described binding site molecule point.Recombinant tissue protectiveness cell factor and albumen to be transported can be expressed as fused polypeptide.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-tire barrier.Suitable molecule by the inventive method transhipment comprises hormone, microbiotic and anticarcinogens such as growth hormone.

Another aspect of the present invention is provided for promoting that the molecule in the mammalian body passes through the composition of the transcytosis of endothelial cell barrier, and described composition comprises the described molecule that associates with above-mentioned recombinant tissue protectiveness cell factor.

In one side more of the present invention; any above-mentioned recombinant tissue protectiveness cell factor is used to prepare the Pharmaceutical composition that the molecule that is used to promote in the mammalian body passes through the transcytosis of endothelial cell barrier, and described composition comprises the described molecule that associates with above-mentioned recombinant tissue protectiveness cell factor.

Described association can be for example unsettled covalent bond, fused polypeptide, stable covalent bond or with the non-covalent association of described binding site molecule point.Endothelial cell barrier can be blood brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier and blood-tire barrier.Suitable molecule by the inventive method transhipment comprises hormone such as growth hormone, neurotrophic factor, microbiotic, antiviral agent or such as usually by antibody, medical substance, dyestuff, label and the anticarcinogen of the excluded antifungal of the organ of brain and other barrier protection, peptide radiomimetic drug, antisense drug, antibiont active factors.

By will be better understood these aspects of the present invention and others with reference to following accompanying drawing and detailed description.

4. description of drawings

Fig. 1 is presented at anti-erythrocyte and generates in the slice of plain antibody staining, the distribution of erythropoietin receptor in normal brain.

Fig. 2 is the image of the more high-amplification-factor of Fig. 1.

Fig. 3 shows the second antibody of using golden mark, and the submicroscopic of erythropoietin receptor distributes.

Fig. 4 by the method preparation of similar Fig. 3, shows the high density erythropoietin receptor of human brain capillary luminal surface and reverse side.

Fig. 5 describes the transhipment of erythropoietin(EPO) in cerebrospinal fluid that stomach and intestine give outward.

Fig. 6 A and Fig. 6 B show the result who uses erythropoietin(EPO) and recombinant tissue protectiveness cell factor K45D and S100E the protection of SK-N-SH neuroblastoma cellular neural to be measured (at rotenone).Y-axis is represented the absorbance reading among the figure, and data are mean value ± replication scopes.Figure among Fig. 6 A clearly illustrates that at K45D and S100E cells in sample vigor and is held, and proves that they have the cytoprotection effect.Fig. 6 B shows the plasmid map of hEPO-6xHis mark-PciNeo.

Fig. 7 comparison erythropoietin(EPO) and take off the external effect of sialic acid erythropoietin(EPO) to the P19 cell viability of serum starvation.

Fig. 8 is the comparison erythropoietin(EPO) and takes off another experiment to the external effect of the P19 cell viability of serum starvation of sialic acid erythropoietin(EPO).

Fig. 9 shows erythropoietin(EPO) and takes off the protective effect of sialic acid erythropoietin(EPO) in the focal cerebral ischemia in rats model.

Figure 10 shows that comparison erythropoietin and people take off the dose response of the effect of sialic acid erythropoietin(EPO) in the middle cerebral artery occlusion of ischemic stroke model.

Figure 11 shows the activity of iodate erythropoietin(EPO) in P19 measures.

Figure 12 shows the biotinylation erythropoietin(EPO) and takes off the effect of sialic acid erythropoietin(EPO) in P19 measures.

Figure 13 compares the external effect of the erythropoietin(EPO) of erythropoietin(EPO) and the modification of phenyl glyoxal to the P19 cell viability of serum starvation.

Figure 14 is presented at the effect of tissue protective cell factor in the water intoxication mensuration.

Figure 15 shows that erythropoietin(EPO) is to being the maintenance of transplanting the cardiac function of preparing.

Erythropoietin(EPO) protection cardiac muscle cell prevented ischemic injury after Figure 16 showed temporary vascular occlusion.

Figure 17 A, Figure 17 B, Figure 17 C and Figure 17 D describe the result of treatment of erythropoietin(EPO) in rat glaucoma model.

Figure 18 shows erythropoietin(EPO) degree of protection to retinal function in rat glaucoma model.

Figure 19 describes the recovery that began to give the cognitive function of erythropoietin(EPO) to being caused by cerebral trauma after the cerebral trauma in 5 days.

Figure 20 describes the recovery that began to give the cognitive function of erythropoietin(EPO) to being caused by cerebral trauma after the cerebral trauma in 30 days.

Figure 21 describes the people and takes off the effect of sialic acid erythropoietin(EPO) in brain toxicity kainic acid model.

Figure 22 describes the effect of tissue protective cell factor in the Spinal Cord Injury in Rats model.

The effect of Figure 23 display organization protectiveness cell factor in the rabbit spinal cord injury model.

Figure 24 A, Figure 24 B and Figure 24 C show the cerebral cortex coronal section of h and E dyeing.

Figure 25 A, Figure 25 B and 25C show the coronal section with cortex before the contiguous infarct of GFAP antibody staining.

Figure 26 A and 26B show the corticocerebral coronal section with the OX-42 antibody staining.

Figure 27 A and 27B show the cerebral cortex coronal section with the contiguous infarct of OX-42 antibody staining.

Figure 28 shows the antiphlogistic effects of erythropoietin(EPO) in the EAE model.

Figure 29 has compared dexamethasone and the antiphlogistic effects of erythropoietin(EPO) in the EAE model.

Figure 30 A shows that with Figure 30 B erythropoietin(EPO) suppresses the inflammation relevant with neuronal death.

Figure 31 is presented at MNDA handle before, erythropoietin and recombinant tissue protectiveness cell factor R130E and R150E are joined in former generation Hippocampal Neuron Cells culture, effectively reduce the cell death that causes by NMDA.Compare with the solvent control cells, show the cell death (p=0.01) of remarkable minimizing with the cell of R103E (5nM) processing.Compare with the solvent control cells, show the cell death (p=0.01) of remarkable minimizing with the cell of R103E (5nM) processing.Compare with the solvent control cell, the cell of handling with R150E (5nM) shows about 20% minimizing (p=0.001) in cell death.Statistics: ANOVA adds that TukeyShi checks afterwards.

Figure 32 shows and removes the neuro-protective in the P19 cell behind the serum.For carrying out pretreated cell with Epo, EpoWT and recombinant tissue protectiveness cell factor S100E, the percent of apoptotic cell death descends.Compare with untreated control cells, the cell of handling with Epo shows about 20% decline on apoptotic cell death.Compare with untreated control cells, the cell of handling with EpoWT and S100E all shows about 10% decline on apoptotic cell death.

Figure 33 A and Figure 33 B are presented at two independently in the experiment, in removing the differentiation PC12 cell of NGF, and the effect of carrying out pre-incubation with S100E.The S100E pre-service of the PC12 cell usefulness prescribed concentration of differentiation 24 hours, Figure 33 A (3pM), Figure 33 B (0.00003pM-3pM).Mensuration vigor in MTT measures.NGF (100ng/ml) is used as positive control, and does not have the NGF nutrient culture media (NGF) as negative control.Data shown in Figure 33 be positive control (+NGF) and the % of vigor (in these two experiments, n=8).Adopt unidirectional ANOVA and Bonferroni to check, (NGF) compare, the vigor of handling cell through S100E has significance,statistical to increase with negative control cell afterwards. ^***p＜0.001， ^*p＜0.05。For rendeing a service and effect, the observed effect of usefulness S100E is similar with usefulness Epo's in such experimental system.

Figure 34 A, Figure 34 B and Figure 34 C are presented in the differentiation PC12 cell of removing NGF, with the effect of Epo pre-incubation.PC12 cell usefulness Epo, the S100E of differentiation or carbamylation Epo (30pM-30nM) pre-service 24 hours.The Epo molecule AA24496 of chemical modification hangs down 10000 times than EPO is active in the UT-7 raji cell assay Raji.Mensuration vigor in MIT measures.NGF (100ng/ml) (NGF) is not used as negative control as positive control and there is the NGF nutrient culture media.

Figure 35 shows Epo, K45D and the concentration-response curve of S100E in the UT-7 cell.Epo, EpoWT, K45D and the S100E of variable concentrations are joined in the UT-7 cell.In WST-1 measures, mensuration vigor after 48 hours.Data are the mean value ± SD that at every turn all carry out twice replication in three different experiments.This curve is the non-linear regression curve fitting.

Figure 36 shows Epo, R103E and the dose-effect curve of R150E in the UT-7 cell.Epo, EpoWT, R103E and the R150E of variable concentrations are joined in the UT-7 cell.In WST-1 measures, mensuration vigor after 48 hours.Data are the mean value ± SD that at every turn all carry out twice replication in three different experiments.This curve is the non-linear regression curve fitting.

Figure 37 is a curve map, proves the sport rank of 42 days time of experience from the rat of spinal cord injury recovery.As seen from the figure, easier also the proof from injury recovery of rat that gives S100E compared according to mouse and the mouse that gives methylprednisolone recovery comprehensively preferably.

Figure 38 shows for different therapeutic schemes, the ratio in the latent period of the latent period of injured eye and normal eye.Showing latent period with the rat of EPO treatment is 1.2, more quite a lot of than the rat with saline treatment.To every kind of this 4 kinds of recombinant tissue protectiveness cell factors, produce the preclinical EPO that is equal to or better than with R103E, R150E and S100E, illustrate better than EPO statistically.

5. embodiment

The present invention relates to mutain recombinant tissue protectiveness cell factor.Specifically, the invention provides comprising the coding contain the isolated nucleic acid molecule of recombinant tissue protectiveness cell factor mutain and comprise the separation of described nucleic acid molecules and/or the composition of recombinant cell and carrier.The present invention also comprises the polypeptide of the separation that lacks at least a mutain recombinant tissue protectiveness cell factor that is selected from following erythropoiesis activity: increase the generation of hematocrit, vasoactive effect (vessel retraction/vasodilation), superactivation blood platelet, procoagulant activity and increase blood platelet, described cell factor has the effector cell who at least aly is selected from protection, keeps, strengthens or recovers mammal effector cell, tissue or organ dysfunction or vigor and protects activity.The present invention also comprises the method for using recombinant tissue protectiveness cell factor mutain of the present invention to protect, keep or strengthen the vigor of isolated cells, tissue or organ in the mammalian body, and the purposes of described mutain in disease and treatment of conditions and prevention.

" effector cell " is meant the mammalian cell that its function or vigor can be kept, promote, strengthen, regenerate or otherwise be benefited by the contact erythropoietin(EPO).The limiting examples of described cell comprises neuronal cell, retina cell, myocyte, core cell, pneumonocyte, liver cell, nephrocyte, small intestine cells, adrenal cortical cell, adrenal medullary cell, capillary endothelial cell, testicular cell, gonad cell, pancreatic cell, osteocyte, Skin Cell and endometrial cell.Specifically, the effector cell will include but not limited to neuronal cell; Purkinje cell; Retina cell: photosensory cell (rod cell and awl cell), gangliocyte, Beale's ganglion cells, horizontal cell, amakrine and muller cell; The myocyte; Core cell: cardiac muscle cell, pacemaker cells, sinus node cells, hole nodal cell and conjunctive tissue cell (atrioventricular node and atrioventircular bundle); Pneumonocyte; Liver cell: liver cell, astrocyte and Kupffer cell; Nephrocyte: mesangial cell, renal epithelial cell and tubulose enterocyte; Small intestine cells: goblet cell, enteraden cell (cryts) and enteroendocrine cell; Adrenal cortical cell: messangial cell, pencil cell and desmacyte; Adrenal medullary cell: chromaffin cell; Capillary cell: pericyte; Testicular cell: interstitial cell, trophocyte and spermatid and initial cell thereof; Gonad cell: ripe follicle cell and original follicular cells; Pancreatic cell: pancreas islet, A cells, beta cell, γ-cell and F-cell; Osteocyte: osteogenic cell, osteoclast and Gegenbaur's cell; Skin Cell; Endometrial cell: endometrial stromal cell and endometrial cell; And be present in stem cell and endothelial cell in the organ listed above.In addition, described effector cell and by the benefit that recombinant tissue protectiveness cell factor provides can expand to for and other cell of indirect effect or the tissue or the organ that contain described non-effector cell indirect protection or humidification is provided.These other cell or tissue or organs that benefit indirectly from the effector cell's that exists as the part cell, tissue or the organ humidification are " being correlated with " cell, tissue and organ.Therefore; existence effector cell a small amount of or small scale can provide the benefit of recombinant tissue protectiveness cell factor as herein described in tissue or the organ; described effector cell for example is present in excitable tissue or the neuronal tissue in the described tissue, or the interstitial cell in the testis of generation testosterone.On the one hand, described effector cell or its relevant cell, tissue or organ be neither excitable cell, tissue or organ, dominant excitable cell or the tissue of also not comprising.

Method of the present invention is provided under the various normal and adverse condition part or the whole body protection or the humidification of the cell in the mammalian body, tissue and organ, and the protective effect of transplanting (relocation) to interior cell, tissue or organ of another mammalian body to predetermined perhaps is provided.In addition, also provide handicapped recovery or regeneration.As mentioned above; the ability that erythropoietin(EPO) mutain or recombinant tissue protectiveness cell factor are passed through endothelial cell barrier closely and the effector cell's (and cell of other type) who leaves vascular system is played a positive role; the potentiality (otherwise described illness and disease cause tangible primary cellular defect and tissue damage in comprising people's animal) that prevention are provided and treated various illnesss and disease, and make up to now without attempt, think that risk succeeds greater than the operation of interests traditionally.The duration and the degree of the autotelic adverse condition that causes for final benefit, for example the ischaemic cycle is induced in the operation of the stripped transplanting survival phase of high dose chemotherapy and radiotherapy, prolongation and prolongation, can be overcome by advantage of the present invention.Yet the present invention is not limited to this, as comprising that also its effector cell that hits leaves the method or the composition of vascular system because endothelial cell barrier closely is connected with endothelium on the one hand.The present invention relates generally to can be from any effector cell and relevant cell, tissue and the organ that benefits by contact recombinant tissue protectiveness cell factor.In addition, cell, tissue or organ dysfunction can recover by contact recombinant tissue protectiveness cell factor in acute adverse events (for example wound) back or regenerate.

Therefore, the present invention relates generally to recombinant tissue protectiveness cell factor and is used for the purposes of the Pharmaceutical composition of above-mentioned purpose in preparation, and wherein cell function is kept, promotes, strengthens, regenerated or benefits by any way.The present invention also relates to by giving the mammal effective dose recombinant tissue protectiveness cell factor as described herein with keep, strengthen, the method for promotion or regenerative cell's function.The invention still further relates to by making cell, tissue or organ contact recombinant tissue protectiveness cell factor to keep, promote, to strengthen or the method for the isolated cells function of regenerating.The present invention also relates to comprise recombinant tissue protectiveness cell factor, be used for the perfusion composition that organ or tissue preserves.

The whole bag of tricks of the present invention uses a kind of Pharmaceutical composition; described composition comprises the recombinant tissue protectiveness cell factor of the effective dose that is used for concrete route of exposure and duration at least, makes to bringing into play positive effect or benefit in the mammalian body or from the effector cell in the mammalian body.When target cell, tissue or the organ of expection treatment needed recombinant tissue protectiveness cell factor to pass through endothelial cell barrier, described Pharmaceutical composition comprised that its concentration can the pairing effect cell after passing through endothelial cell barrier brings into play the recombinant tissue protectiveness cell factor of its required effect.The molecule that can interact and regulate cell protection activity in this article with erythropoietin receptor in described cell can be used for the present invention.

5.1. nucleic acid of the present invention

The recombinant tissue protectiveness cell factor that comprises nucleic acid molecules of the present invention comprises that coding comprises and lacks that at least a to be selected from following erythropoiesis active or show the nucleic acid of tissue protective cell factor of erythropoietin(EPO) mutain of the described activity of reduction: increase hematocrit; vasoactive effect (vessel retraction/vasodilation); the superactivation blood platelet; the generation of procoagulant activity and increase blood platelet, described cell factor has at least a protection that is selected from; keep; strengthen or recovery mammal effector cell; the effector cell of tissue or organ dysfunction or vigor protects activity.The tissue protective cell factor that comprises nucleic acid molecules of the present invention comprises that coding has the nucleic acid of the erythropoietin(EPO) mutain of above-mentioned activity, and described nucleic acid is included in the amino acid residue of one or more changes of [SEQ ID NO:4] between the position 146-151 of [SEQ ID NO:3] between the position 100-108 of [SEQ ID NO:2], SEQ ID NO between the position 44-51 of [SEQ ID NO:1], SEQ ID NO:10 between the position 11-15 of SEQ ID NO:10 or SEQ ID NO 10.The tissue protective cell factor that comprises nucleic acid molecules of the present invention comprises that coding has the nucleic acid of the erythropoietin(EPO) mutain of above-mentioned activity, and the one or more positions of described nucleic acid in following SEQ ID NO:10 comprise the amino acid residue of change: 7,20,21,29,33,38,42,59,63,67,70,83,96,126,142,143,152,153,155,156 or 161.The tissue protective cell factor that comprises nucleic acid molecules of the present invention comprises that coding has the nucleic acid of the erythropoietin(EPO) mutain of above-mentioned activity, and described nucleic acid comprises the amino acid sequence with one or more following SEQ ID NO:10 that change: the alanine of SEQ ID NO:10 residue 6; the alanine of SEQ ID NO:10 residue 7; the serine of SEQ ID NO:10 residue 7; the isoleucine of SEQ ID NO:10 residue 10; the serine of SEQ ID NO:10 residue 11; the alanine of SEQ ID NO:10 residue 12; the alanine of SEQ ID NO:10 residue 13; the alanine of SEQ ID NO:10 residue 14; the glutamic acid of SEQ ID NO:10 residue 14; the glutamine of SEQ ID NO:10 residue 14; the alanine of SEQ ID NO:10 residue 15; the phenylalanine of SEQ ID NO:10 residue 15; the isoleucine of SEQ ID NO:10 residue 15; the glutamic acid of SEQ ID NO:10 residue 20; the alanine of SEQ ID NO:10 residue 20; the alanine of SEQ ID NO:10 residue 21; the lysine of SEQ ID NO:10 residue 24; the serine of SEQ ID NO:10 residue 29; the tyrosine of SEQ ID NO:10 residue 29; the asparagine of SEQ ID NO:10 residue 30; 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; the lysine of SEQ ID NO:10 residue 83; the asparagine of SEQ ID NO:10 residue 42; the alanine of SEQ ID NO:10 residue 42; the alanine of SEQ ID NO:10 residue 43; the isoleucine of SEQ ID NO:10 residue 44; the aspartic acid of SEQ ID NO:10 residue 45; the alanine of SEQ ID NO:10 residue 45; the alanine of SEQ ID NO:10 residue 46; the alanine of SEQ ID NO:10 residue 47; the isoleucine of SEQ ID NO:10 residue residue 48; the alanine of SEQ ID NO:10 residue 48; the alanine of SEQ IDNO:10 residue 49; the serine of SEQ ID NO:10 residue 49; the phenylalanine of SEQ IDNO:10 residue 51; the asparagine of SEQ ID NO:10 residue 51; the alanine of SEQID NO:10 residue 52; the asparagine of SEQ ID NO:10 residue 59; the threonine of SEQID NO:10 residue 62; the serine of SEQ ID NO:10 residue 67; the alanine of SEQ ID NO:10 residue 70; the arginine of SEQ ID NO:10 residue 96; the alanine of SEQID NO:10 residue 97; the arginine of SEQ ID NO:10 residue 100; the glutamic acid of SEQID NO:10 residue 100; the alanine of SEQ ID NO:10 residue 100; the threonine of SEQID NO:10 residue 100; the alanine of SEQ ID NO:10 residue 101; the isoleucine of SEQID NO:10 residue 101; the alanine of SEQ ID NO:10 residue 102; the alanine of SEQ ID NO:10 residue 103; the glutamic acid of SEQ ID NO:10 residue 103; the alanine of SEQ ID NO:10 residue 104; the isoleucine of SEQ ID NO:10 residue 104; the alanine of SEQ ID NO:10 residue 105; the alanine of SEQ ID NO:10 residue 106; the isoleucine of SEQ ID NO:10 residue 106; the alanine of SEQ ID NO:10 residue 107; the leucine of SEQ ID NO:10 residue 107; the lysine of SEQ ID NO:10 residue 108; the alanine of SEQ ID NO:10 residue 108; the serine of SEQ ID NO:10 residue 108; the alanine of SEQ ID NO:10 residue 116; the alanine of SEQ ID NO:10 residue 126; the alanine of SEQ ID NO:10 residue 132; the alanine of SEQ ID NO:10 residue 133; the alanine of SEQ ED NO:10 residue 134; the alanine of SEQ ID NO:10 residue 140; the isoleucine of SEQ ID NO:10 residue 142; the alanine of SEQ IDNO:10 residue 143; the alanine of SEQ ID NO:10 residue 146; the lysine of SEQ IDNO:10 residue 147; the alanine of SEQ ID NO:10 residue 147; the tyrosine of SEQ IDNO:10 residue 148; the alanine of SEQ ID NO:10 residue 148; the alanine of SEQ IDNO:10 residue 149; the alanine of SEQ ID NO:10 residue 150; the glutamic acid of SEQ IDNO:10 residue 150; the alanine of SEQ ID NO:10 residue 151; the alanine of SEQ IDNO:10 residue 152; the tryptophane of SEQ ID NO:10 residue 152; the alanine of SEQ IDNO:10 residue 153; the alanine of SEQ ID NO:10 residue 154; the alanine of SEQ IDNO:10 residue 155; the alanine of SEQ ID NO:10 residue 158; the serine of SEQ IDNO:10 residue 160; the alanine of SEQ ID NO:10 residue 161 or the alanine of SEQID NO:10 residue 162.

Nucleic acid molecules of the present invention comprises that also coding has with one of above-mentioned erythropoietin(EPO) mutain and has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or the nucleotide sequence of the recombinant erythropoietin mutain of homoamino acid sequence homogeneity more.In order to measure two amino acid sequences or the % homogeneity of two nucleic acid of the erythropoietin(EPO) mutain of encoding, for the best comparison purpose is carried out sequence alignment (for example in order to carry out the best comparison with second amino acid sequence or nucleotide sequence, can introduce the room in the sequence of first amino acid sequence or nucleotide sequence).The amino acid residue or the nucleotide of more corresponding then amino acid position or nucleotide position.When position of first sequence by with the second sequence relevant position on identical amino acid residue or nucleotide when occupying, then this molecule is identical in this position.% homogeneity between two sequences is the function (being % homogeneity=identical lap position number/lap position sum x100%) that described sequence is shared the same position number.In one embodiment, these two sequence lengths equate.

Nucleic acid molecules of the present invention also comprises the nucleotide sequence of coding recombinant erythropoietin mutain, and wherein being comprised with SEQ ID NO:7 by the nucleotide sequence of the coding erythropoietin(EPO) of one or more replacements, disappearance or above-mentioned modification change has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence homogeneity.Nucleic acid molecules of the present invention also comprises the nucleotide sequence of coding recombinant erythropoietin mutain, is the nucleic acid of coding non-human erythropoietin(EPO) by the nucleotide sequence of the coding erythropoietin(EPO) of one or more replacements, disappearance or above-mentioned modification change wherein.

Also can use mathematical algorithm to finish the mensuration of % homogeneity between two sequences.A preferred limiting examples that is used for the mathematical algorithm of two sequences of comparison is Karlin and Altschul, 1990, Proc.Natl.Acad.Sci.USA 87:2264-2268 algorithm and by Karlin and Altschul, 1993, Proc.Natl.Acad.Sci.USA 90:5873-5877 revises.Described algorithm is attached to Altschul etc., and 1990, in NBLAST that describes among the J.Mol.Biol.215:403-410 and the XBLAST program.Can carry out the BLAST nucleotide search with the NBLAST program, score value=100, word length=12 are to obtain the nucleotide sequence with nucleic acid molecules homology of the present invention.Can carry out BLAST albumen search with the XBLAST program, score value=50, word length=3 are to obtain the amino acid sequence with protein molecular homology of the present invention.For relatively purpose obtains introducing the comparison in room, can be as Altschul etc., 1997, NucleicAcids Res.25:3389-3402 is described, adopts Gapped BLAST.Perhaps, PSI-Blast can be used for carrying out iterative search, to measure intermolecular distance relation (Altschul etc., 1997, referring to above).When using BLAST, Gapped BLAST and PSI-Blast program, can use the default parameters (referring to http://www.nobi.nlm.nih.gov) of each program (for example XBLAST and NBLAST).Another limiting examples that is used for the preferred mathematical algorithm of sequence comparison is Myers and Miller, 1988, and the algorithm of CABIOS 4:11-17.Described algorithm is attached in the ALIGN program (2.0 editions), and described program is the part of GCG sequence alignment software package.When using the ALIGN program to carry out the amino acid sequence comparison, can use PAM120 weight residue table (weight residue table), room length point penalty is 12, gap penalty is 4.

Can use above-mentioned technology similar techniques, allow or do not allow the room, measure the % homogeneity of two sequences.When calculating % homogeneity, only count accurately coupling usually.

Nucleic acid molecules of the present invention also comprises: (a) under stringency with the nucleotide sequence of the making nucleic acid molecular hybridization of coding erythropoietin(EPO) mutain of the invention described above or recombinant tissue protectiveness cell factor, for example in conjunction with the DNA of filter membrane in 6x sodium chloride/sodium citrate (SSC) in about 45 ℃ of hybridization, then in 0.2xSSC/0.1%SDS, wash one or many at about 50-65 ℃, or (b) under high stringency, for example in conjunction with the nucleic acid of filter membrane in 6xSSC in 45 ℃ of hybridization, then in 0.1xSSC/0.2%SDS, wash one or many at about 68 ℃, perhaps under other hybridization conditions that it will be apparent to those skilled in the art (referring to chief editors such as for example Ausubel F.M., 1989, Current Protocols in Molecular Biology, the I volume, Green Publishing Associates, Inc. and John Wiley﹠amp; Sons, Inc., NewYork, 6.3.1-6.3.6 page or leaf and 2.10.3 page or leaf).The nucleic acid molecules that is preferably in the coding erythropoietin(EPO) mutain of hybridizing under (a) and condition (b) as mentioned above is a kind of complementary nucleic acid molecule that comprises the nucleic acid molecules of coding erythropoietin(EPO) mutain.In a preferred embodiment, at above (a) and the nucleic acid molecule encoding protein product of (b) hybridizing under the condition, for example be equal to the protein product that promptly has one or more above-mentioned erythropoietin activities with erythropoietin(EPO) sudden change egg on the function.Nucleic acid of the present invention is human nucleic acid preferably.

Nucleic acid molecules of the present invention also comprises the above-mentioned nucleotide sequence with above-mentioned erythropoietin(EPO) mutain or the hybridization of recombinant tissue protectiveness cell factor; and above-mentioned erythropoietin(EPO) mutain or recombinant tissue protectiveness cell factor also lack, and at least a to be selected from following erythropoiesis active or show the described activity of reduction: increase hematocrit; vasoactive effect (vessel retraction/vasodilation); the superactivation blood platelet; the generation of procoagulant activity and increase blood platelet, described cell factor or mutain comprise at least a protection that is selected from; keep; strengthen or recovery mammal effector cell; the effector cell of tissue or organ dysfunction or vigor protects activity.Described reduction can be to reduce a little or almost lack a kind of erythropoiesis activity.Described minimizing can be by measured by standard techniques known in the art (Gruber etc., 2002, J.Biol Chem.277 (81): 27581-27584; Page etc., 1996, Cytokine 8 (1): 66-69; Park etc., 1997, Mol.Cells 7 (6): 699-704; Wolf etc., 1997, ThrombHaemost 78:1505-1509; With Dale etc., 2002, Nature 415:175-179.The UT-7 raji cell assay Raji is described in 6.17 trifles, is a limiting examples measuring the active technology of erythropoiesis that reduces or lower.

Nucleic acid molecules of the present invention also comprises the complementary nucleic acid of above-mentioned nucleic acid.

Erythropoietin(EPO) mutain nucleic acid molecule fragment is meant above-mentioned erythropoietin(EPO) mutain nucleotide 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 a plurality of continuous nucleotide.Perhaps, described fragment can comprise the sequence of at least 10,20,30,40,50,60,70,80 of coding erythropoietin(EPO) mutains or more a plurality of continuous amino acid residues.In one embodiment, described erythropoietin(EPO) mutain nucleic acid molecule encoding shows the gene outcome of at least a corresponding biologic activity of erythropoietin(EPO) mutain.Erythropoietin(EPO) mutain nucleic acid molecule fragment also can refer to the to encode erythropoietin(EPO) mutain code area part of erythropoietin(EPO) mutain domain or encoding mature erythropoietin(EPO) mutain.

Derive from other biological erythropoietin(EPO) and can be used for producing erythropoietin(EPO) mutain of the present invention.As for erythropoietin(EPO) mutain or recombinant tissue protectiveness cell factor nucleic acid with from the homologue of other species with directly to the clone of the variant of homologue, the erythropoietin(EPO) nucleotide sequence of separation disclosed herein openly can add mark and be used to screen the cDNA library that makes up from the mRNA that derives from the suitable cell or tissue that comes from target organism.When the biochron that the cDNA library is derived from and flag sequence source is dissimilar, used hybridization conditions is low severity normally, and can carry out conventional determining to the cDNA library according to for example target with reference to biological relativeness.

Perhaps, re-use suitable stringency, described labeled fragment also can be used for screening the genomic library that is derived from target organism.Suitable stringency is that those skilled in the art are well-known as mentioned above, and expection will change according to the particular organisms that library and flag sequence are originated.The guide of relevant described condition is referring to for example Sambrook etc., 1989, Molecular Cloning, A Laboratory Manual, the 2nd edition, Cold Spring HarborPress, N.Y.; With Ausubel etc., 1989-1999, Current Protocols in MolecularBiology, Green Publishing Associates and Wiley Interscience, N.Y, these two documents all are attached to herein by reference.

