CN103421094A - Polypeptide compound with EPO-like activity - Google Patents

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

A kind of polypeptide compound with EPO similar activity

field of invention

The invention belongs to the field of biochemistry, and in particular relates to a polypeptide compound with EPO-like activity. and the use of the compound in the treatment of diseases associated with insufficient or defective production of red blood cells.

Background of the invention

Erythropoietin (EPO) is a glycoprotein hormone of 165 amino acids with a molecular weight of approximately 34 kDa. The identification, cloning and expression of the gene encoding erythropoietin has been described in U.S. Patent 4,703,008 to Lin, the specification of which is incorporated by reference in its integrated into this article as a whole.

EPO is usually produced in the kidney (90%) and liver, enters the peripheral blood circulation after secretion, and is mainly metabolized in the liver, only a small amount is excreted in the urine. It is the main factor for promoting the growth, proliferation, differentiation and maturation of bone marrow erythroid progenitor cells stimulating factor. The biological effect of EPO is mediated through the specific EPO receptor (EPOR) located on the surface of bone marrow erythroid progenitor cells. EPO and EPOR combine to form dimers, and then regulate the proliferation and differentiation of red blood cells through signal transduction pathways. There are many pathways in the EPO signal transduction process, among which the EPOR-JAK2-STAT5 pathway is more thoroughly studied, and other proven signal transduction mechanisms include: EPOR-JAK2-PI3K pathway, EPOR-JAK2-ERKs pathway, EPOR-JAK2 -NF-KappaB pathway, EPOR-JAK2-Ras protein-MAPK pathway, etc. It can be seen that EPO intracellular signal transduction is not a single cascading pathway, but a complex transduction process in which multiple pathways are cross-linked, complementary, and form a network.

For cancer patients with chronic renal failure and advanced stage of radiotherapy and chemotherapy, their normal hematopoietic function is inhibited, leading to absolute or relative deficiency of EPO, often accompanied by anemia. At present, recombinant human EPO injection is often used clinically to improve the anemia of patients, and remarkable results have been achieved. However, there are still the following problems in practical application: it often causes cardiovascular complications, such as hypertension, myocardial infarction, etc.; it promotes the development of some solid tumors; pure red blood cell aplasia occurs; Inject 1-3 times a week, etc. Therefore, there is an urgent need to develop new drugs for the treatment of anemia. Chinese patent ZL94109128.7 discloses a highly glycosylated recombinant erythropoiesis-stimulating protein. Clinical studies have shown that the half-life in vivo is significantly longer than that of recombinant human EPO. Chinese patent ZL00809895.6 discloses an erythropoietin and poly Ethylene glycol conjugates, injected once a month on average, can be equivalent to injecting recombinant human EPO 1-3 times a week. However, drugs with this type of mechanism will cross-react with EPO antibodies in vivo, reducing the efficacy of this type of drug. Through a lot of experiments, the inventor unexpectedly discovered a compound completely unrelated to the sequence and structure of EPO, which has significant EPO-like activity and will not cross-react with EPO antibodies, and can be used for the treatment of Insufficient or defect-related diseases, thus completing the present invention.

Invention content:

The present invention relates to a polypeptide compound, which is composed of two polypeptide sequences, wherein the 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; 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 the first sequence and the The two sequences are joined by a linking amino acid (X8) in the first sequence to form a dimer.

The polypeptide compound of the present invention binds to the EPO receptor and exerts a receptor agonizing effect, thereby treating diseases related to insufficient, defective or excessive consumption of red blood cells.

The amino acids used in the present invention include not only twenty common amino acids well known to those skilled in the art, but also some uncommon amino acids. The names and abbreviations of common amino acids are summarized in Table 1; uncommon amino acids, names, abbreviations, and their structures are plotted in Table 2.

Table 1 The names and abbreviations of common amino acids

Table 2 Names, abbreviations and structures of uncommon amino acids

The amino acids in Table 1 and Table 2 used in the present invention are all L-type amino acids unless the D or L configuration is particularly limited; unless otherwise specified, all amino acids are α-amino acids. Unless otherwise specified, the directions of all the sequences in the present invention are from N-terminus to C-terminus from left to right.

The present invention firstly provides a polypeptide compound consisting of two polypeptide sequences, wherein the 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; 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 the first sequence and the second sequence are connected to form a dimer by connecting amino acids (X8) in the first sequence; X1 is selected from G or AcG; X2, X5 and X6 are 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, and the α-amino group in its structure participates in the first sequence synthesis, and the ε-amino group in its structure Link with the C-terminal amino acid of the second sequence to form a dimer, preferably using Lys; X9 is lysine or a short peptide consisting of 2-10 amino acids whose C-terminus is lysine, more preferably, X9 consists of 3- Composed of 7 amino acids, particularly preferably, X9 is selected from GK, GGK, GGARRAGK, GGAGAGK, GADEAGGKK or GADEGGAK; X10 is selected from G or AcG; X11, X14, X16 or X19 are 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, the first sequence of the present invention includes but not limited to the 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 the 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

The polypeptide compound of the present invention can be modified, such as N-terminal acetylation, intramolecular disulfide bond formation, C-terminal amidation, and the like. Preferably, N-terminal acetylation and intramolecular disulfide bond formation can occur simultaneously in the first sequence and the second sequence, and optionally C-terminal amidation occurs in the first sequence. The intramolecular disulfide bond described herein refers to the disulfide bond formed by the 6th and 15th cysteines in the first sequence and the disulfide bond formed by the 6th and 15th cysteines in the second sequence. In a specific embodiment of the present invention, the amino acid sequence of the first sequence is selected from SEQ ID NO: 1, the 6th and 15th cysteines form an intramolecular disulfide bond, and the C-terminus is amidated; the amino acid sequence of the second sequence The amino acid sequence is selected from SEQ ID NO: 37, the 6th and 15th cysteines form intramolecular disulfide bonds.

