CN109336963B - Erythropoietin mimetic peptide dimer and preparation method and application thereof - Google Patents

Abstract

The application relates to a novel erythropoietin mimetic peptide dimer, a preparation method thereof, a pharmaceutical composition containing the erythropoietin mimetic peptide dimer and application of the erythropoietin mimetic peptide chemical dimer in medicines for preventing or treating diseases and/or symptoms related to low activity of EPO or EPO receptors or characterized by EPO deficiency or red blood cell deficiency or defect. The dimer can significantly improve the EPO receptor agonistic activity and the cell proliferation promoting activity. In addition, the dimer designed by the application does not need to protect a connecting arm in the synthesis process, so that the step of deprotection is not involved, the synthesis method is greatly simplified, the environmental pollution is reduced, and the dimer has obvious advantages in industrial production.

Description

Erythropoietin mimetic peptide dimer and preparation method and application thereof

Technical Field

The application relates to the field of biotechnology, in particular to a novel erythropoietin mimetic peptide dimer, a preparation method thereof, a pharmaceutical composition containing the same and application of the erythropoietin mimetic peptide dimer in medicines for preventing or treating diseases and/or symptoms related to low activity of EPO or EPO receptor or characterized by EPO deficiency or red blood cell deficiency or defect.

Background

Erythropoietin (EPO), a Hematopoietic Growth Factor (HGF), is a glycoprotein essential to the erythropoiesis. Contains 165 amino acids, has a molecular weight of about 30.4kD, and has a glycosylation modification result at 40% of the molecular weight, wherein the glycosylation sites are asparagine side chain amino N-glycosylation at positions 24,38 and 83 and serine side chain hydroxyl O-glycosylation at position 126. In humans, EPO is produced primarily in the kidney and fetal liver, synthesized and released by peritubular capillary endothelial cells of the renal tubules. EPO, as a cytokine, acts to promote erythropoiesis in the hematopoietic tissues of the bone marrow, thereby increasing the concentration of hemoglobin to ensure adequate oxygen transport from the lungs to aerobic tissues. Normally, EPO production is inversely related to the oxygen concentration in tissues, and increased EPO production during tissue hypoxia ensures that each tissue organ is adequately supplied with oxygen. When plasma EPO concentration is in the range of 10-25mU/mL, normal level of hemoglobin (12-17g/dL) can be maintained, and half-life is about 5 h. In addition to promoting the generation of red blood cells in hematopoietic tissues, EPO plays an important role in non-hematopoietic tissues and organs, such as promoting the growth and development of some tissues and organs, participating in the processes of formation, repair and repair of cardiovascular diseases and myocardial damage, and participating in the response process of brain to neuronal damage and the wound healing process.

In erythropoiesis, hematopoietic progenitor cells are first produced from bone marrow, and formed into Granulocyte (G) -erythroblast (Erythroid, E) -Monocyte (Monocyte, M) -megakaryocyte (Megakaryocytic, M) colony-forming units under the action of various cytokines, and then further differentiated into Erythroid colony-forming units (CFUe) through Erythroid burst-forming units (BFUe). EPO binds to EPO receptor (EPOR) on the surface of CFUe, forming erythroblasts, which further differentiate into reticulocytes, finally forming erythrocytes. This process requires not only nutrients such as iron, folic acid, vitamin B12, etc., but also many cytokines, and EPO is the most important regulator in erythropoiesis. Therefore, anemia is readily induced in the absence of EPO. Many secondary anemias are caused by insufficient production of the cytokine EPO. Studies show that EPO can be used for treating anemia caused by various reasons (Dailan, Chengning, research progress of recombinant human erythropoietin for treating anemia, Zhejiang clinical medicine, 9 th 6 th volume of 2004, 9 th phase, 827-.

In the 80 s of the 20 th century, the research on the treatment of secondary anemia has drawn attention, and through the research on the pathophysiology thereof, some Erythropoietic-stimulating Agents (ESAs) have been developed, mainly EPO receptor agonists. Since this time, EPO drugs have been a hot spot in drug research, the sales of EPO products continue to be in the fourth place of the best drug sold, and the EPO market continues to grow. The ESAs class of drugs has dominated the therapeutic market for secondary anemia.

