TWI344964B - Novel peptides that bind to the erythropoietin receptor - Google Patents

Description

Nine, invention description: [Yu ^ Ming belongs to the _45" field] The cross-references of the relevant applications for the United States Provisional Patent Application Nos. 60/469,993 and 60/470,244 request priority 'two cases are all in 2003 5 12 months. The contents of these prior matters are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION The present invention relates to peptide compounds which are erythropoiesis-derived || (EPO-R) agonists. The invention is also directed to a method of treating such a deficiency or defect-related disorder in the treatment of red blood cells using such a peptide compound. A pharmaceutical composition comprising the peptide compound of the present invention is also provided. [Prior Art 3 Background] Erythropoietin (EPO) is a glycoprotein hormone containing 165 amino acids having a molecular weight of about 34 kDa, and a preferred saccharification position at the amino acid position 24, 38 , 83 and 126. Originally produced as a precursor protein with a signal peptide containing 23 amino acids. EPO can appear in three forms: 邙, and asialo. The carbohydrate components of the alpha form and the yS form are slightly different, but have the same strength, biological activity and molecular weight. The Asialo form is 〇; or the 10,000-type terminal carbohydrate (sialic acid) is removed. The DNA sequence of the bat code EP0 has also been reported [U.S. Patent No. 4,703,008 to Lin]. EP0 stimulates mitosis and differentiation of red blood cell precursor cells' to ensure the production of red blood cells. When a tissue hypoxia occurs, 'erythropoietin is produced in the kidney. When Ep〇 induces red blood cell precursor cell differentiation, it induces protein synthesis, stimulates the synthesis of blood complexes, and increases the number of ferritin. These changes allow the cells to take up a larger amount of iron, and the successful hemoglobin, which combines mature red blood cells with oxygen. Such red blood cells and their hemoglobin play a key role in the body's oxygen supply. These changes are triggered by the interaction of EPO with appropriate receptors on the surface of the red blood cell precursor cells [eg eg Graber and Krantz (1978) Arm. Rev. Med. 29.51-66] » Although the body is in a healthy state, the number of tissues is from the number of existing red blood cells When adequate oxygenation is acceptable, EPO is present in the plasma at very low concentrations. This normal low concentration is sufficient to stimulate the red blood cells that are normally lost through aging. Under hypoxic conditions, when the oxygen transported by the blood cells in the blood circulation is reduced, the EPO content in the blood circulation is increased. Tissue hypoxia may be caused, for example, by substantial blood loss through bleeding, red blood cells destroyed by excessive exposure to radiation, decreased oxygen levels due to high altitude or prolonged coma, or various forms of anemia. In response to this tissue hypoxia stress, the EPO concentration rises and increases the production of red blood cells by stimulating red sputum cell proliferation. When the number of red blood cells circulating in the blood is greater than that required for normal tissue oxygen demand, the EPO concentration of the blood circulation decreases. Because EPO is required for erythropoiesis, this Epo hormone may be used for the diagnosis and treatment of blood diseases characterized by low red blood cell production or defects. Recent studies provide the basis for predicting the therapeutic efficacy of EPO for a variety of disease states, disorders, and blood irregularities, including: 10,000 Mediterranean anemia [Ref. Vedovato 荨人 (1984) Acta. Haematol. 71:211_213]; Fibrosis [Ref. vichinsky et al. (1984)] 1344964

Pediatric 105:15-21]; gestational disorders and menstrual disorders [Ref. Cotes et al. (193) Brit. J. Ostet. Gyneacol. 90:304-311]; early anemia in preterm infants [Ref. Haga et al. (1983) Acta Pediatr Scnad. 72; 827-831]; spinal cord injury [cf. Claus-walker et al. (1984) Arch Phys. Med. 5 Rehabil. 65: 370-374]; space flight [cf. Dunn et al. (1984) Eur. J Appl. Physiol. 52: Π 8-182]; acute blood loss [Ref. Miller et al. (1982) Brit. J. Haematol. 52: 545-590]; aging [cf. Udupa et al. (1984) J. Lab. Clin. Med. 103:574-580 and 581-588 and Lipschitz et al. (1983) Blood 63:502-509]; a variety of tumors associated with abnormal erythropoiesis 10 disease states [Reference Dainiak et al. (1983) ACNcer 5:1101- 1106 and Schwartz et al. (1983) Otolaryngol. 109:269-272]; and renal insufficiency [cf. Eschblach et al. (1987) N. Eng. J. Med. 316: 73-78]. The homogenized EPO after purification has been characterized (Hewick's U.S. Patent No. 4,677,195). The DNA sequence encoding EPO is purified, cloned, and expressed to produce recombinant polypeptides and natural EPO having the same biochemical and immunological properties. Recombinant EPO molecules having the same oligosaccharides as native EP0 have also been produced [see Sasaki et al. (1987) J. Biol. Chem. 262: 12059-12076]. The biological effects of Ε Ρ 显然 are clearly partly through interaction with the cell membrane receptor. Preliminary studies using immature red 20 cells isolated from mouse spleens, preliminary studies suggest that EPO-binding cell surface proteins contain two polypeptides, each with an approximate molecular weight of 85,000 Torr and 1 〇〇, aw 尔 吞 [Sawyer Et al. (1987) Proc. Natl. Acad. Sci. USA 84:3690-3694]. The number of EPO binding sites was calculated to average 800 to 1000 per cell surface. Of these binding positions, approximately 300 bound EP0 have 7 1344964 Κ3^90ρΜ (pico molar concentration), while the remaining bound EP〇 have a lower affinity of about 57〇pM [Sawyer et al. (1987) J. Biol. Chem 262:5554-5562]»Independent research suggests that the mouse is injected with the affluent germline (FVA) of Friend leukemia virus to prepare an EPO response, 5 spleen red mother cells, and a total of about 400 high EPOs. Combined position and low affinity

^ EPO binding sites have Kd values of approximately 100 pM to 800 pM, respectively

[Landschulz et al. (1989) Blood 73: 1476-1486]. p Subsequent research indicates that the two forms of EPO receptor (EPO-R) are encoded by a single gene. This gene has been selected for breeding [eg reference to J〇nes et al. 10 (1990) Blood 76, 31-35; Noguchi et al. (1991) Blood 78: 2548-2556; Maouche et al. (1991) Blood 78: 2557-2563 ]. example

The DNA sequences and coding sequences of the mouse EPO-R protein and the human EPO-R protein are described in PCT Publication No. WO 90/18822 to D'Andrea et al. This model suggests that EPO binds to EPO-R, resulting in dimerization and activation of two 15 EPO-R molecules, resulting in subsequent signal transduction steps [eg, see, for example, Watowich et al. (1992) Proc. Natl. Acad. Sci USA 89:2140-2144]. The EPO-R selection gene can be obtained to facilitate the study of agonists and antagonists of such important receptors. Recombinant receptor proteins can be obtained, allowing for the study of receptor-coordination interactions in a variety of random and semi-random peptide diversity production systems, including "peptides in colony" systems U.S. Patent No. 6,270,170; "Peptide in Phage" System [described in U.S. Patent No. 5,432,018 and Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382]; Synthetic Repository (ESL) system [described in U.S. Patent No. 8, 1 344,964, U.S. Patent Application Serial No. 946, 239, filed on Sep. 16, 1992; and "Extremely Large Scale Brake Polymer Synthesis System [described in U.S. Patent No. 5,143,854; PCT Notice Case No. 90/15070; Fodor et al. (1991) Science 251: 767-773; Dower and Fodor (1991) Ann. Ep. Med. Chem. 5 26:271-180; and U.S. Patent No. 5,424,186]. At least some degree of peptides of the EPO-R interaction have been identified, for example, in U.S. Patent Nos. 5,773,569, 5,830,851 and 5,986,047 to Wrighton et al.;\¥1^匕〇11, etc. (:1' Bulletin No. *0 96/40749; US Patent Nos. 5,767,078 and 10 PCT by Johnson and Zivin Prosecution No. 96/40772; PCT Bulletin No. WO 01/38342 to Balu; and WO 01/91780 by Smith-Swintosky et al. A special set of peptides containing a peptide moiety is identified and its members are bound to EPO-R, and stimulates EPO-dependent cell proliferation. However, the peptides that have been identified to date with the peptides can be stimulated by EPO-dependent cells in a test tube with an EC50 value of about 20 nanomolar (nM). Up to about 250 nM. This requires a peptide concentration of 20 nM to 250 nM to stimulate 50% of the maximum cell proliferation stimulated by EPO. It is known that EP Ο-R agonists have broad potential for the study of important biological activities of this receptor media, As well as for the treatment of diseases, it is still necessary to identify a peptide PEO ER-R agonist having enhanced ability and activity. The present invention provides such a compound. The references cited and/or discussed in this section and the entire specification are not clarified. The description of the invention is by no means an admission that the reference is a "prior art" of the present invention. 9 1344964 [Inventive 3 Summary of the Invention The present invention provides a novel peptide compound which is an EPO-R agonist with a sharp increase in strength and activity. The peptide compound is a homodimer having a amino acid sequence * 5 (AcG) GLYACHMGPIT (l-nal) VCQPLRK (SEQ ID NO: 1), a peptide monomer, or an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: φ 2) The homodimer of the peptide monomer' has an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR (MeG) (SEQ ID NO: 10 3) The homodimer of the peptide monomer; here each amino acid is represented by a standard one-letter abbreviation. "(AcG)" is N-ethinylglycine, "(i-nai)" is 1-naphthylalanine and "(MeG)" is N-mercaptoglycine also known as sarcosine. Each peptide monomer of the peptide monolayer contains an intramolecular disulfide bond between the cysteine residues of the monomer. 15 peptide monomers can be dimerized by covalent attachment to the branched third amine linkage. The third indole linkage can be expressed as: -c1o-ch2-x-ch2-c2o- ★ where x is nc〇-(CH2)2_n1h-; the c1 of the linkage group and the c-terminus of the first peptide monomer The ε-amine group of the lysine residue forms a guanamine bond; the bond group [2] forms a guanamine bond with the ε amine group of the amino acid residue at the c-terminus of the second -20 peptide monomer: and X The lanthanide is attached to the activated polyethylene glycol (PEG) moiety via an amine phthalate linkage or a guanamine linkage. Here PEG has a molecular weight of from about 20 000 to about 4 Torr. The term indicates that some molecules are heavier or lighter than the molecular weight when preparing PEG. 10 1344964 When each monomer of a homomeric binary body has an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and a N1 line of a linkage group is attached via an amine phthalate linkage By the activation of the polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention are represented as follows:

When the individual monomers of the homogeneous binary have an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the N1 system of the linkage is attached to the activated polyethylene via a guanamine linkage In the case of an alcohol (PEG) moiety, the novel peptide compounds of the invention are represented as follows:

(AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 11 1344964

2) When the N1 line of the bonding group is attached to the activated polyethylene glycol (PEG) moiety through the urethane linkage, the novel peptide compound of the present invention is expressed as follows:

-PEGjo^ok When the individual monomers of the homomorphic entity have the amino acid sequence 5 (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) and the N1 line of the linkage group through the guanamine When the linkage is attached to the activated polyethylene glycol (peg) moiety, the novel peptide compounds of the invention are expressed as follows:

The peptide monomer can be dimerized by covalent attachment to the branched third guanamine linkage. The third indole linkage can be expressed as: -c]o-ch2-x-ch2-c2o- where X is nco-(ch2)2-nh-c3o; the linkage of c1 with the first peptide The C-terminus of the body 12 1344964 forms a guanamine bond with the epsilon amine group of the amine acid residue; the C2 of the bond group forms a serotonin bond with the ε amine group of the amine acid residue at the C-terminus of the second peptide monomer. The peptide peptide of the present invention further comprises a spacer moiety of the formula: -βΗ-ΚΗΑ-θΗ-Ν2!!- 5 where C4 is a spacer covalently bonded to C3 of X; Covalently attached to the activated polyethylene glycol (PEG) moiety via a urethane linkage or a guanamine linkage; and the N2 linkage of the spacer is covalently attached via an amine phthalate linkage or a guanamine linkage Connected to the activated PEG moiety, where PEG has a molecular weight of about 10, 〇〇〇 to about 50,000 Torr (the term "about" means that certain molecules can be heavier or lighter than the molecular weight when preparing PEG. ). Each PEG moiety is individually 10,000 Doha (10 kD), 20 kD, 30 kD, 40 kD or 50 kD. When each monomer of the homodimer has an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the spacers N1 and N2 are attached via an amine phthalate linkage By the activation of the polyethylene glycol diol (PEG) moiety, the novel peptide compounds of the invention are represented as follows:

In a preferred embodiment, the C-terminal amino acid of the dipeptide monomer is an amine acid. Those skilled in the art are aware of two linear 15 £s from the aforementioned chemical structural formula; (3 part 13 1344964 is linked by an aminic acid (for example, mPEGrLys-NHS or mPEG2-Lysinol-NPC), also preferably L-amine For the acid, the following stereochemistry was obtained.

(AcGJGLYA^HMGPnXl-NaOviQPLR-NH^L.^ A

5 Further, one or more of the amino acid residues may be a stereo compound which is known to those skilled in the art from D to the amine acid. When each monomer of the homomorphic entity has an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the spacers N1 and N2 are attached to the activated poly In the case of the ethylene glycol® 10 (PEG) moiety, the novel peptide compounds of the present invention are expressed as follows: • '

V x^-PEGk^cok ο PEGi〇^〇k 14 1344964 Again, the lysine molecules of the present compound are preferably L-amino acids, and the following stereochemistry is obtained.

Further, the one or more lysine residues may be a stereo compound known to those skilled in the art from D to the amine acid. When each monomer of the homodimer has an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) and a spacer N] and N2 are all permeable to the uric acid When the ester linkage is attached to the activated polyethylene glycol (PEG) moiety, the novel Lai Sheng skin compound of the present invention is expressed as follows:

Preferably, the molecules of the conjugated peptide monomer and the linear PEG moiety are all from the amine acid residue of the L-amino acid. The following stereochemistry is obtained: 15 1344964

P£G10_5dk

Further, one or more of the amino acid residues may be a stereo compound which is known to those skilled in the art from D to the amine acid. When each monomer of the homodimer has an amino acid sequence 5 (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) and the spacers N1 and N2 are linked through a guanamine linkage When attached to an activated polyethylene glycol (PEG) moiety, the novel peptide compound of the invention is represented as follows: 0 (AcGXH-YACHMGPrr(l-ittl)VCQPLR(Mei

〇 Ο V NH

Ε^ιο^οκ

NH ·· ΗΝ xj^^PEGi tte〇K Ο (AcOjOLYAiptMGHTO^naDV^QPLRtMeGy^H)/ Preferably, the amino acid residues of the conjugated peptide monomer and the linear PEG moiety are L-amino acid. The following stereochemistry. 16 1344964

In other embodiments, one or more of the lyophilic acid residues may be D-amino acid obtained from other stereochemical compounds known to those skilled in the art. The peptide monomer can also be dimerized by attachment to an amide linkage, whereby the C-terminus of the 5 peptide monomers is attached to the epsilon amine group of the amine acid, and the second peptide monomer The C-terminus is attached to an alpha amine group of an amine acid. The peptide peptide of the present invention further comprises a spacer moiety of the formula: -N1H-(CH2)2-0-(CH2)2-N2H- 10 at one end, and the spacer N1 is attached via a guanamine bond Attached to the carbonyl carbon of the amine linkage. At the opposite end, the N2 of the spacer is attached to the activated polyethylene glycol (PEG) moiety via an amine phthalate linkage or a urethane linkage, where PEG has a molecule of about 20, 〇〇〇 to about 40,000 channels. Er (the term "about" means that some molecules may be heavier or lighter than the molecular weight when preparing pEG). 15 When the spacer is attached to the activated polyethylene glycol (PEG) moiety via an amino phthalate linkage, the novel peptide compound of the invention is represented by: 17 1344964

〇-peg2IM0K

Mwt When the spacer is attached to the activated polyethylene glycol (peg) moiety via a chiral linkage, the novel peptide compound of the invention is represented by:

The invention further provides pharmaceutical compositions comprising such peptide compounds, and methods of using the peptide compounds to treat various medical conditions. I: Embodiments The detailed description of the preferred embodiment is defined as follows: The amino acid residue of the peptide is abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is I 〖e or I; methionine is Met4M; retic acid is Val or V; serine is Ser or S; proline is pro or p; threonine is Thr or T; alanine is Ala or A; Tyrosine is Tyr or Y; histidine is His or Η; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Arg or D; glutamine The acid is Glu or E; the cysteine is Cys or C; the tryptophan is Trp or w; the arginine is Arg or R; and the glycine is Gly or G. The non- conventional amino acid abbreviation of the peptide is as follows: naphthylalanine is Ι-nal or Np; N-methylglycine (also known as sarcosine) is]yieG or 18 15 1344964

Sc; and acetylated glycine (N-ethinylglycine) are AcG. As used herein, the terms "polypeptide" or "protein" refer to a polymer of an amino acid group in which an alpha amino acid is bonded via a guanamine linkage. Thus the polypeptide is at least two amino acid residues in length, usually longer. Generally, the word "peptide" is a polypeptide having a length of only a few amino acid residues. The novel EPO-R agonist peptide of the present invention preferably has a length of no more than 50 amino acid residues. More preferably, the length is from about 17 to about 40 amino acid residues. In contrast to the peptide, the polypeptide may comprise any number of amino acid residues. Thus the term polypeptide includes both the peptide and the longer amino acid sequence. 10 As used herein, the term "pharmaceutically acceptable" means that the molecular entity and composition "usually considered safe" is, for example, physiologically acceptable, and typically does not produce an undesired reaction, such as an allergic reaction. It does not cause stomach upset, dizziness, etc. when given to the human body. Preferably used herein, the term "pharmaceutically acceptable:" means approved by the federal or state government, or listed in the US Pharmacopoeia or other recognized pharmacopoeia for use by animals, particularly humans. The term "carrier" means a diluent, adjuvant, excipient or vehicle with which the compound is administered. The Zhuo Le learning carrier can be a non-functional liquid such as water and oil, including oils, animals, plants or oils of various origins, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or an aqueous solution, an aqueous salt solution and an aqueous dextrose solution and a 20 glycerin solution are preferably used as a carrier, particularly for injectable solutions. The appropriate pharmaceutical carrier is described in "Remington Pharmaceutical Sciences" by E.w Martin. The term "agonist" as used herein refers to a biologically active ligand that binds to its complementary biologically active receptor and activates the receptor, initiates a biological response of the receptor, or enhances the existing receptor biological activity. 19 1344964 Novel oxime peptides belonging to ΕΡΟ-R agonists The present invention provides novel peptide compounds which are EPO-R agonists with a dramatic increase in strength and activity. These peptide compounds are homodimers of a *5 peptide monomer having an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1), or have an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) is a homodimer of the peptide monomer; here each amino acid is represented by the standard single | letter abbreviation. "(AcG)" is N. acetylglycolic acid, "(Ι-nal)" is 1-naphthylalanine and "(MeG)" is N-methylglycine, also called sarcosine. Each peptide monomer of the 10 peptide peptide contains an intramolecular disulfide bond between the cysteine residues of the monomer. These monomers can be schematically represented as follows: (AcG)GLYA<!:HMGPrr(l*Ml)V(!;〇PLRK(AcG)GLYACHMGPIT(l-nal)VCQPLRK^ _ or - ;

(AcG)GLYA(lHMGPm(lHial)viQPLRiMeaiK(AcG)GLYACHMGPIT(ln#l)VCQPLK<MeG)K or these peptide monomers are dimerized to provide a peptide II with enhanced EP〇_R agonist activity Yuan body. The linker (LK) moiety is a branched third guanamine which bridges the C-terminus of the dipeptide monomer via simultaneous attachment to the C-terminal amine acid residue of each monomer. The third indole linkage can be expressed as: m -C,0-CH2-X-CH2-C20- / where X is ΝΟΟ-βΗ2)2-:^!!-; the linkage group c1 and the first The c-terminus of the peptide monomer forms a guanamine bond from the epsilon amine group of the amine acid residue; the c2 of the bond group forms a guanamine bond with the ε amine group of the second 20-peptide monomer terminal and the amino acid residue And X Ν 1 is attached to the activated polyethylene glycol (PEG) moiety via a urethane linkage or a guanamine linkage. Here PEG has a molecular weight of from about 20,000 to about 40,000 channels 20 1344964 ernt (" The term "about" indicates that some molecules are heavier or lighter than the molecular weight when preparing PEG). The hydrazine amine linkage can be expressed as: -C^O-CHyX-CHa-C2. - 5 where X is NC0-(CH2)2-NH-C30; the C1 of the bond group forms a guanamine bond with the amine group of the C-terminus of the first peptide monomer from the amine acid residue; C2 forms a guanamine linkage with the epsilon amine group of the amino acid residue at the C-terminus of the second peptide monomer. The peptide peptide of the present invention further comprises a spacer moiety of the following structural formula: -βίΗα^νθΗ-Ν2^ 10 where the c4 group of the spacer is covalently bonded to C3 of X; the Ν1 of the spacer is permeable to the amine group An acid ester linkage or a guanamine linkage is attached to the activated PEG moiety, where PEG has a molecular weight of from about 20,000 to about 40,000 Dolphant (the term "about" means that some molecules may be heavier than the molecular weight when preparing the PEG. Or lighter). Thus, the novel peptide of the present invention also contains a PEG moiety which is covalently attached to the third guanamine linkage of the peptide binary via a urethane linkage or a guanamine linkage. PEG is a pharmaceutically acceptable water soluble polymer. The PEG used in the present invention may be a linear non-branched pEG having a molecular weight of about 20 kD (20K) to about 60 F (the term "about" is used in the preparation of PEG, and some molecules are heavier than the molecular weight, some Lighter than the molecular weight). The preferred PEG 20 has a molecular weight of from about 30K to about 40K. A skilled artisan can select the desired polymer size based on the following considerations: for example, the desired dose; cycle time; resistance to protein breakdown; growth; effects on biological activity (if any); ease of treatment; degree of antigenicity or lack of antigen And other effects known to be therapeutic peptides. The peptide of the present invention, the peptide binary and other peptide-based molecules can be used in any of a variety of chemistries to bond a water-soluble polymer to a receptor binding moiety of a molecule (eg, a peptide + spacer). Attached to a water soluble polymer (eg PEG). A typical embodiment uses a single attachment joint to covalently attach a water soluble polymer to the receptor binding portion, but in other embodiments, multiple attachments can be used, including various variations, among which different types of water soluble polymers A separate attachment joint attached to the jaw engagement portion can include covalent attachment to the spacer and/or to the one or two peptide chain. In a number of specific examples, a binary or a more versatile polymorph will contain a plurality of different winning chains (i.e., non-homologous or other non-homogeneous plurals). By way of example and not limitation, the binary may comprise a first peptide chain having a PEG attachment junction, and a second peptide chain lacking a PEG attachment linkage, or utilizing a first peptide chain Different linkage chemistry 'and in some variations, the spacer may contain or lack a pEG attachment junction, and the spacer may utilize a different bond than the first peptide chain and/or the second peptide chain based on PEGylation Another specific example uses a PEG attached to the spacer portion of the acceptor binding 15 moiety, a different water soluble polymer (e.g., carbohydrate) | a branch that is conjugated to one of the amino acids of the molecular peptide moiety. A variety of polyethylene glycols (PEG) can be used for PEGylation of the receptor binding moiety (peptide + spacer). Essentially any suitable reactive PEG reagent can be used. In a preferred embodiment, when the reactive PEG reagent is conjugated to the acceptor binding moiety, hydrazine 20 will result in the formation of a urethane linkage or a guanamine linkage. Suitable reactive PEGs include, but are not limited to, the NOF Corporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome, Shibuya-ku 'Tokyo 150-6019) drug delivery system catalogue (2003), and Molecular Engineering Catalogue (2003) by Nektar Therapeutics Inc. (490 Discovery Avenue, Huntsville, Alabama) purchased 22 1344964

