WO2017113445A1

WO2017113445A1 - Erythropoietin peptide, derivatives and polymers, preparation method and application

Info

Publication number: WO2017113445A1
Application number: PCT/CN2016/070888
Authority: WO
Inventors: 张晓�; 厉保秋; 路杨; 杨东晖; 何南海; 沈柯; 周俊
Original assignee: 杭州阿诺生物医药科技股份有限公司
Priority date: 2015-12-31
Filing date: 2016-01-14
Publication date: 2017-07-06
Also published as: CN106928338A

Abstract

Provided is an erythropoietin peptide, which has the sequence SEQ ID No.1: R₁YR₂CR₃R₄GPR₅TWVCR₆R₇R₈, wherein R₁, R₂, R₅, R₆, R₇, and R₈ are separately either L- or D-amino acids; R₃ is Lys, Glu, Asp, Gln, Asn, Met, Ser, Tyr, Pro, or Ile, and R₃ is an L- or D-amino acid; R₄ is Met, Phe, or Ile, and R₄ is an L-amino acid. The erythropoietin peptide of the present invention can be used for treating anemia and anemia-related diseases.

Description

Erythropoietin peptide and derivative and polymer, preparation method and application thereof

Technical field

The invention relates to the field of protein polypeptides, in particular to a kind of erythropoietin peptide (EPO peptide) and derivatives and polymers thereof, preparation method and application thereof, and further relates to such erythropoietin peptides for treating and preventing anemia The use of related diseases.

Background technique

Erythropoietin (EPO) is a glycoprotein hormone that regulates erythroid hematopoiesis. EPO can only exert biological effects if it binds to the erythropoietin receptor (EPOR) active on the surface of erythroid progenitor cells. EPO is a sialic acid glycoprotein with a molecular weight of 196 amino acids, an isoelectric point of 4.5, a wide pH range (pH 3-9), and heat resistance (80 ° C for 5-15 minutes still active). The mature EPO molecule contains 166 amino acid residues. The development of red blood cells is through the processes of stem cells, progenitor cells, precursor cells, reticulocytes, etc., and the corresponding cytokine cells must be used to proliferate and differentiate. Erythropoietin is the most important cytokine of red blood cells. It not only stimulates the proliferation, transformation and maturation of bone marrow erythroid progenitor cells, but also produces reticulocytes in the bone marrow and releases them into the blood, and stimulates the synthesis of hemoglobin, which eventually leads to an increase in the number of peripheral red blood cells. EPO works by: (1) as a mitogen, stimulating mitosis and promoting proliferation of progenitor cells. (2) Delay DNA cleavage, reduce apoptosis, and maintain cell transition from G0/G1 phase to S phase. (3) Activation of erythroid specific genes (such as globin genes) to induce cell differentiation. The concentration of human EPO in plasma is usually low (10-26 U/L). When the oxygen concentration of kidney and hepatic arteries decreases, the synthesis reactivity of EPO increases.

The sensitivity of EPO is closely related to EPOR. EPO must bind to its receptor and dimerize it to cause signal down-transfer to exert its biological effects. The sensitivity of erythroid progenitor cells to EPO is related to the number of EPORs. EPOR is mainly expressed in the CFU-E phase, which decays with the maturation of red blood cells, and there is no EPOR on the surface of reticulocytes and mature red blood cells. Many studies have shown that the number of EPOR of CFU-E can be increased in the absence of oxygen to increase the sensitivity of EPO. The mouse EPOR cDNA was successfully cloned in 1989. In 1991, human EPOR cDNA was also successfully cloned. Both human and murine EPO receptors are composed of 507 amino acids. The EPO receptor can be divided into three regions, namely an extracellular ligand binding region, a transmembrane region, and a cytosolic signaling region. Among the amino acids constituting EPOR, 24 amino acids form a single peptide chain, and 223 amino acids form a cytoplasm In the outer portion, 24 amino acids form a transmembrane portion and 236 amino acids form a cytosolic portion. Its extracellular domain has a 5-amino structure, WSXWS. Deficiency and insertion studies indicate that it is an essential component of the EPO binding site, and this site changes, which can change the sensitivity of EPO. Another study found that amino acids 40-90 of the carboxyl terminus of EPOR have the effect of down-regulating the signal. Studies have shown that the reactivity of EPO does not depend on the primary structure of EPOR, and the secondary structure and higher structure are more important for EPO receptor function. When the secondary structure and advanced structure are changed, the sensitivity of the EPO can be affected. In addition, P85 and P100 are also coupled with EPO. EPOR can be activated by EPO and the like to form a homodimer, which leads to autophosphorylation of the EPO receptor, participates in signal transduction, and transmits information to the nucleus. There are two homeoboxes in the cytoplasmic region of the EPO receptor, which are the delivery sites for proliferation information. Its changes can cause information transmission changes, so it has an important impact on EPO sensitivity. EPO receptors are classified into high affinity and low affinity, and their ratio changes can directly affect the sensitivity of EPO.

Since the discovery of EPO by French scientists Carnot and Deflandre in 1906, people's understanding of EPO has been deepened and achieved many great achievements in this century. In June 1989, Amgen successfully developed and marketed the world's first recombinant human erythropoietin (rhEPO) product, Epogen, using genetic engineering technology, mainly in the treatment of renal anemia and malignant tumors. Symptoms such as anemia caused by chemotherapy are effective. In September 2001, Amgen's "high glycosylation" long-acting recombinant erythropoietin product, Arnesp, was approved by the FDA and officially launched in 2002. In November 2007, Roche launched another long-acting recombinant erythropoie product, Mircera (pegylated betaxetine). In the field of peptides, there are still no agonist drugs for EPO receptors listed, and there is huge research and development space and market potential.

Summary of the invention

The prior art problem solved by the present invention is that the existing EPO polypeptide has low biological activity, low bioavailability, short half-life, and poor clinical compliance, and the stability of such protein molecules is poor and easy to decompose. Not easy to store, transport and use.