In one embodiment of the present invention, the amino acid sequence of the first sequence of the polypeptide compound is selected from SEQ ID NO: 1, the 6th and 15th cysteines form an intramolecular disulfide bond, and the C-terminus is amidated; the second The amino acid sequence of the sequence is selected from SEQ ID NO: 37, the 6th and 15th cysteines form an intramolecular disulfide bond, and form a dimer through the 21st L-lysine in the first sequence, hereinafter referred to as It is polypeptide dimer 1, the structure is as follows:

In another embodiment of the present invention, the amino acid sequence of the first sequence of the polypeptide compound is selected from SEQ ID NO: 2, the 6th and 15th cysteines form an intramolecular disulfide bond, and the C-terminus is amidated; The amino acid sequence of the second sequence is selected from SEQ ID NO: 37, the 6th and 15th cysteines form an intramolecular disulfide bond, and form a dimer through the 21st L-lysine in the first sequence, below It is referred to as polypeptide dimer 2 for short.

In another embodiment of the present invention, the amino acid sequence of the first sequence of the polypeptide compound is selected from SEQ ID NO: 3, the 6th and 15th cysteines form an intramolecular disulfide bond, and the C-terminus is amidated; The amino acid sequence of the second sequence is selected from SEQ ID NO: 37, the 6th and 15th cysteines form an intramolecular disulfide bond, and form a dimer through the 21st L-lysine in the first sequence, below It is referred to as polypeptide dimer 3 for short.

In another embodiment of the present invention, the amino acid sequence of the first sequence of the polypeptide compound is selected from SEQ ID NO: 4, the 6th and 15th cysteines form an intramolecular disulfide bond, and the C-terminus is amidated; The amino acid sequence of the second sequence is selected from SEQ ID NO: 37, the 6th and 15th cysteines form an intramolecular disulfide bond, and form a dimer through the 21st L-lysine in the first sequence, below Abbreviated as polypeptide dimer 4.

In yet another embodiment of the present invention, the amino acid sequence of the first sequence of the polypeptide compound is selected from SEQ ID NO: 5, the 6th and 15th cysteines form an intramolecular disulfide bond, and the C-terminus is amidated; The amino acid sequence of the second sequence is selected from SEQ ID NO: 37, the 6th and 15th cysteines form an intramolecular disulfide bond, and form a dimer through the 21st L-lysine in the first sequence, below Abbreviated as polypeptide dimer 5.

In yet another embodiment of the present invention, the amino acid sequence of the first sequence of the polypeptide compound is selected from SEQ ID NO: 6, the 6th and 15th cysteines form an intramolecular disulfide bond, and the C-terminus is amidated; The amino acid sequence of the second sequence is selected from SEQ ID NO: 37, the 6th and 15th cysteines form an intramolecular disulfide bond, and form a dimer through the 21st L-lysine in the first sequence, below Abbreviated as polypeptide dimer 6.

In yet another embodiment of the present invention, the amino acid sequence of the first sequence of the polypeptide compound is selected from SEQ ID NO: 26, the 6th and 15th cysteines form an intramolecular disulfide bond, and the C-terminus is amidated; The amino acid sequence of the second sequence is selected from SEQ ID NO: 41, the 6th and 15th cysteines form an intramolecular disulfide bond, and form a dimer through the 21st L-lysine in the first sequence, below Abbreviated as polypeptide dimer 7.

In yet another embodiment of the present invention, the amino acid sequence of the first sequence of the polypeptide compound is selected from SEQ ID NO: 32, the 6th and 15th cysteines form an intramolecular disulfide bond, and the C-terminus is amidated; The amino acid sequence of the second sequence is selected from SEQ ID NO: 42, the 6th and 15th cysteines form an intramolecular disulfide bond, and form a dimer through the 21st D-lysine in the first sequence, below Abbreviated as polypeptide dimer 8.

The polypeptide compound of the present invention is also preferably a dimer composed of a combination of the first sequence and the second sequence shown in the following table, optionally amidated at the C-terminus of the first sequence of the polypeptide dimer:

The polypeptide compound of the present invention can also include a polyethylene glycol (PEG) moiety, and polyethylene glycol can be conjugated to the lysine in the C-terminal X9 of the first sequence, thereby forming a polypeptide compound conjugated with PEG. wherein the PEG has a molecular weight of from about 5,000 to about 60,000 Daltons (the term "about" means that in a preparation of PEG, some molecules will have a molecular weight greater than that stated and some molecules will have a molecular weight less than that stated), which is the same as in paragraph The epsilon-amino group of the C-terminal lysine of a sequence is covalently linked. The activated PEG of the present invention can be connected to the ε-NH ₂ of the terminal lysine of the first sequence through a carbamate bond or an amide bond, thereby performing PEG modification on the polypeptide dimer of the present invention. When the short peptide X9 of the first sequence contains more than two lysines, the polypeptide dimer of the present invention can also be modified with double or multiple PEGs.

The PEG used in the present invention can be a linear or branched PEG with a molecular weight of about 5,000 Daltons (5k) to about 60,000 Daltons (60k), preferably, the PEG has a molecular weight of about 20k to about 40k. The skilled artisan will be able to select an appropriate polymer size based on considerations such as desired dose, circulation time, resistance to proteolysis and other known effects of PEG on therapeutic peptides.

