CN103421094A - Polypeptide compound with EPO-like activity - Google Patents
Polypeptide compound with EPO-like activity Download PDFInfo
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Abstract
The invention belongs to the field of biochemistry, and particularly relates to a polypeptide compound with EPO-like activity and applications of the compound in treating diseases related to insufficient or faulty red blood cell production. The compound is formed by two polypeptide sequences, wherein a first sequence is X1-G-X2-Y-X3-C-X4-M-G-P-X5-T-W-X6-C-Q-P-L-X7-G-X8-X9, a second sequence is X10-G-X11-Y-X12-C-X13-M-G-P-X14-X15-W-X16-C-X17-X18-X19-X20-G, and the first sequence and the second sequence are connected by a connecting amino acid (X8) of the first sequence to form a dimer.
Description
Invention field
The invention belongs to biochemical field, in particular to a kind of polypeptide compound with the similar activity of EPO.And the application in this compound disease relevant with erythrocyte generation deficiency or defect in treatment.
Background of invention
Erythropoietin (EPO) is to have 165 amino acid whose glycoprotein hormoneses, its molecular weight is about 34kDa, authorize the United States Patent (USP) 4 of Lin, 703,008 has described evaluation, clone and the expression of erythropoietin encoding gene, and its specification sheets is attached to herein with its integral body by reference.
EPO results from kidney (90%) and liver usually, after secretion, enters Peripheral Circulation, and mainly metabolism in liver, only has on a small quantity and discharges through urine, and it is to promote the growth of marrow CFU-E, hyperplasia, differentiation and ripe main stimulating factor.The biological effect of EPO is that the specificity EPO acceptor (EPOR) by being positioned at marrow CFU-E surface has mediated.EPO is combined rear formation dimer with EPOR, then regulates erythrocytic hyperplasia and differentiation by signal transduction path.EPO signal transduction process has many approach, what wherein research and comparison was thorough is the EPOR-JAK2-STAT5 approach, and other certified signal transduction mechanisms also comprise: EPOR-JAK2-PI3K approach, EPOR-JAK2-ERKs approach, EPOR-JAK2-NF-KappaB approach, EPOR-JAK2-Ras albumen-MAPK approach etc.Visible, the conduction of EPO intracellular signal is not single cascade path, but a plurality of path is crosslinked, complementary, the complexity conduction work process of formation network.
For the cancer patient in chronic kidney hypofunction and chemicotherapy late period, because its normal hemopoietic function is suppressed, cause EPO definitely or relative deficiency, anaemia has often occurred together.Present stage adopts input recombinant human epo injection liquids to improve patient's Status of Anemia clinically, and has obtained significant effect.Yet still there is in actual applications following problem: usually cause cardiovascular complication, such as hypertension, myocardial infarction etc.; Some solid tumor is promoted development; Single pure red cell aplasia occurs; Half-life in vivo is shorter, need inject weekly 1-3 inferior.Therefore, need the newtype drug of exploitation treatment anaemia badly.Chinese patent ZL94109128.7 discloses a kind of restructuring erythropoiesis stimulating protein of high-glycosylation, clinical study shows, Half-life in vivo is than recombinant human epo's significant prolongation, Chinese patent ZL00809895.6 discloses the conjugate of a kind of erythropoietin and polyoxyethylene glycol, on average injects per month the effect that can be equivalent to inject weekly 1-3 recombinant human epo for 1 time.Yet, the medicine of this class mechanism in vivo all can with EPO antibody generation cross reaction, reduce the curative effect of this class medicine.The inventor passes through lot of experiments, accident has been found a kind of and EPO sequence and the complete incoherent compound of structure, this compound have the similar activity of significant EPO and not can with EPO antibody generation cross reaction, and can be used in the disease that treatment is relevant to erythrocyte generation deficiency or defect, thereby complete the present invention.
Summary of the invention:
The present invention relates to a kind of polypeptide compound, consist of two peptide sequences, wherein First ray is: X1-G-X2-Y-X3-C-X4-M-G-P-X5-T-W-X6-C-Q-P-L-X7-G-X8-X9; The second sequence is: X10-G-X11-Y-X12-C-X13-M-G-P-X14-X15-W-X16-C-X17-X18-X19-X20-G; And First ray and the second sequence are connected to form dimer by the connection amino acid (X8) in First ray.
Polypeptide compound of the present invention and EPO receptors bind, the performance receptor agonism, treatment produces deficiency, defect or consumes relevant disease excessively to erythrocyte by this.
Amino acid used in the present invention, except comprising 20 kinds of common amino acids well-known to those skilled in the art, also comprises the non-common amino acid of part.The title of common amino acid and abbreviation will be summarized in table 1; Non-common amino acid, title, the abbreviation with and structure, be drawn in table 2.
The title of table 1 common amino acid and abbreviation
The non-common amino acid whose title of table 2, abbreviation and structure
If the amino acid in table 1 of the present invention and table 2, without particular limitation of D or L configuration, is L-type amino acid; If without particular limitation of, all amino acid is a-amino acid.If the direction of all sequences of the present invention does not specify, be from left to right the direction of N-terminal-C-terminal.
At first the present invention provides a kind of polypeptide compound, two peptide sequences, consists of, and wherein First ray is: X1-G-X2-Y-X3-C-X4-M-G-P-X5-T-W-X6-C-Q-P-L-X7-G-X8-X9; The second sequence is: X10-G-X11-Y-X12-C-X13-M-G-P-X14-X15-W-X16-C-X17-X18-X19-X20-G; And First ray and the second sequence are connected to form dimer by the connection amino acid (X8) in First ray; X1 is selected from G or AcG; X2, X5 and X6 are respectively independently selected from L, I or V; X3 is selected from A or G; X4 is selected from H or D-His; X7 is selected from R, HomoArg or Pal-Lys; X8 is selected from Lys or D-Lys, participates in First ray with alpha-amino group in its structure synthetic, with the C-terminal amino acid of epsilon-amino in its structure and the second sequence, is connected to form dimer, preferably uses Lys; X9 is the small peptide that Methionin or the C-terminal that is comprised of 2-10 amino acid are Methionin, and preferred, X9 is comprised of 3-7 amino acid, and particularly preferred, X9 is selected from GK, GGK, GGARRAGK, GGAGAGK, GADEAGGKK or GADEGGAK; X10 is selected from G or AcG; X11, X14, X16 or X19 are respectively independently selected from L, I, V or Nle; X12 is selected from A, Aib or G; X13 is selected from H or D-His; X15 is selected from T or S; X17 is selected from Q or N; X18 is selected from P or Hyp; X20 is selected from R, HomoArg, Cit or Pal-Lys.