In order to solve the above problems, the present invention provides an erythropoietin peptide and related derivatives thereof, which can significantly improve the activity and efficacy of EPOR, and have a longer half-life, better clinical compliance, and higher stability than EPO than EPO. , transport preservation and use are better than EPO.

Specifically, the present invention provides the following technical solutions:

In a first aspect, the present invention provides an erythropoietin peptide having the sequence of SEQ ID No. 1: R ₁ YR ₂ CR ₃ R ₄ GPR ₅ TWVCR ₆ R ₇ R ₈ ,

Wherein R ₁ , R ₂ , R ₅ , R ₆ , R ₇ and R ₈ are each independently an L-form or a D-form amino acid;

R ₃ is Lys, Glu, Asp, Gln, Asn, Met, Ser, Tyr, Pro or Ile, and the R ₃ is an L-form or a D-form amino acid;

R ₄ is Met, Phe or Ile, and R ₄ is an L-form amino acid.

Preferably, R ₁ is Leu; R ₂ is Ala; R ₃ is Lys, Met, Ser, Tyr or Ile; R ₄ is Met or Phe; R ₅ is Ile; R ₆ is Pro; R ₇ is Leu; R ₈ For Arg.

Preferably, the amino acids in the erythropoietin peptide are each independently a protected amino acid modified; the protecting group is preferably a tert-butoxycarbonyl group, an oxygen t-butyl group, a trityl group, and 2 One or more of 2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl or allyl.

In a second aspect, the present invention provides a erythropoietin peptide derivative which is modified by polyethylene glycol at any position of the C-terminus, N-terminus or side chain of the above-mentioned cytopoietin peptide .

In a third aspect, the present invention provides a pro-poietin peptide derivative, wherein the erythropoietin peptide derivative has the formula (I),

R ₉ -R ₁₀ -(CH ₂ ) _n1 -R ₁₁ -(CH ₂ ) _n2 -R ₁₂ -R ₁₃ (I)

Wherein R ₉ and R _{13 are} selected from the erythropoietin peptides according to

claim

1 or 2; n1 and n2 are each independently selected from an integer of 0 to 10; and R ₁₀ and R ₁₂ are each independently selected from CO or CH _{2 .} R _{11 is} selected from N(CH ₂ ) _n3 NHR ₁₄ , NCO(CH ₂ ) _n3 NHR ₁₄ , CHOCONH(CH ₂ ) _n3 NHR ₁₄ , CHSCON(CH ₂ ) _n3 NHR ₁₄ or CHNHCON(CH ₂ ) _n3 NHR ₁₄ One of them, wherein n3 is selected from an integer of from 2 to 10, and R ₁₄ is a methoxypolyethylene glycol.

Preferably, the methoxypolyethylene glycol has a molecular weight of from 5,000 to 100,000 Daltons, preferably from 5,000 to 50,000 Daltons, more preferably from 5,000 to 30,000 Daltons.

In a fourth aspect, the present invention provides a cytopoietin peptide polymer, which comprises the erythropoietin peptide according to any one of the above, or The erythropoietin peptide derivative acts as a repeating structural unit.

Preferably, the erythropoietin peptide is a dimer of an erythropoietin peptide or a tropin-producing peptide derivative or a multimer of 2 to 10 repeating structural units.

Preferably, the erythropoietin peptide polymer is polymerized in a manner selected from the group consisting of S-S, C-N, One of C-O, C=N, C-C, C=C, C-S, CO-S, CO-NH bond.

More preferably, the erythropoietin peptide polymer is polymerized in the form of a CO-NH, CO-S or S-S bond.

In a fifth aspect, the present invention also provides a method for preparing an erythropoietin peptide, comprising the steps of:

(1) using a solid phase synthesis method, in accordance with the order of attachment of erythropoietin peptides, under the action of a coupling agent and a reaction solvent, sequentially coupling amino acids to synthesize a fully protected erythropoietin peptide;

(2) Cleavage to obtain erythropoietin peptide.

Preferably, the coupling agent is selected from one of the following combinations: (1) N, N'-diisopropylcarbodiimide and 1-hydroxybenzotriazole,

(2) benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 1-hydroxybenzotriazole and N,N'-diisopropylethylamine,

(3) 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N'-diisopropylethylamine,

(4) benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 1-hydroxybenzotriazole and N-methylmorpholine,

(5) 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N-methylmorpholine;

The reaction solvent is one or more selected from the group consisting of N,N'-dimethylformamide, dichloromethane, N-methylpyrrolidone, and dimethyl sulfoxide.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the erythropoietin peptide as defined in any one of the above forms in free form or in a pharmaceutically acceptable salt form, or any of the above The erythropoietin peptide derivative or the erythropoietin peptide polymer of any of the above, as an active ingredient: one or more pharmaceutically acceptable carrier materials and/or diluents.

In a seventh aspect, the present invention provides the erythropoietin peptide according to any of the above, or the erythropoietin peptide derivative according to any one of the above, or the erythropoietin peptide polymerization according to any one of the above Use of a medicament for the treatment of a disease of a erythropoietin deficiency or a low or defective red blood cell population.

In an eighth aspect, the present invention provides the erythropoietin peptide of any one of the above, or the erythropoietin peptide derivative according to any one of the above, or the erythropoietin peptide polymerization according to any one of the above Use of the substance in the treatment of diseases in which the erythropoietin is insufficient or has a low or defective red blood cell population.

The beneficial effects of the present invention are that the erythropoietin peptides and derivatives and polymers of the present invention can be used for treating anemia or related diseases caused by anemia, in particular, erythropoietin peptides and derivatives and polymers for It would be useful to prepare a medicament for treating anemia or a related disease caused by anemia with a more effective side effect.

DRAWINGS

Figure 1 is a graph showing the binding activity of rhEPO to recombinant EPO receptors by a 125I-EPO competitive binding assay.

Figure 2 is a graph showing the binding activity of differently numbered EPO peptides to recombinant EPO receptors.

