Novel peptides that bind to the erythropoietin receptor

The present invention relates to peptide compounds that are erythropoietin receptor (EPO-R) agonists. The invention also relates to therapeutic methods using such peptide compounds to treat disorders associated with insufficient or defective red blood cell production. The invention also relates to pharmaceutical compositions that contain the peptide compounds of the invention.

BACKGROUND OF THE INVENTION [0002] Erythropoietin (EPO) is a 165 amino acid glycoprotein hormone with a molecular weight of about 34 kilodaltons (kD) and preferred glycosylation sites at amino acid positions 24, 38, 83, and 126. Initially, it is produced as a precursor protein from a signal peptide of 23 amino acids. EPO can come in three forms: α, β, and asialo. Forms α and β differ slightly in their carbohydrate components, but have the same potency, biological activity, and molecular weight. The asialo form means the α or β form with the carbohydrate (sialic acid) group removed. The DNA sequences encoding EPO have been described [US Patent No. 4,703,008 to Lin].

[0003] EPO stimulates mitotic division and differentiation of erythrocyte precursor cells, thus ensuring the production of erythrocytes. When hypoxia dominates, it is produced in the kidney. During EPO-induced differentiation of erythrocyte precursor cells, globin synthesis is induced; heme complex synthesis is stimulated; and the number of ferritin receptors increases. These changes allow the cell to take up more iron and synthesize working hemoglobin, which binds oxygen in mature erythrocytes. Thus, erythrocytes and their hemoglobin play a key role in providing oxygen to the body. These changes are initiated by the interaction of EPO with the appropriate receptor on the cell surface of erythrocyte precursor cells [see, e.g., Graber and Krantz (1978) Ann. Rev. Med. 29.51-66].

[0004] When the body is in a healthy state, in which tissues receive sufficient oxygenation from the existing number of erythrocytes, EPO is present in plasma at very low concentrations. This normal low concentration is sufficient to stimulate red blood cell replacement, which is normally lost through aging.

[0005] The amount of EPO in the circulation is increased under hypoxic conditions when oxygen transport through the blood cells in the circulation is reduced. Hypoxia can be caused, for example, by significant blood loss due to hemorrhage, destruction of red blood cells by excessive radiation exposure, reduction of oxygen intake due to high altitude (above sea level) or prolonged loss of consciousness, or various forms of anemia. In response to such hypoxic stress, elevated EPO levels increase red blood cell production by stimulating the proliferation of erythroid daughter cells. When the circulating red blood cell count is greater than needed for normal tissue oxygen demand, then circulating EPO levels decrease.

[0006] Because EPO is essentially important in the formation of red blood cells, this hormone has potentially useful applications in both the diagnosis and treatment of blood disorders characterized by

Low or defective production of red blood cells. Recent studies have provided the basis for designing efficacy of EPO therapy for a variety of disease states, disorders, and hematological abnormalities, including beta-thalassemia [see, Vedovato, et al. (1984) Acta Haematol. 71: 211-213]; cystic fibrosis [see, Vichinsky, et al. (1984) J. Pediatrie 105: 15-21]; pregnancy and menstrual disorders [see, Cotes, et al. (193) Brit. J. Ostet. Gyneacol. 90: 304-311]; early prematurity anemia [see, Haga, et al. (1983) Acta Pediatr. Scand. 72; 827-831]; spinal cord injury [see, Claus-Walker, et al. (1984) Arch. Phys. Med. Rehabil. 65: 370-374]; space flight [see, Dunn, et al. (1984) Eur. J. Appl. Physiol. 52: 178-182]; acute blood loss [see, Miller, et al. (1982) Brit. J. Haematol. 52: 545-590]; aging [see, Udupa, et al. (1984) J. Lab. Clin. Med. 103: 574-580 and 581-588, and Lipschitz, et al. (1983) Blood 63: 502-509]; various states of cancer associated with abnormal erythropoiesis [see, Dainiak, et al. (1983) Cancer 5: 1101-1106 and Schwartz, et al. (1983) Otolaryngol. 109: 269-272]; and renal failure [see, Eschbach, et al. (1987) N. Eng. J. Med. 316: 73-78].

[0007] Purified, homogeneous EPO has been characterized [US Patent No. 4,677,195 to Hewick]. The DNA sequence encoding EPO was purified, cloned, and expressed to obtain recombinant polypeptides with biochemical and immunological properties that are the same as natural EPO. A recombinant EPO molecule having oligosaccharides identical to natural EPO has also been produced [see, Sasaki, et al. (1987) J. Biol. Chem. 262: 12059-12076].

[0008] The biological effect of EPO appears to be mediated, in part, by interaction with a cell membrane-bound receptor. Preliminary studies using murine immature erythroid cells isolated from the spleen suggest that EPO binding proteins on the cell surface include two polypeptides having approximate molecular weights of 85,000 daltons and 100,000 daltons, respectively [Sawyer, et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3690-3694]. The calculated number of EPO binding sites is on average between 800 and 1000 per cell surface. Of these binding sites, approximately 300 bind EPO to K_d of approximately 90 pM (picomol / liter), while the others bind EPO with a reduced affinity of approximately 570 pM [Sawyer, et al. (1987) J. Biol. Chem. 262: 5554-5562]. An independent study suggested that EPO-responsive spleen erythroblasts, produced in mice injected with anemia-inducing Friend leukemia virus (FVA) strain, have a total of approximately 400 high and low affinity EPO binding sites that have K values_d of approximately 100 pM and 800 pM, respectively [Landschulz, et al. (1989) Blood 73: 1476-1486].

[0009] Another work indicated that two forms of the EPO receptor (EPO-R) are encoded by a single gene. This gene has been cloned [see, e.g., Jones, et al. (1990) Blood 76, 31-35; Noguchi, et al. (1991) Blood 78: 2548-2556; Maouche, et al. (1991) Blood 78: 2557-2563]. For example, DNA sequences and encoded peptide sequences for murine and human EPO-R proteins are described in PCT Publication No. WO 90/08822 by D'Andrea, et al. Current models suggest that binding of EPO to EPO-R causes dimerization and activation of two EPO-R molecules, resulting in subsequent signal transduction steps [see, e.g., Watowich, et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2140-2144].

[0010] The availability of cloned genes for EPO-R enables the search for agonists and antagonists of this important receptor. The availability of a recombinant receptor protein enables the study of receptor-ligand interaction in a variety of random and semi-random systems to generate peptide diversity. These systems include the "peptides on plasmids" system [described in US Patent No. 6,270,170]; the "phage peptides" system [described in US Patent No. 5,432,018 and Cwirla, et al. (1990) Proc. Natl. Acad.

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Sci. USA 87: 6378-6382]; the "encoded synthetic library" (ESL) system [described in US Patent Application Serial No. 946.239, filed on September 16, 1992]; and the "large scale synthesis for polymer immobilization" system [described in US Patent No. 5,143,854; PCT Publication No. 90/15070; Fodor, et al. (1991) Science 251: 767-773; Dower and Fodor (1991) Ann. Rep. Med. Chem. 26: 271-180; and US Patent No. 5,424,186].

[0011] Peptides that interact at least to some extent with EPO-R have been identified and are described, for example, in US Patent Nos. 5,773,569; 5,830,851; and 5,986,047 to Wrighton, et al .; PCT Publication No. WO 96/40749 to Wrighton, et al .; in US Patent No. 5,767,078 and PCT Publication No. 96/40772 to Johnson and Zivin; PCT Publication No. WO 01/38342 for Bal; and WO 01/91780 for Smith-Swintosky, et al. In particular, a group of peptides containing a peptide motif has been identified whose members bind to EPO-R and stimulate EPO-dependent cell proliferation. However, peptides identified to date that contain this motif stimulate EPO-dependent cell proliferation in vitro with EC50 values ​​of about 20 nanomolar / liter (nM) to about 250 nM. Thus, peptide concentrations of 20 nM to 250 nM are needed to stimulate 50% of the maximum level of cell proliferation stimulated by EPO.

[0012] In view of the huge potential of EPO-R agonists, both in studies of important biological activities mediated by this receptor and in the treatment of diseases, there is a continuing need to identify EPO-R peptide agonists with increased potency and activity. The present invention relates to such compounds.

[0013] The citation and / or discussion of the cited references in this section and throughout the description merely serves to clarify the description of the present invention and does not imply that any such reference is "prior art" for the present invention.

SUMMARY OF THE INVENTION [0014] The present invention relates to new peptide compounds that are EPO-R agonists with significantly increased potency and activity. These peptide compounds are homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1), or homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1nal) VCQPLR (MeG) K ( KNOT. SEQ ID NO: 2), homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) (SEQ ID NO: 3); where each of the amino acids is indicated by a normal single letter abbreviation, "(AcG)" means N-acetylglycine, "(1-nal)" means 1-naphthylalanine, and "(MeG)" means N-methylglycine also known as sarcosine. Each of the peptide dimer monomers contains an intramolecular disulfide bond between monomer cysteine ​​residues.

[0015] Peptide monomers may be dimerized by covalent attachment to a branched tertiary amide linker. The tertiary amide linker can be represented as:

-C¹O-CH₂-X-CH_2-C²Each: X is NCO- (CH2) 2-N¹H; C¹ the linker forms an amide bond with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; C² the linker forms an amide bond with the ε-amino group of the C-terminal lysine residue of the second peptide monomer; and N¹ of the X group is attached by

A carbamate or amide bond to an activated polyethylene glycol (PEG) moiety, where PEG has a molecular weight of about 20,000 to about 40,000 daltons (the term "about" indicates that in PEG preparations some molecules will have a larger mass, and some lower than the specified molecular weight).

[0016] When each of the homodimer monomers has an amino acid sequence, (AcG) GLYACHMGPIT (1nal) VCQPLRK (SEQ ID NO: 1) and N¹ the linker is attached via a carbamate bond to an activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0001.tif" />

[0017] When each of the homodimer monomers has an amino acid sequence, (AcG) GLYACHMGPIT (1nal) VCQPLRK (SEQ ID NO: 1) and N¹ the linker is attached via an amide bond to the activated polyethylene glycol (PEG) moiety, the new peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0002.tif" />

[0018] When each of the homodimer monomers has an amino acid sequence, (AcG) GLYACHMGPIT (1nal) VCQPLR (MeG) K (SEQ ID NO: 2) and N¹ the linker is attached via a carbamate bond to an activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

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<img file="PL1629007T3_D0003.tif" />

[0019] When each of the homodimer monomers has an amino acid sequence, (AcG) GLYACHMGPIT (lnal) VCQPLR (MeG) K (SEQ ID NO: 2) and N¹ the linker is attached via an amide bond to the activated polyethylene glycol (PEG) moiety, the new peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0004.tif" />

[0020] Peptide monomers may also be dimerized by covalent attachment to a branched tertiary amide linker. The tertiary amide linker may be represented as:

-C¹O-CH₂-X-CH₂-C²Each: X is NCO- (CH2) 2-NH-C³ABOUT-; C¹ the linker forms an amide bond with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; and C.² the linker forms an amide bond with the ε-amino group of the C-terminal lysine residue of the second peptide monomer. The peptide dimers of the invention further comprise a spacer (spacer) moiety having the following structure:

-N¹H- (CH₂)₄-C⁴HN²Hgdzie: C⁴ spacer is covalently attached to C.³ from group X; N¹ the spacer is covalently attached via a carbamate or amide bond to the activated polyethylene glycol (PEG) moiety; and N² the spacer is covalently attached via a carbamate or amide bond to the activated PEG moiety, where the PEG has a molecular weight of about 10,000 to about 50,000 daltons (wherein "about" indicates that in PEG preparations some molecules

EP 1629007 B1 will have a greater mass and some less than the stated molecular weight). Each of the PEG moieties can have 10,000 Daltons (10 kD), 20 kD, 30 kD, 40 kD, or 50 kD, separately.

[0021] When each of the homodimer monomers has an amino acid sequence, (AcG) GLYACHMGPIT (1nal) VCQPLRK (SEQ ID NO: 1) and both N¹ and N² spacers are covalently attached via a carbamate bond to an activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0005.tif" />

In preferred embodiments, the C-terminal lysine of the two peptide monomers is L-lysine. Based on the above chemical structures, specialists will also note that two linear moieties

PEGs are connected via lysine (e.g., as mPEG2-Lys-NHS or as mPEG2-Lysine-NPC), which is also preferably L-lysine and contributes to the following stereochemistry.

<img file="PL1629007T3_D0006.tif" />

[0022] Alternatively, as those skilled in the art will easily recognize, one or more of the lysine residues may be D-lysine, contributing to alternative stereochemistry.

[0023] When each of the homodimer monomers has an amino acid sequence, (AcG) GLYACHMGPIT (1nal) VCQPLRK (SEQ ID NO: 1) and both N¹ and N² spacers are covalently attached via an amide bond to an activated PEG moiety, the novel peptide compounds of the invention may be represented as follows:

EP 1629007 B1

<img file="PL1629007T3_D0007.tif" />

Again, preferably all L-lysine molecules in this compound are L-lysine, contributing to the following stereochemistry.

<img file="PL1629007T3_D0008.tif" />

Alternatively, as is readily apparent to those skilled in the art, one or more of the lysine residues may be D-lysine, contributing to alternative stereochemistry.

[0024] When each of the homodimer monomers has an amino acid sequence, (AcG) GLYACHMGPIT (1nal) VCQPLR (MeG) K (SEQ ID NO: 2) and both N¹ and N² spacers are covalently attached via a carbamate bond to an activated PEG moiety in which Y is a carbamate group, the new peptide compounds of the invention may be represented as follows:

<img file="PL1629007T3_D0009.tif" />

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Preferably, all lysine residues connecting peptide monomer moieties and linear PEG in this molecule represent L-lysine, contributing to the following stereochemistry:

<img file="PL1629007T3_D0010.tif" />

[0025] Alternatively, as will be readily apparent to those skilled in the art, one or more of the lysine residues may be D-lysine, contributing to alternative stereochemistry.

[0026] When each of the homodimer monomers has an amino acid sequence, (AcG) GLYACHMGPIT (1nal) VCQPLR (MeG) K (SEQ ID NO: 2) and both N¹ and N² spacers are covalently attached via an amide bond to an activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0011.tif" />

Preferably, all lysine residues connecting peptide monomer moieties and linear PEG in this molecule are L-lysine, contributing to the following stereochemistry.

EP 1629007 B1 (AsG) GLYA &lt;

PEGiwok

CAcGX? LYAip &amp; 10PtTfί 44 «l&gt; VOQPIR (Meq&gt; -HN

In other embodiments, as will be readily apparent to those skilled in the art, one or more of the lysine residues may be D-lysine, contributing to alternative stereochemistry.

[0027] Peptide monomers can also be dimerized by attachment to a lysine linker, whereby one of the peptide monomers is attached at its C-terminus to the ε-amino group of lysine, and the other of the peptide monomers is attached to its C-terminus to the group α-amino lysine.