Preferably, First ray of the present invention includes but not limited to following sequence:
SEQ?ID?NO：1(AcG)GLYACHMGPITWVCQPLRGKGK
SEQ?ID?NO：2(AcG)GLYACHMGPITWVCQPLRGKGGK
SEQ?ID?NO：3(AcG)GLYACHMGPITWVCQPLRGKGGARRAGK
SEQ?ID?NO：4(AcG)GLYACHMGPITWVCQPLRGKGGAGAGK
SEQ?ID?NO：5(AcG)GLYACHMGPITWVCQPLRGKGADEAGGKK
SEQ?ID?NO：6(AcG)GLYACHMGPITWVCQPLRGKGADEGGAK
SEQ?ID?NO：7(AcG)GLYACHMGPITWICQPLRGKGK
SEQ?ID?NO：8(AcG)GLYACHMGPITWICQPLRGKGGK
SEQ?ID?NO：9(AcG)GLYACHMGPITWICQPLRGKGGARRAGK
SEQ?ID?NO：10(AcG)GLYACHMGPITWICQPLRGKGGAGAGK
SEQ?ID?NO：11(AcG)GLYACHMGPITWICQPLRGKGADEAGGKK
SEQ?ID?NO：12(AcG)GLYACHMGPITWICQPLRGKGADEGGAK
SEQ?ID?NO：13(AcG)GLYAC(D-His)MGPITWVCQPLRGKGK
SEQ?ID?NO：14(AcG)GLYAC(D-His)MGPITWVCQPLRGKGGK
SEQ?ID?NO：15(AcG)GLYAC(D-His)MGPITWVCQPLRGKGGARRAGK
SEQ?ID?NO：16(AcG)GLYAC(D-His)MGPITWVCQPLRGKGGAGAGK
SEQ?ID?NO：17(AcG)GLYAC(D-His)MGPITWVCQPLRGKGADEAGGKK
SEQ?ID?NO：18(AcG)GLYAC(D-His)MGPITWVCQPLRGKGADEGGAK
SEQ?ID?NO：19(AcG)GLYACHMGPLTWVCQPLRGKGK
SEQ?ID?NO：20(AcG)GLYACHMGPLTWVCQPLRGKGGK
SEQ?ID?NO：21(AcG)GLYACHMGPLTWVCQPLRGKGGARRAGK
SEQ?ID?NO：22(AcG)GLYACHMGPLTWVCQPLRGKGGAGAGK
SEQ?ID?NO：23(AcG)GLYACHMGPLTWVCQPLRGKGADEAGGKK
SEQ?ID?NO：24(AcG)GLYACHMGPLTWVCQPLRGKGADEGGAK
SEQ?ID?NO：25(AcG)GIYGCHMGPITWVCQPLRGKGK
SEQ?ID?NO：26(AcG)GIYGCHMGPITWVCQPLRGKGGK
SEQ?ID?NO：27(AcG)GIYGCHMGPITWVCQPLRGKGGARRAGK
SEQ?ID?NO：28(AcG)GIYGCHMGPITWVCQPLRGKGGAGAGK
SEQ?ID?NO：29(AcG)GIYGCHMGPITWVCQPLRGKGADEAGGKK
SEQ?ID?NO：30(AcG)GIYGCHMGPITWVCQPLRGKGADEGGAK
SEQ?ID?NO：31(AcG)GIYACHMGPLTWVCQPL(Pal-Lys)GKGK
SEQ?ID?NO：32(AcG)GIYACHMGPLTWVCQPL(Pal-Lys)GKGGK
SEQ?ID?NO：33(AcG)GIYACHMGPLTWVCQPL(Pal-Lys)GKGGARRAGK
SEQ?ID?NO：34(AcG)GIYACHMGPLTWVCQPL(Pal-Lys)GKGGAGAGK
SEQ?ID?NO：35(AcG)GIYACHMGPLTWVCQPL(Pal-Lys)GKGADEAGGKK
SEQ?ID?NO：36(AcG)GIYACHMGPLTWVCQPL(Pal-Lys)GKGADEGGAK
Preferably, the second sequence of the present invention includes but not limited to following sequence:
SEQ?ID?NO：37(AcG)GLYACHMGPITWVCQPLRG
SEQ?ID?NO：38(AcG)GLYACHMGPITWICQPLRG
SEQ?ID?NO：39(AcG)GLYAC(D-His)MGPITWVCQPLRG
SEQ?ID?NO：40(AcG)GLYACHMGPLTWVCQPLRG
SEQ?ID?NO：41(AcG)GIYGCHMGPITWVCQPLRG
SEQ?ID?NO：42(AcG)GIYACHMGPLTWVCQPL(Pal-Lys)G
SEQ?ID?NO：43(AcG)GLY(Aib)CHMGPITWVCQ(Hyp)L(Cit)G
SEQ?ID?NO：44(AcG)GLYACHMGPISWVCNPLRG
SEQ?ID?NO：45(AcG)GLYACHMGPITWVCNPL(HomoArg)G
SEQ?ID?NO：46(AcG)GLY(Aib)CHMGPISWVCQP(Nle)RG
Polypeptide compound of the present invention can be modified, such as N-terminal acetylize, formation intramolecular disulfide bond, C-terminal amidation etc.Preferably, N-terminal acetylize and formation intramolecular disulfide bond can exist in First ray and the second sequence simultaneously, and optionally in First ray, carry out the C-terminal amidation.Intramolecular disulfide bond as herein described refers in First ray that in disulfide linkage that the 6th and the 15th halfcystine forms and the second sequence, the 6th and the 15th halfcystine forms disulfide linkage.In a specific embodiments of the present invention, the aminoacid sequence of First ray is selected from SEQ ID NO:1, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, C-terminal amidation; The aminoacid sequence of the second sequence is selected from SEQ ID NO:37, and the 6th and the 15th halfcystine forms intramolecular disulfide bond.
In one embodiment of the invention, the aminoacid sequence of the First ray of polypeptide compound is selected from SEQ ID NO:1, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, C-terminal amidation; The aminoacid sequence of the second sequence is selected from SEQID NO:37, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, and forms dimer by the 21st L-type Methionin in First ray, and hereinafter referred is polypeptide dimer 1, and structure is as follows:
In another embodiment of the present invention, the aminoacid sequence of the First ray of polypeptide compound is selected from SEQ ID NO:2, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, C-terminal amidation; The aminoacid sequence of the second sequence is selected from SEQID NO:37, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, and forms dimer by the 21st L-type Methionin in First ray, and hereinafter referred is polypeptide dimer 2.
In another embodiment of the present invention, the aminoacid sequence of the First ray of polypeptide compound is selected from SEQ ID NO:3, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, C-terminal amidation; The aminoacid sequence of the second sequence is selected from SEQID NO:37, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, and forms dimer by the 21st L-type Methionin in First ray, and hereinafter referred is polypeptide dimer 3.
In another embodiment of the present invention, the aminoacid sequence of the First ray of polypeptide compound is selected from SEQ ID NO:4, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, C-terminal amidation; The aminoacid sequence of the second sequence is selected from SEQID NO:37, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, and forms dimer by the 21st L-type Methionin in First ray, and hereinafter referred is polypeptide dimer 4.
In another embodiment of the present invention, the aminoacid sequence of the First ray of polypeptide compound is selected from SEQ ID NO:5, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, C-terminal amidation; The aminoacid sequence of the second sequence is selected from SEQID NO:37, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, and forms dimer by the 21st L-type Methionin in First ray, and hereinafter referred is polypeptide dimer 5.