Figure 3 is a graph showing the proliferation of rhEPO and rhEPO-free FDCP-1/rhEPOR.

Figure 4 is a graph showing the FDCP-1/rhEPOR proliferation curve of the EPO peptide numbered 1331.

Figure 5 is a graph showing the phosphorylation of EPO peptide and EPO numbered 1331.

detailed description

As described above, it is an object of the present invention to provide an erythropoietin peptide, a polymer and a derivative, a preparation method and use thereof.

Wherein, the erythropoietin peptide of the present invention can be used as an agonist of the erythropoietin receptor, and the agonist defined in the present invention has a strong affinity for the receptor and a strong intrinsic activity. a substance that acts by receptor excitability, that is, a substance that binds to the action receptor and can promote the activity of the receptor; wherein the erythropoietin peptide described in the present invention specifically has a strong affinity for EPOR, thereby A polypeptide that renders EPOR active.

The present inventors have found that erythropoietin peptides can interact with EPOR to a certain extent, as shown by the fact that such peptides bind to EPOR and stimulate the proliferation of EPO-dependent cells. Such EPOR-derived peptides can be used as a substitute for EPO, and thus have great potential for treating related diseases.

The erythropoietin peptide is a monomeric peptide agonist comprising a length of 16 amino acids.

Wherein, in a preferred embodiment of the present invention, the sequence of the erythropoietin peptide is SEQ ID No. 2: H-Leu-Tyr-Ala-Cys-Lys-Met-Gly-Pro-Ile- Thr-Trp-Val-Cys-Pro-Leu-Arg-OH;

In another preferred embodiment of the present invention, the sequence of the erythropoietin peptide is: SEQ ID No. 3: H-Leu-Tyr-Ala-Cys-Tyr-Met-Gly-Pro-Ile- Thr-Trp-Val-Cys-Pro-Leu-Arg-OH;

In still another preferred embodiment of the present invention, the sequence of the erythropoietin peptide is: SEQ ID No. 4: H-Leu-Tyr-Ala-Cys-Ile-Met-Gly-Pro -Ile-Thr-Trp-Val-Cys-Pro-Leu-Arg-OH;

In another preferred embodiment of the present invention, the sequence of the erythropoietin peptide is SEQ ID No. 5: H-Leu-Tyr-Ala-Cys-Ser-Met-Gly-Pro-Ile-Thr -Trp-Val-Cys-Pro-Leu-Arg-OH;

In still another preferred embodiment of the present invention, the sequence of the erythropoietin peptide is SEQ ID No. 6: H-Leu-Tyr-Ala-Cys-Met-Met-Gly-Pro-Ile-Thr -Trp-Val-Cys-Pro-Leu-Arg-OH;

In still another preferred embodiment of the present invention, the sequence of the erythropoietin peptide is SEQ ID No. 7: H-Leu-Tyr-Ala-Cys-Lys((PEG)10)-CH ₂ CH _2- Phe-Gly-Pro-Ile-Thr-Trp-Val-Cys-Pro-Leu-Arg-OH.

Meanwhile, the present invention provides an erythropoietin peptide derivative, wherein the erythropoietin peptide derivative refers to a polyethylene glycol at any position of the N-terminal, C-terminal or amino acid side chain of the monomeric peptide amino acid. Make modifications.

Meanwhile, the present invention provides an erythropoietin peptide polymer wherein the polymer is a dimer or a polymer, and the polymer uses the erythropoietin peptide as a repeating structural unit, and the number of repeating structural units is preferably 2 to 10, the monomer peptide and the monomer peptide are connected by SS, CN, CO, C=N, CC, C=C, CS, CO-NH bond, etc., preferably SS or CO-NH The key is polycondensed. Moreover, such polypeptide compounds can also exhibit significant potency and activity by way of dimerization or polymerization.

Moreover, the present invention provides an erythropoietin peptide derivative having the formula of formula (I),

R ₉ -R ₁₀ -(CH ₂ ) _n1 -R ₁₁ -(CH ₂ ) _n2 -R ₁₂ -R ₁₃ (I)

claim

1 or 2; n1 and n2 are each independently selected from an integer of 0 to 10; and R ₁₀ and R ₁₂ are each independently selected from CO or CH _{2 .} R _{11 is} selected from N(CH ₂ ) _n3 NHR ₁₄ , NCO(CH ₂ ) _n3 NHR ₁₄ , CHOCONH(CH ₂ ) _n3 NHR ₁₄ , CHSCON(CH ₂ ) _n3 NHR ₁₄ or CHNHCON(CH ₂ ) _n3 NHR ₁₄ One of them, wherein n3 is selected from an integer of from 2 to 10, and R ₁₄ is a methoxypolyethylene glycol. The methoxypolyethylene glycol has a molecular weight of from 5,000 to 100,000 Daltons, preferably from (5,000 to 50,000 Daltons), more preferably from (5,000 to 30,000 Daltons).

The invention also provides a preparation method of erythropoietin peptide, but is not limited to the following methods:

The route of the present invention is described by taking the following sequence as an example: H-Leu-Tyr-Ala-Cys-Lys-Met-Gly-Pro-Ile-Thr-Trp-Val-Cys-Pro-Leu-Arg-OH

Using a solid phase synthesis method, CTC resin/king resin is used as a starting resin, and an N-terminal Fmoc-protected and side-chain-protected amino acid is sequentially coupled according to the erythropoietin peptide main chain peptide sequence; finally, the peptide resin is cleaved. Purified and lyophilized to give the target compound.

To this end, the present invention provides a method for synthesizing erythropoietin peptides, the steps of which are as follows:

Step 1, in the presence of an activator system, a resin solid phase support and Fmoc-Arg (Pbf)-OH coupling to obtain Fmoc-Arg (Pbf)-resin;

Step 2, sequentially, by solid phase synthesis, according to the erythropoietin peptide main chain peptide sequence, an amino acid having N-terminal Fmoc protection and side chain protection;

Step 3, cleavage, purification, and lyophilization to obtain erythropoietin peptide and analogs thereof.