A variety of polyethylene glycol species can be used to PEGylate polypeptide dimers of the invention, for example, including 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 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, Peptide Dimer 17, Peptide Dimer 18, Peptide Dimer 19, Peptide Dimer 20, Peptide Dimer 21, Peptide Dimer 22, Peptide Dimer 23 by carbamic acid An ester bond is attached to the activated PEG.

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. Peptide dimer 8, Peptide dimer 9, Peptide dimer 10, Peptide dimer 11, Peptide dimer 12, Peptide dimer 13, Peptide dimer 14, Peptide dimer 15, Peptide dimer 16, Peptide dimer 17, Peptide dimer 18, Peptide dimer 19, Peptide dimer 20, Peptide dimer 21, Peptide dimer 22, Peptide dimer 23 via amide bond Linked to activated PEG.

In a specific embodiment of the present invention, when polypeptide dimer 1 is covalently linked to activated PEG _20k through a carbamate bond, the structure is as follows:

In a specific embodiment of the present invention, when polypeptide dimer 6 is covalently linked to activated PEG _20k through an amide bond, the structure is as follows:

In a specific embodiment of the present invention, when polypeptide dimer 5 is covalently linked to activated PEG _20k through a carbamate bond, the structure is as follows:

In another aspect, the preparation method of the polypeptide compound of the present invention is a classical solid-phase synthesis method well known in the art, directly preparing the first sequence and the second sequence of the polypeptide dimer of the present invention; using an oxidizing reagent (for example, DMSO) forming said intramolecular disulfide bond; and optionally covalently linking with an activated PEG (eg, mPEG-SPA-NHS) via a carbamate bond or an amide bond, etc. Specifically, the X8 linking amino acid (lysine) in the first sequence uses Fmoc (9-fluorenyl-methylcarbonyl) as the protecting group of α-amino or ε-amino, and Alloc (allyloxypropylcarbonyl) can also be used. ) as the protecting group of the lysine ε-amino group. When Fmoc is used as the protecting group of the α-amino group and ε-amino group of this lysine, the sequence of the first 20 amino acids of the second sequence is preferably the same as that of the first sequence. At this time, the Fmoc can be removed at the same time, thereby carrying out the two steps simultaneously. Solid-phase synthesis of the sequence; when using Fmoc as the protecting group of the α-amino group of the lysine, and using Alloc as the protecting group of the ε-amino group of the lysine, the first sequence and the second sequence of the present invention Synthesize sequentially. During the synthesis, a weakly basic deprotection reagent (such as 25% piperidine/DMF) is used to remove the Fmoc protecting group. At this time, the Alloc protecting group of the ε-amino group of lysine is not affected. After the synthesis of the first sequence is completed, use a suitable reagent (such as Pd(PPh ₃ ) ₄ , ie tetrakistriphenylphosphine palladium) to remove the Alloc protecting group, and then synthesize the second sequence.

Specifically, the steps for preparing the compounds of the present invention are as follows:

(1) Solid-phase synthesis of the first sequence and the second sequence

According to whether the C-terminus of the first sequence needs to be amidated, a suitable resin can be selected for the preparation of the polypeptide compound of the present invention. When the C-terminus of the first sequence is modified by amidation, Rink Amide MBHA resin can be selected; when the C-terminus of the first sequence does not need to be amidated, Wang resin coupled with the C-terminal amino acid can be selected.

The direction of solid-phase synthesis is C-terminal-N-terminal, starting with the C-terminal lysine of the X9 part in the first sequence, extending the sequence to the connecting amino acid (lysine) of the first sequence, and then removing the connection simultaneously or sequentially The α-amino protecting group and ε-amino protecting group of body lysine were used to carry out the continuous synthesis of the first sequence and the synthesis of the second sequence. When removing the α-amino protecting group and ε-amino protecting group of the linker amino acid at the same time, the steps are as follows: soak the resin, remove the amino protecting group of the resin, wash and monitor, and couple the first part of the X9 part of the first sequence. Amino acid (i.e. lysine for linking PEG), wash and monitor, remove the α-amino protecting group and sequentially couple other amino acids of the X9 moiety, remove the α-amino protecting group and ε-amino of the linker amino acid The protecting group is used to continue the synthesis of the remaining amino acids of the first sequence and the synthesis of the amino acid sequence of the second sequence, and optionally perform acetylation modification on the last amino acid in each sequence. When successively removing the α-amino protecting group and ε-amino protecting group of the linker amino acid, the steps are as follows: soak the resin, remove the amino protecting group of the resin, wash and monitor, and couple the first part of the X9 part of the first sequence. Amino acids (i.e., lysine for linking to PEG), washed and monitored, removal of the α-amino protecting group and sequential coupling of other amino acids in the X9 moiety, removal of the α-amino protecting group of the linker amino acid and the first To continue the synthesis of the remaining amino acids in the sequence, the ε-amino protecting group of the linker amino acid is removed and the second amino acid sequence is synthesized, and the last amino acid in each sequence is optionally modified by acetylation.

The "amino-protecting group", "α-amino-protecting group" and "ε-amino-protecting group" used in the present invention refer to chemical groups introduced to protect amino groups involved in condensation reactions. The amino protecting groups include, but are not limited to: tert-butoxycarbonyl (Boc), benzyloxycarbonyl (cbz), trichloroethoxycarbonyl (Troc), fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc) etc. Preference is given to using Fmoc as α-amino protecting group, preferably Fmoc or Alloc as ε-amino protecting group.