In another embodiment of the present invention, the aminoacid sequence of the First ray of polypeptide compound is selected from SEQ ID NO:6, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, C-terminal amidation; The aminoacid sequence of the second sequence is selected from SEQID NO:37, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, and forms dimer by the 21st L-type Methionin in First ray, and hereinafter referred is polypeptide dimer 6.
In another embodiment of the present invention, the aminoacid sequence of the First ray of polypeptide compound is selected from SEQ ID NO:26, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, C-terminal amidation; The aminoacid sequence of the second sequence is selected from SEQID NO:41, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, and forms dimer by the 21st L-type Methionin in First ray, and hereinafter referred is polypeptide dimer 7.
In another embodiment of the present invention, the aminoacid sequence of the First ray of polypeptide compound is selected from SEQ ID NO:32, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, C-terminal amidation; The aminoacid sequence of the second sequence is selected from SEQID NO:42, and the 6th and the 15th halfcystine forms intramolecular disulfide bond, and forms dimer by the 21st D type Methionin in First ray, and hereinafter referred is polypeptide dimer 8.
Polypeptide compound of the present invention, the dimer constituted of the First ray shown in preferred according to the form below and the second sequence also, optionally in the First ray C-terminal amidation of described polypeptide dimer:
Polypeptide compound of the present invention can also comprise polyoxyethylene glycol (PEG) part, and polyoxyethylene glycol can hold the Methionin in X9 to put together with the C of First ray, thereby forms the polypeptide compound of puting together with PEG.Wherein PEG has approximately 5,000 to approximately 60, (term " about " refers in the prepared product of PEG 000 daltonian molecular weight, some molecule will have and be greater than stated molecular weight, some molecule have be less than stated molecular weight), the epsilon-amino of the C-terminal Methionin of itself and First ray is covalently bound.The PEG of activation of the present invention can be connected to by amino-formate bond or amido linkage the ε-NH of First ray end Methionin
2Upper, thus polypeptide dimer of the present invention is carried out to the PEG modification.When the small peptide X9 of First ray contains two above Methionin, also can carry out two or many PEG to polypeptide dimer of the present invention and modify.
For PEG of the present invention, can be the PEG of straight chain or branch, its molecular weight be that approximately 5,000 dalton (5k) arrive approximately 60,000 dalton (60k), and preferably, PEG has the molecular weight of about 20k to about 40k.The technician can consider to select based on all dosage as desired, cycling time, on the resistance of proteolysis and PEG the size of suitable polymkeric substance on other known impacts for the treatment of peptide etc.
Multiple polyoxyethylene glycol kind can be used for PEGization polypeptide dimer of the present invention, for example, include but not limited to mPEG2-NHS, mPEG2-ALD, mPEG (MAL) 2, mPEG2 (MAL), mPEG-NH2, mPEG-SPA, mPEG-SBA, mPEG-BTC, mPE-ACET, mPEG-SPA-NHS etc.
In one embodiment of the invention, polypeptide dimer 1, polypeptide dimer 2, polypeptide dimer 3, polypeptide dimer 4, polypeptide dimer 5, polypeptide dimer 6, polypeptide dimer 7, polypeptide dimer 8, polypeptide dimer 9, polypeptide dimer 10, polypeptide dimer 11, polypeptide dimer 12, polypeptide dimer 13, polypeptide dimer 14, polypeptide dimer 15, polypeptide dimer 16, polypeptide dimer 17, polypeptide dimer 18, polypeptide dimer 19, polypeptide dimer 20, polypeptide dimer 21, polypeptide dimer 22, polypeptide dimer 23 is connected with the PEG of activation by amino-formate bond.
In another embodiment of the present invention, polypeptide dimer 1, polypeptide dimer 2, polypeptide dimer 3, polypeptide dimer 4, polypeptide dimer 5, polypeptide dimer 6, polypeptide dimer 7, polypeptide dimer 8, polypeptide dimer 9, polypeptide dimer 10, polypeptide dimer 11, polypeptide dimer 12, polypeptide dimer 13, polypeptide dimer 14, polypeptide dimer 15, polypeptide dimer 16, polypeptide dimer 17, polypeptide dimer 18, polypeptide dimer 19, polypeptide dimer 20, polypeptide dimer 21, polypeptide dimer 22, polypeptide dimer 23 is connected with the PEG of activation by amido linkage.
In a specific embodiments of the present invention, polypeptide dimer 1 is by the PEG of amino-formate bond and activation
20kWhen covalently bound, structure is as follows:
In a specific embodiments of the present invention, polypeptide dimer 6 is by the PEG of amido linkage and activation
20kWhen covalently bound, structure is as follows:
In a specific embodiments of the present invention, polypeptide dimer 5 is by the PEG of amino-formate bond and activation
20kWhen covalently bound, structure is as follows:
On the one hand, the preparation method of polypeptide compound of the present invention is classical solid phase synthesis process well known in the art, directly prepares First ray and second sequence of polypeptide dimer of the present invention again; Use oxidising agent (for example, DMSO) to form described intramolecular disulfide bond; And optional PEG (such as mPEG-SPA-NHS) with activation passes through amino-formate bond or amido linkage is covalently bound etc.Particularly; in First ray, X8 connects the protecting group of amino acid (Methionin) use Fmoc (9-fluorenyl-methyl carbonyl) as alpha-amino group or epsilon-amino, also can use the protecting group of Alloc (alkene oxygen propyl group carbonyl) as this Methionin epsilon-amino.When using Fmoc as the protecting group of the alpha-amino group of this Methionin and epsilon-amino, preferably the second sequence is identical with front 20 amino acid whose sequences of First ray, now can remove Fmoc simultaneously, thereby carry out the solid phase synthesis of two sequences simultaneously; When the alpha-amino protecting group of using Fmoc as this Methionin; and while using Alloc as the protecting group of the epsilon-amino of this Methionin; First ray of the present invention and the second sequence are synthetic successively; when synthetic, first adopt weakly alkaline deprotecting regent (as 25% piperidines/DMF) to remove the Fmoc protecting group, now the Alloc protecting group of this Methionin epsilon-amino is unaffected.After the First ray end of synthesis, use suitable reagent (as Pd (PPh
3)
4, i.e. tetra-triphenylphosphine palladium) and remove Alloc protecting group, resynthesis the second sequence.
Particularly, prepare the step of the compounds of this invention as follows:
(1) solid phase synthesis First ray and the second sequence
Whether need to carry out the amidation modification according to the First ray C-terminal, can select the preparation of suitable resin for polypeptide compound of the present invention.When the C-terminal of First ray carries out the amidation modification, can select Rink Amide mbha resin; When the C-terminal of First ray does not need to carry out the amidation modification, the optional amino acid whose Wang resin of connection C-terminal of choosing spouse.