Got it. For example, but not by way of limitation, the following PEG reagents are often preferred in a number of specific examples: mPEG2-NHS, mPEG2-ALD, multi-arm PEG, mPEG (MAL) 2, mPEG2 (MAL), mPEG-NH2, mPEG-SPA , mPEG-SBA, mPEG-sulfuric acid, mPEG-complex vinegar, mPEG-BTC, mPEG-ButyrALD, 5 mPEG-ACET, heterofunctional PEGs (NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS , NHS-PEG-VS, NHS-PEG-MAL), PEG acrylate (ACRL-PEG-NHS), PEG-phospholipid (eg mPEG-DSPE), SUNBRITE series of multi-arm PEGs including cooked 谙Chemically activated glycerol-based GL series of 10 PEGs, glycerol-activated PEGs (including but not limited to carboxyl-PEGs 'p-NP-PEGs, Tresyl-PEGs, aldehyde PEGs, acetaldehyde) -PEGs, Amino-PEGs, Mercapto-PEGs, Maleimide-PEG-PEGs, Hydroxy-PEG-amines, Amino-PEG-COOH, Hydroxy-PEG-aldehydes, Carboxylic anhydrides-PEG, Functional PEG-phospholipids and other similar and/or suitable reactive PEGs selected by those skilled in the art for specific applications and uses. The novel peptide of the present invention also contains a diPEG moiety which is covalently attached to the spacer moiety via an aminoester linkage or a guanamine linkage, wherein the spacer moiety is covalently bonded to the peptide II The third indoleamine linkage of the moiety. The two PEG moieties used in this particular embodiment of the invention are each linear and can be bonded at 20 single attachment points. Each PEG moiety preferably has a molecular weight of from about 1 kilogram to 10 kilograms (about 10K) to about 60K (the term "about" means that some molecules may be heavier or lighter than the molecular weight when preparing peg) and the linear PEG moiety It is especially good. More preferably, the two PEG moieties each have a molecular weight of from about 20K to about 40K, and more preferably from about 20K to about 40K. Still more preferably, the two PEG moieties each have a molecular weight of about 20K. A skilled artisan can select the desired polymer size based on considerations such as desired dosage, cycle time; resistance to protein breakdown; effects on biological activity (if any); ease of handling; degree of antigenicity or lack of antigenicity; The effect of PEG on therapeutic peptides is known. 5 The present invention also encompasses a peptide agonist which is a homodimeric monomer having a peptide monomer having an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR (MeG) (SEQ ID NO: 3), each of which is The amino acid is represented by a standard abbreviation, "(ACG)" is N-acetylglycine, "(1 _nai)" is 1-naphthylalanine and "(MeG)" is N-methyl. Glycine, also known as myosin 10 acid. Each peptide monomer of the peptide binary contains an intramolecular disulfide bond between the cysteine residues of the monomer. Such a monomer can be schematically represented as follows: (AcG)OLYA<!HMGPIT(l-Ml)viQl>LR(MeG) or (Ac〇)GLYACHMGFrr(lP«l)VCQPm(MeO) Dimerization provides a peptide peptide with enhanced EPO-R agonist activity. The linking group (LK) moiety is an amino acid residue, and the linking group 15 is partially attached to the C-terminal amino acid of each monomer to bridge the C-terminal peptide monomer of the two peptide monomers. The C-terminus is attached to the epsilon-amine group of the amine acid, and the C-terminus of the second peptide monomer is attached to the a-amine group of the amine acid. For example, a binary body can be shown in formula I and the summary is shown in formula II:

Formula II

24 20 1344964 In the formulae I and II, N2 represents a nitrogen atom of the epsilon amine group of the amine acid, and Ν1 represents a nitrogen atom of the α-amino group of the amine acid. The peptide peptide of the present invention further comprises a spacer moiety of the formula: 5 ^^-(^2)2-0-(^2)2^3⁄4 at one end, the spacer N1 is linked through a guanamine linkage Attached to the carbonyl carbon of the amine linkage. At the opposite end, the spacer N2 is attached to the activated polyethylene glycol (PEG) moiety via an amine phthalate or guanamine linkage, where PEG has a molecular weight of from about 10,000 to about 60,000 dolphine ("about" one The words indicate that some molecules may be heavier or lighter than the molecular weight when preparing peg 10.) More preferred PEGs have a molecular weight of from about 20,000 to 40,000 dolphins. Thus, the novel peptide of the present invention also contains a PEG moiety which is covalently attached to the peptide binary. PEG is a pharmaceutically acceptable water soluble polymer. The PEG used in the present invention may be a linear non-branched PEG having a molecular weight of from about 20 kilo Torr 15 (20K) to about 60K (the term "about" is used in the preparation of pEG" and some molecules are heavier than the molecular weight. Some are lighter than the molecular weight). The preferred PEG has a molecular weight of from about 20K to about 40K, and more preferably has a molecular weight of from about 30K to about 40K. A skilled artisan can select the desired polymer size based on the following considerations: for example, the desired dose; cycle time; resistance to protein breakdown 20; effect on biological activity (if any); ease of handling; degree of antigenicity or lack of antigenicity And other effects known to be therapeutic peptides. When the individual monomers of the homogeneous binary have an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the N1 line of the linkage is attached to the activation via a urethane linkage In the case of the polyethylene glycol 25 1344964 (PEG) moiety, the novel peptide compounds of the invention are represented as follows:

When the individual monomers of the homogeneous binary have an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and the N1 system of the linkage is attached to the activated polyethylene via a guanamine linkage In the case of the alcohol (PEg) moiety, the novel peptide compounds of the present invention are expressed as follows: (AcO)OLYACHMGPIT(l-naJ)VCQPLS:

NH t^EG2〇-4〇k

(AcG)GLYA<&iMGPIT(l*nel)viQPI When the individual monomers of the homomorphic entity have an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 10 2) And the N1 line of the linkage group is attached to the activated polyethylene glycol (PEG) moiety through a urethane linkage, and the novel peptide compound of the present invention is represented by the latter 26 2634464

-PEG2O40K When the individual monomers of the homoduplex have an amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K (SEQ ID NO: 2) and the N1 system of the linkage group is attached via a guanamine bond Upon addition to the activated polyethylene glycol (PEG) 5 moiety, the novel peptide compounds of the present invention are represented as follows: Ο (AeG)OLYA<iHMGPrT(I-nal)vlQPLR(MeG). ο Ο 〇20-ΜΚ

Ο (AcO) GLYACHMGPm^n^) VCQPLR (MeG) The preferred peptide binary of the present invention includes, but is not limited to: 27 1344964

(AcG)GLYACHMGPrT(l^iaI)VCQPI Ο I 〇

V NH -FEGjok

(AcG)GL YACHMOPITY1 -naQVCQPI

(AcOGLYAdHMGPrTil-nalJVCQPI

O

°y NH 0 〇 -PEGsok (AcG)GLYACHMGPIT( l -nal) VCQPLR-

O

(AcG)GLYA<!hMGPIK1 -naJ)viQPLR-NH JL 〇 〇

(AcG)GLYACHMGPIT(t-nal)VCQPLR-HN ^-NH2 28 1344964 (AcG)GLYACHMGPrr(l -nal)V6QPLR- 0

NH (AcG)OLYACHMGPIT(l-Dal)VajPLR-HN ^

V NH

Ni£G

20K (AcG)GLYA(iHMGPIT(l-nal)VCQ]

0 PLR-NH

NH

°v NH 〇 0

EG

30K (AcG)GLYACHMOPIT(l-nal)VCQPLR-

O

(AcG)GLYA(iHMGPriXl-nel)viQPLR-NH JL

NH2

NH (AcG)GLYACHMGPIT(l-nal)VCQPLR-

O

V NH 〇 0

4QK 29 1344964

30 1344964 (AcG)GLYAiHMOPnr(卜mW:QPLR(McGVnh Ο Ν °ν ,ΝΗ Ο

〇〇2〇κ (AcG)GLYACHMqPlT(l-nal)VCQPLR(MeG)-

Ο

(AcG)GLYACHMGPIT(l-nBl)viQPLR(MeG)-NH Ο (AcG)GLYACHMGPIT(l-na〇VCQPLR(MeG) ~ΗΝ

Ο V , ΝΗ U ° EGsqk

(AcG}GLYA(£M(3Tr(I-Ml)viQI»LR(MeG)-NH JL

V ΝΗ π ° (AcG)OLYACHMGPrr(l-na〇VfQPU^MeG)~HN_

Ο When each monomer of the homodimer has an amino acid sequence (AcG) GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1) and 31 1344964, the β and N2 of the spacer are all permeable to the urethane bond. When attached to an activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention are expressed as follows:

0 —PEG10h5〇K Ο V ,ΝΗ

, O-PEG1 < W0K When each monomer of the homomorphic entity has an amino acid sequence 5 (AcG) GLYACHMGPIT (l-nal) VCQPLRK (SEQ ID ΝΟ _ 1) and the spacers Ν 1 and Ν 2 are all passed through the guanamine When the linkage is attached to the activated polyethylene glycol (PEG) moiety, the novel lysin compound of the present invention is expressed as follows:

(AcG)GLYA<lHMGPniCl-iial)V<!:QPLR-NH IL PEGi〇^s〇k \ HNG

阳T(Ac〇)GLYACHMGPIT(ln«l)VCQPLR--HN/ When the individual monomers of the homomorphic entity have the amino acid sequence 10 (AcG) GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO 2) and when the spacers N1 and N2 are attached to the activated polyethylene glycol (PEG) moiety through a guanamine linkage, the novel peptide compounds of the present invention are expressed as follows: 32 1344964

(AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 5 2) and the spacers N1 and N2 are attached to the activated polyethylene glycol (PEG) moiety via a pull amine linkage. The novel peptide compounds of the present invention are expressed as follows:

Preferred peptide peptides of the invention include, but are not limited to:

33 1344964

34 1344964

(AcO)GLYA(iHMGPIT(l-Da))V(!:QPL Ο 〇 〇 PEG PEG50K ΝΗ O'/O—EGsok ^ΝΗ 〆 ΗΝ, one n_f

(AcG)GLYACHMGPIT(l-na])VCQPLR-HN

Ο τ (AcG)GLYA« dHMGPIT(l-nal)viQPL]

R-NH Ο ΝΗ ΝV ΝΗ Ο

(AcG)GLYACHMGPrr(l-iial)VCQPLR-HN ^

ο °γhn^,peg10K Ο

PEG10K (AcG)GLYA<fHMGPm(i-iial)viQPLR-h Ο

ΝΗ,.〇 9 0 〇yPEG-, Ν, Λ\, 八/ΝΗ (AcG) GLYACHMGPIT(1-dsI)VCQ; »PLR—ΗΝ

Ο V ΝΗ

ΗΝ^ΡΕ〇2〇κ 35 1344964 (AcG)GLYA^HMGPITXl-nal)V<!x?PLR- 0 Q 0 (AcG)GLYACHMGPIT(i-nal)VCQPLR-HN )‘

V々 Y HN 丫 PEG

V / NH

SQK

(AcG)GLYA(!HMOPIT(l-iial)V(i:Q!»LR-NH^ JL °v m (AcG)GLYACHMGPIT(l-na1)VCQPLRr

o (AcO)GLYA(iHMGPIT(l^al)VCQ]

• PLRHNH

O

NH

(AcG)GLYACTMGPrr(l-nal)VCQPLR—HN

peg40K

V Ο o PEG40k

X 〇 0

V PEG.

NV NH

O

, NH

HN^PEG50K O 36 1344964

37 1344964 (AcC)GLYAc!HMGPrr(l-nal)VCQPLR(MeG)—NH,

NH 9 0 V* • peg4 Ϊ HN^o—PEG40K 0

(AcO)GLYACHMGPIT(l-nal)VCQPLR(MeG>-HN (AcG)GLYAcWGPIT(l-naI)VCQPLR(McG)-

〇 O O-PEGsok

0Y HNx^o-peG5〇K

(AcG)GLYACHM〇Prr(l-f»al)VCQPLR(McG)—HN (Ai^^YACHMGPIT(l-naDV([QPLR(McG)-

O V°〇 O VY hn^peg10K o (AcG)GLYACHMGPIT(l -naJ: ^VCQPLR(MeG)-HNy 38 1344964

39 1344964

When the spacer is attached to the activated polyethylene glycol (PEG) moiety through a urethane linkage, the novel peptide compound of the invention is represented by: (AcG)GLYACHMGPIT(l -Ml)V(!:QPLR( MeG). (AcG)GLYA<piMGPrr〇~Ml>V〒QPLR(MeG),

ο -ο Ο Λ ο -PEG2M0k

When the spacer is attached to the activated polyethylene glycol (PEG) moiety via a chiral linkage, the novel peptide compound of the invention is represented by: (AcG)GLYA(?HMGPnXl-nal)viQPLR(MeG)-

(AcG)GLYACHMGPIT(l-nal)VCQPLIKMeG)-HN

Κκ\

PEG20"40K-Ο This binary structure can be written as [Ac-peptide, disulfide] 2Lys-spacer-PEG2 (M()K to indicate N-terminal acetylated peptide bonding to lysine Both the alpha amine group and the 10 ε amine group, each peptide contains an intramolecular disulfide loop and a spacer molecule forms a covalent linkage between the C-terminus of the amine acid and the PEG moiety, where the PEG has a molecular weight of about 20 , 〇〇〇 to about 40,000 dolphine. 40 1344964 Preferably, the peptide peptide of the present invention includes, but is not limited to: (AcO) GLYACHMGPIT (

Unal)V(!:QPLR(MeG)-NH Ο人

O-PEG

20K Ο Ο

(AcG)GLYACHMGPnxl-naI)VCQPUt(MeG) -HN -ο (AcG)GLYACHNTCPIT(l.iial)V0QPLRCMeG)- (AcG)GLYACHMGPIT(i-nal)va3PLR(MeG) •ΗΝ ΝΗ

Ο -ο Λ-,

EG30K ο (Αςσ)ΟΙΎΑ<ίΗΜΟΡ1Τ(1-ω1>νί(3ΡΙΛ(ΜβΟ)·又 〇-peg4〇k ο

(AcG)GLYACHMGPrr(l -nal)VCQPLR(McG)-HN -Ο (AcG)GLYAi

c!HMGPlT(l-nal)viQPLR(MeG)H (AcG)GLYACHMGPIT(l-iud)VCQPLR(MeG) -ί

·〇 ερε, ©20Κ Ο -ο (AcQ)GLYACHMGPm(l-nal) VCQPLR(MeG)- Ο人 (AcG)GLYACHOVlGPnXl-nal)VCQPLR(McG) ·ί

ο ο O^EG2〇k •ο (AcG)GLYACHMGPrT(l-nal)VCQPLR(McO)- 〇Λρ

EG40K Ο Ο (AcO) GLYACHMGPrT (l-naJ) VCQPLR (McG) · • Ο 41 20 stereoisomers of conventional amino acids (such as D amino acids), unnatural amino acids such as a, a- Disubstituted amino acids, N-alkylamino acids, lactic acid, and other non-conventional amino acids are also suitable components of the compounds of the present invention. Non-proprietary amino acids include, for example, but are not limited to, cold-alanine, 3-pyridyl alanine, 4-hydroxyproline, 〇-phosphosine, N-methylglycine, N-ethyl basal acid 'N-methyl methionine, 3-methylhistamine, 5-hydroxy lysine, neoleucine and other similar amino acids and imino acids. Other modifications are also possible, including modification of the amino terminus, modification of the carboxy terminus, substitution of one or more of the native gene encoding amino acids with a non-conventional amino acid, modification of one or more amino acid residue branches, Peptide phosphorylation and the like. The peptide sequence of the present invention may be present alone or in combination with the N-terminal elongation and/or C-terminal elongation of the peptide. Such extension may be such that the naturally-encoded peptide sequence optionally contains a non-native sequence or is substantially free of non-native sequences; elongation may include any addition, censoring, point mutation or other sequence modification or combination as desired by those skilled in the art. For example and without limitation, the native sequence can be a full length sequence or a partial length sequence, and can include amino acid substitutions to provide attachment of carbohydrates, PEG, other polymers, and the like through branching conjugates. In a variant, the amino acid substitution results in a sequence that is humanized to become compatible with the human immune system. All types of fusion proteins can be provided, including immunoglobulin sequences that are adjacent to or near the EPO-R activation sequence of the invention with or without non-immunoglobulin spacer sequences. A specific example is the immunoglobulin chain containing an EPO-R activation sequence in place of the variable region of the heavy and/or light chain). The preparation of the peptide compound of the present invention - Synthesis of the peptide 1344964 The peptide of the present invention can be prepared by a conventional method known in the art. Standard methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, conventional solution synthesis, and recombinant DNA techniques [see, for example, j. Am. Chem. Soc. 1963 85:2149]. In one embodiment, the peptide monomers of the peptide binary can be synthesized separately and polymerized after synthesis. In another embodiment, the peptide monomer of the binary form a branched third amine linkage through its C-terminus. Partial linkage, the linkage has a difunctional group as the initiation site for peptide synthesis, and a third functional group (eg, a slow or amine 10 group) that can bind to another molecule moiety (eg, can be present in a solid Supporting the surface of the body. Under the condition of the solid phase synthesis technique, the two victoric monomers can be directly synthesized from the two reactive nitrogen groups of the LK moiety of the linking group. This synthesis can be carried out sequentially or simultaneously. In the 15 20 〃 system, in the variation technique of the solid phase synthesis technique, the two peptide precursors can be directly synthesized from the two reactive nitrogen groups of the linked SLk moiety. Such synthesis can be carried out sequentially or simultaneously. This specific use, the use of a kind of amine-free S夂 linkage (LK) moiety has two amine-based peptides for the initiation of the synthesis of the peptide 'and a third functional group (such as the reduction of amines or amines) The amine group is separated from the 16 acid residue in which the slow group has been converted to the indole moiety -C〇NH2) which allows for binding to another molecular moiety (for example, may be present on the surface of the solid support). "When a human is ordered to synthesize a peptide chain of a binary body to a one-linking group, the two amine functional groups of the linking molecule can be protected by two different orthogonally removable amine groups. The third official of the county, the first official of the county, 43 1344964, is coupled to a solid support. The first amino protecting group is removed, the first peptide of the dimer is removed from the first amine protecting group, and the first peptide of the binary is synthesized in the first deprotected amine moiety. The second amine protecting group is then removed. The second peptide of the binary is synthesized in the second deprotected amine moiety. For example, the first amine moiety of the 5-bonding group can be Alloc protected, and the second amine moiety can be