The removal of the α-amino protecting group and the resin amino protecting group Fmoc uses piperidine (PIP) at a concentration of 20-25% (PIP:DMF) for 20-50 minutes. The removal of the Alloc protecting group of the ε-amino group is selected, for example, Pd(PPh ₃ ) ₄ , the concentration is 10-15% (Pd(PPh ₃ ) ₄ : chloroform), and the time is 30-60 min.

As an advantage of solid-phase peptide synthesis technology, the side chains of some amino acids can be protected by introducing chemical groups, for example, Arg can use pentamethylbenzofuran-5-sulfonyl (Pbf); His, Cys, Gln, Asn can use trityl (Trt); Lys can use tert-butoxycarbonyl (Boc); Thr, Tyr, Ser can use tert-butyl (tBu); Asp, Glu can use tert-butyl ester (Otbu). The protecting group is not limited thereto, and can be reasonably selected according to conventional schemes in the art.

The solvent used in the synthesis process is selected from dimethylformamide (DMF) or dichloromethane (DCM).

Coupling reagent is selected from: a kind of in carbodiimide type reagent or benzotriazolium salt type reagent, and 1-hydroxybenzotriazole (HOBt) or N, N-diisopropylethylamine ( A combination of one of DIEA). Among them, carbodiimide-type reagents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and N-diaminopropyl-N-ethylcarbodiimide (EDC) . Benzotriazolium salt-type reagents include 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O- Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), benzotriazole-1-oxytris(dimethylamino)phosphorus hexafluorophosphate (BOP), Benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP) and the like.

The coupling reagent is preferably a combination of DCC and HOBt, a combination of DCC and DIEA, a combination of TBTU and HOBt, a combination of TBTU and DIEA, most preferably a combination of DCC and HOBt.

As the acetylation reagent, acetic acid, acetic anhydride or its halogenated derivatives (such as α-chloroacetic acid, etc.) can be selected, and pyridine or DIEA can be used to create an alkaline reaction environment. Preferably, pyridine and acetic anhydride are selected in the present invention. Alternatively, acetic anhydride and DIEA are selected in the present invention.

The monitoring method is ninhydrin detection method.

(2) Cleavage of the polypeptide dimer from the resin

Cleavage can be carried out in trifluoroacetic acid or other strong acid medium, adding 5-20% V/V side chain protection group scavengers, such as thioanisole, triisopropylsilane, phenol, water, ethanedithiol, m-formazol phenol etc. The selection and ratio of side chain protecting group scavengers are flexibly determined according to the type and quantity of side chain protecting groups in the sequence. The polypeptide compound obtained by cleavage is obtained by precipitation with glacial ether.

(3) Purify the polypeptide dimer and dry it

The resulting polypeptide dimer is purified using reverse phase chromatography or ion exchange chromatography, preferably using reverse phase chromatography. The purified polypeptide dimers were vacuum freeze-dried and stored.

It is particularly advantageous to be able to obtain intramolecular disulfide bond modified polypeptides of the invention, which can be achieved by oxidative methods. The oxidizing reagent is selected from DMSO, potassium cyanide, hydrogen peroxide, iodine and the like. Preferably DMSO is used as the oxidizing agent. It is reported that it tends to form intramolecular disulfide bonds, so that highly purified intramolecular disulfide bond-modified polypeptides of the present invention can be obtained. The concentration of DMSO is 10-40%, preferably 20-30%.

It is particularly beneficial that in order to obtain PEG-modified polypeptides of the present invention, PEG reagents that modify amino groups can be selected for modification. For example, methoxypolyethylene glycol propionate NHS ester (mPEG-SPA-NHS, Beijing Jiankai), the reaction system used for PEG modification is DMF, the molar ratio of polypeptide compound to PEG is controlled at 1:1.2-1.5, and the reaction concentration It can be flexibly determined according to the molecular weight of the polypeptide and PEG used. PEG-modified products can be purified using reverse phase, macroporous adsorption or ion exchange resins, preferably reverse phase chromatography. The purified polypeptide compound is vacuum freeze-dried and stored.

In another aspect of the present invention, the present invention provides a pharmaceutical composition containing a polypeptide compound as an active ingredient, which may also include suitable pharmaceutically acceptable excipients, and those skilled in the art may select suitable excipients according to the route of administration. agent. The pharmaceutical compositions of the present invention may be administered in any suitable manner, including parenteral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, direct infusion, and the like. The polypeptide compound of the present invention is preferably in the form of lyophilized powder, and can be mixed with an appropriate isotonic solution in an appropriate dose before administration to a subject in need. Dosage and frequency of administration will depend on age, sex, and patient's condition, other drugs given at the same time, contraindications and other parameters considered by clinicians, for example, the dosage can be 1ug/kg-80mg/kg, and the administration frequency It can be once a day, once a week, or once a month, etc., and the dosage and frequency of administration are not particularly limited, and those skilled in the art can choose appropriately by considering the above factors.

Another aspect of the present invention provides the use of said polypeptide compound for diseases associated with underproduction, defect or over consumption of red blood cells. For example, the polypeptide compound of the present invention can be used to treat anemia caused by renal insufficiency and/or end-stage renal failure/dialysis, anemia after kidney transplantation, anemia caused by radiotherapy and chemotherapy in tumor treatment, anemia associated with AIDS, anemia associated with chronic inflammatory Anemia associated with disease (eg, rheumatoid arthritis and chronic enteritis) and for increasing a patient's red blood cell count prior to surgery. The polypeptide compounds of the present invention can also be used in beta-thalassemia, cystic fibrosis, pregnancy and menopausal disorders, anemia of prematurity, spinal cord injury, space flight, acute blood loss, aging, stroke, ischemia (CNS and heart) and various neoplastic conditions with abnormal erythropoiesis, among others. Preferably, the polypeptide compound of the present invention is especially suitable for anemia caused by renal insufficiency and/or end-stage renal failure/dialysis, anemia after kidney transplantation, and anemia caused by radiotherapy and chemotherapy in tumor treatment.

