CN1318084C

CN1318084C - Neuroprotective peptides

Info

Publication number: CN1318084C
Application number: CNB018131794A
Authority: CN
Inventors: V·史密斯－斯文托斯基; M·伦兹; C·普拉塔-萨拉曼; L·乔利菲; F·法雷尔; D·L·约翰森
Original assignee: 3 Dimensional Pharmaceuticals Inc
Current assignee: Janssen Pharmaceuticals Inc; 3 Dimensional Pharmaceuticals Inc
Priority date: 2000-05-26
Filing date: 2001-05-23
Publication date: 2007-05-30
Anticipated expiration: 2021-05-23
Also published as: EP1296702A4; CN1452492A; MXPA02011727A; IL153079A0; JP2003534384A; AU7490401A; NZ522924A; BR0111182A; HK1058484A1; WO2001091780A1; ZA200210304B; CA2410453A1; AU2001274904B2; EP1296702A1

Abstract

Methods of treating diseases of the nervous system by administration of compositions having the neurological therapeutic activity of human erythropoietin are disclosed. These compositions include therapeutic agents such as peptides, peptide dimers, polypeptides, and proteins that have the full range of biological activity of human erythropoietin or only certain biological activities of erythropoietin. Improved therapeutic regimens where the erythropoietin is administered at concentrations below those required to stimulate hematopoiesis are also provided.

Description

Neuroprotective peptide

The cross reference of related application

The application requires the rights and interests at the U.S. Provisional Application serial number 60/207654 of application on May 26th, 2000.

Invention field

The combination treatment that the present invention relates to the therapeutic activity by having erythropoietin relates to the method for neural disease and disease.These compositionss comprise having all biological activitys of erythropoietin or only have some bioactive therapeutic agent of erythropoietin, for example peptide, peptide dimer, polypeptide and albumen.The present invention also provides the therapeutic scheme of improvement, and wherein said therapeutic agent stimulates the needed concentration of erythropoiesis to give to be lower than.

Background of invention

Erythropoietin (EPO) is histanoxia to be replied and the glycoprotein hormones that produces by kidney, in the generation of bone marrow moderate stimulation erythrocyte.As U.S. Patent number 4,703, described in 008, the gene of erythropoietin has been cloned and has been expressed in Chinese hamster ovary cell (CHO).Recombinant human erythropoietin (rHuEPO or Epoetin α) has the aminoacid sequence identical with people's urinary erythropoietin, and they can't be distinguished on chemical detection, physical detection and immune detection.The recombinant human erythropoietin by increase can break up the cell quantity that becomes mature erythrocyte, the differentiation that triggers them and increase grow in erythroblastic hemoglobin synthesize (Krantz SB.Blood (1991) 77:419-434 that works, Beckman BS, Mason-Garcia M.The Faseb Journal (1991) 5:2958-2964).

Epoetin α can well be tolerated in the research of carrying out up to now.In dialysis patient, have the record of hypertensive encephalopathy and epilepsy once in a while with Epoetin α treatment, at length say it is (the Eschbach JW when hematocrit raises in early days that is treating, EgrieJC, Downing MR, Browne JK, Adamson JW.New Engl JMed (1987) 316:73-78, Winearls CG, Oliver DO, Pippard MJ etc., Lancet (1986) 2 (8517): 1175-1177).This class public lecture becomes rarer because obtain gradually to use the experience of described chemical compound.Occasionally, the cancer patient of Epoetin α treatment can experience one with the obviously relevant hypertension of rising of hematocrit.Yet in fact described risk demonstrates and is lower than patients with chronic renal failure.

Can not illustration in reach the patient in 2 years with Epoetin α treatment and the tiring of the anti-Epoetin Alpha antibodies of conclusive evidence, this shows the immune sensitivity of shortage to Epoetin α.Erythra and rubella seldom are observed, and in fact be slight and of short duration when report, but these incidents have hinted the pleoergy to some component in the Epoetin α preparation.

Epoetin α the listing of many state approvals be used for the treatment of chronic renal failure anemia (before dialysis and the dialysis), zidovudine treatment HTV positive patient anemia (US), accept anemia, as the promoter before body is contributed blood with carrying out among the patient of plastic operation as enclosing the probability that the operation adjuvant requires allos to transfuse blood with minimizing based on the cancer patient of platinum chemotherapy.

EPO is perhaps in embryo development procedure and first stem cell that may affect the nerves in vitro differentiation experiment Pediatr Dev Pathol (1999) 2 (2) 148-158 such as (, Pediatr Res such as Juul (1998) 43 (1) 40-49) Juul.The neonate of the CNS damage that in addition, suffers to cause owing to hypoxia, meningitis and ventricle internal hemorrhage and baby demonstrate the inductive neuroprotective of EPO Ped Res (1999) 46 (5) 543-547 such as () Juul.

In case damage causes hypoxia, EPO can help prevent the apoptosis of nervous tissue.This might be result Neuroscience (1996) 76 (1) 105-116 such as () Morishita of the local EPO of generation of astrocyte.Neuroprotective has obtained illustration (PNAS (1998) 95 (8) 4635-4640 such as Sakanaka, Biochem Biophys Res Commun (1998) 253 (1) 26-32 such as Sadamoto) in pallasiomy hippocampal tissue and rat cerebral cortex tissue.

EPO induces the biological effect of PC12 cell, comprises Ca ²⁺Variation, transmembrane potential variation and promote neuronal survival.This is interpreted as EPO can excite nerve function and survival ability (J.Neurochem (1999) 72 (6) 2565-2572 such as Koshimura.Int JDev Neurosci (1995) 13 (3/4) 241-252 such as Tabria).

Propositions such as O ' Brien, 17 amino acid peptide sequences of EPO can be brought out biological activity by EPO-R (erythropoietin receptor) in NS20Y, SK-N-MC and PC12 cell, comprise sprout, differentiation and neuroprotective.Surprisingly, this peptide does not promote the propagation of hemocyte, so it it seems non-activity in knowing the cell line active responsive for EPO.(Int J Mol Med (1998) 1 (1) 235-241 such as Campana and O ' Brien etc. in 12/23/1999 U.S. Patent number of authorizing 5,700,909, in 11/5/1996 U.S. Patent number of authorizing 5,571,787, authorize 5,714 in 2/3/1998,459 and in 12/9/1997 U.S. Patent number of authorizing 5,696,080).

First stem cell typing promotes neuron not to astrocyte or oligodendrocyte differentiation thereby EPO can affect the nerves.This and EPO promote that the similar activity of hematopoietic stem cell typing generation erythrocyte (RBC) is comparable.Causing producing EPO promotion neuronal stem cell from astrocyte in the damage of CNS hypoxia is divided into neuron and also the neuron that exists is brought into play obviously relevant (WIPO publication No. WO99/21966 between the neuroprotective simultaneously; announce Weiss etc. in 5/6/1999).

Summary of the invention

The present invention relates to treat the method that relates to neural disease and disease by having the active compositions of erythropoietin Neurotherapeutic.

In first embodiment, the present invention relates to a kind of method that is used for the treatment of the patient who suffers from the disease that is characterized as neurotoxicity, neural degeneration or nerve injury, described method comprises the peptide of described patient being treated effective dose, this peptide contain one or more length 8 to about 40 aminoacid and with the monomeric peptide of EPO receptors bind, each monomeric peptide comprises an aminoacid sequence X ₄X ₅X _aX _bX ₆X _cX _dX ₇(SEQ ID NO:47), wherein

X _aBe G or A;

X _bBe P or A;

X _cBe T or A;

X _dBe selected from W, A and F;

X ₄Be selected from R, H, Y, L and W, perhaps X ₄Do not exist;

X ₅Be selected from F, M and I;

X ₆Independently be selected from the L-aminoacid or the stereomeric D-aminoacid of 20 kinds of genetic codings; With

X ₇Be selected from D, V, E, I and L.

Say that at length described sequence is X ₃X ₄X ₅GPX ₆TWX ₇X ₈(SEQ ID NO:1), wherein

X ₃Be selected from C, E, A, alpha-amido-γ-bromo-butyric acid and homocysteine (Hoc); With

X ₈Be selected from C, K, A, alpha-amido-γ-bromo-butyric acid and homocysteine (Hoc).

In second embodiment, the present invention relates to as the agonist of cell surface receptor and the peptide of antagonist, and the dimer and the polymer that show this class peptide of binding growth factor receptoroid and startup somatomedin receptoroid signal.In one embodiment, the invention provides peptide as the EPO agonist.These peptides can be the dimer or the polymers of this class peptide, and preferably long 14 to about 20 residues, comprise an X ₃X ₄X ₅GPX ₆TWX ₇X ₈The core aminoacid sequence of (SEQ ID NO:1), wherein each aminoacid is abridged by the standard single-letter and is represented; X ₃Can be C, E, A, alpha-amido-γ-bromo-butyric acid or Hoc, wherein Hoc be a homocysteine; X ₄Can be R, H, Y, L or W, perhaps X ₄Do not exist; X ₅Can be M, F or I; X ₆Be any in the L-aminoacid of 20 kinds of genetic codings or the stereoisomerism D-aminoacid independently; X ₇Can be D, E, I, L or V; And X ₈Can be C, K, A, alpha-amido-γ-bromo-butyric acid or Hoc, wherein Hoc be a homocysteine.

Preferred described dimer or polymeric monomeric peptide unit comprise a YX ₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈(SEQ ID NO:2) aminoacid core sequence, wherein X ₂And X ₆In each be any in the L-aminoacid of 20 kinds of genetic codings independently; X ₃Be C; And X ₈Be C.

Preferred described dimeric monomeric peptide unit comprises an X ₁YX ₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈X ₉X ₁₀X ₁₁(SEQ ID NO:3) aminoacid core sequence, wherein X ₁, X ₂, X ₆, X ₉, X ₁₀And X ₁₁In each independently be selected from the L-aminoacid of 20 kinds of genetic codings.At length say X ₃Can be C, E, A; X ₄Can be R, H or Y, perhaps X ₄Do not exist; X ₅Can be M, F or I; X ₇Can be D or V; And X ₈Can be C, K or A.

In a preferred embodiment, X ₃And X ₈All be C, therefore described dimeric monomeric peptide unit comprises an X ₁YX ₂CX ₄X ₅GPX ₆TWX ₇CX ₉X ₁₀X ₁₁(SEQ ID NO:4) aminoacid core sequence.Say that at length described monomeric peptide unit comprises an X ₁YX ₂CX ₄X ₅GPX ₆TWX ₇CX ₉X ₁₀X ₁₁(SEQ ID NO:5) aminoacid core sequence, wherein X ₄Can be R or H; X ₅Can be F or M; X ₆Can be I, L, T, M or V; X ₇Be D or V; X ₉Can be G, K, L, Q, R, S or T; And X ₁₀Can be A, G, P, R or Y.More particularly, described dimeric monomeric peptide unit can comprise an X ₁YX ₂CX ₄X ₅GPX ₆TWX ₇CX ₉X ₁₀X ₁₁(SEQ ID NO:6) aminoacid core sequence, wherein X ₁Can be D, E, L, N, S, T or V; X ₂Can be A, H, K, L, M, S or T; X ₄Be R or H; X ₉Can be K, R, S or T; And X ₁₀Be P.