Wherein the resin solid carrier in step 1 is a 2-CTC resin, the activator system is selected from DIEA, TMP or NMM, and the Fmoc-Arg (Pbf)-resin is Fmoc- at a degree of substitution of 0.10 to 0.80 mmol/g. Arg(Pbf)-CTC resin can be synthesized by itself or purchased directly.

Wherein, the resin solid carrier in step 1 is a king resin, the activator system is composed of DIC, HOBt and DMAP, and the Fmoc-Arg (Pbf)-resin is Fmoc-Arg having a degree of substitution of 0.10 to 0.80 mmol/g ( Pbf) - King resin can be synthesized by itself or directly through purchase.

The solid phase synthesis method described in step 2 includes the following steps:

1) removing the Fmoc protecting group on the Fmoc-Arg(Pbf)-resin using a deprotecting solution consisting of piperidine and DMF in a volume ratio of 1:4 to obtain an H-Arg(Pbf)-resin;

2) H-Arg(Pbf)-resin and Fmoc-protected and side-chain-protected leucine are coupled to obtain Fmoc-Leu-Arg(Pbf)-resin under the action of a coupling agent and a reaction solvent;

3) Repeat steps 1) and 2) to sequentially perform amino acid coupling according to the erythropoietin peptide main chain peptide sequence. The amino acid sequence is as follows:

Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH,

Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH , Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH;

The coupling agent is selected from one of the following combinations: (1) N, N'-diisopropylcarbodiimide and 1-hydroxybenzotriazole,

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, the examples are only intended to illustrate the invention and are not intended to limit the scope of the invention.

Among them, the manufacturer models of the reagents and instruments used in the examples are as follows:

Fmoc-Arg (Pbf)-King resin, purchased from Jill Biochemical (Shanghai) Co., Ltd.;

Amino acid, purchased from Jill Biochemical (Shanghai) Co., Ltd.;

Dimethylformamide, purchased from Zhejiang Jiangshan Chemical Co., Ltd.;

Piperidine, purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.;

DCM (dichloromethane), purchased from Zhejiang Suihua Fluorine Chemical Co., Ltd.;

MeOH (methanol), purchased from (Zhehai Zhedong Special Reagent Factory)

Anisole, purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.;

TFA (trifluoroacetic acid), purchased from Zhejiang Chemical Industry Technology Co., Ltd.;

DIC (N,N'-diisopropylcarbodiimide), purchased from Zibo Tianshan Chemical Co., Ltd.;

HOBT (1-hydroxybenzotriazole), purchased from Suzhou Haofan Biotechnology Co., Ltd.;

DIEA (N,N'-diisopropylethylamine), purchased from Suzhou Wufan Biotechnology Co., Ltd.;

C18 column, C8 column, purchased from Daisogel;

Mass spectrometer, model MALDI-TOF 4700, manufacturer AB SCIEX;

Preparative high performance liquid chromatography, model LC3000, manufacturer Beijing Innovation Tongheng Technology Co., Ltd.

Among them, the cells used in the present invention: Chinese hamster ovary (CHO) and mouse bone marrow cells (FDCP-1) were purchased from ATCC.

RPMI medium, purchased from: Thermo Fisher SCIENTIFIC;

Some commonly used abbreviations in the present invention have the following meanings;

Fmoc: fluorenylmethoxycarbonyl

Fmoc-AA: Aminocarbonyl protected amino acid

DIC: N, N'-diisopropylcarbodiimide

DCC: N, N'-dicyclohexylcarbodiimide

PyBOP: benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate

HATU: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate

HOBt: 1-hydroxybenzotriazole

tBu: tert-butyl

OtBu: tert-butoxy

Trt: trityl

Boc: tert-butoxycarbonyl

Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

Cys: Cysteine

Pro: Proline

Leu: Leucine

Gly: glycine

Arg: arginine

Ala: Alanine

Lys: Lysine

Tyr: Tyrosine

Met: methionine

Ile: Isoleucine

Thr: threonine

Trp: tryptophan

Val: Valine

DMF: N, N'-dimethylformamide

MeOH: methanol

DCM: dichloromethane

NMP: N-methylpyrrolidone

DMSO: dimethyl sulfoxide

TFA: trifluoroacetic acid

Piperidine: Hexahydropyridine

DMAP: 4-dimethylaminopyridine

DIEA: N,N'-diisopropylethylamine

TMP: 2,4,6-trimethylpyridine.

The type and manufacturer of the freeze-drying equipment used in vacuum drying and freeze-drying mentioned in the examples are as follows:

Freeze-drying equipment: lyophilizer FD-3 (Beijing Bo Yikang Experimental Instrument Co., Ltd.);

Freeze-drying conditions: The lyophilized tray was placed in a freezer (-20 ° C) and pre-frozen for 6 h. Turn on the freeze dryer, turn on the refrigeration, pre-cool for more than 30min, set the freeze-drying curve as follows:

First stage: 16h operation at -27 °C; second stage: 4h operation at -5 °C; third stage: 2h operation at 5 °C; fourth stage: 16h operation at 30 °C.

(1) Preparation of erythropoietin peptide (EPO peptide)

Embodiment 1

Synthesis of erythropoietin peptide, the sequence of which is SEQ ID No. 2: H-Leu-Tyr-Ala-Cys-Lys-Met-Gly-Pro-Ile-Thr-Trp-Val-Cys-Pro-Leu-Arg -OH synthesis

(1) Weigh 0.1 mmol of Fmoc-Arg(Pbf)-Wang resin with a degree of substitution of 0.52 mmol/g, add it to the solid phase reaction column, wash it once with DMF, and swell Fmoc-Arg(Pbf)-wang resin with DMF. After 30 minutes, Fmoc protection was removed with a mixed solution of DMF:pyridine in a volume ratio of 4:1, and then washed 6 times with DMF, and Fmoc-Leu-OH 0.5 mmol and HOBt 0.5 mmol were added to a volume ratio of 1:1. The mixed solution of DCM and DMF was activated by adding 80 μl of DIC (0.5 mmol) in an ice water bath, and then added to the reaction column containing the resin, and reacted at room temperature for 2 hours, and then judged by the ninhydrin method, if the resin is colorless. Transparent means that the reaction is complete; if the resin develops color, it means that the reaction is incomplete and needs to be reacted for another hour. This criterion is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method.