In 1996, the EPO receptor was scanned with a combinatorial library of random peptide sequences using phage display technology to generate a number of short peptides that were not sequence related to endogenous EPO, were able to bind to EPO receptor, triggered a series of signal transduction, promoted proliferation of the corresponding cell line, and acted on the same mechanism as EPO. However, half the Effective Concentration (EC) of these short peptides₅₀) A minimum of 200nM, well above the EC of EPO measured at the same time₅₀(20pM) and, in addition, they are inferior in vivo stability and, therefore, cannot satisfy the drug-forming conditions. However, the discovery of these peptide sequences has been a guide for subsequent studies.

Chinese patent 201210170307.7 discloses an erythropoietin mimetic peptide represented by formula (1a) which has an excellent effect of promoting the proliferation of red blood cells, hemoglobin and reticulocytes:

since EPO receptor is activated by ligand-induced dimerization of EPO receptor, Oded Livnah was 1999 to establish from the crystal structure that the dimeric conformation of the ligand (i.e., erythropoietin herein) is also the key to the biological effect of agonizing EPO receptor (Science 1999, 283: 987-.

The subject group designs and synthesizes a series of dimers of the erythropoietin mimetic peptide with novel structures and the formula (1a), wherein the dimer is formed by connecting the mimetic peptide by connecting arms with different lengths and flexibility, and the obtained dimer has higher EPO receptor agonistic activity and obvious erythropoiesis, hemoglobin and/or reticulocyte proliferation activity.

Summary of The Invention

The subject finds that the length and flexibility of the erythropoietin mimetic peptide dimer connecting arm have important influence on the activity in the research process. It has also been surprisingly found that selection of specific linker arms significantly enhances EPO receptor agonistic activity as well as pro-cell proliferative activity. In addition, the dimer designed by the application does not need to protect a connecting arm in the synthesis process, so that the step of deprotection is not involved, the synthesis method is greatly simplified, the environmental pollution is reduced, and the dimer has obvious advantages in industrial production.

It is therefore an object of the present application to provide a polypeptide of formula (I):

wherein, P is erythropoietin mimic peptide 1 a:

R-(COOH)₂is a linker arm selected from succinic acid, glutaric acid, adipic acid, phthalic acid, isophthalic acid, terephthalic acid, phthalic acid, isophthalic acid, terephthallic acid, or diglycolic acid;

wherein the bridge between the two Cys in 1a represents a linkage via a disulfide bond; and the C-terminal Lys side chain amino group of each 1a forms an amide bond with the linker arm.

It is another object of the present application to provide a pharmaceutical composition comprising a polypeptide as described herein, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

It is another object of the present application to provide the use of a polypeptide as described herein, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament having EPO receptor agonist activity.

It is another object of the present application to provide the use of a polypeptide as described herein or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention and/or treatment of diseases and/or conditions associated with low EPO or EPO receptor activity or characterized by EPO deficiency or erythrocyte deficiency.

It is another object of the present application to provide a method for preparing a polypeptide of formula (I), comprising the steps of:

(1) preparing erythropoietin mimic peptide 1a by adopting a conventional polypeptide synthesis method;

(2) mixing R- (COOH)₂And erythropoietin mimetic peptide 1a in a ratio of 1: and (2-2.5) carrying out condensation to obtain the polypeptide.

Detailed Description

In one aspect, the application provides a polypeptide of formula (I):

wherein, P is erythropoietin mimic peptide 1 a:

R-(COOH)₂is a linker arm selected from succinic acid, glutaric acid, adipic acid, phthalic acid, isophthalic acid, terephthalic acid, phthalic acid, isophthalic acid, terephthallic acid, or diglycolic acid;

wherein the bridge between the two Cys in 1a represents a linkage via a disulfide bond; and the C-terminal Lys side chain amino group of each 1a forms an amide bond with the linker arm.