Preferred described dimeric monomeric peptide unit can comprise an X ₁YX ₂CX ₄X ₅GPX ₆TWX ₇CX ₉X ₁₀X ₁₁(SEQ ID NO:6) aminoacid core sequence, wherein X ₁Can be D, E, L, N, S, T or V; X ₂Can be A, H, K, L, M, S or T; X ₄Be R or H; X ₉Can be K, R, S or T; And X ₁₀Be P.

Described dimeric particularly preferred monomeric peptide unit comprises:

GGLYLCRFGPVTWDCGYKGG (SEQ ID NO：7)；

GGTYSCHFGPLTWVCKPQGG (aka EMP-1)(SEQ ID NO：8)；

GGDYHCRMGPLTWVCKPLGG (SEQ ID NO：9)；

VGNYMCHFGPITWVCRPGGG (SEQ ID NO：10)；

GGVYACRMGPITWVCSPLGG (SEQ ID NO：11)；

VGNYMAHMGPITWVCRPGG (SEQ ID NO：12)；

GGTYSCHFGPLTWVCKPQ (aka EMP-16)(SEQ ID NO：13)；

GGLYACHMGPMTWVCQPLRG (aka EMP-36)(SEQ ID NO：14)；

TLAQYICYMGPETWECRPSPKA (aka EMP-38)(SEQ ID NO：15)；

YSCHFGPLTWVCK (aka EMP-20(SEQ ID NO：16)；

YCHFGPLTWVC (aka EMP-23)(SEQ ID NO：17)；

SCHFGPLTWVCK (aka EMP-24)(SEQ ID NO：18)；

GGTASCHFGPLTWVCKPQGG (aka EMP-6)(SEQ ID NO：19)；

GGTYSCHFAPLTWVCKPQGG (aka EMP-9)(SEQ ID NO：20)；

GGTYSCFGPLTWVCKPQGG (aka EMP-27)(SEQ ID NO：21)；

TYSCHFGPLTWVCKPQGG (aka EMP-17)(SEQ ID NO：22)；

TYSCHFGPLTWVCKPQ (aka EMP-18)(SEQ ID NO：23)；

YSCHFGPLTWVCKP (aka EMP-19)(SEQ ID NO：24)；

YSCHFGPLTWVC (aka EMP-21)(SEQ ID NO：25)；

YSCHFGALTWVCK (aka EMP-22)(SEQ ID NO：26)；

GGCRIGPITWVCGG (aka EMP-25)(SEQ ID NO：27)；

HFGPLTWV (aka EMP-26)(SEQ ID NO：28)；

GGTTSCHFGPLTWVCKPQGG (aka EMP-7)(SEQ ID NO：29)；

GGTFSCHFGPLTWVCKPQGG (aka EMP-8)(SEQ ID NO：30)；

GGTYSCHFGALTWVCKPQGG (aka EMP-10)(SEQ ID NO：31)；

GGTYSCHFGPATWVCKPQGG (aka EMP-11)(SEQ ID NO：32)；

GGTYSCHFGPLAWVCKPQGG (aka EMP-12)(SEQ ID NO：33)；

GGTYSCHFGPLTVCKPQGG (aka EMP-13)(SEQ ID NO：34)；

GGTYSCHFGPLTFVCKPQGG (aka EMP-14)(SEQ ID NO：35)；

GGTYSCHFGPLTWVCKAQGG (aka EMP-15)(SEQ ID NO：36)；

GGTXSCHFGPLTWVCKPQGG (asa EMP-28，X＝D-Tyr)(SEQ ID NO：37)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-29，X＝p-NO ₂-Phe)

(SEQ ID NO：38)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-30，X＝p-NH ₂-Phe)

(SEQ ID NO：39)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-31，X＝p-F-Phe)(SEQ ID NO：40)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-32，X＝p-I-Phe)(SEQ ID NO：41)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-33, X=3,5-two bromo-Tyr

(SEQ ID NO：42)；

Ac-GGTYSCHFGPLTWVCKPQGG (aka EMP-34)(SEQ ID NO：43)；

GGLYACHMGPMTWVCQPLGG (aka EMP-35)(SEQ ID NO：44)；

LGRKYSCHFGPLTWVCQPAKKD (aka EMP-37) (SEQ ID NO:45); With

GGTYSEHFGPLTWVKKPQGG (aka EMP-39)(SEQ ID NO：46).

Preferred described dimeric monomeric peptide unit comprises:

GGTYSCHFGPLTWVCKPQGG (aka EMP-1)(SEQ ID NO：8)；

GGTASCHFGPLTWVCKPQGG (aka EMP-6)(SEQ ID NO：19)；

GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO:20); With

YCHFGPLTWVC (aka EMP-23)(SEQ ID NO：17).

According to the present invention, described dimeric monomer unit can be identical or different.

In a preferred embodiment, Polyethylene Glycol (PEG) can be used as joint and forms dimer peptide of the present invention by covalent bond.

In another embodiment, the present invention relates to comprise at least a peptide of the present invention and a kind of pharmaceutical carrier, be used for the treatment of or prevent Pharmaceutical composition in the neurovirulent method.

In a further embodiment, the invention provides one comes therapeutic treatment to suffer from mammiferous method by neurotoxicity incident, neural degeneration incident or caused disease of nerve injury incident or disease by giving at least a peptide of the present invention.

In another embodiment, provide one by using at least a peptide of the present invention to come therapeutic treatment to have the mammiferous method of neurotoxicity disease, nerve injury sexually transmitted disease (STD) disease or the neurodegenerative disease that can regulate by EPO.

The accompanying drawing summary

Fig. 1 picture A and picture B demonstration EPO receptor are expressed in rat hippocampus culture and cortex culture.

Fig. 2 shows that the EPO receptor at neuronal cell is: express in PC12 and the SK-N-MC cell.

Fig. 3 shows EPO inducible gene expression in the PC12 cell.(top) comes the variation of quantitative particular B CL family member gene expression from carrying out RT-PCR with isolating total RNA 24 hours the PC-12 cell of 1nm EPO processing.Cause anti-apoptotic gene BCL with the EPO pretreatment _XLExpression increase by 6 times, and cause the expression of short cell apoptogene Bak to reduce above 5 times.These results are consistent with the gene chip result of the possibility mechanism of hint EPO protective effect.(bottom) shown description Bcl _XLAgarose gel with the RT-PCR product of the adjusting of Bak.M-label, 1-RT-PCR negative control, 2 roads-be untreated, 3 roads-50ng/ml NGF, 4 roads-1nm EPO.

Fig. 4 shows that rhEPO protects the rat brain cortex neuron to avoid glutamate toxicity.

Fig. 5 shows that rhEPO protects P of Rats C12 cell to avoid the cell death that glutamic acid brings out.7 days cultures of PC-12 cell are being exposed to the glutamic acid of toxic concentration (200 μ m) before with erythropoietin processing 24 hours.Allow culture recover 24 hours, utilize trypan blue to get rid of algoscopy then and determine cell survival.1 to 10pm erythropoietin was handled 24 hours, was exposed to glutamic acid then 15 minutes, obviously increased cell survival (p＜0.001, StudentShi t-check).The protection of described EPO is active under high dose more reduces.

Fig. 6 shows that rhEPO protects P of Rats C12 cell to avoid the cell death that the NGF drug withdrawal is brought out.The PC12 cell culture was grown 7 days in the presence of NGF, handled 24 hours with EPO before then in transferring to the culture medium that does not have NGF.Cell survival by after removing NGF immediately the living cell counting number and with its with the somatomedin drug withdrawal after the viable count of 12hr, 24hr, 48hr and 72hr compare definite.The cell survival ability comprises differing brightness, have aixs cylinder and not having the film bubble to form to determine based on morphological feature.EPO handles the viable count that has increased each time point after the somatomedin drug withdrawal, and optimum concentration is 10pm.

Fig. 7 shows that rhEPO promotes neurite projection (outgrowth) in the rat brain culture.

Fig. 8 shows that rhEPO promotes the neurite projection in the rat hippocampus culture.

Fig. 9 shows that EMP-1 promotes the neurite projection in the rat brain cortex culture.

Figure 10 shows that EMP-1 promotes the neurite projection in the rat hippocampus culture.

Figure 11 shows that EMP-6 promotes the neurite projection in the rat brain cortex culture.

Figure 12 shows that EMP-6 promotes the neurite projection in the rat hippocampus culture.

Figure 13 shows that EMP-9 promotes the neurite projection in the rat brain cortex culture.

Figure 14 shows that EMP-9 promotes the neurite projection in the rat hippocampus culture.

Figure 15 shows that EMP-23 promotes the neurite projection in the rat brain cortex culture.

Figure 16 shows that EMP-23 promotes the neurite projection in the rat hippocampus culture.

Figure 17 shows that EMP-27 promotes the neurite projection in the rat brain cortex culture.

Figure 18 shows that EMP-27 promotes the neurite projection in the rat hippocampus culture.

Figure 19 shows that EPO prevents ischemic injury: I is by the continuous venoclysis of miniature osmotic pumps in research.

Figure 20 shows the determination of plasma of studying I.

Figure 21 shows that EPO does not prevent ischemic injury: the heavy dose of azygos vein of research II instils.

Figure 22 shows the determination of plasma of studying II.

Figure 23 shows that EPO prevents ischemic injury: research III repeats heavy dose of intravenous drip.

Figure 24 shows the determination of plasma of studying III.