The common and most direct pathological manifestation of the diseases related to insufficient, defective or excessive consumption of red blood cells in the present invention is the relative or absolute deficiency of red blood cells and/or the reduction of red blood cell function. It is very beneficial that the activity of the polypeptide compound of the present invention is determined and confirmed by the principle of the reticulocyte method. The specific method is as follows: the mouse is subcutaneously injected with the polypeptide compound of the present invention, and an automatic reticulocyte analyzer is used to count each Ratio of the number of reticulocytes to the total number of red blood cells (Ret%) in the blood of only mice. The erythropoietic activity of the experimental samples was represented by the increase rate of Ret%.

Detailed ways

The invention is illustrated by the following examples. However, these and other examples in the present invention are for illustration only and do not limit the scope of the invention or the examples. Likewise, the invention is not limited to any specific preferred embodiments described herein. In fact, many modifications and variations of the present invention are apparent to those skilled in the art upon reading this specification, and can be made without departing from the rights and scope of the present invention.

Embodiment 1 prepares polypeptide dimer 1

(1) Materials and reagents

Rink Amide MBHA resin, substitution value 0.29mmol/g.

Desired protected amino acids 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.

Reagents: HOBt, DIC, DMF, piperidine, acetic anhydride, pyridine

(2) Instrument

PSI300 peptide synthesizer, Waters high performance liquid chromatography, magnetic stirrer

(3) Operation steps

Take 0.15mmol as an example:

a. Weigh 0.52g of Rink Amide MBHA resin, put it in the reactor of the peptide synthesizer, add 15mL of DMF, soak for 2h.

b. Removal of resin amino protecting group

Add 15 mL of 20% PIP (DMF) solution, mix for 30 min, and wash the resin 7 times with DMF.

c.Coupling reaction

Add 211mg Fmoc-L-Lys(Boc)-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and 0.2mol/L DIC each 2.5ml to react in the mixing reactor, reaction temperature is room temperature, with ninhydrin Reaction The reaction progress was monitored to ensure that Lys was coupled to the resin, and the resin was washed 7 times with DMF.

d. Peptide chain extension

After Lys is connected to the resin, add 15mL of 20% PIP (DMF) solution, mix for 30min, remove the α-amino protecting agent Fmoc of Lys, wash the resin 7 times with DMF, add 134mg Fmoc-Gly-OH (0.45mmol) 1. Coupling reagent 0.2mol/L HOBt and 0.2mol/L DIC each 2.5ml were reacted, the reaction temperature was room temperature, and the reaction progress was detected by ninhydrin reaction to ensure that Gly was coupled to the resin, and the resin was washed 7 times with DMF.

When Gly is connected to the resin, add 15mL of 20% PIP (DMF) solution, mix for 30min, remove the α-amino protecting agent Fmoc of Gly, wash the resin 7 times with DMF, add 266mg Fmoc-L-Lys(Fmoc)- OH (0.45mmol), 2.5ml of coupling reagent 0.2mol/L HOBt and 0.2mol/L DIC were reacted, the reaction temperature was room temperature, and the reaction progress was detected by ninhydrin reaction to ensure that Gly was coupled to the resin. Wash the resin 7 times.

When the connecting amino acid Lys is connected to the resin, add 30mL of 20% PIP (DMF) solution, mix for 40min, remove the α-amino and ε-amino protecting group Fmoc of Lys at the same time, wash the resin 7 times with DMF, add 268mg Fmoc- Gly-OH (0.90mmol), 5.0ml of coupling reagent 0.2mol/L HOBt and 0.2mol/L DIC were reacted, the reaction temperature was room temperature, and the reaction progress was detected by ninhydrin reaction to ensure that Gly was coupled to the resin. The resin was washed 7 times with DMF. Then continue the synthesis of the first sequence and the subsequent amino acids of the second sequence according to the same steps until all the amino acids of the first sequence and the second sequence are linked to the resin.

e. Acetylation modification

After the synthesis of the peptide sequence was completed, 5 ml of piperidine and 5 ml of acetic anhydride were added to react for 30 minutes to remove the α-amino protecting group Fmoc of the first sequence and the second sequence.

f. Lysis and precipitation

After acetylation, the resin was vacuum dried and weighed. Add the cleavage reagent according to the ratio of 1g resin to 10mL cleavage reagent (reagent ratio: TFA/thioanisole/triisopropylsilane/phenol/water/TIS=85/5/5/4/1), stir and react at room temperature for 3 hours , suction filtration. Add glacial ether to the lysed filtrate to precipitate the peptide, centrifuge, discard the supernatant, dry it in vacuum, and weigh the crude peptide.

g. Oxidation to form disulfide bonds

The crude peptide was dissolved in DMSO at a concentration of 5 mg/ml, followed by the addition of 4 volumes of water. Stand still at room temperature (≥10°C) for 36 hours.

h. Purification by reverse phase chromatography

The above crude peptide was purified by preparative HPLC using reverse phase chromatography.

HPLC column: C18 preparative column

Flow rate: 10mL/min

A: Aqueous solution containing 0.1% TFA

B: Acetonitrile solution containing 0.1% TFA

Use 18-38% phase B to elute for 90 minutes.

The purified product was homogeneous with a purity of 95% and an overall yield of 18%. MS: m/z = 4693.3 [M+H].