(2) repeating the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH Coupling of Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH. After the coupling, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH and dried to give 0.751 g of crude peptide resin.

(3) Cleavage: 0.751 g of fully protected peptide resin was weighed, added to a 25 mL three-neck round bottom flask, and 10 mL of lysate was placed in a volume ratio of TFA: anisole = 95:5, and the lysate was added to the above resin. The mixture was reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA. The filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for 1 hour, centrifuged, washed with anhydrous diethyl ether for 6 times, and dried under vacuum. The crude peptide was obtained in 186.2 mg.

(4) Purification, lyophilization: The above crude peptide was dissolved in 50 mL of water, and then purified twice by a C18 column, desalted, concentrated by rotary evaporation, and lyophilized to give the desired product (18.7 mg, 10.11%). The purification conditions were as follows: mobile phase: phase A: 0.1% TFA; phase B: acetonitrile; gradient procedure: 15% B, 60 minutes to 60% B; detection wavelength 220 nm; Desalination conditions are flow Phase: Phase A: 20 mmol/L ammonium acetate in water: acetonitrile = 95:5; Phase B: water: acetonitrile = 95:5; Phase C: 0.03% acetic acid in water: acetonitrile = 95:5; D phase: 0.03% Aqueous solution of acetic acid: acetonitrile = 50:50; gradient program: elution with mobile phase A gradient for 15 minutes, conversion to mobile phase B, gradient elution for 10 minutes, conversion to mobile phase C, gradient elution for 10 minutes, conversion Gradient elution in mobile phase D for 25 minutes; detection wavelength 220 nm; collection of peak fractions of interest. At the same time, the product was analyzed by MALDI-TOF, and it was found that the m/z value of the protonated molecular ion peak was 1850.3627 (theoretical amount was 1849).

Embodiment 2

Synthesis of erythropoietin peptide with the sequence of SEQ ID No. 3: H-Leu-Tyr-Ala-Cys-Tyr-Met-Gly-Pro-Ile-Thr-Trp-Val-Cys-Pro-Leu-Arg -OH synthesis

(2) repeating the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH Coupling of Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH. After the coupling, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH and dried to give 0.726 g of crude peptide resin.

(3) Cleavage: Weigh 0.726 g of fully protected peptide resin, add it to a 25 mL three-neck round bottom flask, arrange 10 mL of lysate according to the volume ratio of TFA: anisole = 95:5, and add the lysate to the above tree. The mixture was reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA. The filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for 1 hour, centrifuged, and washed with diethyl ether for 6 times. Drying in vacuo gave 167.9 mg of crude peptide.

(4) Purification, lyophilization: The crude peptide was dissolved in 50 ml of water, and then purified by C8 column twice, transferred to salt, and lyophilized to obtain the target product (17.5 mg, 9.26%), and the product was analyzed by MALDI-TOF. The m/z value of the protonated molecular ion peak is 1885.8624 (theoretical amount is 1885).

Embodiment 3

Synthesis of erythropoietin peptide, the sequence of which is SEQ ID No. 4: H-Leu-Tyr-Ala-Cys-Ile-Met-Gly-Pro-Ile-Thr-Trp-Val-Cys-Pro-Leu-Arg -OH synthesis

(2) repeating the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Ile-OH, Fmoc-Cys(Trt)-OH, Fmoc- Coupling of Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH. After the coupling, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH, and dried to give 0.689 g of crude peptide resin.

(3) Weigh 0.689 g of fully protected peptide resin, add it to a 25 mL three-neck round bottom flask, dispose 10 mL of the lysate according to the volume ratio of TFA: anisole = 95:5, and add the lysate to the above resin. The mixture was reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA. The filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for 1 hour, centrifuged, washed with diethyl ether for 6 times, and dried under vacuum. The crude peptide was obtained in 163.7 mg.

(4) Purification, lyophilization: The crude peptide was dissolved in 50 ml of water, and then purified twice by a C18 column, transferred to a salt, and lyophilized to give the object product (17.3 mg, 9.40%), and the product was analyzed by MALDI-TOF. The m/z value of the protonated molecular ion peak is 1835.8972 (theoretical amount is 1835).

Embodiment 4:

Synthesis of erythropoietin peptide with the sequence of SEQ ID No. 5: H-Leu-Tyr-Ala-Cys-Ser-Met-Gly-Pro-Ile-Thr-Trp-Val-Cys-Pro-Leu-Arg -OH synthesis

(2) repeating the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH Coupling of Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH. After the coupling, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH, and dried to give 0.689 g of crude peptide resin.

(3) Cleavage: Weigh 0.689 g of fully protected peptide resin, add to a 25 mL three-neck round bottom flask, dispose 10 mL of lysate according to the volume ratio of TFA: anisole = 95:5, and add the lysate to the above tree. The mixture was reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA. The filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for 1 hour, centrifuged, and washed with diethyl ether for 6 times. Drying in vacuo gave 175.4 mg of crude peptide.

(4) Purification, lyophilization: The crude peptide was dissolved in 50 ml of water, and then purified twice by a C18 column, transferred to a salt, and lyophilized to obtain the target product (19.8 mg, 10.94%), and the product was analyzed by MALDI-TOF. The protonated molecular ion peak has an m/z value of 1809.9533 (theoretical amount is 1809).