Detailed Description Of The Invention

(EPO) comprise (for example having all biologically actives of erythropoietin with here " erythropoietin(EPO) ", hematopoiesis activity and neural activity) or (for example only have some particular organisms activity of erythropoietin(EPO), only have the hematopoiesis active or only have neural activity) those peptides, peptide dimer, polypeptide and albumen, and erythropoietin analogue, the erythropoietin(EPO) isotype, the erythropoietin(EPO) analogies, the erythropoietin(EPO) fragment, the heterozygosis erythropoietin protein, fusion, oligomer recited above and polymer, homologue recited above, glycosylation form variant recited above and mutain recited above, no matter and whether biologically active is identical, no matter the method for also synthesizing or making with which kind of, described method include but not limited to produce from cDNA or from the genomic DNA restructuring, synthetic, transgenosis and gene activation method. The object lesson of erythropoietin(EPO) comprises: Epoetin α (EPREX^、ERYPO ^、 PROSCRIT ^); NEORECORMON; Novel erythropoiesis stimulating protein (NESP or ARANESP are described in the high-glycosylation analog of the recombinant human erythropoietin (Epoetin) in the European patent application EP 640619); Erythropoietin-for example those are described in the human serum albumin fusion proteins in the International Patent Application WO 99/66054; For example those are described in the erythropoietin mutant in the International Patent Application WO 99/38890; The ω erythropoietin(EPO), it can be by U.S. Patent number 5,688, and the Apa I restriction fragment of the human epo gene described in 679 produces; For example those are described in the glycosylated human erythropoietin(EPO) of the change in the International Patent Application WO 99/11781; For example those PEG that are described in WO 98/05363 or the U.S. Patent number 5,643,575 put together erythropoietin analogue. The object lesson of modified clone for expressing endogenous erythropoietin is described in International Patent Application WO 99/05268 and WO 94/12650. The common preferred form of EPO is the recombinant human epo (rhEPO) behind the purifying, at present with EPREX^、ERYPO ^Or PROSCRIT^Trade mark preparation and distribution.

Be used for the peptide mimics that herein abbreviation " EMP " refers to EPO, particularly be described in U.S. Patent number 5,767, some peptide in 078 and 5,773,569.

Following is be used to the amino acid abbreviations tabulation that various peptides are described in detail in detail. The single amino acids residue is to know and single-letter code and the trigram code of usefulness identify according to those skilled in the art.

Amino acid		Abbreviation
Amino acid		Abbreviation	Alanine valine leucine isoleucine proline phenylalanine tryptophan methionine glycine serine threonine cysteine tyrosine asparagine glutamine aspartic acid glutamic acid lysine arginine histidine	3 alphabetical ala val leu ile pro phe trp met gly ser thr cys tyr asn gln asp glu lvs arg his	1 alphabetical A V L I P F W M G S T C Y N Q D E K R H

In first embodiment, the present invention relates to utilize comprise as the therapeutic activity peptide of cell surface receptor activator and show the combination of growth factor receptoroid and the dimer of this class peptide of signal enabling effect and the method that polymeric Pharmaceutical composition is processed neuronal cell. In one embodiment, the invention provides peptide as the EPO activator. Say that at length these peptides can be to have two long 8 to 40 or amino acids more, preferred long 14 dimer or polymers to ' monomer ' peptide units of about 20 residues, described monomeric peptide unit comprises an X₃X ₄X ₅GPX ₆TWX ₇X ₈(SEQ ID NO:1) core amino acid sequence, wherein each amino acid abridges to represent by the standard single-letter; X₃Can be C, E, A, alpha-amido-γ-bromo-butyric acid or Hoc, wherein Hoc be homocysteine; X₄Can be R, H, Y, L or W, perhaps X₄Do not exist; X₅Can be M, F or I; X₆Any in the L-amino acid of 20 kinds of genetic codings or the alloisomerism D-amino acid independently; X₇Can be D, E, I, L or V; And X₈Can be C, K, A, alpha-amido-γ-bromo-butyric acid or Hoc, wherein Hoc be homocysteine, and condition is X₃Or X₈C or Hoc. Preferred described dimer or polymeric monomeric peptide unit comprise a YX₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈(SEQ ID NO:2) core sequence, wherein each amino acid abridges to represent by the standard single-letter; X₁、X ₂、X ₆、 X ₉、X ₁₀And X₁₁In each independently be selected from the L-amino acid of 20 kinds of genetic codings; X₃Can be C, E, A, alpha-amido-γ-bromo-butyric acid or Hoc, wherein Hoc be homocysteine; X₄Can be R, H, Y, L or W, perhaps X₄Do not exist; X₅Can be M, F or I; X₇Can be D, E, I, L or V; And X₈Can be C, K, A, alpha-amido-γ-bromo-butyric acid or Hoc, wherein Hoc be homocysteine. More preferably X₃Or X₈C or Hoc.

Preferred described dimer or polymeric monomeric peptide unit comprise a YX₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈(SEQ ID NO:2) amino acid core sequence, wherein X₂And X₆In each be any in the L-amino acid of 20 kinds of genetic codings independently; X₃C; And X₈C.

Preferred described dimeric monomeric peptide unit comprises an X₁YX ₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈X ₉X ₁₀X ₁₁(SEQ ID NO:3) amino acid core sequence, wherein X₁、X ₂、X ₆、X ₉、X ₁₀And X₁₁In each independently be selected from the L-amino acid of 20 kinds of genetic codings. At length say X₃Can be C, E, A; X₄Can be R, H or Y, perhaps X₄Do not exist; X₅Can be M, F or I; X₇Can be D or V; And X₈Can be C, K or A.

In a preferred embodiment, X₃And X₈All be C, therefore described dimeric monomeric peptide unit comprises an X₁YX ₂CX ₄X ₅GPX ₆TWX ₇CX ₉X ₁₀X ₁₁(SEQ ID NO:4) amino acid core sequence. Say that at length described monomeric peptide unit comprises an X₁YX ₂CX ₄X ₅GPX ₆TWX ₇CX ₉X ₁₀X ₁₁(SEQ ID NO:5) amino acid core sequence, wherein X₄Can be R or H; X₅Can be F or M; X₆Can be I, L, T, M or V; X₇D or V; X₉Can be G, K, L, Q, R, S or T; And X₁₀Can be A, G, P, R or Y. More particularly, described dimeric monomeric peptide unit can comprise an X₁YX ₂CX ₄X ₅GPX ₆TWX ₇CX ₉X ₁₀X ₁₁(SEQ ID NO:6) amino acid core sequence, wherein X₁Can be D, E, L, N, S, T or V; X₂Can be A, H, K, L, M, S or T; X₄R or H; X₉Can be K, R, S or T; And X₁₀P.

Preferred described dimeric monomeric peptide unit can comprise an X₁YX ₂CX ₄X ₅GPX ₆TWX ₇CX ₉X ₁₀X ₁₁(SEQ ID NO:6) amino acid core sequence, wherein X₁Can be D, E, L, N, S, T or V; X₂Can be A, H, K, L, M, S or T; X₄R or H; X₉Can be K, R, S or T; And X₁₀P.

Described dimeric particularly preferred monomeric peptide unit comprises:

GGLYLCRFGPVTWDCGYKGG (SEQ ID NO：7；

GGTYSCHFGPLTWVCKPQGG (aka EMP-1)(SEQ ID NO：8)；

GGDYHCRMGPLTWVCKPLGG (SEQ ID NO：9)；

VGNYMCHFGPITWVCRPGGG (SEQ ID NO：10)；

GGVYACRMGPITWVCSPLGG (SEQ ID NO：11)；

VGNYMAHMGPITWVCRPGG (SEQ ID NO：12)；

GGTYSCHFGPLTWVCKPQ (aka EMP-16)(SEQ ID NO：13)；

GGLYACHMGPMTWVCQPLRG (aka EMP-36)(SEQ ID NO：14)；

TLAQYICYMGPETWECRPSPKA (aka EMP-38)(SEQ ID NO：15)；

YSCHFGPLTWVCK (aka EMP-20(SEQ ID NO：16)；

YCHFGPLTWVC (aka EMP-23)(SEQ ID NO：17)；

SCHFGPLTWVCK (aka EMP-24)(SEQ ID NO：18)；

GGTASCHFGPLTWVCKPQGG (aka EMP-6)(SEQ ID NO：19)；

GGTYSCHFAPLTWVCKPQGG (aka EMP-9)(SEQ ID NO：20)；

GGTYSCFGPLTWVCKPQGG (aka EMP-27)(SEQ ID NO：21)；

TYSCHFGPLTWVCKPQGG (aka EMP-17)(SEQ ID NO：22)；

TYSCHFGPLTWVCKPQ (aka EMP-18)(SEQ ID NO：23)；

YSCHFGPLTWVCKP (aka EMP-19)(SEQ ID NO：24)；

YSCHFGPLTWVC (aka EMP-21)(SEQ ID NO：25)；

YSCHFGALTWVCK (aka EMP-22)(SEQ ID NO：26)；

GGCRIGPITWVCGG (aka EMP-25)(SEQ ID NO：27)；

HFGPLTWV (aka EMP-26)(SEQ ID NO：28)；

GGTTSCHFGPLTWVCKPQGG (aka EMP-7)(SEQ ID NO：29)；

GGTFSCHFGPLTWVCKPQGG (aka EMP-8)(SEQ ID NO：30)；

GGTYSCHFGALTWVCKPQGG (aka EMP-10)(SEQ ID NO：31)；

GGTYSCHFGPATWVCKPQGG (aka EMP-11)(SEQ ID NO：32)；

GGTYSCHFGPLAWVCKPQGG (aka EMP-12)(SEQ ID NO：33)；

GGTYSCHFGPLTAVCKPQGG (aka EMP-13)(SEQ ID NO：34)；

GGTYSCHFGPLTFVCKPQGG (aka EMP-14)(SEQ ID NO：35)；

GGTYSCHFGPLTWVCKAQGG (aka EMP-15)(SEQ ID NO：36)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-28，X＝D-Tyr)(SEQ ID NO：37)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-29，X＝p-NO ₂-Phe)

(SEQ ID NO：38)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-30，X＝p-NH ₂-Phe)

(SEQ ID NO：39)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-31，X＝p-F-Phe)(SEQ ID NO：40)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-32，X＝p-I-Phe)(SEQ ID NO：41)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-33, X=3,5-two bromo-Tyr)

(SEQ ID NO：42)；

Ac-GGTYSCHFGPLTWVCKPQGG (aka EMP-34)(SEQ ID NO：43)；

GGLYACHMGPMTWVCQPLGG (aka EMP-35)(SEQ ID NO：44)；

LGRKYSCHFGPLTWVCQPAKKD (aka EMP-37) (SEQ ID NO:45); With

GGTYSEHFGPLTWVKKPQGG (aka EMP-39)(SEQ ID NO：46).

Most preferably described dimeric monomeric peptide unit comprises:

GGTYSCHFGPLTWVCKPQGG (aka EMP-1)(SEQ ID NO：8)；

GGTASCHFGPLTWVCKPQGG (aka EMP-6)(SEQ ID NO：19)；

GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO:20); With

YCHFGPLTWVC (aka EMP-23)(SEQ ID NO：17).