[0028] The peptide dimers of the invention further include a spacer moiety with the following structure:

-N¹H- (CH₂)₂-O- (CH₂)₂-O- (CH₂)₂-N²H10 At one end, N¹ the spacer is attached via an amide bond to the carbonyl carbon atom of the lysine linker. At the opposite end, N² the spacer is attached via a carbamate or amide bond to an activated polyethylene glycol (PEG) moiety, where PEG has a molecular weight of about 20,000 to about 40,000 daltons (with the term "about" indicating that some PEG preparations will have a larger mass, and some lower than the given molecular weight).

[0029] When the spacer is attached via a carbamate bond to an activated polyethylene glycol (PEG) moiety, the new peptide compounds of the invention (SEQ ID NO: 3) can be represented as follows:

<img file="PL1629007T3_D0012.tif" />

[0030] Another object of the invention are pharmaceutical compositions containing such peptide compounds, and methods of treating various medical conditions using such peptide compounds.

EP 1629007 B1

DETAILED DESCRIPTION OF THE INVENTION

definitions:

[0031] The amino acid residues in the peptides are shortened as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Valine is Val or V; Serine is Cheese 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 H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic acid is Asp or D; Glutamic acid is Glu or E; Cysteine ​​is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G. Unnatural amino acids in peptides are shortened as follows: 1-naphthylalanine is 1-nal or Np; N-methylglycine (also known as sarcosine) is MeG or Sc; and acetylated glycine (N-acetylglycine) is AcG.

[0032] As used herein, the term "polypeptide" or "protein" refers to a polymer of amino acid monomers that are alpha-amino acids linked together through amide bonds. Thus, the polypeptides are at least two amino acid residues long and are usually longer. Generally, the term "peptide" refers to a polypeptide that is only a few amino acid residues in length. The novel EPO-R agonist peptides of the present invention are preferably no more than about 50 amino acid residues in length. They are more preferably about 17 to about 40 amino acid residues in length. The polypeptide, unlike a peptide, can include any number of amino acid residues. Hence, the term polypeptide includes peptides as well as longer amino acid sequences.

[0033] As used herein, the phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are "generally recognized as safe", e.g., which are physiologically tolerable and typically do not produce an allergic or similar adverse reaction, such as stomach upset, dizziness heads and the like when they are given to man. Preferably, the term "pharmaceutically acceptable" as used herein means as approved by a federal or state government regulatory agency or included in the US Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, especially humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous glucose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Useful pharmaceutical carriers are described in the EW handbook Martin "Remington's Pharmaceutical Sciences".

[0034] The term "agonist" as used herein refers to a biologically active ligand that binds to the corresponding biologically active receptor and activates the latter either elicits a biological response in the receptor or increases already existing biological activity of the receptor.

New peptides that are EPO-R agonists [0035] The present invention relates to new peptide compounds that are EPO-R agonists with significantly increased potency and activity. These peptide compounds are homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1), or homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (110

EP 1629007 B1) VCQPLR (MeG) K (SEQ ID NO: 2); where each of the amino acids is indicated by a normal single letter abbreviation, "(AcG)" means N-acetylglycine, "(1-nal)" means 1-naphthylalanine, and "(MeG)" means "(MeG)" means N-methylglycine known also as sarcosine. Each of the peptide dimer monomers contains an intramolecular disulfide bond between monomer cysteine ​​residues. Such monomers can be represented schematically as follows:

(SEQ ID NO: 1): and (^ glyaS ^^ plwmkijk _andand"₀(SEQ ID NO: 2) [0036] These monomeric peptides are dimerized to form peptide dimers with increased EPO-R agonist activity. Linker grouping (L_K) is a branched tertiary amide that bridges the C-terminus of two peptide monomers by simultaneously attaching to the C-terminal lysine residues of each of the monomers. The tertiary amide linker may be represented as:

-C¹O-CH₂-X-CH₂-C²Each: X is NCO- (CH2) 2-N¹H; C¹ the linker forms an amide bond with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; C² the linker forms an amide bond with the ε-amino group of the C-terminal lysine residue of the second peptide monomer; and N¹ from the X moiety is attached via a carbamate or amide bond to an activated polyethylene glycol (PEG) moiety, where the PEG has a molecular weight of about 20,000 to about 40,000 daltons (wherein the term "about" indicates that in PEG preparations some molecules will have a mass higher and some lower than the specified molecular weight).

[0037] The tertiary amide linker may also be represented as:

-C¹O-CH_2-X-CH₂-C²Each: X is NCO- (CH2) 2-NH-C³ABOUT-; C¹ the linker forms an amide bond with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; and C.² the linker forms an amide bond with the ε-amino group of the C-terminal lysine residue of the second peptide monomer. The peptide dimers of the invention further include a spacer moiety with the following structure:

-N¹H- (CH₂)₄-C⁴HN²Hgdzie: C⁴ the walk is covalently linked to C³ X; N¹ the spacer is covalently attached via a carbamate or amide bond to the activated PEG moiety; and N²the spacer is covalently attached via a carbamate or amide bond to the activated PEG moiety, where the PEG has a molecular weight of about 10,000 to about 60,000 daltons (wherein the "about" indicates that in PEG preparations some molecules will have a larger mass and some less than given molecular weight).

[0038] Thus, the new peptides of the invention may also contain a PEG moiety that is covalently attached via a carbamate or amide bond to the tertiary amide linker of the peptide dimer. PEG means a water-soluble polymer that is pharmaceutically acceptable. The PEG for use in the present invention may be linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20K) to about 60K (wherein the term "about" indicates that in PEG preparations some molecules will have a larger mass and some smaller than the given molecular weight). Most preferably, PEG has a molecular weight of about 30K to about 40K. The skilled person will be able to choose the desired size of the polymer having regard to the desired dosage; circulation time; resistance to proteolysis; possible effects on biological activity; ease of use; degree or lack of antigenicity; and other known effects of PEG on a therapeutic peptide.

[0039] Peptides, peptide dimers and other molecules based on the peptides of the invention may be attached to water-soluble polymers (e.g., PEG) using any of a variety of chemistry for linking the water-soluble polymer (s) to the receptor-binding portion (e.g. ., peptide + spacer). A typical embodiment uses a single attachment site for covalent attachment of the water-soluble polymer (s) to the receptor-binding portion, however, in alternative embodiments, multiple attachment sites can be used, including further variants where different types of water-soluble polymer attach to the portion receptor binding at various attachment sites, which may include the covalent attachment site (s) for the spacer and / or one or both of the peptide chains. In some embodiments, the higher order dimer or multimer will contain different types of peptide chain (i.e., a heterodimer or other heteromultimer). For example, but without limitation, the dimer may contain a first peptide chain having a PEG attachment site and the second peptide chain may either have no PEG attachment site or use different binding chemistry than the first peptide chain, and in some embodiments the spacer may or may not contain a site PEG attachments and said spacer if it is PEGylated, it may use binding chemistry other than the chemistry used for the first and / or second peptide chains. An alternative embodiment uses PEG attached to the spacer portion in the receptor binding portion and another water-soluble polymer (e.g., carbohydrate) conjugated to the side chain of one of the amino acids of the peptide portion of the molecule.

[0040] Various types of polyethylene glycol (PEG) can be used to PEGylate the receptor binding portion (peptides + spacer). In principle, any suitable reactive PEG reagent may be used. In preferred embodiments, the reactive PEG reagent will result in the formation of a carbamate or amide bond upon conjugation with the receptor binding portion. Useful reactive types of PEG include, but are not limited to, those types that are available for sale in the Drug Delivery Systems (2003) catalog from NOF Corporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome, Shibuya-ku, Tokyo 150-6019) and Molecular Engineering catalog (2003) from Nektar Therapeutics (490 Discovery Drive, Huntsville, Alabama 35806). For example, but not limited to, in various embodiments, the following PEG reagents are often preferred: mPEG2-NHS, mPEG2-ALD, multi-Arm PEG, mPEG (MAL) 2, mPEG2 (MAL), mPEG-NH2, mPEG- SPA, mPEG-SBA, mPEG-thioesters, mPEG-double esters, mPEG-BTC, mPEG-ButyrALD, mPEG-ACET, heterofunctional PEG (NH2-PEG-COOH, Boc-PEG12

EP 1629007 B1

NHS, Fmoc-PEG-NHS, NHS-PEG-VS, NHS-PEG-MAL), PEG acrylates (ACRL-PEG-NHS), PEG phospholipids (e.g., mPEG-DSPE), multi-arm PEG from the SUNBRITE series, including GL PEG series based on glycerin activated by chemistry selected by specialists, any of activated PEG SUNBRITE (including, but not limited to, carboxyl-PEG, p-NP-PEG, tresyl-PEG, aldehyde PEG, acetal PEG, amine-PEG , thiol-PEG, maleimide-PEG, hydroxyl-PEG-amine, amino-PEG-COOH, hydroxyl-PEG-aldehyde, Carboxylic anhydride-type PEG, functionalized phospholipid PEG, and other similar and / or useful reactive PEGs that are chosen by those skilled in the art for their specific use and use.

[0041] The new peptides of the invention may also contain two PEG moieties that are covalently attached via a carbamate or amide bond to the spacer moiety, where the spacer moiety is covalently attached to the tertiary amide peptide dimer linker. Each of the two PEG moieties used in such embodiments of the present invention may be linear, and they may be joined together at a single point of attachment. Each of the PEG moieties preferably has a molecular weight of about 10 kilodaltons (10K) to about 60K (with the term "about" indicating that in PEG preparations some molecules will have a larger mass and some less than the specified molecular weight). Linear PEG moieties are particularly preferred. More preferably, each of the two PEG moieties has a molecular weight of about 20K to about 40K, and even more preferably between about 20K and about 40K. Even more preferably, each of the two PEG moieties has a molecular weight of about 20K. The skilled person will be able to choose the desired size of the polymer having regard to the desired dosage; circulation time; resistance to proteolysis; possible effects on biological activity; ease of use; degree or lack of antigenicity; and other known effects of PEG on a therapeutic peptide.

The present invention also includes peptide agonists, which are homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) (SEQ ID NO: 3), where each amino acid is indicated by a normal abbreviation single letter, "(AcG)" means N-methylglycine, "(1-nal)" means 1-naphthylalanine, and "(MeG)" means N-methylglycine, also known as sarcosine. Each of the peptide dimer monomers contains an intramolecular disulfide bond between monomer cysteine ​​residues. Such monomers can be represented schematically as follows (SEQ ID NO: 3):

[0043] These monomeric peptides are dimerized to form peptide dimers with increased EPO-R agonist activity. Linker grouping (L_K) is a lysine residue that bridges the C-terminus of two peptide monomers by simultaneously linking to the C-terminal amino acid of each of the monomers. One of the peptide monomers is attached at its C-terminus to the ε-amino group of lysine, and the other of the peptide monomers is attached at its C-terminus to the α-amino group of lysine. For example, the dimer can be structurally illustrated as shown in Formula I, and in a simplified manner shown in Formula II:

EP 1629007 B1

Formula I

Template II

<img file="PL1629007T3_D0013.tif" />

In formula I and formula II, N² is the nitrogen atom of the ε-amino group of lysine and N¹ is the nitrogen atom of the α-amino group of lysine.

[0044] The peptide dimers of the invention further include a spacer moiety with the following structure:

-N¹H- (CH₂)₂-O- (CH₂)₂-O- (CH₂)₂-N²H At one end, N¹ the spacer is attached via an amide bond to the carbonyl carbon atom of the lysine linker. At the opposite end, N² the spacer is attached via a carbamate or amide bond to an activated polyethylene glycol (PEG) moiety, where PEG has a molecular weight of about 10,000 to about 60,000 daltons (with the term "about" indicating that some PEG preparations will have a larger mass, and some lower than the given molecular weight). More preferably, PEG has a molecular weight of about 20,000 to 40,000 daltons.

[0045] Thus, the new peptides of the invention also contain a PEG moiety that is covalently attached to the peptide dimer. PEG means a water-soluble polymer that is pharmaceutically acceptable. The PEG for use in the present invention may be linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20K) to about 60K (wherein the term "about" indicates that in PEG preparations some molecules will have a larger mass and some smaller than the given molecular weight). Most preferably, PEG has a molecular weight of about

0 20K to about 40K, and even more preferably a molecular weight of about 30K to about 40K.

The skilled person will be able to choose the desired size of the polymer having regard to the desired dosage; circulation time; resistance to proteolysis; possible effects on biological activity; ease of use; degree or lack of antigenicity; and other known effects of PEG on a therapeutic peptide.