In some preferred embodiments, the polypeptide is selected from the group consisting of:

SEQ－01:

SEQ－02

SEQ－03

SEQ－04

SEQ－05

SEQ－06

SEQ－07

SEQ－08

SEQ－09

SEQ－10

in the polypeptides of the above formulae, the various amino acids or unnatural amino acids, if not specifically designated as chiral, are all in the L-form, the amino acid symbols are expressed according to the general rules, and the unnatural amino acid symbols are described in the specification.

Herein, the natural amino acids include alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met), glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (gin), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu).

In another aspect, the present application provides a pharmaceutical composition comprising a polypeptide as described herein, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier or excipient.

The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with the other ingredients of the formulation and not deleterious to the subject. The carrier herein refers to a substance for improving the selectivity, effectiveness and/or safety of a drug during delivery. The carriers are primarily used to control drug release and may also be used to improve the pharmacokinetic properties of the drug, particularly bioavailability. The excipient means other substances than the active ingredient in the pharmaceutical preparation, mainly for long-term stability, filling solid preparations (therefore, often used in particular as "fillers"), enhancing the therapeutic effect of the product (e.g., promoting absorption, reducing viscosity or improving solubility, etc.). Pharmaceutically acceptable carriers that may be employed in the pharmaceutical compositions of the present application include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Such excipients as binders, fillers, disintegrants, lubricants in tablets; wine, vinegar, etc. in the Chinese medicinal pill; base portion in semisolid formulations ointments, creams; preservative, antioxidant, correctant, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator, colorant, etc. in the liquid preparation.

Water is an exemplary carrier when the pharmaceutical compositions described herein are administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The pharmaceutical composition may also optionally contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents.

Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

The pharmaceutical compositions of the present application may act systemically and/or locally. Thus, it may be administered by a suitable route, for example by injection, intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or transdermal administration; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation. For these routes of administration, the pharmaceutical compositions of the present application may be administered in a suitable dosage form. Such dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, decoctions, oral liquids, powders, teas, vinuses, granules, pills, extracts, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

The dosage of the polypeptide or pharmaceutically acceptable salt thereof to be administered in the present application will depend on a number of factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, sensitivity and individual response of the patient or animal, the particular polypeptide or pharmaceutically acceptable salt thereof used, the route of administration, the number of administrations and the therapeutic effect desired. The above dosage may be administered in a single dosage form or divided into several, e.g., two, three, four dosage forms.

In another aspect, the present application provides the use of the polypeptide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament having EPO receptor agonist activity.

In some embodiments, the polypeptides of the present application have the effect of increasing red blood cell number and hemoglobin content in normal mice. Thus, in some preferred embodiments, the medicament is for promoting proliferation of red blood cells, hemoglobin, and/or reticulocytes.

In another aspect, the present application provides the use of said polypeptide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and/or treatment of diseases and/or conditions associated with low EPO or EPO receptor activity or characterized by EPO deficiency or red blood cell deficiency or deficiency.

In another aspect, the present application provides said polypeptide or a pharmaceutically acceptable salt thereof for use in the prevention and/or treatment of diseases and/or disorders associated with low activity of EPO or EPO receptor, or characterized by EPO deficiency or red blood cell deficiency or deficiency.

In another aspect, the present application provides a method for preventing and/or treating a disease and/or disorder associated with low EPO or EPO receptor activity, or characterized by EPO deficiency or red blood cell deficiency or deficiency, comprising the step of administering to a subject in need thereof an effective amount of a polypeptide as described herein or a pharmaceutically acceptable salt thereof.

In embodiments of the present application, the disease and/or disorder is anemia.

In some preferred embodiments, the anemia is selected from anemia arising from: erythrocyte deficiency, low erythrocyte count, low hemoglobin content, myelodysplastic syndrome, HIV infection, autologous blood collection, bone marrow transplantation, anemia resulting from hemoglobinopathy, renal anemia, anemia associated with tumors or cancer, anemia of prematurity, post-surgical anemia, maternal anemia, aplastic anemia, anemia resulting from chronic inflammation or infection.