The direction of solid phase synthesis is C-terminal-N-terminal; initial with X9 part C end Methionin in First ray; extend the connection amino acid (Methionin) of sequence to First ray; then simultaneously or successively remove alpha-amino group protecting group and the epsilon-amino protecting group of this linker Methionin, carry out the synthetic of synthetic and the second sequence of the continuation of First ray.When removing the amino acid whose alpha-amino group protecting group of linker and epsilon-amino protecting group simultaneously, step is as follows: soak resin, the amino protecting group of deresinate, wash and monitor, first amino acid (for connecting the Methionin of PEG) of X9 part in the coupling First ray, wash and monitor, remove alpha-amino group protecting group other amino acid of coupling X9 part successively, remove the protecting group of the amino acid whose alpha-amino group protecting group of linker and epsilon-amino, carry out the synthetic of synthetic and the second sequence amino acid sequence of the continuation of First ray remaining amino acid, optionally last amino acid in each sequence is carried out to acetylation modification.When successively removing the amino acid whose alpha-amino group protecting group of linker and epsilon-amino protecting group, step is as follows: soak resin, the amino protecting group of deresinate, wash and monitor, first amino acid (for connecting the Methionin of PEG) of X9 part in the coupling First ray, wash and monitor, remove alpha-amino group protecting group other amino acid of coupling X9 part successively, remove the amino acid whose alpha-amino group protecting group of linker and carry out the continuation of First ray remaining amino acid synthetic, remove the amino acid whose epsilon-amino protecting group of linker and carry out the synthetic of the second sequence amino acid sequence, optionally last amino acid in each sequence is carried out to acetylation modification.
" amino protecting group " used in the present invention, " alpha-amino group protecting group " and " epsilon-amino protecting group " refer to the chemical group of introducing for the amino of protection participation condensation reaction.Described amino protecting group includes but not limited to: tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (cbz), trichloro-ethoxycarbonyl (Troc), fluorenyl methoxy carbonyl (Fmoc), allyloxy carbonyl (Alloc) etc.Preferably use Fmoc as the alpha-amino group protecting group, preferably use Fmoc or Alloc as the epsilon-amino protecting group.
Piperidines (PIP) is selected in removing of alpha-amino group protecting group and resin amino protecting group Fmoc, concentration 20-25% (PIP:DMF), and the time is 20-50min.The removing of Alloc protecting group of epsilon-amino selected, for example Pd (PPh
3)
4, concentration 10-15% (Pd (PPh
3)
4: chloroform), the time is 30-60min.
As an advantage of solid-phase polypeptide synthetic technology, to the side chain of partial amino-acid, can be protected by introducing chemical group, for example Arg can adopt pentamethyl-benzo furans-5-alkylsulfonyl (Pbf); His, Cys, Gln, Asn can adopt trityl (Trt); Lys can adopt tertbutyloxycarbonyl (Boc); Thr, Tyr, Ser can adopt the tertiary butyl (tBu); Asp, Glu can adopt the tert-butyl ester (Otbu).Described blocking group is not limited to this, can carry out choose reasonable according to this area conventional scheme.
In building-up process, solvent for use is selected from dimethyl formamide (DMF) or methylene dichloride (DCM).
Coupling reagent is selected from: a kind of in carbodiimide type reagent or benzotriazole salt type reagent, and with a kind of combination in I-hydroxybenzotriazole (HOBt) or DIPEA (DIEA).Wherein, carbodiimide type reagent comprises dicyclohexylcarbodiimide (DCC), DIC (DIC) and N-diamino propyl group-N-ethyl carbodiimide (EDC).Benzotriazole salt type reagent comprises 2-(1H-benzo trisazo-L-1-yl)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester (TBTU), O-benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate (HBTU), phosphofluoric acid benzotriazole-1-oxygen base three (dimethylamino) phosphorus (BOP), phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus (PyBOP) etc.
The combination of the preferred DCC of coupling reagent and HOBt, the combination of DCC and DIEA, the combination of TBTU and HOBt, the combination of TBTU and DIEA, the most preferably combination of DCC and HOBt.
But acetylation reagent choice for use acetic acid, diacetyl oxide or its halide derivative (as α-Mono Chloro Acetic Acid etc.) select pyridine or DIEA etc. to build the alkali reaction environment simultaneously.Preferably, select pyridine and diacetyl oxide in the present invention.Alternative, select diacetyl oxide and DIEA in the present invention.
Monitoring method is selected the triketohydrindene hydrate detection method.
(2) cracking polypeptide dimer from resin
Cracking can be carried out in trifluoroacetic acid or other strong acid media, adds 5-20%V/V Side chain protective group scavenging agent, as thioanisole, tri isopropyl silane, phenol, water, dithioglycol, meta-cresol etc.The selection of Side chain protective group scavenging agent and proportioning are determined flexibly according to classification and the quantity of Side chain protective group in sequence.The polypeptide compound that cracking obtains is precipitated and is obtained by the ice ether.
(3) purified polypeptide dimer dry
Use reverse-phase chromatography or ion exchange chromatography to carry out purifying the gained polypeptide dimer, preferably use reverse-phase chromatography.To after the polypeptide dimer vacuum lyophilization after purifying, store.
Useful especially is the polypeptide of the present invention of modifying in order to obtain intramolecular disulfide bond, can lead to snperoxiaized method and reach.Oxidising agent is selected from DMSO, potassium cyanide, hydrogen peroxide, iodine etc.Preferably adopt DMSO as oxidising agent.It is reported, it tends to form intramolecular disulfide bond, thereby can obtain the polypeptide of the present invention that highly purified intramolecular disulfide bond is modified.The concentration of DMSO is 10-40%, preferably 20-30%.
Useful especially is the polypeptide of the present invention of modifying in order to obtain PEG, can select the PEG reagent of being modified for amino to be modified.As methoxy poly (ethylene glycol) propionic acid NHS ester (mPEG-SPA-NHS, Beijing key is triumphant), it is DMF that PEG modifies the reaction system adopted, and it is 1: 1.2～1.5 that the mol ratio of polypeptide compound and PEG is controlled, and reaction density can be determined flexibly according to the molecular weight of polypeptide and PEG used.That the product that PEG modifies can be used is anti-phase, macroporous absorption or ion exchange resin carry out purifying, preferably uses reverse-phase chromatography.To after the polypeptide compound vacuum lyophilization after purifying, store.
In another aspect of this invention, the invention provides the pharmaceutical composition of polypeptide compound as activeconstituents, it also can comprise pharmaceutically acceptable appropriate excipients, and those skilled in the art can be selected from suitable vehicle according to route of administration.Pharmaceutical composition of the present invention can give in any suitable manner, comprise parenteral, intravenously, intramuscular, intraperitoneal, subcutaneous, through skin, direct infusion etc.Polypeptide compound of the present invention preferably exists with the form of freeze-dried powder, can be before administration with suitable dosage with give needed experimenter after suitable isotonic solution mixes.Dosage and frequency will depend on age, sex and patient's the patient's condition, the other drug simultaneously given, taboo and other parameters of being considered by the clinician etc., for example dosage can be 1ug/kg-80mg/kg, administration frequency can be every day 1 time, 1 time or 1 inferior per month weekly, dosage and administration frequency be restriction especially not, and those skilled in the art can consider above-mentioned factor and select suitably.