As for those skilled in the art apparent, EPO can be by any appropriate method administration of the particular patient that is suitable for being treated. The phrase " treatment effectively " that is used for this is different between patient and patient, and depends on the bioactive particular range that the EPO molecule of intending giving has. Usually, for the EPO with hematopoiesis activity, the treatment effective dose will be from about 1 to 500I.U./kg body weight, more preferably from 50 to 300I.U./kg body weight, especially when by subcutaneous when giving erythropoietin(EPO). For the EPO molecule that does not have the hematopoiesis activity, the treatment effective dose can be greater or less than the dosage of the EPO molecule with hematopoiesis activity. Preferred medication is intravenously administrable (iv) and subcutaneous administration (sc), usually preferred subcutaneous administration. The active EPO of hematopoiesis is weekly 1 to 5 administration in the scope of every dose of about 50-1000U/kg. In the another one embodiment, described EPO composition directly gives nervous system. This method of administration includes but not limited to method of administration, the interior method of administration of spinal cord and/or all methods of administration of spinal cord in method of administration in the brain, indoor method of administration, the interior method of administration of the ventricles of the brain, intrathecal drug delivery approach, the pond, they can use the conduit of encephalic pin and the interior pin of vertebra and with or without pump installation. The scope of infusion dosage can for for example a few minutes to several days the time range from about 1.0 to 50,000U/kg/min EPO composition. Surpass about 15g/dL if the male sex or female patient show hemoglobin level, then the active EPO administration of hematopoiesis will postpone or drug withdrawal.

The present invention provides the method for the acute and chronic neurodegenerative disease for the treatment of therein in embodiment, and described method comprises and gives EPO or its analogies. The acute neurodegenerative disease includes but not limited to nerve cell death or comprises various types of acute neurodegenerative diseases that the infringement of cerebrovascular deficiency, focus or dispersivity brain trauma, dispersivity brain damage and spinal cord injury is relevant. The example of acute neurodegenerative disease is: cerebral ischemia or comprise embolic and thrombus occlusion, acute ischemia after after again perfusion, perinatal period hypoxemia-anoxia-induced apoptosis, heartbeat stops and various types of intracranialing hemorrhage (for example extradural hemorrhage, subdural hemorrhage, subarachnoid hemorrhage and ICH) and encephalic infringement and backbone in infringement (for example dampen, penetrate, cut wounds, oppress and tear) and ACTS tremble baby head jolting syndrome. Can include but not limited to alzheimer's disease with the chronic neurodegenerative disease of one or more method treatments of the present invention, the Pick disease, diffusivity thunder dimension corpusculum disease, paralysis (Steel-Richardson syndrome) on the carrying out property nuclear, multisystem sex change (shy-Drager syndrome), the Chronic Epilepsy venereal disease disease relevant with neurodegeneration, motor neuron disease comprises ALS, the sex change incoordination, corticobasal degeneration, Guam ALS-Parkinson's-chronic brain syndrome, subacute sclerosing panencephalitis, Huntingtons chorea, Parkinson's, synucleinopathies (comprising MSA), primary progressive aphasia disease, SND, Machado-JosephShi disease/3 type spinocebellar ataxia and olivopontocerebellar degenerations, Gilles De La TouretteShi disease, bulbar paralysis and pseudobulbar palsy, Duchenne-Arandisease and spinal cord oblongata muscular atrophy (KennedyShi disease), primary lateral sclerosis, familial spastic, the Werdnig-Hoffmann disease, the Kugelberg-Welander disease, the Tay-SachShi disease, the Sandhoff disease, the familial spastic disease, the Wohlfart-Kugelberg-Welander disease, spastic paraparesis, many focuses of carrying out property property leukoencephalopathy, dysautonomia (Riley-Day syndrome) and prion disease (include but not limited to the Creutzfeldt-Jakob disease, Gerstmann-Str  ussler-Scheinker disease, kuru and fatal familial insomnia).

Owing to the neuroprotective that is caused by rhEPO and the combination of neural process projection; can treat other clinical diseases with one or more methods of the present invention; comprise that treating and/or preventing the neurology relevant with peripheral diseases performance (including but not limited to cognitive) and psychiatry shows and (include but not limited to psychopathology; depression or anxiety); include but not limited to that EPO lacks (being ephrosis); losing blood of any type (includes but not limited to hemodialysis; peritoneal dialysis; diagnostic sampling; recessive hemorrhage of gastrointestinal tract); kidney failure and end-stage renal disease; kidney transplant and other and anaemia; neurology performance and the relevant illness of neuropsychiatry performance include but not limited to blood malignant tumour/cancer and non-blood malignant tumour/cancer; accept chemotherapy (including but not limited to cis-platinum) and other drug (including but not limited to Zidovudine) treatment patient's symptom or complication; other blood diseases (including but not limited to sicklemia and thalassemia); diseases associated with inflammation and communicable disease (include but not limited to human immunodeficiency virus's sexuality dye); chronic Autoimmune diseases (including but not limited to systemic loupus erythematosus); Henoch Schonlein purpura and hemolytic uremic syndrome. Comprise equally in the present invention be to treat and/or prevent because neural chemical lesion, toxic damages, infectious damage and radioactive damage and because the precocious neurology performance that causes and neuropsychiatry performance, and treat and/or prevent and include but not limited to because neurology sequelae and the neuropsychiatry sequelae of the encephalopathic that those Hypoxic ischaemics, hepatopathy, glucohemia, uremia, electrolyte and endocrine cause.

Equally; owing to the neuroprotective that is caused by rhEPO and the combination of neural process projection; this molecule equally also can be applied to treat and/or prevent clump sick (comprising the neuropile paralysis); many focuses property neuropathy; esthesioneurosis; motor neuropathy; sensation-motor neuropathy; the infectiousness neuropathy; AN; sensation-AN; demyelinating neuropathy (including but not limited to Guillain-Barre syndrome and chronic inflammation demyelinate polyneural root neuroma); other inflammatory neuropathies and immunity neuropathy; drug-induced neuropathy; the neuropathy that drug therapy causes; the neuropathy that toxin causes; traumatic neuropathy (includes but not limited to the pressurized neuropathy; crushing property (crush) neuropathy; lancinating (laceration) neuropathy and segmental neuropathy); metabolic neuropathy; incretion neuropathy and paraneoplastic neuropathy and other neuropathy; charcot-Marie-Tooth atrophy (1a type for example; the 1b type; 2 types; the 4a type; 1-X linked (linked)); the Friedreich incoordination; metachromatic leukodystrophy; the Refsum disease; adrenomyeloneuropathy (adrenomyeloneuropathy); Dejerine-Sottas neuropathy (A type and Type B), Lambert-Eaton syndrome and cranial nerve disease.

The following example illustration the present invention, yet do not limit the present invention.

Embodiment 1

RhEPO former generation rat neuron culture and neuronal cell system in expression

Former generation neuronal cell cultivation

(Mattson etc., 1994) as described previously, isolating hippocampal cell culture and cortex cell culture begin to set up from 18 days rat fetus of period of fetus.Say that briefly the tire Mus is taken out by cesarotomy according to the AVMA Panel on Euthanasia from the conceived parent (Sprague-Dawley) of halothane anesthesia.With young Mus broken end, get brain, put into the buffered HankShi balanced salt solution of HEPES (HBSS; Gibco) in.Dissect out Hippocampus and cortex, and merge according to types of organization.Tissue 15 minutes (1mg/ml trypsin-HBSS of trypsinization; Worthington), with fresh HBSS rinsing, then at trypsin inhibitor (1mg/ml; Sigma) hatched in 5 minutes, the fresh HBSS rinsing of reuse is chiseled glass pipet with fire then tissue is smashed to pieces in the fresh HBSS of 1ml.Isolated cells is inoculated on 96 orifice plates (Collaborative BioScience) of poly-D-lysine bag quilt with 30,000 cells/well.The NaHCO by 26mM is contained in every hole ₃(Sigma), 100 additional μ l EagleShi minimal essential medium (MEM of 56mM glucose (Sigma), 15mM KCl (Sigma), 1mM Sodium Pyruvate (Sigma), 1.1mM L-glutaminate (Sigma), 10% (v/v) heat-inactivated fetal bovine serum (Hyclone) and 0.001% gentamycin sulfate (Sigma); Gibco) (pH 7.4).Before handling, experiment allows the 37 ℃ 5%COs of cell in humidity ₂In the incubator adherent 24 hours.Changed with described culture medium sucking-off and with fresh culture in per three days.

Immunocytochemistry

Parallel Hippocampus culture and cortex culture be according to top described the processing, and according to former described method (Smith Swintosky etc., 1997) processing to carry out immunocytochemistry (ICC).Say briefly, seed cells into the poly-D-lysine bag quilt in Room 4 glass slide (LabTek, Napersville, IL) on.At the 7th day that cultivates, take out culture medium, with Dulbecco phosphate-buffered saline (DPBS; Sigma) washed cell is once at room temperature used 10% phosphoric acid buffer formalin fixed 15 minutes then.After fixing, sealing serum (notmal horse sera is put in culture DPBS rinsing then; Be diluted among the DPBS at 1: 50; Vector Labs, Burlingame, CA) in 10 minutes.Culture is rinsing once more, and be diluted to the antibody dilution agent then (Zymed, South San Francisco CA) were hatched 30 minutes in the anti-mouse monoclonal antibody of the specificity at EPO receptor (EBP-7) at 1: 75.Culture is exposed in the biotinylated second antibody (Vector Labs) 30 minutes with the DPBS rinsing several times then.The last rinsing of culture was once hatched 30 minutes in Avidin-biotinylated horseradish peroxidase complex (mice IgG ABC test kit, Vector Labs) then.The existence of first antibody is to utilize 3 ' 3-diaminobenzidine, four hydrochlorates (Foster City CA)-expose twice, detected in each 5 minutes for DAB, Biomeda.Cell is with haematoxylin redyeing, dehydration, clear and bright (clear), lid coverslip with take pictures under Olympus BX-2 optical microscope then.

The result

In the neuron of Hippocampus culture and cortex culture and neuroglia, all found the sane dyeing (Fig. 1 a and Fig. 1 b) of EPO receptor.It seems that EPO receptor expression level increase with incubation time.

Discuss

These results show EPO at nervous system, at length say it is to work Hippocampus and corticocerebral early development stage.