In a preferred embodiment; in order to prepare recombinant tissue protectiveness cell factor; can pass through PCR (PCR) amplification; use is from the primer of the known array design of the recombinant tissue protectiveness cell factor of relevant or homology, from genome or cDNA (being SEQID NO:7) DNA amplification.PCR is used for the required sequence in DNA amplification clone or genomic library or cDNA library, selects then.For example by use thermal cycler and Taq polymerase (

), can carry out PCR.(PCR) is generally used for obtaining target gene or genetic fragment in the PCR.Use in abutting connection with the PCR primer of the nucleotide sequence of coding open read frame, can produce the nucleotide sequence of the recombinant tissue protectiveness cell factor of any Len req of for example encoding.Perhaps, can cut recombinant tissue protectiveness cytokine gene sequence (if such site is arranged) with restriction endonuclease, discharge the dna fragmentation of coding recombinant tissue protectiveness cytokine gene in appropriate site.If there is not conventional restriction site, can pass through direct mutagenesis and/or DNA cloning method known in the art, produce described site (referring to for example Shankarappa etc., 1992, PCR Method supplementary issue 1:277-278) at correct position.Separate the dna fragmentation of coding recombinant tissue protectiveness cell factor then, and be connected on the suitable expression vector, careful operation is read frame to guarantee suitable translation.

In order to carry out aminoacid replacement in the expression of peptides sequence, perhaps for produce/lack restriction site so that further operation can be used any induced-mutation technique known in the art, the single nucleotide in the modified dna sequence.Described technology include but not limited to mutagenesis, external direct mutagenesis (Hutchinson etc., 1978, J.Biol.Chem.253:6551), oligonucleotide-directed mutagenesis (Smith, 1985, Ann.Rev.Genet.19:423-463; Hill etc., 1987, Methods Enzymol.155:558-568) and as the overlap extension (Ho etc. of the PCR-based described of 6.3 trifles, 1989, Gene 77:51-59), big primer mutagenesis of PCR-based (Sarkar etc., 1990, Biotechniques 8:404-407) etc.By for example double-stranded di-deoxynucleoside acid dna sequencing, can confirm to modify.

The present invention also comprises and the nucleic acid molecules of the nucleotide sequence complementation of above-mentioned paragraph, dna molecular preferably.

In certain embodiments, nucleic acid molecules of the present invention is to comprise containing or the nucleic acid molecules part of the nucleotide sequence of the allos of encoding (for example carrier, expression vector or fusion) sequence.

5.2. recombinant tissue protectiveness cell factor of the present invention

Recombinant tissue protectiveness cell factor of the present invention comprises the erythropoietin(EPO) mutain of retaining part or whole erythropoiesis activity.Erythropoietin(EPO) is a glycoprotein hormones, and the molecular weight in human body is about 34kDa.Its maturation protein comprises 165 amino acid, and saccharide residue accounts for 40% of molecular weight.The form that is used for the recombinant tissue protectiveness cell factor of the present invention practice comprises the natural existence form that generates plain correlation molecule as servant and other mammalian erythropoietin; at least one amino acid change of synthesized form and recombinant forms: erythropoietin(EPO); take off the sialic acid erythropoietin(EPO); the deglycosylation erythropoietin(EPO); erythropoietin analogue; the erythropoiesis mimetics; the erythropoietin(EPO) fragment; the hybrid erythropoietin molecule; the erythropoietin receptor binding molecule; the erythropoietin(EPO) activator; the kidney erythropoietin(EPO); the brain erythropoietin(EPO); its oligomer and polymer ex hoc genus anne thing.The described recombinant tissue protectiveness cell factor that is equal to comprises the saltant erythropoietin(EPO); it can contain replacement, comprise inner disappearance disappearance, comprise produce the interpolation of melting in being added on of albumen or in amino acid sequence and/or near conservative replacement of amino acid residue; but they but produce " silence " variation, because described variation produces erythropoietin(EPO) mutain or the recombinant tissue protectiveness cell factor that is equal on the function.In a preferred embodiment, described recombinant tissue protectiveness cell factor right and wrong are erythropoietic, promptly lack the erythropoiesis activity or show the erythropoiesis activity of attenuating.Can on polarity, electric charge, dissolubility, hydrophobicity, water wettability and/or the amphipathic similar basis of the residue that relates to, carry out conserved amino acid and replace.For example nonpolar (hydrophobicity) amino acid comprises alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophane and methionine; Polar neutral amino acid comprises glycocoll, serine, threonine, halfcystine, tyrosine, asparagine and glutamine; Positively charged (alkalescence) amino acid comprises arginine, lysine and histidine; Comprise aspartic acid and glutamic acid with electronegative (acidity) amino acid.Perhaps, non-conserved amino acid variation and insertion greatly and disappearance can be used for producing the recombinant tissue protectiveness cell factor that function changes.Described mutant can be used for changing in the mode of needs the characteristic of erythropoietin(EPO).For example in one embodiment, the erythropoietin(EPO) that is used for the present invention practice can be the recombinant tissue protectiveness cell factor at following 4 one or more amino acid changes of functional domain of the erythropoietin(EPO) that influences receptors bind: 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, can use the erythropoietin(EPO) that contains sudden change in the molecule peripheral region of dynamics that influences described molecule or receptor-binding characteristic.Which kind of change or will influence combination in which position of domain, this can determine by standard method.For example, can pass through paired alanine mutation (ala scanning mutagenesis) and change domain, the binding kinetics of measuring mutant then is to determine influence (Bernat etc., 2003, the PNAS 100:952-957 to receptors bind; Wells etc., 1989, Science 244:1081-1085).

Term " recombinant tissue protectiveness cell factor " and " recombinant tissue protectiveness cell factor " are used interchangeably or are used in combination; comprise recombinant tissue protectiveness cell factor of the present invention and further modify for example deglycosylation of recombinant tissue protectiveness cell factor, asialylated and other parts glycosylation form or amino acid whose chemical modification.The limiting examples of described variant is described in Tsuda etc., and 1990, Eur.J.Biochem.188:405-411, described document is attached to herein by reference.Cell factor is a highly flexible, with regard to human growth hormone (HGH), known flexible be the activation needed (Wells etc., 1989, Science244:1081-1085).Therefore, the sudden change of the three-dimensional structure of the stabilized cell factor, the normal activation of prevention erythropoietin receptor comprises in the present invention.In addition; various host systems can be used for recombinant tissue protectiveness cytokine expression and generation, and described system includes but not limited to the mammal cell line system that bacterial cell system, yeast cells system, insect cell system, plant cell are unified and comprised people's cell.For example; the bacteriogenic non-glycosylated form that does not have glycosylation, the asialylated or glycosylated recombinant erythropoietin of part to can be used for producing recombinant tissue protectiveness cell factor; maybe can be with the further glycosylation of means known in the art, what described technology included but not limited to be disclosed in following document adjusts the technology of protein glycosylation with fucosylation: Application No. US 2003/0040037 A1 and US2003/0003529.Perhaps, can make at other and produce recombinant tissue protectiveness cell factor in glycosylated system of expressing protein, described system is vegetable cell and human cell for example.

As mentioned above, the present invention includes any and whole erythropoietin receptor active regulator molecule that the pairing effect cell can be played a positive role, no matter the structural relation of described molecule and erythropoietin(EPO) how.

In addition, can modify recombinant tissue protectiveness cell factor, to customize its activity to particular organization.Can take some non-limiting strategies to reach this required tissue specificity; comprise shorten the circulation half life and therefore reduce recombinant tissue protectiveness cell because of with the modification of precursor interaction time of class red blood cell, perhaps to the modification of the primary structure of erythropoietin(EPO) mutain or recombinant tissue protectiveness cell factor molecule.A kind of method that shortens the half life that circulates is that three N connection sugar and O that removal or modification erythropoietin(EPO) have joins sugared glycosylation part.Can produce the described variant of glycosylation recombinant tissue protectiveness cell factor in every way.For example; modifying the erythropoietin(EPO) primary structure is unnumbered with the technology that produces tissue protective cell factor of the present invention, and the replacement that comprises one or more specific amino acids is promptly by making N connection sugar or O join amino acid mutation and/or one or more amino acid whose chemical modification of sugared glycosylation site or adding other structure of disturbing erythropoietin(EPO) and its acceptor interaction.Use the described form of recombinant tissue protectiveness cell factor all to comprise in the present invention.Can by special sialidase, sialic acid be removed according to the sialic acid of sugar-chain end and the chemical bonding of sugar chain.Perhaps, can pass through different modes, use other enzyme of cutting particular key, remove the glycosylation structure.In a preferred embodiment, the half life of the non-erythropoietic recombinant tissue protectiveness cell factor of the present invention, shorten about 90% than natural erythropoietin(EPO).

Yet some in these recombinant tissue protectiveness cell factor molecules will be simulated the effect of erythropoietin(EPO) itself in other tissue or organ.For example, the 17-mer that contains natural erythropoietin(EPO) amino acid sequence 31-47 is a non-activity on red blood cell takes place, but whole activity (Campana﹠amp are arranged in external neurocyte; O ' Brien, 1998:Int.J.Mol.Med.1:235-41).

In addition, the recombinant tissue protectiveness cell factor molecule of deriving that purposes as described herein is required can produce by the following method: guanidineization (guanidination), amidation, carbamylation, trinitrophenylization, acidylate such as acetylation or succinylation, nitrated, perhaps at other method limited proteolysis for example, in the amino removal to arginine, aspartic acid, glutamic acid, lysine, tyrosine, the modification of tryptophane or cysteine residues or carboxyl, and/or by Protocols in Molecular Biology to arginine, lysine, tyrosine, the replacement that suddenlys change of tryptophane or cysteine residues, with produce to certain organs and tissue keep proper level (but not to other cell for example red blood cell keep proper level) erythropoietin(EPO) mutain or recombinant tissue protectiveness cell factor (Satake etc. for example; 1990, Biochim.Biophys.Acta1038:125-9; Described document all is attached to herein by reference).A limiting examples as described below be by with glyoxal such as the phenyl glyoxal reaction, the erythropoietin(EPO) arginine residues is modified (according to Takahashi, 1977, the scheme of J.Biochem.81:395-402).It will be appreciated that as following described recombinant tissue protectiveness cell factor molecule keeps the neurotrophy effect of erythropoietin(EPO) fully.Described recombinant tissue protectiveness cell factor molecule all is included in various uses as herein described and the composition.In addition, these chemical modifications can be further used for strengthening any variation of the branch charge of the electron that the protective effect of recombinant tissue protectiveness cell factor or neutralization brought by natural erythropoietin(EPO) amino acid mutation.Described modification is described in the copending application serial number PCT/US01/49479 of application on Dec 28 calendar year 2001; The reel number KW00-009C02-US of agency of the serial number 09/753,132 of application on Dec 29th, 2000 and application on July 3rd, 2002, all described patented claims all are attached to herein by reference.

This paper provides synthetic molecules and recombinant molecule, such as brain erythropoietin(EPO) and kidney erythropoietin(EPO) etc., the recombinant mammalian form of erythropoietin(EPO), and natural isotype, tumour source isotype and reorganization isotype, such as recombinant expressed molecule and the molecule by the homologous recombination preparation.In addition, the present invention includes the molecule that contains in conjunction with the erythropoietin receptor peptide, and recombinant precursor or other have the part or all of structure of erythropoietin(EPO) and/or the molecule of biological characteristics, comprises the fragment and the polymer of erythropoietin(EPO) or its fragment.The present invention includes erythropoietin(EPO) mutain or other recombinant tissue protectiveness cell factor of the glycosylation site of glycosylation site with increase or minimizing.As mentioned above, term " erythropoietin(EPO) " and " analogies " and other term are used interchangeably at this paper, all are meant the effector cell protection relevant with the molecule that can pass through endothelial cell barrier with erythropoietin(EPO) and strengthen molecule.In addition, the present invention also comprises the molecule that is produced by transgenic animals.It should be noted, the erythropoietin molecule that this paper comprises is not necessarily similar with erythropoietin(EPO) on structure or alternate manner, except interacting with erythropoietin receptor or regulating the ability of erythropoietin receptor activity or activation erythropoietin(EPO) activation signal as described herein cascade.

As limiting examples, the form that is used for the recombinant tissue protectiveness cell factor of the present invention's practice comprises recombinant tissue protectiveness cell factor, for example has the amino acid whose form of change at its c-terminus, be described in United States Patent (USP) 5,457,089 and United States Patent (USP) 4,835,260; Take off the erythropoietin(EPO) isotype that has the sialic acid residues of different numbers in sialic acid erythropoietin(EPO) and each molecule, for example be described in United States Patent (USP) 5,856,298; Polypeptide is described in United States Patent (USP) 4,703,008; Activator is described in United States Patent (USP) 5,767,078; The peptide that combines with erythropoietin receptor is described in United States Patent (USP) 5,773, and 569 and 5,830,851; And small molecule mimetics, be described in United States Patent (USP) 5,835,382; And erythropoietin analogue, be described in WO 9505465, WO 9718318 and WO 9818926.All above-mentioned citing documents relate in its disclosure on relevant various alternative forms that are used for recombinant tissue protectiveness cell factor form of the present invention or preparation method's the meaning and being attached to herein by reference.

Erythropoietin(EPO) is commercially available, and for example derives from Ortho Biotech Inc., Raritan, and NJ, trade mark is PROCRIT, and derives from Amgen, Inc., Thousand Oaks, CA, trade mark are EPOGEN.

The activity (in unit) of erythropoietin(EPO) (EPO) and erythropoietin(EPO) sample molecule usually defines the source of the international standard of erythropoietin(EPO) (also as) according to it in effectiveness that rodent model moderate stimulation red blood cell produces.A unit (U) of common erythropoietin(EPO) (MW is～30,000-34,000) is about 8ng albumen, and (1mg albumen is about 125,000U).Yet; because erythrogenic effect is accompanied by the required activity of this paper; and the detected characteristic of recombinant tissue protectiveness cell factor not necessarily of the present invention, so the activity that defines some tissue protective cell factor of the present invention according to the erythropoiesis activity is unsuitable.Therefore, the active unit of erythropoietin(EPO) used herein or erythropoietin(EPO) correlation molecule is defined as in neurocyte system or other effector cell system and causes the required protein content of identical activity that causes with WHO international standard erythropoietin(EPO) in same system.The technician will easily determine the unit of non-erythrogenic recombinant tissue protectiveness cell factor or correlation molecule according to the guidance of this paper.

Recombinant tissue protectiveness cell factor mutain includes but not limited to albumen and the polypeptide by the erythropoietin(EPO) nucleic acid sequence encoding that is described in 6.3 trifles.The present invention includes the mutain that is equal to the erythropoietin gene product that is described in 6.3 trifles on the function.Described erythropoietin gene product can contain one or more disappearances, interpolation or the replacement of erythropoietin(EPO) amino acid residue at the amino acid sequence by the erythropoietin(EPO) nucleic acid sequence encoding, but they produce reticent the variation, therefore produce the erythropoietin gene product that is equal on the function.Can on polarity, electric charge, dissolubility, hydrophobicity, water wettability and/or the amphipathic similar basis of the residue that relates to, carry out aminoacid replacement.

Recombinant tissue protectiveness cell factor mutain of the present invention can produce for example discontinuous point mutation or brachymemma through mutagenesis.Recombinant tissue protectiveness cell factor mutain of the present invention keeps the cytoprotection biologic activity of native form, but can lack one or more erythropoiesis activity of described albumen native form.Therefore, can cause the particular biological effect by the mutain that adds limited function.

Can carry out recombinant tissue protectiveness cell factor mutain structural modification (for example changing the phosphorylation pattern of mutain) in order to strengthen purposes such as effect, stability or posttranslational modification.The recombinant tissue protectiveness cell factor mutain of described modification; when designing to keep at least a cell protection activity of described albumen native form; or when producing its specific antagonists, can consider it is the function equivalent of recombinant tissue protectiveness cell factor mutain.For example can pass through aminoacid replacement, disappearance or interpolation, produce this modified recombinant tissue protectiveness cell factor mutain.

For example, have reason to believe, replace leucine, replace aspartic acid, replace threonine or will can be the biologic activity generation significant impact of gained molecule with the similar amino acid of aminoacid replacement relevant on the structure (promptly with joining sudden change and/or waiting electricity to suddenly change) with serine with glutamic acid with isoleucine or valine.

Whether the variation at the amino acid sequence of recombinant tissue protectiveness cell factor mutain produces function homolog or NOT-function homolog (one or more activity that promptly lack not mutated cell factor); can in cell, produce effect or the competitive ability that suppresses described effect in similar wild-type cytokines mode by estimating the variation mutain, come easily to determine.The recombinant tissue protectiveness cell factor mutain that a more than replacement wherein takes place can easily detect by same way as.

Can identify the mutain of the present invention of the function that shows change by screening mutant combinatorial libraries, described mutant for example has required activity or lacks the truncated mutant of these active recombinant tissue protectiveness cell factors of the present invention.In one embodiment,, produce piebald (variegated) library of variant, and encode by the gene library of piebald by the combinatorial mutagenesis on the nucleic acid level.Can be by for example synthetic oligonucleotide potpourri enzymatic being connected on the nucleotide sequence, produce the piebald library of variant, make the degenerate sequence of potential protein sequence can be expressed as not homopolypeptide, perhaps, be expressed as one group of bigger fusion (for example being used for phage display).Can make in all sorts of ways, produce the potential variant library of recombinant tissue protectiveness cell factor of the present invention from degenerate oligonucleotide sequence.The method of synthetic degenerate oligonucleotide be known in the art (referring to for example Narang, 1983, Tetrahedron 39:3; Itakura etc., 1984, Annu.Rev.Biochem.53:323; Itakura etc., 1984, Science 198:1056; Ike etc., 1983, Nucleic Acids Res.11:477).

In addition, the fragment library of recombinant tissue protectiveness cell factor coded sequence of the present invention can be used for producing the piebald colony of recombinant tissue protectiveness cell factor, selects to use for screening and mutain thereafter.For example, breach is only had an appointment in per molecule under once the condition therein, can be by double-stranded PCR fragment with nuclease processing target coded sequence, produce the library of coded sequence fragment, denatured double stranded dna, renaturation DNA can include the right double-stranded DNA of justice/antisense to form from different band breach products, removes the strand part from the duplex that forms again by handling with the S1 nuclease, and gained fragment library is connected on the expression vector.By this method, can obtain encode the target recombinant tissue protectiveness cell factor mutain N end of different sizes and the expression library of interior segments.

Some are used to screen by point mutation or brachymemma and combinatorial libraries gene outcome that produces and the technology that is used to screen the cDNA library of the gene outcome with selected characteristic are known in the art.The most widely used technology of high throughput analysis that is applicable to the big gene library of screening generally includes is cloned into gene library in the science expression vector, transform suitable cell with the gained vector library, and therein the detection of required activity is convenient to separate under the condition of carrier of the gene that its product of coding can be detected and expresses combination gene.Recurrence group mutagenesis (the Recursive ensemble mutagenesis) technology (REM) that increases the frequency of functional mutants in the library can be used in combination with Screening test, to identify mutain (Arkin and the Yourvan of recombinant tissue protectiveness cell factor of the present invention, 1992, Proc.Natl.Acad.Sci.USA 89:7811-7815; Delgrave etc., 1993, Protein Engineering 6 (3): 327-331).

Can replace, add or disappearance by in the erythropoietin(EPO) nucleotide sequence, introducing one or more nucleotide; produce the isolated nucleic acid molecule of encoding mutant albumen, so that one or more aminoacid replacement, interpolation or disappearance are introduced in the recombinant tissue protectiveness cell factor of coding.Can introduce sudden change by such as standard techniques such as direct mutagenesis and PCR mediation mutagenesis.In brief, design PCR primer, the amino acid whose trinucleotide codon that this primer disappearance remains to be changed and with the amino acid whose trinucleotide codon replacement of desiring to comprise.This primer be used to encode pcr amplification of DNA of target recombinant tissue protectiveness cell factor.Separate this fragment then and be inserted in the full-length cDNA of coding destination organization protectiveness cell factor and make it recombinant expressed.Gained recombinant tissue protectiveness cell factor promptly comprises described aminoacid replacement.

Conservative or non-conserved amino acid replaces and can carry out on one or more amino acid residues.Can guard and non-conservative replacement.Conservative replacement is the replacement that occurs in the amino acid family relevant in their side chain.Usually amino acids coding can be divided into 4 families: (1) acidity=aspartic acid, glutamic acid; (2) alkalescence=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophane; (4) uncharged polar=glycocoll, asparagine, glutamine, halfcystine, serine, threonine, tyrosine.In a similar manner, the amino acid repertoire can be divided into (1) acidity=aspartic acid, glutamic acid; (2) alkalescence=lysine, arginine, histidine, (3) aliphatic series=glycocoll, alanine, valine, leucine, isoleucine, serine, threonine, and the optional aliphatic series-hydroxyl that is divided into of serine and threonine; (4) aromatics=phenylalanine, tyrosine, tryptophane; (5) acid amides=asparagine, glutamine; (6) sulfur-bearing=halfcystine and methionine (referring to for example Biochemistry, the 4th edition, L.Stryer writes, WH Freeman and Co., 1995).

Perhaps, can introduce at random in all or part of coded sequence of recombinant tissue protectiveness cell factor, and the gained mutant can screen according to biologic activity, to identify the mutant of retention activity by suddenling change such as saturation mutagenesis.After the mutagenesis, can recombinant expressed encoded protein and can measure the activity of recombinant tissue protectiveness cell factor.

Except the above-mentioned erythropoietin(EPO) that is used for this paper is modified, below discuss various recombinant tissue protectiveness cell factors of the present invention have been described in detail in detail.As described in above-mentioned Elliott etc., Boissel etc. and Wen etc., following erythropoietin(EPO) mutain is used for purpose described herein, and can provide in Pharmaceutical composition to be used for the method for this paper.In this article in the mutain nomenclature of Cai Yonging, the amino acid of change with at first be natural amino acid one-letter code, secondly be its position on erythropoietin molecule, be that substituted amino acid one-letter code is represented at last.For example, " erythropoietin S100E " or " recombinant tissue protectiveness cell factor 100E " is meant that the serine of amino acid/11 00 wherein becomes the erythropoietin molecule of glutamic acid.The mutain that is used for the present invention's practice includes but not limited to have the erythropoietin that at least one 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, the 44-49 disappearance,

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.

In preferred embodiments, erythropoietin(EPO) mutain of the present invention or recombinant tissue protectiveness cell factor comprise one or more above replacements.In other embodiments, erythropoietin(EPO) mutain or another recombinant tissue protectiveness cell factor of the present invention comprise replacement or its combination more than one.

In an alternate embodiment; recombinant tissue protectiveness cell factor of the present invention, Pharmaceutical composition, purposes and methods of treatment comprise one or more above replacements, and condition is that they do not comprise one or more following replacements: 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 relevant embodiment of the present invention; recombinant tissue protectiveness cell factor of the present invention, Pharmaceutical composition, purposes and methods of treatment comprise one or more above replacements, and condition is that they do not comprise any following replacement combination: N24K/N38K/N83K or A30N/H32T.

In certain embodiments, can make up more than one and change, produce mutain with upper amino acid.The example of described combination includes but 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, recombinant tissue protectiveness cell factor mutain of the present invention does not comprise one or more above polysubstituted.In certain embodiments, the Pharmaceutical composition of the present invention that comprises recombinant tissue protectiveness cell factor mutain of the present invention does not comprise one or more above polysubstituted.In certain embodiments, use the purposes of the present invention of recombinant tissue protectiveness cell factor mutain of the present invention and methods of treatment not to comprise one or more above polysubstituted.

The combination of some modification or modification can influence the flexibility of erythropoietin(EPO) mutain to the bind receptor effect, second acceptor of described acceptor such as erythropoietin receptor or erythropoietin(EPO) or the combination of erythropoietin(EPO) mutain.Be used for the described modification of the present composition and method or the example of its combination and include but not limited to K152W, R14A/Y15A, I6A, C7A, D43A, P42A, F48A, Y49A, T132A, I133A, T134A, N147A, F148A, R150A, G151A, G158A, C161A and R162A.It is (Wells etc.) that are harmful in human growth hormone (HGH) that known these suddenly change accordingly.In certain embodiments, recombinant tissue protectiveness cell factor mutain of the present invention does not comprise one or more above-mentioned replacements.In certain embodiments, the Pharmaceutical composition of the present invention that comprises recombinant tissue protectiveness cell factor mutain of the present invention does not comprise one or more above-mentioned replacements.In certain embodiments, use the purposes of the present invention of recombinant tissue protectiveness cell factor mutain of the present invention and methods of treatment not to comprise one or more above-mentioned replacements.

Except one above-mentioned amino acid modified, recombinant tissue protectiveness cell factor of the present invention also can not have the sialic acid part, is called to take off sialic acid erythropoietin(EPO) mutain.Of the present invention take off sialic acid erythropoietin(EPO) mutain preferably the people take off the sialic acid erythropoietin(EPO).In alternate embodiment, recombinant tissue protectiveness cell factor of the present invention can have at least 1,2,3,4,5,6,7,8,9,10,11,12 or 13 sialic acid residueses.They can use recombinant tissue protectiveness cell factor asialylated and prepare with sialidase, and described enzyme is for example by ProZyme Inc., and San Leandro describes on the sialidase A manufacturer packing of California.Usually,

Order-checking level sialidase A ^TM(N-n acetylneuraminic acid n glycosyl hydrolase, EC 3.2.1.18) be used for from complicated sugar and glycoprotein for example erythropoietin(EPO) cut all non-reducing end sialic acid residueses.It also can cut branching sialic acid (connecting inner residue).Sialidase A separates from the clone who produces urea arthrobacterium (Arthrobacter ureafaciens).

The sialylated limiting examples of glycoprotein can be referring to Application No. US2003/0040037, and described document discloses with mammal sialyltransferase or the sialylated method of bacterium sialyltransferase.The change of sialic acid pattern can be referring to Application No. US 2002/0160460A1 and US 6,399,336 B1 on the limiting examples of another sialylated method and the glycoprotein.Disclose the external method that is used for sialylated recombinant glycoprotein in this application, wherein the sialic acid donor part combines with the glycoprotein with galactose or N-acetylgalactosamine acceptor portion.In this way, the sialyltransferase of connection acceptor and donor is connected to sialic acid on the sugar.

Recombinant tissue protectiveness cell factor of the present invention can have the N connection sugar of decreased number at least.In order to remove N connection sugar, can be according to for example Hermentin etc., 1996, the method that Glycobiology6 (2): 217-30 describes is handled recombinant tissue protectiveness cell factor with hydrazine.As mentioned above, erythropoietin(EPO) has 3 N connection sugar moieties; The present invention includes have 2,1 or do not contain the erythropoietin(EPO) of N connection sugar.

Recombinant tissue protectiveness cell factor of the present invention can have the sugared content that reduces at least because of using at least a glycosidase to handle recombinant tissue protectiveness cell factor.For example can adopt Chen and Evangelista, 1998, Electrophoresis 19 (15): the method that 2639-44 describes.In addition, can be according to Hokke etc., 1995, the method that Eur.J Biochem.228 (3): 981-1008 describes is removed O connection sugar.

Because express recombinant erythropoietin(EPO) mutain in the nonmammalian cell, the sugar moieties of recombinant tissue protectiveness cell factor molecule can have nonmammalian glycosylation pattern at least.Recombinant tissue protectiveness cell factor of the present invention is preferably in insect cell or the vegetable cell to be expressed.As limiting examples, can be according to Quelle etc., 1989, Blood 74 (2): 652-657, with baculovirus expression system in expressed in insect cells recombinant tissue protectiveness cell factor.Another kind method is described in United States Patent (USP) 5,637,477.

Can be according to Matsumoto etc., 1993, Biosci.Biotech.Biochem.57 (8): the method for 1249-1252, in botanical system, express.Perhaps, the expression in bacterium will produce the recombinant tissue protectiveness cell factor of non-glycosylated form.These only are the exemplary recombinant tissue protectiveness production of cytokines methods of the present invention and restrictive anything but that is used for.

The limiting examples that glycosylation pattern is modified is to use fucosylation, as is disclosed in Application No. US2003/0040037 A1 and Application No. US2003/0003529 A1.Wherein disclose the method for the glycosylation pattern that is used to modify glycopeptide, promptly, modified the glycosylation pattern of glycopeptide by making reaction mixture contact have the glycopeptide of the acceptor portion of fucosyltransferase with fucose donor part.Also disclose and used the reorganization glycopeptide to come the method for modification of glycosylation patterns.

Recombinant tissue protectiveness cell factor of the present invention can have at least one or a plurality of also can be by the oxosugar of electronation.For example, recombinant tissue protectiveness cell factor can be the erythropoietin(EPO) mutain of periodate oxidation; The also available borohydride salt of the erythropoietin(EPO) mutain of periodate oxidation carries out electronation such as sodium borohydride or sodium cyanoborohydride.The periodate oxidation of erythropoietin(EPO) mutain can be for example by Linsley etc., 1994, the method that Anal.Biochem.219 (2): 207-17 describes is carried out.Can be according to Tonelli and Meints, 1978, J.Supramol.Struct.8 (1): the method for 67-78, carry out electronation earlier, then carry out periodate oxidation.

It should be noted, to some of natural erythropoietin(EPO) above-mentioned and following amino acid modified be impossible change because in natural molecule, be used for the particular target amino acid of chemical modification, form recombinant tissue protectiveness cell factor of the present invention.Certainly, the amino acid of change can rely on self experience chemical modification, the present invention includes all such molecules.Those skilled in the art will determine amino acid residue that recombinant tissue protectiveness cell factor of the present invention can be used and easily to its available modification.