[0046] When each of the homodimer monomers has the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1) and N¹ the linker is attached via a carbamate bond to an activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

EP 1629007 B1

<img file="PL1629007T3_D0014.tif" />

[0047] When each of the homodimer monomers has the amino acid sequence (AcG) GLYACHMGPIT (lnal) VCQPLRK (SEQ ID NO: 1) and N¹ the linker is attached via an amide bond to the activated polyethylene glycol (PEG) moiety, the new peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0015.tif" />

[0048] When each of the homodimer monomers has the amino acid sequence (AcG) GLYACHMGPIT (1nal) VCQPLR (MeG) K (SEQ ID NO: 2) and N¹ the linker is attached via a carbamate bond to an activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0016.tif" />

[0049] When each of the homodimer monomers has an amino acid sequence, (AcG) GLYACHMGPIT (1nal) VCQPLR (MeG) K (SEQ ID NO: 2) and N¹ the linker is attached via an amide bond to the activated polyethylene glycol (PEG) moiety, the new peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0017.tif" />

[0050] Preferred peptide dimers of the present invention include, but are not limited to

<img file="PL1629007T3_D0018.tif" />

limited to this: (SEQ ID NO: 1)

<img file="PL1629007T3_D0019.tif" />

(SEQ ID NO: 1)

EP 1629007 B1 (SEQ ID NO: 1)

<img file="PL1629007T3_D0020.tif" />

<img file="PL1629007T3_D0021.tif" />

<img file="PL1629007T3_D0022.tif" />

<img file="PL1629007T3_D0023.tif" />

(SEQ ID NO: 1) (SEQ ID NO: 1) (SEQ ID NO: 1)

EP 1629007 B1 (SEQ ID NO: 2)

<img file="PL1629007T3_D0024.tif" />

(SEQ ID NO: 2) (SEQ ID NO: 2) (SEQ ID NO: 2)

EP 1629007 B1 (SEQ ID NO: 2)

<img file="PL1629007T3_D0025.tif" />

(SEQ ID NO: 2) [0051] When each of the homodimer monomers has the amino acid sequence (AcG) GLYACHMGFIT (1nal) VCQPLRK (SEQ ID NO: 1) and both N¹ and N² spacers are covalently attached via a carbamate bond to an activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0026.tif" />

[0052] When each of the homodimer monomers has the amino acid sequence (AcG) GLYACHMGPIT (1nal) VCQPLRK (SEQ ID NO: 1) and both N¹ and N² spacers are covalently attached via an amide bond to an activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

EP 1629007 B1

<img file="PL1629007T3_D0027.tif" />

[0053] When each of the homodimer monomers has the amino acid sequence (AcG) GLYACHMGPIT (1nal) VCQPLR (MeG) K (SEQ ID NO: 2) and both N¹ and N² spacers are covalently attached via a carbamate bond to an activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0028.tif" />

[0054] When each of the homodimer monomers has the amino acid sequence (AcG) GLYACHMGPIT (1nal) VCQPLR (MeG) K (SEQ ID NO: 2) and both N¹ and N² spacers are covalently attached via an amide bond to an activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="PL1629007T3_D0029.tif" />

[0055] Preferred peptide dimers of the present invention include, but are not limited to:

EP 1629007 B1 (SEQ ID NO: 1)

<img file="PL1629007T3_D0030.tif" />

(SEQ ID NO: 1) (SEQ ID NO: 1) (SEQ ID NO: 1)

EP 1629007 B1 (SEQ ID NO: 1)

<img file="PL1629007T3_D0031.tif" />

(SEQ ID NO: 1) (SEQ ID NO: 1) (SEQ ID NO: 1)

EP 1629007 B1 (SEQ ID NO: 1)

<img file="PL1629007T3_D0032.tif" />

(SEQ ID NO: 1) (SEQ ID NO: 1) (SEQ ID NO: 2)

EP 1629007 B1 (SEQ ID NO: 2)

<img file="PL1629007T3_D0033.tif" />

(SEQ ID NO: 2) (SEQ ID NO: 2) (SEQ ID NO: 2)

EP 1629007 B1 (SEQ ID NO: 2)

<img file="PL1629007T3_D0034.tif" />

(SEQ ID NO: 2) (SEQ ID NO: 2) (SEQ ID NO: 2)

[0056] When the spacer is attached via a carbamate bond to an activated polyethylene glycol (PEG) moiety, the new peptide compounds of the invention (SEQ ID NO: 3) can be represented as follows:

(AcGXłLYA &lt;inMOt&gt; lTtboel) viQiLR (M (X}) - NH 'PEGjo ^ (AiO) aLYAtjHafOWT (l-nfl3) VQFUŁ (MgG) -HN [0057] When the spacer is attached via an amide bond to the activated glycol polyol moiety PEG), the new peptide compounds of the invention (SEQ ID NO: 3) can be represented as follows:

<img file="PL1629007T3_D0035.tif" />

[0058] This dimeric structure can be written [Ac-peptide, disulfide] 2Lys-spacer-PEG20_-40K to indicate the N-terminally acetylated peptide associated with both the α- and ε-amino groups of lysine, where each peptide contains an intramolecular disulfide loop and a spacer molecule forming a covalent bond between the C-terminus of lysine and the PEG moiety, where PEG has a molecular weight of about 20,000 to about 40,000 daltons.

[0059] Preferred peptide dimers of the present invention include, but are not limited to:

(SEQ ID NO: 3)

<td>(AffljOLYAtiHMGFlTfl-TiiJjyioPLRiMeGJ-NH</td><td>0 NłiAą-PEGiaK</td>

<td>(AflOXiLYACHMGP] T (l-Mt) VCQPUl (MeG) -IIN</td><td> « 1°</td>

<td>(AcO) GL YA άωΟΡΙΙΐ 1-nal) V (!: QFUŁ (MsG) -l®</td><td> - 0</td>

<td>(AsCjGLYA ^ HMaPmC 1 -nal) VCQPLR (Mii]) -HN ^</td><td>Ł j&gt;</td>

<td>(AuOJGLYAtklMGf IT (1-u]) V ^ QPUUMeG) -] W</td><td>0 PEGljok</td>

<td>and (AiGJOLYAaHMGPITt 1-naJ) VCQK J &lt;&lt;McO&gt; 4 {N</td><td></td>

(SEQ ID NO: 3) (SEQ ID NO: 3)

EP 1629007 B1 (SEQ ID NO: 3)

<img file="PL1629007T3_D0036.tif" />

(SEQ ID NO: 3) (SEQ ID NO: 3) [0060] Components useful for compounds of the present invention may also be stereoisomers (e.g., D-amino acids) of twenty conventional amino acids, non-naturally occurring amino acids, such as α, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids. Examples of unconventional amino acids include, but are not limited to: β-alanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N-methylglycine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, norleucine, and other similar amino acids and imino acids. Other modifications are also possible, including amino terminal modification, carboxy terminal modification, unconventional amino acid replacement of one or more of the genetically coded natural amino acids, side chain modification of one or more amino acid residues, peptide phosphorylation, and the like.

[0061] The peptide sequences of the present invention may be present alone or in combination with N-terminal and / or C-terminal extensions of the peptide chain. Such extensions may be naturally encoded peptide sequences optionally with or substantially without non-naturally occurring sequences; extensions may contain any additions, deletions, point mutations, or other sequence modifications or combinations as desired by those of skill in the art. For example, but not limited to, naturally occurring sequences may be full length or partial length and may contain amino acid substitutions to form a site for attachment of a carbohydrate, PEG, other polymer, or the like by side chain conjugation. In a variation, amino acid substitution humanizes the sequence to render it compatible with the human immune system. All types of fusion proteins are provided, including immunoglobulin sequences adjacent to or in close proximity to the activating EPOR sequences of the present invention with or without a non-immunoglobulin spacer sequence. One type of embodiment is an immunoglobulin chain having an EPO-R activating sequence instead of the variable (V) region of the heavy and / or light chain.

EP 1629007 B1

Preparation of peptide compounds according to the invention:

Peptide synthesis [0062] The peptides of the invention can be produced by classical methods known in the art. These normal methods include solid phase synthesis only, partially solid phase synthesis methods, fragment condensation, classical solution synthesis, and recombinant DNA technology [see, e.g., Merrifield J. Am. Chem. Soc. 1963 85: 2149].

[0063] In one embodiment, the peptide dimer monomers are synthesized separately and dimerized after synthesis.

[0064] In another embodiment, dimer peptide monomers are linked via their C-terminus via a branched L-tertiary amide linker group._K having two functional groups that can serve as an initiation site for peptide synthesis and a third functional group (e.g. carboxy or amino group) that allows binding to another molecular moiety (e.g., which may be present on the surface of the solid support). In this case, two peptide monomers can be synthesized directly on the two reactive nitrogen groups of the linker group L_K in a variation of the solid phase synthesis technique. Such synthesis can be sequential or simultaneous.

[0065] In another embodiment, two peptide monomers can be synthesized directly on the two reactive nitrogen groups of the Lk linker moiety in a variation of the solid phase synthesis technique. Such synthesis can be sequential or simultaneous. This embodiment uses a lysine linker (Lk) moiety having two amino groups that can serve as an initiation site for peptide synthesis and a third functional group (e.g., lysine carboxyl group; or the lysine amide amino group, the lysine residue in which the carboxyl group has been transformed into the amide -CONH moiety₂), which allows binding to another molecular moiety (e.g., which may be present on the surface of a solid support).

[0066] When sequential synthesis of dimer peptide chains on a linker must be carried out, the two amino functional groups on the linker molecule are protected using two different amino protecting groups that can be removed independently of each other. The protected linker is coupled to the solid support via the linker's third functional group. The first amino protecting group is removed, and the first dimer peptide is synthesized on the first deprotected amino moiety. The second amino protecting group is then removed, and a second dimer peptide is synthesized on the second deprotected amino moiety. For example, the first amino moiety of the linker may be protected by the Alloc group and the second by the Fmoc group. In this case, the Fmoc group (but not the Alloc group) can be removed by treatment with a mild base [e.g., 20% piperidine in dimethylformamide (DMF)], and the first peptide chain synthesized. After which the Alloc group can be removed using a useful reagent [e.g., Pd (PPh₃) / 4-methylmorpholine and chloroform], and synthesize the second peptide chain. It should be noted that when different cysteine ​​thiol protecting groups must be used to control the formation of a disulfide bond (as discussed below), this technique must be used even when the terminal amino acid sequences of the dimer peptide chains are identical.

[0067] When the simultaneous synthesis of dimer peptide chains on the linker must be carried out, the two amino functions of the linker molecule are protected using the same

EP 1629007 B1 removal of an amino protecting group. The protected linker is coupled to the solid support via the linker's third functional group. In this case, the two protected functional groups of the linker molecule are deprotected simultaneously, and on the deprotected amines simultaneously two peptide chains are synthesized. It should be noted that using this technique, the dimer peptide chain sequences will be identical, and all thiol protecting groups for cysteine ​​residues are the same.

[0068] A preferred method for peptide synthesis is solid phase synthesis. Solid phase peptide synthesis procedures are known in the art [see, e.g., Stewart Solid Phase Peptide Synthesis (Freeman and Co .: San Francisco) 1969; 2002/2003 General Catalog of Novabiochem Corp, San Diego, USA; Goodman Synthesis of Peptides and Peptidomimetics (Houben-Weyl, Stuttgart) 2002]. In solid phase synthesis, synthesis typically begins at the C-terminus of the peptide using a resin with protected α-amino groups. A suitable starting material can be prepared, for example, by attaching the required α-amino acid to a chloromethylated resin, hydroxymethyl resin, polystyrene resin, benzhydrylamine resin, or the like. One of these chloromethylated resins is sold under the trade name BIO-BEADS SX-1 by Bio Rad Laboratories (Richmond, CA). The production of hydroxymethyl resin has been described [Bodanszky, et al. (1966) Chem. Indium. London 38: 1597]. Benzhydrylamine (BHA) resin has been described [Pietta and Marshall (1970) Chem. Commun. 650] and its hydrochloride form is commercially available from Beckman Instruments, Inc. (Palo Alto, CA). For example, an α-amino protected amino acid can be coupled to a chloromethylated resin with cesium bicarbonate as a catalyst, according to the method described by Gisin (1973) Helv. Chim. Acta 56: 1467.

[0069] After the initial coupling, the α-amino protecting group is removed, for example, using solutions of trifluoroacetic acid (TFA) or hydrochloric acid (HCl) in organic solvents at room temperature. Then, amino acids with an α-amino protected group are subsequently coupled to the growing peptide chain bound to the substrate. The α-amino protecting groups are groups known to be useful in the field of stepwise peptide synthesis, which include: acyl type protecting groups (e.g., formyl, trifluoroacetyl, acetyl), aromatic urethane protecting groups [e.g., benzyloxycarbyl (Cbz) and substituted Cbz], aliphatic urethane protecting groups [e.g., tert-butyloxycarbonyl ( Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl], and alkyl type protecting groups (e.g., benzyl, triphenylmethyl), fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), and 1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde).

[0070] Side chain protecting groups (typically ether, ester, trityl, PMC, and the like) remain intact during coupling and are not cleaved during deprotection of the amino-terminal protecting group or during coupling. The side chain protecting group must be removable at the end of the synthesis of the final peptide and under reaction conditions that will not change the target peptide. The side chain protecting groups for Tyr include tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl. Side chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl, and cyclohexyl. Side chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and Cbz. The side chain protecting groups for Arg include nitro, tosyl (Tos), Cbz, adamantyloxycarbonyl, mesitylsulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), 4-methoxy-2,3 6-trimethyl-benzenosulfonylo29

EP 1629007 B1 (Mtr) or Boc. Lys side chain protecting groups include Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-Br-Cbz), Tos, or Boc.

[0071] After removal of the α-amino protecting group, the remaining protected amino acids are coupled in stages in the desired order. Each of the protected amino acids is generally reacted in excess of about three times using a suitable carboxyl group activator such as 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium (HBTU) or dicyclohexylcarbodiimide (DCC) hexafluorophosphate (DCC) in solution, for example in methylene chloride (CH₂cl₂), N-methylpyrrolidone, dimethylformamide (DMF), or mixtures thereof.

[0072] After completing the desired amino acid sequence, the desired peptide is detached from the resin substrate by treatment with a reagent such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF), which not only cleaves the peptide from the resin, but also cleaves all other side chain protecting groups . When chloromethylated resin is used, the action of hydrogen fluoride causes the formation of free peptide acids. When a benzhydrylamine resin is used, the hydrogen fluoride treatment directly gives the free peptide amide. Alternatively, when using a chloromethylated resin, the side chain protected peptide can be detached by treating the resin with the peptide ammonia to give the desired side chain protected amide or alkylamine to give the side chain protected alkylamide or dialkylamide. Side chain protection is then removed in the usual manner by treatment with hydrogen fluoride to give free amides, alkylamides, or dialkylamides.

[0073] In preparing the esters of the invention, the resins used to make the peptide acids are used, and the side chain protected peptide is cleaved using a base and the appropriate alcohol (e.g., methanol). The side chain protecting groups are then removed in the usual manner by treatment with hydrogen fluoride to obtain the desired ester.

[0074] These procedures can also be used to synthesize peptides in which amino acids other than the 20 genetically coded natural amino acids are substituted at one, two or more positions for any of the compounds of the invention. Synthetic amino acids that can be substituted into the peptides of the present invention include, but are not limited to, Nmethyl, L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, δ-amino acids such as L---hydroxylyzyl and D-δ-methylalanyl, La -methylalanyl, β-amino acids, and isoquinolyl. D-amino acids and non-naturally occurring synthetic amino acids can also be included in the peptides of the present invention.

Peptide Modifications [0075] To prepare other compounds of the invention, the amino and / or carboxy termini of the peptide compounds of the invention can also be modified. For example, the amino terminus can be acetylated with acetic acid or its halogenated derivative, such as α-chloroacetic acid, abromoacetic acid, or α-iodoacetic acid).

[0076] The side chains of 20 genetically encoded amino acids found in nature (or stereoisomeric D-amino acids) can be replaced with other side chains, for example, groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered groups alkyl, amide, lower alkylamide, di (lower alkyl) amide, lower alkoxy, hydroxyl, carboxyl and their lower ester derivatives, and 4-, 5-, 6-, to 7-membered heterocyclic groups. In particular, 30

Proline analogs in which the size of the proline residue ring varies from 5 members to 4, 6, or 7 members can be used. Cyclic groups can be saturated or unsaturated, and if they are unsaturated, they can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholine group), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidinyl), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl 1-pyrrolidinyl), pyrrolyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. When the group is substituted, the substituent may be an alkyl, alkoxy group, halogen, oxygen atom, or a substituted or unsubstituted phenyl group.

[0077] It is also possible to easily modify peptides by phosphorylation, and by other methods [e.g., as described in Hruby, et al. (1990) Biochem J. 268: 249-262].