Embodiment 2 determines the reaction time of DMSO oxidation to form disulfide bond

According to the method of Example 1, polypeptide dimer 6 was similarly synthesized, wherein the additionally added protected amino acids were Fmoc-L-Asp(Otbu)-OH and Fmoc-L-Glu(Otbu)-OH.

(1) Materials and reagents

Crude peptide before oxidation of peptide dimer 6, DMSO, water, acetonitrile, TFA

(2) Instrument

Waters high performance liquid chromatography, 25 ℃ thermostat.

(3) Oxidation method

Weigh 9 crude peptide samples of polypeptide dimer 6 before oxidation, each about 2 mg. Add 1ml of 20% DMSO solution, place in a 25°C incubator, and take samples of 0h, 1h, 2h, 4h, 8h, 12h, 24h, 30h, and 36h for liquid phase analysis.

(4) Analysis method

Apply the oxidation process and trend of RP-HPLC samples, using chromatographic column-Boston PHlex ODS C18 (5μm) 4.6×250mm, mobile phase (A) is 0.05% TFA aqueous solution, (B) is 0.05% TFA acetonitrile solution. The elution conditions are phase B gradient from 20% to 30% for 10 minutes, 30% to 45% for 25 minutes, and a detection wavelength of 215 nm.

(5) Oxidation result:

The main peak of the sample before the oxidation reaction is 26.5min, and the main peak of the sample after the oxidation reaction is 18.7min. Considering the influence of impurity peaks and baseline noise, it can be considered that the reaction has been completed in 36h.

^* (Sample concentration has been converted to 2mg/ml)

Embodiment 3 prepares polypeptide dimer 9

(1) Materials and reagents

Rink Amide MBHA resin, substitution value 0.29mmol/g.

Desired protected amino acids 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.

Reagents: HOBt, DIC, DMF, piperidine, acetic anhydride, pyridine, chloroform, Pd(PPh ₃ ) ₄

(2) Instrument

PSI300 peptide synthesizer, Waters high performance liquid chromatography, magnetic stirrer

(3) Operation steps

Take 0.15mmol as an example:

a. Weigh 0.52g of Rink Amide MBHA resin, put it in the reactor of the peptide synthesizer, add 15mL of DMF, soak for 2h.

b. Removal of resin amino protecting group

Add 15 mL of 20% PIP (DMF) solution, mix for 30 min, and wash the resin 7 times with DMF.

c.Coupling reaction

Add 211mg Fmoc-L-Lys(Boc)-OH (0.45mmol), coupling reagent 0.2mol/L HOBt and 0.2mol/L DIC each 2.5ml to react in the mixing reactor, reaction temperature is room temperature, with ninhydrin Reaction The reaction progress was monitored to ensure that Lys was coupled to the resin, and the resin was washed 7 times with DMF.

d. Peptide chain extension

After Lys is connected to the resin, add 15mL of 20% PIP (DMF) solution, mix for 30min, remove the α-amino protecting agent Fmoc of Lys, wash the resin 7 times with DMF, add 134mg Fmoc-Gly-OH (0.45mmol) 1. Coupling reagent 0.2mol/L HOBt and 0.2mol/L DIC each 2.5ml were reacted, the reaction temperature was room temperature, and the reaction progress was detected by ninhydrin reaction to ensure that Gly was coupled to the resin, and the resin was washed 7 times with DMF.

When Gly is connected to the resin, add 15mL of 20% PIP (DMF) solution, mix for 30min, remove the α-amino protecting agent Fmoc of Gly, wash the resin 7 times with DMF, add 204mg Fmoc-L-Lys(Alloc)- OH (0.45mmol), 2.5ml of coupling reagent 0.2mol/L HOBt and 0.2mol/L DIC were reacted, the reaction temperature was room temperature, and the reaction progress was detected by ninhydrin reaction to ensure that Gly was coupled to the resin. Wash the resin 7 times.

After connecting the amino acid Lys to the resin, add 15 mL of 20% PIP (DMF) solution, mix for 30 min, remove the α-amino protecting group Fmoc of Lys, wash the resin 7 times with DMF, add 134 mg of Fmoc-Gly-OH (0.45 mmol), coupling reagent 0.2mol/L HOBt and 0.2mol/L DIC each 2.5ml were reacted, the reaction temperature was room temperature, and the reaction process was detected by ninhydrin reaction to ensure that Gly was coupled to the resin, and the resin was washed with DMF 7 Second-rate. Then continue to synthesize the subsequent amino acids of the first sequence according to the same steps until all the amino acids of the first sequence are linked to the resin.

Then add 10% Pd(PPh ₃ ) ₄ (chloroform) 10mL, mix for 45min, remove the ε-amino protecting group Alloc, wash the resin 7 times with DMF, add 134mg Fmoc-Gly-OH (0.45mmol), coupling reagent 0.2 5.0ml each of mol/L HOBt and 0.2mol/LDIC were reacted at room temperature, and the reaction progress was detected by ninhydrin reaction to ensure that Gly was coupled to the resin, and the resin was washed 7 times with DMF. Then continue to synthesize the subsequent amino acids of the second sequence according to the same steps until all the amino acids of the second sequence are linked to the resin.

e. Acetylation modification

After the synthesis of the peptide sequence was completed, 5 ml of piperidine and 5 ml of acetic anhydride were added to react for 30 minutes to remove the α-amino protecting group Fmoc of the first sequence and the second sequence.

f. Lysis and precipitation

After acetylation, the resin was vacuum dried and weighed. Add the cleavage reagent according to the ratio of 1g resin to 10mL cleavage reagent (reagent ratio: TFA/thioanisole/triisopropylsilane/phenol/water/TIS=85/5/5/4/1), stir and react at room temperature for 3 hours , suction filtration. Add glacial ether to the lysed filtrate to precipitate the peptide, centrifuge, discard the supernatant, dry it in vacuum, and weigh the crude peptide.

g. Oxidation to form disulfide bonds

The crude peptide was dissolved in DMSO at a concentration of 5 mg/ml, followed by the addition of 4 volumes of water. Stand still at room temperature (≥10°C) for 36 hours.

h. Purification by reverse phase chromatography

The above crude peptide was purified by preparative HPLC using reverse phase chromatography.