In some preferred embodiments, the anemia resulting from hemoglobinopathy is, for example, thalassemia, sickle cell anemia.

In some preferred embodiments, the renal anemia is primarily anemia arising from chronic renal failure.

In some preferred embodiments, the tumor-or cancer-associated anemia can be caused by a variety of factors, which collectively include factors related to the tumor (e.g., blood loss, hemolysis, bone marrow invasion) or factors related to the treatment of the tumor (e.g., myelosuppression by chemotherapy, tumor radiotherapy, etc.).

The polypeptides of the present application have an agonist effect on the EPO receptor at the cellular level. Thus, in another aspect, the present application provides the use of the polypeptide or a pharmaceutically acceptable salt thereof for the manufacture of an agent for agonizing the EPO receptor in a cell. The polypeptides described herein or pharmaceutically acceptable salts thereof can promote the EPO receptor to exert its biological activity. In some preferred embodiments, the polypeptide or a pharmaceutically acceptable salt thereof is used in an in vivo method. In some preferred embodiments, the polypeptide or a pharmaceutically acceptable salt thereof is used in an in vitro method.

In another aspect, the present application provides said polypeptide, or a pharmaceutically acceptable salt thereof, for use in agonizing the EPO receptor in a cell. In some preferred embodiments, the polypeptide or a pharmaceutically acceptable salt thereof is used in an in vivo method. In some preferred embodiments, the polypeptide or a pharmaceutically acceptable salt thereof is used in an in vitro method.

In another aspect, the present application provides a method of stimulating an EPO receptor in a cell, comprising the step of administering an effective amount of the polypeptide or a pharmaceutically acceptable salt thereof to the cell.

In some preferred embodiments, the method is performed in vivo. In some preferred embodiments, the method is performed in vitro.

In another aspect, the present application provides a method for preparing a polypeptide of formula (I), comprising the steps of:

(1) preparing erythropoietin mimic peptide 1a by adopting a conventional polypeptide synthesis method;

(2) mixing R- (COOH)₂And erythropoietin mimetic peptide 1a in a ratio of 1: and (2-2.5) carrying out condensation to obtain the polypeptide.

In some preferred embodiments, the conventional polypeptide synthesis method in step (1) is selected from the group consisting of a solid phase polypeptide synthesis method, a liquid phase polypeptide synthesis method, and a solid-liquid phase polypeptide synthesis method. In some preferred embodiments, the conventional polypeptide synthesis method in step (1) is a solid phase polypeptide synthesis method. In some preferred embodiments, the solid phase polypeptide synthesis method comprises the steps of: 1) rink-amide resin is used as a solid phase carrier, HBTU-HOBt is used as a condensing agent, corresponding amino acid protected by Fmoc is used as a raw material according to the amino acid sequence of the erythropoietin mimic peptide 1a, and the peptide resin is synthesized according to a standard Fmoc solid phase polypeptide synthesis method; 2) reacting trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: a mixture of water (e.g., such five in a volume ratio of 8.25:0.5:0.5:0.25:0.5) as a cleavage solution, deprotecting the peptide and cleaving it from the resin at 0-40 ℃ (e.g., reaction at about 0 ℃ for about 30 minutes followed by reaction at room temperature for about 90 minutes); 3) dissolving the obtained peptide in a mixed solution of 20% DMSO/H2O, reacting for 1-3 days at room temperature, and purifying to obtain the erythropoietin mimetic peptide 1 a.

In some preferred embodiments, the condensation in step (2) is carried out in the presence of a condensing agent; preferably, the condensing agent is DCC, HOSu. In some preferred embodiments, the condensation in step (2) may be carried out under the following conditions: under ice-bath condition, 1 eq.R- (COOH)₂Dissolving in DMF, adding 1eq v.DCC and 1eq v.HOSu, reacting at room temperature for 6-12 hr, adding 2-2.5eq v. DMF solution of erythropoietin mimetic peptide 1a, reacting for 1-4 hr, and purifying.