The recombinant tissue protectiveness cell factor that is used for such use can have at least one or a plurality of modification arginine residues.For example, recombinant tissue protectiveness cell factor can comprise R-glyoxal part on one or more arginine residues, and wherein R is aryl, heteroaryl, low alkyl group, lower alkoxy or naphthenic base, or α-desoxysugar alcohol radical.Term used herein rudimentary " alkyl " is meant the straight or branched radical of saturated aliphatic alkyl that preferably contains 1-6 carbon atom.Representational described group is methyl, ethyl, isopropyl, isobutyl, butyl, amyl group, hexyl etc.Term " alkoxy " is meant the above-mentioned low alkyl group that connects described molecule remainder by oxygen.The example of alkoxy comprises methoxyl, ethoxy, propoxyl group, isopropoxy etc.Term " naphthenic base " is meant 3 naphthenic base of about 8 carbon extremely at the most, comprises for example cyclopropyl, cyclobutyl, cyclohexyl etc.Term aryl is meant phenyl and naphthyl.The term heteroaryl is meant and contains 4-10 annular atoms and 1-3 heteroatomic heterocyclic radical that is selected from oxygen, nitrogen and sulphur.Example includes but not limited to isoxazolyl, phenyl-isoxazole azoles base, furyl, pyrimidine radicals, quinolyl, tetrahydric quinoline group, pyridine radicals, imidazole radicals, pyrrolidinyl, 1,2,4-triazolyl, thiazolyl, thienyl etc.The R group can be substituted, and for example 2,3 of the 3-deoxyglucosone, 4-three hydroxyl butyl.The representative instance of R-glyoxalated compound be glyoxal, methyl-glyoxal, 3-deoxyglucosone and phenyl glyoxal.Preferred R-glyoxalated compound is methyl-glyoxal or phenyl glyoxal.The illustrative methods of using the phenyl glyoxal to carry out described modification is found in Werber etc., and 1975, Isr.J.Med.Sci.11 (11): 1169-70.

In another embodiment, be preferably in the borate buffer solution of about 50 mMs, at pH 8-9, can be by with for example 2,3-diacetyl or cyclohexanedione etc. connect two reactive ketones, modify at least one arginine residues.With 2, a back method of modifying of 3-diacetyl can be according to Riordan, and 1973, Biochemistry 12 (20): the method for 3915-3923 is carried out; And can be according to Patthy etc. with the method for cyclohexanedione, 1975, J.Biol.Chem 250 (2): the method for 565-9 is carried out.

It is amido modified that recombinant tissue protectiveness cell factor of the present invention can comprise a N end of at least one or a plurality of modification lysine residue or erythropoietin molecule, and described modification is for example reacted resulting modification from lysine residue with amino modified dose.In another embodiment, lysine residue can form corresponding α-carboxyalkyl derivant by being modified with glyoxal reaction (for example with glyoxal, the reaction of methyl-glyoxal or 3-deoxyglucosone).Be found in Glomb and Monnier with glyoxal reaction with the example that forms carboxymethyl-lysine, 1995, J.Biol.Chem.270 (17): 10017-26, perhaps be found in Degenhardt etc. with the example that forms (1-carboxyethyl) lysine with the methyl-glyoxal reaction, 1998, Cell.Mol.Biol. (Noisy-le-grand) 44 (7): 1139-45.The lysine residue of modifying also can be further by electronation.For example; recombinant tissue protectiveness cell factor can be by the lysine group by biotinylation; wherein D-biotin acyl-EACA-N-hydroxy-succinamide ester and erythropoietin(EPO) reaction; then remove unreacted biotin: Wojchowski and Caslake by gel filtration on Centricon 10 posts according to the description of following document; 1989, Blood 74 (3): 952-8.In the document, the author has used the biotinylated 3 kinds of distinct methods of erythropoietin(EPO), and every kind of method all can be used to prepare the erythropoietin(EPO) that is used for this paper purposes.Biotin can join on (1) sialic acid part (2) carboxyl or (3) amino.

In a further preferred embodiment, described lysine can form imines with aldehyde or reducing sugar reaction, described imines can by formed by sodium cyanoborohydride reduction N-alkylation lysine residue for example glucose alcohol radical lysine stablize, perhaps under the situation of reducing sugar, can stablize in erythropoietin molecule, to form α-deoxidation-for example α-deoxidation of alpha-amido sugar-α-fructosyl lysine residue by Amadori or Heyns rearrangement.As an example, be incubated 60 days by sodium phosphate buffer (pH 7.4) with 0.5M glucose, can prepare the albumen of fructosyl polylysine modification, this method is described in Makita etc., and 1992, J.Biol.Chem.267:5133-5138.In another example; the lysine group can be for example by with the cyanic acid ion reaction by carbamylation; perhaps with alkyl-isocyanate or aryl-isocyanate, alkyl-isothiocyanate or aryl-isothiocyanic acid reactant salt and by alkyl-carbamylation or aryl-carbamylation or alkyl-thiocarbamoylization or aryl-thiocarbamoylization; perhaps can be and acidylate by active alkyl carboxylic acid derivative or arylcarboxylic acid derivative, for example by with acetic anhydride or succinic anhydride or phthalic anhydride.Example is to modify the lysine group with 4-S-PITC or acetic anhydride, and the two all is described in Gao etc., 1994, and Proc Natl AcadSci USA 91 (25): 12027-30.The lysine group also can be by with trinitro-benzene-sulfonic acid or preferably and its reactant salt and being modified by trinitrophenyl.

At least one tyrosine residue of recombinant tissue protectiveness cell factor can be modified by for example nitrated or iodate in the aromatic ring position by electrophilic reagent.As limiting examples, erythropoietin(EPO) can be reacted (Nestler etc., 1985, J.Biol.Chem.260 (12): 7316-21 with tetranitromethane; Perhaps as described in the embodiment 4 by iodate.

At least one asparagicacid residue of recombinant tissue protectiveness cell factor or glutaminic acid residue can be for example by with the carbodiimide reaction after again by with amine (such as but not limited to glycine amide) reaction and modified.

In another example, a trp residue of recombinant tissue protectiveness cell factor can be for example by with positive bromine succinimide or positive chloro-succinimide reaction after again by for example being described in Josse etc., Chem Biol Interact 1999 May 14; The method of 119-120 and being modified.

In another example, recombinant tissue protectiveness cell factor can be by removing at least one amino preparation, this can by with ninhydrin reaction after finish by the carbonyl that produces subsequently with hydroborate reaction reduction again.

In another example; be provided at the recombinant tissue protectiveness cell factor that has at least one to open at least one halfcystine key in the erythropoietin molecule; wherein erythropoietin molecule by with the reaction of reductive agent such as for example dithiothreitol (DTT), then form again with the prevention disulfide bond with iodoacetamide, iodoacetic acid or the reaction of another kind of electrophilic reagent by the sulfydryl that produces subsequently.As mentioned above, can eliminate disulfide bond by one of following two kinds of methods or two method couplings, promptly change participate in other amino acid residue of the disulfide bond that actual crosslinked halfcystine molecule or at least one cause the erythropoietin(EPO) mutain not form existing at least one natural molecule.

Can prepare recombinant tissue protectiveness cell factor by the limited chemical proteolysis (for example behind trp residue, cutting) that erythropoietin(EPO) is carried out the specific residue of target.Gained recombinant tissue protectiveness cell factor fragment is included in herein.

As mentioned above, the recombinant tissue protectiveness cell factor that is used for this paper purpose can have at least one above-mentioned modification, but also can have more than modification more than.The sugar moieties at its molecule as an example has one to be modified and has the recombinant tissue protectiveness cell factor of a modification in its amino part, and recombinant tissue protectiveness cell factor can be to take off the sialic acid erythropoietin(EPO) and its lysine residue of 45 becomes aspartic acid.

Therefore, comprise various recombinant tissue protectiveness cell factor molecules that are used for purposes described herein and the Pharmaceutical composition that contains them.As mentioned above; described erythropoietin molecule includes but not limited to mutain; described mutain is to take off the sialic acid erythropoietin(EPO); N-deglycosylation erythropoietin(EPO); O-deglycosylation erythropoietin(EPO); has the erythropoietin(EPO) that reduces sugared content; has the erythropoietin(EPO) that glycosylation pattern changes; erythropoietin(EPO) with the sugar that is reduced again after the oxidation; the erythropoietin(EPO) that aryl glyoxal is modified; the erythropoietin(EPO) that alkyl glyoxal is modified; 2; the erythropoietin(EPO) that the 3-diacetyl is modified; the erythropoietin(EPO) that cyclohexanedione is modified; the biotinylation erythropoietin(EPO); N-alkylation lysyl erythropoietin(EPO); glucose alcohol radical lysine erythropoietin(EPO); α-deoxidation-α-fructosyl lysine-erythropoietin(EPO); the carbamylation erythropoietin(EPO); the acetylation erythropoietin(EPO); the succinylation erythropoietin(EPO); α-carboxyalkyl erythropoietin(EPO); nitrated erythropoietin(EPO); the iodate erythropoietin(EPO), this is representational but be nonrestrictive example according to some of defining named of the present invention.Be preferably based on the above-mentioned modified forms of erythropoietin.

In addition, the present invention includes above-mentioned recombinant tissue protectiveness cell factor and the Pharmaceutical composition that comprises described compound.As limiting examples, described recombinant tissue protectiveness cell factor comprises that erythropoietin(EPO) mutain, glucose alcohol radical lysine erythropoietin(EPO) mutain, fructosyl lysine erythropoietin(EPO) mutain, 3-deoxyglucosone erythropoietin(EPO) mutain and the carbamylation of periodate oxidation take off sialic acid erythropoietin(EPO) mutain.

5.3. expression system

Various host expresses carrier systems can be used for producing the recombinant tissue protectiveness cell factor that comprises erythropoietin(EPO) mutain molecule of the present invention.Described host expression system is represented carrier; but can produce target recombinant tissue protectiveness cell factor and at purifying thereafter by described carrier; and described system also represents cell, and described cell is when transform or show in position during transfection the erythropoietin gene product of modification with suitable nucleotide coding sequence.These include but not limited to bacterium, insect, plant, comprise people's mammalian hosts system, such as but not limited to the insect cell system that infects with the recombinant virus expression vector (for example baculoviral) that contains recombinant tissue protectiveness cytokine product coded sequence; With the recombinant virus expression vector that contains recombinant tissue protectiveness cell factor coded sequence (cauliflower mosaic virus for example, CaMV; Tobacco mosaic virus (TMV), TMV) the vegetable cell system of Gan Raning, or with the recombinant plasmid expression vector that contains recombinant tissue protectiveness cell factor coded sequence (for example Ti-plasmids) plant transformed cell system; Or comprise and carrying from the genomic promoter of mammalian cell (for example metallothionein promoter) or from mammalian disease virus promoter (gland virus stage starting for example; Vaccinia virus 7.5K promoter) recombinant expression construct body comprise the mammal cell line system (for example HT1080, COS, CHO, BHK, 293,3T3) that the human cell line unites.

Expression construct used herein is meant the effective recombinant tissue protectiveness cell factor coding nucleotide sequence that is connected of the regulatory region of expressing with one or more permission recombinant tissue protectiveness cell factors in the suitable host cell." effectively connect " is meant regulatory region wherein and treats express recombinant tissue protective cell factor peptide sequence being connected of combination and location as follows: allow to transcribe, and finally translate described recombinant tissue protectiveness cell factor sequence.Various expression vectors all can be used for express recombinant tissue protective cell factor, include but not limited to plasmid, clay, bacteriophage, phasmid or modification virus.Example comprises such as bacteriophages such as λ derivants, or such as pBR322 or pUC plasmid derivative thing or Bluescript vector plasmids such as (Stratagene).Usually; described expression vector comprises and is used for duplicating the functional origin of replication of described carrier, one or more restriction endonuclease site and one or more selected marker that is used to insert recombinant tissue protectiveness cytokine gene sequence at the suitable host cell.

In preferred embodiments, the pCI-neo carrier is used for making oligonucleotides to be annealed to original people EPO cDNA clone, to introduce said mutation.The pCI-neo carrier contains neomycin phosphotransferase gene, and this gene is the selected marker that is used for mammalian cell.By selecting transfectional cell with microbiotic G-418, the pCI-neo carrier can be used for transient expression or stably express.(Brondyk，1995，New?Mammalian?Expression?Vector?with?a?selectablemarker：pCI-neo.Promega?Notes?51，10-14)。

For the expression of recombinant tissue protectiveness cell factor in mammalian host cell; can use various regulatory regions, for example SV40 is early stage and late promoter, cytomegalovirus (CMV) immediate early promoter and Rous sarcoma virus long terminal repeat (RSV-LTR) promoter.The inducible promoter that can be used for mammalian cell includes but not limited to the promoter (Williams etc. that are connected with alpha-interferon genes with metallothionein II gene, mouse mammary tumor virus glucocorticoid effect long terminal repeat (MMTV-LTR), 1989, Cancer Res.49:2735-42; Taylor etc., 1990, Mol.Cell.Biol.10:165-75).

Can be by in expression vector, comprising suitable transcriptional enhancer element, enhancing is recombinant tissue protectiveness cytokine expression effect in host cell, described enhancer element is such as being present in enhancer element in SV40 virus, hepatitis B, cytomegalovirus, immunoglobulin gene, metallothionein, the α-Ji Dongdanbai (referring to Bittner etc., 1987, Methodsin Enzymol.153:516-544; Gorman, 1990, Curr.Op.in Biotechnol.1:36-47).

Expression vector also can contain the sequence that allows described carrier to keep and duplicate in more than one type host cell, perhaps with described vector integration to host chromosome.Described sequence can include but not limited to origin of replication, autonomously replicating sequence (ARS), centromeric DNA and telomeric dna.Also can advantageously use the shuttle vector that at least two types host cell, duplicates and keep.

In addition, expression vector can contain and is useful on selectable marker gene or the selection markers gene that initial gross separation or evaluation contain the host cell of recombinant tissue protectiveness cell factor coding DNA.For long-term, high yield ground produce recombinant tissue protectiveness cell factor, can use expression stable in mammalian cell, vegetable cell, bacterial cell or fungal cell.Various selective systems all can be used for mammalian cell, include but not limited to herpes simplex virus thymidine kinase (Wigler etc., 1977, Cell 1l:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski and Szybalski, 1962, Proc.Natl.Acad.Sci.U.S.A.48:2026) and adenine phosphoribosyl transferase (Lowy etc., 1980, Cell 22:817) gene can be respectively applied for tk-cell, hgprt-cell or aprt-cell.In addition, the antimetabolite resistance can be used as the basis of selecting below the basis: dihyrofolate reductase (dhfr), and it gives the resistance to methotrexate (MTX) (Wigler etc., 1980, Natl.Acad.Sci.U.S.A.77:3567; O ' Hare etc., 1981, Proc.Natl.Acad.Sci.U.S.A.78:1527); Gpt, it give resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc.Natl.Acad.Sci.U.S.A.78:2072); Neomycin phosphotransferase (neo), it give to the resistance of aminoglycoside G-418 (Colberre-Garapin etc., 1981, J.Mol.Biol.150:1); And hygromix phosphotransferase (hyg), it gives the resistance to hygromycin (Santerre etc., 1984, Gene 30:147).Also can use other selected marker, such as but not limited to histidinol and Zeocin ^TM

For recombinant tissue protectiveness cell factor coded sequence being inserted into the cloning site of carrier, dna sequence dna such as the promoter that has regulatory function must be connected with coded sequence.In order to accomplish this point; provide the joint of proper compatibility restriction site can be by technology well-known in the art; be connected to the end (Wu etc., 1987, Methods Enzymol.152:343-349) of the synthetic DNA of cDNA or coding recombinant tissue protectiveness cell factor.Can modify before connecting with restriction enzyme cutting back, by reverse digestion or mend flat single stranded DNA end and produce flush end.Perhaps, can contain the primer of required restriction enzyme sites,, required restriction enzyme sites is incorporated in the dna fragmentation by pcr amplified dna by use.

Can directly import the expression construct that comprises the recombinant tissue protectiveness cell factor coded sequence that effectively is connected with regulatory region in the proper host cell; be used for recombinant tissue protectiveness cytokine expression of the present invention and generation; need not further clone (referring to for example United States Patent (USP) the 5th; 580, No. 859).Expression construct also can contain to be convenient to coded sequence is incorporated into dna sequence dna in the host cell gene group, for example passes through homologous recombination.In this case, not necessarily use to comprise the expression vector of the origin of replication that is suitable for the suitable host cell, so that in host cell, duplicate and express recombinant tissue protective cell factor.

Can pass through various techniques known in the art; the expression construct that will contain clone's recombinant tissue protectiveness cell factor coded sequence imports in the mammalian host cell; described technology includes but not limited to the transfection (Wigler etc. of calcium phosphate mediation; 1977; Cell 11:223-232), liposome-mediated transfection (Schaefer-Ridder etc.; 1982; Science 215:166-168), electroporation (Wolff etc.; 1987; Proc.Natl.Acad.Sci.84:3344) and microinjection (Cappechi; 1980, Cell 22:479-488).

In addition, can select to regulate the expression of insetion sequence or the host cell strain of modification and processed gene product with required ad hoc fashion.Described modification of protein product (for example glycosylation) and processing (for example cutting) may be important to protein function.Different host cells are to albumen and gene outcome translation back processes and modification has distinctive peculiar mechanism.Suitable clone or host system be can select, the correct modification and the processing of the foreign protein of expression guaranteed.For this reason, can use the eukaryotic host cell of the cell machine of the glycosylation of correct processing to primary transcript, gene outcome and phosphorylation.The mammalian host cell that comprises the human host cell includes but not limited to HT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293,3T3 and WI38.

For long-term, high yield ground produce recombinant protein, preferably stably express.For example, can carry out engineered to the clone of stably express recombinant tissue protectiveness cell factor correlation molecule gene outcome.Do not use the expression vector that contains the virus replication starting point, can use by the DNA and the selected marker of suitable expression control element (for example promoter, enhancer, sequence, transcription terminator, polyadenylation site etc.) control and come transformed host cell.After importing foreign DNA, can allow engineering cell in enriched medium, grow 1-2 days, transfer in the selective medium then.Selected marker in the recombinant plasmid is given the resistance to candidate, and allow cell with the plasmid stable integration in its chromosome, allow its growth to form transforming focus, can be cloned and be increased into clone subsequently.This method can be advantageously used in carries out the clone of express recombinant tissue protective cytokine gene product engineered.Described engineering cell ties up in the compound that screens and estimate the endogenous activity that influences recombinant tissue protectiveness cytokine gene product particularly useful.

Any described cloning vector and expression vector can be by technology well-known in the art, the synthetic and assembling from known dna sequence.Regulatory region and enhancer element can be various sources, and natural and synthetic can.Some carrier and host cell are commercially available.The limiting examples of useful carrier is described in the appendix 5,1988 of Current Protocols in MolecularBiology, and Ausubel etc. write, Greene Publish.Assoc.﹠amp; WileyInterscience, described document is attached to herein by reference; With such as ClontechLaboratories, Stratagene Inc. and Invitrogen are on suppliers' such as Inc the products catalogue.

Perhaps, various expression systems based on virus also are used in recombinant expressed tissue protective cell factor in the mammalian cell.Use the carrier of dna virus skeleton to derive from simian virus 40 (SV40) (Hamer etc., 1979, Cell 17:725), adenovirus (Van Doren etc., 1984, Mol.Cell Biol.4:1653), adeno-associated virus (McLaughlin etc., 1988, J.Virol.62:1963) and bovine papilloma virus (Zinn etc., 1982, Proc.Natl.Acad.Sci.79:4897).Use therein under the situation of adenovirus as expression vector, the donor dna sequence can be connected to adenovirus and transcribe/translate the control zone, for example late promoter and tripartite leader[.This mosaic gene can be inserted in the adenoviral gene group by reorganization in external or the body.The insertion of viral genome nonessential region (for example distinguishing E1 or E3) will produce live and can be in infection host the recombinant virus of expressing heterologous product (referring to for example Logan and Shenk, 1984, Proc.Natl.Acad.Sci.U.S.A.81:3655-3659).

Perhaps, can use vaccinia virus 7.5K promoter (referring to for example Mackett etc., 1982, Proc.Natl.Acad.Sci.U.S.A.79:7415-7419; Mackett etc., 1984, J.Virol.49:857-864; Panicali etc., 1982, Proc.Natl.Acad.Sci.U.S.A.79:4927-4931).Under the situation of end user's host cell, can use therein based on Epstein-Barr virus (EBV) starting point (OriP) and EBV nuclear antigen 1 (EBNA-1; The trans-acting replicator) carrier.Described carrier can be used for various human host cells, for example EBO-pCD (Spickofsky etc., 1990, DNA Prot.Eng.Tech.2:14-18), pDR2 and α DR2 (can derive from Clontech Laboratories).

Can carry out recombinant tissue protectiveness cytokine expression by based on the retrovirus expression system.Different with transfection, retrovirus can effectively infect gene and be transferred to various cell types, comprises for example former generation hematopoietic cell.In such as retrovirus such as Moloney muroid leukemia virus, most of virus gene sequences can be removed and be replaced by recombinant tissue protectiveness cell factor coded sequence, and can supply the viral function that it loses conversely.The host range that retroviral vector infects also can be operated by the coating of selecting package carrier for use.

For example, retroviral vector can comprise 5 ' long terminal repeat (LTR), 3 ' LTR, packaging signal, bacterium origin of replication and selected marker.Recombinant tissue protectiveness cell factor DNA is inserted on the position between 5 ' LTR and the 3 ' LTR, makes and to transcribe cloning DNA from transcribing of 5 ' LTR promoter.5 ' LTR comprises promoter, includes but not limited to LTR promoter, Zone R, U5 district and primer binding site successively.The nucleotide sequence of these LTR elements is well-known in the art.Allogeneic promoter and multiple medicines selected marker are also included within the expression vector, so that the selection of carrying out infection cell is (referring to McLauchlin etc., 1990, Prog.Nucleic Acids Res. and Molec.Biol.38:91-135; Morgenstern etc., 1990, Nucleic Acids Res.18:3587-3596; Choulika etc., 1996, J.Virol70:1792-1798; Boesen etc., 1994, Biotherapy 6:291-302; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; With Grossman and Wilson, 1993, Curr.Opin.in Genetics and Devel.3:110-114).

In one embodiment of the invention, sialic acid residues recombinant tissue protectiveness cell factor not enough or that lack sialic acid residues fully can be produced by the mammalian cell that comprises people's cell.Described cell can add sialic enzyme, i.e. beta galactose glycosides-α-2 to reduce or to lack through through engineering approaches, 3-sialyltransferase (α-2,3-sialyltransferase @) and beta galactose glycosides-α-2,6-sialyltransferase (α-2,6-sialyltransferase @) activity.In one embodiment, use wherein α-2,3-sialic acid transferase gene and/or α-2, one or two mammalian cell that all lacks in the 6-sialic acid transferase gene.Can adopt gene knochout technique well-known in the art to make up described disappearance.In another embodiment, dihyrofolate reductase (DHFR) deficiency Chinese hamster ovary (CHO) cell is as the host cell that produces recombinant tissue protectiveness cell factor.Therefore Chinese hamster ovary celI is express alpha-2 not, and the 6-sialyltransferase does not add the sialic acid that connects the N connection oligosaccharides of the glycoprotein that these cells produce with 2,6 keys.As a result, the recombinant protein of Chinese hamster ovary celI generation lacks sialic acid (Sasaki etc., (1987 that connect galactose with 2,6 keys; Takeuchi etc. are referring to above; Mutsaers etc., Eur.J. Biochem.156,651 (1986); Takeuchi etc., J. Chromotgr.400,207 (1987).In one embodiment, be used to produce the host cell that takes off the sialic acid erythropoietin(EPO), make coding for alpha in Chinese hamster ovary celI-2, the gene delection of 3-sialyltransferase in order to produce.Described rejecting α-2, the Chinese hamster ovary celI of 3-sialyltransferase lacks the sialyltransferase activity fully, and the result can be used for taking off the recombinant expressed of sialic acid erythropoietin(EPO) mutain and produces.

In another embodiment, can produce asialoglycoprotein, Eckhardt etc. for example, 1998, J.Biol.Chem.273:201 89-95) by disturbing sialic acid be transported to golgiosome.Adopt the well-known method of those skilled in the art (Oelmann etc. for example, 2001, J.Biol.Chem.276:26291-300), can finish the mutagenesis of nucleotide sugar cmp sialic acid transport protein, produce the mutant of Chinese hamster ovary cell.These cells can not for example add sialic acid residues on the recombinant tissue protectiveness cell factor to glycoprotein, take off sialic acid erythropoietin(EPO) mutain so can only produce.

The transfection mammalian cell that produces the erythropoietin(EPO) mutain also produces the kytoplasm sialidase, can efficient degradation sialic acid erythropoietin(EPO) mutain if described enzyme leaks in the nutrient culture media (for example Gramer etc., 1995 Biotechnology 13:692-698).Adopt the well-known method of those skilled in the art (for example by Ferrari etc., 1994, the information that Glycobiology 4:367-373 provides), clone can be transfected, sudden change or other method produce sialidase in the composing type mode.By this way, taking off sialic acid erythropoietin(EPO) mutain can produce in taking off sialic acid erythropoietin(EPO) mutain production run.

Recombinant cell can be cultivated under the standard conditions that temperature, incubation time, optical density and nutrient culture media are formed.Perhaps, improved condition of culture and nutrient culture media can be used for increasing recombinant tissue protectiveness production of cytokines.For example, recombinant cell can be grown under the condition that starts induction type recombinant tissue protectiveness cytokine-expressing.Any known technology in this area all can be used for setting up top condition for reorganization tissue protective cytokine production.The cell lysate or the extract that comprise recombinant tissue protectiveness cell factor can be used for being further purified, to separate recombinant tissue protectiveness cell factor.

For the ease of the purifying of recombinant tissue protectiveness cell factor, marker amino acid sequence is six histidine peptides, such as the mark (QIAGEN that provides in the pQE carrier; Inc., 9259 EtonAvenue, Chatsworth; CA, 91311) and other mark, they are many all to be commercially available.For example, as Gentz etc., 1989, PNAS 86:821 is described, is provided for six histidines of the conventional purifying of fusion.Be used for other peptide-labeled hemagglutinin " HA " mark that includes but not limited to of purifying, described mark is corresponding to the epi-position (Wilson etc., 1984, Cell 37:767) from influenza hemagglutinin protein, and " flag " mark.Can use any purification process known in the art (referring to for example International Patent Publication No. W WO 93/21232; EP 439,095; Naramura etc., 1994, Immunol.Lett.39:91-99; United States Patent (USP) 5,474,981; Gillies etc., 1992, PNAS 89:1428-1432; With Fell etc., 1991, J Immunol 146:2446-2452).

5.4. the mensuration of recombinant tissue protectiveness cell factor organization protection characteristic

Preparation recombinant tissue protectiveness cell factor and further tissue protective cell factor of the present invention is carried out chemical modification in certain embodiments after, those of ordinary skills can use well-known mensuration to detect organization protection's characteristic of described cell factor and to the shortage of bone marrow effect.

For example, can measure the non-erythrogenic effect of checking recombinant tissue protectiveness cell factor by using TF-1.In this is measured, allow the TF-1 cell at 37 ℃ of CO ₂In having replenished the complete RPMI nutrient culture media of 5ng/ml GM-CSF and 10%FCS, cultivated 1 day in the incubator.Then with the washing and with 10 in the hungry nutrient culture media (5%FCS does not have GM-CSF) of described cell ⁶The density of cell/ml suspended 16 hours in hungry nutrient culture media (5%FCS does not have GM-CSF).Prepare 96 orifice plates by following steps: (1) adds 100 μ l sterilized waters to keep humidity in periphery holes; (2) in 5 holes, add nutrient culture media (10%FCS, acellular or GM-CSF); (3) in all the other holes, inoculate the nutrient culture media (every kind of cell factor to be measured 5 holes) that contains 10%FCS and described recombinant tissue protectiveness cell factor with 25,000 cells/well.If cell proliferation, then recombinant tissue protectiveness cell factor can be erythrogenic.In measuring in the body that can increase then, test the vivo effect of described compound at the hematocrit that monitoring is caused by recombinant tissue protectiveness cell factor.The cell of negative findings-in the TF-1 external test do not breed and/or measure in vivo in hematocrit not increase-show recombinant tissue protectiveness cell factor right and wrong erythrogenic.

As the alternative method that above-mentioned TF-1 measures, those skilled in the art can adopt other erythropoiesis known in the art to measure, and include but not limited to the UT-7 raji cell assay Raji, the mensuration of partly describing such as following embodiment.

Can adopt the P-19 external test or measure in mouse water intoxication body, check organization protection's characteristic of recombinant tissue protectiveness cell factor, these two kinds have general introduction in more detail below being determined at.The mensuration that substitutes includes but not limited to other mensuration of summarizing in following examples, measures such as PC-12 and hypocampal slice.More than measuring only as an example, determine that the tissue protective effect of recombinant tissue protectiveness cell factor and/or other suitable mensuration of bone marrow effect are that those of ordinary skills are known, also is that the present invention is desired.

5.5. Pharmaceutical composition of the present invention

In the practice of one aspect of the present invention; any approach of recombinant tissue protectiveness cell factor that can be by enough levels are provided in vascular system; give mammal with the above-mentioned Pharmaceutical composition that contains recombinant tissue protectiveness cell factor, pass through endothelial cell barrier and the useful influence of pairing effect cell to allow transhipment.When being used for perfused tissue or organ, can obtain similar results.The erythropoietin(EPO) mutain is used under the situation of ex vivo perfusion therein, and recombinant tissue protectiveness cell factor can be any type of erythropoietin(EPO) mutain, for example above-mentioned recombinant tissue protectiveness cell factor.At cell or tissue is that non-vascularization and/or administration are by soaking under the situation of cell or tissue with the present composition, and described Pharmaceutical composition provides the recombinant tissue protectiveness cell factor of the useful effective dose of pairing effect cell.The endothelial cell barrier that the transhipment of recombinant tissue protectiveness cell factor is passed through comprises that tight connection, perforation (perforated) connect, break-through (fenestrated) connects and the endothelial barrier of interior other any kind that exists of mammalian body.Preferred barrier is that endothelial cell closely connects, but the present invention is not limited to this.

Above-mentioned recombinant tissue protectiveness cell factor is generally used for the therapeutic treatment or the prophylactic treatment of following human diseases: the central nervous system disease or peripheral nervous disease and illness in eye, angiocardiopathy, heart and lung diseases, breathing problem, ephrosis, urethral disease and genital diseases, enterogastric diseases and cryptorrhea and the metabolic disorder that mainly have neurology symptom or psychiatric symptom.Specifically, described illness and disease comprise the hypoxemia illness, described illness has adverse effect to excitable tissue, and described excitable tissue is the excitable tissue in brain, the heart, retina/central nervous system tissues such as eye, peripheral nervous system tissue or heart tissue or retinal tissue for example.Therefore, the present invention's infringement of can be used for treating or preventing the hypoxemia illness by various illnesss and situation to cause to excitable tissue.The limiting examples such as the following table of described illness and situation provide.