The immunohistochemistry of neuronal cell system

The neuronal cell that use derives from the adrenal pheochromocytoma of rat is PC-12 (Greene and Tischler, 1976) and is SK-N-MC (Spengler etc., 1973) from the neuronal cell that the neuroepithelioma of human brain obtains; The PC-12 cell exists under the situation and can reversibly be induced the neuron phenotype at nerve growth factor (NGF).Allow the PC-12 cell in the DMEM that in the presence of 0.1 μ g/ml NGF, comprises 10% horse serum and 5%FBS on tissue culture's ware of poly-D-lysine bag quilt, grow 7 days, induce the neuron phenotype.The SK-N-MC cell was cultivated 4 days in the minimal essential medium of having replenished 1.0mM Sodium Pyruvate, 1.5g/L sodium bicarbonate, 2mM glutamine and 10%FBS.PC-12 and SK-N-MC cell are cultivated on 96 orifice plates from Greiner, to help microscopy., in 10% formalin that comprises 10% sucrose, and in sealing buffer (40mM Tris HCl, pH8.0,27mM NaCl and 0.2%Tween 20), hatch in experiment cell fixation on the same day.Erythropoietin receptor be by with rabbit polyclonal anti--second antibody incubated cell that erythropoietin receptor antibody (from the C-20 of Santa Cruz) and FITC put together detects.Utilize fluorescence microscope (ATTO) visual inspection labeled cell.

The result

The polyclonal antibody that generates plain receptor as anti-erythrocyte seen at Fig. 2 (right part) both labelling the SK-N-MC cell, also labelling the PC-12 cell.All under low power is amplified visible SK-N-MC cell all by described antibody labeling.Great majority can be resisted-the erythropoietin receptor antibody labeling by detected PC-12 cell.Complete PC 12 cells of minority that remain with characteristic neuron phenotype in this preparation process demonstrate whole aixs cylinder and cell space all is colored.Independent second antibody is this cell of two types of labelling (Fig. 2, left part) not.

Therefore, these as a result illustration these cells-from the SK-N-MC cell of people's neuroepithelioma with from the described erythropoietin receptor of PC-12 cellular expression of rat pheochromocytoma.Therefore these cell lines can be made a response to erythropoietin and also can be provided a good system to neuronic effect for the research erythropoietin.

Embodiment 2

The inductive gene expression of EPO in PC 12 cells

Cell culture

PC-12 cell (from the rat pheochromocytoma) is incubated on the tissue culturing plastic's goods that gather D-lysine bag quilt and comprises among the DMEM of 10%FBS and 5% horse serum.In order in the PC-12 cell, to induce the neuron phenotype, remove serum and handle described cell with NGF (50ng/ml).Cell is grown in the presence of NGF and was used for experiment in 7 days then.

EPO handles and separates with RNA

The PC-12 cell is cultivated in the 10cm tissue culture ware of a poly-D-lysine bag quilt as described in example 1 above.Cell was hatched 24 hours in the presence of 1U/ml EPO.Utilize Qiagen RNAeasy to prepare test kit in a small amount then and separate total RNA, be used further to RT-PCR.Quantitative RT-PCR

Be used in a reaction, carry out real-time reverse transcription and PCR from light circulation instrument (light cycler) and the RNA amplification kit of Roche Mo1ecular Biochemicals.Quantitatively RNA joins the RNA of equivalent in the reactant mixture that comprises dsDNA specificity dyestuff SYBR green I then.Come quantitative specific PCR product by the fluorescence volume that detects in each PCR circulation reaction.Last analysis and utilization comprises that the data analysis software that described light circulating device has carries out.

General introduction

As seen in Figure 3, caused bcl-2 family member bcl in 24 hours with EPO (1U/ml) pretreatment PC-12 cell _XLSignificant change with bak gene expression.Handle cell with EPO and demonstrate anti--apoptosis gene bcl _XLThe expression of expressing 6 times of growths and short-apoptosis gene bak reduce by 5 times.EPO had demonstrated by increasing bcl in the past _XLExpress the survival (Silva etc., 1996) that increases the erythrocyte CFU-GM.These results show that EPO utilizes similar mechanism neuroprotective unit to make it not experience the apoptosis that damage is replied.The adjusting of bak shows that EPO can influence other expression of gene to bring out this effect.

Embodiment 3

RhEPO to the neuroprotective of rat hippocampus cell, cortex cell and PC 12 cells and The effect of neurite projection

Former generation neuronal cell cultivation

(Mattson etc., 1994) as described previously, isolating hippocampal cell culture and cortex cell culture begin to set up from 18 days rat fetus of period of fetus.Say that briefly the tire Mus is taken out by cesarotomy according to the AVMA Panel on Euthanasia from the conceived parent (Sprague-Dawley) of halothane anesthesia.With young Mus broken end, get brain, put into the buffered HankShi balanced salt solution of HEPES (HBSS; Gibco) in.Dissect out Hippocampus and cortex, and merge according to types of organization.Tissue 15 minutes (1mg/ml trypsin-HBSS of trypsinization; Worthington), with fresh HBSS rinsing, then at trypsin inhibitor (1mg/ml; Sigma) hatched in 5 minutes, the fresh HBSS rinsing of reuse is chiseled glass pipet with fire then tissue is smashed to pieces in the fresh HBSS of 1ml.Isolated cells is inoculated on 96 orifice plates (Collaborative BioScience) of poly-D-lysine bag quilt with 30,000 cells/well.The NaHCO by 26mM is contained in every hole ₃(Sigma), 100 additional μ l EagleShi minimal essential medium (MEM of 56mM glucose (Sigma), 15mM KCl (Sigma), 1mM Sodium Pyruvate (Sigma), 1.1mM L-glutaminate (Sigma), 10% (v/v) heat-inactivated fetal bovine serum (Hyclone) and 0.001% gentamycin sulfate (Sigma); Gibco) (PH7.4).Before handling, experiment allows 37 ℃ 5% CO of cell in humidity ₂In the incubator adherent 24 hours.Changed with described culture medium sucking-off and with fresh culture in per three days.

Glutamate toxicity is measured

Cortex cell is inoculated on the 35mm culture dish of polymerization aziridine bag quilt with 200,000 cell/culture dishs.Each culture dish comprises the MEM that 1.5ml replenished as mentioned above.At the 7th day that cultivates, observe each four visuals field and photograph before experiment is handled of labelling culture dish in advance with Nikon Diaphot inverted microscope (10 times of amplifications).And then, with solvent or recombinant human erythropoietin (rhEPO; Lot number 41C514; 0.2M 50 μ M liquid storages in the citrate, 0.585g.L NaCl is diluted to (DPBS in the phosphate buffered saline(PBS) with debita spissitudo; Sigma)+0.1% bovine serum albumin (BSA; Sigma)) handle described culture.Described culture is handled with 100 μ M glutamic acid (Sigma) after 24 hours.Glutamic acid was handled after 24 hours, took a picture once more in four visuals field of each plate.Measure cell survival rate with the living cells of testing after handling by counting before experiment is handled in each visual field.If neuron has the neurite of on diameter unanimity and smooth in appearance and shape is slick and sly and for circular to ovate cell space, neuron just is considered to live.Data are with percentage expression (solvent, the meansigma methods ± SD) of contrast.

The PC12 cell culture

Glutamate toxicity

By top described cultivation PC-12 cell.Damaged preceding 24 hours, cell is handled with the rhEPO of concentration range at 1pm to 1nm.On experiment same day, cellular exposure was in 200 μ M glutamic acid 30 minutes.Cell cleans twice with fresh culture and comprises NGF but do not contain in the fresh culture of EPO to remove glutamic acid, to be incubated at then.After 24 hours, utilize trypan blue to get rid of the viability that algoscopy is measured cell.Say briefly, remove culture medium and in 0.4% trypan blue, hatched described cell 5 minutes then.Clean cell lightly with PBS then, reuse 10% formalin fixed.Cell viability is recently determined by total cell number of counting and trypan blue positive cell (dead cell) number.

The NGF drug withdrawal

As mentioned above, the PC-12 cell culture gathers on the porous plate of D-lysine bag quilt in 96 holes, and handles 24 hours with rhEPO (1pm to 10nm) before the NGF drug withdrawal.On experiment same day, described cell, is incubated in the fresh culture that does not contain NGF to remove NGF then with buffer solution for cleaning 3 times.Count NGF immediately and wash out cell (t=0) to determine living cells quantity.Cell viability comprises differing brightness, have aixs cylinder and steeping formation based on morphological feature.At 12 hours, 24 hours, 48 hours and 72 hours counting cells and note down living cells quantity.

The neurite projection is measured

Behind the plating 24 hours, culture was with solvent (PBS+0.1%BSA), somatomedin (Brain Derived Neurotrophic Factor (BDNF that 100ng is different; Promega), neurogliocyte derived neurotrophic factor (GDNF; Promega), nerve growth factor (NGF; BoehringerMannheim), basic fibroblast growth factor (bFGF; Boehringer Mannheim), insulin-like growth factor-i (IGF-1; Boehringer Mannheim), neurotrophin-3 (NT3; Calbiochem), neurotrophin-4 (NT4; Calbiochem), ciliary neurotrophic factor (CNTF; Calbiochem), epidermal growth factor (EGF; Calbiochem), VEGF (VEGF; Calbiochem)) or rhEPO (with top the same preparation; 10fM-10nM)) handle.Every kind of treatment conditions are carried out 4 times or 8 times and are repeated.When the 3rd day of cultivating, the described culture medium of sucking-off replaces and detection compound with fresh culture.When cultivating a week, described cell is used DPBS (Sigma) rinsing then with the formalin fixed of 10% phosphoric acid buffer 15 minutes, puts into and seals serum (horse serum; Be diluted among the DPBS at 1: 50; VectorLabs) in 30 minutes.Reuse DPBS rinsing culture, (microtubule-associated protein-2 (MAP-2) is as the selected marker of dendron to hatch 2 hours then in first antibody; Anti-mouse monoclonal (Chemicon); MAP-2 to be diluted in antibody dilution agent (Zymed) at 1: 1000) in.Negative control hole is only hatched in the antibody dilution agent.Background signal is by determining with antibody incubation or without the blank hole (acellular) of antibody incubation.1 hour (FITC of fluorescein is put in culture reuse DPBS rinsing then; Anti-mice IgG; Rat absorption; Be diluted among the DPBS at 1: 50; Vector Labs).Culture is last with the DPBS rinsing, then with described flat board at the dull and stereotyped readout meter reading of Cytofluor 4000 fluorescence.The neurite projection is represented (solvent with the percent that changes compared with the control; Mean fluorecence ± SD).

The result

With the neuroprotective research carried out of neuron culture of former generation:Before giving glutamic acid, use rhEPO pretreatment culture 24 hours, caused neuron survival rate significantly to increase (Fig. 4).Cell survival rate is than the parallel maximum increase of only handling with glutamic acid about 200% of culture.The described neuroprotective of rhEPO is a concentration dependent, and the pM concentration place that is greater than or equal to solvent (no glutamic acid) processing culture at cell survival rate has found maximum effect.