Fmoc protection. In this case, the Fmoc group (but not the Alloc group) can be removed by treatment with a weak base such as 20% piperidine in dimercaptocaramine (DMF) to synthesize the first &gt; peptide chain. Subsequently, the Alio group can be removed by a suitable reactant [e.g., Pd(PPh3)/4-methylmorpholine and chloroform] to synthesize a second peptide chain. Note that when different thiol 10 protecting groups are used to protect the cysteine from the formation of disulfide bonds (described in detail later), this technique must even be used for the final amino acid sequence of the binary peptide chain. For the same time. If the dipeptide chain of the binary is to be synthesized simultaneously with the linkage, the two amine functional groups of the linkage are protected with the same removable amine protecting group. The protected linkage is coupled to the solid support steroid via a third functional group of the linkage. In this case, the two protected functional groups of the linking molecule are simultaneously deprotected from the synthesis of the two peptide chains simultaneously with the deprotected amine. Note that the use of the present technology 'the binary peptide sequence is the same, and the thiol protecting groups of the cysteine residues must be the same. * 20 The preferred peptide synthesis method is solid phase synthesis. Solid phase peptide synthesis procedures are well known in the industry [eg reference to Stewart solid phase peptide synthesis (Freeman's 'San Francisco) 1969; Nyva Bi〇chem's 2002/2003 general catalogue 'San Diego, USA; Goodman peptide and the peptide of the peptide (Houben-Weyl, Stuttgart) 2002]. For solid phase synthesis, a typical system is synthesized. 44 1344964 The use of hydrazine: an amine-protected resin begins at the c-terminus of the peptide. A suitable starting material can be prepared, for example, by attaching a desired α-amino acid to a gas methylated resin, a hydroxymethyl resin, a polystyrene resin, a benzhydryl resin, or the like. Such a gas methylated resin is sold under the trade name Biobeads (BIO-BEADS) SX-1 from the Bi〇 5 Rad laboratory (Liqimen, Calif.). The preparation of hydroxymethylated resins has also been described [Bodonszky et al. (1966) Chem. Ind. London 38: 1597]. The benzhydrylamine (BHA) resin is also described [Piett^Marshall (1970) Chem. Commun. 650]' hydrochloride form available from Beckman Instruments Inc. (Paul Otto, Calif.). For example, the alpha amine-protected amino acid root 10 is coupled to the chloromethylated resin by means of a cesium hydrogencarbonate catalyst according to the procedure described by Gisin (1973) Helv. Chim. Acta 56:1467. After the preliminary coupling, the [alpha]amino protecting group is removed, for example, using a solution of trifluoroacetic acid (TFA) or hydrochloric acid (HC1) in an organic solvent at room temperature. Subsequently, the alpha amino-protected amino acid is sequentially coupled to the growing peptide 15 chain which binds to the support. The alpha-amino protecting group is a protecting group known to be useful in the stepwise synthesis of the peptide industry, including: fluorenyl protecting groups (eg, formazan, trifluoroethenyl, ethenyl), aromatic urethanes. Protecting group [eg benzyloxycarbonyl ((: 1) phantom and substituted Cbz] 'aliphatic amino phthalate protecting group [eg third butoxycarbonyl (Boc), isopropoxy, cyclohexyloxycarbonyl) And alkyl protecting groups (eg 20-membered, trityl), fluorenyl oxycarbonyl (Fmoc), acryloxycarbonyl (Alloc) and 1-(4,4-dimercapto-2, 6-diketocyclohex-1-ylidene)ethyl (Dde). Branched protecting groups (typically ethers, esters, triphenylsulfonyl, PMC, etc.) remain intact during the coupling process at the amine end The protecting group is not cleaved or removed during the deprotection or during the coupling. The branched protecting group must be removed when the peptide synthesis is completed 45, and the target peptide is not changed. The branch protecting group of Tyr includes tetrahydrogen. Pyranyl, tert-butyl, triphenylsulfonyl, benzyl, Cbz, Z-Br-Cbz and 2,5-digasbenzyl. The branched protecting group of Asp includes benzyl, 2 6 -diqibenzyl , 曱基,乙The base and cyclohexyl group. The branch protecting group 5 of Thr and Ser includes an ethyl, benzoyl, trityl, tetrahydro 0, benzyl, 2,6-dibenzyl and cbz. The branched chain protecting group of Arg includes nitro, tosyl sulphonate (Tos), Cbz adamantyl oxycarbonyl, toluene sulfonyl sulfonyl (Mts), 2,2,4,6,7-pentamethyl Dihydro benzofuran-5-sulfonyl (Pbf), 4-decyloxy-2,3,6-trimethyl-benzoinyl (Mtr) or B〇(^ Lys branched protective group 10 Including Cbz, 2-benzyl benzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-Br-Cbz), Tos or Boc. After removal of the α-amino protecting group, the remaining protected amine group The acid is coupled stepwise in the desired order. Each protected amino acid is typically reacted in about a 3-fold excess using a suitable carboxyl activator such as 2-(lH-benzotriazole 15 based broad 1,1,3 , 3 · tetradecyl hexafluorophosphate (HBTU) or dicyclohexyl propylene diamine (DCC) in solution, for example, in dichloromethane (CH2C12), Ν-methyl ° ratio Reaction with dimercaptocaramine (DMF) or a mixture thereof. After completion of the predetermined amino acid sequence, the desired peptide is made of resin. Holder decoupling, decoupling is carried out using reagents such as trifluoroacetic acid (TFA) or fluorinated diazoxide (HF). These reagents not only cleave the peptide by the resin, but also cleave all remaining branch protecting groups. When a chloromethylated resin is used, the hydrogen fluoride treatment results in the formation of a free-form peptide. When a benzhydrylamine resin is used, the result of hydrogen fluoride treatment directly obtains the free-form peptide guanamine. In addition, when a gas-based group is used. In the case of a resin, the branched protected peptide can be ammonia treated with a peptide 46 1344964 resin decoupled to obtain the desired branched chain protected guanamine, or alkylamine decoupled to obtain a branched protected alkyl guanamine Or dialkyl decylamine. The branching protection is then removed in a conventional manner using hydrogen fluoride treatment to obtain free-form guanamines, deuterated guanamines, or dialkyl decylamines. 5 When preparing the ester of the present invention, a resin for preparing a peptide acid is used, and the peptide-protected peptide is cleaved using a base and a suitable alcohol such as methanol. The bond protecting group is then removed in a conventional manner by treatment with hydrogen fluoride to obtain the desired ester. These procedures can also be used to synthesize peptides in which 20 natural genes encoding amino acids other than 10 amino acids are substituted at one, two or more positions of any of the compounds of the present invention. Synthetic amino acids which may be substituted for the peptides of the present invention include, but are not limited to, N-methyl, L-propylpropyl, L-3,4-di-phenylphenylalaninium, 5-amino acids Such as L-5-hydroxyl-aminopurine and D-(5-mercaptopropylamine sulfhydryl, L-α-mercaptopropylamine sulfhydryl, arylamino acid and isoindolyl. D_amino 15 acid and non Natural synthetic amino acids can also be incorporated into the peptides of the present invention. The peptide modification can also modify the amine end and/or carboxyl end of the peptide compound of the present invention to produce other compounds of the invention. For example, the amine end can be acetic acid. Or a dentate derivative thereof such as α-gas acetic acid, α-bromoacetic acid or α-mothranacetate. 20 Other natural branches may be substituted for the amino acid encoding the amino acid (or stereoisomeric D-amino acid). Naturally branched, for example, substituted with: for example, alkyl, lower alkyl, cyclic 4-, 5- to 6- to 7-membered alkyl, decylamine, decylamine, lower alkyl, decyl Lower alkyl), lower alkoxy, hydroxy, carboxy, and lower aliphatic ester derivatives thereof; and 4-, 5-, 6- to 7-membered heterocyclic substitutions. Special 47 1344964 may be similar to lysine Object, of which The ring size of the acid residue is changed from 5 to 4, 6 or 7. The cyclic group may be saturated or unsaturated, and if it is unsaturated, it may be aromatic or non-aromatic. Containing one or more nitrogen, milk, and/or heteroatoms. Heterocyclic groups include, for example, d-n-n-decyl, n-n-Nan, • 5 imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, iso Carbazolyl, aryl. (eg morpholine ring), carbazolyl, piperage (eg 1-pipe "4 base", "bottom bite (eg 1-piperidinyl, piperidine)), pyridyl Meryl, pyridyl, pyridazole, benzoxyl, pyrazolyl, pyridyl, pyridyl, pyrimidinyl, pyridinyl (eg 1-pyrrolidyl), pyrrolinyl, Pyrrolyl, thiadiazolyl, succinyl, 10 phenyl, thiophene (for example, thiourapine) and tris(s). These heterocyclic rings may be substituted or unsubstituted. When substituted, the substituent may be a pendant group, an alkoxy group, an atom, an oxygen or a substituted or unsubstituted phenyl group. Phosphorylation and other methods may be used to facilitate the modification of the peptide [for example, in H ru by Et al. (1990) Bioche m J. 268:249-262]. 15 The peptide compounds of the present invention can also be used as structural models of non-peptide compounds having similar biological activities. Those skilled in the art understand that a number of techniques can be used to form the same or with a peptide compound. A similar bioactive compound is intended to have a more favorable activity than the lead compound in terms of solubility 'stability' and sensitivity to hydrolysis and proteolysis. [Ref. M〇rgan -20 and Gainor (1989) Ann. Rep. Med. Chem. 22: 243-252]. These techniques include phosphonates, phthalates, urethanes, sulfonamides, secondary amines, and N-decylamines. The backbone of the base acid composition replaces the peptide backbone. Formation of Disulfide Bonds The compounds of the invention contain two intramolecular disulfide bonds. Such a disulfide bond can be formed by oxidation of a semi-deaminating acid residue of each peptide monomer. In a specific example, the control of the formation of cysteine bond formation can be carried out by selecting and purifying the isomerization of the isomer. For example, when the oxidizing agent is DMSO or hydrazine (12), (comparing the formation of intramolecular bismuth bonds) preferentially oxidizes the dacombinide to form two intramolecular disulfide bonds (one disulfide in each peptide chain) key). In another specific example, the formation of a cysteine bond is formed by appropriately selecting a thiol protecting group during the synthesis of the winning form. For the sputum, when a binary body having two intramolecular disulfide bonds is required, the first monomeric peptide chain system uses a thiol protecting group as a core sequence [eg, a tris-f-group, and an HM-dimethyl group). - Recognition of two small subunits, etc.] The two cysteine residues are protected, and then the 'second monomer peptide is a second thiol protecting group that uses a core sequence to differ from the first thiol protecting group. [For example, two cysteine residues protected by anthranyl sulfhydryl (Acm), tert-butyl (tBu), etc.] are synthesized. Subsequent removal of the &lt;RTI ID=0.0&gt;&gt;&gt; </RTI> thiol protecting group is carried out to carry out the disulfide cyclization of the first monomer, and then the second thiol protecting group is removed, and the disulfide cyclization of the second monomer is carried out. Other specific examples of the invention provide disulfide derivative analogs in which one sulfur has been replaced by a CH2 group or other sulfur iStere. Such analogs can be prepared from the compounds of the invention wherein each peptide monomer contains at least one c residue or a high half-depleted residue, and alpha-aminobutyric acid is substituted for the second anthracene residue, which is used in the industry. The method is known to be carried out by intramolecular or intermolecular ectopic [see, for example, Barker et al. 1992] J. Med. Chem. 35:2040-2048 and Or et al. (1991) J. Org. Chem. 56:3146-3149]. Skilled person 1344964 It is understood that this alternative can also be carried out using alpha-amino-r-butyric acid and other homologues of homocysteine. In addition to other cyclization strategies, other non-disulfide peptide cyclization strategies may be employed, such as a guanidine cyclization strategy and a %ification strategy involving thio-ether Α 5 bond generation. Thus, the compounds of the present invention may exist in the form of intramolecular or cyclized forms of the molecule (tetra). For example, a peptide can be synthesized in which one cysteine of the nuclear w sequence is replaced with an amine acid, and the second + deaminating acid is replaced with a face acid. Subsequently, the cyclic monomer can be formed via a guanamine linkage between the branches of the two residues. In addition, it is possible to synthesize Shengyuetai, in which the core sequence is substituted with aminic acid (or seric acid). The cyclic monomer is then formed via a sulphur-ether bond between the branch of the amine (or 4 fe) residue and the second cysteine residue of the core sequence. Thus, in addition to the disulfide cyclization strategy, the indole cyclization strategy and the thio-ether cyclization strategy are conveniently used to cyclize the fluorinated product. In addition, the amino group of the peptide can be substituted with an acetic acid plus a terminal group. The alpha substituent in I5 is a leaving group such as alpha haloacetic acid such as hydrazine chloroacetic acid, alpha bromoacetic acid or alpha-iodoacetic acid. Addition of a branched third indole linkage The peptide monomer can be dimerized by a branched third indole linkage moiety. In a specific example, the linkage is incorporated into the peptide based on the peptide synthesis process. For example, when the moiety of the L20 linkage group contains two functional groups, which can be used as the initiation site for the synthesis of the peptide, and one or more other functional groups (such as a carboxyl group or an amine group), which can be bonded to one or more other In the molecular moiety, the linking group can be conjugated to a solid support. Subsequently, the two peptide monomers can be directly synthesized into two reactive nitrogen groups of the LK moiety of the linking group by various solid phase synthesis techniques. 50 1344964 In another embodiment, the 'bonding group' can be conjugated to the two peptide monomers of the peptide dimer after synthesis of the peptide. Such conjugates can be achieved by methods established by the industry. In one embodiment, the linking group contains two functional groups which are suitable for attachment to the target functional group of the synthetic peptide monomer. For example, a linking group containing two suspending groups can be preactivated or reacted with the respective target of the dipeptide monomer alone from the branched amine amine group in the presence of a suitable coupling agent. For example, the peptide monomer can be chemically coupled to the third guanamine linkage, A*-C10-CH2-X-CH2-C20-B* where X is NCO-(CH2)2-NH-Y and γ is A suitable protecting group such as a third 10 butyloxycarbonyl (Boc) protecting group; A* is a suitable functional group such as N-oxybutylenimide, and A* is used to conjugate a first peptide monomer of a bonding group a c-terminal amide group of a second peptide monomer for c2 conjugated to a bonding group, and a suitable functional group such as N-oxybutaneimine Ε-amino group of the residue. In addition, for example, a peptide monomer can be chemically coupled to a third guanamine linkage, A^-C^-C^-XC^-C'OB* where: X is NCO-(CH2)2-NH- C3〇- ; A* is a suitable functional group such as N-oxybutaneimine, and A* is used to bond the C-terminal amino acid residue of the first peptide monomer of the bond group. And B* is a suitable functional group such as &amp; 20 oxybutaneimine, which is used to conjugate the C-terminal of the second peptide monomer of the bond C2 to the amine group of the amine acid residue; The third indole linkage is chemically bonded to the spacer moiety,

Y-NH-(CH2)4-C4H-NH-Y where: C3 of X is covalently bonded to the C4 of the spacer; and γ is suitably protected 51 1344964, such as a third butoxycarbonyl (Boc) protecting group The addition of a sulphate linkage can be carried out by partial condensation of the amino acid linkage group LK. In one embodiment, the lysine linkage is incorporated into the peptide during the peptide synthesis process. For example, when the 5 moiety of the bond contains two functional groups which can serve as (10) a synthetic initiation site, and a third functional group (such as a carboxyl group or an amine group) which can be bonded to another molecular moiety, the linkage can be Lightly fit to a solid building. Subsequently, the dipeptide monomer can be directly synthesized to the di-reactive nitrogen group of the lk moiety of the amine linkage by a solid phase synthesis technique.

In another specific example, the peptide binary system herein is partially dimerized by an amine acid linkage LK. The linkage can be conjugated to the peptide peptide of the peptide binary after synthesis of the peptide. . Such conjugates can be achieved by methods established by the industry. In one embodiment, the 'bonding group' contains at least two functional groups suitable for attachment to the target functional group of the synthetic peptide monomer. For example, two free amine groups from the amine acid can react with the respective C-terminal carboxyl groups of the 15 dipeptide monomer. I. Addition of Spacer The peptide compound of the present invention further comprises a spacer moiety. In a specific example, the spacer can be incorporated into the peptide during the peptide synthesis process. For example, when the spacer contains a free amine group and a second functional group (e.g., a carboxyl group or an amine group) which can bind to another molecular moiety, the spacer can be conjugated to the solid support. In one embodiment, the spacer containing a difunctional group is first coupled to the solid support through the first functional group. Secondly, the difunctional group of the amine acid linkage LK has a difunctional group as a peptide synthesis initiation site, and a third functional group (such as a carboxyl group or an amine group) which can bind to another molecular moiety, the isoleucine linkage group. The third functional group of the bonding group is conjugated to the spacer through the second functional group of the 52 spacer. Subsequently, the dipeptide monomer can be directly synthesized in the solid phase synthesis technique by two reactive nitrogen groups in the LK moiety of the linkage group. For example, a solid support couples a free amine group spacer which is reactive with an amine 5 linkage through a free moieving group of the linkage. In another embodiment, the spacer can be conjugated to the peptide dimer after synthesis of the peptide. Such conjugates can be achieved by methods established by the industry. In one embodiment, the linker contains at least one g-energy group suitable for attachment to a target functional group of the synthetic peptide. For example, a spacer having a free amine group can be reacted with a C-terminal carboxyl group of the peptide. In another example, a bond having a free carboxyl group can be reacted with a free amine group of a guanamine amine. Attachment of polyethylene glycol (PEG) In recent years, water-soluble polymers such as polyethylene glycol (PEG) have been used to covalently modify peptides of therapeutic and diagnostic importance. Attachment of this polymer is believed to provide high biological activity, prolonged blood circulation time, reduced immunogenicity, increased solubility in water, and increased resistance to protease digestion. For example, covalent attachment of PEG to therapeutic polypeptides such as the white matter class [Knauf et al. (1988) J. Biol. Chem. 263; 15064; Tsutsumi et al. (1995) Controlled Release Journal 33:447], Interferon (Kita Et al. (1990) Drug Delivery 6: 157), Catalytic 20 Enzyme (Abuchowski et al. (1977) J. Biol. Chem. 252: 582), Superoxide Dismutase (Beauchamp et al. (1983) Anal. Biochem. 131:25) and adenosine deaminase (Chen et al. (1981) Biochim. Biophy. Acta 660:293) have been reported to prolong in vivo half-life and/or reduce immunogenicity and antigenicity. 53 1344964 The peptide compound of the present invention may comprise a polyethylene glycol (PEG) moiety which is covalently attached to the branch of the peptide binary by a urethane linkage or an acid amine linkage to a third amine linkage. Base or spacer. The PEG used in the present invention is, for example, a linear unbranched PEG 'component of about 20 kilo Torr (20K) to about 40K ("about" Λ 5 words means that in PEG preparation, some molecules are higher than the molecular weight, some molecules Lower than the molecular weight). Preferably, the PEG has a molecular weight of from about 30K to about *40K.

&amp; Another example of PEG used in the present invention is a linear PEG having a molecular weight of from about ιοκ to about 60K (the term "about" means that in the preparation of peg, some molecules are more molecular than the molecular weight of 10, some molecules are lower than the molecular weight) . Preferably, the PEG has a molecular weight of from about 20K to about 40K. More preferably, the molecular weight of PEG is about 20K. An example of a method of covalently attaching PEG (PEGylation) is as follows. The examples are not intended to be limiting. Those skilled in the art understand that the industry has established a variety of methods for covalently attaching a wide range of PEGs. Thus, it is known in the art that peptides which have been attached to PEG by a variety of attachment methods are encompassed by the scope of the present invention. For example, PEG can be covalently bonded to a linking group through a reactive group, and the reactive group can bind to an activated PEG molecule (e.g., a free amine group or a slow group). &quot; PEG molecules can be attached to the amino acid using methacrylic PEG ("mPEG _j)-20 with different reactive moieties. Such polymers include mPEG-butyl succinimide succinate, mPEG-butyl Di-i-imine carbonate, mPEG-hydrazide vinegar, mPEG-4-nitrophenyl carbonate S and mPEG-cyanuridine chloride. Similarly, peg molecules can be used with free amine-based methacrylic PEG (mPEG-NH2) Attached to Rebel 0 54 1344964 In several embodiments, the linker or spacer contains a terminal amine group (ie, located at the end of the spacer). This terminal amine group can be suitably activated with a PEG molecule such as mPEG- The p-nitrophenyl carbonate (mPEG-NPC) is reacted to produce a stable covalent amine phthalate linkage. In addition, the terminal amine group can be reacted with a suitable activated PEG, for example, containing a reactive N-hydroxy-butane An imine (NHS) group of mPEG-butanediamine-based butyrate (mPEG-SB A) or mPEG-butanediamine-based propionic acid vinegar (mPEG-SPA) to form a stable covalent amine Further oxime bond. In other embodiments, the linker reactive group contains a carboxyl group which can be activated to form a covalent bond with the amine-containing PEG molecule under appropriate reaction conditions. The appropriate PEG molecule 10 includes mPEG-NH2, and the appropriate reaction conditions include the indoleamine production reaction conditions of the indole diimide medium, etc. E_PO-R agonist activity assay: In-tube functional assay analysis In-tube competition binding assay can be quantified The test peptide competes with EPO for the ability to bind to HPO-R. For example (see, for example, U.S. Patent No. 5,773,569), the extracellular domain of human EPO-R (EPO binding protein EBP) can be produced recombinantly in E. coli, recombinant protein. Coupling to a solid support such as a micro-valve or synthetic bead [eg, Sulfolink beads from Pierce Chemical Company (Rockford, Ill.)] ^ then braked EBP 20 and labeled recombination EPO co-cultured or co-cultured with labeled recombinant EPO and test peptides. A series of dilutions of the test peptide was used for this experiment. The assay point without the addition of the test peptide defines the total binding of EPO to EBP. Containing the test peptide reaction, the EP0 binding amount is quantified and expressed as a percentage of the control (total = 100%) binding. This value is plotted against the peptide concentration. IC50 55 1344 The 964 value is defined as the concentration of the test peptide that reduces EPO binding to EBP up to 50% (ie, 50% inhibition of EPO binding). Different test tubes are tested for competitive binding assays to determine two beads: EP0 beads and The change in the proximity of EP〇-R|fe beads is measured by the light signal of the ginseng. The proximity of the beads can be produced by EPO binding to EPO-R. The test for competing with EPO to EPO-R The peptide prevents the junction from causing a decrease in luminescence. The test peptide concentration obtained by reducing the luminescence by 5% is defined as the IC50 value. The peptide of the present invention is extremely effective in competing with EP0 for binding to EP0_R. Such a 〇 enhancing function can be represented by a compound of the present invention which inhibits the binding of EPO at a substantially lower concentration of a peptide (i.e., having a very low IC50 value). The biological activity and strength of the monomeric peptides and the binary peptide EPO_R agonists of the present invention, in particular in combination with the biological activity and strength of the EPO receptor, can be determined by assays based on cell-based functional assays in vitro. 15 One assay was based on a pre-B cell that expresses human EP0-R. The system further utilizes the fOS promoter-driven luciferase reporter gene constitutive transfer infection. Such cells are responded to by synthetic luciferase when exposed to EP 〇 or other EP0-R agonists. It causes luminescence when worm fluorescein is added. The degree of EP0-R activation of such cells can be quantified by measuring luciferase activity. The measurement of the activity of the test is carried out by adding a serial dilution of the test peptide to the cells, and then culturing for 4 times. After the culture, the luciferase substrate was added to the cells, and luminescence was measured. The test peptide concentration that results in half-maximal luminescence is recorded as the EC50. The peptides of the present invention have been shown to have an increased ability to promote the expression of EP0-R signaling-dependent 56 1344964 luciferase in this assay. Such enhanced function may result in a semi-maximal luciferase activity at a substantially lower peptide concentration (i.e., having a very low EC50 value). This assay is a preferred method for estimating the strength and activity of the EPO-R agonist peptide of the present invention. 5 FDC-P1/ER cells can be used [Dexter et al. (1980) J. Exp. Med. 152: 1036-1047 Another assay was performed, which was a well-characterized untransformed murine bone marrow-derived cell line that had been stably metastatically infected with EPO-R. These cells have EPO-dependent proliferation. In one assay, cells were grown to a 10 semi-static density in the presence of the desired growth factor (see, e.g., U.S. Patent 5,773,569). The cells were then washed in PBS, hungry for -24 h in growth medium without growth factors, and assayed for cell viability (eg, by trypan blue staining) to prepare a stock solution (without growth factor) Full medium) obtained a series of 15 dilutions of each of the peptides of the peptide EpO_R agonist (containing about 105 cells per 5 microliters) (typically a free-form liquid phase peptide, rather than a phage-binding or other binding peptide or The brake peptide was prepared in a 96-well tissue culture well plate to a final volume of 50 μL per well. Cells (5 μL) were added to each well and the cells were cultured for 24-48 hours. At this point the negative control will die or quiescent. The cell proliferation is then determined by techniques known in the art, such as MTT assay, and the MTT assay is assayed for H3-20 thymidine. The binding amount is used as an indicator of cell proliferation [Ref. Mosmann (1983) Immunol. Journal of 65:55 63p peptide system for the comparison of the sand 〇 尺 表现 表现 表现 亲 亲 亲 亲 亲 亲 。 。 。 。 。 。 。 。 。 。 。 。 。 The test peptide concentration at which the half maximal cell growth was obtained was recorded as the EC50. In the present assay, the peptide of the present invention showed an increase in the ability to promote EPO-dependent cells. Such enhanced function may be obtained by obtaining a semi-maximal cell proliferative stimulating activity at a substantially low concentration peptide (i.e., having a very low Ec5 〇 value). This assay is a preferred method for estimating the strength and activity of the peptide of the present invention. In another assay, the cells were grown in a medium supplemented with EP 至 to a steady state, and the cells were collected and cultured for 18 hours in an EP-free medium. The cells were divided into three groups of equal cell density: one group of unadded factors (negative control), one group containing EPO (positive control), and one experimental group containing test peptides. The cultured cells are then collected, fixed, and stained with DNA-binding fluorescent dyes (e.g., propidium iodine or Hoechst dye, all from sigma) at various time points. Fluorescence is then measured, for example, using a FACS scan flow cytometer. The percentage of cells in each phase of the cell cycle is then determined, for example, using the SOBR model of CellFIT software (Becton Dickinson). Cells treated with EPO or active peptides compared to the negative control group showed a larger percentage of cells in the S phase (as measured by an increase in fluorescence as an indicator of increased DNA content). Similar assays can be performed using FDCP-1 [for example, see Dexter et al. (1980) J. Exp. Med. 152: 1036-1047] or TF-1 [Kitamura et al. (1989) Blood 73: 375-380]. FDCP-1 is a growth factor-dependent murine multi-potential primitive hematopoietic progenitor cell line that proliferates but does not differentiate when cultured in WEHI-3 conditioned medium (IL-3 containing medium ATCC number TIB-68). For this experiment, the FDCP-1 cell line was infected with human EPO-R or murine EP0-R to produce FDCP-1-hEPO-R cell line or FDCP-1-mEPO-R cell line, which can be used in the presence of EPO. Proliferation but not divided into 1344496. TF-1 is an epo-dependent cell line. TF-1 can also be used to determine the effect of peptide EPO-R agonists on cell proliferation. For yet another assay, the compound of the invention can be used as a Ε Ρ Ο 可采用 可采用 5 ( ( 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 1983 胸 胸 胸The ability of the agent. Briefly, B6C3FN and rats were injected daily with phenylhydrazine (60 mg/kg) for 2 days. On the third day, spleen cells were removed and assayed for their ability to proliferate in 24 hours using a sputum assay. The EPO-R, which binds to the erythropoietin-reactive cell line, induces 10 tyrosine phosphorylation receptors and a variety of intracellular proteins, including She, vav and JAK2 kinases. Thus, another tube assay assay analyzes the ability of the peptides of the invention to induce tyrosine phosphorylation of EPO-R and downstream intracellular signal transducer proteins. By the above-mentioned binding and proliferative assay analysis, the active peptide was extracted, and the phosphorylation pattern was similar to that of EPO-like erythropoietin-reactive phosphorylation. For the assay, FDC-P1/ER cells [Dexter et al. (1980) J. Exp Med 152:1036-47] were maintained and grown to a stationary phase in supplemented EPO medium. The cells were then cultured in an EPO-free medium for 24 hours. A specific number of cells are then incubated with the test peptide at 37 ° C for about 1 minute. Cell control samples containing EP0 were also performed simultaneously in each assay. 2〇 The treated cells were then collected by centrifugation and resuspended in SDS Lysis Buffer' subjected to SDS polypropylene gel electrophoresis. The protein subjected to gel electrophoresis in the gel was transferred to nitrocellulose, and the protein containing phospholamic acid on the ink dot was observed by standard immunological techniques. For example, the ink dot can be tested with an anti-sphingic acid antibody (for example, mouse anti-squaric acid IgG 'from Upstate 59 Biotech A), washed, and then used a secondary antibody. [For example, the peroxidase standard ^^ goat anti-mouse 1gG, obtained from Kirkegaard &amp; Perry to A Division (Washington, DC detection. Subsequently, protein containing lysine Borrowing techniques include colorimetric assays for chemiluminescence assays 5 or fluorescence assays. For example, chemiluminescence assays can be performed using the Western Western blot system from Amersham. To evaluate the activity of the peptide of the present invention, the cell-based test s internal assay D is a community assay, which uses murine bone marrow, cells or peripheral blood cells. The rat bone can be obtained from the mouse femur, human peripheral blood cell sample 1 〇 can be paid from healthy donors. Taking peripheral blood as an example, mononuclear cells are first isolated from the blood, for example, by a Ficoll-Hypaque gradient [Stem Cell Technology Corporation (Vancouver, Vancouver, Canada). In The number of cells containing the nucleus is calculated to determine the concentration of nucleated cells in the original blood sample. A specific number of cells are in accordance with the manufacturer's instructions [Stem Cell Technology Inc. (Canada 15 Vancouver is inoculated with methylcellulose. The experimental group is treated with the test peptide) The 1% control group was treated with EP0, and the negative control group was not treated. Then the growth community amount of each group was usually scored from 1〇曰 to 18曰 after the specific culture time. The active peptide can promote the formation of the community. A test tube bioassay that can be used to verify the activity of a compound of the invention is disclosed in Greenberger et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2931-2935 (EPO-dependent hematopoietic progenitor cell line); Queue and