In the example by the pathologic conditions protection of the medicable neuronal tissue of the present invention, described pathologic conditions comprises the illness that is caused by neuronal tissue's oxygenate reduction.Can be by method of the present invention, treatment reduce the oxygen availability of neuronal tissue and cause stress, damage and finally cause any illness of neuronal cell death.Be commonly referred to hypoxemia and/or ischemic these illnesss and be cause by following disease or include but not limited to following disease: apoplexy, vascular occlusion, antenatal anoxic or postpartum anoxic, (suffocation) suffocates, the plug of choking, approximate drowning, anthracemia, smog sucks, the wound that comprises operation and radiotherapy, (asphyxia) suffocates, epilepsy, hypoglycemia, chronic obstructive disease of lung, pulmonary emphysema, adult respiratory distress syndrome (ARDS), hypotensive shock, septic shock, anaphylactic shock, insulin shock, sickle cell crisis, asystole, dysrhythmia, neurologically handicapped due to nitrogen narcosis and the cardiovascular shunt operation.

In one embodiment, for example can give concrete recombinant tissue protectiveness composition of cellular factors, to prevent damage or caused damage of tissue damage risk or tissue damage in operations such as for example tumor resection or aneurysm excision.Can causing or other pathologic conditions of causing comprises insulin excessive (being also referred to as iatrogenic hyperinsulinism), insulinoma, GHD, hypoadrenocorticism, overdose and some tumour by methods described herein treatments by hypoglycemia.

But other pathologic conditions that is caused by the tissue damage of excitor nerve unit comprises epilepsy, for example epilepsy, convulsions or Chronic Epilepsy.Other can treat optic nerve lesion and neurone loss that illness and disease include but not limited to for example apoplexy, multiple sclerosis, low blood pressure, asystole, alzheimer's disease, Parkinson's, cerebral palsy, cerebral trauma or spinal cord injuries receptor, acquired immune deficiency syndrome (AIDS) dementia, relevant cognitive decrease of age, failure of memory, amyotrophic lateral sclerosis, epilepsy, alcoholism, treat retinal ischemic, caused by glaucoma.

Composition that the present invention is concrete and method can be used for treating the inflammation that is caused by illness or various wound, for example physics or chemically induced inflammation.Described wound can comprise vasculitis, chronic bronchitis, pancreatitis, osteomyelitis, rheumatoid arthritis, glomerulonephritis, optic neuritis, temporal arteritis, encephalitis, meningitis, transverse myelitis, dermatomyositis, polymyositis, necrotizing fasciitis, hepatitis and NEC.

Evidence suggests that the activation astroglia can be brought into play cytotoxicity to neuron by producing neurotoxin.Can discharge nitrogen monoxide during Deiter's cells response cerebral ischemia; reactive oxygen species and cell factor are (referring to Becker; K.J.2001.Targeting the centralnervous system inflammatory response in ischemic stroke (the inflammation of the central nervous system reaction of targeted local ishemic stroke) .Curr Opinion Neurol 14:349-353 and Mattson; M.P.; Culmsee; C. and Yu; Z.F.2000.Apoptotic and Antiapoptoticmechanisms in stroke (Apoptosis of apoplexy and anti-apoptosis mechanism) .Cell Tissue Res301:173-187.) .Research further shows; in the neurodegeneration model; and neuroglial activation and produce inflammatory cytokine subsequently and depend on that the primary neure damage is (referring to 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 neurocyte activates neuroglia by discharging the proteinase 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 ofproinflammatory cytokines (inhibition of the Guang winter protease-1 sample activity that is caused by Ac-Tyr-Val-Ala-Asp-chloromethane ketone is included in the cerebral ischemia by the minimizing of apoptotic reduction and pro-inflammatory cytokine, long-time continuation neuroprotective) .J Neurosci.20:4398-4404). Inflammation and neuroglial activation are common for the multi-form of the neurodegenerative disease that comprises cerebral ischemia, cerebral trauma and EAE, erythropoietin (EPO) performance cell protective effect in these diseases.The inhibition that the pair cell factor that is caused by erythropoietin (EPO) produces can mediate its protective effect to small part.Yet, be different from that direct inhibition TNF produces such as " classics " anti-inflammatory cytokines such as IL-10 and IL-13, erythropoietin (EPO) be it seems only just activity when having neuronal death.

As if though do not wish to be subjected to the constraint of any concrete theory, this anti-inflammatory activity can be explained with some non-limiting theory hypothesis ground.At first, because erythropoietin(EPO) stops Apoptosis, so will stop the inflammatory events that triggers by Apoptosis.In addition, erythropoietin(EPO) can stop dying neuron to discharge molecular signal, can excite nerve spongiocyte or can directly act on Deiter's cells to reduce their reactions to these products of described signal.Another possibility is that the erythropoietin(EPO) target is more approaching and trigger the inflammation cascade member (for example Guang winter proteinase 1, active oxygen or nitrogen intermediate product) of Apoptosis and inflammation.

As if in addition, erythropoietin(EPO) provides anti-inflammatory protection, and not usually with other anti-inflammatory compound relevant effect of rebounding of dexamethasone for example.And, though do not wish to be subjected to the constraint of any concrete theory, this seemingly because erythropoietin(EPO) to the multiple goal neurotoxin for example due to the effect of nitrogen monoxide (NO).Although the NO of activation astroglia and microglia generation neurotoxicity amount when responding different wound, NO has many purposes in body, comprise the adjusting of necessary physiological function.Therefore, although use anti-inflammatory agent to come amelioration of inflammation,, also can disturb these chemical actions in the injury repair that causes by wound that causes inflammation if anti-inflammatory agent has oversize half life by suppressing NO or other neurotoxin.Suppose recombinant tissue protectiveness cell factor energy amelioration of inflammation of the present invention and do not disturb for example repair ability of NO of neurotoxin.

Composition that the present invention is concrete and method can be used for treating the illness and the infringement of retinal tissue.Described disease includes but not limited to treat retinal ischemic, macular degeneration, retinal detachment, retinitis pigmentosa, arteriosclerotic retinopathy, hypertensive retionpathy, retinal arterial obstruction, retinal vein obstruction, low blood pressure and diabetic retinopathy.

In another embodiment, method of the present invention and principle can be used for protecting or treat the infringement that the radiation damage of excitable tissue causes.The further purposes of the inventive method is to be used for the treatment of such as neurotoxins such as domoic acid mytilotoxism, lathyrisms to poison and Guam disease, amyotrophic lateral sclerosis and Parkinson's.

As mentioned above, the present invention also relates to strengthen the method for mammal excitable tissue function, promptly by the above-mentioned recombinant tissue protectiveness of administered peripherally cell factor.Various diseases and illness all are fit to treat with this method, and this method strengthens cognitive function when being used in no any illness or disease.These purposes of the present invention have more detailed description hereinafter, are included in the study of people and non-human mammal and the enhancing in the training.

Method treatment that can be by this aspect of the present invention, the illness and the disease that relate to central nervous system include but not limited to mood disorder, anxiety disorder, depression, autism, scatterbrained hyperactivity disorder and cognition dysfunction.These illnesss are benefited from the enhancing of neuronal function.Comprise for example interruptions of sleep, sleep apnea and travelling associated disorders according to medicable other obstacle of the present invention; Subarachnoid hemorrhage and aneurysm are hemorrhage, hypotensive shock, concussion damage, septic shock, anaphylactic shock and various encephalitis and meningitis sequela, for example connective tissue disease (CTD) correlativity encephalitis (cerebritide) lupus for example.Other purposes comprises prevention or protective effect, in order to avoid poison and Guam disease, amyotrophic lateral sclerosis, Parkinson's such as neurotoxins such as domoic acid mytilotoxism, lathyrisms; The aftertreatment of embolic damage or ischemic injury; Full brain irradiation; Sickle cell crisis; And convulsions.

The illness of other type that can be by method of the present invention treatment comprises heredity or acquired mitochondria dysfunction, and described obstacle is to be the various neuropathic reason of characteristic feature with neure damage and death.For example the feature of SNE (subacute necrotizing encephalopathy) is carrying out property vision loss and encephalopathic, is because neurone loss and myopathy.In these cases, the metabolism of deficiency mitochondria can not be supplied sufficiently high energy substrate, to give excitable cell metabolism energize.The erythropoietin receptor active regulator can make the function optimization of destruction in various mitochondrial diseases.As mentioned above, hypoxemia illness negative effect excitable tissue.Described excitable tissue includes but not limited to central nervous system tissue, peripheral nervous system tissue and heart tissue.Except above-mentioned illness, method of the present invention can be used for treating the imbedibility poisoning and for example sucks carbon monoxide and smog, serious asthma, adult respiratory distress syndrome (ARDS), suffocates and approximate drowning.Produce the hypoxemia illness or induce other illness of excitable tissue's damage to comprise and to occur in the improper tumour (insulinoma) that gives the hypoglycemia of insulin or produce insulin by alternate manner.

Think that the various neural mental handicape (neuropsychological disorder) that originates from excitable tissue damage can treat by the inventive method.The chronic obstacle that wherein relates to neure damage and treat with the present invention comprises central nervous system and/or peripheral nervous system associated disorders, comprise relevant cognitive decrease of age and senile dementia, Chronic Epilepsy, alzheimer's disease, Parkinson's, dull-witted, failure of memory, amyotrophic lateral sclerosis, multiple sclerosis, tuberous sclerosis, the Wilson disease, benumb on cerebral palsy and the carrying out property nuclear, the Guam disease, thunder dimension corpusculum dementia (Lewy body dementia), prion disease is spongiform encephalopathy infectiousness spongiform encephalopathy for example for example, Huntington (Huntington ' s disease), myotonia atrophica, Friedreich ataxia (Freidrich ataxia) and other incoordination and tourette's syndrome (Gilles de la Tourettle ' s syndrome), epilepsy is epilepsy and Chronic Epilepsy for example, apoplexy, cerebral trauma or spinal cord injuries receptor, the acquired immune deficiency syndrome (AIDS) dementia, alcoholism, autism, treat retinal ischemic, glaucoma, the autonomic function obstacle is hypertension and sleep-disorder for example, and includes but not limited to following neuropsychiatric disorders (neuropsychiatric disorder): schizophrenia, schizoaffective disorder, scatterbrained hyperactivity disorder, dysthymic disorder, heavy melancholy sexual dysfunction, mania, obsessive-compulsive disorder, mentation drug use obstacle, anxiety disorder, panic disorder and single phase property and two-phase disposition sense obstacle.Other neuropsychiatric disorders and neurodegenerative disease comprise the obstacle of listing in phrenoblabia (DSM) diagnostic and statistical manual of American Psychiatric Association (American Psychiatric Association) for example, and the latest edition IV of described document all is attached to herein by reference.

In another embodiment, the reorganization chimeric toxin molecule that comprises recombinant tissue protectiveness cell factor can be used for therapeutic and gives toxin, with treatment proliferative disease cancer for example, or viral disease subacute sclerosing panencephalitis for example.

Following table is listed extra exemplary, the non-limiting indication as various illnesss and disease that can be by above-mentioned recombinant tissue protectiveness cytokine therapy.

As mentioned above, these diseases, obstacle or illness only are the scopes that explanation recombinant tissue protectiveness cell factor of the present invention provides benefit.Therefore, the present invention generally provides therapeutic treatment or the prophylactic treatment to the result who is caused by mechanical trauma or human diseases.Be preferred for disease, obstacle or the treatment of conditions treatment or the prophylactic treatment of CNS and/or peripheral nervous system.Be provided for having disease, obstacle or the treatment of conditions treatment or the prophylactic treatment of psychiatry problem.Be provided for including but not limited to following disease, obstacle or treatment of conditions treatment or prophylactic treatment: illness in eye, angiocardiopathy, heart and lung diseases, breathing problem, ephrosis, urethral disease, genital diseases, enterogastric diseases, cryptorrhea and metabolic disorder.

In one embodiment, can system give the Pharmaceutical composition of recombinant tissue protectiveness cell factor, with protection or intensifier target cell, tissue or organ.Described administration can be outside stomach and intestine, by sucking or mucosal, in for example oral, via intranasal application, rectum, the vagina, hypogloeeis, mucous membrane be down or through skin.Administration preferably outside stomach and intestine, for example by intravenous or intraperitoneal injection, and also includes but not limited to intra-arterial, intramuscular, intracutaneous and subcutaneous administration.

By using perfusion liquid, being expelled to other methods of administration such as organ or other topical approach, the Pharmaceutical composition of the similar level that produces aforesaid recombinant tissue protectiveness cell factor will be provided for for example.Preferred levels is about 0.01pM-30nM.

Pharmaceutical composition of the present invention can comprise the compound and the pharmaceutically acceptable carrier for the treatment of effective dose.In a specific embodiment, term " pharmaceutically acceptable " is meant by federal government or state government's approved by management, perhaps lists American Pharmacopeia in or other is generally acknowledged foreign pharmacopeia, is used for animal, especially people.Term " carrier " is meant thinning agent, auxiliary material, excipient or the solvent that gives with therapeutic agent.Described pharmaceutical carrier can be a sterile liquid, for example brine solution and the oil that comprises oil, animal oil, vegetable oil or synthetic oil, for example peanut oil, soybean oil, mineral oil, sesame wet goods.When Pharmaceutical composition during through intravenous administration, preferably brine solution is as carrier.Brine solution and G/W and glycerite also can be used as liquid-carrier, are particularly useful for parenteral solution.Suitable pharmaceutical excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, odium stearate, glyceryl monostearate, talcum powder, sodium chloride, skimmed milk power, glycerine, propylene glycol, water, ethanol etc.If desired, described composition also can contain a small amount of wetting agent or emulsifying agent or pH buffering agent.These compositions can be following form: solution, supensoid agent, emulsion, tablet, pill, capsule, powder, sustained release preparation etc.Described composition can with conventional adhesive and carrier for example triglyceride be mixed with suppository.Compound of the present invention can be mixed with neutral form or salt form.Pharmaceutically acceptable salt comprises the salt that forms with free amine group, for example derived from the salt of hydrochloric acid, phosphoric acid, acetate, oxalic acid, tartrate etc.; With the salt that forms with free carboxy, for example derived from the salt of NaOH, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine etc.The case description of suitable pharmaceutical carrier is in E.W.Martin " Remington ' s Pharmaceutical Sciences ".Described composition can contain the compound for the treatment of effective dose, preferably with pure form, and with an amount of carrier, so that the form that is fit to give the patient is provided.Described preparation should be fit to mode of administration.

The Pharmaceutical composition that is suitable for oral administration can be capsule or tablet; Powder or granule; Solution, syrup or supensoid agent (in water or on-aqueous liquid); Edible foaming agent or whipping agent (whip); Or emulsion.Tablet or hard-gelatin capsules can comprise lactose, starch or derivatives thereof, dolomol, saccharin sodium, cellulose, magnesium carbonate, stearic acid or its salt.Gelseal can comprise vegetable oil, wax, fat, semisolid or liquid polyol etc.Solution and syrup can comprise water, polyvalent alcohol and sugar.

The activating agent that expection is used for oral administration can mix (for example can use glyceryl monostearate or glycerol distearate) with the material dressing of this activating agent of delay disintegration and/or absorption in intestines and stomach or with described material.Therefore, the lasting release of activating agent can reach a few hours, and can protect described activating agent to avoid being degraded under one's belt in case of necessity.Can prepare Pharmaceutical composition for oral administration, so that activating agent is because specific pH or enzymatic hydrolysis condition and discharge at concrete intestines and stomach position.

The Pharmaceutical composition that is suitable for percutaneous dosing can be that expection keeps and the long-time discrete patch that contacts closely of acceptor epidermis.The Pharmaceutical composition that is suitable for topical can be ointment, cream, supensoid agent, lotion, powder, solution, paste, gel, spray, gasoloid or finish.For the topical of skin, oral cavity, eye or other outside organization, preferably adopt local with ointment or cream.When being mixed with ointment, active component can use with paraffin or water-miscible ointment base.Perhaps, active component can be formulated in the cream with oil-in-water matrix or Water-In-Oil matrix.The Pharmaceutical composition that is suitable for a topical comprises eye drops.In these compositions, active component dissolves in or is suspended in suitable carrier for example in the aqueous solvent.The Pharmaceutical composition that is suitable for oral cavity local medication comprises dragee, pastille and collutory.

The Pharmaceutical composition that is suitable for nasal cavity and pulmonary administration can comprise for example pulvis solid carriers such as (preferably particle size range are the 20-500 micron).Pulvis can suck (promptly by the container of splendid attire pulvis is sucked fast the mode of nasal cavity near nose) administration.Perhaps, the composition that is suitable for nasal-cavity administration can comprise liquid-carrier, for example nasal mist or nasal drop.Perhaps, can suck or by leading to pharyngeal oral-cavity device, reach and directly be drawn into lung by the deep.These compositions can comprise the aqueous solution agent or the oily solution agent of active component.Inhalation can provide with special suitable device with composition, and described device includes but not limited to the gasoloid, sprayer or the insufflator that pressurize, can produce described device, so that the active component of predetermined close is provided.In a preferred embodiment, with Pharmaceutical composition of the present invention by nasal cavity or pharyngeal nasal cavity or the lung of directly giving.

The Pharmaceutical composition that is suitable for rectally can be suppository or enema.The Pharmaceutical composition that is suitable for vagina administration can be pessary, cotton balls, cream, gel, paste, foaming agent or spray agent.

The Pharmaceutical composition that is suitable for parenteral comprises and can contain antioxidant, buffering agent, bacteriostatic agent and give described composition and intended recipient blood waits the water-based of the solute that oozes and non-aqueous aseptic injection with solution or supensoid agent substantially.Other composition that can have in the described composition comprises for example water, alcohols, polyvalent alcohol, glycerine and vegetable oil.The composition that is suitable for parenteral can be single dose container or multi-dose container, for example Mi Feng ampoule and bottle, and can be stored under freeze-dried (freeze-drying) condition, only need face with preceding adding sterile liquid carrier for example the Injectable sterile salt solusion get final product.Facing the injection solution agent and the supensoid agent of joining can be from sterile powder injection, granula and preparation tablets.In one embodiment; can be provided under ambulance, emergency ward and the battlefield situation the urgent automatic injector that comprises recombinant tissue protectiveness cytokine injections liquid that uses, in addition the middle automedication that also can be used for being in, particularly may take place traumatic cut-out for example mower use under the careless situation.By as early as possible (even before the medical worker reaches the spot; perhaps the cut the wounded of toe arrives before the emergency ward) give recombinant tissue protectiveness cell factor at place of incision multidigit point; after the pin that cuts off or toe were planted again, the possibility of its cell and tissue survival can improve.

In a preferred embodiment, described composition is mixed with the Pharmaceutical composition that is suitable for the intravenous administration of human according to conventional method.Usually, the composition that is used for intravenous administration is the solution that is dissolved in sterile isotonic water-based buffering agent.If necessary, described composition also can comprise for example lidocaine of solubilizer and local anesthetic, to alleviate the pain of injection site.Usually, described component can provide separately or mix in unit dosage forms and provide, for example at the airtight container of indication activating agent quantity freeze dried powder or the anhydrous concentrating agents in ampoule or the sachet for example.When infusion gives described composition, it can be formulated in the infusion bottle that sterile pharmaceutical grade water or salt solution are housed.When injection gives described composition, can provide the ampoule of Sterile Saline, before administration so that described component can be mixed.

Suppository contains scope usually at the active component of 0.5% (weight) to 10% (weight); Oral formulations preferably contains 10% to 95% active component.

Can be provided for transplant organ immersion, situ perfusion or before the results organ, give the perfusion liquid composition of organ donor blood vessel.Described Pharmaceutical composition can comprise and not be suitable for acute or chronic, part or system gives the individual recombinant tissue protectiveness cell factor or the form of recombinant tissue protectiveness cell factor; but the form of described recombinant tissue protectiveness cell factor or recombinant tissue protectiveness cell factor can be before the level of elimination or the wherein contained recombinant tissue protectiveness cell factor of reduction, before treated organ or tissue being exposed or feed back normal circulation, performance this paper predictive role in corpse, organ soak solution, organ perfusion's liquid or situ perfusion liquid.

The present invention also provides a kind of drug packages or medicine box that comprises one or more containers of the component that one or more Pharmaceutical compositions of the present invention are housed.The optional government department that has of described container is to production, the use of medicine or biological products or the notice of selling the prescribed form that requires, and this notice has reflected the approval of production, use or sale to human drugs.

In another embodiment, for example can in controlled release system, give recombinant tissue protectiveness cell factor.For example can adopt intravenous infusion, implantable osmotic pump, transdermal patch, liposome or other administering mode to give described polypeptide.In one embodiment, can use pump (referring to Langer, referring to above; Sefton, 1987, CRC Crit.Ref.Biomed.Eng.14:201; Buchwald etc., 1980, Surgery88:507; Saudek etc., 1989, N.Engl.J.Med. 321:574).In another embodiment, can in carrier, especially liposome, give described compound (referring to Langer, Science 249:1527-1533 (1990); Treat etc., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (writing), Liss, New York, 353-365 page or leaf (1989); WO91/04014; United States Patent (USP) the 4th, 704, No. 355; Lopez-Berestein, ibid, the 317-327 page or leaf; Generally referring to above).In another embodiment, can use polymeric material (referring to Medical Applications of Controlled Release, Langer and Wise (writing), CRC Press:Boca Raton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (writing), Wiley:New York (1984); Ranger and Peppas, J.Macromol.Sci.Rev.Macromol.Chem.23:61,1953; Other sees Levy etc., 1985, and Science 228:190; During etc., 1989, Ann.Neurol.25:351; Howard etc., 1989, J.Neurosurg.71:105).

In another embodiment, controlled release system can be placed on the treatment target be target cell, tissue or organ near, therefore only sub-fraction that need the amount of being administered systemically is (referring to for example Goodson, be stated from Medical Applications of Controlled Release, the 2nd volume, the 115-138 page or leaf, referring to above, 1984).The summary of relevant other controlled release system can be referring to Langer (1990, Science 249:1527-1533).

In another embodiment, can via intranasal application, oral, rectum, vagina or sublingual administration, give the suitably recombinant tissue protectiveness cell factor of preparation.

In a specific embodiment, wish position topical administration recombinant tissue protectiveness of the present invention cell factor composition to the needs treatment; This can such as but not limited to by local infusion, topical application in the operation for example with post-operative wound dressing use, by injection, by conduit, finish by suppository or by implant, described implant is porous, atresia or gelatin class material, comprises film or fiber such as silicone rubber membrane etc.

The technician will easily determine the selection of preferred effective dose according to the known some factors of those of ordinary skills.Described factor comprises pharmacokinetic parameters such as the concrete form of recombinant tissue protectiveness cell factor and bioavilability thereof, metabolism, half life, and these parameters are set up in being generally used for obtaining the conventional development sequence of the legal approval of medicinal compound.Other factors when considering dosage comprises the well-known other factors of illness to be treated or disease or the benefit that seeks out, weight in patients, method of administration (no matter administration is acute or chronic), while administered agents and the influence medicine effect of giving in normal individual.Therefore exact dose will for example decide according to each patient's illness and immune state and according to standard clinical techniques according to doctor's judgement and each patient's situation.

In another aspect of this invention, being provided for the perfusion of transplant organ and the perfusion liquid or the perfusion of preservation uses solution, described perfusion liquid to comprise the recombinant tissue protectiveness cell factor of protective effect cell and relevant cell, tissue or organ effective dose.Transplanting includes but not limited to heteroplastic transplantation and autotransplatntation, and described heteroplastic transplantation is just from donor harvesting organ (comprising cell, tissue or other body part) and be transplanted to not the isoacceptor; Described autotransplatntation just organ is taken out and is replaced another part from the part of body, comprises that wherein organ is removed, the excision of exsomatizing, the bench surgery operation of repairing or carrying out other operational example such as tumor resection, replacing then.In one embodiment, described perfusion liquid is University of Wisconsin (UW) solution (United States Patent (USP) the 4th, 798, No. 824), described solution contain have an appointment 1U/ml to about 25 U/ml erythropoietin(EPO), (molecular weight is about 200 to 5% Hydroxyethyl Starch, 000 to about 300,000, and be substantially free of ethylene glycol, ethylene chlorhydrin, sodium chloride and acetone); 25mM KH ₂PO ₄The 3mM glutathione; The 5mM adenosine; 10mM glucose; 10mM HEPES damping fluid; The 5mM magnesium gluconate; 1.5mM CaCl ₂The 105mM gluconic acid sodium salt; 200,000 unit penicillin; 40 units of insulin; The 16mg dexamethasone; 12mg is phenol red; And pH is 7.4-7.5, and Morie osmolarity is about 320mOSm/l.Described solution is used for keeping the kidney and the pancreas of corpse before transplanting.Use described solution, storage life can prolong to surpass recommends to be used for 30 hours the limit that the corpse kidney is preserved.This concrete perfusion liquid only is used for illustrating that the tank solution of the recombinant tissue protectiveness cell factor that comprises effective dose can be suitable for present use.In another embodiment, described perfusion liquid contains the 0.01pg/ml that has an appointment to about 400ng/ml recombinant tissue protectiveness cell factor, or about 40ng/ml is to about 300ng/ml recombinant tissue protectiveness cell factor.As mentioned above, aspect this, can use any type of recombinant tissue protectiveness cell factor of the present invention.

Though being used for the preferred acceptor of the recombinant tissue protectiveness cell factor of this paper purpose is the people, the method for this paper can be applied to other mammal, especially performing animal, domestic animal, companion animals and zoo animal equally.Yet the present invention is not limited to this, and its benefit can be applicable to any mammal.

5.6. the therapeutical uses and the preventive use of recombinant tissue protectiveness cell factor

As described in following embodiment 1; there is erythropoietin receptor in the human brain capillary endothelium; the target that shows recombinant tissue protectiveness cell factor of the present invention is present in the human brain, and can directly transfer in human treatment or the prevention the zooscopy of these recombinant tissue protectiveness cell factors of the present invention.

In another aspect of this invention; be provided for strengthening not because of the method and composition of endothelial cell barrier with vascular system isolated cells, tissue or organ vitality; promptly by making described cell, tissue or organ directly contact the Pharmaceutical composition that comprises recombinant tissue protectiveness cell factor, the Pharmaceutical composition that perhaps will comprise recombinant tissue protectiveness cell factor gives or contacts the vascular system of described cell, tissue or organ.The activity that the effector cell strengthens in processed tissue or organ is the reason that positive effect plays a role.

As mentioned above, the present invention part is based on following discovery: erythropoietin molecule can be transported to the basilar memebrane surface from the endothelial cell luminal surface of capillary with endothelial cell tight coupler official, and described endothelial cell tight coupler official comprises for example brain, retina and testis.Therefore, the effector cell who passes through barrier is the target that is subject to the useful influence of recombinant tissue protectiveness cell factor, also is the target of the inventive method and contain effector cell and all or part of dependence effector cell's other cell type or tissue or organ.Though do not wish to be subjected to the constraint of any concrete theory; but behind the transcytosis of recombinant tissue protectiveness cell factor; recombinant tissue protectiveness cell factor can interact with erythropoietin receptor on the following effector cell: neuronal cell for example; the retina cell; the myocyte; core cell; pneumonocyte; liver cell; nephrocyte; small intestine cells; adrenal cortical cell; adrenal medullary cell; capillary endothelial cell; testicular cell; gonad cell; pancreatic cell; osteocyte; Skin Cell or endometrial cell; and receptors bind can the enabling signal transductory cascade; cause the activation of gene expression program in described effector cell or the tissue, cause protecting described cell or tissue or organ to exempt from for example toxin; chemotherapeutic; radiotherapy; the hypoxemia equivalent damage.Therefore, be described in more detail below and be used to protect the tissue that contains the effector cell to exempt from damage or hypoxia stress and strengthen the method for described function of organization.As mentioned above, method of the present invention can be used for human and other animal equally.

In the practice of one embodiment of the invention, mammalian subject has experienced the systemic chemotherapy that is used for the treatment of cancer, comprise have nervous lesion usually, the radiotherapy of spinoffs such as injury of lungs, heart injury, ovary damage or injury of testis.Before chemotherapy and/or radiotherapy and comprise the Pharmaceutical composition of above-mentioned recombinant tissue protectiveness cell factor simultaneously, exempt from the damage of chemotherapeutic to protect various tissues and organ, for example protect testis.Treatment can continue until the level of chemotherapeutic in circulation and reduces to mammalian organism is produced under the level of potential danger.

In the practice of another embodiment of the invention, to plan to gather in the crops various organs and be used for transplanting to a plurality of acceptors from the victim of traffic accident, wherein some need be grown distance and transport for a long time for this.Before the results organ, comprise the Pharmaceutical composition of recombinant tissue protectiveness cell factor as described herein for victim's infusion.The organ of the results that are used to transport pours into the perfusion liquid that contains recombinant tissue protectiveness cell factor as described herein, and is stored in the soak solution that comprises recombinant tissue protectiveness cell factor.Some organ continues perfusion with the perfusion liquid that pulsed perfusion equipment, usefulness contain with good grounds recombinant tissue protectiveness cell factor of the present invention.In transportation and transplanting and organ original position refilling process, minimal decay takes place in organ dysfunction.

In another embodiment of the invention, the operation of prosthetic heart valve needs temporary transient cardioplegia and arterial occlusion.Give patient's infusion per kilogram of body weight 4 μ g recombinant tissue protectiveness cell factors before the art.Described treatment prevention hypoxemia ischemic cellular damage, especially after perfusion again.

In another embodiment of the invention, for example in the cardiopulmonary bypass, can use recombinant tissue protectiveness cell factor of the present invention in any operation.In one embodiment, before the bypass, during and/or afterwards, comprise the Pharmaceutical composition of above-mentioned recombinant tissue protectiveness cell factor, with the protection brain, heart and other organ function.