Another aspect of the present invention provides described polypeptide compound to produce deficiency, defect for erythrocyte or has consumed the purposes of relevant disease excessively.For example, polypeptide compound of the present invention can be used for treating anaemia that in renal insufficiency and/or the latter stage renal failure/anaemia that dialysis causes, the anaemia after renal transplantation, oncotherapy, chemicotherapy causes, the anaemia relevant to acquired immune deficiency syndrome (AIDS), the anaemia for example, with chronic inflammatory disease (, rheumatoid arthritis and chronic intestinal inflammations) relevant and for the front increase patient's that performs the operation RBC number.Polypeptide compound of the present invention can also be for β-thalassemia, cystic fibrosis, gestation and menopause illness, the early stage anaemia of prematureness, Spinal injury, space flight, acute bleeding, aging, apoplexy, local asphyxia (CNS and heart) and the kinds of tumors patient's condition of following abnormal erythrocyte to generate etc.Preferably, polypeptide compound of the present invention is specially adapted to the anaemia that in renal insufficiency and/or the latter stage renal failure/anaemia that dialysis causes, the anaemia after renal transplantation, oncotherapy, chemicotherapy causes.
That erythrocyte of the present invention produces is not enough, defect or consume excessively relevant disease its jointly and the most directly pathological manifestations be erythrocyte number relatively or definitely not enough and or the reduction of erythrocytic function.Very usefully confirmation is measured and obtained to the activity of polypeptide compound of the present invention by the reticulocyte ratio juris, concrete grammar is as follows: give mouse subcutaneous injection polypeptide compound of the present invention, adopt reticulocyte counts in every mouse blood of the full-automatic reticulocyte analyser counting ratio (Ret%) to the red corpuscle sum.Increment rate with Ret% means the activity that the promoting erythrocyte of laboratory sample generates.
Embodiment
By the following examples, the present invention is described.Yet in the present invention, these and other embodiment are only for illustrating the scope that does not limit the present invention or embodiment.Equally, the invention is not restricted to any concrete preferred embodiment described herein.In fact, while reading this specification sheets, many modifications of the present invention and work-around solution are apparent for those skilled in the art, and modification and work-around solution and do not deviate from right of the present invention and scope under can making.
Embodiment 1 prepares polypeptide dimer 1
(1) material and reagent
Rink Amide mbha resin, substitution value 0.29mmol/g.
Required protected amino acid Fmoc-L-Ala-OH, Fmoc-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-His (Trt)-OH, Fmoc-L-Leu-OH, Fmoc-L-Ile-OH, Fmoc-L-Cys (Trt)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Pro-OH, Fmoc-L-Met-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Lys (Fmoc)-OH and Fmoc-L-Trp-OH.
Reagent: HOBt, DIC, DMF, piperidines, diacetyl oxide, pyridine
(2) instrument
PSI300 type Peptide synthesizer, Waters high performance liquid chromatograph, magnetic stirring apparatus
(3) operation steps
Take 0.15mmol as example:
A. take Rink Amide mbha resin 0.52g, be placed in the reactor of Peptide synthesizer, add 15mLDMF, soak 2h.
B. deresinate amino protecting group
Add 20%PIP (DMF) solution 15mL, mix 30min, use DMF washing resin 7 times.
C. linked reaction
In mixing reactor, add 211mg Fmoc-L-Lys (Boc)-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and each 2.5ml of 0.2mol/L DIC to be reacted, temperature of reaction is room temperature, with ninhydrin reaction detection reaction process, guarantee that Lys is coupled on resin, use DMF washing resin 7 times.
D. the prolongation of peptide chain
After Lys is connected on resin; add 20%PIP (DMF) solution 15mL; mix 30min; remove the alpha-amino group protective material Fmoc of Lys, use DMF washing resin 7 times, add 134mg Fmoc-Gly-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and each 2.5ml of 0.2mol/L DIC to be reacted; temperature of reaction is room temperature; with ninhydrin reaction detection reaction process, guarantee that Gly is coupled on resin, use DMF washing resin 7 times.
After Gly is connected on resin; add 20%PIP (DMF) solution 15mL; mix 30min; remove the alpha-amino group protective material Fmoc of Gly, use DMF washing resin 7 times, add 266mg Fmoc-L-Lys (Fmoc)-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and each 2.5ml of 0.2mol/L DIC to be reacted; temperature of reaction is room temperature; with ninhydrin reaction detection reaction process, guarantee that Gly is coupled on resin, use DMF washing resin 7 times.
After connection amino acid Lys is connected on resin; add 20%PIP (DMF) solution 30mL; mix 40min; remove alpha-amino group and the epsilon-amino protecting group Fmoc of Lys simultaneously, use DMF washing resin 7 times, add 268mg Fmoc-Gly-OH (0.90mmol), coupling reagent 0.2mol/L HOBt and each 5.0ml of 0.2mol/L DIC to be reacted; temperature of reaction is room temperature; with ninhydrin reaction detection reaction process, guarantee that Gly is coupled on resin, use DMF washing resin 7 times.Then by identical step, proceed First ray and follow-up amino acid whose synthesizing of the second sequence, until all amino acid of First ray and the second sequence all are linked on resin.
E. acetylation modification
After the peptide sequence end of synthesis,, add 5ml piperidines and 5ml acetic anhydride 30 minutes, remove the alpha-amino group protecting group Fmoc of First ray and the second sequence.
F. cracking and precipitation
After acetylize finishes, the vacuum-drying resin, weigh.In the ratio of 1g resin 10mL lytic reagent add lytic reagent (the reagent proportioning: TFA/ thioanisole/tri isopropyl silane/phenol/water/TIS=85/5/5/4/1), stirring at room reaction 3 hours, suction filtration.In cracking suction filtration liquid, add the ice ether, the precipitation polypeptide, centrifugal, abandon supernatant, vacuum-drying, the thick peptide of weighing.
G. oxidation forms disulfide linkage
Concentration with 5mg/ml is dissolved in thick peptide in DMSO, then adds the water of 4 times of volumes.(>=10 ℃) static 36h under room temperature.
H. reverse-phase chromatography purifying
Use preparation HPLC, adopt reverse-phase chromatography, the above-mentioned thick peptide of purifying.
HPLC post: C18 preparative column
Flow velocity: 10mL/min
A: containing the aqueous solution of 0.1%TFA
B: containing the acetonitrile solution of 0.1%TFA
With the B phase of 18-38%, wash-out 90min.
Purified product is homogeneous, and its purity is 95%, and total yield is 18%.MS：m/z＝4693.3[M+H]。
Embodiment 2 determines that the DMSO oxidation forms the reaction times of disulfide linkage
According to the method for embodiment 1, similar synthetic polypeptide dimer 6, the protected amino acid wherein added in addition is Fmoc-L-Asp (Otbu)-OH and Fmoc-L-Glu (Otbu)-OH.
(1) material and reagent
Thick peptide before polypeptide dimer 6 oxidations, DMSO, water, acetonitrile, TFA
(2) instrument
Waters high performance liquid chromatograph, 25 ℃ of thermostat containers.