[0078] The peptide compounds of the invention also serve as structural models for non-peptide compounds with similar biological activity. Those skilled in the art will recognize that a variety of techniques are available for the construction of compounds with the same or similar desired biological activity as the lead peptide compound, but with more favorable activity than the lead compound for solubility, stability, and susceptibility to hydrolysis and proteolysis [see, Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24: 243-252]. These techniques include replacing the peptide backbone with a backbone consisting of phosphonates, amide enolates, carbamates, sulfonamides, secondary amines, and N-methylamino acids.

Formation of disulfide bonds [0079] The compounds of the present invention contain two intramolecular disulfide bonds. Such disulfide bonds can be formed by oxidizing the cysteine ​​residues of each of the peptide monomers.

[0080] In one embodiment, cysteine ​​bond formation is controlled by choosing the type of oxidizing agent and its concentration effective for optimal formation of the desired isomer. For example, oxidation of a peptide dimer to form two intramolecular disulfide bonds (one on each of the peptide chains) is achieved selectively (relative to the formation of intermolecular disulfide bonds) when the oxidizing agent is DMSO or iodine (I2).

[0081] In other embodiments, cysteine ​​bond formation is controlled by the selective use of thiol protecting groups during peptide synthesis. For example, when a dimer with two intramolecular disulfide bonds is desired, then the first monomeric peptide chain is synthesized with two cysteine ​​residues from the core sequence protected by a first thiol protecting group [e.g., trityl (Trt), allyloxycarbonyl (Alloc), and 1 (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) or similar], then a second monomeric peptide is synthesized with two cysteine ​​residues from the core sequence protected by a second thiol protecting group different from the first thiol protecting group [e.g.,

EP 1629007 B1 acetamidomethyl (Acm), tert-butyl (tBu), or the like]. The first thiol protecting groups are then removed to cyclise the disulfide of the first monomer, and then the second thiol protecting groups are removed to obtain the disulfide cyclization of the second monomer.

[0082] Other embodiments of the present invention give analogues of those disulfide derivatives in which one of the sulfur atoms has been replaced by a CH group₂ or other sulfur isoster. These analogs can be prepared from compounds of the present invention in which each of the peptide monomers contains at least one C or homocysteine ​​residue and α-amino-γ-butyric acid instead of the other C residue, by intramolecular or intermolecular substitution using methods known in the art. [see, e.g., Barker, et al. (1992) J. Med. Chem. 35: 2040-2048 and Or, et al. (1991) J. Org. Chem. 56: 3146-3149]. The skilled person will readily recognize that this replacement may also occur using other α-amino-γ-butyric acid and homocysteine ​​homologues.

[0083] In addition to the above cyclization strategies, other strategies for cyclization of non-sulfide peptides may be employed. Such alternative cyclization strategies include, for example, amide cyclization strategies as well as cyclization strategies involving the formation of thioether bonds. Thus, the compounds of the present invention may exist in cyclized form with an amide intramolecular bond or thioether intramolecular bond. For example, a peptide in which one cysteine ​​from the core sequence is replaced with lysine and the other cysteine ​​is replaced with glutamic acid can be synthesized. Then a cyclic monomer can form through the amide bond between the side chains of these two residues. Alternatively, a peptide in which one cysteine ​​from the core sequence is replaced with lysine (or serine) can be synthesized. Then a thioether bond between the side chains of the lysine (or serine) residue and the second cysteine ​​residue from the core sequence can form a cyclic monomer. As such, both amide cyclization strategies and thioether cyclization strategies can easily be used to cyclize compounds of the present invention in addition to disulfide cyclization strategies. Alternatively, the amino terminus of the peptide may be terminated with an α-substituted acetic acid, wherein the α-substituent is a leaving group such as α-halogenoacetic acid, for example, α-chloroacetic acid, α-bromoacetic acid, or α-iodoacetic acid.

Attachment of the branched tertiary amide linker [0084] Peptide monomers may be dimerized by the branched tertiary amide linker. In one embodiment, the linker is incorporated into the peptide during peptide synthesis. For example, when linker group L_K contains two functional groups that can serve as an initiation site for peptide synthesis and one or more other functional groups (e.g. a carboxyl group or amino group) that allows binding to one or more other molecular moieties, the linker can be coupled to a solid support. After which, two peptide monomers can be synthesized directly on two reactive nitrogen groups of the linker group L_K in a variation of the solid phase synthesis technique.

[0085] In alternative embodiments, the linker may be conjugated to two peptide dimer peptide monomers after peptide synthesis. Such coupling can be achieved by methods known in the art. In one embodiment, the linker contains two functional groups useful for attachment to target functional groups of synthesized peptide monomers. For example, a linker containing two carboxyl groups, either pre-activated or in the presence of a useful one

In conjugation reagent, one can react with target lysine side chain groups of each of the two peptide monomers.

[0086] For example, peptide monomers can be chemically coupled to a tertiary amide linker

A * -C¹O-CH₂-X-CH₂-C²OB * where: X is NCO- (CH₂)₂-NH-Y and Y is a suitable protecting group, such as a tert-butyloxycarbonyl (Boc) protecting group; A * represents a useful functional group, such as the N-succinic acid group, used for coupling C¹ linker with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; and B * is a useful functional group, such as the N-oxy succinic acid group, used for coupling C² linker with the ε-amino group of the C-terminal lysine residue of the second peptide monomer.

[0087] Additionally, for example, peptide monomers can be chemically coupled to a tertiary amide linker,

A * -C¹O-CH₂-X-CH₂-C²OB * where: X is NCO- (CH₂)₂-NH-C³ABOUT-; A * represents a useful functional group, such as the N-succinic acid group, used for coupling C¹ linker with the ε-amino group of the C-terminal lysine residue of the first peptide monomer; and B * is a useful functional group, such as the N-oxy succinic acid group, used for coupling C² linker with the ε-amino group of the C-terminal lysine residue of the second peptide monomer; and the tertiary amide linker is chemically bonded to the spacer moiety,

Y-NH- (CH₂)₄-C⁴H-NH-Y where: C³ moiety X is covalently linked to C⁴ the spacer; and Y is a suitable protecting group, such as a tert-butyloxycarbonyl (Boc) protecting group.

Lysine linker attachment [0088] Peptide monomers may be dimerized by the Lysine linker moiety_K. In one embodiment, the lysine linker is incorporated into the peptide during peptide synthesis. For example, when the Lk lysine linker moiety contains two functional groups that can serve as initiation sites for peptide synthesis and a third functional group (e.g. carboxy or amino group) that allows binding to another molecular moiety, the linker may be coupled to a solid support . Two peptide monomers can then be synthesized directly on the two reactive nitrogen groups of the Lk lysine linker moiety in a variation of the solid phase synthesis technique.

[0089] In alternative embodiments, when the peptide dimer is dimerized by the Lk lysine linker moiety, said linker may be conjugated to two peptide dimer dimer monomers after peptide synthesis. Such coupling can be achieved by methods known in the art. In one embodiment, the linker contains at least two functional groups useful for attachment to target functional groups of peptide monomers have been synthesized. For example, two free lysine amino groups can be reacted with the C-terminal carboxyl groups of each of the two peptide monomers.

EP 1629007 B1

Spacer attachment [0090] The peptide compounds of the invention further comprise a spacer moiety. In one embodiment, the spacer may be included in the peptide during peptide synthesis. For example, when the spacer contains a free amino group and a second functional group (e.g., a carboxy group or amino group) that allows bonding to another molecular moiety, the spacer may be coupled to a solid support.

[0091] In one embodiment, the spacer containing the two functional groups is first coupled to the solid support via the first functional group. Then the lysine linker moiety L_K having two functional groups that can serve as an initiation site for peptide synthesis and a third functional group (e.g. a carboxy or amino group) that allows binding to another molecular moiety, coupled to the spacer via the second spacer functional group and the third linker functional group. Then two peptide monomers can be synthesized directly on the two reactive nitrogen groups of the linker group L_K in a variation of the solid phase synthesis technique. For example, a spacer coupled to a solid support having a free amino group can be reacted with a lysine linker through the free carboxyl group of the linker.

[0092] In alternative embodiments, the spacer can be conjugated to a peptide dimer after peptide synthesis. Such coupling can be achieved by methods known in the art. In one embodiment, the linker contains at least one functional group useful for attachment to the target functional group of the synthesized peptide. For example, a spacer with a free amino group can be reacted with the C-terminal carboxyl group of the peptide. In another example, a linker having a free carboxyl group can be reacted with the free amino group of lysine amide.

Attachment of polyethylene glycol (PEG) [0093] In recent years, water-soluble polymers such as polyethylene glycol (PEG) have been used to covalently modify peptides of therapeutic and diagnostic significance. Attachment of such polymers is believed to increase biological activity, extend blood circulation time, reduce immunogenicity, increase water solubility, and increase resistance to protease digestion. For example, it has been described that the covalent attachment of PEG to therapeutic polypeptides, such as interleukins [Knauf, et al. (1988) J. Biol. Chem. 263; 15064; Tsutsumi, et al. (1995) J. Controlled Release 33: 447), interferons (Kita, et al. (1990) Drug Des. Delivery 6: 157), catalase (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. Biophys. Acta 660: 293), extends their half-life in vivo, and / or reduces their immunogenicity and antigenicity.

[0094] The peptide compounds of the invention may contain a polyethylene glycol (PEG) moiety that is covalently attached to a branched tertiary amide linker or peptide dimer spacer via a carbamate bond or via an amide bond. An example of the PEG used in the present invention is a linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20K) to about 40K (wherein the term "about" indicates that in PEG preparations some molecules will have a larger mass and some less than the specified mass Molecular). Preferably, the PEG has a molecular weight of about 30K to about 40K.

[0095] Another example of the PEG used in the present invention is a linear PEG having a molecular weight of about 10K to about 60K (wherein the term "about" indicates that in PEG preparations some molecules will have a larger mass and some less than stated molecular weight). Preferably, the PEG has a molecular weight of about 20K to about 40K. More preferably, PEG has a molecular weight of about 20K.

[0096] Examples of methods for covalent attachment of PEG (PEGylation) are described below. These illustrative descriptions are not intended to be limiting. One skilled in the art will recognize that various methods for covalent attachment of a wide range of PEG are well established in the art. As such, the present invention encompasses peptide compounds to which PEG has been attached by any of a number of attachment methods known in the art.

[0097] For example, PEG may be covalently attached to the linker via a reactive group to which an activated PEG molecule (e.g., a free amino group or a carboxyl group) may be attached. PEG molecules can be attached to amino groups using methoxylated PEG ("mPEG") having different reactive moieties. Such polymers include mPEG succinimidyl succinate, mPEG succinimidyl carbonate, mPEG imine ether, mPEG 4-nitrophenyl carbonate, and mPEG cyanuric acid chloride. Similarly, PEG molecules can be attached to carboxyl groups using methoxylated PEG with a free amino group (mPEG-NH₂).

[0098] In some embodiments, the linker or spacer contains an terminal amino group (i.e., located at the end of the spacer). This terminal amino group can be reacted with a conveniently activated PEG molecule, such as mPEG para-nitrophenyl carbonate (mPEG-NPC), to form a stable covalent carbamate bond. Alternatively, this terminal amino group can be reacted with a conveniently activated PEG molecule such as mPEG succinimidyl butyrate (mPEGSBA) or mPEG succinimidyl propionate (mPEG-SPA) containing the reactive N-hydroxysuccinimidyl (NHS) group to form a stable covalent bond. In other embodiments, the linker reactive group contains a carboxyl group capable of being activated to form a covalent bond with the amino-containing PEG molecule under useful reaction conditions. Useful PEG molecules include mPEG-NH₂and useful reaction conditions include carbodiimide-mediated amide formation or the like.

EPO-R agonist activity tests:

In vitro functional tests [0099] In vitro competitive binding assays quantitatively measure the ability of a test peptide to compete with EPO for binding to EPO-R. For example (see, e.g., as described in US Patent No. 5,773,569), the extracellular domain of human EPO-R (EPO binding protein, EBP) can be recombinantly produced in E. coli and recombinant protein conjugated to a solid support such as a microtiter plate or synthetic bead [e.g., Sulfolink beads from Pierce Chemical Co. (Rockford, IL)]. Then immobilized EBP is incubated with labeled recombinant EPO, or with labeled recombinant EPO and test peptide. Further dilutions of test peptide are made for such experiments. Test points without added test peptide determine the total EPO binding to EBP. For reactions containing the test peptide, the amount of bound EPO is measured and expressed as a percentage of binding comparison test (total = 100%). These values ​​are plotted versus peptide concentration. The IC50 value specifies 35

EP 1629007 B1 is the concentration of test peptide that reduces EPO binding to EBP by 50% (i.e., 50% inhibition of EPO binding).

[0100] Another in vitro competitive binding assay measures the light signal produced as a function of the proximity of two beads: EPO conjugated beads and EPO-R conjugated beads. The proximity of the beads is created by binding EPO to EPO-R. A test peptide that competes with EPO for binding to EPO-R will prevent this binding, resulting in a decrease in light emission. The concentration of test peptide that causes a 50% decrease in light emission is referred to as the IC50 value.

[0101] The peptides of the present invention compete very efficiently with EPO for binding to EPO-R. This increased activity is expressed by their ability to inhibit EPO binding at substantially lower peptide concentrations (i.e., they have very low IC50 values).

[0102] The biological activity and potency of the monomeric and dimeric EPO-R peptide agonists of the invention that specifically bind to the EPO receptor can be measured using in vitro cellular assays.

[0103] One assay is based on a mouse pre-B cell line expressing human EPO-R and further transfected with a luciferase reporter gene construct driven by the phosp promoter. When exposed to EPO or another EPO-R agonist, such cells respond by synthesizing luciferase. Luciferase emits light when adding its substrate luciferin. Thus, the level of EPO-R activation in such cells can be quantified by measuring luciferase activity. Test peptide activity is measured by adding successive dilutions of the test peptide to the cells, which are then incubated for 4 hours. After incubation, luciferin substrate is added to the cells, and light emission is measured. The concentration of test peptide that produces half the maximum light emission is recorded as EC50.

[0104] The peptides of the present invention exhibit a significantly increased ability to promote luciferase expression in a manner dependent on EPO-R signaling in this assay. This increased activity is expressed by their ability to achieve half the maximum luciferase activity at substantially lower peptide concentrations (i.e., they have very low EC50 values). This assay is a preferred method for estimating the potency and activity of an EPO-R agonist peptide of the invention.

[0105] Another assay can be performed using FDC-P1 / ER cells [Dexter, et al. (1980) J. Exp. Med. 152: 1036-1047], a well-characterized cell line derived from untransformed mouse bone marrow into which EPO-R has been stably transfected. These cells exhibit EPO-dependent proliferation.