Embodiment 5

Synthesis of erythropoietin peptide with the sequence of SEQ ID No. 6: H-Leu-Tyr-Ala-Cys-Met-Met-Gly-Pro-Ile-Thr-Trp-Val-Cys-Pro-Leu-Arg -OH synthesis

(2) repeating the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Met-OH, Fmoc-Met-OH, Fmoc-Cys(Trt)-OH, Fmoc- Coupling of Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH. After the coupling, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH and dried to give 0.730 g of crude peptide resin.

(3) Cleavage: Weigh 0.730 g of fully protected peptide resin, add it to a 25 mL three-neck round bottom flask, arrange 10 mL of lysate according to the volume ratio of TFA: anisole = 95:5, and add the lysate to the above tree. The mixture was reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA. The filtrate was combined, concentrated, and the concentrated liquid was added to ice diethyl ether for 1 hour, centrifuged, and washed with diethyl ether for 6 times. Drying in vacuo gave 165.7 mg of crude peptide.

(4) Purification, lyophilization: The crude peptide was dissolved in 50 ml of water, and then purified twice by a C18 column, transferred to a salt, and lyophilized to give the object product (17.1 mg, 9.24%), and the product was analyzed by MALDI-TOF. The m/z value of the protonated molecular ion peak was 1853.8677 (theoretical amount was 1853).

Embodiment 6

Synthesis of erythropoietin peptide, the sequence of which is SEQ ID No. 7: H-Leu-Tyr-Ala-Cys-Lys((PEG)10)-CH ₂ CH ₂ -Phe-Gly-Pro-Ile-Thr- Synthesis of Trp-Val-Cys-Pro-Leu-Arg-OH

(1) Weigh 0.1 mmol of Fmoc-Arg(Pbf)-Wang resin with a degree of substitution of 0.52 mmol/g, add it to the solid phase reaction column, wash it once with DMF, and swell Fmoc-Arg(Pbf)-wang resin with DMF. After 30 minutes, Fmoc protection was removed with a mixed solution of DMF:pyridine in a volume ratio of 4:1, and then washed 6 times with DMF, and 0.177 g of Fmoc-Leu-OH (0.5 mmol) and 0.068 g of HOBt (0.5 mmol) were weighed. Add a mixed solution of DCM and DMF in a volume ratio of 1:1, add 80 μl of DIC (0.5 mmol) to the reaction column in an ice water bath, add to the reaction column containing the resin, react at room temperature for 2 hours, and then detect by ninhydrin method. Judging the end of the reaction, if the resin is colorless and transparent, it means the reaction is complete; if the resin develops color, it means that the reaction is incomplete and needs to be reacted for another hour. This criterion is applicable to the subsequent amino acid coupling and the end point of the reaction is determined by the ninhydrin method. .

(2) repeating the above steps of removing Fmoc protection and adding the corresponding amino acid coupling, and sequentially completing Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH, Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Lys(Dde)-OH, Fmoc-Cys(Trt)-OH Coupling of Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Boc-Leu-OH, adding 3% hydrazine hydrate DMF solution to the resin for 30 min, coupling Fmoc-NH-PEG10-CH ₂ CH ₂ COOH. After the coupling, the peptide resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, 3 times with MeOH, and dried to give 0.825 g of crude peptide resin.

(3) Cleavage: Weigh 0.825 g of fully protected peptide resin and add to a 25 mL three-neck round bottom flask. In the volume ratio of TFA: anisole = 95:5, 10 mL of the lysate was placed, and the lysate was added to the above resin, reacted at room temperature for 2 hours, filtered, and the cracked resin was washed 3 times with a small amount of TFA, and the filtrate was concentrated. The concentrated liquid was added to ice diethyl ether for precipitation for 1 hour, centrifuged, washed with diethyl ether for 6 times, and dried under vacuum to give 193.7 mg of crude peptide.

(4) Purification, lyophilization: The crude peptide was dissolved in 50 ml of water, and then purified twice by C18 or C8 column, transferred to salt, and lyophilized to obtain the target product (19.4 mg, 8.16%), and the product was analyzed by MALDI-TOF. The m/z value of the protonated molecular ion peak was found to be 2377.7428 (theoretical amount was 2377).

(II) Determination of activity of erythropoietin peptide (EPOR peptide)

Experimental methods and results

1. Preparation of EPO peptide

EPO peptide: The target polypeptides obtained in Example 1 to Example 5 were numbered 1329, 1330, 1331, 1332, 1333, respectively, and used.

2. Expression, purification and immobilization of EPO receptor

We ligated the carboxy-terminal signal sequence of HPAP (human placental alkaline phosphatase) at the carboxy terminus of the EPOR extracellular domain (EPO ECD), and added a thrombin cleavage site before the two sequences, and fused it to CHO cells. . A HPAP antibody is immobilized on the polyethylene plate and can be used to identify the HPAP moiety, thereby immobilizing the EPO-R in the detection well.

The RPMI culture solution required for cell culture contains 1% BSA. The HPAP signal sequence can anchor the fusion protein to the cell surface via a phosphatidyl glycan. After the recombinant EPOR was treated with phospholipase c, the fusion protein was immobilized in polystyrene wells with an anti-HPAP region antibody (mAb179).

3. 125I-EPO competitive binding assay to detect the binding activity of rhEPO to recombinant EPO receptor

EPOR (rhEPOR) was recombinantly expressed according to the above procedure, and rhEPOR was fixed in polystyrene microwells, followed by radiolabeled 125I-rhEPO (600 ci/mmol), in which only one molecule of rhEPO was added as a non-specific control well. The wells were washed after incubation at 4 ° C for 2 h, and the data were analyzed by the ALLFIT method. The results are shown in Figure 1. 125I-rhEPO competitively binds rhEPOR to rhEPO, and the rhEPO solution is added, the radioactivity decreases, and the concentration of rhEPO increases (from 10 ^-12 M, 10 ^-11 M). , 10 ^-10 M, 10 ^-9 M, 10 ^-8 M, 10 ^-7 M, 10 ^-6 M), the lower the radioactivity binding rate of the solution. The dissociation coefficient of rhEPO and recombinant EPOR calculated by the ALLDFIT analysis method by the curve of Fig. 1 is about 200 pM, which is close to the dissociation value of the natural receptor.