Study with the neuroprotective that PC 12 cells carry out:In the PC-12 cell, caused significantly reducing (Fig. 5 and Fig. 6) by the cell death that glutamate toxicity and somatomedin drug withdrawal are brought out with the EPO pretreatment.Peak concentration in neuroprotective described in two experiments is 10pm.The dose-effect curve of the described neuroprotective of EPO is a two-phase, and the Cytotoxic ability of EPO protection cell resistance is low greater than the 10pm lowering of concentration, and not significantly effect under 1nm concentration.These results show that EPO can prevent to be exposed to the neuronic apoptosis reaction of various cytotoxicity infringements.

With the neurite projection research carried out of neuron culture of former generation:Culture is handled with rhEPO, and causing significantly increases with the neurite projection that the MAP2-FITC immunofluorescence is measured.Described neurite projection facilitation is a concentration dependent, finds maximum activity (Fig. 7 and Fig. 8) in the pM level.Described result shows that rhEPO handles (to be higher than and to contrast 12-44%) than (be higher than contrast 15-29%) in the cortex culture and induces bigger projection reaction in the Hippocampus culture.Relatively demonstrating them and on neurite projection promotion ability, represented regional difference between rhEPO and the known somatomedin.RhEPO increases the neurite projection in the cortex culture ability is greater than or equals the ability of those known somatomedin.Owing to have only a few factors that cortex cell is had such effect, so this discovery is noticeable and important.On the other hand, many somatomedin are brought into play powerful projection reaction (being higher than contrast 38-86%) in Hippocampus.Compare with these somatomedin, rhEPO demonstrates medium but firm projection and promotes activity, yet its activity will be higher than several somatomedin and comprise BDNF, NGF and VEGF

Discuss

Described neuroprotective studies confirm that previous evidence, and the protective effect of opposing glutamate toxicity and serum drug withdrawal is promptly arranged under pM concentration at external rhEPO.

Surprisingly, we have found that rhEPO promotes the neurite projection in the first cell of former foster nursing breast animal nerve.Described effect is firm for hippocampal cell and cortex cell.Described effect is effectively, is checked through effect under Asia-picomole concentration, and is effective more than any previous observed result relevant with EPO.In addition, in the cerebral cortex neurons to the reaction of minority somatomedin only, rhEPO is better than most of known somatomedin inducing on the neurite projection.

From the treatment prospect, rhEPO promotes that in cerebral cortex neurons neuroprotective and neurite projection are very important.In neurodegenerative process, neurocyte can be in different developmental stage.Some may be in stress state, and other experiences significant neurite retraction and forfeiture synapse input, and all affected cells are with death at last.The therapeutic agent that can intervene this process on multilevel can have very big benefit to the recovery of neurocyte and the recovery of final function of nervous system.Notebook data support rhEPO by protect described cell, by increasing them survival rate, by promoting synapse contact and synaptic contact reconstruction and finish this task by stablizing neuronal circuit and neural circuit.

Should particularly point out equally; consider that the only a few somatomedin is effective in cerebral cortex neurons; and the only a few somatomedin demonstrates in cortical neuron as the neuroprotective of neurite projection and the double activity of promoter, and described data are even more important.Especially relevant is that this double activity of rhEPO is checked through under Asia-picomole/picomole concentration.

Embodiment 4

The thumping of exciting nerve of EPO simulating peptide rises

Cell culture

(Mattson etc., 1994) as described previously, isolating hippocampal cell culture and cortex cell culture begin to set up from 18 days rat fetus of period of fetus.Say that briefly the tire Mus is taken out by cesarotomy according to the AVMA Panel on Euthanasia from the conceived parent (Sprague-Dawley) of halothane anesthesia.With young Mus broken end, get brain, put into the buffered HankShi balanced salt solution of HEPES (HBSS; Gibco) in.Dissect out Hippocampus and cortex, and merge according to types of organization.Tissue 15 minutes (1mg/ml trypsin-HBSS of trypsinization; Worthington), with fresh HBSS rinsing, then at trypsin inhibitor (1mg/ml; Sigma) hatched in 5 minutes, the fresh HBSS rinsing of reuse is chiseled glass pipet with fire then tissue is smashed to pieces in the fresh HBSS of 1ml.Isolated cells is inoculated on 96 orifice plates (Collaborative BioScience) of poly-D-lysine bag quilt with 30,000 cells/well.The NaHCO by 26mM is contained in every hole ₃(Sigma), 100 additional μ l EagleShi minimal essential medium (MEM of 56mM glucose (Sigma), 15mM KCl (Sigma), 1mM Sodium Pyruvate (Sigma), 1.1mM L-glutaminate (Sigma), 10% (v/v) heat-inactivated fetal bovine serum (Hyclone) and 0.001% gentamycin sulfate (Sigma); Gibco) (pH 7.4).Before handling, experiment allows 37 ℃ 5% CO of cell in humidity ₂In the incubator adherent 24 hours.Changed with described culture medium sucking-off and with fresh culture in per three days.

The neurite projection is measured

Behind the plating 24 hours, culture was with solvent (PBS+0.1%BSA), somatomedin (Brain Derived Neurotrophic Factor (BDNF that 100ng is different; Promega), neurogliocyte derived neurotrophic factor (GDNF; Promega), nerve growth factor (NGF; BoehringerMannheim), basic fibroblast growth factor (bFGF; Boehringer Mannheim), insulin-like growth factor-i (IGF-1; Boehringer Mannheim), neurotrophin-3 (NT3; Calbiochem), neurotrophin-4 (NT4; Calbiochem), ciliary neurotrophic factor (CNTF; Calbiochem), epidermal growth factor (EGF; Calbiochem), VEGF (VEGF; Or EPO simulating peptide (EMP1, EMP6, EMP9, EMP23 and EMP27 Calbiochem)); Be diluted among the DPBS+0.1%BSA; 10fM-10nM; Table 1) handles.Every kind of treatment conditions are carried out 4 times and are repeated.When the 3rd day of cultivating, the described culture medium of sucking-off is changed with fresh culture and experimental compound.When cultivating a week, described cell is used DPBS (Sigma) rinsing then with the formalin fixed of 10% phosphoric acid buffer 15 minutes, puts into and seals serum (horse serum; Be diluted among the DPBS at 1: 50; VectorLabs) in 30 minutes.Reuse DPBS rinsing culture, (microtubule-associated protein-2 (MAP-2) is as the selected marker of dendron to hatch 2 hours then in first antibody; Anti-mouse monoclonal (Chemicon); MAP-2 was diluted in the antibody dilution agent (Zymed) with 1: 1000).Negative control hole is only hatched in the antibody dilution agent.Background signal is by determining with antibody incubation or without the blank hole (acellular) of antibody incubation.1 hour (FITC of fluorescein is put in culture reuse DPBS rinsing then; Anti-mice IgG; Rat absorption; Be diluted among the DPBS at 1: 50; Vector Labs).Culture is last with the DPBS rinsing, then with described flat board reading on the dull and stereotyped readout meter of Cytofluor 4000 fluorescence.The neurite projection is represented (solvent with the percent that changes compared with the control; Mean fluorecence ± SEM).

Table 1: erythropoietin simulating peptide (EMP)
Table 1: erythropoietin simulating peptide (EMP)			SEQ.ID.NO	The sequence title	Sequence
7	EMP-1	GGTYSCHFGPLTWVCKPQGG	SEQ.ID.NO	The sequence title	Sequence
7	EMP-1	GGTYSCHFGPLTWVCKPQGG	19	EMP-6	GGTASCHFGPLTWVCKPQGG
20	EMP-9	GGTYSCHFAPLTWVCKPQGG	19	EMP-6	GGTASCHFGPLTWVCKPQGG
20	EMP-9	GGTYSCHFAPLTWVCKPQGG	17	EMP-23	Y-CHFGPLTWVC
21	EMP-27	GGTYSC-FGPLTWVCKPQGG	17	EMP-23	Y-CHFGPLTWVC

The result

The research of neurite projection:Handling culture with EMP causes the neurite projection of MAP2-FITC immunofluorescence measurement obviously to increase.Described neurite projection facilitation is a concentration dependent, finds maximum activity (Fig. 9-Figure 18) in the pM level.Described result shows that EMP shows different activity distribution curves.Under the concentration that is detected, EMP-1 and EMP-6 show typical bell dose response distribution curve in the Hippocampus culture, are checked through peak activity (increasing 83-117% than solvent) between 30-300pM.EMP-9 and EMP-27 show smooth reaction profile curve in the Hippocampus culture, be checked through peak activity to the EMP-1 concentration place similar with EMP-6, but reaction range reduces (increasing 29-32% than solvent) greatly.EMP-23 has a medium reaction to EPO in the Hippocampus culture, be checked through peak activity between 30pM-1 nM, thereby causes increasing 43-46% than the solvent reaction.In described cortex culture, the reaction profile curve similar to most of known somatomedin-maximum activity occurs in the smooth reaction profile curve that is higher than solvent reaction 32-40% between the 30-300pM on the whole thereby EMP-1, EMP-9 and EMP-27 show.EMP-6 and EMP-23 show typical bell dose response distribution curve, detect peak activity between 30-300pM, thereby cause increasing 68-87% than solvent reaction level on projection.On the whole, EMP-6 promotes firm neurite projection activity in Hippocampus culture and cortex culture, yet, with in the cortex culture, compare, EMP-1 demonstrates selectively acting in the Hippocampus culture, and the effect of EMP-23 in the cortex culture is higher than the effect in hippocampal cell.Generally speaking EMP-9 and EMP-27 neurite projection react not too deep to people's impression.

Relatively demonstrating between described EMP and known somatomedin, they show regional difference on neurite projection promotion ability.Described EMP increases the neurite projection in the cortex culture ability will be greater than or equal to ability (Fig. 9-Figure 18) of known somatomedin.Owing to have only a few factors that cortex cell is had such effect, so this discovery is noticeable and important.On the other hand, many somatomedin are brought into play powerful projection reaction (being higher than contrast 38-86%) in Hippocampus.Compare with these somatomedin, described EMP demonstrates medium but firm projection and promotes activity, yet they show the activity that is higher than BDMF, NGF and VEGF.

Discuss

EMP promotes the neurite projection in mammalian cell.Described effect is firm for hippocampal cell and cortex cell.Described neurite projection facilitation is better than the effect of various somatomedin.Described effect is effectively, is checked through effect under picomole concentration.

Should particularly point out equally, consider only a few somatomedin or analogies are promoting on the neurite projection it is that effectively described data are even more important in cerebral cortex neurons.Especially relevant is that this activity of described EMP is checked through under Asia-picomole/picomole concentration.