HPLC column: C18 preparative column

Flow rate: 10mL/min

A: Aqueous solution containing 0.1% TFA

B: Acetonitrile solution containing 0.1% TFA

Use 18-38% phase B to elute for 90 minutes.

The purified product was homogeneous with a purity of 96% and an overall yield of 16%. MS: m/z = 4706.2 [M+H].

Example 4 The PEG (molecular weight is about 20K) modification of polypeptide dimer 1

The compound of Example 1 (ie, polypeptide dimer 1) was modified with PEG (molecular weight of about 20K), and the effects of temperature, time, and concentration on the modification reaction were investigated respectively.

(1) Materials and reagents

DMF, DIEA, methoxypolyethylene glycol propionate NHS (about 20k, Beijing Jiankai Technology Co., Ltd.).

(2) Instrument

Waters high performance liquid chromatograph, 28 ℃ thermostat, 37 ℃ thermostat.

(3) Operation process

a. According to 1:1.5 (molar ratio), weigh a certain amount of polypeptide and PEG respectively, and dissolve them in DMF to obtain a clear solution.

b. Add 10 μl DIEA after the peptide and PEG are completely dissolved, and inject and analyze every 30 minutes. The effects of temperature, time, and concentration on the modification reaction were investigated respectively, and the results were expressed in modification rate.

(4) Experimental results

a. When the reaction temperature is 28°C and the reaction concentration is 3mg/ml, the results are as follows:

b. When the reaction temperature is 28°C and the reaction concentration is 2 mg/ml, the results are as follows:

c. When the reaction temperature is 28°C and the reaction concentration is 1mg/ml, the results are as follows:

d. When the reaction temperature is 37°C and the reaction concentration is 2mg/ml, the results are as follows:

e. When the reaction temperature is 4°C and the reaction concentration is 2 mg/ml, the results are as follows:

It can be seen from the above results that within the range of conditions selected in this example, the modification reaction is positively correlated with temperature, concentration and reaction time. When the temperature is 28°C and the concentration is 2mg/ml or 3mg/ml, the reaction is complete within 2 hours. When the temperature was 37°C and the concentration was 2 mg/ml, the reaction was complete in 1.5 hours.

The effect of temperature, reaction concentration and reaction time on PEG (about 5k or about 40k) modified polypeptide dimers can be similarly performed.

Example 5 Using PEG with a molecular weight of about 20k to modify polypeptide dimer 1

(1) Materials and reagents.

Methoxypolyethylene glycol propionate NHS ester (about 20k, mPEG-SPA-NHS, Beijing Jiankai Technology Co., Ltd.), DMF, DIEA, wherein mPEG-SPA-NHS (about 20k) is a linear type, and its structure as follows:

(2) Instrument

Waters high performance liquid chromatography, ultrafilter, constant temperature box

(3) Operation steps

a. Weigh 114mg of mPEG-SPA-NHS of about 20k, dissolve it in 5.7ml DMF, and make reaction mother liquid A

b. Weigh about 6.0mg of the polypeptide compound and add 2ml of mother solution A. Add 10ul DIEA after 5min. Place in a thermostat at 28°C. Samples were taken at half hour intervals and the progress of the reaction was monitored using HPLC.

c. After 2 hours, the reaction is basically complete. The reaction solution was diluted with 10 times volume of purified water, and then the modified product was purified by reverse phase chromatography.

The reversed-phase chromatographic conditions are as follows:

HPLC column: YMC-Pack C4 (10μm) 10×250mm

Flow rate: 2mL/min

A: Aqueous solution containing 0.1% TFA

B: Acetonitrile solution containing 0.1% TFA

The diluted reaction solution was passed through a C4 preparative column, followed by washing 10 column volumes with mobile phase A/B (95/5), and then using mobile phase A/B (30/70) to separate the target product to obtain Collection of the target product.

d. Remove TFA and acetonitrile in the collected solution by distillation under reduced pressure, and then use an ultrafiltration membrane with a molecular weight cut-off of 3000 to remove residual DMF.

e. Freeze-dry the obtained target product, and the purity of the compound after freeze-drying is 99.14%. Molecular weights were determined by MALDI-TOF. The molecular weight of the target product should be centered at 24692 and normally distributed within a certain range. The theoretical value is consistent with the mass spectrometry results.

Example 6 Using PEG with a molecular weight of about 40k to modify polypeptide dimer 1

(1) Materials and reagents.

Methoxy polyethylene glycol propionate NHS ester (about 40k, mPEG-SPA-NHS, Beijing Jiankai Technology Co., Ltd.), DMF, DIEA, wherein mPEG-SPA-NHS (about 40k) is branched, each The molecular weight of the branched chain is about 20k, and its structure is as follows:

The apparatus and operation steps are with reference to embodiment 5, but the reaction time in operation step c is 2.5 hours.

The obtained target product was freeze-dried, and the purity of the compound after freeze-drying was 99.96%. Molecular weights were verified by MALDI-TOF. The molecular weight of the target product should be centered at 44692 and form a normal distribution within a certain range, and the theoretical value is consistent with the mass spectrometry results.