Among them, the radioactive binding rate refers to the ratio of the radiolabeled 125I-EPO combined with the total concentration of 125I-EPO when the radiolabeled 125I-EPO competes with the EPO peptide or rhEPO for EPOR binding, ie [125I-EPO] binding concentration/ [125I-EPO] total concentration * 100%; wherein, the dissociation coefficient refers to the concentration of the ligand when 50% of the receptor is bound by the ligand, and is used to reflect the affinity of the reaction.

4. Determination of binding activity of EPO peptide

EPO peptides (peptide Nos. 1329, 1330, 1331, 1332, 1333) were each dissolved in 100% DMSO to a final concentration of 50 mM, respectively, as an EPO peptide stock solution. EPO peptide stock solution was diluted with the binding solution (phosphate solution, Na2CO31.59g, NaHCO32.94g, plus distilled water to 1000mL) to the use concentration (10 ^-10 M, 10 ^-9 M, 10 ^-8 M, 10 ^-7 M,10 ^-6 M,10 ^-5 M,10 ^-4 M,10 ^-3 M), then added to the polystyrene microwells fixed with EPOR, and then the radiolabeled 125I-rhEPO is competitive with the EPO peptide. In combination with the EPOR receptor, one well was only added with 1 uM rhEPO as a non-specific detection control well. The wells were washed after incubation at 4 ° C for 2 h, and the data were analyzed by the ALLFIT method. The results are shown in Figure 2. The radiolabeled 125I-rhEPO was added, and 125I-rhEPO competed with the EPO short peptide for competitive binding to the EPOR receptor. As can be seen from Figure 2, the radioactivity of the solution containing the five peptides was different. The degree of decline, and the higher the concentration of short peptide, the lower the radioactivity binding rate; among them, the peptide solution numbered 1331 has the fastest decline in radioactivity, and the radioactivity is when the concentration of peptide 1331 is 10 ^-6 M. It fell below 20%, and when the concentration of the peptide 1331 was 10 ^-4 M, almost no radioactivity was detected. The experimental results of Figure 2 indicate that peptide 1331 is most active.

5. Determination of in vitro activity of EPO peptide

To determine if an EPO peptide can act on EPOR eukaryotic cells. We transfected FDCP-1 cells with full-length human EPOR to prepare FDCP-1/hEPOR cells. Among them, FDCP-1 cells are a hematopoietic progenitor of mouse bone marrow.

FDCP-1/rhEPOR proliferation curve of EPO: FDCP-1/hEPOR cells were cultured in 10% FCS and 1 nM rhEPO RPMI to a density of 10 ⁶ /mL, washed with PBS to remove rhEPO, and continued with rhEPO-free medium. Cultivate overnight. Cells were plated at 10 ⁵ cells per well and rhEPO (10 ^-12 M, 10 ^-11 M, 10 ^-10 M, 10 ^-9 M) was added as indicated, and MTT assay was used at 570 nm after 48 hours. Cell proliferation was measured by colorimetry, and the results are shown in Fig. 3. The curves in Figure 3 represent the meaning of cell proliferation curves after stimulation of FDCP-1/rhEPOR cells with rhEPO and cell proliferation curves without rhEPO stimulation. The results in Figure 3 indicate that the culture of rhEPO-containing medium can promote the proliferation of FDCP-1/rhEPOR cells compared with rhEPO-containing medium, and the proliferation of FDCP-1/rhEPOR cells increases with the concentration of rhEPO. . The results in Figure 3 indicate that rhEPO can stimulate proliferation of recombinant FDCP-1/rhEPOR cells.

FDCP-1/rhEPOR proliferation curve of EPO short peptide numbered 1331: EPO short peptide 1331 was dissolved in 100% DMSO to a final concentration of 50 mM. It was diluted with serum-free RPMI to the use concentration (10 ^-7 M, 10 ^-6 M, 10 ^-5 M, 10 ^-4 M). FDCP-1/hEPOR cells were cultured to a density of 10 ⁶ /mL in 10% FCS and 1 nM rhEPO RPMI, washed with PBS to remove rhEPO, and cultured overnight with rhEPO-free medium. The cells were plated at 10 ⁵ cells per well, and EPO short peptide 1331 was added as indicated. After 48 hours, cell proliferation was measured by MTT assay at a wavelength of 570 nm, and the results are shown in Fig. 4 . From the results in Figure 4, EPO stimulated cell line proliferation with an EC50 value of ^10-20 pM. The results in Figure 4 indicate that peptide 1331, as an EPOR agonist, stimulates cell proliferation. The occurrence of proliferation depends on the presence of EPO.

The experimental results of Figures 3 and 4 indicate that the occurrence of cell proliferation activity is dependent on the EPO short peptide, not the EPO contamination. At the same time, we added 1% rabbit polyclonal serum to the FDCP-1/hEPOR proliferation experiment of EPO short peptide. Polyclonal serum completely inhibited EPO activity, and the results showed that the addition of polyclonal serum did not affect the proliferative activity of EPO short peptide.

6, tyrosinase phosphorylation analysis

After EPO binds to EPOR, EPOR forms a dimer and then regulates the proliferation and differentiation of erythroid cells through signaling pathways, which involve multiple signaling pathways. During this process, EPO can activate a variety of protein receptors, resulting in phosphorylation activation such as JAK2, VAV, EPOR, SHC. 2×10 ⁶ /mL FDCP-1/rhEPOR cells were cultured with 10% FCS, 2nm rhEPO, collected and resuspended in rhEPO-free medium for 24h. The cells were then counted and plated at 0.5 x 10 ⁶ /mL per well. The cells were stimulated with rhEPO (10 ^-5 M) and EPO peptide numbered 1331 (10 ^-5 M) for 10 min, respectively, and sterile water was used as a control group. Phosphorylation was detected by immunoblotting. The antibody uses an anti-tyrosine phosphorylated monoclonal antibody as a detection antibody.