Embodiment 5

EPO prevents ischemia injury

Object of study

Weigh for the male spontaneous hypertensive rat (Charles River) of weight between 250-300g, use ketamine (100mg/ml)/xylazine (20mg/ml) intermixture (1.2ml/kg then; I.p.) anesthesia.Narcotic level is by corneal reflex (air is blown in the eye) and afterbody or foot are squeezed the shank of pinching reflect and assess.In case rat is anaesthetized, it is placed on the little animal surgery operation plate, and fixing in operation process.With the body temperature of the described rat of transrectal probe continuous monitoring and utilize homoiostasis heating cushion (homeostatic heating pad) that body temperature is remained on 37 ℃.

The experimental model of cerebral ischemia

Caused the rat ischemia in 2 hours by closed left common carotid artery of connecting (common carotid artery) and left brain medium-sized artery (middle cerebral artery), then utilize improving one's methods of Brint and his partner institute description technique (J.Cereb Blood Flow Metab 8:474-485,1988) to carry out 22 hours reindoctrinating.Specifically, described left CCA separates by the otomy in the cervical region ventral surface.For the separation of homonymy MCA, between the outer canthus of left eye and corresponding external auditory meatus, make the head of second otch below exposing.Described MCA is by exposing in the 5mm hole of zygomatic arch and squamosum fusion site mouth side 2-3mm brill under Zeiss operating microscope direct observation.Open dura mater with aseptic 26g stylus printer, platinum alloy tinsel (diameter 0.1mm) is inserted into just has been higher than following cortex vein below the described MCA.As (Stroke, 25: the 2235-2240 pages or leaves, 1994) as described in Aronowski and he's the colleague, described MCA comes temporary transient closed by the blood vessel of raising and described alloying metal silk (across the alloy wire) is passed in compressing.Simultaneously, described CCA aneurysm clamp closure.The closing period of described CCA and described MCA is 2 hours.When finish this period, described tinsel and described tumor folder are carefully removed, make described blood vessel reindoctrinate, and sew up described incision tract.Described rat is put into one with it before in turning back to its original cage and separates cage and recover.

Research design

Research I: send EPO and solvent by miniature osmotic pumps

Start preceding 20 hours of ischemia, be filled with miniature osmotic pumps (the model 1003D of solvent or EPO; Alza) put between the scapula of described rat.The conduit that connects described pump is inserted and fastens in right jugular vein speed continuous infusion medicine with 1 μ l hour.Rat is divided into 5 groups: (1) sham-operation vehicle treated, and (2) ischemia vehicle treated, (3) ischemia 1.32U/ days EPO handle, and (4) ischemia 132U/ days EPO handle and (5) EPO processing in ischemia 1321U/ days.At second day, cause the rat ischemia as mentioned above.After 22 hours, assess the behavior performance of described rat, collect the terminal blood plasma level that blood sample is used for EPO, allow described rat euthanasia then, get brain, section also dyeing to be used for histologic analysis.

Research II: send EPO and solvent with the azygos vein large bolus injection

At first day, cause the rat ischemia as mentioned above.Rat is divided into 4 groups: (1) ischemia vehicle treated, and (2) ischemia 1000U/kg EPO handles, and (3) ischemia 2500U/kgEPO handles and (4) 5000U/kg EPO handles.Closed back 15 minutes, described rat was accepted solvent or EPO with the intravenous large bolus injection.After 22 hours, assess the behavior performance of described rat, collect the terminal blood plasma level analysis that blood sample is used for EPO, allow described rat euthanasia then, get brain, section also dyeing to be used for histologic analysis.

Research III: send EPO and solvent to repeat the vein large bolus injection

At first day, cause the rat ischemia as mentioned above.Rat is divided into 2 groups: (1) ischemia vehicle treated and (2) ischemia 2500U/kg EPO handle.With the heavy dose of administration of intravenous, accumulated dose was 10 to medicine, 000U/kg closure back 15 minutes, 2 hours, 4 hours and 6 hours.After 22 hours, assess the behavior performance of described rat, collect the terminal blood plasma level analysis that blood sample is used for EPO, allow described rat euthanasia then, get brain, section also dyeing to be used for histologic analysis.

Outcome measurement

Determination of plasma:When putting to death, collect blood sample from every rat by the socket of the eye hole.Separated plasma, freezing and determine plasma concentration (U/ml) by the EPO elisa assay.

Infarct volume:Get brain, be enclosed in and also at room temperature use chlorination 2,3,5-triphenyltetrazolium  dyestuff (TTC on the 1mm sheet; Sigma) dyeing is 15 minutes.Section after the dyeing is stored in 10% buffered formalin at 4oC.By these sections of Nikon SMZ-U microdissection sem observation.Utilize each brain section image of CCD captured by camera and utilize Image Pro Phase III software processes to calculate infarct volume.

The result

Research I:Significantly reduce infarct volume (Figure 19) by miniature osmotic pumps with the EPO that 132U/ days or 1321U/ days continuous infusions give.Plasma concentration relevant with described protective effect (Figure 20).

Research II:The EPO that gave with heavy dose in 1000U/kg, 2500U/kg or the 5000U/kg azygos vein in closed back 15 minutes in this model does not protect prevention ischemic injury (Figure 21).Plasma concentration in every group low (Figure 22).

Research III:Closure back 15 minutes, 2 hours, 4 hours and the EPO that gave with 2500U/kg repetition intravenous heavy dose in 6 hours cause infarct volume significantly to reduce (Figure 23).Measuring plasma concentration (Figure 24) at present.

Discuss

Data are supported in by closed described CCA of of short duration series connection and MCA and cause in the ischemic spontaneous hypertensive rat, and can significantly reduce with the EPO successive administration that is low to moderate intermediate concentration or repeat administration can infarct volume.Same amount and the critical relation of supporting for protective effect of last existence of time that EPO gives that occur in of described result.The EPO administration of low dosage ratio is with the same manner high dose administration or more useful with single heavy dose of infusion administration in the long term.This with demonstrate observe when low dosage (pM) effect of EPO maximum protection and in fact when higher dosage (μ M) vitro data of forfeiture effect be consistent.

List of references

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Claims

1. a peptide is used for the treatment of application in the medicine of the disease of being damaged mediation by neurotoxicity, neural degeneration or neurological in preparation, wherein said peptide comprise one or more length 8 to about 40 aminoacid and with the monomeric peptide of EPO receptors bind, each monomeric peptide comprises an aminoacid sequence X ₄X ₅X _aX _bX ₆X _cX _dX ₇(SEQ ID NO:47), wherein

X _aBe G or A;

X _bBe P or A;

X _cBe T or A;

X _dBe selected from W, A and F;

X ₄Be selected from R, H, Y, L and W, perhaps X ₄Do not exist;

X ₅Be selected from F, M and I;

X ₆Independently be selected from the L-aminoacid and the stereomeric D-aminoacid of 20 kinds of genetic codings; With

X ₇Be selected from D, V, E, I and L.

2. the application described in the claim 1, wherein said sequence is X ₄X ₅GPX ₆TWX ₇(SEQ ID NO:48); Wherein

X ₄Be selected from R, H, Y, L and W, perhaps X ₄Do not exist;

X ₅Be selected from F, M and I;

X ₇Be selected from D, V, E, I and L.

3. the application described in the claim 2, wherein said sequence is X ₃X ₄X ₅GPX ₆TWX ₇X ₈(SEQ ID NO:1), wherein

X ₃Be selected from C, E, A, alpha-amido-γ-bromo-butyric acid and homocysteine (Hoc);

X ₄Be selected from R, H, Y, L and W, perhaps X ₄Do not exist;

X ₅Be selected from F, M and I;

X ₆Independently be selected from the L-aminoacid and the stereomeric D-aminoacid of 20 kinds of genetic codings;

X ₇Be selected from D, V, E, I and L; With

4. the application described in the claim 3, condition is X ₃Or X ₈Be C or homocysteine (Hoc).

5. the application described in the claim 4, wherein X ₃Or X ₈Be C.

6. the application described in the claim 3, wherein

X ₃Be selected from C, E and A;

X ₄Be selected from R, H or Y, perhaps X ₄Do not exist;

X ₆Be selected from V, L, I, M, E and A; With

X ₇Be D or V; With

X ₈Be selected from C, K and A.

7. the application described in the claim 3, wherein said peptide are each self-contained YX ₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈The dimer of the described monomeric peptide of (SEQ ID NO:2) aminoacid sequence, wherein

X ₂And X ₆Be selected from the L-aminoacid of 20 kinds of genetic codings independently of one another;

X ₃Be C; With

X ₈Be C.

8. the application described in the claim 7, wherein

X ₂Be selected from L, S, H, M, A and I, or X ₂Do not exist; With

X ₆Be selected from V, L, I, M, E and A.

9. the application described in the claim 7, wherein each described monomeric peptide comprises an X ₁YX ₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈X ₉X ₁₀X ₁₁The aminoacid sequence of (SEQ ID NO:3), wherein X ₁, X ₂, X ₆, X ₉, X ₁₀And X ₁₁In each be independently selected from the L-aminoacid of 20 kinds of genetic codings.

10. the application described in the claim 9, wherein

X ₃Be selected from C, E and A;

X ₄Be selected from R, H and Y, perhaps X ₄Do not exist;

X ₇Be D or V;

X ₈Be C or K;

X ₉Be K, G, L, Q, R, S or T; With

X ₁₀Be A, G, P, R or Y.

11. the application described in the claim 10, wherein

X ₁Be D, E, L, N, S, T or V;

X ₂Be selected from L, S, H, M, A and I, perhaps X ₂Do not exist;

X ₉Be selected from K, Q, R, S and G; With

X ₁₀Be selected from P, Y and A.