(3) method for oxidation
Take the front thick peptide sample of 9 parts of polypeptide dimer 6 oxidations, every part of about 2mg.Respectively add 1ml 20%DMSO solution, be placed in 25 ℃ of thermostat containers, 0h is answered in negate, 1h, and 2h, 4h, 8h, 12h, 24h, 30h, the sample of 36h carries out liquid phase analysis.
(4) analytical procedure
Oxidising process and the trend of application RP-HPLC sample, adopt chromatographic column-Boston PHlex ODS C18 (5 μ m) 4.6 * 250mm, and moving phase (A) is the 0.05%TFA aqueous solution, is (B) the 0.05%TFA acetonitrile solution.Elution requirement is B phase gradient from 20% to 30% wash-out 10min, and 30% to 45% wash-out 25min detects wavelength 215nm.
(5) oxidation results:
Sample main peak before oxidizing reaction is 26.5min, and after oxidizing reaction, the sample main peak is 18.7min, considers the impact of impurity peaks and baseline noise, can think, 36h reacts completely.
*(sample concentration all being scaled to 2mg/ml)
Embodiment 3 prepares polypeptide dimer 9
(1) material and reagent
Rink Amide mbha resin, substitution value 0.29mmol/g.
Required protected amino acid Fmoc-L-Ala-OH, Fmoc-Gly-OH, Fmoc-L-Arg (Pbf)-OH, Fmoc-L-Gln (Trt)-OH, Fmoc-L-His (Trt)-OH, Fmoc-L-Leu-OH, Fmoc-L-Ile-OH, Fmoc-L-Cys (Trt)-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Pro-OH, Fmoc-L-Met-OH, Fmoc-L-Thr (tBu)-OH, Fmoc-L-Tyr (tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Lys (Alloc)-OH and Fmoc-L-Trp-OH.
Reagent: HOBt, DIC, DMF, piperidines, diacetyl oxide, pyridine, chloroform, Pd (PPh
3)
4
(2) instrument
PSI300 type Peptide synthesizer, Waters high performance liquid chromatograph, magnetic stirring apparatus
(3) operation steps
Take 0.15mmol as example:
A. take Rink Amide mbha resin 0.52g, be placed in the reactor of Peptide synthesizer, add 15mLDMF, soak 2h.
B. deresinate amino protecting group
Add 20%PIP (DMF) solution 15mL, mix 30min, use DMF washing resin 7 times.
C. linked reaction
In mixing reactor, add 211mg Fmoc-L-Lys (Boc)-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and each 2.5ml of 0.2mol/L DIC to be reacted, temperature of reaction is room temperature, with ninhydrin reaction detection reaction process, guarantee that Lys is coupled on resin, use DMF washing resin 7 times.
D. the prolongation of peptide chain
After Lys is connected on resin; add 20%PIP (DMF) solution 15mL; mix 30min; remove the alpha-amino group protective material Fmoc of Lys, use DMF washing resin 7 times, add 134mg Fmoc-Gly-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and each 2.5ml of 0.2mol/L DIC to be reacted; temperature of reaction is room temperature; with ninhydrin reaction detection reaction process, guarantee that Gly is coupled on resin, use DMF washing resin 7 times.
After Gly is connected on resin; add 20%PIP (DMF) solution 15mL; mix 30min; remove the alpha-amino group protective material Fmoc of Gly, use DMF washing resin 7 times, add 204mg Fmoc-L-Lys (Alloc)-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and each 2.5ml of 0.2mol/L DIC to be reacted; temperature of reaction is room temperature; with ninhydrin reaction detection reaction process, guarantee that Gly is coupled on resin, use DMF washing resin 7 times.
After connection amino acid Lys is connected on resin; add 20%PIP (DMF) solution 15mL; mix 30min; remove the alpha-amino group protecting group Fmoc of Lys, use DMF washing resin 7 times, add 134mg Fmoc-Gly-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and each 2.5ml of 0.2mol/L DIC to be reacted; temperature of reaction is room temperature; with ninhydrin reaction detection reaction process, guarantee that Gly is coupled on resin, use DMF washing resin 7 times.Then by identical step, proceed First ray follow-up amino acid whose synthetic, until all amino acid of First ray all are connected on resin.
Then add 10%Pd (PPh
3)
4(chloroform) 10mL; mix 45min; remove epsilon-amino protecting group Alloc; with DMF washing resin 7 times; add 134mg Fmoc-Gly-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and each 5.0ml of 0.2mol/LDIC to be reacted, temperature of reaction is room temperature, with ninhydrin reaction detection reaction process; guarantee that Gly is coupled on resin, use DMF washing resin 7 times.Then by identical step, proceed the second sequence follow-up amino acid whose synthetic, until all amino acid of the second sequence all are connected on resin.
E. acetylation modification
After the peptide sequence end of synthesis, add 5ml piperidines and 5ml acetic anhydride 30 minutes, remove the alpha-amino group protecting group Fmoc of First ray and the second sequence.
F. cracking and precipitation
After acetylize finishes, the vacuum-drying resin, weigh.In the ratio of 1g resin 10mL lytic reagent add lytic reagent (the reagent proportioning: TFA/ thioanisole/tri isopropyl silane/phenol/water/TIS=85/5/5/4/1), stirring at room reaction 3 hours, suction filtration.In cracking suction filtration liquid, add the ice ether, the precipitation polypeptide, centrifugal, abandon supernatant, vacuum-drying, the thick peptide of weighing.
G. oxidation forms disulfide linkage
Concentration with 5mg/ml is dissolved in thick peptide in DMSO, then adds the water of 4 times of volumes.(>=10 ℃) static 36h under room temperature.
H. reverse-phase chromatography purifying
Use preparation HPLC, adopt reverse-phase chromatography, the above-mentioned thick peptide of purifying.
HPLC post: C18 preparative column
Flow velocity: 10mL/min
A: containing the aqueous solution of 0.1%TFA
B: containing the acetonitrile solution of 0.1%TFA
With the B phase of 18-38%, wash-out 90min.
Purified product is homogeneous, and its purity is 96%, and total yield is 16%.MS：m/z＝4706.2[M+H]。
The PEG of embodiment 4 polypeptide dimers 1 (molecular weight is about 20K) modifies
The compound (being polypeptide dimer 1) of embodiment 1 is carried out to PEG (molecular weight is about 20K) and modify, investigate respectively the impact on modification reaction of temperature, time, concentration.
(1) material and reagent
DMF, DIEA, methoxy poly (ethylene glycol) propionic acid NHS ester (about 20k, Jiankai Science and Technology Co., Ltd., Beijing).
(2) instrument
Waters high performance liquid chromatograph, 28 ℃ of thermostat containers, 37 ℃ of thermostat containers.
(3) operating process
A. take respectively polypeptide and the PEG of certain mass according to 1: 1.5 (mol ratio), be dissolved in DMF, obtain settled solution.