The results are shown in Figure 5, where C is the protein molecular mass of the marker, and the marker is purchased from Beijing Boaosen Biotechnology Co., Ltd.

The results showed that the molecular weight of the phosphorylated protein induced by rhEPO was approximately 140, 95, 70, 55 KD. These proteins may correspond to JAK2, VAV, EPOR, SHC. Peptide 1331 produced the same phosphorylation class as EPO, so peptide 1331 was consistent with the regulation of EPO signaling in humans.

It is to be understood that the above-described preferred embodiments are only illustrative of the technical solutions of the present invention, and are not intended to be limiting, although the present invention has been described in detail by the foregoing preferred embodiments, those skilled in the art Various changes are made in the details without departing from the scope of the invention as defined by the appended claims.

Claims

An erythropoietin peptide having the sequence of SEQ ID No. 1: R 1 YR 2 CR 3 R 4 GPR 5 TWVCR 6 R 7 R 8 ,

Wherein R 1 , R 2 , R 5 , R 6 , R 7 and R 8 are each independently an L-form or a D-form amino acid;

R 3 is Lys, Glu, Asp, Gln, Asn, Met, Ser, Tyr, Pro or Ile, and the R 3 is an L-form or a D-form amino acid;

R 4 is Met, Phe or Ile, and R 4 is an L-form amino acid.
The erythropoietin peptide according to claim 1, wherein R 1 is Leu; R 2 is Ala; R 3 is Lys, Met, Ser, Tyr or Ile; and R 4 is Met or Phe; 5 is Ile; R 6 is Pro; R 7 is Leu; and R 8 is Arg.
The erythropoietin peptide according to claim 1 or 2, wherein the amino acids in the erythropoietin peptide are each independently a protected amino acid modified; the protective group is preferably One or more of tert-butoxycarbonyl, oxy-tert-butyl, trityl, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl or allyl.
An erythropoietin peptide derivative characterized in that the C-terminal, N-terminal or side chain of the cytopoietin peptide according to any one of claims 1 to 3 is modified with polyethylene glycol .
A cytopoietin peptide derivative, wherein the erythropoietin peptide derivative has the formula of formula (I),

R 9 -R 10 -(CH 2 ) n1 -R 11 -(CH 2 ) n2 -R 12 -R 13 (I)

Wherein R 9 and R 13 are selected from the erythropoietin peptides according to claim 1 or 2; n1 and n2 are each independently selected from an integer of 0 to 10; and R 10 and R 12 are each independently selected from CO or CH 2 . R 11 is selected from N(CH 2 ) n3 NHR 14 , NCO(CH 2 ) n3 NHR 14 , CHOCONH(CH 2 ) n3 NHR 14 , CHSCON(CH 2 ) n3 NHR 14 or CHNHCON(CH 2 ) n3 NHR 14 One of them, wherein n3 is selected from an integer of from 2 to 10, and R 14 is a methoxypolyethylene glycol.
The erythropoietin peptide derivative according to claim 5, wherein the methoxypolyethylene glycol has a molecular weight of from 5,000 to 100,000 Daltons, preferably from 5,000 to 50,000 Daltons, more preferably from 5,000 to 30,000. Dalton.
A cytopoietin peptide polymer characterized in that the cytopoietin peptide is polymerized The erythropoietin peptide according to any one of claims 1 to 3, or the erythropoietin peptide derivative according to any one of claims 4 to 6 as a repeating structural unit, preferably erythropoietin A dimer of a peptide or a cytopoietin peptide derivative or a multimer of 2 to 10 repeating structural units.
The erythropoietin peptide polymer according to claim 7, wherein the polymerization mode is selected from the group consisting of SS, CN, CO, C=N, CC, C=C, CS, CO-S, and CO-NH bonds. Preferably, it is a CO-NH, CO-S or SS bond.
A method for preparing an erythropoietin peptide, comprising the steps of:

(1) using a solid phase synthesis method, in accordance with the order of attachment of erythropoietin peptides, under the action of a coupling agent and a reaction solvent, sequentially coupling amino acids to synthesize a fully protected erythropoietin peptide;

(2) Cleavage to obtain erythropoietin peptide.
The production method according to claim 9, wherein the coupling agent is selected from one of the following combinations: (1) N, N'-diisopropylcarbodiimide and 1-hydroxyl group. Benzotriazole,

(2) benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 1-hydroxybenzotriazole and N,N'-diisopropylethylamine,

(3) 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N'-diisopropylethylamine,

(4) benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate, 1-hydroxybenzotriazole and N-methylmorpholine,

(5) 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N-methylmorpholine;

The reaction solvent is one or more selected from the group consisting of N,N'-dimethylformamide, dichloromethane, N-methylpyrrolidone, and dimethyl sulfoxide.
A pharmaceutical composition comprising a therapeutically effective amount of the erythropoietin peptide as defined in any one of claims 1-3 in free form or in a pharmaceutically acceptable salt form, or as claimed in any one of claims 4-6 The erythropoietin peptide derivative or the erythropoietin peptide polymer of any one of claims 7-8 as an active ingredient: one or more pharmaceutically acceptable carrier materials and/or diluents.
The erythropoietin peptide according to any one of claims 1 to 3, or the erythropoietin peptide derivative according to any one of claims 4 to 6 or the erythropoiesis production according to any one of claims 7-8 Prime Use of peptide polymers in medicaments for the treatment of diseases of erythropoietin deficiency or low or defective red blood cell populations.
The erythropoietin peptide according to any one of claims 1 to 3, or the erythropoietin peptide derivative according to any one of claims 4 to 6 or the erythropoiesis production according to any one of claims 7-8 Use of a peptide polymer in the treatment of diseases in which erythropoietin is insufficient or has a low or defective red blood cell population.