Recombinant tissue protectiveness cell factor of the present invention therein is used for exsomatize using or Treatment Effects cell neuronal tissue for example; retinal tissue; core cell; pneumonocyte; liver cell; nephrocyte; small intestine cells; adrenal cortical cell; adrenal medullary cell; capillary endothelial cell; testicular cell; in the above-mentioned example of gonad cell or endometrial cell or tissue; the invention provides a kind of like this Pharmaceutical composition; its dosage unit form is applicable to protection or strengthens the effector cell who leaves vascular system; tissue or organ; the dosage range that described composition comprises every dosage unit is about 0.01pg-5mg; 1pg-5mg; 500pg-5mg; 1ng-5mg; 500ng-5mg; 1 μ g-5mg; the recombinant tissue protectiveness cell factor of 500 μ g-5mg or 1mg-5mg, and pharmaceutically acceptable carrier.In a preferred embodiment, the amount of described recombinant tissue protectiveness cell factor is in about 1ng-5mg scope.In a preferred embodiment, the recombinant tissue protectiveness cell factor right and wrong of above-mentioned composition are erythrogenic.

In another aspect of this invention, find that the animal EPO that experiences cerebral trauma can recover cognitive function.Recombinant tissue protectiveness cell factor of the present invention is expected to have the cell protective effect identical with EPO.Postpone or after 5 days or 30 days, compare with the dummy treatment animal, giving EPO still can restore funcitons, the ability that this shows EPO regeneration or recovers cerebration.Therefore, the present invention also relates to recombinant tissue protectiveness cell factor and be used for the treatment of purposes in the Pharmaceutical composition of cerebral trauma (for example 3 days, 5 days, 1 week, January or the treatment of longer time for a long time after being included in damage) and other cognition dysfunction in preparation.The present invention also relates to be used for after damage, treating the method for cognition dysfunction by the recombinant tissue protectiveness cell factor that gives effective dose.Any recombinant tissue protectiveness cell factor as described herein all can be used for this one side of the present invention.

In addition; any recombinant tissue protectiveness cell factor that this recovery aspect of the present invention relates to this paper is used for the purposes of Pharmaceutical composition of the recovery of cell, tissue or organ dysfunction in preparation, treatment and begin treatment for a long time afterwards wherein will begin in a minute after causing described handicapped initial injury.In addition, treat and to stride across in the course of disease of disease or illness in acute stage and chronic phase with recombinant tissue protectiveness cell factor of the present invention.

Recombinant tissue protectiveness cell factor of the present invention therein has under the situation of erythropoiesis activity; in a preferred embodiment, the each administration of recombinant tissue protectiveness cell factor can with between about 0.01pg and about 100 μ g/kg body weight, preferably about 1-50 μ g/kg body weight, the most preferably from about sosimetric system administration of 5-30 μ g/kg body weight.After giving recombinant tissue protectiveness cell factor, this effective dose should be enough to reach in the serum greater than about 10,000mU/ml, 15,000mU/ml or 20, the serum levels of the recombinant tissue protectiveness cell factor of 000mU/ml.Described serum levels can reach after administration in about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours.If necessary, described administration can repeat.For example, as long as clinical needs, can every day repeat administration, perhaps repeat administration after appropriate interval, for example every 1-12 week but preferably every 1-3 week.In one embodiment, the recombinant tissue protectiveness cell factor of effective dose and pharmaceutically acceptable carrier can be packaged into single dose bottle or other container.In another embodiment, the recombinant tissue protectiveness cell factor right and wrong that are used for this paper purpose are erythropoietic, can bring into play activity described herein but do not cause hemoglobin concentration or hematocrit increases.Expection therein provides under the situation of method of the present invention the recombinant tissue protectiveness cell factor of preferred non-erythropoiesis form for a long time.In another embodiment, to give recombinant tissue protectiveness cell factor greater than maximal stimulation red blood cell generation required dosage.As mentioned above, recombinant tissue protectiveness cell factor of the present invention not necessarily has the erythropoiesis activity, only is exemplary with the above-mentioned dosage with unit representation concerning reorganization tissue protective cell factor therefore; In this article, be provided for the molar equivalent of the dosage of any recombinant tissue protectiveness cell factor.

The invention still further relates to the method that endothelial cell barrier is passed through in the molecule transhipment that is used to promote in the mammalian body, promptly by comprising the composition of the specific molecular that associates with recombinant tissue protectiveness cell factor mentioned above.As mentioned above, closely connect between endothelial cell in some organ of body some molecule entered the generation barrier.For various treatment of conditions in the barrier organ, need the means that promote that pharmaceutical formulation passes through.Recombinant tissue protectiveness cell factor of the present invention is used to transmit other molecule and passes through blood brain barrier or other similar barrier as carrier.Preparation comprises molecule and the recombinant tissue protectiveness composition of cellular factors that barrier is passed through in hope, and described composition is peripherally administered, causes described composition to pass through the transcytosis of barrier.Waiting to transport with the molecule that passes through barrier and the association between the recombinant tissue protectiveness cell factor can be unsettled covalent bond, in this case after passing through barrier described molecule from the association of recombinant tissue protectiveness cell factor be released.If the association between described molecule and recombinant tissue protectiveness cell factor still can keep or not influence the required pharmacological activity of described molecule, can give described compound.

The technician knows the variety of way that is used for molecule and recombinant tissue protectiveness cell factor of the present invention and the association of above-mentioned other medicines, promptly by covalency, non-covalent and alternate manner.In addition, in experimental system, can easily carry out the evaluation of described compound efficacy.Can be unsettled by comprising, covalent bond, crosslinked etc. variety of way reach the association of molecule and tissue protective cell factor.Can use the interaction of biotin/avidin.As mentioned above, can prepare hybrid molecules, for example comprise the domain of molecule and cause the active domain of regulating of erythropoietin receptor with required pharmacological activity by reorganization or synthesis mode.

A molecule can be that multifunctional crosslinking chemical and recombinant tissue protectiveness cell factor are puted together by multifunctional molecule.Term used herein " multifunctional molecule " comprises the molecule formaldehyde for example with functional group of successive reaction more than once, and the molecule with more than reactive group.Term used herein " reactive group " be meant on the crosslinking chemical with molecule (for example remain to pass through endothelial cell barrier and transmit peptide, albumen, sugar, nucleic acid, especially hormone, microbiotic or anticarcinogen) on functional group reactions to form the functional group of crosslinking chemical and described intermolecular covalent bond.Term " functional group " keeps its standard implication in organic chemistry.Operable described multifunctional molecule is the biocompatibility coupling agent preferably, and promptly they are non-carcinogenic, nontoxic in vivo and do not have immunogenic basically.Test case multifunctional crosslinking chemical as known in the art and that describe in this article easily in animal model is to determine their biocompatibility.Described multifunctional molecule is preferably dual functional.Term used herein " bifunctioanl molecule " is meant the molecule with two reactive groups.Described bifunctioanl molecule can be special-shaped bifunctioanl molecule or homotype bifunctioanl molecule.Special-shaped bifunctional cross-linker allows the vector combination.Preferred especially multifunctional molecule has enough dissolubilities in water, because cross-linking reaction occurs in aqueous solution for example in the aqueous buffer solution of pH 6-8, and for more effective bio distribution, it is water-soluble that the gained conjugate keeps.Usually, multifunctional molecule and amino or mercapto functional group covalent bonding.Yet, with other functional group for example the multifunctional molecule of carboxylic acid or hydroxyl reaction be also included among the present invention.

The homotype bifunctioanl molecule has at least two identical active function groups.Active function groups on the homotype bifunctioanl molecule comprises for example aldehyde radical and active ester group.Homotype bifunctioanl molecule with aldehyde radical comprises for example glutaraldehyde and subaraldehyde.Glutaraldehyde is disclosed in Poznansky etc. as the application of crosslinking chemical, and Science 223,1304-1306 (1984).Homotype bifunctioanl molecule with at least two active ester unit comprises the ester and the N-hydroxy-succinamide of dicarboxylic acid.Some examples of described N-succinimido ester comprise two succinimidos, two succinimido suberates and two sulfo-s-two-(succinyl phosphorons amino propyl acid ester) and solubility thereof two-sulfonic acid and two-sulfonate their sodium salt and sylvite for example.These homotype difunctionality reagent can derive from various commercial source (Pierce, Rockford, Illinois).

Special-shaped bifunctioanl molecule has at least two different reactive groups.Described reactive group and different functional groups (for example erythropoietin(EPO) mutain and described molecule) reaction.These two different functional groups that go up the reactive group reaction with special-shaped bifunctional cross-linker are normally amino, for example the ε amino of lysine; Sulfydryl, for example sulfydryl of halfcystine; Carboxylic acid, for example carboxylic acid on the aspartic acid; Or hydroxyl, for example hydroxyl on the serine.Certainly, recombinant tissue protectiveness cell factor of the present invention can lack particular amino acid residue, and this will promote the crosslinked of natural erythropoietin(EPO), but one skilled in the art will appreciate that residue part and therefore crosslinked available in the mutain of the present invention.

In addition, various recombinant tissue protectiveness cell factor molecule of the present invention can not be suitable for the group with the reaction of certain crosslinking chemical; Yet those skilled in the art will fully understand, select crosslinking chemical according to being used for the crosslinked useful group of erythropoietin(EPO) of the present invention.

When special-shaped bifunctioanl molecule reactive group formed covalent bond with amino, described covalent bond is amido bond or imido key normally.The reactive group that forms covalent bond with amino can be carboxylate group, halogen carbonyl or the ester group that for example activates.Described halogen carbonyl is the chloroformyl base preferably.Ester group is N-hydroxyl-succinimide ester group isoreactivity ester group for example preferably.

Usually, other functional group can be sulfydryl, the group that can change into sulfydryl or the group that can form covalent bond with sulfydryl.Usually, described covalent bond is thioether bond or disulfide bond.The reactive group that forms covalent bond with sulfydryl can be for example with two keys of the disulfide reaction of sulfydryl or activation.Contain can be with the reactive group of two keys of sulfydryl reaction for example maleimide and other group such as vinyl cyanide.Active disulfide bond is 2-pyridine two thio groups or 5,5 '-two sulfo-s-two-(2-nitrobenzoic acid) group for example.Some examples that contain the special-shaped difunctionality reagent of active disulfide bond comprise N-succinimido 3-(2-pyridine-two sulfo-) propionic ester (Carlsson etc., 1978, Biochem J., 173:723-737), S-4-succinimido oxo carbonyl-α-Jia Jibianji sodium thiosulfate and 4-succinimido oxo carbonyl-Alpha-Methyl-(2-pyridine two sulfo-s) toluene.Preferred N-succinimido 3-(2-pyridine two sulfo-s) propionic ester.Comprise and have and some examples of the special-shaped difunctionality reagent of the reactive group of two keys of sulfydryl reaction comprise maleimide benzoic ether between succinimido 4-(N-maleimide methyl) cyclohexane-1-carboxylate and succinimido.

Other special-shaped bifunctioanl molecule comprises succinimido 3-(maleimide) propionic ester, thiosuccimide base 4-(to maleimide-phenyl) butyric ester, thiosuccimide base 4-(N-maleimide methyl-cyclohexyl alkane)-1-carboxylate, maleimide benzoyl-N-hydroxyl-succinimide ester.The sulfonate sodium of maleimide benzoic ether between preferred succinimido.Many above-mentioned special-shaped difunctionality reagent and sulfonate thereof can derive from Pierce ChemicalCo., Rockford, Illinois USA.

The technician can easily determine to above-mentioned puting together be reversible or unsettled needs.Can be at the recombinant tissue protectiveness cell factor and the required pharmacological activity of in vitro test conjugate.If described conjugate keeps two specific characters, then its suitability can be tested in vivo.If the described molecule of puting together need be separated from recombinant tissue protectiveness cell factor by activity, preferably with the unstable bonding or the reversible association of recombinant tissue protectiveness cell factor.Before the test, also can test unstable characteristic in vivo with the standard in-vitro method.

About how preparing and using the extraneous information of they and other multifunctional reagent from following publication or in other available information of this area, to obtain:

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

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

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

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

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

6.Liu, F.T. etc., 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. etc., 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. etc., 1984, Biochem.81:7772-7776.

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

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

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

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

15.Novick, D. etc., 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. etc., 1984, J.Applied Biochem.6:56-63.

In addition, cross-linking method is summarized in Means and Feeney, and 1990, Bioconjugate Chem.1:2-12.

The barrier that said method and composition of the present invention passed through includes but not limited to blood brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier, blood-heart barrier, blood-kidney barrier and blood-uterus barrier.

Be used to transport the candidate molecules that passes through endothelial cell barrier and for example comprise hormone for example growth hormone, neurotrophic factor, microbiotic, antiviral agent or usually by the antibody of the excluded antifungal of the organ of brain and other barrier protection, peptide radiomimetic drug, antisense drug, antibiont active factors and antiviral agent, medical substance and anticarcinogen.The limiting examples of described molecule comprises for example growth hormone of hormone, nerve growth factor (NGF), neurotrophic factor derived from brain (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), transforminggrowthfactor-(TGF β 1), transforming grouth factor beta 2 (TGF β 2), transforming growth factor 3 (TGF β 3), interleukin-11, interleukin-22, interleukin-13 and interleukin 6, AZT, the antibody of anti-tumor necrosis factor and immunosuppressive drug be cyclosporin for example.In addition, dyestuff or mark can be connected on the erythropoietin(EPO) or on a kind of tissue protective cell factor of the present invention, so that diagnostic purpose manifests cell, tissue or organ in brain and other barrier organ.As an example, the mark that is used to manifest scab in the brain can be connected to erythropoietin(EPO) or tissue protective cell factor, so that determine the process of alzheimer's disease in patient's body.

The present invention also relates to treat that passing through endothelial cell by transcytosis closely connects molecule and the above-mentioned recombinant tissue protectiveness composition of cellular factors that barrier is transported a kind of comprising.The conjugate that the invention still further relates between molecule and above-mentioned recombinant tissue protectiveness cell factor is used for sending the purposes that described molecule passes through the Pharmaceutical composition of above-mentioned barrier in preparation.

Following as the exemplary non-limiting example of the present invention by reference, the present invention may be better understood.It is in order more to prove absolutely embodiment preferred of the present invention that following examples are provided.Yet they never should limit wide region of the present invention by any way.

6. embodiment

6.1. embodiment 1: the distribution of erythropoietin receptor in human brain

The normal brain that will cut in operation (no borderline tumor for example is provided in tumorectomy) is placed on immediately in the 0.1 M phosphate buffer (pH 7.4) of 5% acryl aldehyde and fixes 3 hours.Be cut into 40 microns slabs with vibratome.With free-floating section and adopt indirect antibody PAP method, with 1: 500 dilution erythropoietin receptor antiserum (deriving from Santa Cruz Biotechnology) carry out immunohistochemical staining.Cut into slices with the endogenous peroxidase activity of quencher (3% hydrogen peroxide in methanol was handled 30 minutes) with hydrogen peroxide pre-service tissue.Also omitting by first antibody and contrast by using suitable closure peptide (deriving from Santa Cruz Biotech.) to handle tissue, is specific to confirm dyeing to erythropoietin(EPO) (EPO) acceptor.

Fig. 1 shows that carrying out immunohistochemistry by the antibody with the anti-EPO acceptor of specificity measures, and the human brain capillary is expressed very high-caliber EPO acceptor.This provides EPO why can penetrate the mechanism of brain from the body circulation, although blood-brain barrier is arranged.

Fig. 2 shows that the EPO acceptor is concentrated and is arranged in human brain and forms the capillary of blood-brain barrier and on every side.

Carried out among Fig. 3 and Fig. 1 and the similar scheme of Fig. 2, except 10 microns sections be section with paraffin section, embedding be immersed in 4% paraformaldehyde fixing.Fig. 3 shows the high density EPO acceptor of human brain capillary luminal surface and reverse side, constitutes EPO is transported to anatomical basis in the brain from circulation.

Fig. 4 derives from the similar scheme with Fig. 3, and mirror is used, second antibody is used the colloid gold particle mark with powering after the ultramicrotome section except organizing.This figure shows, find the EPO acceptor the inner skin surface of human brain ( ^*) go up, in kytoplasm vesicle (arrow) and in neuroglia synaptic knobs (+), provide EPO to be transported to anatomical basis in the brain from circulation.

6.2. embodiment 2: erythropoietin(EPO) is passed through the tight barrier of blood-cerebrospinal fluid

With Sprague-Dawley bull rat anesthesia, give the recombinant human erythropoietin of 5000U/kg body weight in the peritonaeum.Got the cerebrospinal fluid sample up to 4 hours from Da Chi at interval at 30 minutes,, measure erythropoietin(EPO) concentration with sensitive enzyme-specific linked immunoassay.As shown in Figure 5, baseline erythropoietin(EPO) concentration is 8mU/ml among the CSF.After a few hours postponed, the erythropoietin(EPO) level began to rise among the CSF of measurement, to 2.5 hours and later significantly different with baseline concentrations (p＜0.01 level).The peak level of about 100mU/ml (0.1-100mU/ml) in the scope of the external protective effect of known performance.Time to peak occurs in about 3.5 hours, and this time interval peak serum levels significantly postpones (less than 1 hour).This experimental result shows, the level of signifiance of erythropoietin(EPO) can be passed through tight cell by the erythropoietin(EPO) that heavy dose of stomach and intestine give suitable concn outward and be connected and reach.

6.3. embodiment 3: recombinant tissue protectiveness cell factor

Adopt the following erythropoietin construct of following method preparation.Use is based on disclosed human cDNA sequence (registration number NM_000799), by the cDNA of PCR from human brain cDNA human cloning erythropoietin(EPO).Primer is introduced the Xba I site of Xho I site and the 3 ' end of cDNA 5 ' end.Primer sequence is:

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

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

With the PCR product cloning between the Xho I site and Xba I site of pCiNeo mammalian expression vector (Promega).Described clone is checked order, confirm except a base sequences match of this sequence and NM_000799.Base 418 in the coded sequence (from the ATG open numbering) is C rather than G, and the Arg that begins amino acid/11 40 from first methionine in total length EPO sequence becomes Gly.Yet this is from the normal sequence variation of original series and is present in the erythropoietin(EPO) of most of forms.

The coded sequence of erythropoietin cdna is as follows:

ATGGGGGTGCACGAATGTCCTGCCTGGCTGTGGCTTCTCCTGTCCCTGCTGTCGCTCCCTCTGGGCCTCCCAGTCCTGGGCGCCCCACCACGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGACGGGCTGTGCTGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAGAAGTCTGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTTGGTCAACTCTTCCCAGCCGTGGGAGCCCCTGCACTGCATGTGGATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTTCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGATGA(SEQ?IDNO：7).

The full length amino acid sequence of the erythropoietin(EPO) that this cDNA coding is following:

MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR(SEQ?ID?NO：10).

Preceding 27 amino acid residues of SEQ ID NO:10 comprise targeting sequencing.Unless indicate prefix term " total length ", otherwise the sudden change of all amino acid positions in the erythropoietin(EPO) sequence and wherein record is meant the position in the maturation protein.

By designing new oligonucleotides, the 6xHis mark is introduced the C end of people's epo protein, make and adopt following oligonucleotides that this 6 histidine is connected with Asp 192 in the full length sequence:

3’-6xhis-hEPO?5’-

GGTCTAGATCAATGGTGATGGTGATGATGGTCCCCTGTCCTGCAGGCC-3’(SEQID?NO：134)

Use HEPO-5-Xho I oligomer and 6xHis-mark oligomer,, and it is cloned between the Xho I site and Xba I site of pCiNeo mammalian expression vector by pcr amplification EPO cDNA.Again insertion is checked order and confirm sequence.

In 5.2 trifles,, introduce by the described site-directed oligonucleotide mutagenesis of this trifle to the sudden change that has C end 6xHis mark of people EPO cDNA sequence description.To mutant clon check order and confirm the sudden change.

Can use the whole bag of tricks of purifying recombinant tissue protectiveness of the present invention cell factor, include but not limited to the following scheme that is used in combination with the recombinant expressed tissue protective cell factor of the present invention of histidine mark.Recombinant cell (CHO-K1) supernatant (for example from (Ni-CAMHC RESIN:High Capacity Nickel Chelate Affinity Matrix, Sigma, the resin of catalog number N 3158)) thoroughly suspends and gentle upset again.Then, 100 μ l resin suspending liquid (being equivalent to 50 μ l packaging resins) are joined in the microcentrifugal tube (big or small 1.7ml).With potpourri with 8,000rpm in 4 ℃ centrifugal 5 minutes, the precipitation resin, abandoning supernatant then.This microcentrifuge is Megafuge 1.0R (Heraeus Instruments).Potpourri with 1ml deionized water (0.2 μ m filtration) washing 2 times, is removed ethanol.Resin is suspended in the 500 μ l level pads (50mM sodium phosphate, pH 8.0,0.3M NaCl, 10mM imidazoles) again, then potpourri is transferred in the 50ml tapered tube.The effective 500 μ l level pads flushing of microcentrifugation joins this quantity in the potpourri in the 50ml tapered tube then.With potpourri with 3,000rpm in 4 ℃ centrifugal 5 minutes, the precipitation resin.Supernatant is poured out and discarded.In conjunction with before, with 3,800rpm is centrifugal 5 minutes in 4 ℃ with sample (CHO-KI supernatant).Cell conditioned medium liquid is joined in this resin.Sample loading buffer (50mM sodium phosphate, pH 8.0,3M NaCl, 100mM imidazoles) is joined among the 1X, and on rotation platform, mixed 1 hour in 4 ℃ of gentlenesses.The example of used platform is Nutator (rotation platform) (Clay AdamsBrand).With 3,000rpm is centrifugal 5 minutes in 4 ℃ with potpourri.Supernatant taking-up and reservation are analyzed and ELISA (not combination) usefulness to be used as SDS-PAGE.Resin is suspended in the 500 μ l lavation buffer solutions again, then potpourri is transferred in the microcentrifugal tube.The 50ml tapered tube is washed with 500 μ l level pads, then this quantity is joined in the potpourri in the microcentrifugal tube.Then resin suspending liquid was mixed 10 minutes in 4 ℃ on rotation platform.With 8,000rpm is in 4 ℃ centrifugal 5 minutes (cleansing solution stays and does ELISA usefulness for the first time) with suspending liquid.Resin is suspended in the 1ml lavation buffer solution again, and resin suspending liquid mixed 10 minutes in 4 ℃ on rotation platform then, again washing resin.Cleansing solution is discarded.Then, add 75 μ l elution buffers (50mM sodium phosphate, pH 8.0,0.3M NaCl, 500mM imidazoles).Resin was mixed 10 minutes in 4 ℃ on rotation platform.8,000rpm is centrifugal 5 minutes in 4 ℃ with potpourri.Supernatant is taken out and reservation.Histidine tagged protein is just in this flow point.For wash-out goes out greater protein, add 75 μ l elution buffers (50mM sodium phosphate, pH 8.0,0.3M NaCl, 500mM imidazoles) again.Resin was mixed 10 minutes in 4 ℃ on rotation platform again.8,000rpm is centrifugal again 5 minutes in 4 ℃ with potpourri.The wash-out flow point is kept as a amalgamation liquid or the flow point that separates.

Perhaps, adopt the histidine mark cell factor of following method separation and purification.The collection condition nutrient culture media also filters through 0.45 μ m filter.Then 50ml equal portions sample pipetting volume is arrived with 20mM sodium phosphate pH 7.4 balances also with 2.5ml 100mM NiSO ₄The 5ml HiTrap chelate column (Amersham biosciences) of activation.With pillar with 20 mM sodium phosphate pH 7.4 washing, and with 0M-0.5M imidazoles (being dissolved in 20mM sodium phosphate pH 7.4) gradient elution above 25 times of column volumes.Flow velocity is the 5ml/ branch, and the flow point volume is 5ml.

By the recombinant tissue protectiveness cell factor that exists in SDS-PAGE and the EPO elisa assay flow point.Merge positive flow point, and to 10mM Tris pH 7.0 dialysis.The amalgamation liquid application of sample is to the 1ml HiTrap Q HP (Amershambiosciences) with 10mM Tris pH 7.0 balances.After the level pad washing, sample is used above 10 times of column volume NaCl gradients to the 0.5M wash-out with the flow velocity that 1ml/ divides.(anti-His6, antibody ROCHE) are analyzed by SDS-PAGE, EPO ELISA and Western blotting at six His marks to collect 1m flow point and use.Merging the flow point that contains recombinant tissue protectiveness cell factor, and concentrate with centricon, is 10kDa (Amicon) by size, and final volume is 1-2ml.

By SDS-PAGE (NuPage 4-12%; Invitrogen) analyze the final amalgamation liquid of recombinant tissue protectiveness cell factor, and, develop the color with NOVEX colloid indigo plant (Invitrogen) with the scheme that the manufacturer recommends.Can judge the purity of recombinant tissue protectiveness cell factor according to the gained gel.A band is only arranged corresponding to glycosylation recombinant tissue protectiveness cell factor on each swimming lane of gel, illustrate that the isolated cells factor is a high-purity.

Use fat transfection amine, make all plasmids all transfection in CHO-1 cell or COS-7 cell.After the transfection 48-72 hour, the nutrient culture media of collecting cell.This nutrient culture media can be directly or is measured or neuron be used for after measuring measuring by ELISA and detects EPO at erythropoiesis behind the purifying.

Use following oligonucleotides, introduce sudden change K45D, S100E and the A30N/H32T of people EPO cDNA sequence by site-directed oligonucleotide mutagenesis:

HEPO-S100E-cochain 5 '-

CATGTGGATAAAGCCGTCGAGGGCCTTCGCAGCCTCACCACTCTG-3’(SEQ?IDNO：11)

Chain 5 under the HEPO-S100E-'-

CAGAGTGGTGAGGCTGCGAAGGCCCTCGACGGCTTTATCCACATG-3’(SEQ?IDNO：12)

HEPO-K45D-cochain 5 '-

GAGAATATCACTGTCCCAGACACCGACGTTAATTTCTATGCCTGG-3’(SEQ?IDNO：13)

Chain 5 under the HEPO-K45D-'-

CCAGGCATAGAAATTAACGTCGGTGTCTGGGACAGTGATATTCTC-3’(SEQ?IDNO：14)

HEPO-A30N/H32T-cochain 5 '-

GAATATCACGACGGGCTGTAATGAAACCTGCAGCTTGAATGAG-3’(SEQ?ID?NO：132)

Chain 5 under the HEPO-A30N/H32T-'-

CTCATTCAAGCTGCAGGTTTCATTACAGCCCGTCGTGATATTC-3’(SEQ?ID?NO：133)

For other erythropoietin(EPO) mutain and recombinant tissue protectiveness cell factor of the present invention, employed oligonucleotide sequence comprises in the site-directed oligonucleotide mutagenesis:

For the R150E mutain:

R150E-F?GTCTACTCCAATTTCCTCGAGGGAAAGCTGAAGCTG，(SEQ?IDNO：120)

R150E-R?GCTTCAGCTTTCCCTCGAGGAAATTGGAGTAGAC(SEQ?ID?NO：121)

For the R103E mutain:

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

R103E-R?CAGAGTGGTGAGGCTCTCAAGGCCACTGACGG(SEQ?ID?NO：123)

For R103E/L108S (103) combinatorial mutagenesis albumen:

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)

Lack for 44-49:

d44-49F?GAATATCACTGTCCCAGACGGTGGTGCCTGGAAGAGGATG，(SEQID?NO：128)

d44-49R?CATCCTCTTCCAGGCACCACCGTCTGGGACAGTGATATTC(SEQID?NO：129)

For the K20A mutain:

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

K20A-R?GATATTCTCGGCCTCCGCGGCCTCCAAGAGGTA(SEQ?ID?NO：131)

For the K140A mutain:

K140A-F?GCTGACACTTTCCGCGCACTCTTCCGAGTCTACTC，(SEQ?ID?NO：132)

K140A-R?GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC(SEQ?IDNO：133)

For the K152A mutain:

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

K152A-R?CTGTGTACAGCTTCAGCGCTCCCCGGAGGAAAT(SEQ?ID?NO：135)

For the K154A mutain:

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

K154A-R?TCCCCTGTGTACAGCGCCAGCTTTCCCCGGAG(SEQ?ID?NO：137)

For the K45A mutain:

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

K45A-R?CAGGCATAGAAATTAACTGCGGTGTCTGGGACAGT(SEQ?ID?NO：139)

For the K52A mutain:

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

K52A-R?CGACCTCCATCCTCGCCCAGGCATAGAAATTAACT(SEQ?ID?NO：141)

For the K97A mutain:

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

K97A-R?GGCCACTGACGGCTGCATCCACATGCAGCTGCA(SEQ?ID?NO：143)

For the K116A mutain:

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

K116A-R?AGGGGAGATGGCTTCCGCCTGGGCTCCCAGAG(SEQ?ID?NO：145)

For K140A/K52A combinatorial mutagenesis albumen:

K140A-F?GCTGACACTTTCCGCGCACTCTTCCGAGTCTACTC，(SEQ?ID?NO：146)

K140A-R?GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC(SEQ?IDNO：147)

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

K52A-R?CGACCTCCATCCTCGCCCAGGCATAGAATTAACT(SEQ?ID?NO：149)

For K140A/K52A/K45A combinatorial mutagenesis albumen:

K140A-F?GCTGACACTTTCCGCGCACTCTTCCGAGTCTACTC，(SEQ?ID?NO：150)

K140A-R?GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC(SEQ?IDNO：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 K97A/K152A combinatorial mutagenesis albumen:

K97A-FTGCAGCTGCATGTGGATGCAGCCGTCAGTGGCC，(SEQ?ID?NO：156)

K97A-R?GGCCACTGACGGCTGCATCCACATGCAGCTGCA(SEQ?ID?NO：157)

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

K152A-RCTGTGTACAGCTTCAGCGCTCCCCGGAGGAAAT(SEQ?ID?NO：159)

For K97A/K152A/K45A combinatorial mutagenesis albumen:

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 combinatorial mutagenesis albumen:

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 combinatorial mutagenesis albumen:

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?GCTGACACTTTCCGCGCACTCTTCCGAGTCTACTC，(SEQ?ID?NO：182)

K140A-R?GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC(SEQ?IDNO：183)

For K97A/K152A/K45A/K52A/K140A/K154A combinatorial mutagenesis albumen:

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?GCTGACACTTTCCGCGCACTCTTCCGAGTCTACTC，(SEQ?ID?NO：192)

K140A-R?GAGTAGACTCGGAAGAGTGCGCGGAAAGTGTCAGC(SEQ?IDNO：193)

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

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

For N24K/N38K/N83K combinatorial mutagenesis albumen:

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

N24K-R?ACAGCCCGTCGTGATTTTCTCGGCCTCCTTG(SEQ?ID?NO：197)

N38K-F?ACTGCAGCTTGAATGAGAAATCACTGTCCCAGACAC，(SEQ?IDNO：198)

N38K-R?GTGTCTGGGACAGTGATTTTCTCATTCAAGCTGCAGT(SEQ?IDNO：199)

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

N83K-R?CACGGCTGGGAAGATTTGACCAACAGGGCCT(SEQ?ID?NO：201)

For the K152W mutain:

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

K152W-R?CTGTGTACAGCTTCAGCCATCCCCGGAGGAAAT(SEQ?ID?NO：203)

For R14A/Y15A combinatorial mutagenesis albumen:

RY14AA-F?AGCCGAGTCCTGGAGGCGGCCCTCTTGGAGGCCAA，(SEQ?IDNO：204)

RY14AA-RTTGGCCTCCAAGAGGGCCGCCTCCAGGACTCGGCT(SEQ?IDNO：205)

Y15A-F?AGCCGAGTCCTGGAGAGGGCCCTCTTGGAGGCCAA(SEQ?ID?NO：206)

Y15A-R?TTGGCCTCCAAGAGGGCCCTCTCCAGGACTCGGCT(SEQ?ID?NO：207)

Below be the example of the construct of preparation: people EPO (hEPO)-6xHis mark-pCiNeo sequence (SEQ ID NO:208); HEPO6xHis mark-A30N/H32T-pCiNeo (SEQ IDNO:209); HEPO-6xHis mark-K45D-pCiNeo sequence (SEQ ID NO:210); HEPO-6xHis mark-S100E-pCiNeo sequence (SEQ ID NO:211); With hEPO-6xHis mark-K45D/S100E-pCiNeo sequence (SEQ ID NO:212).The early stage immediately enhancers/promoters district of pCI-neo mammalian expression vector carrier cytomegalovirus (CMV) inserts the constitutive expression of fragment with the cloned DNA that starts mammalian cell.