Wojchowski (1991) J. Β〇1· Chem. 266: 609-614 (protein cool acid nucleotification of B6SUt.EP cells); Dusanter-Fourt et al. (1992) J. Biol. Chem. 287:10670- 10678 (EPO of human EPO-reactive cells is phosphorylated by tyrosine of 60 1344964); Quelle et al. (1992) J. Biol. Chem. 267:17055-17060 (FDC-ER cell lysate protein pp 100 Tyrosine acid acidification); Worthington et al. (1987) Exp. Hernatol. 15:85-92 (colorimetric assay for hemoglobin); Kaiho and Miuno (1985) 5 Anal. Biochem. 149:117-120 (hemoglobin use) Detection of 2,7-diaminopurine); Patel et al. (1992) J. Biol. Chem. 267:21300·21302 (performance of c-myb); Withuhn et al. (1933) cells 74:227-236 ( Combination of JAK2 and phosphorylation of tyrosine); Leonard et al. (1993) Blood 82: 1071-1079 (performance of GATA transcription factors); and Ando et al. (1993) 10 Proc. Natl· Acad. Sci. USA 90: 9571-9575 (adjustment of G through loop D2 and D3). An instrument designed by Molecular Devices to be used as a microphysiometer has been reported to be successfully used to determine the effects of agonists and antagonists on various receptors. The basis of the device is the determination of the rate of acidification of the extracellular medium in response to activation of the receptor. In vivo functional assay analysis An in vivo functional assay that can be used to assess the strength of a peptide in a test. Bioassay for mice with erythrocytosis. For this assay analysis, mice received alternating conditioning cycles for several days. During this cycle, mouse 20 alternates between subatmospheric conditions and ambient atmospheric conditions. The mice were then maintained at ambient pressure 2-3 days prior to the administration of the test sample. The test peptide samples of the positive control mice or the positive control mice were injected subcutaneously into the conditioned mice. Radiolabeled iron (e.g., &amp; 59) was administered 2 days after the administration of radiolabeled iron. The blood volume and radioactivity measurements of each blood sample are then determined by standard techniques 61 1344964. The blood sample of the mouse injected with the activity test peptide (due to erythrocyte hemoglobin binding to Fe59) showed higher radioactivity than the mice that did not receive the test Shengtai or EPO. Ίο Another in vivo functional assay that can be used to assess peptide activity is a reticulocyte assay used in this assay. Normal untreated mice were injected subcutaneously with EPO or test peptides for 3 consecutive days. In the third episode, mice were also injected intraperitoneally with iron glucomannan. On day 5, blood samples were taken from mice. The percentage (%) of reticulocytes in the blood was determined by thiazole orange staining and flow cytometry analysis (lattice counting). In addition, the blood volume is measured manually. The corrected percentage of reticulocytes was determined using the following formula: % RETIC correction = % RETIC observation x (blood volume _ / normal blood volume) The activity test compound showed a % RETICft concentration higher than that of the untested peptide or Ερ〇 mice. Use of the agonist of the present invention 15 20 The peptide compound of the present invention can be used in a test tube as a tool for understanding the role of sputum organisms' including a number of factors that are considered to affect the yield of sputum and the binding of hydrazine to EPO-R. (eg EPO/EPO-R signal transduction/receiver activation) 'either by the yield and the effect of hydrazine binding to EPO-R. The peptide is useful for the development of other compounds by providing important structure-activity-relationship information. Thus, the peptide of the present invention can also be used to develop other compounds that can bind to EPO-R. In addition, based on the ability of the peptide of the present invention to bind to EPO-R, the peptide of the present invention can be used as a reagent for detecting EPO-R in living cells, cells after fixation, biological fluids, tissue homogenates, purified natural biological substances, and the like. . Example 62 1344964 By labeling such peptides, cells having a ΕΡΟ-R surface can be identified. Further, based on its ability to bind EPO-R, the peptide of the present invention can be used for in situ coloring 'FACS (fluorescence activated cell screening) analysis, Western blot analysis, ELISA (enzyme-linked immunosorbent assay), and the like. Furthermore, based on the ability of the compound of the present invention to bind to EPO-R, the peptide of the present invention can be used for purification of the receptor, or for purifying cells expressing EPO-R on the cell surface (or inside the osmotic cell). The peptide of the present invention can also be used as a commercial reagent for various pharmaceutical research and diagnostic purposes. Such uses include, but are not limited to, (1) quantification as a calibration target for multiple functional assays, activity of candidate EPO-R agonists, and (2) use as a blocker for random peptides Screening, that is, for finding a new EPO-R peptide peptide ligand group, the peptide can be used to block the recovery of the EPO peptide of the present invention; (3) for co-crystallization with EPO-R, Inventive peptides bind to the crystal of EPO-R, so X-ray crystallography can be used to determine the receptor 15 / peptide structure; (4) used to determine the red blood cell precursor cells through the initiation of differentiation, and induced globulin synthesis and blood quality Complex synthesis and the ability to increase the number of ferritin receptors; (5) to maintain proliferation and growth of EPO-dependent cell lines such as FDCP-1-mEPO-R and TF-1 cell lines; (6) use of radioactive chromophores The group is labeled for use with the peptide of the present invention; and other research and diagnostic applications, wherein EP〇-R is preferably activated, or such activation is conveniently calibrated to a known amount of EPO-R agonist. In yet another aspect of the invention, methods of treatment and manufacture of medicaments are provided. The peptide compounds of the present invention can be administered to warm-blooded animals including humans to mimic the binding of EP to EPO-R in vivo. Thus, the present invention encompasses a therapeutic treatment of Ep〇 deficiency phase 63 1344964's' which comprises administering a compound of the invention in an amount sufficient to stimulate the EP to improve the symptoms associated with EPO deficiency in vivo. For example, the peptide of the present invention can be used for the treatment of renal insufficiency and/or terminal renal abortion/dialysis, AIDS-related anemia, chronic inflammatory diseases (such as rheumatoid arthritis and chronic intestinal inflammation), and autoimmune diseases. Anemia; and increase the number of red blood cells in the patient before surgery. Other disease states, conditions, and blood irregularities that can be treated by the peptide treatment of the present invention include: no type I thalassemia; cystic fibrosis; pregnancy and menstrual conditions; immature early anemia; spinal cord injury; space flight Acute blood loss; aging; 10 stroke, ischemia (including the central nervous system and heart); and a variety of tumor diseases accompanied by abnormal red blood cell production. In other embodiments, the peptide compounds of the present invention are useful for treating conditions that are not characterized by low or low red gold balls, e.g., for pre-treatment prior to transfusion. In addition, administration of a compound of the invention may result in a shortened bleeding time, 15 so that it can be administered to a patient prior to surgery, or for an appropriate condition for which bleeding may be expected. Furthermore, the compounds of the invention are useful for activating the activation of megakaryocytes. Because EPO shows mitogenic and chemotactic effects on vascular endothelial cells' and effects on central choline-induced neurons [eg reference to Amagnostou (1990) Proc. Natl. Acad. Sci. USA. 20 87:5978- 5982 and Konishi et al. (1993) Brain Research 609: 29-35], the compounds of the invention may also be used to treat a variety of vascular disorders, such as promoting wound healing, promoting collateral vessel growth (eg, may occur after myocardial infarction); sores Treatment; and post-vascular graft treatment. The compounds of the invention are also useful in the treatment of a variety of neurological disorders 'generally at a lower concentration of acetylcholine than other nerves 64 1344964 active substances (eg, neurotransmitters), or the relative volatility of the bile test is relative to other neuroactive substances. Low concentration is characteristic. Pharmaceutical Compositions In yet another aspect of the present invention, there is provided a pharmaceutical composition of the aforementioned Ep〇R agonist. Conditions that can be improved or adjusted by administering such compositions include the conditions listed above. Such pharmaceutical compositions can be, for example, by oral, parenteral (muscle, intra-abdominal, intravenous (IV) or subcutaneous injection), transdermal (passive infiltration, or using ion electrophoresis or electrophoresis), transmucosal (transnasal, transvaginal) , transrectal or sublingual route of administration, or the use of biodegradable implants, 10 which can be formulated for a variety of administration routes. Generally, the pharmaceutical compositions of the present invention comprise an effective amount of an EPO-R agonist peptide or derivative product of the present invention, together with a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and/or Or carrier. Such compositions include 豨15 release agents containing various buffering agents (eg, Tris-HCl, acetate, phosphate), different pH and ionic strength; additives such as detergents and solubilizers (eg, Tween 20, Thon 80, Polysorbate 80), antioxidants (such as ascorbic acid, sodium metabisulfite), preservatives (such as mercapto, benzyl alcohol) and bulk substances (eg lactose, mannitol) The mash compound is mixed with the granule preparation of the polymer compound granule preparation such as polylactic acid, polyglycolic acid or the like or mixed with the vesicle. 20 Hyaluronic acid can also be used. Such a composition affects the physical state, stability of the protein and its derivatives, the rate of release in vivo, and the rate of clearance in vivo. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (Murck Publishing Company, 1990, Easton, TX 18042), pages 1435-1712, incorporated herein by reference. The composition can also be prepared in liquid form, or in the form of a dry powder (e.g., lyophilized). 65 1344964 Oral delivery of oral solid dosage forms is intended for use herein, and oral solid dosage forms are outlined in Remington's Pharmaceutical Sciences, 18th Edition (1990 Merck Publishing Company, Easton, Pennsylvania 18042), Chapter 89, by reference Enter here. Solid dosage forms include ingots, capsules, pills, buccal or diamond ingots, lenticular capsules, tablets, powders or granules. In addition, liposome or protein-like encapsulation can also be used to formulate » this composition (eg, protein-like microspheres, as reported in U.S. Patent No. 4,925,673). Liposomes can also be used, and the vesicles can be derivatized using various polymers (e.g., U.S. Patent No. 5,013,556). A description of possible solid therapeutic dosage forms is described in Marshall, K.: Modern Medicine G.s. Banker and C.T. Rhodes Chapter 10, 1979, incorporated herein by reference. Typical formulations include the Ep0_R agonist peptide (or a chemically modified form thereof) and an inert ingredient which allows protection against the stomach environment and release of the biologically active substance in the intestinal tract. It is contemplated herein to also encompass oral pharmaceutical dosage forms, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain &amp; the ingredients 'including inert diluents; adjuvants such as wetting agents , emulsifiers and suspensions; and sweeteners, dwarfs and fragrances. The peptide can be chemically modified to allow oral delivery of the derivative to be effective. Generally, it is contemplated that the chemical modification will attach at least a portion to the component molecule itself, where this moiety allows (a) inhibition of protein breakdown; and (b) absorption into the bloodstream by the stomach or intestine. It is also desirable to increase the overall stability of each component' and extend the time in the body. As previously discussed, PEGylation is preferred for chemical modification for pharmaceutical use. Other modifiable parts include: propylene glycol, a copolymer of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrole 66 1344964. Ketone, polylysine, poly-1,3-dioxolane, and poly-1,3,6-trioxocyclo [see, for example, Abuchowski and Davis (1981) "Water Soluble Polymer-Enzyme Adducts "Enzymes as drugs. Edited by Hocenberg and Roberts (Willie Technology, Inc., New York, NY), pp. 367-383; and Newmark et al. (1982) J. Appl. 5 Biochem. 4:185-189]. For oral formulation, the release site can be the stomach, small intestine (duodenum, jejunum or ileum) or large intestine. Those skilled in the art can already obtain a formulation that does not dissolve in the stomach but can release material in the duodenum or other parts of the intestine. The preferred release of the drug avoids deleterious effects on the stomach environment by protecting the peptide (or derivative) or by releasing the peptide 10 (or derivative) beyond the stomach environment, such as the intestinal tract. In order to ensure total gastric resistance, a coating that is at least impervious to pH 5.0 is required. The more common inert ingredients that can be used as casings are cellulose acetate trimellitate (CAT), hydroxypropyl decyl cellulose (HPmcP), HPMCP 15 5 , HPMCP 55, polyvinyl acetate phthalate. (PVAP), Eudragit L30D, Aquateric, cellulose acetate (CAP), yuraji, yuraji S and shellac. These coatings can be used as a hybrid film. The coating or coating mixture can also be used to protect the 20-key agent from contact with the stomach. It includes sugar coatings or makes it easier to swallow the coating. Capsules may be composed of a hard shell (e.g., gelatin) for delivery of a dry therapeutic agent (i.e., a powder) for use in a liquid therapeutic drug. A soft gelatin shell may be used. The shell material of the bean-shaped capsule may be thick paper or other edible paper. For pellets, diamond ingots, molded ingots or ingot wet abrasives, wet block technology can be used. 67 1344964 The peptide (or derivative) may be finely multiparticulate, in the form of granules or granules of about 1 mm in size. The formulation of the material for capsule administration can be in the form of a powder, a micro-compressed plunger or even an ingot. These therapeutic agents can be prepared by compression. Coloring agents and/or flavoring agents may also be included. For example, the peptide (or derivative) can be formulated (e.g., by liposome or microsphere encapsulation) and then further included in an edible product such as a frozen beverage containing a coloring agent and a flavoring agent. The inert peptide can be used to dilute or increase the volume of the peptide (or derivative). These φ diluents include carbon hydrates, particularly mannitol, alpha lactose, anhydrous lactose cellulose, sucrose, modified dextran, and starch. Some inorganic salts can also be used as a filler, including tri-dead acid, carbonic acid and sodium chloride. Several commercially available thinners are FastFo F. (Fast_F1), Emdex, STA-Rx 1500, Emc〇mpress and Avicell. The powder ΊΓ 3 is included in the formulation of the therapeutic agent formulated into a solid dosage form. Materials used as disintegration θ丨 include, but are not limited to, temple powders, including commercially available disintegrating agents with a powder of the main powder, Expl〇tab, sodium starch glycolate, Amber Wright (Ambei*lite) ϋ fiber money, ultra-pentose candy gel, sodium alginate, gelatin, scalp, acid buffered sodium cellulose, natural sponge and bauxite ~ 纟 can be used. The disintegrating agent can also be an insoluble cation exchange resin. Powdered gum can be used as a disintegrating agent and a binder, as well as a powdery tree, such as the soil, carrageenan or scutellaria. The brown acid and the sodium alginate salt can also be disintegrating agents. The binder can be used to bind the peptide (or derivative) to form a hard ingot d, including products derived from nature such as acacia, scutellaria, and gelatin. Its money-reducing agents include methyl cellulose (MC), ethyl cellulose tons 68 1344964 and slow methyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can also be used in alcoholic solutions to granulate peptides (or derivatives). Anti-friction agents can also be included in peptides (or derivatives). ) Formulations to prevent sticking during the dispensing process. Lubricants can be used as the layer between the peptide (or derivative) and the wall of the stamp. Lubricants include (but without limitation, stearic acid including its magnesium and about salts, polytetrafluoroethylene (PTFE), liquid paraffin, Vegetable oils and waxes. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, Carbowax 4000 of various molecular weights and Carbows 10 6000 can also be used. The agent can improve the drug flow properties of the formulation process and can assist in the rearrangement of the compression process. The slip agent includes starch, talc, pyrogen, and hydrated yttrium aluminate. To assist the peptide (or derivative) to dissolve in water. Environment, interface 15 active agent can be added as a wetting agent. Surfactant includes anionic detergents such as sodium lauryl sulfate 'dioctyl sulfonate sodium succinate and dioctyl benzoate. Can use cationic detergents, including chlorine Benzalkanal or chlorinated phenethyl hydrazine. Possible nonionic detergent forms that can be included as a surfactant in the formulation include lauromacol 400, Boliox (p〇ly) Xyi) 40 2 〇 stearate, polyoxyethylidene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, borissol 20 '40, 60 '65 and 80 'salt fatty vinegar, Methylcellulose and carboxymethylcellulose. These surfactants may also be present in a protein or derivative formulation, either alone or in a mixture of different ratios. Additives that promote absorption of the peptide (or derivative) are, for example, fatty acids 69. Classes such as oleic acid, linoleic acid and linolenic acid. It is also desirable to use controlled release oral formulations "peptides (or derivatives) which can be mixed with an inert matrix which allows for the release of, for example, gum by diffusion or percolator. The slowly denatured matrix can also be mixed with the formulation. Several casings also have an extended release effect. Another controlled release form is based on the method of the Oros (0r0s) treatment system (Alza), in other words, the drug package. Within the semipermeable membrane, the semipermeable membrane allows water to enter due to osmosis and pushes the drug out through a single small opening. Other coatings can also be used in the formulation. These coatings include a variety of sugars that can be applied to the coating tray. ( The derivative can also be made into a film-coated tablet. The materials used in this example are divided into two groups. The first group is a non-enteric material, including thiol cellulose, ethyl cellulose, hydroxyethyl cellulose, methyl hydroxy group. - ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium thioglycolate, providone and polyethylene glycol. The second group includes enteric materials , usually an ester of phthalic acid. A mixed material can be used to provide an optimum film coat. The coat can be carried out in a pan coater or in a fluidized bed or by a compression coating. The preparation for parenteral administration of the present invention comprises a sterile aqueous or non-aqueous solution, suspension or emulsion. The non-aqueous solvent or vehicle is, for example, propanol, I-ethyl alcohol, vegetable oil such as sein oil and corn oil, Organic vinegar such as ethyl oleate for bright kicking and injection. These dosage forms may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The sterilization method can be carried out, for example, by filtering through a filtration membrane that retains bacteria, sterilizing the composition i344_ via a mash-mixing sterilizing agent, sterilizing by using a glazing composition, or sterilizing β by heating the composition. Several other sterile injectable media are formulated just prior to use. Transrectal or transvaginal delivery 5 The composition for rectal or vaginal administration is preferably a suppository which contains excipients such as cocoa butter or a suppository in addition to the active substance. Compositions for nasal or lingual administration can also be prepared using standard excipients well known in the art. Transpulmonary delivery 1〇 The invention also encompasses delivery of an EPO-R agonist peptide (or a derivative thereof) via the lung. The peptide (or derivative) is delivered to the mammalian lung when inhaled by the mammal and enters the bloodstream through the lung epithelial lining [see, for example, Adjei et al. (1990) Pharmaceutical Research 7:565_569; Adjei et al. (1990) International Pharmaceutical Journal 63: 135-144 (leuprolide acetate); Braquet et al. (1989) 15 Journal of Cardiovascular Pharmacology 13 (sup5): 143-146 (endothelin-1); Hubbard et al. (1989) Annual Report of Internal Medicine , VOL. III, 206-212 (a 1-antitrypsin); Smith et al. (1989) J. Clin. Invest 84: 1145-1146 (α-l-protease); Oswein et al. (1990) "Protein Aerosolization, the second session of the Respiratory Drug Delivery Symposium, Colorado Chiss (Recombinant Human Growth Hormone); Debs et al. (1988) J. Immunol. 140:3482-3488 (interferon-r and tumor) Necrosis factor a); and U.S. Patent No. 5,284,656 to Platz et al. (granulocyte community stimulating factor). The method and composition of the system for the delivery of a drug through the lungs is described in U.S. Patent No. 5,451,569 to Wong et al. 71 1344964 The teachings encompassed by the present invention are a wide variety of mechanical devices for the delivery of therapeutic products via the lung, including, but not limited to, nebulizers, metered dose inhalers, and dry powder inhalers&apos;, all of which are well known to those skilled in the art. A number of commercially available special devices suitable for the practice of the present invention are Yazhevent (10) ravem) 5 • (Mallindaodt, St. Louis, Montana); Acorn I1 atomization (Marquest Medical Products, Inc., Yingwu, CO), Ventolin metered dose inhaler (Glax〇 φ, Research Triangle Park, North Carolina); and Spencer Spinhaler dry powder inhaler (Fis〇ns, Bedford, MA). 10 All such devices require the use of a formulation suitable for dispensing peptides (or derivatives). Typically, such formulations will depend on the type of device used, and in addition to the usual diluents, adjuvants, and/or therapeutic carriers, the use of suitable propellant materials. The use of vesicles, microcapsules or microspheres, including complexes or other types of carriers, is also contemplated. Depending on the type of chemical modification or the type of device used, the chemically modified peptides are also prepared in different formulations. φ Formulations suitable for nebulizers including spray atomization or ultrasonic atomization typically comprise a peptide (or derivative) dissolved in water at a level of from about 1 to 25 mg of bioactive protein per ml of solution. Formulations also include buffers and monosaccharides (e.g., for protein stability and osmotic pressure regulation). The nebulizer formulation also contains an interfacial agent to reduce or prevent agglomeration of the peptide (or derivative) due to solution atomization when the aerosol is formed. Formulations for metered dose inhaler devices typically comprise a finely divided powder comprising a propellant peptide (or derivative) suspended by means of a surfactant. The propellant can be any material used in this project, such as carbon fluorinated carbon, hydrochlorofluorocarbon 72 1344964 carbonized carbon hydrofluorocarbon or hydrocarbon 'including chlorotrifluoromethane, chlorodifluoromethane, two Chlorotetrafluoroethane, and U,l,2-tetrafluoroethane or combinations thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid can also be used as a surfactant. 5 The formulation delivered by the dry powder inhaler device comprises a finely divided dry powder containing a win (or derivative), and also includes a bulking agent such as lactose, sorbitol, sucrose and mannitol, in an amount to assist in dispersing the dry powder by the device. For example, 50 to 90% by weight of the formulation. The peptide (or derivative) is preferably prepared in a granular form having an average particle size of less than 10 mm (or micrometers), preferably from 0.5 mm to 5 mm 10 for most efficient delivery to the distal lung. Nasal delivery The present invention also encompasses transnasal delivery of EPO-R agonist peptides (or derivatives). Nasal delivery allows the therapeutic product to be administered to the nose, and the peptide enters the bloodstream directly, and the product does not need to be deposited in the lungs. Nasal delivery formulations include formulations formulated with 15 dextran or cyclodextrin. Dosage For all peptide compounds, further studies will be conducted to understand the appropriate doses for various patients to treat various conditions, and those skilled in the art will consider the treatment content 'recipient age and general health status will determine the appropriate dose of 20 doses. The choice of dosage is based on the desired therapeutic effect, the route of administration, and the time of the scheduled treatment. Usually, 0.001 to 10 mg/kg of body weight per dose is administered to the mammal. Usually for intravenous or infusion, the dose can be reduced. These dosing schedules may vary depending on the cycle half-life and the formulation used. The peptide of the present invention (or a derivative thereof) can be administered in combination with one or more other activities into a fraction of 73 1344964 or a pharmaceutical composition. Next, the present invention will be described by way of the following examples. However, the use of these and other embodiments in the specification is for illustrative purposes only and is not intended to limit the scope and the scope of the invention. In the meantime, the invention is not limited to any particularly specific examples described herein. It is to be understood that many modifications and variations of the present invention will be apparent to those skilled in the art. Therefore, the present invention is limited only by the scope of the following claims and the full scope of the equivalent of the patent application. Example 1: Synthesis of EPO-R agonist peptide peptide by solid phase synthesis Step 1 - Synthesis of Cbz-TAP: Contains commercially available diamine (rTAp from Aldrich Chemical Company) (1 The solution of gram, 67.47 mmoles in anhydrous DCM (1 mL) was cooled to 〇 °C. A solution of benzyl plutonate (4.82 ml, 33.7 15 mmol) in anhydrous DCM (50 mL) was added to a funnel over 6-7 s _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ (About 25. 〇. After another 16 hours, DCM was removed by vacuum. The residue was dissolved in 3N hydrochloric acid and ether. The aqueous layer was collected and neutralized to pH 8-9 with 50% aqueous sodium hydroxide and Extracted with ethyl acetate. The ethyl acetate layer was dried <RTI ID=0.0></RTI> to <RTI ID=0.0> One reaction. NHCbz

DCM

74 1344964 Step 2 - Synthesis of Cbz-TAP-Boc: Dibutyl ether (3.86 g, 17.7 mmol) was added to a vigorously stirred suspension of Cbz-TAP (5 g, 17.7 mmol) in hexanes (25 mL) Ear), stirring continuously at room temperature overnight. The reaction mixture was diluted with DCM (25 mL) and washed with EtOAc EtOAc (EtOAc) The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product (5 g) was used directly in the next reaction.