[0106] In one such assay, cells are grown to half stationary density in the presence of essential growth factors (see, e.g., as described in US Patent No. 5,773,569). The cells are then washed in PBS and starved for 16-24 hours in complete medium without growth factors. After determining cell viability (e.g., by trypan blue staining), stock solutions are prepared (in complete medium without growth factors) to give about 10⁵ cells for 50 pl. Serial dilutions of test compounds that are EPO-R peptide agonists (typically free peptides in the solution phase as opposed to phage-bound or otherwise bound or immobilized peptide) are prepared in 96-well tissue culture plates to a final volume of 50 µl per well. Cells (50 pL) are added to each well and incubated for 24-48 hours after which

At the time, negative comparative tests should die out or be inactive. Cell proliferation is then measured by techniques known in the art, such as the MTT assay, which measures H incorporation³-thymidine as an indication of cell proliferation [see, Mosmann (1983) J. Immunol. Methods 65: 55-63]. Peptides are assessed on both the EPO-R expressing cell line and the parent EPO-R expressing cell line. The concentration of test peptide required to obtain half the maximum cell proliferation is recorded as EC50.

[0107] The peptides of the present invention exhibit a significantly enhanced ability to promote EPO-dependent cell growth in this assay. This increased activity is expressed by their ability to achieve half the maximum stimulation activity of cell proliferation at substantially lower peptide concentrations (i.e., they have very low EC50 values). This assay is a preferred method for estimating the potency and activity of an EPO-R agonist peptide of the invention.

[0108] In another assay, cells are grown to stationary phase in medium supplemented with EPO, harvested, and then cultured for an additional 18 h in medium without EPO. The cells are divided into three groups with equal cell density: one group without the addition of factor (negative control), the group with EPO (positive control), and the experimental group with test peptide. The cultured cells are then harvested at various times, fixed, and stained with a DNA binding fluorescent dye (e.g., propidium iodide or Hoechst dye, both of which are available from Sigma). Fluorescence is then measured, for example, using a FACS Scan Flow cytometer. The percentage of cells in each phase of the cell cycle can then be determined, for example, using the SOBR model from CellFIT (Becton Dickinson). With respect to the negative control group, cells treated with EPO or active peptide will show a greater proportion of cells in the S phase (determined by increased fluorescence as an indicator of increased DNA content).

[0109] Similar tests can be performed using FDCP-1 cell lines [see, e.g., Dexter et al. (1980) J. Exp. Med. 152: 1036-1047] or TF-1 [Kitamura, et al. (1989) Blood73: 375-380]. FDCP-1 is a growth factor-dependent mouse cell line of initial hematopoietic precursors capable of multidirectional differentiation that can proliferate but not differentiate when supplemented with WEHI-3 conditioned medium (medium that contains IL-3, ATCC No. TIB -68). For such experiments, the FDCP-1 cell line is transfected with human or mouse EPO-R to obtain FDCP-1-hEPO-R or FDCP-1-mEPO-R cell lines, respectively, which can proliferate but do not differentiate in the presence of EPO . The EPO-dependent TF-1 cell line can also be used to measure the effect of EPO-R peptide agonists on cell proliferation.

[0110] In yet another test, the procedure set out in Krystal (1983) Exp. Hematol. 11: 649660 for a microtest based on the inclusion of H³thymidine for spleen cells can be used to determine if the compounds of the present invention can serve as EPO agonists. In short, B6C3F mice₁ phenylhydrazine (60 mg / kg) is injected daily for two days. On the third day, spleen cells are removed and their ability to proliferate over a 24 hour period is assessed using an MTT test.

[0111] Binding of EPO to EPO-R in an erythropoietin-responsive cell line induces tyrosine phosphorylation of both the receptor and numerous intracellular proteins, including Shc, vav and JAK2 kinases. Thus, another in vitro assay measures the ability of peptides of the invention to induce tyrosine phosphorylation in EPO-R and intracellular signal forward proteins. Active peptides, as identified by the binding and proliferation assays described above, trigger a phosphorylation pathway almost identical to pathway 37

EP 1629007 B1

EPO in erythropoietin responsive cells. For this test, FDC-P1 / ER cells [Dexter, et al. (1980) J. Exp. Med. 152: 1036-47] is kept in medium supplemented with EPO and grown to stationary phase. These cells are then cultured in medium without EPO for 24 h. A specified number of such cells are then incubated with the test peptide for approximately 10 min at 37 ° C. A comparative EPO cell sample is also tested with each assay. The treated cells are then harvested by centrifugation, resuspended in SDS lysis buffer, and subjected to SDS polyacrylamide gel electrophoresis. Proteins after gel electrophoresis are transferred to nitrocellulose, and phosphotyrosine-containing proteins on the print are visualized by normal immunological techniques. For example, the print can be probed with anti-phosphotyrosine antibody (e.g., mouse anti-phosphotyrosine IgG from Upstate Biotechnology, Inc.), washed and then probed with a second-order antibody [e.g., anti-mouse IgG goat peroxidase labeled from Kirkegaard &amp; Perry Laboratories, Inc. (Washington, DC)]. Subsequently, phosphotyrosine-containing proteins can be visualized by normal techniques including colorimetric, chemiluminescent, or fluorescent tests. For example, a chemiluminescent test can be performed using the Western Blotting System ECL system from Amersham.

[0112] Another cell-based in vitro assay that can be used to assess the activity of the peptides of the present invention is a colony assay using mouse bone marrow cells or human peripheral blood cells. Mouse bone marrow can be obtained from mouse femurs, and a sample of human peripheral blood can be obtained from a healthy donor. In the case of peripheral blood, mononuclear cells are first secreted from the blood, for example, by centrifugation through a Ficoll-Hypaque gradient [Stem Cell Technologies, Inc. (Vancouver, Canada)]. For this test, nucleated cell counts are performed to determine the number and concentration of nucleated cells in the original sample. A specified number of cells are plated on methylcellulose according to the manufacturer's instructions [Stem Cell Technologies, Inc. (Vancouver, Canada)]. The experimental group is treated with the test peptide, the positive control group is treated with EPO, and the negative control group receives no treatment. Then, after specific incubation periods, generally 10 days and 18 days, the number of colonies for each group is evaluated. The active peptide will promote colony formation.

[0113] Other in vitro bioassays that can be used to demonstrate the activity of the compounds of the present invention are disclosed in Greenberger, et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2931-2935 (EPO dependent hematopoietic precursor cell line); Quelle and Wojchowski (1991) J. Biol. Chem. 266: 609-614 (protein tyrosine phosphorylation in B6SUt.EP cells); Dusanter-Fourt, et al. (1992) J. Biol. Chem. 287: 10670-10678 (EPO receptor tyrosine phosphorylation in human EPO responsive cells); Quelle, et al. (1992) J. Biol. Chem. 267: 17055-17060 (tyrosine phosphorylation of cytosolic protein pp 100 in FDC-ER cells); Worthington, et al. (1987) Exp. Hematol. 15: 85-92 (hemoglobin colorimetric test); Kaiho and Miuno (1985) Anal. Biochem. 149: 117-120 (detection of hemoglobin using 2,7-diaminofluoren); Patel, et al. (1992) J. Biol. Chem. 267: 21300-21302 (c-myb expression); Witthuhn, et al. (1993) Cell 74: 227-236 (association and phosphorylation of tyrosine in JAK2); Leonard, et al. (1993) Blood 82: 1071-1079 (expression of GATA transcription factors); and Ando, ​​et al. (1993) Proc. Natl. Acad. Sci. USA 90: 95719575 (transition adjustment G₁ through circuit D2 and D3).

[0114] It has been reported that an instrument constructed by Molecular Devices Corp., known as a microfiziometer, was successfully used to measure the effect of agonists and antagonists on various receptors. Basis

The operation of this apparatus is to measure changes in the acidification rate of the extracellular environment in response to receptor activation.

In vivo functional tests [0115] One of the in vivo functional tests that can be used to assess the potency of a test peptide is the bioassay in hypoxic after polycythemia mice. For this test, mice are subjected to an alternating conditioning cycle for several days. In this cycle, the mice alternate during periods of vacuum and ambient pressure conditions. The mice are then kept under ambient pressure for 2-3 days prior to administration of the test samples. Test peptide samples, or the EPO standard, in the case of a positive mouse control test, are injected subcutaneously in conditioned mice. Radiolabeled iron (e.g., Fe⁵⁹) is given after 2 days, and blood samples are taken two days after administration of radiolabeled iron. Next, hematocrits and radioactivity measurements are determined by normal techniques for each blood sample. Blood samples from mice injected with active test peptides will show greater radioactivity (due to Fe binding⁵⁹by erythrocyte hemoglobin) than from mice that did not receive test peptides or EPO.

[0116] Another in vivo functional test that can be used to assess the potency of a test peptide is a reticulocyte test. To perform this test, normal untreated mice are injected subcutaneously for three consecutive days with either EPO or test peptide. On the third day, mice are also injected intraperitoneally with iron dextran. On the fifth day, blood samples are taken from the mice. The percentage (%) of reticulocytes in the blood is determined by thiazole orange staining and flow cytometric analysis (retic-count program). In addition, hematocrits are manually determined. The corrected reticulocyte percentage is determined using the following formula:

%^RheticusCorrected. =%^RhaetianMEASURE. ^{x Hematokr}ytINDYWIDUALNY^{/ Hematokr}Y^tNORMAL⁾

Active test compounds will show% RETYKskorYG levels relative to mice that did not receive test peptides or EPO.

Use of EPO-R Agonist Peptides of the Invention [0117] The peptide compounds of the invention are useful in vitro as tools for understanding the biological role of EPO, including the assessment of many factors considered to affect or be associated with EPO production and binding of EPO to EPO-R (e.g., EPO / EPOR signal transduction / receptor activation mechanism). The present peptides are also useful in the development of other compounds that bind to EPO-R, because the present compounds provide important information on structure-activity relationships that enable this development.

[0118] In addition, based on their ability to bind to EPO-R, the peptides of the present invention can be used as reagents for detecting EPO-R on live cells; fixed cells; in biological fluids; in tissue homogenates; in purified, natural biological materials; etc. For example, by labeling such peptides, cells having EPO-R on their surface can be identified. In addition, based on their ability to bind to EPO-R, peptides of the present invention can be used for in situ staining, FACS (fluorescence-activated cell sorting) analysis, Western blotting, and ELISA (enzyme-linked) immunosorbent assay), etc. In addition, based on their ability to bind to EPO-R, the peptides of the present invention can be

EP 1629007 B1 can be used to purify receptors, or to purify cells that express EPO-R on the cell surface (or inside cells after being permeable).

[0119] The peptides of the invention can also be used as commercial reagents for a variety of medical and diagnostic research purposes. Such applications may include, but are not limited to: (1) use as a calibration standard for quantitative measurement of the activity of candidate EPO-R agonists in various functional tests; (2) use as blocking reagents for random peptide screening, i.e., when seeking new families of EPO-R peptide ligands, the peptides can be used to block the recovery of the EPO peptides of the present invention; (3) use for co-crystallization of EPO-R, i.e., crystals of the EPO-R-related peptides of the present invention can be made, enabling receptor / peptide structure determination by X-ray crystallography; (4) use to measure the ability of erythrocyte precursor cells to induce globin synthesis and heme complex synthesis, and to increase the number of ferritin receptors, by initiating differentiation; (5) use for maintaining the proliferation and growth of EPO-dependent cell lines, such as FDCP-1-mEPO-R and TF-1 cell lines; (6) use associated with the meaning of the peptides of the invention using a radioactive chromophore; and (7) other research and diagnostic applications in which EPO-R is preferably activated or such activation is conveniently calibrated against a known amount of EPO-R agonist, and the like.

[0120] In yet another aspect of the present invention, methods for treating and making a medicament are provided. The peptide compounds of the invention can be administered to warm-blooded animals, including humans, to mimic the binding of EPO to EPO-R in vivo. Thus, the present invention includes methods for the therapeutic treatment of disorders associated with EPO deficiency, the methods comprising administering the peptide of the invention in amounts sufficient to stimulate EPO-R, and thus alleviating the symptoms associated with EPO deficiency in vivo. For example, the peptides of the present invention will find use in the treatment of renal failure and / or end-stage renal disease / dialysis; AIDS-related anemia; anemia associated with chronic inflammatory diseases (e.g., rheumatoid arthritis and chronic colitis) and autoimmune disease; and for increasing a patient's red blood cell count before surgery. Other disease states, disorders, and haematological abnormalities that can be treated by administering the peptides of the present invention include: beta-thalassemia; cystic fibrosis; pregnancy and menstrual disorders; early prematurity anemia; spinal cord injury; space flight; acute blood loss; aging; stroke, ischemia (both CNS and heart muscle); and various states of cancer associated with abnormal erythropoiesis.

[0121] In other embodiments, the peptide compounds of the invention can be used to treat disorders that are not characterized by low or deficient red blood cells, such as pre-transfusion preparation. In addition, administration of the compounds of the present invention may result in a reduction in bleeding time and thus find use for administration to patients prior to surgery or for indications of when bleeding is expected to occur. In addition, the compounds of the present invention will find use for activating megakaryocytes.

[0122] Since EPO has been shown to have mitogenic and chemotactic effects on vascular endothelial cells, as well as affecting central cholinergic neurons [see, e.g., Amagnostou, et al. (1990) Proc. Natl. Acad. Sci. USA 87: 5978-5982 and Konishi, et al. (1993) Brain Res. 609: 29-35] are compounds according to

EP 1629007 B1 of the present invention will also find use in the treatment of various vascular disorders such as: promoting wound healing; supporting the growth of collateral coronary vessels (such as those that may appear after myocardial infarction); injury treatment; and treatment after vascular transplants. The compounds of the present invention will also find use in the treatment of various neurological disorders, generally characterized by low absolute levels of acetylcholine or low relative levels of acetylcholine to other neuroactive substances, e.g., neurotransmitters.

Pharmaceutical compositions [0123] In yet another aspect, the present invention relates to pharmaceutical compositions of the above EPO-R agonist peptide compounds. Conditions alleviated or modulated by administration of such compositions include those indicated above. Such pharmaceutical compositions may be intended for oral, parenteral (intramuscular, intraperitoneal, intravenous (iv) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), mucous membranes (nose, vagina, rectum, or sublingually) or using bio-erosive pads, and may be formulated in dosage forms appropriate for each of these routes of administration. In general, the invention encompasses pharmaceutical compositions comprising effective amounts of an EPO-R agonist peptide, or derived products of the invention, together with pharmaceutically acceptable diluents, preservatives, dissolving agents, emulsifiers, adjuvants and / or carriers. Such compositions contain diluents with various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH values ​​and ionic strength; additives such as detergents and dissolving agents (e.g., Tween 20, Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol) and fillers (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Hyaluronic acid can also be used. Such compositions may affect the physical state, stability, release rate in vivo, and in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, ed. 18 (1990, Mack Publishing Co., Easton, PA 18042), pp. 1435-1712.

[0124] The compositions may be made in liquid form, or may be in the form of dried powder (e.g., lyophilized).