The documents cited in the present application, the entire contents of which are incorporated herein by reference, and the statements of the present application shall control if the meaning of the documents expressed is inconsistent with the present application. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even so, it is intended that the present application be more fully described and interpreted herein, to the extent that such terms and phrases are not inconsistent with this known meaning and from the context in which such terms and phrases are expressed.

The term "about" as used herein, means that the numerical value so described is within a tolerance range allowed in the art, e.g., ± 10%, e.g., ± 5%, e.g., ± 2%.

As used herein, the term "effective amount" refers to a dose that achieves treatment, prevention, alleviation and/or amelioration of a disease or disorder described herein in a subject.

As used herein, the term "subject" or "patient" refers to an animal, particularly a mammal, e.g., a human, dog, monkey, cow, horse, etc., that receives a polypeptide or pharmaceutical composition of the present application to treat, prevent, alleviate and/or alleviate a disease, disorder, symptom described herein.

As used herein, the term "disease or condition" refers to a physical state of the subject that is associated with the disease or condition described herein.

As used herein, "%" refers generally to weight/weight percentages for total material being solid and weight/volume percentages for total material being liquid, unless otherwise specified. Of course, for the case where the total material is liquid and the solute is liquid, the percentages that characterize the liquid solute generally refer to volume/volume percentages.

The preparation of the polypeptide adopts a conventional polypeptide synthesis method, which comprises a solid-phase polypeptide synthesis method, a liquid-phase polypeptide synthesis method and a solid-liquid-phase polypeptide synthesis method, amino acid adopts Fmoc-/tBu-or Boc-/Bzl-protection strategy, the connection mode adopts the mode of sequentially connecting from N-terminal to C-terminal, or the mode of firstly synthesizing fragments and then connecting the fragments, the solid-phase synthesis adopts various resins capable of forming amide terminal as carriers (such as MBHA, PAL, Rink amide resins and the like), condensation reaction is carried out by various common condensing agents (such as DCC/HOBT, BOP/DIEA, HBTU/HOBt, TBTU and the like), and after the reaction is finished, the peptide is cut off from the resins by trifluoroacetic acid or without HF. Oxidizing two sulfydryl groups in a molecule to form cyclic peptide, separating and purifying to obtain single-chain peptide, then carrying out chemical reaction with a proper connecting arm to form a dimer product, and finally determining MALDI-TOF-MS (matrix-assisted laser Desorption-time of flight-Mass Spectrometry) of the product.

The term "pharmaceutically acceptable salts" as used herein refers to salts which retain the desired physiological activity of the parent compound without any unexpected toxic or side effects, or compositions containing them, such as: hydrochloride, hydrobromide, sulfate, phosphate, nitrate, and acetate, oxalate, tartrate, succinate, malate, benzoate, pamoate, alginate, methanesulfonate, naphthalenesulfonate, and the like.

Some abbreviations used in the present invention have the following meanings:

sar denotes N-methylglycine

Cit represents citrulline

1-Nal represents 3- (1-naphthyl) -alanine

Fmoc represents fluorenylmethyloxycarbonyl

Boc represents tert-butyloxycarbonyl

DMF means dimethylformamide

DCC represents dicyclohexylcarbodiimide

HOBt stands for 1-hydroxybenzotriazole

HOSu represents N-hydroxysuccinimide

DMAP for 4-N, N-dimethylaminopyridine

TFA represents trifluoroacetic acid

EDT stands for mercaptoethanol

HBTU represents 2- (1H-1-hydroxybenzotriazole) -1,1,3, 3-tetramethyluronium hexafluorophosphate

RP-HPLC stands for reversed phase high performance liquid chromatography

Other abbreviations not indicated have the meaning known in the art.