Step 3 - Synthesis of Boc-TAP: The crude product from the previous reaction was dissolved in methanol (25 mL) and hydrogenated at balloon pressure for 16 hours in the presence of 5% palladium/carbon (5% w/w). The mixture was filtered, washed with EtOAc (EtOAc)EtOAc. The total estimated yield of Boc-TAP after steps 1-3 was 44% (calculated based on the amount of Cbz-Cl).

Step 4 - Synthesis of TentaGel Bonding Group: Brominated Tianta Gum 15 (2.5 g '〇.48 mmol/g' from Rapp Polymers, phenolic bond) 5 equivalents of the base and potassium carbonate (5 equivalents) were heated to 70 ° C in 2 mL of DMF for 14 hours. After cooling to room temperature, the resin was washed (0·1 N Ha, water, ACN, DMF, MeOH). And drying to obtain an amber resin.

75 1344964 Step 5 - Synthesis of Tianta Glue-TAP (Boc): 2.5 g of the above-obtained resin and H-TAP-Boc (1.5 g, 5 equivalents) and glacial acetic acid AcOH (34 μL, 5 equivalents) Mix in 1:1 MeOH-THF and shake overnight. A solution of 1 M sodium cyanoborohydride (5 eq.) in THF was added thereto and shaken again for 7 hours. The resin was washed with filtration (DMF, THF, 0.1NH.sub.1 water, MeOH) and dried. A small amount of resin was deuterated in DCM using Bz-Cl and DIEA, cleavage in 70% TFA-DCM, and by LCMS and HPLC.

Step 6 - Synthesis of Tianta Glue-TAP-Lys: Resin 10 obtained above was prepared by dissolving Fmoc-Lys(Fmoc)-OH active solution (5 equivalents of amino acid and 5 equivalents of HATU in 0.5 Torr in DMF). Then 10 equivalents of DIEA treatment) was added and allowed to shake gently for 14 hours. The resin was washed (DMF, THF, DCM, MeOH) and dried to give a purified resin. The residual amine group was treated with a 1% by weight acetic anhydride &lt; 20% pyridine in DCM solution for 20 minutes, followed by washing as before. The Fmoc group was removed by gently shaking the resin with 30% piperidine in DMF for 20 minutes, followed by washing (DMF, THF, DCM, MeOH) and dried.

Step 7 - Synthesis of tyrosin-linked TAP-Lys (peptide) 2: as above 76 1344964 Resin accepts Fmoc-amino acid coupling, activates with HBTU/HOBt, and repeats the cycle of removal of Fmoc with piperidine Processing, to build a second peptide chain at the same time. It is conveniently carried out from Applied Biosystems' ABI 433 automated peptide synthesizer. After the final Fmoc removal, the terminal amine groups were deuterated in acetic acid (1 当量 equivalent) and DIEA (20 eq.) in DMF for 20 minutes, followed by washing as previously described.

Ίο Step 8 - Cleavage by resin: The resin obtained above was suspended at room temperature in TFA (82.5%), aged (5%), ethanedithiol (2.5%), water (5%) and thiocyanate (5%). ) The solution was 3 hours. Another cleavage mixture can also be used, such as TFA (95%), 7]c (2.5%), and triisopropyldecyl (2 5%). The TFA solution was cooled to 5 ° C and poured into diethyl ether to precipitate the peptide. Filtration and dehydration under reduced pressure gave the desired peptide. Purification by preparative HPLC using a C19 column gave a pure peptide.

Step 9 - Oxidation of the oxime to form an intramolecular disulfide bond: The peptide peptide was dissolved in 20% DMSO/water (1 mg dry weight peptide/ml) and allowed to stand at room temperature for 36 hours. The peptide was purified by loading the reaction mixture on a C18 HPLC column (Waters Corporation Delta-Pak C18, 15 77 1344964 micron particle size, 300 angstrom aperture, 40 mm x 200 Millimeter length) followed by a linear ACN water/0.01% TFA gradient from 5% to 95% ACN for 40 minutes. The elution fraction containing the desired peptide was lyophilized to give the product as a flocculent white solid.

Binary peptides (XYZ) containing reduced disulfide bonds

DMSO/H2O

S-S

Binary peptides containing oxidized biguanide bonds (XYZ) 5 Step 10 - PEGylation of terminal NH2 groups: PEGylation via amino phthalate linkages: peptide equivalents mixed 1.5 equivalents (mole basis) activity PEG species (mPEG-NPC, available from Sakamoto NOF) A clear solution was obtained in anhydrous DMF. After 5 minutes, 4 equivalents of DIEA were added to the above solution. The mixture was stirred at ambient temperature for 14 hours then purified by C18 reverse phase 10 HPLC. The structural formula of the PEGylated peptide was confirmed by MALDI mass spectrometry. The purified peptide was also purified by cation exchange chromatography, as described later. (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)ί Ο ?ΝΗ- (AcGjGLYACHMGPrrCI-naQVCQPLRtMeO^HN^

mPEG-NPC DIEA, DMF (AcG)GLYACHMGPIT(l.na1)viQPLR(MeG)—). NH/ O A. O-PEGjok 〇(AcQGLYACHMGPnXI-aaQVCQPLR^MeGHIN^NH-VQ^ 78 0 1344964 via guanamine bond PEGylation: peptide peptide mixed L5 equivalent (mole reference) active PEG species (PEG-SPA-NHS 'from Shearwater, USA) to obtain a clear solution in anhydrous DMF. After 5 minutes, 4 Equivalent DIEA was added to the above solution. The mixture was mixed at ambient temperature for 2 hours, and then purified by C18 reverse phase HPLC. The structure of PEGylation was confirmed by MALDI mass spectrometry. The purified form was also subjected to cation exchange chromatography. Purification, as described below.

PEG.SPA.NHS

D1EA.DMF

Step 11 - Ion exchange purification: In addition to the ability to retain the initial binary peptide, several exchange supports were also investigated for their ability to separate the aforementioned peptide-PEG conjugates from unreacted (or hydrolyzed) pEG. The ion exchange resin (2_3 g) was loaded on a 1 cm column and then converted to sodium form (〇2 N sodium hydroxide was placed on the column until elution was divided into pH 14, about 5 times the column volume) instead of Hydrogen form (extracted with 0.1 N hydrochloric acid or 〇.1 M acetic acid until the elution fraction matches the loading cation of about 15 15 column volumes)' then washed to ρ Η 6 with 25 % AC Ν/water. Pre-Conjugation The peptide or peptide-PEG light compound is dissolved in 25% ACN/water (1 mg/ml) and the pH is adjusted to less than 3 with THA and the load is applied to the column. After washing with 2_3 tube volume 79 1344964 25% ACN/water and collecting 5 ml of elution fraction, the peptide was released from the column by 0.1 Μ ammonium acetate in 25% ACN/water, and 5 ml of the elution fraction was collected again. . Analysis by HPLC showed that the elution fraction contained the desired peptide. Analysis by gasification light scattering detector (ELSD), indicating that when the peptide remains in the column and is eluted with 5 ammonium acetate solution (usually eluted in elution 4 and elution 10), it is not observed. Contaminants are not PEG. When the peptide was eluted in the initial elution buffer ' (usually the first two washes), no separation of the desired PEG-conjugate and excess PEG were observed. The following columns successfully retained the peptide and peptide-PEG complexes and were successfully purified from the non-light compound peptides. PEG roll compounds: Table 1: Ion exchange resin support source Mono S HR 5 /5 strong cation exchange preloaded column Amoshan (AmershairO Biotech SE53 cellulose, fine particle cation exchange support body Wattman SP Sepharose fast flow strong cation exchange support Amoshan Biotech company' 2: Synthesis of EPO-R agonist peptide peptide by fragment condensation Step 1 - Synthesis of (Cbz)2-Lys: Amino acid is reacted with chlorine and 15 benzyl acetate solution under standard conditions to obtain two amines. The base is protected with an amine acid using a Cbz group.

Step 2 - Synthesis of Boc-TAP: Boc-TAP was synthesized as described in steps 1 to 3 of Example 1. 80 1344964 Step 3 - Coupling of (Cbz)-Lys with Boc-TAP: (cbz)2-Lys coupled with Boc-TAP under standard coupling conditions to obtain (Cbz)2-Lys-TAP-Boc.

Step 4 _ Lys-TAP-Boc: The crude product obtained from the previous reaction was dissolved in methyl alcohol (25 ml) and nitrided at 5% palladium on carbon (5% w/w) under balloon pressure for 16 hours. The mixture was filtered, washed with EtOAc (EtOAc)EtOAc.

10 Step 5 - Synthesis of peptide monomer by fragment condensation: The peptide fragments of the four peptide monomers are synthesized by standard techniques. Part of the protected smear piece is subjected to two separate coupling reaction rounds. In the first round, the monomer is called the half-coupled with the formation of the two peptide fragments, and the c-terminal of the monomer is coupled with the other two: = formation, the second coupling round, and the N-terminal half and the c-terminal semi-coupling Follow the monomer. Nilai relied on standard performance (4) (10) to solve the problem. ’, 81 1344964

OtBu Tit coupling

FhiocU t-f-d-na^-V-όγΡ-Οί OlBu Trt

Coupling S(Aan) CAeC)-G-L-Y-A-C-H-M Offiu Tit

Offlu Trt Pbf

Coupling

Standby protection (AcG)-G-

S(Acm) -Tj-( l-nal)-V&gt;CQPLR-(MeG)-OH OtBu Tit Pbf Step 6 - Oxidation of the peptide monomer to form an intramolecular disulfide bond: then 〇Bn removes the protective condensation The peptide monomer is oxidized with iodide to form an intramolecular disulfide bond between the Acm-protected cysteine residues of the monomer. S(Acm)

Trt Pbf

OlBu ΤΠ

Offlu

t-P-I-T-(l-oaI)-V-C-Q-P-L.R^MeG)-OH 1 II 5 (AcG&gt;G.L-yA-C-H-M-0-l OtBu Tit

Ofiu Tit Pbf Step 7 - Lys-TAP-Boc coupling to the oxidized oxime Bn deprotected monomer to form a peptide binary: Lys-TAP-Boc coupled to twice the molar excess under standard conditions The oxidized OBn removes the protected monomer to form a peptide binary. The peptide binary is then deprotected under standard conditions. 82 1344964

(Ai^)-G-L.yA&lt;:.^M.G.P-I.T&lt;l^).vAQ.p.L-R^MeG)-OH

OtBu Trt W&gt;£

OiBu Tr I i,. Oxidation :\ NHBoc

HjH NH • 0 inOBoc o

NH· _0 °®u ^ C®« in kt (AcG&gt;04^yAX-HMGPIT^i -naJ&gt;V-&lt;iQp^L*R^McG; 咖u Tn Ofiu Tff Pbf Deprotection (Αϋ〇ΗΗ^ Υ-Α·€-Η-Μ«&lt;3·Ρ·Ι·Τ&lt;1-ι»1)ΛΛ&lt;^&lt;}·ρ·ΐ^(Μβ〇: (AcG)-G*LY-ΑΌ -Η-Μ-GPlT-{I ^al)^*(L&lt;^P-L4lr&lt;MeG)--HN'

NH- 0 0 Step 8 - Pegification of the deprotected binary: then deprotected peptide The PEGylation was carried out as described in Step 10 of Example 1. Step 9 - Ion exchange purification: PEGylated peptide peptides were then purified as described in Example 1, Step 1, Step 11. Example 3: In-tube activity assay analysis This example illustrates a plurality of in-vitro assay assays that can be used to assess the activity and strength of the EP0-R agonist peptide of the present invention. The results of the assay confirmed that the novel peptide of the present invention binds to EPO-R and activates EPO-R signaling. In addition, the results of the assay showed that the novel peptide composition compared to the aforementioned EPO mimetic peptide, the EP0-R binding affinity and biological activity unexpectedly increased. 83 1344964 Ε Ο R - R agonist peptide binary system was prepared according to the method provided in Example 丨 or Example 2. The strength of these winning binary bodies was evaluated using a series of test tube activity assays, including: notification subtest analysis, hyperplastic assay analysis, repeated binding assay analysis, and C/BFU-e assay analysis. Four assays analyzed β 5 further Explain its details as follows. The results of these in vitro assays are summarized in Table 2. • 1. Notification of sub-assay analysis • This assay is based on the reporter cell Baf3/EpoR/GCSFR fos/lux derived from the rat sputum cell line. This reporter cell line can represent a chimera, including the internal portion of the human GC0 receptor from the human EP0 receptor to the extracellular portion. This cell line was further transfected with the f0S promoter gene-driven luciferase reporter gene assembly. This chimera receptor is activated by the addition of a red blood cell generator&apos; resulting in the expression of the luciferase reporter gene, thus emitting light when the luciferase enzyme matrix luciferin is added. The extent of 15 EPO-R activation of such cells can be quantified by measurement of luciferase activity. Moxibustion Baf3/EpoR/GCSFR fos/lux cells were cultured in DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (fbs; Hyclone), 10% WEHI-3 supernatant The supernatant (by WEHI-3 cells, • ATCC # TIB-68) was cultured to obtain penicillin/streptomycin. The assay was divided into -20 minutes before the precipitation, and the cells were transferred to DMEM/F12 medium to supplement 10% FBS and 0.1% WEHI-3 supernatant and ft. On the day of assay analysis, the cells were washed with DMEM/F12 medium supplemented with 10% FBS (without WEHI-3 supernatant), and then tested at a known concentration of lxlO6 cells/ml. Culture in the presence of peptides, or use EP0 (R&amp;D Systems, Minneapolis, Minnesota) for 84 1344964 as a positive control, supplemented with DMEM/F12 supplemented with 10% FBS (without WEHI-3 supernatant) A series of dilutions of the test peptide was tested simultaneously in this assay. The assay plate was assayed at 37. (: incubated for 4 hours in a 5% carbon dioxide atmosphere. Subsequent addition of luciferin (Steady_G1〇) Promega 5 (Marison, Wisconsin) to each hole. After 5 minutes of incubation, at Parker (Packard) company's desktop photometer (Parker Instruments, Donna Gloria, Ill.) measures luminescence. Light intensity values are plotted against test peptide concentration's using Graph Pad software analysis. The result is a semi-maximum luminescence test. The skin wave was recorded as EC50 [Ref. Table 2: Bulletin EC50]. 10 2. Proliferation assay This assay is based on the Bf3 metastasis of the pre-B cell line to express human EPO-R. The resulting cell line Baf3/Gal4/Elk The proliferation of /EPOR is associated with EPO-R activation. The degree of cell proliferation is quantified using MTT, where the signalling of the MTT assay is directly proportional to the number of viable cells. 15 BaF3/Gal4/Elk/EPOR cells are replenished in a rotating vial 1〇% FBS (Hylon) and 2% WEHI-3 supernatant (ATCC # TIB-68) in DMEM/F12 medium (Jibo Co.) culture. After culture, the cells were centrifuged to a cell density of lxlO6 cells. /ml, DMEM/F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant was lacking overnight. Then the depleted fine cells were washed twice with Dubec PBS (Gibb) and resuspended in supplement 10%. FBS (without WEHI-3 supernatant) to cell density lxl〇6 Cells / ml was dispensed .50 l (about 5 0,000 cells) of the cell suspension was inoculated in triplicate in 96 hole plate assay. A full 50 microliter test EPO mimetic peptide series dilution or 50 microliter EPO (R&amp;D Systems, Minneapolis, Minnesota 85) or Arny & Aranesp A: (darbepoeitin α), purchased from Amgen's EPO-R agonist) added to DMEM/F12 medium supplemented with 10% fbs (without WEHI-3 supernatant I) to 96-well assay Orifice plate (final volume of each hole is 100 microliters). For example, test 12 different dilutions' test traits (or control EPO peptides) to a final concentration ranging from 8 l 〇pM to 0.0045 ΡΠ1. The plate cells after inoculation were then at 37. (: culture for 48 hours. Next, 10 μl of MTT (Rose Diagnostics Inc.) was added to each well of each culture dish, and then allowed to culture for 4 hours. The reaction was stopped by adding 1% SDS + 0.01 N HCl. The plate was at 37°. C overnight. Then the absorbance ratio of each well at 595 nm was measured by spectroscopic spectroscopy. The absorbance ratio reading was plotted against the test peptide concentration, and the EC50 was calculated using Graph Pad software. The test score of the semi-maximum absorbance ratio was obtained. As EC50 [Reference Table 2: Proliferation EC50] 3. Competitive binding assay analysis uses competitive assays to perform competitive binding calculations. The optical signal in the assay is based on a change in the proximity of the two beads: Streptavidin-resistant biotin donor beads The pellet carries a biotinylated EP〇_r binding peptide tracer and an acceptor bead that incorporates EPO-R. The non-radiative energy shifts the luminescence, at which time the solitary oxygen is released from the first bead when illuminated. The second bead contacts the orphaned oxygen to cause luminescence. Such a bead collection can be obtained commercially (Pike's Co., Ltd. bead-proximate system is produced by EPO-R binding peptide tracer binding to Ep〇R. Test peptide and EP0 -R combined with peptide Competition for binding to Ep〇_R will prevent this binding, resulting in reduced luminescence. Further details of this method are as follows: Add 4 μl of test EPO-R agonist peptide one dilution or positive control or negative Control to each well of 384-well plate 1344. 4. Subsequently, add 2 μl/receptor bead mixture per well. The composition of the acceptor bead mixture is: 15 μl of 5 mg/ml Streptavidin Receptor Beads (Pike), 15 μl of 5 g/ml monoclonal antibody abl79 (this antibody recognizes the human placental alkaline phosphatase protein fraction contained in recombinant EPO-R), protein 5 A coated cap beads ( Protein A binds to abl79 antibody; Parker), 112.5 μl of 1:6.6 recombinant EPO-R dilution (Chinese hamster ovary cell manufacturing 'binding to human placental alkaline phosphatase protein fraction (which contains abl79 target epitope) Fusion protein, and 607.5 μl Alphaquest buffer 4 mM HEPES, pH 7.4; 1 Mm MgCl2; 10 0.1% BSA, 0.05% ton 20). Tap the tabletop mix. Add 2 μl /hole biotinylated EPO-R combined with skin refining agent, AF330 68 (30 nM final concentration) AF33068 is an EPO-R binding peptide (refer to Table 3 "Notifier EC50 (pM)") prepared according to the method described in Example 1. AF33068 15

Biotin*GGLYAi CHMGPITWVijQPLRG^ K-NH,

Bioda^3GLYACHMGPrTWVCQPLRG 混合 Mix by centrifugation for 1 minute. The plate is sealed with a Parker top seal and wrapped in a metal foil. Gently incubate the instrument. After 18 hours, use Alpha Chastain to read the cry (Pike) to read the luminescence. Luminescence is plotted against peptide concentration and analyzed using Graph Pad or Excel. Comparing the observations without the test peptide, the luminescence was reduced by 5%. 2 The test peptide concentration was recorded as IC50 [Ref. Table 2: AQ IC50]. 4. C/BFU-e assay analysis EPO-R signaling simulates the differentiation of bone marrow stem cells into proliferating red blood cells 87 precursor cells. This assay analyzes the test peptide to mimic the proliferation and differentiation of red blood cell precursor cells derived from a single human bone marrow multiplexed stem cell. For the purpose of this assay, a series of dilutions of one of the test peptides were prepared by supplementing 10% FBS (Hai Keng) IMDM medium (Jibo Co.). A serial dilution or positive control EPO peptide was then added to the thiol cellulose to obtain a final volume of 1.5 ml. The methylcellulose and peptide combination are then thoroughly inverted. Whole (100,000 cells/ml) human bone marrow derived from CD34+ cells (Poietics/Cambrex) was thawed. Then the cells after the bed was gently added to a 50 ml test tube. 1 ml of 1 mg/ml 10 DNAse (stem cells h followed by 40-50 ml of IMDM medium gently added to the cells: the first 10 ml of the medium was passed alongside the 50 ml tube Add dropwise, then the rest of the medium is slowly distributed along the tube. Then the cells are centrifuged at 9 rpm for 20 minutes, and the medium is carefully removed by gentle aspiration. The cells are resuspended in 1 ml of IMDM medium and counted per ml of cells on a hemocytometer slide. Density (15 is a whole 10 microliters of cell suspension on a slide with an average cell density of 0 〇〇 cells/ml). The cells are then diluted to 15 in IMDM medium at a cell density of 〇〇〇 cells/ml. One microliter of diluted cells were then added to each 15 ml sample of methylcellulose conjugated peptide (the final cell concentration in the assay was 丨〇〇〇 cells/ml). The mixture was inverted. The bubbles disappeared and then 20 ml was pipetted with a blunt end needle. 0.25 ml of the extracted mixture from each sample was added to each of the four wells of a 24-well plate (Falcon brand). The inoculation mixture was cultured for 14 days at 37 ° C in 5% incubator in a humidifier. The red blood cell population was calculated using a phase microscope (5 times -10 times objective lens, final magnification 1 〇〇). Comparison using the EP0 positive control group, Generated group = number 88 1344964 The test peptide concentration of 90% of the maximum value was recorded as EC9 〇 [Reference Table 2: C/BFU-e EC90] 5. Radioactive ligand competition binding assay analysis of other radioactive ligands The competitive binding assay can also be used to determine the 1 Cso value of the peptide of the present invention. This assay analyzes the binding of I-EPO to EPOR. The assay is preferably performed according to the following exemplary protocol:

A. Material Recombinant Human EPOR/Fc Chimera • Recognition: Recombinant Human EPOR/Fc Chimera: Lingshen Ning 9:6 Alliance System Corporation (Minneapolis, Minnesota, USA) • Boundary: EOK033071 • Storage: 4° C moth recombination human red blood cells. Identification: (3[125!] iodotyrosamine) erythropoietin, human weight, high specificity, 370 kBq, 10 μα • Supplier: Amoshan Biotech (USA) Pitzkawi, Jersey) • Model: Μ219-10μα • Accusation: • Storage: 4°C Protein-G Sifaros • Identification: Protein-G Sifaros 4 Fast Flow • Supplier: Amoshan Biotech ( Pittsburgh, New Jersey, USA • Model: 17-0618-01 • Batch number: • Storage: 4°C assay buffer • Phosphorylation buffered saline (PBS), pH 7.4, containing 0.1% bovine blood : 杳 albumin and 0.1% stacked gas Nadine orphaned • Storage: 4eC

B. Determination of the appropriate receptor concentration 10 Reconstitute a vial of 50 μg in 1 ml assay buffer; Concans recombinant EPOr extracellular domain was fused to the human igG1 Fc fraction. In order to determine the correct amount of the assay for the assay, 1 〇〇 microliter of a series of diluted preparations of this receptor was combined with a 12x75 mm polypropylene tube for about 2 〇, 〇〇〇cpm at 200 μl of iodinated recombinant human erythropoietin ( Ιι'ΕΡ0). The tubes were capped and gently mixed overnight at 4 °C on a LabQuake rotary shaker. The next day, 50 μl of 50% protein-G sifaros slurry was added to each test 89 1344964 tube. The tubes were then incubated at 4 ° C for 2 hours with gentle mixing. The tube was then centrifuged at 4000 RPM (3297XG) for 15 minutes to pellet the protein-G sifaros. Carefully remove the supernatant and discard it. Take 1 ml of 4. (: After the assay analysis buffer was washed three times, the small pieces were counted in the Wallac Wizard gamma counter to calculate the number k 5 mesh. Then the results were analyzed to determine the degree of dilution required to reach 50% of the maximum binding value. Determination of peptide IC5Q. For the determination of AF37702's IC5〇' 1〇〇 microliter peptide series of dilutions 100 μl recombinant erythropoietin receptor (1 〇〇 picogram / tube) in 10 12x75 mm polypropylene Tubes. Then 100 microliters of iodinated recombinant human erythropoietin (125I-EPO) was added to each tube's tube-capped and gently mixed overnight at 4 ° C. The next day, the combined 125i-epo was quantified as described above. For analysis, IQ was calculated using Graphpad 15 Prism version 4.0 from GraphPad Software, Inc. (San Diego, Calif.). Repeated assay analysis for each test peptide was repeated two or more times for a total of 3 or more replicates. Lc5 〇 measured value. 90 1344964 ^&lt;^^^±i-^M^^wll^M^^:fs&lt;C/BFU-e EC90 (nM) &lt;N (N 卜&lt;N IC50 (pM) I_ Ο r—^ hyperplasia EC50 (nM) II reporter EC50 (pM) II 150 peptide binary o (AcQ)OLYA (!HMGPrr( ln*〇 V&lt;!x3PLR(MeG)_mi NH^-PEQsok 〇° (AcG)aLYACTMGPIT(l-nal)VCQPLR(MeG)*HN/^VNH--y_^/r^ UJ 〇=^ Ό 1 i 1 I ri I ! i ! LI I ^ r | έ 1 l Compound nomenclature AF35645 AF35527

91 1344964 Example 4: In vivo activity assay analysis This example validates an in vivo assay for assessing the activity and intensity of the EPO-R agonist peptide of the present invention. The Ep〇R agonist peptide binary system was prepared according to the method provided in Example 1 or Example 2. Peptide monomer and binary ‘5 d in vivo live (IV) use—series assay analysis includes multiple blood cell extracellular deficiency • oxygen mouse bioassay analysis and reticular cell bioassay analysis. Further details of the two assays are described below. • 丨·Multiple Hematocrit Extracellular Hypoxia Mice Bioassay Test The succulent test is based on the bioassay of multiple blood cells in extracellular hypoxic mice. The assay is determined by Ctoes and Bangham (1961). Natural method 191: 1065-1067 method correction. This assay analyzes the test peptide as an EPO mimetic: that is, the ability to activate Ep〇_R and induce new red blood cells to synthesize. The synthesis of red blood cells is based on the quantification of radiolabeled iron bound to synthetic hemoglobin. 15 BDF1 mice were domesticated for 7-10 days. All mouse body weights were determined, and mice that were too light (less than 15 grams) were not used. The mice received a continuous conditioning cycle in a low pressure chamber for a total of 14 days ^ 24 hours period with 18 hours in 〇 4 bauxite • 0.02% atmospheric pressure and 6 hours in ambient pressure. After conditioning, the mice were maintained at ambient pressure for 72 hours prior to administration. -20 Test peptide or recombinant human EP standard was diluted in PBS + 〇.1% BSA vehicle PBS/BSA). The Shengfu early body backup solution was first solubilized in the dimethyl submine (dms〇). The negative control group included a group of mice injected with PBS/BSA alone, and a group of mice injected with 1% DMS0 alone. Each dose group had 1 taro mouse. Mice were injected subcutaneously (neck back) with 0.5 ml of the appropriate sample. 92 48 hours after the injection of the sample, the mice were intraperitoneally injected with 毫升2 ml of Fe, 枉邦 2 Division NEN) at a dose of about 0.75 microcures per mouse. Mice were weighed 24 hours after administration &amp; 59, and sacrificed 48 hours after administration of sputum. Blood samples were taken from each animal by cardiac puncture, and the measurement was also performed (heparin was used as an anticoagulant). Each blood sample 5 = 2 ml was analyzed using a Parker gamma counter... binding amount. Unresponsive small milk (i.e., mice with less radioactive binding than the negative control group) were excluded from the appropriate data collection. Mice with a blood volume lower than 53% of the negative control group were also excluded. The results were derived from a collection of 10 mice at each experimental dose. The average amount of radioactivity (counting per minute (CpM)) of each group and the blood sample was determined. · 10 2♦ Reticulocyte assay analysis Normal BDF1 mice were given 3 consecutive doses (〇 5 ml 'subcutaneous injection) Ερ〇 for makeup or test peptides. On the third day, mice (〇1 ml, intraperitoneal injection) of iron glucomannan (1 mg/ml) were administered. On the fifth day, the mice were bled with carbon dioxide and by heart puncture. The percentage of reticular cells (%) of each blood sample was measured by artificial sputum 15 by orange staining and flow cytometry analysis (grid counting program). The corrected number of reticulocytes was determined using the following formula: % RETIC corrected ==% RETICfewX (blood volume (four) / blood volume μ) — 3. Hematology assay analysis of normal CD1 mice four times a week high dose intravenous Epo Positive pair, 2 photos, test traits or vehicle. A range of positive controls and test peptides -%· expressed in mg/kg are modified by varying the concentration of active compound in the formulation. The left shot is 5 ml / kg. The vehicle control group contained 12 animals, and each of the remaining drug-administered groups contained 8 animals each. Survival was recorded daily, and body weight was recorded weekly. 93 1344964 The mice were vaccinated with fasting mice followed by inhalation of isoflurane, on the first sputum (media control mice) and on the 15th day. And 29th (4 mice/group/曰) blood samples were taken by cardiac puncture or abdominal aortic puncture. The blood is transferred to the Vacutainer brand test tube. A preferred anticoagulant is exoethyldiaminetetraacetic acid (EDTA). Refer to the well-known automated clinical analyzers (such as those manufactured by Coulter Inc.) to evaluate the erythrocyte synthesis endpoints and physiology of the blood samples such as Rong (1^1;), hemoglobin (Hgb), and total red blood cell count (rbc).实施 Example 5: Amino acid sequence (AcG) GLYACHMGPnXl-nal) VCQPLRK (SEQ ID NO: 1) peptide peptide monomer Ep〇_j^activator peptide homodimer synthesis step 1 - win Synthesis of Peptide Monomers: The peptide single system was synthesized using standard Fm〇c chemistry on an eight-61431 eight-peptide synthesizer using Dingqi 7:8 14 resin (0.18 mmol/Krepp polymer, Germany). For the synthesis of 15 peptide monomers with a guanidine carboxyl end, the fully assembled peptide was cleaved from the resin using 82.5% TFA, 5% &gt; water, 6.25% anisole, 6.25% ethanedithiol. The deprotected product was filtered by resin and precipitated using a puzzle. After thorough drying, the product was purified by C18 reverse phase high performance liquid chromatography eluting with acetonitrile/water eluting with 0.1% trifluoroacetic acid. The peptide structure was confirmed by electrospray ionization mass spectrometry. The peptide was dissolved in -20 DMS0: water 1:1 solution at a concentration of 1 mg/ml to affect the formation of disulfide bonds. The product was purified by C18 reverse phase high performance liquid chromatography eluting with EtOAc/water eluting The peptide monomer is shown as follows. (AcG)GLYACHMGPrT(l-nal)VCXJPLRK.NH2 94 1344964 15 Step 2 - Synthesis of trifunctional linkages: imino group (10) di(tetra) (10). gram, 52.8 millimolar and Bg adenine (sharp , 52 8 mM) Diisopropyl hydrazine iodide (8.0 ml, 511 mmol) was added to the solution of MgSO. The reaction mixture was warmed to about HTC' during the addition and then cooled back to room temperature over a period of (four) minutes. The reaction mixture was allowed to stir overnight and transitioned to the diisopropyl vein of the coffee. The solvent was removed by evaporation under reduced pressure to give a gum, which was dissolved in ethyl acetate. The organic phase was placed in a separatory funnel, washed with sodium (saturated sodium sulphate, brine, 0.5 N hydrochloric acid, brine), dehydrated (sulphuric acid) and evaporated under reduced pressure to give the product as a colorless oil. The mixture was taken up in methanol: THF 1:1 mixture (100 ml), water (25 ml) was added and then sodium hydroxide (5 g, 125 mM) was added. The pH was measured to be greater than 1 Torr. The reaction mixture was stirred at room temperature for 2 hours and then acidified to pH with EtOAc. The aqueous phase was saturated with sodium chloride and extracted 4 times with ethyl acetate. The combined organic phases were washed (salt water) &apos; dehydrated (MgSO4) and concentrated under reduced pressure to afford white semi solid. The solid was dissolved in 50 ml of DCM, and 3 ml of hexane was added thereto to form a white slurry. The solvent was removed under reduced pressure to give the diacid as a white solid (14 g, 91. Add N-hydroxybutaneimine (77 mg, 6 69 mmol) and diisopropyl hydrazine to a solution of 2 mM 20 DMF of diacid (1 g, 3 29 mM) Imine (1 ml, 6 38 mmol) and 4-didecylaminopyridine (3 mg '0.02 mmol). The reaction mixture was stirred overnight and the solvent was evaporated under reduced pressure. The residue was taken up in ethyl acetate and filtered to remove the precipitated urea. The organic phase was placed in a separatory funnel, washed (saturated sodium hydrogen carbonate, brine, 0.5 N hydrochloric acid, brine), dried (MgSO4), filtered, and concentrated under reduced pressure 95 1344964 to give the bis-NHS ester product. White solid (1.12 g, 68% yield).

Step 3 - Trifunctional linkages are coupled to the peptide monomer: for coupling to the linkage, 2 equivalents of the peptide are mixed with 1 equivalent of a trifunctional linkage based on anhydrous DMF to give a clear solution, and 2 equivalents of DIEA are added after 2 minutes. The mixture was stirred at ambient temperature for 14 hours. The solvent was removed under reduced pressure and the crude material was dissolved in &lt;RTI ID=0.0&gt;&gt; The binary structure was confirmed by electrospray ionization mass spectrometry. This coupling reaction attaches a bond to the nitrogen atom of the epsilon amine group of the amine acid residue of each monomer. 96 1344964 (AoG)GLYACHMGPrT(l-naI)VCQPLRK-NH2 Ο

Ο

Ο V ΝΗ

ΝΗ2

(AcO)GLYACHMGPIT(l-nBl)VCQPLR-HN

Ο 97 1344964 Step 4 _Pepsylation of peptide peptides

PEGylation through urethane linkage: peptide peptide mixed equal amount (mole reference) active PEG species (mPEG-NPC, obtained from Sakamoto NOF) after obtaining clear solution in anhydrous DMF for 5 minutes ' 4 Equivalent DIEA is added to the aforementioned solvent. The mixture was stirred at ambient temperature for 14 hours then purified by C18 reverse phase HPLC. The structural formula of the PEGylated peptide was confirmed by MALDI mass spectrometry. The purified peptide was also purified by cation exchange chromatography and summarized as follows.

(AcG)GLYACHMGPIT(l-iBl)VCQPLR-i

NH 0

O

N V (AcO) GLYACHMGPir (l.nal) VCQPLR-

inPEQ-NPC

DOEA, DMP

(AcG)GLYACHMGPrT(l-nal)V(!QPLR-NH NH c

〇 O

, NH 〇-pEGa〇K

N V NH

O 98 1344964 PEGylation via guanamine linkage: peptide peptide mixed equal (mole reference) active PEG species (PEG-SPA-NHS, available from Shearwater, USA) in anhydrous DMF Clarify the solution. After 5 minutes, 4 equivalents of DIEA was added to the aforementioned solution. The mixture was stirred at ambient temperature for 2 hours 5 and then purified by C18 reverse phase HPLC. The structural formula of the pEG-derived peptide was confirmed by MALDI mass spectrometry. The purified peptide was also purified by cation exchange chromatography and summarized as follows.

NH2 O (AoG)QLYACHMC3&gt;rr(l-naI)VCQPLR- NH,

I I (AcO)OLYACHMGPrT(l-oaI)VCQPLR- ΡΒΟθ: DIEA,

ΡΑ-NHS

DMF

NH

(AcG)GLYA0aMGHT(l-naI)V(lQPLR-NH JL

(AcG)GLYACHMGPrr(l-na])VCQPLR-HN °v m

NH 99 0 1344964 Step 5 - Ion exchange purification of peptides: In addition to the ability to retain the initial binary peptide, several exchange supports were also investigated for their separation of the aforementioned peptide-PEG yoke from unreacted (or hydrolyzed) PEG. The ability of the compound. The ion exchange resin (2-3 g) was loaded on a 1 cm column and then converted to sodium form (〇2 N sodium hydroxide • 5 on the column until the elution was divided into PH 14, about 5 times the tube volume) Instead of converting, the hydrogen form (extracted with 0.1 N hydrochloric acid or 0.1 hydrazine acetic acid until the elution fraction matches the loading pH, about 5 times the column volume)' is then washed with 25% ACN/water to pH 6. The peptide or peptide-PEG complex before compounding is dissolved in 25% ACN/water (10 mg/ml) pH adjusted to less than 3 with THA and then loaded onto the column. After washing with 2-3 10 column volume 25% ACN/water and collecting 5 ml of elution fraction, the peptide was released from the column by 0.1 μ ammonium acetate in 25% ACN/water, and 5 ml of the elution was collected again. Minute. By HPLC analysis, the elution fraction contains the desired peptide. Analysis by gasification light scattering detector (ELSD), indicating that when the skin is retained on the column 'and using ammonium acetate solution (usually in elution 4 and elution) When eluted in 1 minute, the PEG without contaminant was not observed. The separation of the desired PEG-conjugate and excess PEG were not observed when the precursor was eluted from the initial elution buffer (usually the first two washes). The following columns successfully retained the peptide and peptide-PEG conjugates, successfully purified from the non-conjugate peptides. PEG conjugates: -2 〇 Table 3: Sources of ion exchange resin supports Mono S HR 5/5 strong cation exchange preloaded column Amorshan (Amersham) Bios company SE53 cellulose 'fine particles strong cation exchange support body Wattman company SP West Faros (Sepharose) fast flow strong cation exchange support Amo Shansheng 100 1344964 Example 6: Synthesis of EPO-R agonist peptide homologous binary with amino acid sequence (AcG) GLYACHMGPIT (l-nal) VCQPLRK (SEQ ID NO: 1) Amino acid sequence (AcG) GLYACHMGPITU-nal) EPO-R agonist peptide of the peptide peptide monomer of VCQPLR (MeG)K (SEQ ID NO: 2) is synthesized as described in Example 1, but Step 1 'The synthesized peptide monomer is: 10 (AcG) GLYACHMGPIT (l-nal) VCQPLR(MeG)K Here, the PEG is attached to the bonding group through a urethane linkage, and the final product of the synthesis is The structural display is as follows:

If the PEG is attached to the linkage via a guanamine linkage, the structural formula of the synthetic end product is shown as follows:

101 1344964 Example 7: Intra-tube activity assay analysis This example illustrates a plurality of in-vitro assay assays that can be used to assess the activity and strength of the EPO-R agonist peptide of the present invention. The results of the assay confirmed that the novel peptide of the present invention binds to EPO-R and activates EPO-R signaling. In addition, the results of the analysis showed that the novel peptide composition was compared with the aforementioned EPO simulation, and the EPO-R binding affinity and biological activity unexpectedly increased. The EPO-R agonist peptide monomer and binary system were prepared according to the method provided in Example 1 or Example 2. The strength of these peptide peptides was assessed using a series of tube activity assays, including: reporter assay, hyperplasia assay, repeated binding assay, and C/BFU-e assay. The four verification analyses further illustrate the details as follows. The results of these in vitro assays are summarized in Table 4. 1. Bullet assay analysis This assay is based on the pre-murine B cell line-derived reporter cells 15 Baf3/EpoR/GCSFR fos/lux. This reporter cell line can represent a chimera receptor, which contains the internal portion of the human GCSF receptor from the human EPO receptor to the extracellular portion. This cell line was further infected with a fos-inducing gene-driven luciferase reporter gene assembly. This chimera receptor is activated by the addition of a red blood cell-forming agent, resulting in the expression of the luciferase reporter gene, so that it emits light when the luciferase enzyme matrix is added. The degree of EP0-R activation of such cells can be quantified by measurement of luciferase activity.

Baf3/EpoR/GCSFR fosMux cells were cultured in DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone) and 10% WEHI-3 supernatant (by WEHI-3 cells, 102 1344964 ATCC # TIB-68) The resulting supernatant was cultured for penicillin/streptomycin. Approximately 18 hours prior to assay analysis, cells were transferred to DMEM/F12 medium supplemented with 10% FBS and 0.1% WEHI-3. The supernatant was scarce. On the day of the assay, the cells were washed with DMEM/F12 medium supplemented with 10% FBS (with no WEHI-3 supernatant), and then 1x106 cells/ml in the presence of a known concentration of the test peptide. Culture, or use epo (r&amp;d Systems, Minneapolis, Minnesota) as a positive control, supplemented with DMEM/F12 supplemented with 10% FBS (without WEHI-3 supernatant). The dilutions were tested simultaneously in this assay. The assay plates were incubated at 37 ° C in a 5% carbon dioxide atmosphere for 4 hours and 10 hours. Then add luciferin (Steady-Glo; Promega ( Promega) company "Marison, Wisconsin" to each hole. After 5 minutes of incubation, at the Packard table Surface luminometer (Pike Instruments, Donna Gloria, Ill.) measured luminescence. Light intensity values were plotted against test peptide concentration and analyzed using Graph Pad software. Results were obtained for semi-maximal luminescence test 15 peptide concentration record As EC50. 2. Proliferation assay This assay is based on the Bf3 metastasis of the murine pre-B cell line to express human EP0-R. The resulting cell line Baf3/Gal4/Elk/EPOR is associated with EPO-R activation. The degree is quantified using MTT, where the signalling of the MTT 20 assay is directly proportional to the number of viable cells.

BaF3/Gal4/Elk/EP〇R cells were supplemented with DMEM/F12 medium supplemented with 10% FBS (Haylon) and 2% WEHI-3 supernatant (ATCC # TIB-68) in a rotating flask (Jibo Co., Ltd.) )to cultivate. After the culture, the cells were depleted in a rotating flask at a cell density of lxlO6 cells/ml in DMEM/F12 medium supplemented with 10% FBS and 0.1% 103 1344964 WEHI-3 supernatant overnight. The depleted cells were then washed twice with Dubec PBS (Giboco) and resuspended in 1% FBS (without WEHI-3 supernatant) to a cell density of 1χΐ〇6 cells/ml. 50 microliters of liquid fraction (approximately 50,000 cells) of the cell suspension was inoculated three times in 96 wells. A full 50 microliter test EPO mimetic peptide series dilution or 50 microliters EPO (R &amp; D Systems, Minneapolis, Minnesota) or Aranesp (Island Bismuth) : (darbepoeitin α ), purchased from _ Angen (Amgen) EPO-R agonist) added to 1%% FBS (without WEHI-3 supernatant I) in DMEM/F12 medium added to % well assay 10 well plates (final volume of each hole is 1 μL). For example, testing 12 different dilutions, the test peptide (or control EPO peptide) has a final indifference ranging from 81 〇pM to 〇.〇〇45Pm. The plate cells after inoculation were then at 37. (: culture for 48 hours. Next, 10 μl of MTT (Rose Diagnostics Inc.) was added to each well of each culture, and then allowed to culture for 4 hours. The reaction was stopped by adding 1% SDS + 0.01 N HC1. Orifice plate 15 at 37 C overnight. Then the absorbance ratio of each well at 595 nm is measured by spectrophotometry. The absorbance ratio reading is plotted against the test winner concentration, and the EC50 is calculated using GraphPad software. The test peptide concentration of the half maximum absorbance ratio can be obtained. Record as EC50. 3. Competing binding assay analysis 20 Using the profiling analysis to perform competitive binding calculations, the optical signal in the assay is based on a change in the proximity of the two beads: Streptococcus avidin donor beads contain biotin The phlegm-R-binding peptide tracer and the acceptor beads combined with EPO-R. The non-radiative energy shifts the luminescence, when the luminescence releases the solitary oxygen from the first bead, and the second bead contacts the lone Oxygen causes 104 1344964 light. This bead collection is commercially available (Pike). The bead is close to the ΕΡΟ-R-binding peptide tracer binding to EP〇_R. Test peptide and EPO- R-binding peptide Competition for binding to EP〇_R will prevent this binding and result in reduced luminescence. 5 Further details of this method are as follows: Add 4 μl of test ΕΡΟ-R agonist peptide to a series of dilutions or positive control Or a negative control to each well of a 384-well plate. Subsequently, a 2 μl/receptor bead mixture per well is added. The composition of the acceptor bead mixture is: 15 μl of 5 mg/ml Streptavidin Body Beads (Parker) '15 μl of 5 mg/ml monoclonal antibody abl79 (this antibody 10 recognizes the human placental alkaline phosphatase protein fraction contained in recombinant ΕΡΟ-R), protein A coated receptor beads (protein A binds to abl79 antibody; Parker), 112.5 microliters of 1:6.6 recombinant ΕΡΟ-R dilution (manufactured by Chinese hamster ovary cells, which binds to the human placenta-tested acid-breaking peak protein fraction (which contains the 179 target epitope) Fusion protein, and 607.5 μl Alphaquest buffer (4 mM HEPES, pH 7.4; IMm MgCl2; 0.1% BSA, 0.05% ton 20). Tap the tabletop mix. Add 2 μl/ Pore biotinylated ΕΡΟ-R binding peptide tracking , AF33068 (30nM final degree desert). AF33068 is a ΕΡΟ-R binding peptide (see Table 3 "communications sub EC50 (pM)") is prepared according to the method described in Example 1. AF33068

Biotin-OGLYACHMGPrrWVCQPLRG^ Biotiii-GGLYACHMGPnWVCQPLRG ^ Centrifuge for 1 minute to mix. The panels are sealed with a Parker top seal and wrapped in a metal box. Incubate overnight at room temperature. After 18 hours, the Alpha Quest reader 105 20 (Pike) was used to read the luminescence. Luminescence is plotted against peptide concentration and analyzed using Graph Pad or Excel. Comparing the observations without the test peptide, the test peptide concentration at which the luminescence was reduced by 50% was recorded as IC50. 4. C/BFU-e assay analysis EPO-R signaling simulates the differentiation of bone marrow stem cells into proliferating red blood cell precursor cells. This assay analyzes the test peptide to mimic the proliferation and differentiation of red blood cell precursor cells derived from a single human bone marrow multiplexed stem cell. For the purpose of this assay, a series of dilutions of the test peptide was prepared by supplementing 10% FBS (Hai Keng) IMDM medium (Jibo Co.). Serial dilutions or positive control EPO peptides were then added to methylcellulose to obtain a final volume of 1.5 ml. The methylcellulose and peptide combination are then thoroughly inverted. A whole (100,000 cells/ml) of human bone marrow derived from CD34+ cells (Paulie/Kenbras) was thawed. The thawed cells were then gently added to 0.1 ml of 1 mg/ml DNAse (stem cells) in a 50 ml test tube. - 40-50 ml of IMDM medium was gently added to the cells: the first 1 ml of the culture system was added dropwise alongside the 50 ml tube, and the remaining medium was slowly dispensed alongside the test tube. The cells were then centrifuged at 900 rpm for 20 minutes and the medium was carefully removed by gentle aspiration. The cells were resuspended in 丨ml of IMDM medium, and the cell density of the mother cells was calculated on the hematocrit of the blood cells (one whole microliter of cell suspension on the slide, the cell length was an average of X10 000 cells/ml) ^ then the cells Dilute to 1.5 cells in IMDM medium at a cell density of 〇〇〇 cells/ml. Then, 100 μl of the diluted cells were added to each 15 ml of methylcellulose vasopeptide sample (the final cell concentration in the assay was 1 〇〇〇 cells/ml), and 1344964 was mixed and inverted. Let the bubbles in the mixture disappear, then use a blunt end needle and take 1 ml. The 0.25 ml of the extracted mixture from each sample was added to each of the four wells of a 24-well plate (Falcon brand). The inoculation mixture was cultured in a humidified incubator for 14 days at 5%. Red blood cell populations were calculated using a phase microscope 5 (5x-10x objective, final magnification 100x). When compared with the EPO-positive control group, the test peptide concentration at which the number of colonies generated was 9% by maximum was recorded as EC90.