12. the application described in the claim 1, wherein each described monomeric peptide comprises one and is independently selected from following sequence:

GGLYLCRFGPVTWDCGYKGG (SEQ ID NO：7)；

GGTYSCHFGPLTWVCKPQGG (aka EMP-1)(SEQ ID NO：8)；

GGDYHCRMGPLTWVCKPLGG (SEQ ID NO：9)；

VGNYMCHFGPITWVCRPGGG (SEQ ID NO：10)；

GGVYACRMGPITWVCSPLGG (SEQ ID NO：11)；

VGNYMAHMGPITWVCRPGG (SEQ ID NO：12)；

GGTYSCHFGPLTWVCKPQ (aka EMP-16)(SEQ ID NO：13)；

GGLYACHMGPMTWVCQPLRG (aka EMP-36)(SEQ ID NO：14)；

TLAQYICYMGPETWECRPSPKA (aka EMP-38)(SEQ ID NO：15)；

YSCHFGPLTWVCK (aka EMP-20(SEQ ID NO：16)；

YCHFGPLTWVC (aka EMP-23)(SEQ ID NO：17)；

SCHFGPLTWVCK (aka EMP-24)(SEQ ID NO：18)；

GGTASCHFGPLTWVCKPQGG (aka EMP-6)(SEQ ID NO：19)；

GGTYSCHFAPLTWVCKPQGG (aka EMP-9)(SEQ ID NO：20)；

GGTYSCFGPLTWVCKPQGG (aka EMP-27)(SEQ ID NO：21)；

TYSCHFGPLTWVCKPQGG (aka EMP-17)(SEQ ID NO：22)；

TYSCHFGPLTWVCKPQ (aka EMP-18)(SEQ ID NO：23)；

YSCHFGPLTWVCKP (aka EMP-19)(SEQ ID NO：24)；

YSCHFGPLTWVC (aka EMP-21)(SEQ ID NO：25)；

YSCHFGALTWVCK (aka EMP-22)(SEQ ID NO：26)；

GGCRIGPITWVCGG (aka EMP-25)(SEQ ID NO：27)；

HFGPLTWV (aka EMP-26)(SEQ ID NO：28)；

GGTTSCHFGPLTWVCKPQGG (aka EMP-7)(SEQ ID NO：29)；

GGTFSCHFGPLTWVCKPQGG (aka EMP-8)(SEQ ID NO：30)；

GGTYSCHFGALTWVCKPQGG (aka EMP-10)(SEQ ID NO：31)；

GGTYSCHFGPATWVCKPQGG (aka EMP-11)(SEQ ID NO：32)；

GGTYSCHFGPLAWVCKPQGG (aka EMP-12)(SEQ ID NO：33)；

GGTYSCHFGPLTAVCKPQGG (aka EMP-13)(SEQ ID NO：34)；

GGTYSCHFGPLTFVCKPQGG (aka EMP-14)(SEQ ID NO：35)；

GGTYSCHFGPLTWVCKAQGG (aka EMP-15)(SEQ ID NO：36)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-28，X＝D-Tyr)(SEQ ID NO：37)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-29，X＝p-NO ₂-Phe)

(SEQ ID NO：38)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-30，X＝p-NH ₂-Phe)

(SEQ ID NO：39)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-31，X＝p-F-Phe)(SEQ ID NO：40)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-32，X＝p-I-Phe)(SEQ ID NO：41)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-33, X=3,5-two bromo-Tyr)

(SEQ ID NO：42)；

Ac-GGTYSCHFGPLTWVCKPQGG (aka EMP-34)(SEQ ID NO：43)；

GGLYACHMGPMTWVCQPLGG (aka EMP-35)(SEQ ID NO：44)；

LGRKYSCHFGPLTWVCQPAKKD (aka EMP-37) (SEQ ID NO:45); With

GGTYSEHFGPLTWVKKPQGG (aka EMP-39)(SEQ ID NO：46)。

13. the application described in the claim 12, wherein each described monomeric peptide is independently selected from:

GGTYSCHFGPLTWVCKPQGG (SEQ ID NO：8)；

GGTASCHFGPLTWVCKPQGG (SEQ ID NO：19)；

GGTYSCHFAPLTWVCKPQGG (SEQ ID NO:20); With

YCHFGPLTWVC (SEQ ID NO：17)。

14. the application described in the claim 1, wherein said peptide are the dimers that is formed by covalent bond by the Polyethylene Glycol joint.

15. it is terminal covalently bound to the N-end that the application described in the claim 14, wherein said dimeric each monomeric peptide are N-.

16. it is terminal covalently bound to the C-end that the application described in the claim 14, wherein said dimeric each monomeric peptide are N-.

17. the application described in the claim 1, wherein said monomeric peptide is dimerization on activatory benzodiazepine (benodiazepins), oxazalones, azalide, aminimide or diketopiperazine.

18. it is terminal covalently bound to the N-end that the application described in the claim 17, wherein said monomeric peptide are N-.

19. it is terminal covalently bound to the C-end that the application described in the claim 17, wherein said monomeric peptide are N-.

20. the application described in the claim 2, wherein said peptide comprise a peptide dimer at least.

21. the dimer of peptide is used for inducing the application of the bioactive medicine of described neuroprotective in preparation in the claim 2, the sequence of wherein said peptide is X ₄X ₅GPX ₆TWX ₇(SEQ ID NO:48); Wherein

X ₄Be selected from R, H, Y, L and W, perhaps X ₄Do not exist;

X ₅Be selected from F, M and I;

X ₇Be selected from D, V, E, I and L.

22. the application described in the claim 21, wherein said cell surface receptor are the EPO receptors.

23. the application described in the claim 21, wherein said peptide dimer are to be selected from following a kind of cell surface receptor agonist: GH agonist, PDGF agonist, EGF agonist, G-CSF agonist, TPO (thrombopoietin) agonist, VEGF agonist, FGF agonist, igf agonist, IL-3 agonist, IL-5 agonist, IL-6 agonist and IL-2 agonist.

24. the application described in the claim 21, wherein said agonist comprise a YX ₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈The aminoacid sequence of (SEQ ID NO:2), wherein X ₂And X ₆In each independently be selected from the L-aminoacid of 20 kinds of genetic codings; X ₃Be C; X ₄Be R, H, L or W; X ₅Be M, F or I; X ₇Be D, E, I, L or V; And X ₈Be C.

25. the application described in the claim 21, wherein said agonist comprise an X ₁YX ₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈X ₉X ₁₀X ₁₁The aminoacid sequence of (SEQ ID NO:3), wherein X ₁, X ₂, X ₆, X ₉, X ₁₀And X ₁₁In each independently be selected from any in the L-aminoacid of 20 kinds of genetic codings; X ₃Be C; X ₄Be R, H, L or W; X ₅Be M, F or I; X ₇Be D, E, I, L or V; And X ₈Be C.

26. the application described in the claim 21, wherein said agonist comprise an X ₁YX ₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈X ₉X ₁₀X ₁₁The aminoacid sequence of (SEQ ID NO:3), wherein X ₁, X ₂And X ₁₁In each independently be selected from any in the L-aminoacid of 20 kinds of genetic codings; X ₃Be C; X ₄Be R or H; X ₅Be F or M; X ₆Be I, L, T, M or V; X ₇Be D or V; X ₉Be G, K, L, Q, R, S or T; And X ₁₀Be A, G, P, R or Y.

27. the application described in the claim 21, wherein said agonist comprise an X ₁YX ₂X ₃X ₄X ₅GPX ₆TWX ₇X ₈X ₉X ₁₀X ₁₁The aminoacid sequence of (SEQ ID NO:3), wherein X ₁Be D, E, L, N, S, T or V; X ₂Be A, H, K, L, M, S or T; X ₃Be C; X ₄Be R or H; X ₅Be M, F or I; X ₆And X ₁₁Be any in the L-aminoacid of 20 kinds of genetic codings independently; X ₇Be D, E, I, L or V; X ₈Be C; X ₉Be K, R, S or T; And X ₁₀Be P.

28. the application described in the claim 21, wherein said agonist is selected from:

GGLYLCRFGPVTWDCGYKGG (SEQ ID NO：7)；

GGTYSCHFGPLTWVCKPQGG (aka EMP-1)(SEQ ID NO：8)；

GGDYHCRMGPLTWVCKPLGG (SEQ ID NO：9)；

VGNYMCHFGPITWVCRPGGG (SEQ ID NO：10)；

GGVYACRMGPITWVCSPLGG (SEQ ID NO：11)；

VGNYMAHMGPITWVCRPGG (SEQ ID NO：12)；

GGTYSCHFGPLTWVCKPQ (aka EMP-16)(SEQ ID NO：13)；

GGLYACHMGPMTWVCQPLRG (aka EMP-36)(SEQ ID NO：14)；

TIAQYICYMGPETWECRPSPKA (aka EMP-38)(SEQ ID NO：15)；

YSCHFGPLTWVCK (aka EMP-20(SEQ ID NO：16)；

YCHFGPLTWVC (aka EMP-23)(SEQ ID NO：17)；

SCHFGPLTWVCK (aka EMP-24)(SEQ ID NO：18)；

GGTASCHFGPLTWVCKPQGG (aka EMP-6)(SEQ ID NO：19)；

GGTYSCHFAPLTWVCKPQGG (aka EMP-9)(SEQ ID NO：20)；

GGTYSCFGPLTWVCKPQGG (aka EMP-27)(SEQ ID NO：21)；

TYSCHFGPLTWVCKPQGG (aka EMP-17)(SEQ ID NO：22)；

TYSCHFGPLTWVCKPQ (aka EMP-18)(SEQ ID NO：23)；

YSCHFGPLTWVCKP (aka EMP-19)(SEQ ID NO：24)；

YSCHFGPLTWVC (aka EMP-21)(SEQ ID NO：25)；

YSCHFGALTWVCK (aka EMP-22)(SEQ ID NO：26)；

GGCRIGPITWVCGG (aka EMP-25)(SEQ ID NO：27)；

HFGPLTWV (aka EMP-26)(SEQ ID NO：28)；

GGTTSCHFGPLTWVCKPQGG (aka EMP-7)(SEQ ID NO：29)；

GGTFSCHFGPLTWVCKPQGG (aka EMP-8)(SEQ ID NO：30)；

GGTYSCHFGALTWVCKPQGG (aka EMP-10)(SEQ ID NO：31)；

GGTYSCHFGPATWVCKPQGG (aka EMP-11)(SEQ ID NO：32)；

GGTYSCHFGPLAWVCKPQGG (aka EMP-12)(SEQ ID NO：33)；

GGTYSCHFGPLTAVCKPQGG (aka EMP-13)(SEQ ID NO：34)；

GGTYSCHFGPLTFVCKPQGG (aka EMP-14)(SEQ ID NO：35)；

GGTYSCHFGPLTWVCKAQGG (aka EMP-15)(SEQ ID NO：36)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-28，X＝D-Tyr)(SEQ ID NO：37)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-29，X＝p-NO ₂-Phe)

(SEQ ID NO：38)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-30，X＝p-NH ₂-Phe)

(SEQ ID NO：39)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-31，X＝p-F-Phe)(SEQ ID NO：40)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-32，X＝p-I-Phe)(SEQ ID NO：41)；

GGTXSCHFGPLTWVCKPQGG (aka EMP-33, X=3,5-two bromo-Tyr)

(SEQ ID NO：42)；

Ac-GGTYSCHFGPLTWVCKPQGG (aka EMP-34)(SEQ ID NO：43)；

GGLYACHMGPMTWVCQPLGG (aka EMP-35)(SEQ ID NO：44)；

LGRKYSCHFGPLTWVCQPAKKD (aka EMP-37) (SEQ ID NO:45); With

GGTYSEHFGPLTWVKKPQGG (aka EMP-39)(SEQ ID NO：46)。

29. the application described in the claim 21, wherein said peptide dimer is formed by covalent bond by the Polyethylene Glycol joint.