Primitive man's erythropoietin cdna that these oligonucleotides are annealed among the pCiNeo is cloned, to introduce described sudden change.To mutant clon check order and confirm the sudden change.Use fat transfection amine, make all plasmids all transfection in CHO-1 cell or COS-7 cell.After the transfection 48-72 hour, the nutrient culture media of collecting cell.This nutrient culture media can be directly or is measured or neuron be used for after measuring measuring by ELISA and detects erythropoietin(EPO) at erythropoiesis behind the purifying.

Then, K45D and S100E recombinant tissue protectiveness cell factor detect in neuron is measured.Specifically, adopt use SK-N-SH neuroblastoma cells in vitro neuroprotective to measure.The SK-N-SH cell is seeded in the density of 40,000 cells/well reaches 24 hours on 24 orifice plates.Then the concentration of recombinant tissue protectiveness cell factor with 3nM is added, after 24 hours (erythropoietin(EPO)=commercial preparation; The erythropoietin(EPO) of EPO=CHO cellular expression and recombinant tissue protectiveness cell factor; Pure carrier=come to be personal not to have the cell conditioned medium liquid of Chinese hamster ovary celI of the carrier transfection of Epo construct).After the pre-incubation, reach 4 hours for rotenone (5 μ M) cellular exposure, wash and made it to recover 24 hours.In all these steps, shown the existence of EPO variant.When experiment finishes, by with cell with tetrazole dyestuff WST-1 (according to manufacturer's instructions: Roche#1644807) be incubated 2 hours, carry out the cell viability quantitative measurement, vigor absorbance readings signify.

Fig. 6 A and Fig. 6 B show the result who uses erythropoietin(EPO) and recombinant tissue protectiveness cell factor K45D and S100E the protection of SK-N-SH neuroblastoma cellular neural to be measured (at rotenone).Y-axis is represented the absorbance reading among the figure, and data are mean value ± double replication scopes.Figure among Fig. 6 A clearly illustrates that at K45D and S100E cells in sample vigor and is held, proves their cytoprotection effect.Fig. 6 B shows the plasmid map of hEPO-6xHis mark-PciNeo.

6.4. embodiment 4: the tissue protective cell factor

The required recombinant tissue protectiveness cell factor of purposes as described herein can further be modified by the following method: asialylated, guanidineization, carbamylation, amidation, trinitrophenylization, acetylation, succinylation, nitrated, the perhaps modification of arginine residues in other method or carboxyl.Perhaps, these modifications that natural erythropoietin(EPO) or erythropoietin(EPO) derivant are done include but not limited to carry out asialylated, guanidineization, carbamylation, amidation, trinitrophenylization, acetylation, succinylation or nitrated erythropoietin(EPO) before recombinant tissue protectiveness cell factor is introduced in its sudden change.Some case descriptions to the further modification of reorganization tissue protective cell factor are as follows.Those of ordinary skills know that following method also is used in the sudden change introducing to produce the recombinant tissue protectiveness cell factor natural erythropoietin(EPO) or derivatives thereof of chemical modification before.

6.4.1. take off sialic acid recombinant tissue protectiveness cell factor

Recombinant tissue protectiveness cell factor can be taken off sialic acid by following illustrative methods.Sialidase (separating from streptococcus (Streptococcacs sp) 6646K.) derives from SEIKAGAKU AMERICA (numbering 120050).Recombinant tissue protectiveness cell factor is handled 3 hours to take off sialic acid with sialidase (0.025U/mg EPO) in 37 ℃.Reaction mixture also concentrates with Ultrafree centrifugal filtration unit desalination.Sample pipetting volume is to AKTAprime then ^TMThe ion exchange column of system.The selected buffer solution elution of albumen.Carry out the IEF gel analysis with the wash-out flow point that contains a large amount of albumen then.Merge and only contain the flow point (on the IEF gel, moving) of going up two bands most, measure the protein content that merges flow point, add the 10x salt solusion (1M NaCl, 0.2M sodium citrate, 3mM citric acid) of 1/9 volume in pI～8.5 and pI～7.9.Measure sialic acid content then.Do not detect tangible sialic acid content.

Shown in Fig. 7-8, it is equally effective with natural erythropoietin(EPO) in external neurocyte to take off the sialic acid erythropoietin(EPO).Carry out in vitro test with apoptotic neural sample embryonal carcinoma cell (P19) after having experienced removal serum.Removing serum preceding 24 hours, and in culture, adding the 1-1000ng/ml erythropoietin(EPO) or modify erythropoietin(EPO).Remove nutrient culture media next day,, in culture, add the nutrient culture media (serum-free) that contains substances, cultivated again 48 hours with not containing fresh serum nutrient culture media washed cell.In order to measure viable count, (CellTiter 96 to carry out the tetrazole reduction test; Promega, Inc.).Shown in Fig. 7-8, take off the sialic acid erythropoietin(EPO) and demonstrate with this equal authenticity in preventing cell death of erythropoietin(EPO).

With the persistence of neuroprotective activity in the focal ischaemic model validation of the rat body, in described model, can form the reversible infringement in mesencephalic arteries district (Brines etc., 2000, Proc.Nat.Acad. Sci.U.S.A.97:10526-31) as previously mentioned.When the inaccessible outbreak of Sprague-Dawley bull rat artery, take off sialic acid erythropoietin(EPO) or erythropoietin(EPO) (in the 5000U/kgBW peritonaeum) or solvent.After 24 hours, put to death animal and get brain, use for research.Carry out serial section and with tetrazolium dyeing to identify survival district in the brain.As shown in Figure 9, taking off the sialic acid erythropoietin(EPO) provides neuroprotective equally effective with natural erythropoietin(EPO) when 1 hour ischaemic, promptly dwindles infarct volume.Figure 10 shows another focal ischaemic model, wherein with erythropoietin(EPO) with take off the sialic acid erythropoietin(EPO) and compare dose response.When the lowest dose level of 4 μ g/kg, taking off the sialic acid erythropoietin(EPO) provides protection, and the erythropoietin(EPO) of unmodified does not provide protection.Number of rats n is more than or equal to 4 in every group.

Expection the present invention takes off sialic acid recombinant tissue protectiveness cell factor and will obtain similar results.

6.4.2. carbamylation recombinant tissue protectiveness cell factor

According to the following method of describing as Jin Zeng (1991), recombinant tissue protectiveness cell factor can be used for preparing corresponding carbamylation molecule.Carry out polylysine modification by carbamylation and guanidine reaction pair metallothionein.Methods?in?Enzymology?205：433-437。With the potassium cyanate recrystallization.Preparation 1M borate buffer (pH 8.8).Recombinant tissue protectiveness cell factor solution is mixed with the equal-volume borate buffer.Potassium cyanate is directly joined in the reaction tube, to final concentration be 0.5M.Solution mixed and in 37 ℃ of insulations 6-16 hour.Thorough then dialysis solution.From dialysis tubing, take out product, collect in the new test tube.Measurement volumes adds the 10X salt solusion (1M NaCl, 0.2M sodium citrate, 3mM citric acid) of 1/9 volume.Measure protein content, calculate the product recovery.Test assay products by the IEF gel and with the TF-1 cells in vitro.

6.4.3. succinylation recombinant tissue protectiveness cell factor

According to the following method of describing as (2001) such as Alcalde, recombinant tissue protectiveness cell factor can be used for preparing corresponding succinylation molecule.Come the succinylation reaction of the cyclodextrin glycosyl transferases of self-heating anaerobic bacillus(cillus anaerobicus) (Thermoanaerobacter sp.) 501 to strengthen its transferase active as donor with starch.J.Biotechnology?86：71-80。The 0.5M NaHCO of recombinant tissue protectiveness cell factor (100 μ g) ₃(pH 8.0) are incubated 1 hour with the succinic anhydride of 15 molar excess at 15 ℃.By to the distill water dialysis cessation reaction.

The 27mg/ml succinic anhydride is dissolved in the anhydrous propanone.Be reflected in the microcentrifugal tube of 10mM sodium phosphate buffer (pH 8.0) and carry out.The succinic anhydride of recombinant tissue protectiveness cell factor albumen and 50 times of molar weights is added in the test tube.Mix, test tube was rotated 1 hour in 4 ℃.Pass through 10mM sodium phosphate buffer dialysis cessation reaction with dialysis cassette (Slide-A-Laze 7K, Pierce 66373).From dialysis cassette, take out product, collect in the new test tube.Measurement volumes adds the 10 X salt solusions (1M NaCl, 0.2M sodium citrate, 3mM citric acid) of 1/9 volume.Measure protein content, calculate the product recovery.Test assay products by the IEF gel and with the TF-1 cells in vitro.

6.4.4. acetylation recombinant tissue protectiveness cell factor

According to the following method of describing as (1990) such as Satake, recombinant tissue protectiveness cell factor can be used for preparing corresponding acetylation molecule.The chemical modification of erythropoietin(EPO): increase external activity by the guanidine reaction.Biochimica?et?Biophysica?Acta.1038：125-129。

Be reflected in the microcentrifugal tube of 80mM sodium phosphate buffer (pH 7.2) and carry out.The acetic anhydride that adds recombinant tissue protectiveness cell factor and equimolar amounts.Mix the back and be incubated 1 hour on ice.Pass through water dialysis cessation reaction with dialysis cassette (Slide-A-Laze 7K, Pierce 66373).From dialysis cassette, take out product, collect in the new test tube.After the measurement volumes, add the 10 X salt solusions (1M NaCl, 0.2 M sodium citrate, 3mM citric acid) of 1/9 volume.Measure protein content, calculate the product recovery.Test assay products by the IEF gel and with the TF-1 cells in vitro.

6.4.5. the lysine of carboxymethylation recombinant tissue protectiveness cell factor

According to the following method of describing as (1999) such as Akhtar, recombinant tissue protectiveness cell factor can be used for preparing corresponding N ^ε-(ethyloic) lysine (CML) decorating molecule, wherein one or more lysyl-residues of recombinant tissue protectiveness cell factor are modified: Conformational study of N ^ε-(carboxymethyl) the lysine adducts ofrecombinant a-crystallins (N of recombinant alpha-crystalline protein ^εThe conformation research of-(ethyloic) lysine adduct) .Current Eye Research, 18:270-276.

Prepared fresh glyoxalic acid (200mM) and NaBH ₃The sodium phosphate buffer of CN (120mM) (50mM, pH 7.5).In microcentrifugal tube, add erythropoietin(EPO) (being dissolved in phosphate buffer).Lysine equivalent in the calculating solution (about 8 lysine residues/mol).In test tube, add the NaBH more than 3 times ₃CN and more than 5 times or the glyoxalic acid more than 10 times.After every pipe spiral concussion, in 37 ℃ of insulations 5 hours.Sample in 4 ℃ to the phosphate buffer dialysed overnight.After the dialysis, measure the volume of each product.Measure protein concentration, calculate the product recovery.Test assay products by the IEF gel and with the TF-1 cells in vitro.

6.4.6. iodate recombinant tissue protectiveness cell factor

According to as Pierce Chemical Company (Rockford; IL) the following method that the instructions that is used for the pre-embedding iodate of IODO-Gen pipe (catalog number 28601) that provides is described, recombinant tissue protectiveness cell factor can be used for preparing corresponding iodate molecule.

The NaI of preparation 0.1M, total reaction volume is the 0.1ml/ pipe in the pre-embedding iodate of IODO-Gen pipe (Pierce, 28601), in sodium phosphate buffer (40mM, pH 7.4), carries out iodination reaction.Protein substrate (recombinant tissue protectiveness cell factor) is mixed with sodium phosphate buffer, transfer to then in the pre-embedding iodate of the IODO-Gen pipe.Add NaI, making its final concentration is 1-2mM, and making NaI/ albumen mol ratio is 14-20.Mix and insulation 15 minutes in the gentle agitation at room temperature.By removing the reaction mixture cessation reaction, join in the pipe that 3.9ml sodium damping fluid (i.e. 40 times of dilutions) are housed.By wetting in advance Ultrafree centrifugal filtration unit enriched product.Measure the concentrate volume, add the 10X salt solusion (1M NaCl, 0.2M sodium citrate, 3mM citric acid) of 1/9 volume.Measure protein concentration, calculate the product recovery then.Test assay products by the IEF gel and with the TF-1 cells in vitro.

Perhaps, recombinant tissue protectiveness cell factor can be with following method iodate.Containing the free Na of 1mCi ¹²³(Pierce, Rockford Il) reach 5 minutes to be incubated the iodine pearl among the 100 μ l PBS of I (20mM sodium phosphate, 0.15M NaCl, pH 7.5).In potpourri, add the 100 μ g recombinant tissue protectiveness cell factors that are dissolved in 100 μ lPBS then.After at room temperature being incubated 10 minutes, by from reaction vessel, taking out 200 μ l solution cessation reactions (staying the iodine pearl).Remove excessive iodine by on Centricon 10 posts, carrying out gel filtration then.As shown in figure 11, the iodine erythropoietin(EPO) that produces by this method is effective aspect the protection P19 cell after removing serum.It will be recognized by those of ordinary skills, expect that the iodate of recombinant tissue protectiveness cell factor of the present invention will obtain similar results.

A following general introduction of method that is used for iodate recombinant tissue protectiveness cell factor is arranged again.The 100 μ g recombinant tissue protectiveness cell factors that will be dissolved in 100 μ l PBS join 500uCiN ¹²⁵Among the I, then potpourri is mixed in microcentrifugal tube.Add 25 μ l toluene-sodium-sulfonchloramides (2mg/ml) then, potpourri at room temperature is incubated 1 minute.Add 50 μ l toluene-sodium-sulfonchloramide stop buffers (PBS of 2.4mg/ml sodium metabisulfite, 10mg/ml tyrosine, 10% glycerine, 0.1% dimethylbenzene) then.By carrying out gel filtration, iodotyrosine and iodate recombinant tissue protectiveness cell factor are separated then with Centricon 10 posts.

6.4.7. biotinylation recombinant tissue protectiveness cell factor

According to (Rockford IL) describes the following method that is used for EZ-LinkNHS-LC-biotin (catalog number 21336), and recombinant tissue protectiveness cell factor can be used for preparing corresponding biotinylation molecule as Pierce Chemical Company.

Just before reaction, EZ-Link NHS-LC-biotin (pierce, 21336) is dissolved among the DMSO, concentration is 2mg/ml.(react in 17 * 100mm) at the test tube that contains 50mM sodium bicarbonate (pH 8.3) that fills cumulative volume 1ml.Add the EZ-Link NHS-LC-biotin of recombinant tissue protectiveness cell factor and＜10%, the mol ratio of biotin/albumen is about 20.Mix and be incubated 2 hours on ice.With Ultrafree centrifugal filtration unit desalination and concentration response product.Product is collected in the new test tube.Measure volume, add the 10 X salt solusions (1 M NaCl, 0.2 M sodium citrate, 3mM citric acid) of 1/9 volume.Measure protein content, calculate the product recovery then.Test assay products by the IEF gel and with the TF-1 cells in vitro.

The following discloses the biotinylated method of free amine group that is used for recombinant tissue protectiveness cell factor.0.2mg D-biotin acyl-EACA-N-hydroxy-succinamide ester (Boehringer Mannheim#1418165) is dissolved among the 100 μ l DMSO.Solution is mixed in the test tube that aluminium foil is covered with the 400 μ l PBS that contain the 0.2mg recombinant tissue protectiveness cell factor of having an appointment.After at room temperature being incubated 4 hours, isolate unreacted biotin by on Centricon 10 posts, carrying out gel filtration.As shown in figure 12, this biotinylation erythropoietin(EPO) protection removes the p19 cell of serum.It will be recognized by those of ordinary skills, expect that the biotinylation of recombinant tissue protectiveness cell factor of the present invention will obtain similar results.

At last; at Wojchowski etc. " Biotinylated recombinant humanerythropoietins:Bioactivity and Utility as a receptor ligand (biotinylation recombinant human erythropoietin :) " .Blood as the biologically active and the purposes of the part of acceptor; 1989; 74 (3): among the 952-8, the author has adopted and has made the biotinylated three kinds of distinct methods of erythropoietin(EPO).Biotin is joined on (1) sialic acid part (2) carboxyl and (3) amino.The author uses the mouse boosting cell proliferation assay, biotin is joined the biologic activity inactivation that can not make erythropoietin(EPO) on the sialic acid part with proof (1); (2) biotin is joined the basic inactivation of the biology that causes erythropoietin(EPO) on the carboxyl; (3) biotin is joined the biology complete deactivation that causes erythropoietin(EPO) on the amino.These methods and modification all are included in herein.Figure 12 is presented at biotinylation erythropoietin(EPO) and the activity of taking off the sialic acid erythropoietin(EPO) in the serum starvation P19 mensuration.It will be recognized by those of ordinary skills, expect that the iodate of recombinant tissue protectiveness cell factor of the present invention will obtain similar results, referring to 6.15 trifles.

6.5. embodiment 5: modify recombinant tissue protectiveness cell factor by other method

6.5.1. trinitrophenylization:

According to (" Activity of bovine pancreatic deoxyribonuclease Awith modified amino groups (having the activity of modifying amino ox pancreas deoxyribonuclease A) " 1971 such as Plapp; J.Biol.Chem.246; 939-845) the method for Miao Shuing; recombinant tissue protectiveness cell factor (100 μ g) is with 2; 4,6-trinitro-benzene-sulfonic acid salt is modified.

6.5.2. arginine is modified

According to Riordan (" Functional arginyl residues in carboxypeptidase A.Modification with butanedione (the functional arginyl residue in the Carboxypeptidase A.Modify with diacetyl). " Riordan JF, Biochemistry 1973,12 (20): the 3915-3923) method of Miao Shuing, recombinant tissue protectiveness cell factor is with 2, and the 3-diacetyl is modified.

During adorned another of the amino acid residue of erythropoietin(EPO) modified therein, according to Takahashi (1977, J.Biochem.81:395-402) method of Miao Shuing, modify arginine residues with the phenyl glyoxal, the described reaction at room temperature variable time scope between 0.5 hour to 3 hours is carried out.By reaction mixture is dialysed and cessation reaction to water.The application of the erythropoietin(EPO) of this class modified forms all comprises in the present invention.With above-mentioned neural sample P19 raji cell assay Raji, the erythropoietin(EPO) of testing phenyl glyoxal-modification.As shown in figure 13, the erythropoietin(EPO) of this chemical modification keeps its neuroprotective effect fully.Same recombinant tissue protectiveness cell factor of the present invention of modifying obtains similar results.

According to (" Identification of functional arginine residues inribonuclease A and lysozyme (evaluation of the functional arginine residues of ribonuclease A and lysozyme). " Patthy such as Patthy; L; Smith EL; J.Biol.Chem 1,975 250 (2): the 565-9) method of Miao Shuing, recombinant tissue protectiveness cell factor is modified with cyclohexanone.

According to (" Proceedings:Carboxypeptidase B:modification offunctional arginyl residues (procceedings: protaminase: the modification of functional arginyl residue). " Werber such as Werber; MM; Sokolovsky M Isr J Med Sci1 975 11 (11): the 1169-70) method of Miao Shuing, recombinant tissue protectiveness cell factor carries out modifying with the phenyl glyoxal.

6.5.3. tyrosine is modified

According to " Stimulation of rat ovarian cell steroidogenesis byhigh density lipoproteins modified with tetranitromethane (stimulation that the high-density lipoprotein (HDL) of modifying with tetranitromethane produces rat ovary cell steroids). " Nestler JE such as Nestler, Chacko GK, Strauss JF 3rd.J Biol Chem 1985 Jun 25; 260 (12): 7316-21) previously described method, (100 μ g) is incubated with tetranitromethane with recombinant tissue protectiveness cell factor.

6.5.4. glutamic acid (and aspartic acid) is modified

In order to modify carboxyl, according to " Carboxyl group modification in chymotrypsin and chymotrypsinogen (carboxyl modified of chymotrypsin and chymotrypsinogen). " Carraway KL such as Caraway, Spoerl P, Koshland DE Jr.J Mol Biol 1969May 28; 42 (1): the method that 133-7 describes at room temperature is incubated 60 minutes with the 1M glycine amide of recombinant tissue protectiveness cell factor (100 μ g) and 0.02M EDC in pH 4.5.

6.5.5. trp residue is modified

According to Ali etc., J Biol Chem.1995 Mar 3; 270 (9): the method that 4570-4 describes at room temperature is incubated the 20mM kaliumphosphate buffer (pH 6.5) of recombinant tissue protectiveness cell factor (100 μ g) with the positive bromine succinimide of 20 μ M.According to Korotchkina (Korotchkina, LG etc., Protein Expr Purif.1995 Feb; 6 (1): the 79-90) method of Miao Shuing, measure oxidation trp residue number.

6.5.6. amino removal

In order to remove the amino of recombinant tissue protectiveness cell factor; according to (Kokkini such as Kokkini; " Modification of hemoglobin by ninhydrin (triketohydrindene hydrate is to the modification of haemoglobin). " Blood such as G.; the 556th volume; the 4th phase, the 1980:701-705) method of Miao Shuing, with recombinant tissue protectiveness cell factor (100 μ g) with contain 20mM triketohydrindene hydrate (PierceChemical; Rockford, PBS Il) (PH 7.4) arise from 37 ℃ of insulations 2 hours.By making the reaction of product and sodium borohydride or lithium aluminium hydride reduction, finish the reduction of gained aldehyde.Specifically, erythropoietin(EPO) (100 μ g) at room temperature is incubated 30 minutes with the PBS of 0.1M sodium borohydride.By sample is stopped reduction reaction cooled on ice 10 minutes, and, spend the night to PBS dialysis 3 times.(Kokkini, G., Blood, the 556th volume, the 4th phase, 1980:701-705).At room temperature be incubated 30 minutes by PBS, finish reduction with lithium aluminium hydride reduction with recombinant tissue protectiveness cell factor (100 μ g) and 0.1M lithium aluminium hydride reduction.By sample is stopped reduction reaction cooled on ice 10 minutes, and, spend the night to PBS dialysis 3 times.

6.5.7. disulfide bond reduction and stabilization

Recombinant tissue protectiveness cell factor (100 μ g) is incubated 15 minutes with 500mM DTT in 60 ℃.In potpourri, add the aqueous solution of 20mM iodoacetamide and lucifuge insulation at room temperature 25 minutes then.

6.5.8. limited proteolysis

Recombinant tissue protectiveness cell factor can experience the limited chemical proteolysis of the specific residue of target.Recombinant tissue protectiveness cell factor and 2-(2-nitrobenzophenone sulfinyl)-3-methyl-3 '-reaction of bromine indolenine; the described room temperature lucifuge that is reflected at is having under nitrogen pressure in the cap test tube in 50 times of 50% excessive acetate, and the specificity cutting is 48 hours behind trp residue.By using tryptophane quencher and desalination cessation reaction.

As mentioned above, recombinant tissue protectiveness cell factor can be modified, but a plurality of modifications of tissue protective cell factor and extra modification also can be carried out not departing under the spirit of the present invention.

6.6. embodiment 6: the tissue protective cell factor has neuroprotective effect

According to Manley etc., 2000, Aquaporin-4 deletion in mice reduces brainedema after acute water intoxication and ischemic stroke (mouse aquaporin protein-4 disappearance reduces acute water intoxication and ischemic stroke associated with hydrocephalus), Nat Med 2000Feb; 6 (2): the method that 159-63 describes, the neuroprotective effect of the erythropoietin(EPO) of usefulness water intoxication evaluation of measuring chemical modification.Use the C3H/HEN female mice.Give the water and the 400ng/kg bw DDAVP (minirin) of its body weight 20% of mouse in the peritonaeum.Give mouse red blood cell and generate plain (A) or tissue protective cell factor: take off sialic acid erythropoietin(EPO) (B); carbamylation takes off sialic acid erythropoietin(EPO) (C); succinylation takes off sialic acid erythropoietin(EPO) (D); sialic acid erythropoietin(EPO) (E) is taken off in acetylation; sialic acid erythropoietin(EPO) (F) is taken off in iodate; carboxymethylation is taken off sialic acid erythropoietin(EPO) (G); carbamylation erythropoietin(EPO) (H); acetylation erythropoietin(EPO) (I); iodate erythropoietin(EPO) (J) or N ^ε-ethyloic erythropoietin(EPO) (K).When giving preceding 24 hours of water and giving water, peritonaeum gives the mouse 100 micrograms/erythropoietin(EPO) of kg dosage or the erythropoietin(EPO) of chemical modification interior twice.Improvement equal from Manley etc. is used to estimate mouse.Following the listing of scoring of revising:

1. explore cage/desk

Be 0

Deny 1

2. visual pursuit target

Be 0

Deny 1

3. beard motion

Have 0

Do not have 1

4. leg-tail motion

Normal 0

Stiff 1

Paralysis 2

5. misery is recalled (toe folder)

Be 0

Deny 1

6. sports coordination

Normal 0

Unusual 1

7. stop on the table limit

Be 0

Deny 1

Possible overall score: 8

At following time point mouse is marked: 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 the erythropoietin(EPO) mouse of accepting erythropoietin(EPO) or chemical modification, in the percent of control mice neuron defective experiment.Figure 14 display organization protectiveness cell factor is to the protection of the mouse of the neurology wound that caused by water intoxication.The recombinant tissue protectiveness cell factor that expection has similar chemical modification can obtain similar results.Also measured significance,statistical.Compared with the control, these dosage regimens have significant difference, and p＜0.05 is used ^*Expression, and have highly significant difference, p＜0.01 is used ^*Expression.

6.7. embodiment 7: in order to transplanting maintenance with cardiac function

According to Delcayre etc., 1992, the scheme that Amer.J.Physiol.263:H1537-45 describes, for body weight at the Wistar of 300-330g male rat, took out heart preceding 24 hours carrying out vitro study, give cell and generate plain (5000U/kg body weight) or solvent.With amobarbital (0.3mL) execution animal and at intravenous heparinize (0.2mL).Heart begins to allow its balance 15 minutes.Then, make the atrium sinistrum capsule be expanded to the volume that end-diastolic pressure is 8mm Hg.Be expanded to 0.02ml by increasing capsule volume, make up the left ventricular pressure volume curve.Zero volume is defined as wherein, and left ventricular diastolic end pressure is zero point.When finishing the pressure volume curve, after checking that coronary artery flows, make the left ventricle capsule be contracted to that end-diastolic pressure returns to 8mmHg and control cycle continued 15 minutes.Then, heart stops to beat with 50mL Celsior+ molecule, being 60cm H at pressure ₂Under the O in 4 ℃ of tranquillization.Cut heart then and filling same solution and pile on every side in the plastic containers of trash ice and stored 5 hours in 4 ℃.

After finishing storage, heart is transferred on the Langendorf equipment.Ductus bursae is inserted into left ventricle again and expand into again and ischemic identical volume in early stage.In 37 ℃ heart was poured into 2 hours more at least.Reperfusion pressure power is arranged on 50cm H ₂O reaches again and flowed 15 minutes, returns to 100cm H then ₂O carried out 2 hours again.Rebulid heartbeat (320 times/minute).Pouring into again 25 minutes, 45 minutes, 60 minutes and 120 minutes, repeat to carry out for 3 times shrinkage index and diastolic pressure etc. hold and measure.Finish the pressure volume curve at this time point, and collect the coronary artery effluent during 45 minutes and reperfusion, to measure the creatine kinase seepage.With paired t-check two groups of treatment groups relatively, be used for designing with the linear regression of end-diastolic pressure data and comply with curve.As shown in figure 15, after with epo treatment, the obvious improvement that left ventricular pressure changes taking place, and the volume-pressure curve that improves, reduces left ventricular diastolic pressure and reduce the creatine kinase seepage.Treat with recombinant tissue protectiveness cell factor of the present invention, expection can obtain similar results.