Example 7 Using PEG with a molecular weight of about 5k to modify polypeptide dimer 1

(1) Materials and reagents.

Methoxypolyethylene glycol propionate NHS ester (about 5k, mPEG-SPA-NHS, Beijing Jiankai Technology Co., Ltd.), DMF, DIEA, wherein mPEG-SPA-NHS (about 5k) is a linear type, and its structure as follows:

The apparatus and operating steps are with reference to Example 5, but the reaction time in operating step c is 1.5 hours.

The obtained target product was freeze-dried, and the purity of the compound after freeze-drying was 99.96%. Molecular weights were verified by MALDI-TOF. The molecular weight of the target product should be centered at 9692 and form a normal distribution within a certain range, and the theoretical value is consistent with the mass spectrometry results.

Embodiment 8 In vivo activity determination of the polypeptide compound of the present invention

(1) Reagent

a. Dipotassium EDTA anticoagulant

Weigh 100 mg of dipotassium EDTA, add 10 ml of physiological sodium chloride solution to dissolve, mix well, and prepare freshly before use.

b. Diluent

Weigh 0.1g bovine serum albumin, add physiological sodium chloride solution to dissolve and dilute to 100ml.

(2) steps

A total of 68 female BALB/c mice (about 16-18 g in weight, from the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army) were selected and divided into 17 groups, with 4 animals in each group. Among them, the negative control group 1 was given 0.2ml of diluent; the positive control group 3 was given recombinant human erythropoietin injection (Beijing Sihuan Biopharmaceutical Co., Ltd., 3000IU/0.6ml/support), and the doses were 10 and 20 respectively. , 40IU/rat; 13 groups of compound groups of the present invention were administered with polypeptide dimer 1, polypeptide dimer 2, polypeptide dimer 3, polypeptide dimer 4, polypeptide dimer 5, and polypeptide dimer 6 , Polypeptide dimer 7, Polypeptide dimer 8, Polypeptide dimer 9, Polypeptide dimer 10, the compound of Example 5, the compound of Example 6, the lyophilized sample of the compound of Example 7, the dose is 20 μg /mouse.

From the 3rd day after the injection, 3-4 drops of blood were collected from the mouse orbit, and placed in a blood collection tube pre-added with 200 μl EDTA-K ₂ anticoagulant. For the negative control, the days of blood collection are 3, 4, 5, 6, 7, 8, 11, 22, and 28; for the positive control and polypeptide dimer 1-10, the days of blood collection are 3, 4, and 5 , 6, and 7 days; for the compounds of Examples 5, 6, and 7, the days for blood collection were the 4th, 5, 8, 11, 22, and 28 days. The anticoagulated blood was taken, and the ratio (Ret%) of the reticulocytes to the total number of red blood cells in the blood of each mouse was counted with an automatic reticulocyte analyzer (XT-2000IV).

其中样品包括阴性对照、阳性对照和本发明的化合物。多肽二聚体1-10的结果如表3所示，实施例5-7的化合物的结果如表4所示：The erythropoietic activity of the polypeptide compound of the present invention is represented by the increase rate of Ret%.

The samples include negative control, positive control and the compound of the present invention. The results of polypeptide dimers 1-10 are shown in Table 3, and the results of the compounds of Examples 5-7 are shown in Table 4:

table 3

Note: --- means untested.

Table 4

After the compound of the present invention is modified by PEG, the activity does not decrease compared with that before the modification, but the action time is significantly prolonged.

Claims (10)

1. A polypeptide compound, which consists of two polypeptide sequences, wherein the 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; 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 the first sequence and the second sequence are connected to form a dimer by connecting amino acid X8 in the first sequence, wherein X1 is selected from G or AcG; X2, X5 and X6 are 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, the α-amino group in its structure participates in the synthesis of the first sequence, and the ε-amino group in its structure connects with the C-terminal amino acid of the second sequence to form a dimer; X9 is lysine or consists of 2-10 A short peptide whose C-terminus is lysine composed of amino acids; X10 is selected from G or AcG; X11, X14, X16 or X19 are 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. The polypeptide compound of claim 1 or 2, wherein the first sequence is selected from the group consisting of 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 ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID 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 ID NO: 45, or SEQ ID NO: 46. 4. The polypeptide compound according to any one of claims 1-3, wherein the first sequence and the second sequence carry out N-terminal acetylation and formation of intramolecular disulfide bond modification, and optionally carry out C-terminal amidation in the first sequence grooming. 5. The polypeptide compound according to any one of claims 1-4, which is polypeptide dimer 1, polypeptide dimer 2, polypeptide dimer 3, polypeptide dimer 4, polypeptide dimer 5, polypeptide dimer Body 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 according to any one of claims 1-5, further comprising a polyethylene glycol moiety, wherein polyethylene glycol is conjugated to the lysine in the C-terminal X9 of the first sequence. 7. The polypeptide compound of claim 6, wherein the polyethylene glycol is a linear or branched PEG having a molecular weight of about 5,000 Daltons to about 60,000 Daltons. 8. A method for preparing a polypeptide compound, comprising the steps of: (1) solid-phase synthesis of the first sequence and the second sequence; (2) cracking the polypeptide dimer from the resin; (3) Purify the polypeptide dimer and dry it; (4) optionally carry out the modification and purification described in claim 4; and (5) Optionally conjugated with PEG and purified. 9. A pharmaceutical composition comprising the polypeptide compound of claims 1-7 and a pharmaceutically acceptable carrier. 10. Use of the polypeptide compound according to claims 1-7 in the preparation of a medicament for the treatment of diseases associated with insufficient, defective or excessive consumption of red blood cells.