Wherein the structural formulas of Sar, 1-Nal and Cit are respectively as follows:

advantageous effects of the invention

The application provides erythropoietin mimetic peptide dimers, wherein two erythropoietin mimetic peptides are connected through a connecting arm with specific length and flexibility, and the dimers can obviously improve EPO receptor agonistic activity and cell proliferation promoting activity. In addition, the dimer designed by the application does not need to protect a connecting arm in the synthesis process, so that the step of deprotection is not involved, the synthesis method is greatly simplified, the environmental pollution is reduced, and the dimer has obvious advantages in industrial production.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is described in detail herein as possible.

The solid-phase synthesis carrier Rink-amide resin used in the examples is a product of Tianjin Nankai synthesis responsibility Co., Ltd; HOBT, HBTU, DIEA and Fmoc-protected amino acids were supplied by Gill Biochemical, Shanghai.

Example 1: synthesis of SEQ-01

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Peptide resins were synthesized using 1.0g of Rink-amide resin (0.50mmol) as a solid support, Fmoc-Ala-OH, Fmoc-Cit-OH, Fmoc-Cys (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-His (Trt) -OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Met-OH, Fmoc-1-Nal-OH, Fmoc-Pro-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Sar-OH, Fmoc-Val-OH as starting materials, HBTU-HOBt as a condensing agent, and based on the amino acid sequence of 1a standard Fmoc solid phase polypeptide synthesis method. With 30ml of trifluoroacetic acid: thioanisole: m-cresol: ethanedithiol: water (8.25: 0.5:0.5:0.25:0.5, vol.) was used as a lysis buffer, and the reaction was carried out at 0 ℃ for 30 minutes and at room temperature for 90 minutes, to deprotect the peptide and cleave it from the resin. The crude peptide is dissolved in 20% DMSO/H2O solution as a medium for forming disulfide bonds by oxidation, the concentration of the crude peptide is 0.1mM, and the reaction is carried out for 1-3 days at room temperature with stirring. The solution was purified by RP-HPLC. RP-HPLC conditions were: phase a, 0.05% TFA/water; phase B, 0.05% TFA/70% ACN/water; a chromatographic column: C18300A 4.6X 250 mm; gradient: b% 35-85 in 0-17 min, B% 85-35 in 17-21 min, and finishing in 25 min; flow rate: 1 mL/min; column temperature: at 25 ℃. The samples with purity of more than 95% are combined, concentrated and freeze-dried to obtain 280mg of pure peptide. MALDI-Tof-MS: 2353.3.

b) synthesis of SEQ-01

5.9mg (0.05mmol) of succinic acid is taken to be put into a 100ml eggplant-shaped bottle, 15ml of dry DMF is added to be completely dissolved, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed and put into the reaction, ice bath is carried out, and then the reaction is stirred at the temperature. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. The DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give 124mg of pure product of SEQ-01. ESI-MS: 4785.6.

example 2: synthesis of SEQ-02

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Synthesis of 1a was carried out in the same manner as in example 1.

b) Synthesis of SEQ-02

Glutaric acid 6.6mg (0.05mmol) is taken to be put into a 100ml eggplant-shaped bottle, 15ml dry DMF is added to be completely dissolved, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed to be put into the reaction, ice bath is carried out, and then the reaction is stirred at the temperature. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. The DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give 110mg of pure product of SEQ-02. ESI-MS: 4799.3.

example 3: synthesis of SEQ-03

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Synthesis of 1a was carried out in the same manner as in example 1.

b) Synthesis of SEQ-03

7.3mg (0.05mmol) of adipic acid is taken to be put into a 100ml eggplant-shaped bottle, 15ml of dry DMF is added to be completely dissolved, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed and put into a reaction, ice bath is carried out, and then the mixture is stirred for reaction after being warmed. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. The DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give pure product of SEQ-03 (140 mg). ESI-MS: 4813.6.

example 4: synthesis of SEQ-04

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Synthesis of 1a was carried out in the same manner as in example 1.