6.8. embodiment 8: erythropoietin(EPO) protection cardiac muscle cell avoids ischemic injury

Give bull rat recombinant human erythropoietin (5000U/kg body weight) in preceding 24 hours in anesthesia, and prepare to carry out coronary occlusion.When the operation beginning, give the erythropoietin(EPO) of extra dose, and make left main coronary occlusion 30 minutes, and then discharge.The erythropoietin(EPO) that gives same dose after handling every day reached for 1 week.The cardiac function of zoologizeing then.Show that as Figure 16 the animal proof of accepting false injection (salt solution) has big increase at left end-diastolic pressure, show heart that enlarge, stiff after the secondary miocardial infarction.By contrast, compare with sham-operation contrast, the animal of accepting erythropoietin(EPO) does not experience cardiac function go down (significant difference is in the level of p＜0.01).Treat with recombinant tissue protectiveness cell factor of the present invention, expection can obtain similar results.

6.9. embodiment 9: the administered peripherally erythropoietin(EPO) is to the protection of treat retinal ischemic

The retina cell is very responsive to ischaemic, so in the ischemic stress reaction after 30 minutes, many cell deaths.In addition, subacute or chronic ischaemic is to follow the basis of the sex change symptom of a large amount of human common disease (for example diabetes, glaucoma and macular degeneration).Up to now, still do not protect cell to avoid ischemic effective therapy.Closely endothelial barrier is present between blood and the retina, but the big molecule of described barrier exclusion great majority.Can protect cell in order to detect administered peripherally erythropoietin(EPO) whether, use as Rosenbaum etc. (1997 to the ischaemic sensitivity; Vis.Res.37:3443-51) described acute, reversible glaucoma rat model.Specifically, inject saline into the anterior chamber of bull rat, give the pressurization of systemic arterial pressure and kept 60 minutes.Inducing ischaemic preceding 24 hours, give animal salt solution or 5000U erythropoietin(EPO)/kg body weight in the peritonaeum, and give 3 days daily dose more continuously.Treatment 1 week of back is carried out electroretinography to adapting to dark rat.Figure 17-18 explanation is compared with the animal (Figure 17, drawing C) that only gives brine treatment, only keep few function, and giving erythropoietin(EPO) has good reservation on electroretinogram (ERG) (Figure 17, drawing D).Figure 18 has compared the electroretinogram a-ripple and the b-wave-amplitude of epo treatment group and brine treatment group, demonstrates erythropoietin(EPO) obvious protection is provided.Treat with recombinant tissue protectiveness cell factor of the present invention, can obtain similar results.

6.10. embodiment 10: erythropoietin(EPO) is to the recovery Effects by the cognitive function that goes down due to the brain damage

In the cognitive function Research on ability that the proof erythropoietin(EPO) is recovered to go down to mouse after accepting cerebral trauma, as Brines etc., PNAS 2000,97; 10295-10672 is described, allows female Balb/c mouse suffer the blunt cerebral trauma, begins peritonaeum after 5 days and gives 5000U/kg-bw erythropoietin(EPO) interior every day.Damaged back 20 days, and in the Morris water maze, checked the cognitive function of animal, 4 tests every day.Though treat in test animal and untreated animal performance all bad (swimming time is about 80 seconds, rather than possible 90 seconds), Figure 19 is presented at damage and began Morris water maze laboratory with EPO treatment brain blunt wound, every group of mouse n=16 in back 5 days.1 week (damaging back 12 days y) after giving EPO of experiment for the first time.Two treated animals performance all bad (swimming time is about 80 seconds, rather than possible 90 seconds).Animal performance through epo treatment better (in the figure, negative value better).Every day, 4 tests were averaged.Figure 19 shows.Even 30 talentes begin to carry out epo treatment (Figure 20) after being deferred to wound, also can be observed the recovery of cognitive function.In Figure 20, every group of mouse n=7 is in damage beginning in back month, except treating with external application 5000U/kg EPO weekend.The mean value of experiment also is 4 experiments every day.Treat with recombinant tissue protectiveness cell factor of the present invention, expection can obtain similar results.

6.11. embodiment 11: the kainic acid model

In kainic acid neurotoxicity model, according to Brines etc., Proc.Nat.Acad.Sci.U.S.A.2000,97; The scheme that 10295-10672 describes was giving the 25mg/kg kainic acid preceding 24 hours, and what give 5000U/kg-bw dosage in the peritonaeum takes off the sialic acid erythropoietin(EPO), show equally effective with erythropoietin(EPO), shown in the death time (Figure 21).Treat with tissue protective cell factor of the present invention, expection can obtain similar results.

6.12. embodiment 12: spinal cord injury model

6.12.1. the rat spinal cord compressing detects erythropoietin(EPO) and tissue protective cell factor

Operating weight is the Wistar of 180-300g rat (female) in the research.Animal fasting 12 hours and by the humanity restriction before operation, intraperitoneal injection pentothal (40mg/kg-bw) and anaesthetizing then.Penetrate to the skin after (Bupivacaine 0.25%),, carry out complete single level (T-3) laminectomy by the 2cm otch by dissecting microscope.Use temporary transient aneurysm clip by epidural and spinal cord is applied 0.6 newton (65 gram) closing force reach 1 minute, induce traumatic spinal cord injury.After removing clip, skin incision is sewn, and allows animal recover fully from anesthesia, puts back to cage.Every day at least 2 bladder palpations, the continuous monitoring rat is till draining automatically.

40 animals are divided into 5 groups at random.Accepting control group (I) animal (n=8) of common salt solution (by intravenous injection) sews up after incision immediately.Group (II; N=8) accept rhEPO@16 microgram/kg-bw iv, group (III) is accepted the sialic acid tissue protective cell factor of taking off of the present invention and (is taken off sialic acid erythropoietin(EPO)) @16 microgram/kg-bw iv, group (IV) accepts to take off sialic acid tissue protective Xi Baoyinzi @30 microgram/kg-bw iv, and group (V) the acceptance sialic acid tissue protective cell factor of taking off of the present invention (is taken off sialic acid erythropoietin(EPO)) @30 microgram/kg-bw; All all are to carry out the heavy dose of intravenous injection of single after removing the aneurysm clip immediately.

The kinesitherapy nerve function of rat will be estimated by the sport rank scale that uses Basso etc.In this scale, the scoring scope of animal (is not observed the hind leg motion) to 21 (normal gaits) from 0.To check the functional defect of rat by the same supervisory personnel of the treatment of not knowing every animals received in damage back 1 hour, 12 hours, 24 hours, 48 hours, 72 hours and 1 week.

Figure 22 is a width of cloth curve map, proves the sport rank that rat recovers in spinal cord injuries receptor 30 days.As shown in the figure, the rat that gives erythropoietin(EPO) (group II) or tissue protective cell factor (group III-group V) is easy to come from injury recovery, and the proof comparison has better overall the recovery according to mouse.Carry out therapeutic treatment with recombinant tissue protectiveness cell factor of the present invention, expection will obtain similar results.

In second correlative study, animal is pressed the same manner injection.40 animals are divided into 3 groups at random.Accepting the control animals (n=8) of common salt solution (by intravenous injection) sews up after incision immediately.Second group (n=8) accepts Jia Ponilong @30 microgram/kg, every day 3 times, then two the week 1 time, methylprednisolone is the curative commonly used of spinal cord injury; Accept recombinant tissue protectiveness cell factor S100E of the present invention immediately for the 3rd group after damage, its dosage is 10 μ g/kg-bw, and all are to carry out the heavy dose of intravenous injection of single after removing the aneurysm clip immediately.

The kinesitherapy nerve function of rat will be estimated by the sport rank scale that uses Basso etc.In this scale, the scoring scope of animal (is not observed the hind leg motion) to 21 (normal gaits) from 0.To check the functional defect of rat by the same supervisory personnel of the treatment of not knowing every animals received in damage back 1 hour, 12 hours, 24 hours, 48 hours, 72 hours and 1 week.

Figure 37 is a width of cloth curve map, proves the sport rank that rat recovers in spinal cord injuries receptor 42 days.As shown in the figure, the rat that gives S100E is easy to come from injury recovery, and the proof comparison has better overall the recovery according to mouse with the rat that methylprednisolone is treated.

6.12.2. rabbit ischemia of spinal cord check erythropoietin(EPO) and tissue protective cell factor

Operating weight is in 36 New Zealand white rabbit (8-12 monthly age, male) of 1.5-2.5kg in the research.Animal fasting 12 hours and by the genuine restriction of people.100% oxygen by sucking 3% fluothane and maintain 50% oxygen and 50% air mixture in the 0.5-1.5% fluothane in, anaesthetize.Intravenous catheter (No. 22) is placed in the left ear vein.In operation, with 4ml/kg body weight (bw) speed infusion hourly Ringers lactate.Intravenous gives the 10mg/kg-bw Cefazolin in case infect before the art.Animal is placed to right arm reclining puts, use the PVP-I preserved skin, permeate and be parallel to spine with Bupivacaine (0.25%) and open skin incision under the side of body at the 12nd rib.After cutting skin and subcutaneous thoracolumbar fascia, waist eye muscle and iliocostalis lumborum shrink.Abdominal aortic is exposed and transfer under the left renal artery by approach behind the left peritonaeum.Article one, the PE-60 pipe ring around the sustainer that leaves left renal artery and two ends pass big rubber tube.By tension PE pipe, main artery is not had inaccessible 20 minutes of wound ground.Before the sustainer obturation, heavy dose of intravenous gives heparin (400IU).After 20 minutes obturations, remove pipe and conduit, with myometrial suture, the monitoring animal is estimated its nervous function then continuously until recovering fully.

36 animals are divided into 6 groups at random.In control group (I), animal (n=6) sustainer is inaccessible discharge after immediately intravenous accept common salt solution.Group (II) is accepted rhEPO@6.5 microgram/kg-bw; Group (III) is accepted tissue protective cell factor (carbamylation erythropoietin(EPO)) @6.5 microgram/kg-bw; Group (IV) is accepted another kind of tissue protective cell factor and (is taken off sialic acid erythropoietin(EPO)) @6.5 microgram/kg-bw; Group (V) is accepted and group (IV) identical tissue protective cell factor, but is @20 microgram/kg-bw; And group (VI) is accepted another tissue protective cell factor and (is taken off sialic acid carbamylation erythropoietin(EPO)) @20 microgram/kg-bw; All all are to give (for every group of n=6) through intravenous immediately after the perfusion again.

According to the standard of Drummond and Moore, by not knowing to estimate motor function the investigator who poured into again back 1 hour, 24 hours and treated in 48 hours.Followingly each animal is carried out from 0 to 4 scoring: 0=does not have the paraplegia of lower extremity motor function; The 1=lower extremity motor function is poor, and faint antigravity motion is only arranged; 2=has good antigravity strength, but can not step the medium lower extremity motor function of moving leg under health; The good motor function of 3=can drag leg and jump under health, but undesired; 4=proper motion function.The bladder defecation is given in 2 craft every day in the paraplegia animal.

Figure 23 is a width of cloth curve map, the motor function of the rabbit of describing to recover.Proof on the figure, even in 2 day cycle only, erythropoietin(EPO) and tissue protective cell factor of the present invention just can allow rabbit recover fully from spinal cord injury.Carry out therapeutic treatment with recombinant tissue protectiveness cell factor of the present invention, expection will obtain similar results.

6.13. embodiment 13: the anti-inflammatory effect of erythropoietin(EPO)

Research in the body:

Rat MCAO

Weight derives from Charles River, Calco, Italy the male Crl:CD of 250-280g (SD) BR rat.According to Brines, M.L., Ghezzi, P., Keenan, S., Agnello, D., de Lanerolle, N.C., Cerami, C., Itri, L. M. and Cerami, A.2000.Erythropoietincrosses the blood-brain barrier to protect against experimental brain injury (erythropoietin(EPO) is passed through blood brain barrier to prevent experimental brain damage) .[In ProcessCitation] method that Proc Natl Acad Sci USA 97:10526-10531 describes, these rats are undergone surgery.In brief, (low-necked artery is by inaccessible right carotid of delayed suture and incision for 400mg/kg-bw, i.p.) anesthesia with chloral hydrate with rat.Contiguous and allow towards the sawtooth hole of right socket of the eye MCA is clear to be presented, described hole is burnt leaving the arteria nasalis place.In order to produce around this fixing penumbra (border) of MCA infringement, use thin pincers traction, opposing carotid is decontroled after inaccessible 1 hour again.Give immediately behind the MCAO PBS or rhEPO (5,000U/kg-bw, i.p.; Before be presented in this model (1) protectiveness had been arranged).When needs, the TNF and the IL-6 of the homogenate of (8) quantitative test for brain cortex as previously mentioned.(biosource, Camarillo CA), measure the MCP-1 in the homogenate with commercially available ELISA kit.

Behind the MCAO 24 hours, anesthetized rat as mentioned above, and by heart perfusion 100ml salt solution, then be 4% paraformaldehyde solution of 250ml sodium phosphate buffer.Downcut brain fast, fix 2 hours in 4% paraformaldehyde solution of sodium phosphate buffer, the PBS that transfers to 20% sucrose solution spends the night, and is submerged up to them in 30% sucrose solution then, and is freezing at-45 ℃ in the 2-methylbutane then.At-20 ℃, (HM 500 OM Microm) go up to cut into slices (30 μ m) by brain and to select with transverse plane and are used at the histochemistry of synantigen or hematoxylin eosin staining not every 5 section at cryostat.Respectively according to described schemes such as Houser and manufacturer's scheme, with anti-neuroglia fibres acid albumin (GFAP) mouse monoclonal antibody (1: 250, Boehringher Mannheim, Monza, Italy) with anti-cdl11b (MRC OX-42) mouse monoclonal antibody (1: 50, Serotec UK) handles the free-floating section and is used for immunoreactivity.The sheet that soaks that is used for optical microscope is made in all sections in salt solution on the slide of embedding, by dehydration of alcohol step by step, in dimethylbenzene fixing and with DPX mountant (BDH, Poole UK) cover slide.Neighboring slice is used hematoxylin eosin staining as described in (10).

Figure 24 shows the cerebral cortex coronal section through h and E dyeing.Control rats (A), ischemic rat (B) are handled with PBS, ischemic rat (C) usefulness rhEPO (5,000U/kg-bw, i.p. gives behind the MCAO immediately) handle.Section B demonstration is compared with contrast (A), and the loss of neuron composition is followed in the obvious reduction of the tissue staining consistent with inflammation.System gives the ischemic injury that rhEPO greatly reduced the cell death position or the damage (C) of regional area.(enlargement factor 2.5x. engineer's scale=800 μ m.)

Figure 25 shows the coronal section with cortex before the contiguous infarct of GFAP antibody staining.Control rats (A), ischemic rat are handled (B) with PBS, and the ischemic rat is handled (C) with rhEPO.The activation astroglia displays (drawing B) by the positive process of its GFAP.Quantitatively and representational handle all obviously decline (drawing C) of staining power that activates astroglia in the animal through rhEPO.(enlargement factor 10x. engineer's scale=200 μ m.)

Figure 26 shows the corticocerebral coronal section with the OX-42 antibody staining.The ischemic rat is handled (A) with PBS, and the ischemic rat is handled (B) with rhEPO.In ischemic brain hemisphere, the cell dyeing around blocking tissue in two treatment groups is outstanding especially, but more intensive more extensive in the brine treatment group.(enlargement factor 20x; Engineer's scale=100 μ m).

Figure 27 shows the cerebral cortex coronal section with the contiguous infarct of OX-42 antibody staining.Compare with the ischemic rat (B) of handling, observe much higher monokaryon inflammatory cell density in the ischemic rat (A) with the PBS processing with rhEPO.Wellability leucocyte with typical circle will enlarge infarct volume.(enlargement factor 10 x; Engineer's scale=200 μ m).

Carry out therapeutic treatment with recombinant tissue protectiveness cell factor of the present invention, expection will obtain similar results.

Acute experimental allergic encephalomyelitis in the Lewis rat (EAE)

The female Lewis rat in age in 6-8 week available from Charles River (Calco, Italy).By under slight ehterization, two metapedes pad injections to rat are added with hot deactivation Much's bacillus (M.tuberculosis) H37Ra (Difco, Detroit, MI) isopyknic complete Freund's adjuvant (CFA, Sigma) 50 μ g cavy MBP (Sigma of emulsification, St.Louis, MO) aqueous solution (making final volume is 100 μ) causes EAE in rat.Blind method is checked rat EAE symptom and following scoring: 0, there is not disease; 1, tail relaxes; 2, incoordination; 3, hind leg is paralysed and fully with aconuresis.From back 3 days of immunity beginning, in the peritonaeum (i.p.) give the r-Hu-EPO that the rat prescribed dose is dissolved in PBS (EPOetin alfa, Procrit, Ortho Biotech, Raritan, NJ), every day 1 time.Because clinical grade EPO contains human serum albumins, so give the PBS that control-animal contains the human serum albumins of equal number usually.Give 5 every day, the EPO of 000U/kg-bw increases hematocrit and reaches 30%.When needs, from 3 days to 18 days with disodic alkaliine (Sigma) injection (s.c.) rat of the DEX of the 1.3mg/kg-bw that is dissolved in PBS of the dexamethasone that is equivalent to 1mg/kg-bw (DEX).When needs, TNF and the IL-6 in [Agnello, 2000#10] quantitative measurement brain and the spinal cord homogenate as previously mentioned.

Figure 28 shows after the MBP immunity 3 days to 18 days and gives various dose EPO protective effect to the EAE clinical sign.EPO postpones the outbreak of disease in dosage dependence mode and reduces the order of severity of disease, summarizes as table 1.But EPO does not postpone to reach the time of the maximum order of severity.As the table shows, EPO obviously reduces the average accumulated scoring when its dosage is 2,500 and 5 during 000U/kg-bw.

Not continuing with EPO treatment and monitoring rat not observe recurrence in the experiment for February behind the disease recovery, after delaying its administration, cause disease progression (Figure 29) on the contrary with DEX.Carry out therapeutic treatment with recombinant tissue protectiveness cell factor of the present invention, expection will obtain similar results.

In vitro study:

Prepare the Deiter's cells primary culture from the newborn Sprague-Dawley rat of 1-2 age in days.Brain hemisphere is peeled off and physical disturbance from meninx.Cell is dispersed in 2.5% trypsase and the 1%DNA enzyme solutions, also inoculate (140,000 cells/35mm ware) on the EagleShi minimum essential medium that replenishes 10% hyclone, 0.6% glucose, chloromycetin (0.1mg/ml) and penicillin (100Ul/ml) by 100 μ m nylon net filters.Feed the neuroglia culture weekly for 2 times and in 37 ℃ of cultivations at 5%CO ₂The humidification incubator in.All experiments are all carried out on the Deiter's cells culture in age in 2-3 week, and described culture is with the immunochemistry mensuration of GFAP and Griffonia sirnplicifolia isolectin B4, and 97% astroglia and 3% microglia are wherein arranged.Set up the neuron culture from 18 days rat fetal hippocampus.Shift out brain and peel off the separation hippocampus from meninx.By in 2.5% trypsin solution in 37 ℃ of insulations 15-20 minute, cell is disperseed, measure concentration then.Cell suspension is inoculated on the cover glass of poly ornithine embedding in the nutrient culture media dilution that is used for Deiter's cells and with the density of the every cover glass of 160,000 cells.Inoculation is transferred to cover glass and is contained the neurons neuroglia individual layer, that replenish cytarabine 5 μ M and keep on nutrient culture media (having replenished the DulbeccoShi improvement EagleShi nutrient culture media and the HamShi nutrition mixing F12 of 5 μ g/ml insulin, 100 μ g/ml transferrinss, 100 μ g/mlputrescin, 30nM sodium selenite, 20nM progesterone and the 100U/ml penicillin) double dish one day after.The overturn cover slide makes hippocampal neuron towards the neuroglia individual layer.The paraffin point that is bonded on the cover glass supports them on neuroglia, produces slit, prevents that two kinds of cell types from contacting with each other, but allows the solable matter diffusion.These condition of culture allow the growth of differentiated neuron cultures, and described culture has＞98% homology with the immunochemistry mensuration of microtubule-associated protein 2 and GFAP.(10U (80ng/ml) exists or not down, with cell processing 24 hours, supernatant was used for TNF mensuration with 1 μ M tin trimethyl (TMT), and cell viability is estimated as mentioned above at rhEPO then.When needs, in the presence of LPS, when being with or without rhEPO, Deiter's cells was cultivated 24 hours, measure the TNF in the culture supernatant.By 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrabormated azoles (MTT) is measured, and measures cell viability.Denizot, F. and Lang, the colorimetric assay for cellgrowth and survival.Modifications to the tetrazolium dye proceduregiving improved sensitivity and reliability. (rapid colorimetric determination of cell growth and survival R.1986.Rapid.To the modification of tetrazole dye method with improved sensitivity and reliability) .JImmunol Methods 89:27 1-277.In brief, the MTT tetrazolium is dissolved in the serum free medium, making its final concentration is 0.75mg/ml, and joins and be in 37 ℃ of cells of handling 3 hour latter stage.Remove nutrient culture media then, use 1N HCl: isopropyl alcohol (1: 24) extraction first

On the micro plate reader, read the 560nm absorbance.

Figure 30 shows that rhEPO prevents to induce the TNF of neuronal death to produce in composite nerve unit colloid culture.Drawing A: when being with or without rhEPO (10U/ml), the nerve cell death percent of inducing by TMT 1 μ M.Drawing B: have (shade band) at neuron or do not have (filling out the secret note band), be with or without rhEPO when (10 U/ml), the TNF that discharges from the Deiter's cells that is exposed to TMT 1 μ M.Carry out therapeutic treatment with recombinant tissue protectiveness cell factor of the present invention, expection will obtain similar results.

6.14. embodiment 14:NMDA inducing cell death is measured

Exitotoxicity may be defined as the overactivity of glutamate receptor, such as the overactivity of N-methyl-D-aspartate (NMDA) acceptor.Nmda receptor shows the activity (Fauci etc. of increase when response ischemic wound or other wound, 1998, Harrison ' s Principles ofInternal Medicine), (Nishizawa, 2001, Life Sci.69,369-381), (White etc., 2000, J.Neurol.Sci.179,1-33).Therefore, this mensuration is as estimating pair cell damage and the dead model that the compound of effect is arranged.

The scheme of NMDA exitotoxicity in former generation hippocampal neuron

According to the method for description such as Krohn (1998), from newborn mice (less than 24 hours) preparation hippocampal neuron culture of former generation.In brief, hippocampus is cut into slices in containing the DMEM of 0.02%BSA.Tissue is transferred among the DMEM that contains 0.1% papain, and in 37 ℃ of insulations 20 minutes.Suction is removed to contain the nutrient culture media of papain and is added MEMII and stop digestion, and hippocampal cell is played dispersion with 1000 μ l transfer pipets suctions.Fragment of tissue leaves standstill, and the supernatant that will contain individual cells is transferred among the MEMII that contains 1% trypsin inhibitor (typeII-O) and 1%BSA.Inhale and play step repetition 3 times, then with individual cells centrifugal 10 minutes at 600U/ minute, and be suspended in growth medium (MEMII again, 20mM D-glucose, 100U/ml penicillin, 100 μ g streptomysins, the 2mM L-glutaminate, 10%Nu-serum (ox), 2%B27 replenishers, 26.2mM NaHCO ₃) in.Cell from 10 hippocampus is used to inoculate 24 orifice plates.Inoculate after 1 day, cell is handled with cytidine-arabinose-furanoside (1 μ M).At the 2nd day, change nutrient culture media, add cytidine-arabinose-furanoside (1 μ M).

Exitotoxicity is measured

The culture in 12 day age and the test compound of 5nM (solvent, R103E, R150E or EPO) pre-incubation 24 hours.At the 13rd day, nutrient culture media is shifted out and keeps from cell, at room temperature attack culture with 300 μ M NMDA simultaneously and reach 5 minutes.After the exitotoxicity infringement, pre-conditioned medium is turned back in the culture, get rid of the quantitative measurement infringement with trypan blue after being incubated 24 hours again.Every kind of condition is all added up about 300 neurons at least 4 independent holes, and repeated experiments at least 2 (Krohn, A.J., Preis, E. and Prehn, J.H.M. (1998) J.Neurosci.18 (20): 8186-8197).

Before Fig. 31 was presented at the MNDA processing, erythropoietin and recombinant tissue protectiveness cell factor R130E and R150E effectively reduced the cell death that is caused by NMDA when joining in the former generation Hippocampal Neuron Cells cultivation.Compare with the solvent control cells, show the cell death (p=0.01) of remarkable minimizing with the cell of R103E (5 nM) processing.Compare with the solvent control cells, show the cell death (p=0.01) of remarkable minimizing with the cell of R103E (5nM) processing.Compare with the solvent control cell, the cell of handling with R150E (5nM) shows about 20% minimizing (p=0.001) in cell death.Statistics: ANOVA adds that TukeyShi checks afterwards.

6.15. embodiment 15: the neuro-protective behind the removal serum in the P19 cell

In order to detect the neuro-protective effect of recombinant tissue protectiveness cell factor of the present invention, the P19 cell of removing serum is as model.Clone P19S1801A1 is provided by Dr.W.H.Fischer close friend.At 7%CO ₂Air in, in the humidification incubator, cell remained on replenished 2mM L-glutaminate, 100U/ml benzyl penicillin, 100 μ g/ml streptomycin sulphates and 10% hyclone (FCS; All from Gibco, Paisley, Scotland, LTK) and contain 1.2g/L NaHCO ₃, (Carlo Erba, Milano is in Italy) the Dulbecco improvement Eagles nutrient culture media (DMEM) (this is called complete medium hereinafter) for 10mM Hepes damping fluid.Serum free medium (N2) has as above identical composition, but lacks serum, and adds following material: 5 μ g/ml insulin, 100 μ g/ml transferrinss, 20nM progesterone, 100 μ M putrescine and 30nM Na ₂SeO ₃(all from Sigma).For the death experiment, cell is disperseed with 10% pancreatinum (Gibco), with complete medium washing 1 time, with N2 nutrient culture media washing 2 times, and inoculation, except as otherwise noted, otherwise be seeded in 25 cm ²In the tissue culture flasks (FalconBecton Dickinson, Lincoln Park, New Jersey), final concentration is 10 in 5 μ l serum free mediums ⁴Cell/cm ²Nicetile (100 μ M) is as positive control, and after removing serum after 24 hours, described material is given protectiveness and reduced the percent that reaches 50% apoptosis nuclear.After behind the removal serum 24 hours, beaing culture flask makes cell disperse (without trypsase), by with 600rpm rotating centrifugal (Shandon Southern, USA) after 10 minutes, be seeded on the microslide and and fix 10 minutes, dyeed 1 hour in 37 ℃ with Hoechst 33258 (0.1 μ r/ml PBS) with Carnoy solution (methyl alcohol: acetate, 3: 1), with tap water washing 15 minutes, air-dry and install.(Zeiss Germany) observes slide under the 365nm excitation wavelength with fluorescent microscope.The percent of apoptosis nuclear is counted in 100 cells altogether by blind method at least 5 times are detected and is measured.

With P19 cell and 3nM Epo or recombinant tissue protectiveness cell factor S100E pre-incubation 24 hours.Such processing is to producing conspicuousness (p＜0.001) protection by the Apoptosis of removing the serum triggering.Data are the mean value that 3 times are measured in 1 experiment.Experiment carries out twice, and the result is similar.

Figure 32 shows and removes the neuro-protective in the P19 cell behind the serum.For carrying out pretreated cell with Epo, EpoWT and recombinant tissue protectiveness cell factor S100E, the percent of apoptotic cell death descends.Compare with untreated control cells, the cell of handling with EPO shows about 20% decline on apoptotic cell death.Compare with untreated control cells, the cell of handling with EpoWT and S100E all shows about 10% decline on apoptotic cell death.

6.16. embodiment 16: NGF removes in the PC12 cell of differentiation

In order to detect the neuro-protective effect of recombinant tissue protectiveness cell factor of the present invention, the PC12 cell of the differentiation that NGF removes is as model.Described mensuration is a good model of cell apoptosis of setting up.Described PC12 rat cell system is from adrenal medullary pheochromocytoma (phaeochromocytoma), and in the presence of NGF, can be divided into neuron cell (Masuda etc., 1993, J Biol Chem 268,11208-11216).PC 12 clones are neuroendocrine cell systems, but described clone in the presence of NGF expression of differentiation neuron phenotype (Vaudry etc., 2002, Science 296,1648-1649).In case cell breaks up fully, it is dependent that they just become NGF, and can cause Apoptosis when removing NGF.

(Invitrogen, Carlsbad keep in the Dulbecco improvement Eagle nutrient culture media (DMEM) USA) the PC12 cell having replenished 10% hot deactivation horse serum, 5% heat-inactivated fetal bovine serum, 1% Sodium Pyruvate and 1% penicillin-streptomysin (P/S).

In order to experimentize, replenishing 1% hot deactivation horse serum, 1% Sodium Pyruvate, 1%P/S and 100ng/ml NGF (7S nerve growth factor, mouse submandibular gland, available from Calbiochem, catalog number 480354) among the DMEM, the cell that with density is 24,000 cells/well broke up 7 days in 48 orifice plates of collagen G embedding, changed nutrient culture media every 2-3 days.At the 6th day, when substituting nutrient culture media with RPMI1640,1%P/S with after from all cells, removing NGF, will the Epo of amino acid/11 00 mutant (=S100E) join with prescribed concentration and reach 24 hours in the cell.Add S100E again, when NGF (100ng/ml) as positive control (+NGF) time.After 24 hours, come the mensuration vigor by tetrazole (MTT) determination by reduction.

Figure 33 A and Figure 33 B are presented at two independently in the experiment, in removing the differentiation PC12 cell of NGF, and the effect of carrying out pre-incubation with S100E.The S100E pre-service of the PC12 cell usefulness prescribed concentration of differentiation 24 hours, Figure 33 A (3 pM) Figure 33 B (0.00003pM-3pM).Mensuration vigor in MTT measures.NGF (100ng/ml) represents positive control, and does not have the NGF nutrient culture media (NGF) as negative control.Data shown in Figure 33 be positive control (+NGF) and the % (n=8 in two experiments) of vigor.Use unidirectional ANOVA and Bonferroni to check afterwards, ^{* *}P＜0.001, ^*P＜0.05, (NGF) compare, the vigor of handling cell through S100E has significance,statistical to increase with negative control cell.For rendeing a service and effect, with the observed effect of S100E and similar with EPO in such experimental system.