107 1344964

C/BFU-e EC90 (nM) &lt;N 1 AQ 1 IC50 i (PM) hyperplasia EC50 (nM) 1 1 1 ff 〇^03⁄4 ^ w ^ peptide binary _ _ ... ______ ________1 ) 0 ^NH^ O-PEGaoK I 1 rM |l ! IIL| r ^ 3 X» u ^ 1- Compound nomenclature AF36205 108 1344964 Example 8: In vivo activity assay analysis This example validation can be used to evaluate the EP 〇-R agonist peptide of the present invention In vivo assay analysis of activity and intensity. The EPCMU kinetic peptide monomer and binary system were prepared according to the method provided in Example 1. The activity of the peptide monomer and the binary body 5 in vivo activity using a series of assays including multiple blood cell extracellular hypoxic mouse bioassay analysis and reticulocyte bioassay analysis. Further details of the two assays are described below. 1. Multiple blood cell extracellular hypoxia mice bioassay analysis test peptides in multiple blood cells extracellular hypoxic mice bioassay analysis 10 疋 in vivo activity 'The assay was analyzed by Ctoes and Bangham (1961), Nature 191 : 1065-1067 The method is modified. This assay analyzes the test peptide as an EPO mimetic: that is, the ability to activate EP0-R and induce new red blood cells to synthesize. The synthesis of red blood cells is based on the quantification of radiolabeled iron bound to synthetic hemoglobin. 15 BDF1 mice were domesticated for 7-10 days. All mice were measured for body weight&apos; mice that were too light (less than 15 grams) were not used. Mice received a continuous conditioning cycle of 14 Torr in a low pressure chamber. The 24-hour cycle consisted of 18 hours at 0.40 ± 0.02% atmosphere and 6 hours at ambient pressure. After conditioning, the mice were maintained at ambient pressure for 72 hours prior to administration. 20 Test peptides or recombinant human EPO standards were diluted in PBS + 0.1% BSA vehicle PBS/BSA). The peptide monomer monomer solution was first solubilized in the Dioxin (DMS0). The negative control group included a group of mice injected with PBS/BSA alone, and a group of mice injected with 1% DMS0 alone. Each dose group had 1 taro mouse. The mice were injected subcutaneously (neck back) with 5 ml of the appropriate sample. 109 1344964 48 hours after the injection of the sample, the mice were intraperitoneally injected with 2 ml of ^ (NEN), at a dose of about 75.75 microcures per mouse. The body weight of the mice was weighed by the administration of &amp; 59, and the mice were sacrificed 48 hours after the administration of W. Blood samples were taken from each animal by cardiac puncture and blood volume was measured (heparin was used as an anticoagulant). Each ash sample .5 (0.2 liters using the Parker (4) counter to analyze Fe59 binding ^. Non-reactive small mice (ie, mice with radioactive binding below the negative slant group) were excluded from the appropriate data set. Mice with a value lower than 53% of the negative control group were also excluded. Φ Xiaoguo was derived from a collection of 10 mice at each experimental dose. The average amount of radioactivity bound to the blood sample was determined [counts per minute ((: 1&gt] ;]^)]. 10. 2. Reticulocyte assay analysis Normal BDFH, rats were administered for 3 consecutive days (〇5 ml, subcutaneous injection) Ep〇 control or test peptide. On the third day, mice were given (〇1 ml) , intraperitoneal injection) iron glucomannan (100 mg / ml). On the fifth day, the mice were treated with carbon dioxide hemp Sr, blood sampling by heart puncture. The percentage of reticulocytes in each blood sample was stained with 15 oxazolium orange and flow cells. The analysis (grid counting) is done manually to determine the blood volume. The corrected number of reticulocytes is determined using the following formula: % RETIC corrected = % RETICft value x (blood volume / normal blood volume) 3 Hematological examination analysis of normal CD1 mice four times a week high dose intravenous E P〇 positive pair • 2 test, test peptide or vehicle” - a range of positive control and test peptide agent (expressed in mg / kg) is tested by changing the active compound concentration of the formulation. The injection volume is 5 ML/kg. The vehicle control group contained 12 animals, and the remaining drug-administered groups received 8 animals each. Survival was recorded daily, and body weight was recorded every week. 110 1344964 The mice were administered as fasting mice, followed by inhalation of isoflurane. Isoflurane anesthesia, blood samples were taken by cardiac puncture or abdominal aortic puncture on day 1 (media control mice) and 15th and 29th (4 mice/group/曰). Blood transfer to tile fall In the Vacutainer brand test tube, the preferred anticoagulant is exoethyldiaminetetraacetic acid (EDTA). Use the well-known automated clinical analyzer (such as Coulter) to evaluate the red blood cell synthesis of blood samples. End points and physiology such as blood volume (Hct), hemoglobin (Hgb) and total red blood cell count (RBC). Example 9: having an amino acid sequence (AcG) GLYACHMGPIT (1-nal) 10 VCQPLRK (SEQ ID NO: 1) EP0-R agonist peptide of peptide monomer Synthesis of Qualitative Binary Steps 1 - Synthesis of peptide monomer: The peptide single system uses standard Fmoc chemistry on the eight 81431 eight-peptide synthesizer, using Ding 〇-11 octadecyl resin (0.18 mmol/Kray Polymeric polymer [Germany] synthesis. Used to synthesize 15 peptide monomers with amidated carboxyl end. 'Completely assembled peptides are made from resin. 82.5% TFA, 5% water' 6.25% anisole, 6.25% Ethylene Thiol cleavage. The deprotected product was filtered from the resin 'precipitated with diethyl ether. After thorough drying, the product was purified by C18 reverse phase high performance liquid chromatography eluting with acetonitrile/water eluting with 0.1% trifluoroacetic acid. The peptide structure was confirmed by electrospray ionization mass spectrometry. The peptide was dissolved in 20 DMSO:water 1:1 solution at a concentration of 1 mg/ml to affect the formation of disulfide bonds. The product was subjected to C18 reversed-phase high performance liquid chromatography with acetonitrile/water in 0.1% trifluoroacetic acid. Gradient purification. The peptide monomer is shown as follows. (AcG)GLYACHMGPIT(l-nal)VCQPLRK-NH2 Step 2 - Synthesis of trifunctional linkages: diethyl iminoacetate 111 1344964 (10.0 g '52.8 mmoles and Boc-no-alanine (1 〇〇克,52 8 mmoles) Diisopropyl phthalimide (8.0 ml '5 U mmol) was added to a solution of 100 ml of DCM at room temperature over 1 Torr. The temperature of the reaction mixture during the addition. Heat to about thief, then cool back to room temperature in 20 minutes. Let the reaction pan 4 5 纟 overnight 'filter out the diisopropyl urea of the sinking. Evaporate under reduced pressure to remove the solvent to obtain a gel, the residue is dissolved In ethyl acetate, re-filtered 'to remove additional precipitated urea. The organic phase was placed in a separatory funnel, and the strip was washed (saturated carbonate 1 φ sodium, brine, 〇·5 N salt 醆, brine dehydrated (sulfate sulphate), And evaporating under reduced pressure, the obtained product was obtained as a colorless oil. The diacetic acid was taken up in methanol: H) THF 1:1 mixture (100 ml) was added water (25 ml), then sodium oxynitride (5 g, 125 mmol.) The pH was measured to be greater than 1 Torr. The reaction mixture was stirred at room temperature for 2 hours and then acidified with 6 N hydrochloric acid. pH i. The aqueous phase is saturated with gasified sodium, and extracted with acetic acid. The combined organic phase is washed with water (salt brine), dehydrated (magnesium sulfate) and concentrated under reduced pressure to obtain a white semi-solid. Dissolve in 50 ml of DCM, add 3 ml of hexane to give a white poly-form. The solvent is removed under reduced pressure to give the diacid as a white solid (147 g, 91.5% yield a total of two steps). Acid (1 g, 3 29 mM) was added to Ν'hydroxybutaneimine (770 mg, 6.69 mmol) and isopropyl hydrazine in 2 ml of DMF. Amine (1 〇〇 ml, millimolar) and dimethyl-20-amino-amine η than bite (3 mg '0 〇 2 mmol) reaction mixture was mixed overnight, the solvent was removed under reduced pressure. In the acetic acid ethyl acetate, and the sputum removal &quot;L melon. The organic phase is placed in a separatory funnel, washed with saturated sodium bicarbonate, brine, 〇·5 Ν hydrochloric acid, brine), dehydrated (sulfate town), transition Concentrate under reduced pressure to give the bis-NHS product as a white solid (1 12 g, 68% yield). 112 丄] 44% 4

Step 3 - Trifunctional linkages are coupled to the peptide monomer: for coupling to the linkage '2 equivalents of peptides, mixed oxime equivalents, trifunctional linkages based on anhydrous 1) 1^1?, obtained a clear solution' 2 minutes later Add 5 equivalents of DIEA. The mixture was allowed to stand at ambient temperature for 14 hours. The solvent was removed under reduced pressure and the crude material was dissolved in &lt;RTI ID=0.0&gt;&gt; The binary structure was confirmed by electrospray ionization mass spectrometry. This coupling reaction attaches a bond to the nitrogen atom of the epsilon amine group of the amine acid residue of each monomer. Bu MVOQPLRK-NH:

ΝΗχ 113 1344964 Step 4 - Synthesis of a PEG moiety comprising a bilinear PEGS chain linked by an aminic acid mPEG2-isamino alcohol-NPC linkage. The oleyl alcohol is commercially available and is treated with an excess of mPEG2-NPC to obtain mPEG2 from the amine alcohol. - 5 m PEG 2-isoamine-NPC, obtained from an amine alcohol followed by NPC treatment.

mPEG2-Lys-NHS This product is commercially available, for example, from Molecular Engineering Catalogue (2003 | year), Nektar Treatment Company (490, Discovery Avenue, Huntsville, Alabama 35806), project No. 2Z3X0T01. 1〇Step 5 - PEGylation of peptide peptides: PEGylation via urethane linkages: peptide peptides and PEG (mPEG2·isoamine alcohol-NPC) at 1:2 molar ratio to anhydrous DMF was mixed to obtain a clear solution. After 5 minutes, 4 equivalents of DIEA were added to the aforementioned solution. The mixture was stirred at ambient temperature for 14 hours then purified by EtOAc EtOAc. The structural formula of the PEGylated peptide was confirmed by MALDI mass spectrometry. • The purified peptide is also purified by cation exchange chromatography, as described below. 114 1344964 0

(AcG)GLYAOEIMGPIT(l-naI)V&lt;iQPLR-

I nPEG^seven ysinol-NPC D1EA, DMF

PEGylation via a guanamine bond: Peptide Binary and PEG (mPEGr-aminol-NHS available from Schwart, USA) gave a clear nights with a 1:2 molar ratio to anhydrous DMF. After 5 minutes 5, 4 equivalents of DIEA were added to the aforementioned solution. The mixture was stirred at ambient temperature for 14 hours then purified by C18 reverse phase HPLC. The structural formula of the peg-winning peptide was confirmed by MALDI mass spectrometry. The purified peptide was also purified by cation exchange chromatography and summarized as follows. 115 1344964

EG20K

Ο Step 6-Peptide ion exchange purification: In addition to the ability to retain the starting binary peptide, 'several exchange supports also studied to separate the above peptides from unreacted (or hydrolyzed) PEG, PEG light compounds ability. Ion exchange resin 5 (2-3 g) was loaded on a 1 cm column and then converted to sodium form (〇2 n gas oxide was supported on the column until elution was divided into pH Η, approximately 5 times the column volume). Instead of converting to hydrogen form (extracted with 0.1 Ν hydrochloric acid or 0.1 Μ acetic acid until the elution fraction matches the load ΡΗ 'about 5 times the volume of the tube), it is washed with 25% ACN/water to ρΗ6. The light-weight peptide or peptide PEG coating was dissolved in 25% ACN/water (10 ng 116 1344964 g/ml), the pH was adjusted to less than 3 with THA, and then loaded onto the column. After washing with 2-3 column volume 25% ACN/water and collecting 5 ml of elution fraction, the peptide was released from the column by UM ammonium acetate in 25% ACN/water, and 5 ml of the elution fraction was collected again. HPLC analysis showed that the elution fraction contained the desired triumph. 5 Analyzed by gasification light scattering detector (ELSD), indicating that the peptide remained in the column and used ammonium acetate solution (usually in elution fraction 4 and When the elution is washed, the PEG is not observed. The unconjugated PEG is not observed when the peptide is eluted in the initial elution buffer (usually the first two washes). Separation of the desired PEG-conjugate and excess PEG. 10 The following column successfully retained the peptide and peptide-PEG conjugate, successfully purifying the peptide-PEG conjugate from the non-conjugate peptide: Table 5: Ion exchange resin support source Mono S HR 5/5 strong cation exchange preloaded column Amor Hill (Amersham) Biosystems SE53 cellulose, particle strong cation exchange support Wattman SP Sefarose (Sepharose) fast Flowing Strong Cation Exchange Support Amoshan Biotech Company Example 6: Having an Amino Acid Sequence (Ac G) GLYACHMGmXl-nal) 15 EPO-R agonist of the peptide monomer of VCQPLRK (SEQ ID NO: 1) The synthesis of the peptide homogenous binary has an amino acid sequence (AcG) GLYACHMGPIT( 1 -nal) VCQPLR ( The EPO-R agonist peptide of MeG)K (SEQ ID NO: 2) is synthesized as described in Example 1, but in Step 1, the synthesized peptide 20 monomer is:

(AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K 117 1344964

Here, the PEG is attached to the bonding group through the urethane linkage, and the structural formula of the final product of the synthesis is as follows:

(AcG)GLYACHMGPIT(l-nal)VCQPLR(Mt&lt;?&gt;-NH

If the PEG is attached to the hydrazine via a stilbene linkage, the structural formula of the final product is shown as follows:

Example 11: Intra-tube activity assay analysis This example illustrates a plurality of in-vitro assay assays that can be used to assess the activity and strength of the EPO-R agonist peptide of the present invention. The results of the assay confirmed that the novel peptide of the present invention binds to EPO-R and activates EPO-R signaling. In addition, the results of the assay showed that the novel peptide composition compared to the aforementioned EPO mimetic, the EPO-R binding affinity and biological activity unexpectedly increased. 118 1344964 ΕΡΟ-R agonist peptide monomer and binary system were prepared according to the methods provided in Example 实 or Example 2. The strength of these peptide peptides was assessed using a series of tube activity assays, including: reporter assays, proliferative assays, repeated binding assays, and C/BFU_e assays. The four 5 test analyses further illustrate the details as follows. The results of these in vitro assays are summarized in Table 4. 1-Reporter assay This assay is based on the murine pre-B cell line-derived reporter cell Baf3/EpoR/GCSFR fGS/iup. This reporter cell line can represent chimeric IgG receptors, including human EP0 receptors to extracellular Part of the person's intracellular part of the GCSF receptor. This cell line was further infected with a fos-inducing gene-driven luciferase reporter gene assembly. This chimera receptor is activated by the addition of a red blood cell-forming agent, resulting in the expression of the luciferase reporter gene, and thus emits light when the luciferase enzyme matrix luciferin is added. The extent of 15 EPO-R activation of such cells can be quantified by measurement of luciferase activity.

Baf3/EpoR/GCSFR f〇s/lux cells were supplemented with 1% fetal bovine serum (FBS; Hyclone) in DMEM/F12 medium (Gibco's culture medium) on 1%% WEHI_3 The supernatant (by WEHI_3 cells, ATCC # TIB-68) was cultured to obtain penicillin/streptomycin. The assay was divided into 2 minutes before decantation, and the cells were transferred to DMEM/F12 medium supplemented with 10% FBS and 0.1%. WEHI-3 supernatant was scarce. On the day of assay analysis, cells were washed with DMEM/F12 medium supplemented with 10% FBS (without WEHI-3 supernatant), then 1 X 1 〇ό cells/ml at known concentrations Culture in the presence of test peptides or use EPO (R & D Systems, Minneapolis, Minnesota) 119 1344964 as a positive control, supplemented with DMEM/F12 supplemented with 10% FBS (without WEHI-3 supernatant) Culture. A series of dilutions of the test peptide was tested at the same time in this assay. The assay plate was incubated at 37 ° C for 4 hours in a 5% carbon dioxide atmosphere, followed by the addition of σ luciferin (steady gyro -Glo); Pro + 5 meters (Promega) 'Marison, Wisconsin' to each hole. After 5 minutes of incubation, The luminescence was measured by a Packard desktop photometer (Parker Instruments, Inc., Douglas, Ill.). The light intensity values were plotted against the test peptide concentration and analyzed using Graph Pad software. The test peptide concentration was recorded as EC50. 10 2. Proliferation assay This assay is based on the mouse pre-B cell line Baf3 metastasis to express human EPO-R. The resulting cell line Baf3/Gal4/Elk/EPOR proliferation line and EP0 -R activation-related. The degree of cell proliferation was quantified using MTT, where the signalling of the MTT assay was directly proportional to the number of viable cells. 15 BaF3/Gal4/Elk/EPOR cells were supplemented with 1%% &gt; FBS in a rotating vial ( Hypoclosure) and 2% WEHI-3 supernatant (ATCC # TIB-68) in DMEM/F12 medium (Jibo Co.) culture. After culture, the cells were centrifuged at a cell density of lxlO6 cells/ml. 10% FBS and 0.1% * WEHI-3 supernatant DMEM/F12 medium was lacking overnight. Then the depleted fine * 20 cells were washed twice with Dubec PBS (Gibob) and resuspended in 1% FBS ( Contains no WEHI-3 supernatant) to cell density Ιχίο6 cells /ml. 50 μl of the aliquot (approximately 50,000 cells) of the cell suspension was re-inoculated three times in 96 well assay assay plates. A full 50 microliter test EPO mimetic peptide series dilution or 50 microliters EPO (R&amp;D Systems, Minneapolis, Minnesota 120 1344964) or Aranesp (Islandiamycin) (darbepoeitin α), purchased from Amgen's EPO-R agonist) in DMEM/F12 medium supplemented with i〇% FBS (without WEHI-3 supernatant I) to 96-well assay Orifice plate (final volume of each hole is 1 〇〇 microliter). For example, test 12 different dilutions, try 5 test (or control EPO peptide) final concentration of 81 ΟρΜ to 0.0045Pm range. The plated cells after inoculation were then cultured at 37 ° C for 48 hours. Next, 10 μl of MTT (Rose Diagnostics Inc.) was added to each well of each culture and allowed to incubate for 4 hours. The reaction was stopped by the addition of 10% SDS + 0.01 N HC1. The orifice plate is at 37. (: Overnight. Then absorbance spectroscopy is used to determine the absorbance ratio of each well at 595 nm. 10 Absorbance ratio s sells the plot to the test win, and calculate the EC50 using Graph Pad software. The half maximum absorbance ratio can be obtained. The test peptide concentration was recorded as EC5〇. 3. Competitive binding assay analysis uses competitive assays to perform competitive binding calculations. The optical signal in the assay is based on a change in the proximity of the two beads: Streptomyces antibiotic 15 donor beads Contains a biotinylated EPO-R-binding peptide tracer and an acceptor bead that incorporates EPO-R. The non-radiative energy shifts the luminescence, at which point the solitary oxygen is released by the first bead when illuminated, and the second The beads are contacted with orphaned oxygen to cause luminescence. This bead collection is commercially available (Pike). The bead proximity is due to the binding of EPO-R to the peptide tracer to EPO-R. Test peptide 20 and EPO The -R-binding peptide tracker competes for binding to EPO-R, which will prevent this binding, resulting in a decrease in luminescence. Further details of this method are as follows: Add 4 μl of test EPO-R agonist peptide to a series of dilutions Positive pair Or a negative control to each well of 384 well plate. Subsequently, 2 microliters / acceptor bead mixture to each well. Acceptor beads 121 1J44964

&gt; See the composition of the mixture is: 15 μl of 5 mg / ml Streptavidin antibiotic label (Pike), 15 μl of 5 mg / ml monoclonal antibody abl79 (this antibody identifies recombinant EPO-R Human placenta alkaline phosphatase protein fraction), protein A coated receptor beads (protein a binds to abl79 antibody; Parker), U2.5 microliter 1:6.6 recombinant EPO-R dilution (Chinese hamster ovary cells) Manufactured as a fusion protein that binds to the human placental virulence enzyme protein portion (which contains the abl79 target epitope), and 607.5 microliters of Alphaquest buffer 4 mM HEPES, pH 7.4; IMm MgClsO. L% BSA, 0.05% 呑 20). Tap the desktop mix. Add 2 μl/well biotinylated EPO-R binding peptide tracer, AF33068 (30 nM final concentration). AF33068 is an EPO-R binding peptide (refer to Table 3 "Notifier EC50 (PM)") prepared according to the method described in Example 1.