b) Synthesis of SEQ-03

8.3mg (0.05mmol) of phthalic acid is taken to be put into a 100ml eggplant-shaped bottle, 15ml of dry DMF is added to be completely dissolved, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed to be put into the reaction, ice bath is carried out, and then the reaction is stirred at the temperature. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. The DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give 78mg of pure product of SEQ-04. ESI-MS: 4834.6.

example 5: synthesis of SEQ-05

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Synthesis of 1a was carried out in the same manner as in example 1.

b) Synthesis of SEQ-05

8.3mg (0.05mmol) of isophthalic acid is taken to be put into a 100ml eggplant-shaped bottle, 15ml of dry DMF is added to completely dissolve the isophthalic acid, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed and put into a reaction, ice bath is carried out, and then the mixture is stirred at a high temperature for reaction. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give pure product of SEQ-05 (86 mg). ESI-MS: 4834.6.

example 6: synthesis of SEQ-06

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Synthesis of 1a was carried out in the same manner as in example 1.

b) Synthesis of SEQ-06

8.3mg (0.05mmol) of terephthalic acid is taken out and put into a 100ml eggplant-shaped bottle, 15ml of dry DMF is added to completely dissolve the terephthalic acid, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed and put into a reaction, ice bath is carried out, and then the mixture is stirred for reaction after being warmed. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. The DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give 91mg of pure product of SEQ-06. ESI-MS: 4834.6.

example 7: synthesis of SEQ-07

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Synthesis of 1a was carried out in the same manner as in example 1.

b) Synthesis of SEQ-07

9.7mg (0.05mmol) of o-phenylenediacetic acid is taken to be put into a 100ml eggplant-shaped bottle, 15ml of dry DMF is added to be completely dissolved, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed to be put into the reaction, ice bath is carried out, and then the mixture is stirred for reaction after being warmed. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. The DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give 87mg of pure product of SEQ-07. ESI-MS: 4861.6.

example 8: synthesis of SEQ-08

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Synthesis of 1a was carried out in the same manner as in example 1.

b) Synthesis of SEQ-08

9.7mg (0.05mmol) of m-phenylenediacetic acid is taken to be put into a 100ml eggplant-shaped bottle, 15ml of dry DMF is added to be completely dissolved, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed to be put into the reaction, ice bath is carried out, and then the mixture is stirred for reaction after being warmed. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. The DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give pure product of SEQ-08 (103 mg). ESI-MS: 4861.6.

example 9: synthesis of SEQ-09

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Synthesis of 1a was carried out in the same manner as in example 1.

b) Synthesis of SEQ-09

9.7mg (0.05mmol) of p-phenylenediacetic acid is taken to be put into a 100ml eggplant-shaped bottle, 15ml of dry DMF is added to be completely dissolved, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed to be put into the reaction, ice bath is carried out, and then the mixture is stirred for reaction after being warmed. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. The DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give 66mg of pure product of SEQ-09. ESI-MS: 4861.6.

example 10: synthesis of SEQ-10

a) Synthesis of Single-chain erythropoietin mimetic peptide (1a)

Synthesis of 1a was carried out in the same manner as in example 1.

b) Synthesis of SEQ-10

6.7mg (0.05mmol) of diglycolic acid is taken to be put in a 100ml eggplant-shaped bottle, 15ml of dry DMF is added to be completely dissolved, 20.6mg (0.1mmol) of DCC and 11.5mg (0.1mmol) of HOSu are weighed to be put into the reaction, ice bath is carried out, and then the mixture is stirred at the temperature. After 10 hours, 250mg (0.1mmol) of 1a was weighed precisely into a 50ml centrifuge tube, and 20ml of dry DMF was added to dissolve it, and the solution was added to the above reaction flask, and the reaction was stirred at room temperature for 1 hour. DMF was evaporated under reduced pressure and the residue was taken up in 10ml of 50% acetonitrile/water and purified by preparative high performance liquid chromatography to give 107mg of pure product of SEQ-02. ESI-MS: 4801.6.

biological experiments: evaluation of cell proliferation Activity mediated by EPO receptor