B. polypeptide and PEG add 10 μ l DIEA after dissolving fully, every the 30min sample introduction, analyze.Investigate respectively the impact on modification reaction of temperature, time, concentration, result means with modification rate.
(4) experimental result
A. working as temperature of reaction is 28 ℃, and when reaction density is 3mg/ml, result is as follows:
B. working as temperature of reaction is 28 ℃, and when reaction density is 2mg/ml, result is as follows:
C. working as temperature of reaction is 28 ℃, and when reaction density is 1mg/ml, result is as follows:
D. working as temperature of reaction is 37 ℃, and when reaction density is 2mg/ml, result is as follows:
E. working as temperature of reaction is 4 ℃, and when reaction density is 2mg/ml, result is as follows:
As can be seen from the above results, at the present embodiment in selected condition and range, modification reaction is proportionate with temperature, concentration with while reacting.When temperature is 28 ℃, when concentration is 2mg/ml or 3mg/ml, within 2 hours, react completely.When temperature is 37 ℃, when concentration is 2mg/ml, within 1.5 hours, react completely.
Can similarly carry out temperature, reaction density and reaction times to PEG (about 5k or about 40k) the dimeric impact of modified polypeptide.
Embodiment 5 adopts the PEG that molecular weight is about 20k to be modified polypeptide dimer 1
(1) material and reagent.
Methoxy poly (ethylene glycol) propionic acid NHS ester (about 20k, mPEG-SPA-NHS, Jiankai Science and Technology Co., Ltd., Beijing), DMF, DIEA, wherein mPEG-SPA-NHS (about 20k) is straight chain type, its structure is as follows:
(2) instrument
Waters high performance liquid chromatograph, ultra-fine filter, thermostat container
(3) operation steps
A. take the mPEG-SPA-NHS 114mg of about 20k, be dissolved in 5.7ml DMF, make reaction mother liquor A
B. take the about 6.0mg of polypeptide compound, add mother liquor A 2ml.Add 10ul DIEA after 5min.Be placed in 28 ℃ of thermostat containers.At interval of sampling half an hour, use HPLC monitoring reaction process.
C.2 hour, after, react substantially complete.Use the pure water diluting reaction solution of 10 times of volumes, then use the reverse-phase chromatography purifying to modify after product.
The reverse-phase chromatography condition is as follows:
HPLC post: YMC-Pack C4 (10 μ m) 10 * 250mm
Flow velocity: 2mL/min
A: containing the aqueous solution of 0.1%TFA
B: containing the acetonitrile solution of 0.1%TFA
Reaction soln after dilution, by the C4 preparative column, is then used mobile phase A/B (95/5) to rinse 10 column volumes, and then with mobile phase A/B (30/70), target product separated, obtain the collection liquid containing target product.
D. TFA and the acetonitrile of collecting in liquid removed in underpressure distillation, and remaining DMF is removed in the ultra-filtration membrane ultrafiltration that to re-use molecular weight cut-off be 3000.
E. the target product obtained is carried out to freeze-drying, after freeze-drying, compound purity is 99.14%.By the MALDI-TOF determining molecular weight.The molecular weight of target product should be centered by 24692, becomes within the specific limits normal distribution.Theoretical value conforms to the mass spectrum result.
Embodiment 6 adopts the PEG that molecular weight is about 40k to be modified polypeptide dimer 1
(1) material and reagent.
Methoxy poly (ethylene glycol) propionic acid NHS ester (about 40k, mPEG-SPA-NHS, Jiankai Science and Technology Co., Ltd., Beijing), DMF, DIEA, wherein mPEG-SPA-NHS (about 40k) is branched chain type, and the molecular weight of every side chain is about 20k, and its structure is as follows:
Instrument and operation steps be with reference to embodiment 5, but the reaction times in operation steps c is 2.5 hours.
The target product obtained is carried out to freeze-drying, and after freeze-drying, compound purity is 99.96%.Verify molecular weight by MALDI-TOF.The molecular weight of target product should be centered by 44692, becomes within the specific limits normal distribution, and theoretical value conforms to the mass spectrum result.
Embodiment 7 adopts the PEG that molecular weight is about 5k to be modified polypeptide dimer 1
(1) material and reagent.
Methoxy poly (ethylene glycol) propionic acid NHS ester (about 5k, mPEG-SPA-NHS, Jiankai Science and Technology Co., Ltd., Beijing), DMF, DIEA, wherein mPEG-SPA-NHS (about 5k) is straight chain type, its structure is as follows:
Instrument and operation steps be with reference to embodiment 5, but the reaction times in operation steps c is 1.5 hours.
The target product obtained is carried out to freeze-drying, and after freeze-drying, compound purity is 99.96%.Verify molecular weight by MALDI-TOF.The molecular weight of target product should be centered by 9692, becomes within the specific limits normal distribution, and theoretical value conforms to the mass spectrum result.
The activity in vivo of embodiment 8 polypeptide compounds of the present invention is measured
(1) reagent
A. EDTA-2K antithrombotics
Take EDTA-2K 100mg, add physiological sodium chloride solution 10ml and dissolve, mix, fresh preparation before using.
B. diluent
Take the 0.1g bovine serum albumin, add physiological sodium chloride solution and dissolve and be diluted to 100ml.
(2) step
Choosing the suburbs is 6-8 week female BALB/c mouse in age (the about 16-18g of body weight, derive from Test Animal Centre, Academy of Military Medical Sciences, P.L.A) 68, is divided into 17 groups, 4 of every treated animals.Wherein negative control is 1 group, gives the diluent of 0.2ml; Wherein positive control is 3 groups, gives Recombinant Human Erythropoietin injection liquid (Beijing Sihuan Biopharmaceutical Co., Ltd., 3000IU/0.6ml/ props up), and dosage is respectively 10,20, the 40IU/ mouse; 13 groups of compound groups of the present invention, give respectively the freeze-drying sample of compound of compound, the embodiment 7 of compound, the embodiment 6 of polypeptide dimer 1, polypeptide dimer 2, polypeptide dimer 3, polypeptide dimer 4, polypeptide dimer 5, polypeptide dimer 6, polypeptide dimer 7, polypeptide dimer 8, polypeptide dimer 9, polypeptide dimer 10, embodiment 5, dosage is 20 μ g/ mouse.
Within the 3rd day, rise after injection from mouse orbit blood sampling 3-4 and drip, be placed in and add in advance 200 μ l EDTA-K
2In the heparin tube of antithrombotics.For negative control, getting the blood number of days is the 3rd, 4,5,6,7,8,11,22,28 days; For positive control and polypeptide dimer 1-10, getting the blood number of days is the 3rd, 4,5,6,7 days; For the compound of embodiment 5,6,7, the number of days of getting blood is the 4th, 5,8,11,22,28 days.Get anticoagulation, knit erythrocyte analyser (XT-2000IV) with fully automatic network and count reticulocyte in every mouse blood ratio (Ret%) to the red corpuscle sum.
The erythropoietic activity that means polypeptide compound of the present invention with the increment rate of Ret%.