Mix by centrifugation for 1 minute. The plate is sealed with a Parker top seal and wrapped in a metal drop. So that it is cultivated overnight. The luminescence was read after 18 hours using an Alpha Quest reader (Pike). Luminescence is plotted against peptide concentration and analyzed using Graph pad or Excel. Comparing the observations without the test peptide, the concentration of the test peptide obtained by reducing the luminescence by 5〇% was recorded as IC50 » 4. C/BFU-e assay analysis EPO-R signaling to simulate the differentiation of bone marrow stem cells into proliferative red blood cell precursors cell. This assay analyzes the test peptides obtained from the proliferation and differentiation of red blood cell precursor cells from the human bone marrow 122 1344964 multiplexed stem cells. For this assay analysis, a series of dilutions of the test peptide was prepared by supplementing 10% FBS (Hai Keng) IMDM medium (Jibo Co.). A serial dilution or positive control EPO peptide was then added to the methylcellulose to obtain a final volume of L5 ml. The methylcellulose and peptide combination are then thoroughly inverted. A whole (100,000 cells/ml) of human bone marrow derived from CD34+ cells (Paulie/Kenbras) was thawed. The thawed cells were then gently added to 0.1 ml of 1 mg/ml DNAse (stem cells) in a 50 ml test tube. The next 40-50 ml of IMDM medium was gently added to the cells: the first 1 ml of the culture was added 10 bases down the 50 ml tube, and the remaining medium was slowly dispensed alongside the test tube. The cells were then centrifuged at 900 rpm for 20 minutes and the medium was carefully removed by gentle aspiration. The cells were resuspended in 1 ml of IMDM medium and the cell density per ml was calculated on a hemocytometer slide (one whole microliter of cell suspension on the slide, cell density XI〇, 〇〇〇 cells/ml). The 15 cells were then diluted in IMDM medium to a cell density of 15,000 cells/ml. Then, 100 μl of the diluted cells were added to each 1.5 ml of methylcellulose vasopeptide sample (the final cell concentration in the assay was 1 〇〇〇 cells/ml), and the mixture was inverted. Let the bubbles in the mixture disappear and then use a blunt end needle to absorb 1 ml. The 0. 25 ml of the extracted mixture from each sample was added to each of the four wells of a 24 20-well plate (Falcon brand). The inoculated mixture was incubated at 37 C for 5% in a humidified incubator for 14 days. Red blood cell populations were calculated using a phase microscope (5x-10x objective, final magnification). When compared with the EPO-positive control group, the test scent concentration with a maximum number of colonies of 9〇% was recorded as EC90. 123 1344964

茬令^逛^昶^: 伽^) Amount ^-^^荽:9&lt; C/BFU-e EC90 (pM) m Si ^ 〇/-^s Proliferation EC50 (pM) iTi VD regenerant EC50 (PM) m peptide peptide 1 I 〇(AeO)GLYACHMGFnxl mOVOQMJ^MeOKNH^^ Ship 0 〇ο^〇-ρε6.κ 丫—PEGjok 广Γ &lt;AoO)GLYAjMMG?rr(l«nal)Vp}PUl(^ e〇HD/^-NH2 Compound Nomenclature AF37702 124 Example 12: In vivo Activity Assay Analysis This example demonstrates an in vivo assay for assessing the activity and intensity of the EPO-R agonist peptide of the present invention. EPO-R agonist The peptide monomer and binary system were prepared according to the method provided in Example 1. The in vivo activity of the peptide monomer and the binary was analyzed using a series of assays including multiple blood cell extracellular hypoxic mice bioassay analysis and reticular Cell bioassay analysis and evaluation. Further details of the two assays are described as follows. 1. Multiple blood cell extracellular hypoxic mice bioassay analysis test peptides in multiple blood cells extracellular hypoxic mice bioassay analysis test in vivo Activity 'This assay was corrected by the method described by Ctoes and Bangham (1961), Nature 191: 1065-1067. This assay analyzes The test peptide is used as an EP0 mimetic: that is, the ability to activate EPO-R and induce the synthesis of new red blood cells. The synthesis of red blood cells is based on the quantification of radiolabeled iron bound to synthetic red blood cells. Let BDF1 mice acclimate to surrounding conditions 7_10 Days. All mice were weighed 'not using mice that are too light (less than 15 grams). The mice received a continuous conditioning cycle of 14 hours in a low pressure chamber. The 24-hour cycle was 18 hours in 〇.4〇土〇 〇 2% atmospheric pressure and 6 hours at ambient pressure. After conditioning, the mice were maintained at ambient pressure for 72 hours before administration. Test peptide or recombinant human EP0 standard in pbs + 0.1% BSA vehicle PBS/BSA )dilution. The peptide monomer monomer solution was first solubilized in the Dioxin (DMS®). The negative control group consisted of a group of mice injected with pBS/BSA alone, and one group of mice injected with 1% DMSC alone. Each group had 10 mice in each dose group. The mouse was injected subcutaneously (neck back) with 0_5 ml of the appropriate sample. 1344964 At 48 hours after the injection of the sample, the mice were intraperitoneally injected with 2 ml of DuPont NEN) dose of about 0.75 microcures per mouse. The body weight of the mice was weighed 24 hours after administration, and the mice were sacrificed 48 hours after the administration of W. Blood samples were taken from each animal by cardiac puncture and blood volume was measured (heparin was used as an anticoagulant). Each blood sample 5 5 (〇_2 ml was analyzed for the amount of Fe59 binding using a Parker's counter. The small amount of no reaction. I, that is, the mouse with radioactive binding lower than the negative control group) was excluded from the appropriate data collection. Mice with a blood volume lower than 53% of the negative control group were also excluded. The age of the county is derived from the actual amount of _ mice mixed to determine the average amount of radioactivity bound to each blood sample [counts per minute ((: 1 &gt; 1 ^)]. 10 2. Reticulocyte assay analysis of normal BDF1 small The rats were administered for 3 consecutive days (〇5 ml, subcutaneous injection) of Ep〇 control or test peptide. On the third day, mice (〇1 ml, intraperitoneal injection) of iron glucomannan (100 mg/ml) were administered. On the fifth day, the mice were anesthetized with carbon dioxide and collected by blood puncture. The percentage of reticular cells (%) of each blood sample was determined by artificial sputum determination by thioloxyl orange staining and flow cytometry analysis (grid counting). The corrected number of reticulocytes was determined using the following formula: % RETIC corrected = % RETICWx (blood volume / normal blood volume) 3. Hematology analysis

X Normal CD1 mice were given a large dose of Ep〇 positive for 4 times a week, test peptide or vehicle. A range of positive controls and test peptide doses (expressed in mg/kg) are tested by varying the concentration of active compound in the formulation. The injection volume is 5 ml / kg. The vehicle mixture contained 12 animals, and the remaining drug administration groups contained 8 animals each. Survival was recorded daily, and 126 1344964 mice were recorded as empty abdomen mice, followed by inhalation of isoflurane (iS〇flurane) anesthesia, in the first sputum (media control mice) and 15th 曰And 29th (4 mice/group/曰) blood samples were taken by cardiac puncture or abdominal aortic puncture. The blood is transferred to the Vacutainer brand test tube. The preferred anticoagulant is exoethyldiaminetetraacetic acid (EDTA) °. The erythrocyte synthesis endpoint and physiology, such as blood volume, of the blood sample are evaluated using an automated clinical analyzer (such as that manufactured by Coulter). (Hct), hemoglobin (Hgb) and total red blood cell count (RBC). * * * φ) 10 The scope of the present invention is not limited by the specific examples described herein. Indeed, various modifications of the invention are apparent to those skilled in the art and These modifications are intended to fall within the scope of the accompanying patents. It should be understood that all values are approximate and are for illustrative purposes only. Each of the references, including patents, patent applications, and publications are hereby incorporated by reference. The citation and/or discussion of these references is merely illustrative of the invention and is not an admission that any reference is a prior art of the invention. The entire disclosure of the present specification is hereby incorporated by reference. ‘ [Simple description of the diagram] (none) [Description of main component symbols] * (none) 127

Claims (1)

1344964 1 Patent Application No. 93134267 Patent Application Amendment This Amendment Period: December 1999 Announcement Ten, Patent Application Range: 1. A combination of activated red blood 5 f) annual IL month / <日则东) original A compound of Etomycin receptor (EPO-R), which comprises a peptide binary having the following formula: (AcG)GLYA(?HNiGPIT(l&lt;ial)VCQPLR-&gt;NH 5 wherein (i) each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and Ι-nal is 1-naphthyl Amino acid; (ii) each of the peptides of the peptide is composed of an intramolecular disulfide bond between two of the ten cysteine (C) residues of each monomer; (iii) [" PET"] comprises at least one linear polyethylene glycol (PEG) moiety, each having a molecular weight of from about 20, 〇〇〇 to about 4 〇, 〇〇〇Dowagen. 2. A compound as claimed in claim 1 wherein each pEG has a sub-quantity of about 30,000 dolphins. 3. The use of a compound as claimed in claim 1 for the manufacture of a medicament for the treatment of a condition characterized by a deficiency or deficiency of erythropoietin or a red blood cell population, wherein the condition is selected Free dialysis or malignant disease-related anemia; AIDS, autoimmune. Single &amp; 曰, cold thalassemia; cystic fibrosis; blood; =; and tumor disease status accompanied by abnormal red blood cell production. For example, if the application is for the purpose of the _3 item, each of the touches has a molecular weight of about 30, which is 〇〇〇道尔吞. A pharmaceutical composition comprising a compound of the formula (IV), and a pharmaceutically acceptable carrier. For example, the pharmaceutical composition of claim 6 wherein each pEG has a molecular weight of about 30,000 doxantane. A compound that binds to and activates a erythropoietin receptor (Ep〇_R), the compound comprising a peptide binary having the formula: (i) each peptide monomer of the peptide monopeptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, i_naj is 〖-naphthylalanine, and MeG is N-methylglycine; (ii) each peptide monomer of the peptide complex contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; PEG comprises linear unbranched polyethylene glycol molecules having a molecular weight of from about 2 Å to about 40,000 Torr. 9. A compound of the eighth aspect of the patent application wherein each PRO has a molecular weight of about 30,000 dolphins. 1) A use of a compound of claim 8 for the manufacture of a medicament for treating a condition characterized by erythropoietin deficiency or a low red blood cell population or defect. U. If the application is for (4), the use of the disease is selected from the group consisting of: end stage renal failure or dialysis; AIDS, autoimmune = or malignant disease-related anemia; type 6 thalassemia; cystic fiber Change ♦ correct early premature infants with poor ash; chronic inflammatory disease-related anemia; spinal cord injury; blood n aging; and accompanied by the red blood cell disease state. 12.2 Please use the patent scope _, which is about 3 〇, _Doltun. 13:: The product 'includes the compound 14 as set forth in claim 8 and the learned carrier. There are eighty-two pharmacy compositions of the 13th item, in which each PEG has a knife amount of about 3 〇, _Doltun. 15. A compound that binds to and activates a erythropoietin receptor (EPO-R), the compound comprising a peptide binary having the formula: 〇(i) for each peptide monomer of the peptide binary, each amino acid is represented by a standard one-letter abbreviation 'AcG is N-ethinylglycine, and Ι-nal is 1-caffeyl propylamine (ii) each peptide monomer of the peptide binary contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; (iii) the PEG comprises a molecular weight of about 2〇, 〇〇0 to about 4 〇〇〇〇Dolphine linear unbranched polyethylene glycol molecules. 16. A compound according to claim 15 wherein each ρΕ(} has a molecular weight of about 30,000 dolphine. 17. a compound such as f, wherein the compound of claim 15 is used for the manufacture of a medicament, For the treatment of conditions characterized by erythropoietin deficiency or low red blood cell population or defects. 18. The use of claim 17 wherein the condition is selected from the group consisting of: (4) dysfunction or dialysis; AIDS Autoimmune disease or malignant disease-related anemia; point-type thalassemia; cystic fibrosis, early anemia in premature infants; anemia associated with chronic inflammatory disease; spinal cord injury; acute blood loss; aging; and tumors often associated with red blood cells The state of the disease 19. The use of the scope of claim 17 wherein each pEG has about 30 molecules in the molecule, and is a pharmaceutical composition comprising a compound as claimed in claim 15 And a pharmaceutically acceptable carrier. 21. The pharmaceutical composition of claim 20, wherein each pEG has a molecular weight of about 30,000 dolphins. And activation in combination of erythropoietin receptors (Ep〇R) of the compound, the compound comprising a peptide dyad, having the formula: (AcGJGLYAOiMGPnXl -nal) viQPLR (MeG) (AcG)GLYACHMGPIT(I-nal)VCQPLR(MeG) Wherein (i) each of the pheno-monomers of the peptide complex, each of the amino acids is represented by a standard one-letter abbreviation 'AcG is N-ethinylglycine, i_nal is 1-naphthylalanine, and MeG is N-methylglycine; (11) each peptide monomer of the peptide binary contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; (iii) PEG comprises a linear unbranched polyhexylene glycol molecule having a molecular weight of from about 2 Å to about 4 Torr. The compound of claim 22, wherein each ρ Ε〇 has a molecular weight of about 30,000 Dao. 24. The use of a compound as claimed in claim 22 for the manufacture of a drug - a drug for the treatment of a condition characterized by a deficiency of erythropoietin or a low red blood cell population or a defect. The use of 24 items, wherein the condition is selected from the group consisting of: end stage renal failure or dialysis; AIDS, autoimmune disease or malignant disease-related anemia; no type of thalassemia; cystic fibrosis, early premature infants Anemia; chronic inflammatory disease-related anemia; spinal cord injury; ~ ί · raw blood loss; aging; and tumor disease state accompanied by abnormal erythropoiesis. For example, the application of patent scope range 24, wherein each pEG has a molecular weight of about 30, A pharmaceutical composition comprising a compound of claim 22 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 27, wherein each pEG has a molecular weight of about 30,000 dolphins. A compound that binds to and activates a erythropoietin receptor (ep〇r), the compound comprising a peptide binary having Formula: (AcG) GLYA &lt; HMOPrT (l-naI) V &lt;:!! QPLR (MeG). g (AcG)GLYACHMCTIT(l-iia〇VCQPLR(MeG)-HN NH (i) for each peptide monomer of the peptide binary, each amino acid is represented by a standard one-letter abbreviation 'AcG is N-ethinylglycine, i_nai is 1-naphthylalanine, and MeG Is N-methylglycine; each peptide monomer of the (η) peptide complex contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; m) PEG comprises a linear unbranched polyethylene glycol molecule having a molecular weight of from about 2 Torr to about 4 Torr. 3. For example, if the compound of claim 29 is applied, the #咖 has a molecular weight of about 30,000 dolphins. The use of a compound such as the scope of claim 29 for the manufacture of a medicament for treating a condition characterized by a deficiency in erythropoietin or a low or a red blood group. 32·=The use of the 31st item of the profit range, wherein the condition is selected from the second stage of renal failure or dialysis; AIDS, autoimmune:: early childhood: poor also; 10,000 types of thalassemia; cystic Fibrosis; Chronic inflammatory disease associated with poor spinal cord injury; 1 Symptoms = loss; aging; and tumors with abnormal erythropoiesis. Each PEG has a molecule 33. The application amount is about 30,000 Dao as claimed in claim 31. swallow. 34. A pharmaceutical composition comprising a compound as claimed in claim 29 and a pharmaceutically acceptable carrier. 35. The pharmaceutical composition of claim 34, wherein each PEG has a molecular weight of about 30,000 dolphins. 36. A compound that binds to and activates a erythropoietin receptor (ep0_r), the compound comprising a peptide binary having the formula: (AcG)GLYAC&amp;MGPIT(l-iial)viQPLR(MeG)H (AcG) GLYACHMGPIT(l.iial)VCQPLR(MeG) -l NH N=EG s 0 -O (i) For each peptide monomer of the peptide binary, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and i_nai is 1 -naphthylalanine, and MeG is N-mercaptoglycine; (ii) each peptide monomer of the peptide binary is contained between two cysteine (C) residues of each monomer An intramolecular disulfide bond; (iii) PEG comprises a linear unbranched polyethylene glycol (PEG) moiety having a molecular weight of from about 2 Torr to about 4 Torr. 37 'As applied for the compound of the range 36 of the paste, each PEG of the towel has a molecular weight of about 20, 〇〇〇dol. 38. Use of a compound as claimed in claim % for the manufacture of a drug. The drug is used in a disorder characterized by a deficiency in erythropoietin or a low red blood cell population. 39. The use of claim 38, wherein the condition is selected from the group consisting of: end stage renal failure or dialysis; AIDS, autoimmune disease or malignant disease-related anemia; cold thalassemia; cystic Fibrosis; early premature infants are also poor; chronic inflammatory disease-related anemia; spinal cord injury; acute blood loss; aging; and tumor disease state accompanied by abnormal red blood cell production. 40. For the purposes of claim 38, each of the pEGs has a molecular weight of about 30, doldorf. 41. A pharmaceutical composition comprising a compound as claimed in claim 36 and a pharmaceutically acceptable carrier. 42. The pharmaceutical composition of claim 41, wherein each pEG has a molecular weight of about 20,000 dolphins. 43. A compound that binds to and activates a erythropoietin receptor (EPO-R), the compound comprising a peptide binary having the formula: (AcGJGLYACHMGPrrOHBalJVCQPLR-NH (AcO)GLYACHMGPrT(l-oa〇VCQPLR-HN 1-1 Ο Wherein (i) each peptide monomer of the peptide peptide, each amino acid system 15 Lu 20 is represented by a standard one-letter abbreviation, AeG is an ethyl glycosidic acid, and Ι-nal is a 1-naphthyl group. Alanine; (8) Each peptide monomer of the second reading of the winning monomer contains an intramolecular disulfide bond between the two half-proline (C) residues of each monomer; ((1)) ["PEG"] contains at least The two-linear polyethylation bond is attached to a single attachment; A compound having a molecular weight of about 〇 〇 to about 43 of the approximate range, wherein each coffee has a molecular weight of about 20, 〇〇〇dolp. The compound of claim 43 is used for the manufacture of a drug ΙΙ ' 物 于 于 于 于 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Lower disease group 2 end stage renal failure or dialysis; AIDS, autoimmune disease related anemia type 1 thalassemia; cystic fibrosis acute - ^ inflammatory disease associated anemia; spinal cord injury; disease state. 'and tumors associated with abnormal erythropoiesis 47.t application for patent (4) 45 applications, each of which has a molecular weight of about 20, 〇〇〇道尔吞. The pharmaceutically acceptable (tetra) article comprises a compound as described in claim 43 and a grammatically acceptable carrier. The pharmaceutically acceptable composition of claim 48 wherein each PEG has a molecular weight of about 20,000 dolphins. 10 50. A compound that binds to and activates a erythropoietin receptor (EPO-R), the compound comprising a peptide binary having the formula: Wherein (i) each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and 1_nal is 1-naphthylalanine, And MeG is N-methylglycine; (11) each peptide monomer of the peptide complex contains an intramolecular disulfide bond between the two semi-proline (C) residues of each monomer (U1) PEG comprises a dilinear polyethylene glycol (PEG) moiety having a combined molecular weight of about 1 〇, 〇〇〇 to about 30,000 Torr. A compound according to claim 5, wherein each PEG has a molecular weight of about 20,000 doxane. A compound such as the patent application 50 is used to manufacture a drug "drugs are used to treat conditions characterized by erythropoietin deficiency or red blood 53: family: low or defective. 9 month and scope 52 Use, wherein the condition is selected from the group consisting of the lower group, Weiwei end stage renal failure or dialysis; AIDS, autoimmune 1344496 = ill phase _ anemia type 1 thalassemia; cystic fibrosis q early anemia; chronic inflammatory disease related anemia ; spinal cord injury; 2 blood loss, aging; and tumor disease state accompanied by abnormal red blood cell formation. 54 'As claimed in the scope of application of the 52nd, each ship has a molecular weight of about 20, 〇〇〇道尔吞. - Pharmaceutical composition 'comprising a compound of the scope of claim (4) and a pharmaceutically acceptable carrier. 10 The pharmaceutical composition of claim 55, wherein each pEG has a molecular weight of about 20, a sputum 57. A combination and activation of erythropoietin receptor (Ep phantom compound, this compound contains a peptide binary, with the following formula (i) for each singular monomer of the winning binary, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and Ι-nal is 1-caffeyl alanine (ii) each peptide monomer of the peptide peptide contains an intramolecular disulfide bond between two 12 5 cysteine (C) residues of each monomer; _) Department - Ertun. There are &amp; knives 1 about 10,0 (8) to about 30,000. 1:: Compound of the 57th patent range, which contains about 20,000 dolphins. Page knife ^The compound of the 57th item of Shen (7) patent (4) is used for manufacturing 10 15 ^ Use 'This __ treats a condition characterized by a red (four) pheromone deficiency or a low or defective spheroid population. Such as U Lifan ϋ Item 59, wherein the condition is selected from the group consisting of 歹L and ia. End stage renal failure or dialysis; AIDS, autoimmune disease or malignant disease-related anemia type 1 thalassemia; cystic fiber Change (birth, early anemia in premature infants; anemia associated with chronic inflammatory disease) · spinal cord injury; acute blood loss; aging; and tumor disease state accompanied by abnormal red blood cell production. 61. The use of claim 59, wherein each PEG has a molecular weight of about 20, dolphal. 62. A pharmaceutical composition comprising a compound of claim 57 and a pharmaceutically acceptable carrier. 2 〇 63. The pharmaceutical composition of claim 62, wherein each PEG has a molecular weight of about 20, doldorf. 64. A compound that binds to and activates a erythropoietin receptor (EPO-R), the compound comprising a peptide binary having the formula: 13 1344964 5 (i) for each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, i_nal is 1-naphthylalanine' and MeG is N-methylglycine; (ii) each peptide monomer of the peptide binary contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; (m) PEG comprises a dilinear polyethylene glycol (pEG) moiety with a group. For the child, about 1 〇, 〇〇〇 to about 3 〇〇〇 〇〇〇 呑 呑. • A compound of claim 64, wherein each PEG has a molecular weight of about 20, doldorf. • The use of a compound according to item 64 of the Chinese Patent No. 4 for the manufacture of a medicament for the treatment of erythropoietin deficiency or red blood in the i m system Wherein the condition is selected from the group consisting of: anemia associated with a disease or malignant disease; sex; early stage renal failure or dialysis in preterm infants; AIDS, autoimmune human blood; type 5 thalassemia; cystic fibrosis Gray; chronic inflammatory disease-related anemia; spinal cord injury; 14 1^44^4 acute blood loss; aging; and tumor disease state accompanied by abnormal red jk ball formation. 5 10 The team used the application of the 66th scope of the patent, in which each ship has a molecular weight of about 20, 〇〇〇道尔吞. A medicine, a composition, and a compound as claimed in the scope of the patent application and a pharmaceutically acceptable carrier. For example, if you apply for special (4) Wai (10) Pharmacy shouts, the hulls of each towel have a molecular weight of about 20,000 dolphins. 71. A compound that binds to and activates a erythropoietin receptor (Ep〇-R), the compound comprising a triumphant binary having the formula: (AcG)GLYA(}HMGPn&lt;l-niU)vii 〇 V jtm PEG (AcO)GLYA(piMGPlT(i-Ml)VCQPLRWIN Ο Wherein (i) each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and 15 Ι-nal is 1-naphthyl Alanine; (ii) each peptide monomer of the peptide complex contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; 15 1344964 (iii) PEG contains A linear unbranched polyethylene glycol (PEG) moiety having a molecular weight of from about 20,000 to about 40,000 mil. 72. A compound that binds and activates a erythropoietin receptor (EPO-R). The compound comprises a peptide binary having the following formula: (i) each peptide monomer of the peptide peptide, each amino acid is represented by a standard one-letter abbreviation, AcG is N-acetylglycine, and Ι-nal is 1-naphthyl alanine (η) each peptide monomer of the peptide peptide contains an intramolecular disulfide bond between the two cysteine (C) residues of each monomer; (Hi) PEG contains a molecular weight of about 1 〇, 〇〇〇 to a linear unbranched polyethylene glycol (PEG) moiety of about 6 〇〇〇〇. 16