Methods for the in vitro detection of EPO activity are based mainly on the study of EPO-induced proliferation and/or differentiation of EPO-sensitive cells. TF-1 cells (available from GenScript) were just such a cell. The TF-1 cell line was first isolated from human erythroleukemia patients and highly expresses EPOR. TF-1 cells are dependent on granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 3(IL-3) for proliferation. EPO, however, also induces the proliferation of TF-1 cells and has been used as a generally accepted method for detecting EPO activity in vitro (T Kitamura, et al. blood. 1989, 73:375-380.S Chretien, et al. the EMBO J.1996, 15: 4174-4181). The EPO receptor-mediated erythropoietin mimetic peptides were assayed at the cellular level for their pro-cell proliferative activity. The results are shown in Table 1.

TABLE 1 erythropoietin mimetic peptide cell proliferative Activity

As can be seen from Table 1, the erythropoietin mimetic peptide chemical dimers of the present invention all have EPO receptor-mediated cell proliferation promoting activity, and the activity is 5 to 146 times higher than that of 1 a.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (16)

1. A polypeptide of formula (I) or a pharmaceutically acceptable salt thereof:

wherein, P is erythropoietin mimic peptide 1 a:

R-(COOH)₂is a linker arm selected from succinic acid, glutaric acid, adipic acid, phthalic acid, isophthalic acid, terephthalic acid, phthalic acid, isophthalic acid, terephthallic acid, or diglycolic acid;

wherein the bridge between the two Cys in 1a represents a linkage via a disulfide bond; and the C-terminal Lys side chain amino group of each 1a forms an amide bond with the linker arm.

2. The polypeptide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the polypeptide is selected from the group consisting of:

SEQ－01:

SEQ－02

SEQ－03

SEQ－04

SEQ－05

SEQ－06

SEQ－07

SEQ－08

SEQ－09

SEQ－10

3. use of a polypeptide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament having EPO receptor agonistic activity.

4. The use of claim 3, wherein the medicament is for promoting proliferation of red blood cells and/or hemoglobin.

5. The use of claim 3, wherein the medicament is for promoting proliferation of reticulocytes.

6. Use of a polypeptide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention and/or treatment of diseases and/or conditions associated with low EPO or EPO receptor activity or characterized by EPO deficiency or erythrocyte deficiency or defect.

7. The use of claim 6, wherein the disease and/or condition is anemia.

8. The use of claim 7, wherein the anemia is selected from anemia arising from: erythrocyte deficiency, low erythrocyte count, low hemoglobin content, myelodysplastic syndrome, HIV infection, autologous blood collection, bone marrow transplantation, anemia resulting from hemoglobinopathy, renal anemia, anemia associated with tumors or cancer, anemia of prematurity, post-surgical anemia, maternal anemia, aplastic anemia, anemia resulting from chronic inflammation or infection.

9. The use of claim 8, wherein the anemia arising from hemoglobinopathy is selected from the group consisting of thalassemia, sickle cell anemia.

10. The use of claim 8, wherein the renal anemia is anemia resulting from chronic renal failure.

11. The use of claim 8, wherein the tumor-or cancer-associated anemia is anemia arising from a tumor factor or anemia arising during treatment of a tumor.

12. The use of claim 8, wherein the tumor-or cancer-associated anemia is tumor-induced blood loss, hemolysis, or anemia resulting from bone marrow invasion.

13. The use of claim 8, wherein the tumor-or cancer-associated anemia is chemotherapy-induced myelosuppression or tumor radiotherapy-induced anemia.

14. A pharmaceutical composition comprising the polypeptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof.

15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable carrier or excipient.

16. A method for the preparation of a polypeptide according to claim 1 or 2, comprising the steps of:

(1) preparing erythropoietin mimic peptide 1a by adopting a conventional polypeptide synthesis method;

(2) mixing R- (COOH)₂And erythropoietin mimetic peptide 1a in a ratio of 1: and (2-2.5) carrying out condensation to obtain the polypeptide.