Wherein sample comprises negative control, positive control and compound of the present invention.The result of polypeptide dimer 1-10 is as shown in table 3, and the result of the compound of embodiment 5-7 is as shown in table 4:
Table 3
Annotate:---mean not survey.
Table 4
After compound of the present invention is modified by PEG, active with modify before do not compare and descend, but action time significant prolongation.
Claims (10)
1. a polypeptide compound, it consists of two peptide sequences, and wherein First ray is:
X1-G-X2-Y-X3-C-X4-M-G-P-X5-T-W-X6-C-Q-P-L-X7-G-X8-X9; The second sequence is: X10-G-X11-Y-X12-C-X13-M-G-P-X14-X15-W-X16-C-X17-X18-X19-X20-G; And First ray and the second sequence are connected to form dimer by the connection amino acid X8 in First ray, and wherein X1 is selected from G or AcG; X2, X5 and X6 are respectively independently selected from L, I or V; X3 is selected from A or G; X4 is selected from H or D-His; X7 is selected from R, HomoArg or Pal-Lys; X8 is selected from Lys or D-Lys, participates in First ray with alpha-amino group in its structure synthetic, with the C-terminal amino acid of epsilon-amino in its structure and the second sequence, is connected to form dimer; X9 is the small peptide that Methionin or the C-terminal that is comprised of 2-10 amino acid are Methionin; X10 is selected from G or AcG; X11, X14, X16 or X19 are respectively independently selected from L, I, V or N1e; X12 is selected from A, Aib or G; X13 is selected from H or D-His; X15 is selected from T or S; X17 is selected from Q or N; X18 is selected from P or Hyp; X20 is selected from R, HomoArg, Cit or Pal-Lys.
2. the polypeptide compound of claim 1, wherein X9 is selected from GK, GGK, GGARRAGK, GGAGAGK, GADEAGGKK or GADEGGAK.
3. claim 1 or 2 polypeptide compound, wherein First ray is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 or SEQ ID NO:36, the second sequence is selected from SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45 or SEQ ID NO:46.
4. the polypeptide compound of any one in claim 1-3, wherein First ray and the second sequence carry out the N-terminal acetylize and form intramolecular disulfide bond modifying, and optionally in First ray, carry out C-terminal amidation modification.
5. the polypeptide compound of any one in claim 1-4, it is polypeptide dimer 1, polypeptide dimer 2, polypeptide dimer 3, polypeptide dimer 4, polypeptide dimer 5, polypeptide dimer 6, polypeptide dimer 7, polypeptide dimer 8, polypeptide dimer 9, polypeptide dimer 10, polypeptide dimer 11, polypeptide dimer 12, polypeptide dimer 13, polypeptide dimer 14, polypeptide dimer 15, polypeptide dimer 16, polypeptide dimer 17, polypeptide dimer 18, polypeptide dimer 19, polypeptide dimer 20, polypeptide dimer 21, polypeptide dimer 22 or polypeptide dimer 23.
6. the polypeptide compound of any one in claim 1-5, it further comprises polyalkylene glycol moiety, wherein in poly-di-alcohol and First ray, C holds the Methionin in X9 to put together.
7. the polypeptide compound of claim 6, the PEG that wherein polyoxyethylene glycol is straight chain or branch, its molecular weight is about 60,000 dalton of 5,000 dalton-Yue.
8. the method for the preparation of polypeptide compound, it comprises the steps:
(1) solid phase synthesis First ray and the second sequence;
(2) cracking polypeptide dimer from resin;
(3) purified polypeptide dimer dry;
(4) optionally carry out modification claimed in claim 4 purifying; With
(5) optional and PEG puts together also purifying.
9. a pharmaceutical composition, it comprises polypeptide compound and the pharmaceutically acceptable carrier of claim 1-7.
10. the purposes of the polypeptide compound of claim 1-7 in preparation treatment and erythrocyte produce deficiency, defect or consume the medicine of relevant disease excessively.
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| CN106554394A (en) * | 2015-09-30 | 2017-04-05 | 天津药物研究院有限公司 | A kind of Erythropoietin mimetic peptide and its preparation method and application |
| US9670261B2 (en) | 2012-12-21 | 2017-06-06 | Sanofi | Functionalized exendin-4 derivatives |
| US9694053B2 (en) | 2013-12-13 | 2017-07-04 | Sanofi | Dual GLP-1/glucagon receptor agonists |
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| US9932381B2 (en) | 2014-06-18 | 2018-04-03 | Sanofi | Exendin-4 derivatives as selective glucagon receptor agonists |
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| US10758592B2 (en) | 2012-10-09 | 2020-09-01 | Sanofi | Exendin-4 derivatives as dual GLP1/glucagon agonists |
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| CN1823088A (en) * | 2003-05-12 | 2006-08-23 | 阿费麦克斯公司 | Novel peptides that bind to the erythropoietin receptor |
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| US9670261B2 (en) | 2012-12-21 | 2017-06-06 | Sanofi | Functionalized exendin-4 derivatives |
| US9745360B2 (en) | 2012-12-21 | 2017-08-29 | Sanofi | Dual GLP1/GIP or trigonal GLP1/GIP/glucagon agonists |
| US10253079B2 (en) | 2012-12-21 | 2019-04-09 | Sanofi | Functionalized Exendin-4 derivatives |
| US9789165B2 (en) | 2013-12-13 | 2017-10-17 | Sanofi | Exendin-4 peptide analogues as dual GLP-1/GIP receptor agonists |
| US9694053B2 (en) | 2013-12-13 | 2017-07-04 | Sanofi | Dual GLP-1/glucagon receptor agonists |
| US9751926B2 (en) | 2013-12-13 | 2017-09-05 | Sanofi | Dual GLP-1/GIP receptor agonists |
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| US9771406B2 (en) | 2014-04-07 | 2017-09-26 | Sanofi | Peptidic dual GLP-1/glucagon receptor agonists derived from exendin-4 |
| US9775904B2 (en) | 2014-04-07 | 2017-10-03 | Sanofi | Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists |
| WO2015155140A1 (en) * | 2014-04-07 | 2015-10-15 | Sanofi | Dual glp-1 / glucagon receptor agonists derived from exendin-4 |
| US9758561B2 (en) | 2014-04-07 | 2017-09-12 | Sanofi | Dual GLP-1/glucagon receptor agonists derived from exendin-4 |
| US9932381B2 (en) | 2014-06-18 | 2018-04-03 | Sanofi | Exendin-4 derivatives as selective glucagon receptor agonists |
| US10806797B2 (en) | 2015-06-05 | 2020-10-20 | Sanofi | Prodrugs comprising an GLP-1/glucagon dual agonist linker hyaluronic acid conjugate |
| US9982029B2 (en) | 2015-07-10 | 2018-05-29 | Sanofi | Exendin-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists |
| CN106554394B (en) * | 2015-09-30 | 2019-08-16 | 天津药物研究院有限公司 | A kind of Erythropoietin mimetic peptide and its preparation method and application |
| CN106554394A (en) * | 2015-09-30 | 2017-04-05 | 天津药物研究院有限公司 | A kind of Erythropoietin mimetic peptide and its preparation method and application |
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