Oral delivery [0125] Herein, oral solid dosage forms are contemplated for use that are described generally in Remington's Pharmaceutical Sciences, ed. 18 1990 (Mack Publishing Co. Easton PA 18042) in chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, wafers, pellets, powders, or granules. Liposomal or proteinoid encapsulation (such as, for example, proteinoid microspheres described in US Patent No. 4,925,673) can also be used to formulate the present compositions. Liposomal encapsulation can be used, and liposomes can be derivatized using a variety of polymers (e.g., US Patent No. 5,013,556). A description of possible solid dosage forms for therapeutics is provided by K. Marshall in Modern Pharmaceutics, ed. GS Banker and CT Rhodes, chapter 10, 1979. In general, the formulation will contain EPO-R agonist peptides (or their chemically modified forms) and inert ingredients that will allow protection against the environment in the stomach and release of biologically active material in the intestine.

[0126] Liquid dosage forms for oral administration are also contemplated for use, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other ingredients, including inert diluents; adjuvants such as wetting, emulsifying and suspending agents; and sweeteners, flavors and fragrances.

[0127] The peptides can be chemically modified so that the oral delivery of the derivative is effective. Generally, the chemical modification under consideration is the attachment of at least one moiety to the component molecule itself, wherein said moiety allows (a) inhibition of proteolysis; and (b) uptake into the bloodstream of the stomach or intestine. It is also desirable to increase the overall stability of the ingredient or ingredients and increase the circulation time in the body. As discussed above, PEGylation is the preferred chemical modification for pharmaceutical use. Other moieties that can be used include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-thioxokan [see, e.g. ., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. ed. Hocenberg and Roberts (Wiley-Interscience: New York, NY) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem. 4: 185-189].

[0128] For oral formulations, the release site may be the stomach, small intestine (duodenum, jejunum, or ileum), or large intestine. The specialist has at his disposal formulations that will not dissolve in the stomach, but will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid harmful effects of the stomach environment, either by protecting the peptide (or derivative) or by releasing the peptide (or derivative) outside the stomach environment, as in the intestine.

[0129] To ensure full resistance to gastric juice, a coating impermeable to at least pH 5.0 is important. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl phthalate acetate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate ), Eudragit L, Eudragit S, and Shellac. These coatings can be used as mixed layers.

[0130] The coating or mixture of coatings may also be used on tablets that are not intended to protect against gastric juice. This may include sugar coatings, or coatings that facilitate swallowing of the tablet. Capsules may consist of a hard shell (such as gelatin) for delivery of a dry therapeutic agent (i.e. powder), a soft gelatin shell may be used for liquid forms. The wafer shell material may be thick starch or other edible paper. Wet pills can be used for pills, lozenges, molded tablets or powdered tablets.

[0131] The peptide (or derivative) may be included in the formulation as fine aggregates of particulate particles in the form of granules or pellets with a particle size of about 1 mm. The formulation of the material for administration in a capsule can also be in the form of powder, lightly compressed granules, or even tablets. These therapies can be made by pressing.

[0132] Coloring and / or flavoring agents may also be included. For example, a peptide (or derivative) formulation may be prepared (such as by encapsulation in liposomes or microspheres) and then further included in an edible product, such as a cooled beverage containing coloring and flavoring agents.

[0133] By using an inert material, the volume of the peptide (or derivative) can be diluted or increased. These diluents may include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers, including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available thinners include Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0134] Disintegrants may be included in the solid therapeutic formulation. Materials used as disintegrating agents include, but are not limited to, starch, including Explotab, a starch based commercial disintegrating agent. Any of the following may be used: sodium starch glycolate, Amberlite, sodium carboxymethyl cellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite. Disintegrating agents may also be insoluble cation exchange resins. Powdered gums may be used as disintegrating agents and as binders, and may include powdered gums such as agar, karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0135] Binders may be used to hold the peptide agent (or derivative) together with the hard tablet formulation and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Both polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) can be used in alcoholic solutions to granulate the peptide (or derivative).

[0136] A lubricant may be included in the peptide (or derivative) formulation to prevent sticking during the formulation process. Lubricants may be used as a layer between the peptide (or derivative) and the punch wall, and may include, but are not limited to, stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes . Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000 can also be used.

[0137] Glidants may be added that may improve the flow properties of the drug during formulation and to facilitate movement during compression. Lubricants may include starch, talc, fumed silica and hydrated aluminosilicate.

[0138] To facilitate dissolution of the peptide (or derivative) in an aqueous medium, a surfactant may be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents may be used and may include benzalkonium chloride or benzethonium chloride. The list of potential non-ionic detergents that can be included in the formulation as surfactants includes lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene 10, 50 and 60 hydrogenated castor oil, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, fatty acid esters sucrose, methylcellulose and carboxymethylcellulose. These surfactants can be present in the protein or derivative formulation either alone or as a mixture in various proportions.

[0139] Additives that potentially increase the absorption of a peptide (or derivative) are, for example, fatty acids oleic acid, linoleic acid and linolenic acid.

[0140] Controlled release oral formulations may be desirable. The peptide (or derivative) may be incorporated into an inert matrix, e.g., gum, which allows release either by diffusion or by leaching mechanisms. Slowly decomposing matrices can also be incorporated into the formulation. Some enteric coatings also have delayed release effects. Another form of controlled release is a method based on the Oros (Alza Corp.) therapeutic system, i.e. the drug is enclosed in a semi-permeable membrane that allows water to enter and pushes the drug out through a single small hole due to osmotic effects.

[0141] Other coatings may be used for the formulation. They include various sugars that can be applied in the coating pan. The peptide (or derivative) can also be administered in a film-coated tablet, and the materials used in this case are divided into two groups. The first are non-enteric materials that include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methyl hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, povidone and polyethylene glycols. The second group consists of enteric materials, which are commonly phthalic acid esters.

[0142] A mix of materials may be used to form the optimal coating layer. Layer coating can be carried out in a coating pan or in a fluidized bed or by compression coating.

Parenteral delivery [0143] Formulations of the present invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of solvents or non-aqueous vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They can be sterilized, for example, by filtration through a bacterial-retaining filter, by including sterilizing agents in the compositions, by irradiating the composition, or by heating the composition. They can also be prepared using sterile water, or some other sterile injectable liquid, immediately before use.

Rectal or vaginal delivery [0144] Compositions for rectal or vaginal administration are preferably suppositories, which may contain, in addition to the active ingredient, excipients such as cocoa butter or suppository wax. Compositions for nasal or lingual administration are also prepared using standard excipients known in the art.

Pulmonary delivery [0145] Pulmonary delivery of EPO-R agonist peptides (or derivatives thereof) is also contemplated herein. The peptide (or derivative) is delivered to the lungs of a mammal when inhaled and passes through the lung epithelium into the bloodstream [see, e.g., Adjei, et al. (1990) Pharmaceutical Research 7: 565-569; Adjei, et al. (1990) Int. J. Pharmaceutics 63: 135-144 (leuprolide acetate); Braquet, et al. (1989) J. Cardiovascular Pharmacology 13 (sup5): 143-146 (endothelin-1); Hubbard, et al. (1989) Annals of Internal Medicine, Volume III, pp. 206-212 (α1-antitrypsin); Smith, et al. (1989) J. Clin. Invest., 84: 1145-1146 (α-1-proteinase); Oswein, and

EP 1629007 B1 et al. (1990) "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II Keystone, Colorado (recombinant human growth hormone); Debs, et al. (1988) J. Immunol. 140: 3482-3488 (interferon-γ and tumor necrosis factor α); and U.S. Patent No. 5,284,656 to Platz, et al. (granulocyte colony stimulating factor). A method and composition for pulmonary drug delivery for systemic effects is described in US Patent No. 5,451,569 to Wong, et al.

[0146] For use in the practice of the present invention, a wide range of mechanical devices designed for the delivery of pulmonary therapeutic products are contemplated, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are known to those skilled in the art. Some specific examples of commercially available devices useful for practicing the present invention are the Ultravent atomizer (Mallinckrodt Inc., St. Louis, MO); Acorn II atomizer (Marquest Medical Products, Englewood, CO); Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, NC); and the Spinhaler powder inhaler (Fisons Corp., Bedford, MA).

[0147] All such devices require the use of formulations useful for dispensing a peptide (or derivative). Typically, each formulation is specific to the type of device used, and may include the use of a suitable propellant in addition to conventional diluents, adjuvants and / or carriers useful in therapy. The use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is also contemplated. Chemically modified peptides can also be prepared in various formulations, depending on the type of chemical modification or the type of device used.

[0148] Formulations suitable for use with a spray, either jet or ultrasonic, will typically contain a peptide (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of bioactive protein per ml of solution. The formulation may also contain a buffer and simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The atomizer formulation may also contain a surfactant to reduce or prevent surface peptide (or derivative) from accumulating caused by atomizing the solution to form the aerosol.

[0149] Formulations for use with a metered dose inhaler device will generally include a particulate powder containing the peptide (or derivative) suspended in the propellant with the aid of a surfactant. The propellant can be any conventional material used for this purpose, such as a chlorofluorocarbon compound, chlorofluorocarbon, fluorocarbon, or hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid may also be useful as a surfactant.

[0150] Formulations for dispensing from a powder inhaler will include particulate dry powder containing a peptide (or derivative) and may also include a bulking agent such as lactose, sorbitol, sucrose, or mannitol in amounts that will facilitate powder dispensing from the device, e.g., 50 to 90% by weight of the preparation. The peptide (or derivative) should most preferably be produced in the form of solid particles with an average particle size less than 10 mm (or microns), most preferably 0.5 to 5 mm, for the most effective delivery to the distal lung.

EP 1629007 B1

Nasal Delivery [0151] Nasal delivery of EPO-R agonist peptides (or derivatives) is also contemplated. Nasal delivery allows the peptide to enter the bloodstream immediately after administration of the therapeutic product to the nose, without the product having to be deposited in the lungs. Formulations for nasal delivery include those with dextran or cyclodextran.

Dosages [0152] For all of the peptide compounds, as research continues, information will emerge regarding appropriate dosage levels for the treatment of various conditions in a variety of patients, and a well-trained specialist, considering the therapeutic context, age and general health of the recipient, will be able to determine the correct dosage. The dosage chosen depends on the desired therapeutic effect, on the route of administration, and on the desired duration of treatment. Generally, mammals are dosed with 0.001 to 10 mg / kg body weight per day. Generally, the dosage may be lower for intravenous injection or infusion. The dosage regimen may vary depending on the circulating half-life and formulation used.

[0153] The peptides of the present invention (or derivatives thereof) may be administered in combination with one or more additional pharmaceutical ingredients or compositions.

Examples [0154] The present invention is further described by the following examples. However, these and other examples anywhere in the description are for illustrative purposes only and in no way limit the scope or meaning of the invention or any of its forms described in the examples. Similarly, the invention is not limited to any particular preferred embodiments described herein. Indeed, after reading this description, those skilled in the art can recognize many modifications and variations of the invention that can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms in the appended claims, together with the full range of equivalents to which the claims are entitled.

Example 1: Synthesis of EPO-R agonist peptide dimers by solid phase synthesis [0155] Step 1 - Cbz-TAP synthesis: Solution containing commercially available diamine ("TAP" from Aldrich Chemical Co.) (10 g, 67, 47 mmol) in anhydrous DCM (100 ml) cooled to 0 ° C. A solution of benzyl chloroformate (4.82 mL, 33.7 mmol) in anhydrous DCM (50 mL) was added slowly through an addition funnel over a period of

6-7 h, keeping the reaction mixture at 0 ° C during this time, then allowed to warm to room temperature (~ 25 ° C). After a further 16 h, DCM was removed under reduced pressure and the residue was partitioned between 3 N HCl and ether. The aqueous layers were collected and neutralized with 50% aqueous NaOH to pH 8-9 and extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous Na₂SO₄, then concentrated under reduced pressure to obtain crude mono-CbzTAP (5 g, about 50% yield). This compound was used for the next reaction without any further purification.

EP 1629007 B1

<img file="PL1629007T3_D0037.tif" />

[0156] Step 2 - Synthesis of Cbz-TAP-Boc: To a vigorously stirred suspension of Cbz-TAP (5 g, 17.7 mmol) in hexane (25 mL) was added Boc2O (3.86 g, 17.7 mmol) and stirring continued at room temperature overnight. The reaction mixture was diluted with DCM (25 mL) and washed with 10% aqueous citric acid (2x), water (2x) and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product (5 g yield) was used directly in the next reaction.

<img file="PL1629007T3_D0038.tif" />

[0157] Step 3 - Boc-TAP synthesis: The crude product from the previous reaction was dissolved in methanol (25 mL) and hydrogenated in the presence of 5% Pd on carbon (5% by weight) under balloon pressure for 16 h. The mixture was filtered, washed methanol and the filtrate was concentrated under reduced pressure to give the crude product H-TAP-Boc (yield 3.7 g). The approximate total Boc-TAP yield after Steps 1-3 was 44% (calculated based on the amount of Cbz-Cl used).

<img file="PL1629007T3_D0039.tif" />

[0158] Step 4 - Synthesis of TentaGel-linker: TentaGel bromide (2.5 g, 0.48 mmol / g, from Rapp Polymere, Germany), phenol linker (5 equivalents), and K₂WHAT₃ (5 equivalents) was heated in 20 ml DMF to 70 ° C for 14 h. After cooling to room temperature, the resin was washed (0.1 N HCl, water, ACN, DMF,

MeOH) and dried to give an amber resin.

<img file="PL1629007T3_D0040.tif" />

[0159] Step 5 - Synthesis of TentaGel-linker-TAP (Boc): 2.5 g resin from the above step and H-TAP-Boc (1.5 g, 20 equivalents) and glacial AcOH (34 pi, 5 equivalents) placed in a 1: 1 MeOH-THF mixture and shaken overnight. To this was added a 1 M solution of sodium cyanoborohydride (5 equivalents) in THF and shaken for another 7 h. The resin was filtered off, washed (DMF, THF, 0.1 N HCl, water, MeOH) and dried. A small amount of resin was benzoylated with Bz-Cl and DIEA in DCM and cleaved with 70% TFADCM and checked by LCMS and HPLC.

EP 1629007 B1

<img file="PL1629007T3_D0041.tif" />

[0160] Step 6 - Synthesis of TentaGel-linker-TAP-Lys: The resin from the above step was treated with an activated solution of Fmoc-Lys (Fmoc) -OH (made from 5 equivalents of amino acid and 5 equivalents of HATU dissolved in a concentration of 0.5 M in DMF, then 10 equivalents of DIEA) were added and left under gentle shaking for 14 h. The resin was washed (DMF, THF, DCM, MeOH) and dried to give a protected resin. Residual amino groups were blocked by treating the resin with a solution of 10% acetic anhydride, 20% pyridine in DCM for 20 minutes, followed by washing as above. The Fmoc groups are removed by gently shaking the resin in 30% piperidine in DMF for 20 minutes, followed by washing (DMF, THF, DCM, MeOH) and drying.