Figure 34 is presented in the differentiation PC12 cell of removing NGF the effect with the Epo pre-incubation.PC12 cell usefulness Epo, the S100E of differentiation or carbamylation Epo (30 pM-30nM) pre-service 24 hours.The Epo molecule AA24496 of chemical modification hangs down 10000 times than EPO is active in the UT-7 raji cell assay Raji.Mensuration vigor in MIT measures.NGF (100ng/ml) is used as positive control, and does not have the NGF nutrient culture media (NGF) as negative control.

6.17. embodiment 17:EPO biologicall test UT-7 cell proliferation

UT-7 is the leukaemia Epo dependent cell system that is used to measure the red blood cell class effect of recombinant tissue protectiveness cell factor such as K45D.UT-7 cell (Deutsche Sammlungvon Mikroorganismen und Zellkulturen (DSMZ), catalog number ACC 363) normal growth in the presence of 10%FBS and 5ng/ml Epo.Propagation/survival (increase of=vigor) effect that is exposed to the cell of Epo is receptor-mediated by traditional periphery type Epo.The quantitative measurement multiplication effect is also determined multiplication effect and the relation of Epo-variant stimulation traditional E po acceptor ability.

Be used for the method for measuring of UT-7 cell viability

Preparation Epo dependence human leukemia cell line UT7 is to the multiplication effect of the Epo/ recombinant tissue protectiveness cell factor that the adds mensuration as its biologic activity.At the 1st day that measures, with cell transfer to containing 10% serum and containing in the fresh complete RPMI1640 nutrient culture media (10% donor calf serum, 4mM L-glutaminate, additional 5ng/ml ofrhuEPO) of Epo (5ng/ml).Cell is at the 75cm that is loaded with the 20ml culture ²Grow in the culture flask.At the 2nd day that measures, from bottle, transfer to cell in the 50ml tapered tube and at room temperature 1, centrifugal 5 minutes of 000rpm.Discard outmoded nutrient culture media and use the hungry nutrient culture media of 10ml (3% donor calf serum, 4mM L-glutaminate) to wash 2 times in cell.Cell is suspended in the hungry nutrient culture media again, aspirates up and down to obtain single cell suspension with suction pipe.In order to measure cell density, the cell that suspends is again diluted with hungry nutrient culture media, making density is 4 * 10 ⁵Cell/ml, nutrient culture media cumulative volume are 10ml, are inoculated into 25cm then ²In the culture flask.Potpourri is at 5%CO ₂Be incubated 4 hours at 37 C in the humidification incubator.At last 1 hour of insulation, prepare 96 orifice plates.Be incubated latter stage at 4 hours, cell culture is shifted out from incubator, cell is transferred to from bottle in the 50ml tapered tube.With hand moving, keep cell suspension with contents mixed.Add the hungry nutrient culture media of 50ml as the nutrient culture media blank that does not have cell.5 holes are the control cells that do not have reagent.An adjacent round contains the recombinant tissue protectiveness cell factor of least concentration.Each adjacent row's hole adds higher concentration successively later on.At 96 orifice plates, will in the cell culture that contains 3% serum and do not have to be incubated in the nutrient culture media of Epo, inoculate 100 μ l with the every hole of 200,000 cells/ml.Track vibration platform with the agitator disk top mixes content rapidly and carefully.With this plate and variable concentrations Epo variant (from 0.2pM-20nM) RPMI 1640 nutrient culture media that contain 3% serum, at 5%CO ₂The humidification incubator in 37 C insulation 48 hours.Measured 4 times in one day, 96 orifice plates are taken out from incubator, place in the laminar flow hood at room temperature.By measuring the first that cellular metabolism tetrazole dyestuff WST1 forms Product (this product is relevant with cell viability and cell number), quantitative measurement biologically active immediately (the photometer absorption value of 450nm deducts the background absorption value of 620nm).

The result

The UT7 cell is containing on the nutrient culture media of Epo growth 3 months, demonstrates stable and growth reliably.

Induce the K45D of vigor to induce Epo dependence UT-7 cell viability to increase its EC in dose-dependent mode ₅₀Be 294.0.By contrast, for Epo, EC ₅₀Be 58.13 (Figure 35), and be 608 for His mark Epo (EpoWT).When concentration＜50nM (promptly in measurable range), S100E does not increase the vigor (greater than 50%) of Epo dependence UT-7 cell.Therefore, K45D demonstrate with the effectiveness of Epo same order, and S100E demonstrates low 1000 times the effectiveness at least than Epo.

Under up to 20nM concentration, R103E does not increase the survival of Epo dependence UT-7 cell, and promptly its effectiveness is compared low at least 4 orders of magnitude with Epo.R150E induces the survival of Epo dependence UT-7 cell, its EC in dose-dependent mode ₅₀Be 20nM.By contrast, for Epo (Epo#4), EC ₅₀Be 66.5 (Figure 36).Therefore, R150E demonstrates the effectiveness than low at least 3 orders of magnitude of Epo.

Figure 35 shows Epo, K45D and the concentration-response curve of S100E in the UT-7 cell.Epo, EpoWT, K45D and the S100E of variable concentrations are joined in the UT-7 cell.In WST-1 measures, mensuration vigor after 48 hours.Data are the mean value ± SD that at every turn all carry out replication in three different experiments.This curve is the non-linear regression curve fitting.

Figure 36 shows Epo, R103E and the dose-effect curve of R150E in the UT-7 cell.Epo, EpoWT, R103E and the R150E of variable concentrations are joined in the UT-7 cell.In WST-1 measures, mensuration vigor after 48 hours.Data are the mean value ± SD that at every turn all carry out replication in three different experiments.This curve is the non-linear regression curve fitting.

6.18. embodiment 18: administered peripherally recombinant tissue protectiveness cell factor is to the protection of treat retinal ischemic

As described in 6.9 trifles, the retina cell is very responsive to ischaemic, so in the ischemic stress reaction after 30 minutes, many cell deaths.In this experiment, use as Rosenbaum etc. (1997; Vis.Res.37:3443-51) the glaucomatous rat model of described reversibility.Detected recombinant tissue protectiveness cell because of. son is to the effect of ischemic stress reaction.

The embodiment of the anterior chamber that injects saline into the bull rat that provides according to 6.9 trifles injects for the eyes of every rat.When pouring into, promptly when the pressure of anterior chamber discharges, intravenous gives a kind of or salt solution among 4 kinds of recombinant tissue protectiveness cell factor R103E, R150E, S100E and the S100e/K45D of rat 10 μ g/kg EPO again.Damaged back 1 day, 3 days, 5 days and 6 days, injured eye and the normal eye of every rat all carried out electroretinography.Compare the latent period of the latent period of the injured eye of every rat and the normal eye of same rat.Data recording is injured eye latent period and the preclinical ratio of normal eye, has produced a ratio when injured eye has normal function.The result has two factors to damage: amplitude (difference from the peak to paddy is shown in Figure 17, and drawing A represents with ' b ' and latent period), arrive peaking institute's time spent when response stimulates.

Figure 38 shows for the latent period of the injured eye of different therapeutic schemes and the preclinical ratio of normal eye.Showing latent period with the rat of EPO treatment is 1.2, better than the rat with saline treatment.To every kind of this 4 kinds of recombinant tissue protectiveness cell factors, produce the preclinical EPO that is equal to or better than with R103E, R150E and S100E, illustrate better than salt solution statistically.

Scope of the present invention is not used as the method for the restriction of specific embodiments of indivedual explanations of various aspects of the present invention and functional equivalent and is formed all within the scope of the present invention.Even from the above content and accompanying drawing to various modifications of the present invention, except that shown in this paper and described, also will it will be apparent to those skilled in the art that.Described modification all comprises within the scope of the appended claims.

All lists of references cited above all are attached to herein by reference, are used for all purposes.

Claims (80)

1. the Pharmaceutical composition that comprises mutain recombinant tissue protectiveness cell factor, wherein said cell factor is included in the erythropoietin(EPO) shown in the SEQ ID NO:10 amino acid sequences one or more in the following aminoacid replacement: 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, N147K, N147A, F148A, F148Y, L149A, R150A, R150E, G151A, K152A, K152W, L153A, K154A, L155A, G158A, A160S, C161A and/or R162A, wherein said amino acid position generates plain molecule numbering according to mature erythrocyte; And (a) described cell factor at least a is selected from following activity and lacks or weaken: increase the generation of hematocrit, vasoactive effect, superactivation blood platelet, procoagulant activity and increase blood platelet; (b) described cell factor has at least a following activity that is selected from:

I. protect the function or the vigor of mammal effector cell, tissue or organ;

Ii. keep the function or the vigor of mammal effector cell, tissue or organ;

Iii. strengthen the function or the vigor of mammal effector cell, tissue or organ; With

Iv. recover the function or the vigor of mammal effector cell, tissue or organ.

2. the Pharmaceutical composition of claim 1, wherein said mutain recombinant tissue protectiveness cell factor comprise at least one following sudden change or sudden change combination: R14Q, S 100E, R103E, R150E, K45D/S100E or K45D/R150E or R103E/L108S.

3. the Pharmaceutical composition of claim 1; wherein said mutain recombinant tissue protectiveness cell factor is included in the amino acid residue that is selected from the change on following one or more amino acid positions: position 11,12,13,14,15 is SEQ ID NO:1; position 44,45,46,47,48,49,51 is that SEQ ID NO:2, position 100,101,102,103,104,105,106,107,108 are SEQ ID NO:3, and position 146,147,148,149,150 and amino acid/11 51 are SEQ ID NO:4.

4. the Pharmaceutical composition of claim 1, wherein said mutain recombinant tissue protectiveness cell factor is included in one or more amino acid residues that are selected from the change of following amino acid position: 7,20,21,29,33,38,42,59,67,70,83,96,126,142,143,152,153,155 and 161.

5. the Pharmaceutical composition that comprises mutain recombinant tissue protectiveness cell factor, wherein said cell factor comprise the amino acid sequence of the SEQ ID NO:10 with following change:

1) one or more during amino acid residue shown in the SEQ ID NO:15-105 or 119 replaces;

2) amino acid residue 44-49 disappearance, wherein said amino acid position generates plain molecule numbering according to mature erythrocyte; Or

3) at least one in the aminoacid replacement shown in the SEQ ID NO:106-118, and (a) described cell factor at least a is selected from following activity and lacks or weaken: increase the generation of hematocrit, vasoactive effect, superactivation blood platelet, procoagulant activity and increase blood platelet; (b) described cell factor has at least a following activity that is selected from:

I. protect the function or the vigor of mammal effector cell, tissue or organ;

Ii. keep the function or the vigor of mammal effector cell, tissue or organ;

Iii. strengthen the function or the vigor of mammal effector cell, tissue or organ; With

Iv. recover the function or the vigor of mammal effector cell, tissue or organ.

6. each Pharmaceutical composition among the claim 1-5, wherein said mutain recombinant tissue protectiveness cell factor also comprises one or more amino acid whose chemical modifications.

7. the Pharmaceutical composition of claim 6,, wherein said chemical modification causes electric charge to change.

8. the Pharmaceutical composition of claim 7,, wherein charged amino acid residue is modified to uncharged residue.

9. each Pharmaceutical composition among the claim 1-5, wherein said mammal effector cell is selected from neuronal cell, myocyte, core cell, pneumonocyte, liver cell, nephrocyte, small intestine cells, adrenal cortical cell, adrenal medullary cell, capillary cell, endothelial cell, testicular cell, gonad cell, endometrial cell and stem cell.

10. each Pharmaceutical composition among the claim 1-5, wherein said mammal effector cell is selected from photosensory cell, gangliocyte, Beale's ganglion cells, horizontal cell, amakrine, muller cell, the cardiac muscle cell, pacemaker cells, sinus node cells, the hole nodal cell, the atrioventricular node cell, the atrioventircular bundle cell, liver cell, astrocyte, Kupffer cell, mesangial cell, goblet cell, the enteraden cell, enteroendocrine cell, messangial cell, the pencil cell, desmacyte, chromaffin cell, pericyte, interstitial cell, trophocyte, spermatid, the ripe follicle cell, original follicular cells, endometrial stromal cell and endometrial cell.

11. each Pharmaceutical composition among the claim 1-5, wherein said mutain recombinant tissue protectiveness cell factor can be crossed endothelial cell barrier.

12. the Pharmaceutical composition of claim 11, wherein said endothelial cell barrier are selected from blood brain barrier, blood-ocular barrier, blood-testis barrier, blood-ovary barrier and blood-uterus barrier.

13. each Pharmaceutical composition among the claim 1-5, wherein said mutain recombinant tissue protectiveness cell factor is compared to have below at least one with natural erythropoietin molecule and is modified:

I.0,1,2,3,4,5,6,7,8,9,10,11,12 or 13 sialic acid part;

Ii. the N of decreased number connection sugar or O join sugar or do not have N connection sugar or O connection sugar;

Iii. by handle the sugared content that natural recombinant tissue protectiveness cell factor has reduction with at least a glycosidase;

Iv. one or more oxosugars;

V. one or more oxosugars, and described mutain recombinant tissue protectiveness cell factor is also by electronation;

Vi. one or more modification arginine residues;

Vii. one or more modification lysine residues or N end are amido modified;

Viii. one or more modification tyrosine residues;

Ix. one or more modification aspartic acids or glutaminic acid residue;

X. one or more modification trp residues;

Xi. one or more amino groups are removed;

Xii. at least one cystine linkage is opened;

Xiii. brachymemma;

Xiv. connect one or more peg molecules;

Xv. connect one or more fatty acid;

Xvi. nonmammalian glycosylation pattern; With

Xvi. one or more histidine mark amino acid.

14. the Pharmaceutical composition of claim 13, wherein said recombinant tissue protectiveness cell factor is to take off the sialic acid erythropoietin(EPO).

15. the Pharmaceutical composition of claim 1-5, wherein said mutain recombinant tissue protectiveness cell factor is a high sialic acidization.

16. the Pharmaceutical composition of claim 13, wherein said mutain recombinant tissue protectiveness cell factor has 1,2,3,4,5,6,7,8,9,10,11,12 or 13 sialic acid parts.

17. the Pharmaceutical composition of claim 13, wherein said mutain recombinant tissue protectiveness cell factor are the erythropoietin(EPO) that does not contain N connection sugar.

18. the Pharmaceutical composition of claim 17, wherein said mutain recombinant tissue protectiveness cell factor are the erythropoietin(EPO) that does not contain O connection sugar.

19. the Pharmaceutical composition of claim 13, wherein said mutain recombinant tissue protectiveness cell factor are to handle through at least a glycosidase by handle natural recombinant tissue protectiveness cell factor with at least a glycosidase.

20. the Pharmaceutical composition of claim 13, wherein said mutain recombinant tissue protectiveness cell factor is the erythropoietin(EPO) of periodate oxidation.

21. the Pharmaceutical composition of claim 20, the erythropoietin(EPO) of wherein said periodate oxidation is by the sodium cyanoborohydride electronation.

22. the Pharmaceutical composition of claim 13, wherein said mutain recombinant tissue protectiveness cell factor comprises R-glyoxal part on one or more arginine residues, and wherein R is aryl or moieties.

23. the Pharmaceutical composition of claim 22, wherein said mutain recombinant tissue protectiveness cell factor is phenyl glyoxal-erythropoietin(EPO).

24. the Pharmaceutical composition of claim 13, wherein said mutain recombinant tissue protectiveness cell factor be wherein arginine residues by be selected from 2, company's two reactive ketones of 3-diacetyl and cyclohexanedione and adorned erythropoietin(EPO).

25. the Pharmaceutical composition of claim 13, wherein said mutain recombinant tissue protectiveness cell factor are the erythropoietin(EPO) of arginine residues and 3-deoxyglucosone reaction wherein.

26. the Pharmaceutical composition of claim 13, wherein said modification lysine or modification N terminal amino group are that at least one biotinylation lysine or biotinylation N end are amino.

27. the Pharmaceutical composition of claim 13, wherein said modification lysine are glucose alcohol radical lysine or fructosyl lysine.

28. one Pharmaceutical composition among the claim 1-5, wherein said mutain recombinant tissue protectiveness cell factor comprises at least one carbamylation lysine residue.

29. the Pharmaceutical composition of claim 28, wherein said mutain recombinant tissue protectiveness cell factor is selected from: α-N-carbamyl erythropoietin(EPO); N-ε-carbamyl erythropoietin(EPO); α-N-carbamyl, N-ε-carbamyl erythropoietin(EPO); α-N-carbamyl takes off the sialic acid erythropoietin(EPO); N-ε-carbamyl takes off the sialic acid erythropoietin(EPO); α-N-carbamyl, N-ε-carbamyl takes off the sialic acid erythropoietin(EPO); α-N-carbamyl hangs down the sialic acid erythropoietin(EPO); N-ε-carbamyl hangs down the sialic acid erythropoietin(EPO); And α-N-carbamyl, N-ε-carbamyl hangs down the sialic acid erythropoietin(EPO).

30. the Pharmaceutical composition of claim 13, wherein said mutain recombinant tissue protectiveness cell factor comprises at least one acidylate lysine residue.

31. the Pharmaceutical composition of claim 30, wherein said mutain recombinant tissue protectiveness cell factor comprises at least one acetylation lysine residue.

32. the Pharmaceutical composition of claim 31, wherein said mutain recombinant tissue protectiveness cell factor is selected from: α-N-acetyl erythropoietin(EPO); N-ε-acetyl erythropoietin(EPO); α-N-acetyl, N-ε-acetyl erythropoietin(EPO); α-N-acetyl takes off the sialic acid erythropoietin(EPO); N-ε-acetyl takes off the sialic acid erythropoietin(EPO); α-N-acetyl, N-ε-acetyl takes off the sialic acid erythropoietin(EPO); α-N-acetyl hangs down the sialic acid erythropoietin(EPO); N-ε-acetyl hangs down the sialic acid erythropoietin(EPO); And α-N-acetyl, N-ε-acetyl hangs down the sialic acid erythropoietin(EPO).

33. the Pharmaceutical composition of claim 30, the lysine residue of wherein said mutain recombinant tissue protectiveness cell factor is a succinylation.

34. the Pharmaceutical composition of claim 33, wherein said mutain recombinant tissue protectiveness cell factor is selected from: α-N-succinyl erythropoietin(EPO); N-ε-succinyl erythropoietin(EPO); α-N-succinyl, N-ε-succinyl erythropoietin(EPO); α-N-succinyl takes off the sialic acid erythropoietin(EPO); N-ε-succinyl takes off the sialic acid erythropoietin(EPO); α-N-succinyl, N-ε-succinyl takes off the sialic acid erythropoietin(EPO); α-N-succinyl hangs down the sialic acid erythropoietin(EPO); N-ε-succinyl hangs down the sialic acid erythropoietin(EPO); And α-N-succinyl, N-ε-succinyl hangs down the sialic acid erythropoietin(EPO).

35. the Pharmaceutical composition of claim 13, wherein said mutain recombinant tissue protectiveness cell factor comprises at least one by 2,4, the lysine residue that 6-trinitrobenzene sulfonate or its another kind of salt are modified.

36. the Pharmaceutical composition of claim 13, wherein said modification tyrosine residue is nitrated and/or iodate.

37. the Pharmaceutical composition of claim 13 reacts with amine after wherein said modification asparagicacid residue or glutaminic acid residue and the carbodiimide reaction again.

38. the Pharmaceutical composition of claim 37, wherein said amine is glycine amide.

39. each Pharmaceutical composition among the claim 1-5, described composition are mixed with, and confession is oral, nose interior or parenteral is used.

40. each Pharmaceutical composition among the claim 1-5, described composition is mixed with perfusion liquid.

41. mutain recombinant tissue protectiveness cell factor, wherein said cell factor comprises at least one following aminoacid replacement: R150E, K45D/S100E or K45D/R150E or R103E/L108S, and wherein said amino acid position generates plain molecule numbering according to mature erythrocyte; And described cell factor at least a is selected from following activity and lacks or weaken: increase the generation of hematocrit, vasoactive effect, superactivation blood platelet, procoagulant activity and increase blood platelet; Described cell factor has the effector cell who at least aly is selected from protection, keeps, strengthens or recovers mammal effector cell, tissue or organ dysfunction or vigor and protects activity.

42. an isolated nucleic acid molecule, described nucleic acid molecules comprises the nucleotide sequence of coded polypeptide, and described polypeptide comprises the mutain recombinant tissue protectiveness cell factor of claim 41.

43. a carrier, described carrier comprises the nucleic acid molecules of claim 42.

44. the regulatory region that the nucleic acid molecules that an expression vector, described carrier comprise claim 42 and at least one and described nucleic acid molecules effectively are connected.

45. the carrier of claim 43 or 44, described carrier are the pCI-neo carriers.

46. a genetically engineered cell, described cell comprises the nucleic acid molecules of claim 42.

47. cell that comprises claim 43,44 or 45 expression vector.

48. one kind is used for protecting, keeping or strengthens from the method for the vigor of body of mammals isolated cells, tissue or organ, described method comprises the Pharmaceutical composition that makes among described cell, tissue or the organ contact claim 1-5 each.

49. the method for claim 48, wherein said mutain recombinant tissue protectiveness cell factor right and wrong are erythropoietic.

50. the purposes of mutain recombinant tissue protectiveness cell factor in the preparation Pharmaceutical composition, wherein said cell factor is included in the erythropoietin(EPO) shown in the SEQ ID NO:10 amino acid sequences one or more in the following aminoacid replacement: 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, N147K, N147A, F148A, F148Y, L149A, R150A, R150E, G151A, K152A, K152W, L153A, K154A, L155A, G158A, A160S, C161A and/or R162A, wherein said amino acid position generates plain molecule numbering according to mature erythrocyte; And (a) described cell factor at least a is selected from following activity and lacks or weaken: increase the generation of hematocrit, vasoactive effect, superactivation blood platelet, procoagulant activity and increase blood platelet and (b) described cell factor have at least a following activity that is selected from:

I. protect the function or the vigor of mammal effector cell, tissue or organ;

Ii. keep the function or the vigor of mammal effector cell, tissue or organ;

Iii. strengthen the function or the vigor of mammal effector cell, tissue or organ; With

Iv. recover the function or the vigor of mammal effector cell, tissue or organ,

Described Pharmaceutical composition is used for:

1) prevents or prevents to influence in the mammal damage, disease or the illness of effector cell, tissue or organ; Or

2) strengthen, keep, recover or the mammal that regenerates in the function or the vigor of effector cell, tissue or organ.

51. the purposes of claim 50, wherein said mutain recombinant tissue protectiveness cell factor comprise at least one following sudden change or sudden change combination: R14Q, S100E, R103E, R150E, K45D/S100E or K45D/R150E or R103E/L108S.

52. the purposes of claim 50; the white recombinant tissue protectiveness of wherein said kink of preserved egg cell factor is included in the amino acid residue that is selected from the change on following one or more amino acid positions: position 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], position 146,147,148,149,150 and amino acid/11 51[SEQ ID NO:4].

53. the purposes of claim 50, wherein said mutain recombinant tissue protectiveness cell factor are included in the amino acid residue of the change of one or more 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. the purposes of mutain recombinant tissue protectiveness cell factor in the preparation Pharmaceutical composition, wherein said cell factor comprises the amino acid sequence of the SEQ ID NO:10 with following change:

(a) one or more during amino acid residue shown in the SEQ ID NO:15-105 or 119 replaces;

(b) amino acid residue 44-49 disappearance, wherein said amino acid position generates plain molecule numbering according to mature erythrocyte; Or

(c) at least one in the aminoacid replacement shown in the SEQ ID NO:106-118, and (i) described cell factor at least a is selected from following activity and lacks or weaken: increase the generation of hematocrit, vasoactive effect, superactivation blood platelet, procoagulant activity and increase blood platelet; (ii) described cell factor has at least a following activity that is selected from:

I. protect the function or the vigor of mammal effector cell, tissue or organ;

Ii. keep the function or the vigor of mammal effector cell, tissue or organ;

Iii. strengthen the function or the vigor of mammal effector cell, tissue or organ; With

Iv. recover the function or the vigor of mammal effector cell, tissue or organ,

Described Pharmaceutical composition is used for:

1) prevents or prevents to influence in the mammal damage, disease or the illness of effector cell, tissue or organ; Or

2) strengthen, keep, recover or the mammal that regenerates in the function or the vigor of effector cell, tissue or organ.

55. each purposes among the claim 50-54, wherein said mammal suffers from: cognition dysfunction, epileptic attack disease, Chronic Epilepsy, epilepsy, convulsions, nerve root compression, multiple sclerosis, apoplexy, low blood pressure, asystole; Central nervous system disease, obstacle or illness; Neurone loss, ischaemic, subarachnoid hemorrhage, aneurysm, aneurysm is hemorrhage, miocardial infarction, inflammation, the age cognitive decrease of being correlated with, radiation damage, radiotherapy, full brain irradiation, cerebral palsy, benumb on the cerebral nucleus, benumb on the carrying out property nuclear, neurodegenerative disease, alzheimer's disease, Parkinson's, Huntington, tourette's syndrome, SNE, acute febrile polyneuritis, dull-witted, the acquired immune deficiency syndrome (AIDS) dementia, senile dementia, thunder dimension corpusculum dementia, failure of memory, amyotrophic lateral sclerosis, alcoholism, neural spirit or neural mental handicape, mood disorder, the single phase property disturbance of emotion, depression, heavy melancholy sexual dysfunction, dysthymic disorder, mania, two-phase disposition sense obstacle, panic disorder, anxiety disorder, schizophrenia, schizoaffective disorder, obsessive-compulsive disorder, absent minded, scatterbrained hyperactivity disorder, autism, prion disease, the infectiousness spongiform encephalopathy, Friedreich ataxia, the Wilson disease, wound, the concussion damage, cerebral trauma or spinal cord injuries receptor, cerebral ischemia or ischemia of spinal cord, cardiopulmonary bypass, the neurological deficit that causes by cardiopulmonary bypass, the postoperative cognitive disorder, the embolic damage, hypoxa, mitochondria dysfunction, heart injury, chronic heart failure, ocular tissue damage, macular degeneration, diabetic neuropathy, diabetic retinopathy, glaucoma, treat retinal ischemic, the retina wound, retinitis pigmentosa, optic nerve lesion, retinal detachment, arteriosclerotic retinopathy, hypertensive retionpathy, retinal arterial obstruction, retinal vein obstruction, low blood pressure, the illness relevant with hypoglycemia, diabetes, nephrotic syndrome, acute renal failure or hepatitis.

56. each Pharmaceutical composition among the claim 1-5, wherein said mutain recombinant tissue protectiveness cell factor is alkylating.

57. each Pharmaceutical composition among the claim 1-5, wherein said mutain recombinant tissue protectiveness cell factor are carbamylation.

58. each Pharmaceutical composition among the claim 1-5, wherein said mutain recombinant tissue protectiveness cell factor is α-N-carbamyl, N-ε-carbamyl erythropoietin(EPO).

59. each composition in claim 1-5 or 28, wherein said mutain recombinant tissue protectiveness cell factor is connected with at least one peg molecule.

60. the mutain recombinant tissue protectiveness cell factor of claim 41, at least one lysine residue of wherein said cell factor is a carbamylation.

61. the mutain recombinant tissue protectiveness cell factor of claim 41, wherein said cell factor are carbamylation.

62. the mutain recombinant tissue protectiveness cell factor of claim 41, wherein said cell factor is alkylating.

63. the mutain recombinant tissue protectiveness cell factor of claim 41, wherein said cell factor is to take off the sialic acid erythropoietin(EPO).

64. the mutain recombinant tissue protectiveness cell factor of claim 41, wherein said cell factor is α-N-carbamyl, N-ε-carbamyl erythropoietin(EPO).

65. the mutain recombinant tissue protectiveness cell factor of claim 41 or 60, wherein said cell factor is connected with at least one peg molecule.

66. the method for claim 48, at least one lysine residue of wherein said mutain recombinant tissue protectiveness cell factor is a carbamylation.

67. the method for claim 48, wherein said mutain recombinant tissue protectiveness cell factor are carbamylation.

68. the method for claim 48, wherein said mutain recombinant tissue protectiveness cell factor is to take off sialic acid erythropoietin(EPO) or alkylation erythropoietin(EPO).

69. the method for claim 48, wherein said mutain recombinant tissue protectiveness cell factor is α-N-carbamyl, N-ε-carbamyl erythropoietin(EPO).

70. the method for claim 48 or 66, wherein mutain recombinant tissue protectiveness cell factor is connected with at least one peg molecule.

71. each purposes among the claim 50-54, at least one lysine residue of wherein said mutain recombinant tissue protectiveness cell factor is a carbamylation.

72. each purposes among the claim 50-54, wherein said mutain recombinant tissue protectiveness cell factor are carbamylation.

73. each purposes among the claim 50-54, wherein said mutain recombinant tissue protectiveness cell factor is to take off sialic acid erythropoietin(EPO) or alkylation erythropoietin(EPO).

74. each purposes among the claim 50-54, wherein said mutain recombinant tissue protectiveness cell factor is α-N-carbamyl, N-ε-carbamyl erythropoietin(EPO).

75. each purposes among the claim 50-54, wherein said mutain recombinant tissue protectiveness cell factor is connected with at least one peg molecule.

76. being formulated into, each purposes among the claim 50-54, wherein said Pharmaceutical composition be used for administration after described damage, disease or illness begin.

77. being formulated into, each purposes among the claim 50-54, wherein said Pharmaceutical composition be used for administration before described damage, disease or illness begin.

78. the purposes of claim 76 or 77, wherein said damage or illness are cardiopulmonary bypass, radiotherapy or chemotherapy.

79. each purposes among the claim 50-54, wherein said Pharmaceutical composition is used for giving during the acute stage of described damage, disease or illness and/or chronic phase by preparation.

80. each purposes among the claim 50-54, wherein said mutain recombinant tissue protectiveness cell factor is a carbamylation, and is connected with at least one peg molecule.

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