<img file="PL1629007T3_D0042.tif" />

[0161] Step 7 - Synthesis of TentaGel-Linker-TAP-Lys / peptides: The resin from the above step was subjected to repeated cycles of Fmoc-amino acid coupling with HBTU / HOBt activation and Fmoc removal using piperidine to construct both peptide chains simultaneously. This was conveniently done using an automated ABI 433 peptide synthesizer available from Applied Biosystems,

Inc. After final Fmoc removal, terminal amino groups were acylated with acetic anhydride (10 equivalents) and DIEA (20 equivalents) in DMF for 20 minutes and then washed as above.

<img file="PL1629007T3_D0043.tif" />

[0162] Step 8 - Cleavage from resin: The resin from the above step was suspended in a solution of TFA (82.5%), phenol (5%), ethanedithiol (2.5%), water (5%), and thioanisole (5% ) for 3 hours at room temperature.

Alternative cleavage cocktails such as TFA (95%), water (2.5%), and triisopropylsilane (2.5%) can also be used. The TFA solution was cooled to 5 ° C and poured into Et₂O to precipitate the peptide. Filtration and drying under reduced pressure gave the desired peptide. Purification by preparative HPLC with a C18 column gave pure peptide.

EP 1629007 B1

<img file="PL1629007T3_D0044.tif" />

[0163] Step 9 - Oxidation of peptides to form intramolecular disulfide bonds: The peptide dimer was dissolved in 20% DMSO / water (1 mg dry weight of peptide / mL) and allowed to stand at room temperature for 36 h. The peptide was purified by introducing the reaction mixture onto a column

HPLC C18 (Waters Delta-Pak C18, particle size 15 microns, pore size 300 angstroms, 40 mm x 200 mm long), and then treated with a linear ACN / water / 0.01% TFA gradient from 5 to 95% ACN for 40 minutes . Lyophilization of fractions containing the desired peptide gave the product as a flocculent white solid.

<img file="PL1629007T3_D0045.tif" />

Dimeric peptide (XYZ) containing Dimeric peptide (XYZ) containing reduced cysteine ​​residue oxidized cysteine ​​residue [0164] Step 10 - PEGylation of the terminal group -NH2: PEGylation via a carbamate bond:

The peptide dimer was mixed with 1.5 equivalents (molar) of the activated PEG type (mPEG-NPC from NOF Corp., Japan) in dry DMF to obtain a clear solution. After 5 minutes, 4 equivalents of DIEA were added to the above solution. The mixture was stirred at ambient temperature for 14 h and then purified by C18 reverse phase HPLC. The structure of PEGylated peptide was confirmed by the MALDI mass method. The purified peptide was also subjected to cation ion exchange chromatography, as outlined below. The following diagram shows PEGylation of mPEGNPC using SEK. ID NO .: 3.

IGPIT (l-na]) viQPLRiMsO) -_: (AcCOGLYAi (AcO) CLY ACHMGPITf l.nal) VOQPUUMeGb '(ArfflGL Y Α ^ ΗΜΰίΤΓΐ I -piOviGPUl (MeG) -1

J mPEC-NPC DIEA, DMF

EP 1629007 B1

PEGylation via an amide bond: The peptide dimer is mixed with 1.5 equivalents (molar) and 1 equivalent of activated PEG type (PEG-SPA-NHS from Shearwater Corp, USA) in dry DMF to obtain a clear solution. After 5 minutes, 10 equivalents of DIEA are added to the above solution. The mixture is stirred at ambient temperature for 2 h and then purified by HPLC

C18 with reversed phases. The structure of PEGylated peptide was confirmed by the MALDI mass method.

The purified peptide was also subjected to cation ion exchange chromatography, as outlined below. The following diagram shows PEGylation of PEG-SPA-NHS using SEK. ID NO .: 3.

<img file="PL1629007T3_D0046.tif" />

[0165] Step 11 - Ion Exchange Purification: For several types of media their ability to separate the above PEG-PEG conjugate from unreacted (or hydrolyzed) PEG was checked, and in addition their ability to retain the original dimeric peptides. The ion exchange resin (2-3 g) was added to a 1 cm column and then converted to the sodium form (0.2 N NaOH was added to the column until the eluent had a pH of 14, approx. 5 column volumes), and then into the hydrogen form (eluted with either 0.1 N HCl or 0.1 M HOAc, until the eluent had a pH corresponding to the input, about 5 column volumes), and then washed with % ACN / water up to pH 6. Either the peptide prior to coupling or the peptide-PEG conjugate was dissolved in 25% ACN / water (10 mg / mL) and the pH was adjusted to &lt;3 using TFA, then loaded onto the column. After washing with 2-3 column volumes of 25% ACN / water and collecting 5 ml fractions, the peptide was released from the column by elution with 0.1 M NH₄OAc in 25% ACN / water, again collecting 5 ml fractions. HPLC analysis revealed which fractions contained the desired peptide. Analysis using the Evaporative Light-Scattering Detector (ELSD) showed that when the peptide was retained on the column and eluted with NH solution₄OAc (generally between fractions 4 and 10), no unconjugated PEG was observed as impurity. When the peptide

5 eluted with the initial washing buffer (generally the first 2 fractions), no separation of the desired PEG conjugate and excess PEG was observed.

[0166] The following columns effectively retained both the peptide and the PEG-PEG conjugate, and effectively purified the Peptide-PEG conjugate from the unconjugated peptide:

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Table 1: Ion exchange resins

<td>fulfillment</td><td>Source</td>

<td>Column ready, powerful Mono S HR 5/5 cation exchanger</td><td>Amersham Biosciences</td>

<td>Filling made of SE53 microfine cellulose, strong cation exchanger</td><td>Whatman</td>

<td>SP Sepharose Fast Flow filling, a strong cation exchanger</td><td>Amersham Biosciences</td>

Example 2: Synthesis of EPO-R agonist peptide dimers by condensation of fragments [0167] Step 1- Synthesis (Cbzjg-Lys: Lysine is reacted under normal conditions with a solution of benzyl chloroformate to obtain lysine protected on its two amino groups by a group

Cbz.

<img file="PL1629007T3_D0047.tif" />

[0168] Step 2 - Boc-TAP synthesis: Boc-TAP is synthesized as described in Steps 1 to 3 of Example 1.

[0169] Step 3 - Coupling (CbzfrLys and Boc-TAP: (Cbz) 2-Lys and Boc-TAP is coupled under normal coupling conditions to give (Cbz) 2-Lys-TAP-Boc.

<img file="PL1629007T3_D0048.tif" />

[0170] Step 4 - Lys-TAP-Boc: The crude product from the previous reaction is dissolved in methanol (25 ml) and hydrogenated in the presence of 5% Pd on carbon (5% by weight) under balloon pressure for 16 h. The mixture is filtered washed with methanol and the filtrate concentrated under reduced pressure to give the crude product Lys-TAP-Boc.

<img file="PL1629007T3_D0049.tif" />

[0171] Step 5 - Synthesis of peptide monomers by condensation of the fragments: Four peptide fragments of the peptide monomer sequences are synthesized by standard techniques. Then these partially protected fragments are subjected to two independent coupling rounds. In the first round, the N-terminal half of the monomer is formed by coupling two peptide fragments and C51

The final half of the monomer is formed by coupling the remaining two peptide fragments. In the second round of coupling, the N-terminal and C-terminal halves are coupled to give a fully protected monomer. The monomer is then deprotected by standard techniques, removing the OBn group.

S (Acm) (AoOJ-OL.IJ-A-OH Rhk-CHMG-OH

DtBu Trt (SEQ ID NO: 5) (SEQ ID NO: 6)

Alone)

OlEu Trt (SEQ ID NO: 7) faml + l

F-OH coupling

H&gt; NLR- (Niigata&gt; O0a

PW (SEKNRIDl: 8) coupling (ΑβΟ) · 04Χ | -Α · € · ^ Μ- &amp; ΟΗ Offin Trt (SEKNRIDl: 9) and '

FmM-Pl-jr- (l-aaI) -VC- (JP-L - ^ '(MiC) -OBii CJtSu Trt PW coupling (SEQ ID NO: Πλ: 10)

S (Acni)

Acm) (AcGl-OL · γ | juC-V * C ^ PLR ^&gt; Wi&gt; Obn

OtBu ^Trt OtBu Trt Phf (SEKNRIDl: 11) deprotection OBn

S (Aea) (Αια) ϋ-Ε.Υ-ΑΐγΜΛ) -Ρ.Ι-γΐ-ηιΙ&gt; ν-ΐί-0.Ρ-1-ϊ1 ^ ΜιΟ) -0ί1 Crfiu τη οφιι irt Ptr

E (Ahb) (SEKNRID1: 12) [0172] Step 6 - Oxidation of peptide monomers to form intramolecular disulfide bonds: Condensed peptide monomers after deprotection of the OBn group (SEQ ID NO: 12) are then oxidized with iodide, forming between cysteine ​​residues Acm-protected intramolecular disulfide bonds.

(Α «σ) - (ϊΊΓγΑΛ ^ Μ · Ο * Ρ · 1 · γ1 · ΕΒ]) · ν - ^ (ϊ-Ρ-ΐΈΐ&gt; (ΜϊΰΚΐΗ OtBll Trt _ΟϊΡ"Ι / t Pbf (SEQ ID NO: 12)

And oxidation Ij (ΑίΟ) · ίΜ&gt; Υ-Α ^ Η · Μ · ΐ1 · Ρ ·] · ^ 1 · £ ϊ]&gt; ν. ^. Γ ^ Κ &lt;ΜΒΰ&gt; ΟΗ OiBii Trt '' [

OtBd Tit (SEQ ID NO: 13) [0173] Step 7 - Coupling of Lys-TAP-Boc with oxidized monomers after deprotection of OBn groups to form a peptide dimer: Lys-TAP-Boc is coupled under normal conditions with twice the molar excess of oxidized monomers after deprotection of OBn groups to obtain a peptide dimer. The peptide dimer is then deprotected under normal conditions.

EP 1629007 B1

<img file="PL1629007T3_D0050.tif" />

[0174] Step 8 - PEGylation of the deprotected dimer: Then the deprotected peptide dimer is PEGylated as described in Step 10 of Example 1.

[0175] Step 9 - Ion exchange purification: Then the PEGylated peptide dimer is purified as described in Step 11 of Example 1.

Example 3: In vitro activity tests [0176] This example describes various in vitro tests that are useful for assessing the activity and potency of EPO-R agonist peptides of the invention. The results for these tests show that the new peptides of the present invention bind to EPO-R and activate EPO-R signal transduction.

In addition, the results for these tests show that the new peptide compositions exhibit an unexpected increase in binding affinity for EPO-R and biological activity compared to EPO mimetic peptides that have been previously described.

[0177] EPO-R agonist peptide dimers are prepared according to the methods given in Example 1 or Example 2. The potency of these peptide dimers is evaluated using a number of in vitro activity assays, including: reporter assay, proliferation assay, competitive binding assay, and assay C / BFU-e. These four tests are described in more detail below.

[0178] The results of these in vitro activity tests are summarized in Table 2.

1. Reporter assay [0179] This assay is based on Baf3 / EpoR / GCSFR fos / lux reporter cells derived from the pre-B mouse cell line. This reporter cell line expresses a chimeric receptor spanning outside53

The cellular portion of the human EPO receptor with the intracellular portion of the human GCSF receptor. This cell line is further transfected with a luciferase reporter gene construct driven by the phosp promoter. Activation of this chimeric receptor by the addition of an erythropoietic factor results in the expression of a luciferase reporter gene, and thus the production of light by the addition of luciferase substrate luciferase. Thus, the level of EPO-R activation in such cells can be quantified by measuring luciferase activity.

[0180] Baf3 / EpoR / GCSFR fos / lux cells are cultured in DMEM / F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3 supernatant (WEHI-3 cell culture supernatant, ATCC) # TIB-68), and penicillin / streptomycin. Approximately 18 h before the test, cells are starved by transferring them to DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day of the test, cells are washed once with DMEM / F12 medium supplemented with 10% FBS (without WEHI-3 supernatant), then 1 x 10⁶ cells / ml are cultured in the presence of known concentration of test peptide, or from EPO (R&amp;D Systems Inc., Minneapolis, MN) as a positive comparison, in DMEM / F12 medium supplemented with 10% FBS (without WEHI-3 supernatant). In this test, subsequent dilutions of the test peptide are tested simultaneously. Test plates are incubated for 4 h at 37 ° C in an atmosphere containing 5% CO₂and luciferin (Steady-Glo; Promega, Madison, WI) is added to each well. After a five-minute incubation, light emission is measured on a Packard Topcount luminometer (Packard Instrument Co., Downers Grove, IL). Light measurements are plotted versus test peptide concentration and analyzed using the Graph Pad program. The concentration of test peptide that produces half the maximum light emission is recorded as EC50 [See Table 2: EC50 reporter].

2. Proliferation assay [0181] This assay is based on the Baf3 line of mouse pre-B cells transfected to express human EPOR. The proliferation of the resulting cell line, BaF3 / Gal4 / Elk / EPOR, is dependent on EPO-R activation. The degree of cell proliferation is quantified using MTT, where the signal in the MTT test is proportional to the number of viable cells.

[0182] BaF3 / Gal4 / Elk / EPOR cells are grown in culture vessels with stirrers in DMEM / F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3 supernatant (ATCC # TIB-68). The cultured cells are starved overnight in a stirring culture vessel at a cell density of 1 x 10⁶ cells / ml, in DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant. The starved cells are then washed twice with Dulbecco's PBS (Gibco), and resuspended to a density of 1x10⁶ cells / ml in DMEM / F12 supplemented with 10% FBS (without WEHI3 supernatant). Aliquots of 50 pL (~ 50,000 cells) are then placed in three uniform embodiments on 96-well assay plates. Aliquots of 50 pL of subsequent dilutions of EPO mimetic peptide test, or 50 pL EPO (R&amp;D Systems Inc., Minneapolis, MN) or Aranesp ™ (darbapoetin alfa, a commercially available EPO-R agonist from Amgen) in DMEM / F12 medium supplemented with 10% FBS (without supernatant I WEHI-3) is added to 96-well assay plates (final well volume 100 pL). For example, 12 different dilutions can be tested, where the final concentration of test peptide (or comparative EPO peptide) ranges from 810 pM to 0.0045 pM. The cells on the plates are then incubated for 48 h at 37 ° C. Then 10 pL MTT (Roche Diagnostics) is added to each well of the culture plate and left to incubate for 4 h. Then stop the reaction by adding 10% SDS + 0.01 N HCl. The plates are then incubated overnight at tempe54

EP 1629007 B1^:C. Then the absorbance of each well is measured spectrophotometrically at 595 nm. Plots of absorbance readings versus test peptide concentration are created and the EC50 is calculated using the Graph Pad program. Test peptide concentration, which gives half the maximum absorbance, is recorded as EC50 [See Table 2: EC50 proliferation].

3. Competitive binding test [0183] Competitive binding calculations are performed using a test in which the light signal produced depends on the proximity of two beads: streptavidin donor beads having a biotinylated EPO-R binding peptide as an indicator, and the acceptor beads to which it is associated EPO-R. Light is created by the radiationless transfer of energy, during which under the influence of lighting from the first pearl singlet oxygen is released, and contact with the released singlet oxygen causes that the second pearl emits light. These bead sets are commercially available (Packard). The proximity of the beads is caused by the binding of an indicator that is an EPO-R binding peptide to EPO-R alone. A test peptide that competes with the indicator for binding to EPO-R will prevent this binding, resulting in a decrease in light emission.

[0184] More specifically, the method is as follows: 4 pL of subsequent dilutions of the EPO-R agonist test peptide, or positive or negative comparative assays are added to the wells of the 384 well plate. Then 2 pL / well of receptor / pearl cocktail are added. The receptor / pearl cocktail consists of: 15 pL streptavidin donor beads (5 mg / ml; Packard), 15 pL monoclonal antibody ab 179 (5 mg / ml; this antibody recognizes a portion of human alkaline phosphatase protein contained in recombinant EPO-R) , acceptor beads coated with protein A (protein A will bind to antibody ab 179; Packard), 112.5 pL of a 1: 6.6 dilution of recombinant EPO-R (produced in Chinese hamster ovary cells as a fusion protein with part of the human alkaline phosphatase protein that contains the ab 179 target epitope) and 607.5 pL of Alphaquest buffer ( 40 mM HEPES, pH 7.4; 1 mM MgCl2; 0.1% BSA, 0.05% Tween 20). To mix, tap carefully. 2 pL / well of biotinylated indicator being EPO-R binding peptide (30 nM final concentration) is added. According to the methods described in Example 1, a peptide indicator is produced that is an EPO-R binding peptide, with the sequence Biotin-GGLYACHMGPITWVCQPLRG (SEQ ID NO: 4).

Peptide indicator

BioiynaGOLYAlS ^ ^ FrrwyJpPLRG

JK-on

Biot_Lon-GGLYACHMGPm^and. 'VCQPLRG / [0185] Mix on a planetary mixer for 1 min. The plate is sealed with Packard Top Seal and wrapped in foil. Incubate overnight at room temperature. After 18 hours, the light emission is read using an AlphaQuest reader (Packard). Light emission is plotted versus peptide concentration and analyzed by Graph Pad or Excel.

[0186] The concentration of test peptide that causes a 50% decrease in light emission, relative to the emission observed without the test peptide, is recorded as IC50 [See Table 2: AQ IC50].

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4. Test C / BFU-e [0187] Signal transduction by EPO-R stimulates the differentiation of bone marrow stem cells into proliferating red blood cell precursors. This test measures the ability of test peptides to stimulate the proliferation and differentiation of red blood cell precursors from primary human bone marrow stem cells with the ability of multidirectional differentiation.

[0188] For this assay, further dilutions of assay peptide are made in IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These subsequent dilutions, or a positive comparison with EPO peptide, are then added to methyl cellulose to give a final volume of 1.5 ml. Then the methylcellulose and peptide mixture is thoroughly mixed by vortexing. Portions (100,000 cells / ml) of CD34 + cells derived from human bone marrow (Poietics / Cambrex) are thawed. Thawed cells are added gently to 0.1 ml DNAse (1 mg / ml; Stem Cells) in a 50 ml tube. Then 40-50 ml IMDM medium is gently added to the cells: the first 10 ml medium is added dropwise along the wall of the 50 mL tube, after which the remaining volume of medium is slowly dispensed down the wall of the tube. The cells are then centrifuged at 900 rpm for 20 min, and the medium is carefully removed by gentle aspiration. The cells are resuspended in 1 ml IMDM medium and the cell density per ml is counted on the hemocytometer slide (10 µl aliquot of cell suspension on the hemocytometer slide, and the cell density expressed in cells / ml is the average count result x 10,000). The cells are then diluted in IMDM medium to a cell density of 15,000 cells / ml. Then for each 1.5 ml methyl cellulose plus peptide samples, 100 pL of diluted cells are added (final cell concentration in the test medium is 1000 cells / ml), and the mixture is vortexed. After the disappearance of bubbles in the mixture, 1 ml is aspirated using a blunt needle. 0.25 ml of aspirated mixture is added to each of 4 wells of a 24-well plate (Falcon brand) from each sample. The plate mixtures are incubated in a humid incubator at 37 ° C in an atmosphere with 5% CO₂ for 14 days. Using a phase microscope (5X-10X lens, final magnification of 100X) the presence of erythroid colonies is scored in points. The concentration of test peptide at which the number of colonies formed is 90% relative to the maximum number observed for the EPO positive control is recorded as EC90 [See Table 2: EC90 C / BFU-e].

5. Competitive binding ligand assay [0189] An alternative competitive ligand binding assay can also be used to measure the IC50 values ​​of peptides in the present invention. This test measures binding¹²⁵I-EPO to EPOr. The test is preferably carried out according to the following exemplary protocol:

A. Materials [0190]

<td>Recombinant human EPO-R / Fc chimera</td><td>• Identification: Recombinant human EPO R / Fc chimera • Supplier: R&amp;D Systems (Minneapolis, MN, USA) • Catalog number: 963-ER • Lot number: EOK033071 • Storage: 4 ° C</td>

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<td>Iodized recombinant human erythropoietin</td><td>• Identification: (3 [¹²⁵I] iodothyrosyl) erythropoietin, human recombinant, high specific activity, 370 kBq, 10 pCi • Supplier: Amersham Biosciences (Piscataway, NJ, USA) • Catalog number: IM219-10 pCi • Lot number: • Storage: 4 ° C</td>

<td>Protein-G Sepharose</td><td>• Identification: Protein-G Sepharose 4 Fast Flow • Supplier: Amersham Biosciences (Piscataway, NJ, USA) • Catalog number 17-0618-01 • Lot number: • Storage: 4 ° C</td>

<td>Test buffer</td><td>• Phosphate buffered saline (PBS), pH 7.4, containing 0.1% bovine serum albumin and 0.1% sodium azide • Storage: 4 ° C</td>

B. Determining the correct receptor concentration.

[0191] One 50 pg vial of lyophilized recombinant extracellular EPOr domain fused to the Fc portion of human IgG1 is reconstituted in 1 mL assay buffer. To determine the correct amount of receptor for use in the assay, 100 pL of subsequent dilutions of this receptor preparation are combined with 200 pL of iodinated recombinant human erythropoietin (¹²⁵I-EPO, approximately 20,000 cpm) in 12 x 75 mm polypropylene tubes. The tubes are capped and mixed gently at 4 ° C overnight on a LabQuake rotary shaker.

[0192] The next day, 50 pL of 50% G-protein Sepharose suspension is added to each tube. The tubes are then incubated for 2 hours at 4 ° C with gentle agitation. The tubes are then centrifuged for 15 min at 4000 rpm (3297 x G) to obtain a G-protein sepharose pellet. Supernatants are carefully removed and discarded. After washing three times with 1 ml assay buffer at 4 ° C, the pellets are counted in a Wallac Wizard gamma counter. The results are then analyzed and the dilution needed to reach 50% of the maximum binding value is calculated.

C. Determination of IC50 for peptide [0193] To determine the IC50 value for peptide I, 100 pL of subsequent dilutions of peptide are combined with 100 pL of recombinant erythropoietin receptor (100 pg / tube) in 12 x 75 mm polypropylene tubes. Then 100 pL iodinated recombinant human erythropoietin is added to each tube (¹²⁵I-EPO) and tubes are capped and mixed gently at 4 ° C overnight.

[0194] The next day, bound ¹²⁵I-EPO is measured quantitatively as described above. The results are analyzed and the IC50 value calculated using Graphpad Prism, version 4.0, from GraphPad Software, Inc. (San Diego, CA). The test is repeated two or more times for each of the peptides tested, resulting in a total of 3 or more IC replicates₅₀.

EP 1629007 B1

Table 2: In vitro activity tests for peptide dimers

<td>Mark Relationship</td><td>Peptide dimer</td><td>EC50 reporter (PM)</td><td>EC50 proliferation (NM)</td><td>IC50 (PM)</td><td>EC90 C / BFU-e (NM)</td>

<td>Peptide II (SEQ ID NO: 3)</td><td>about (AoCXlLyA (ŁHMCiPlTtl-iMl, viQWJl &lt;McO) - Young NH ^ O-PEGsw &gt; &lt;2? (AcGXX.YACHMGPIT (l-IML) VęQPLR (MeG) -HN^X</td><td></td><td></td><td> 110</td><td> 2,2</td>

<td>Peptide III (SEQ ID NO: 3)</td><td>ABOUT (AcG) GLYAilMGPntl-i »1} V &lt;L-QPIJl &lt;M &lt;&lt;;) - NH nh '^^S'EP &amp; (AcCX} LYACHMGPni ([- Bat) vęQPLIl (MoG) -HN NH - \ __f/</td><td> 150</td><td> 72</td><td></td><td> 2,7</td>

Example 4: In vivo activity assays [0195] This example describes various in vivo assays that are useful for assessing the activity and potency of EPO-R agonist peptides of the invention. Peptide dimers that are EPO-R agonists are prepared according to the methods given in Example 1 or Example 2. The in vivo activity of these peptide monomers and dimers is assessed using a number of tests, including bioassay on hypoxic hypoxia mice and reticulocyte test. These two tests are described in more detail below.

1. Bioassay in polycythemia mice after hypoxia [0196] The activity of test peptides in vivo is tested in a bioassay test on polycythemia mice after hypoxia adapted from a method described by Cotes and Bangham (1961), Nature 191: 1065-1067. This test examines the ability of the test peptide to act as an EPO mimetic, i.e., to activate EPO-R and induce the synthesis of new red blood cells. Red blood cell synthesis is quantified by incorporating radiolabeled iron into the hemoglobin of synthesized red blood cells.

[0197] BDF1 mice are accustomed to ambient conditions for 7-10 days. Body weight is determined for all animals and low weight animals (&lt;15 grams) are not used. Mice undergo subsequent conditioning cycles in a hypobaric chamber for a total of 14 days. Each 24-hour cycle consists of 18 hours at 0.40 ± 0.02% atmospheric pressure and 6 hours at ambient pressure. After conditioning, the mice are kept under ambient pressure for an additional 72 h before dosing.

[0198] Test peptides or recombinant human EPO standards are diluted in PBS + 0.1% BSA medium (PBS / BSA). Stock solutions of peptide monomers are first dissolved in dimethyl sulfoxide (DMSO). Negative comparison groups include one group of mice injected with PBS / BSA alone, and one group of mice injected with 1% DMSO. Each dose group contains 10 mice. Mice are injected subcutaneously (in the neck fold) with 0.5 ml of the appropriate sample.

[0199] Forty-eight hours after injection of the sample, mice are given an intraperitoneal injection of 0.2 ml Fe⁵⁹ (Dupont, NEN), to obtain a dose of approximately 0.75 pCi / mouse. 24 hours after the administration of Fe⁵⁹ the body weight of the mice is determined and 48 h after Fe administration⁵⁹ mice are sacrificed. From

In each animal, blood is collected by cardiac puncture and hematocrit (heparin used as anticoagulant) is determined. Each blood sample (0.2 ml) is analyzed for Fe incorporation⁵⁹ using a Packard gamma counter. Non-responding mice (i.e., those mice with less radioactivity than those in the negative control group) are eliminated from the appropriate data set. Mice that have hematocrit values ​​less than 53% of the negative control group are also eliminated.

[0200] The results are from sets of 10 animals for each experimental dose. The average amount of radioactivity [counts per minute (CPM)] included in the blood samples from each group is calculated.

2. Reticulocyte assay [0201] Normal BDF1 mice are dosed (0.5 mL, subcutaneous injection) for three consecutive days with either EPO control or test peptide. On the third day, mice are also dosed (0.1 ml, intraperitoneal injection) with iron dextran (100 mg / ml). On the fifth day, mice are anesthetized with CO₂ and bleeds by heart puncture. The percentage (%) reticulocytes for each blood sample is determined by thiazole orange staining and flow cytometric analysis (retic-count program). Hematocrites are determined by hand. The corrected reticulocyte percentage is determined using the following formula:

% ^RheticusCorrected. =%^RhaetianMEASURE. ^{x (Hematocr}Y^tINDIVIDUAL^{/ Hematokr}s^tNORMAL⁾

3. Hematology assay [0202] Normal CD1 mice are dosed with four weekly high dose intravenous injections of either a positive EPO control test, or test peptide or vehicle. Dose ranges of the positive control and test peptide, expressed as mg / kg, are tested by varying the concentration of active compound in the formulation. The injected volumes are 5 ml / kg. The vehicle comparison group consists of twelve animals, while each of the other dose groups contains 8 animals. Life is recorded daily and body weight is weekly.

[0203] After dosing, the mice are starved and then anesthetized with inhaled isoflurane, and final blood samples are collected by cardiac or abdominal aortic puncture on day 1 (for mice from vehicle control group) and on days 15 and 29 (4 mice / group / day). Blood is transferred to Vacutainer® brand tubes. The preferred anticoagulant is ethylenediaminetetraacetic acid (EDTA).

[0204] The synthesis and physiology of red blood cells is assessed by measuring the end parameters of blood samples such as hematocrit (Hct), hemoglobin (Hgb) and red blood cell count in mm³ (RBC), using known automated clinical analyzers (e.g., from Coulter, Inc.).

Example 5: Synthesis of EPO-R agonist peptide homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1) [0205] Step 1 - Synthesis of peptide monomers: Peptide monomers are synthesized by using normal Fmoc chemistry on the ABI 431A peptide synthesizer using TG-RAM resin (0.18 mmol / g Rapp Polymere, Germany). In the synthesis of peptide monomers with an amidated carboxy terminus, the fully completed peptide is cleaved from the resin using 82.5% TFA, 5% water, 6.25% anisole, 6.25% ethanedithiol. The deprotected product is filtered off with resin and precipitated with diethyl ether. After thorough drying, the product is purified by C18 reverse phase high performance liquid chromatography using an acetonitrile / water gradient in 0.1% trifluoroacetic acid. structure

The peptide is confirmed by electrospray mass spectrometry. The peptide is dissolved in DMSO: water (1: 1) at a concentration of 1 mg / mL to achieve disulphide formation. The product is purified by high performance liquid chromatography on a C18 reverse phase column using an acetonitrile / water gradient in 0.1% trifluoroacetic acid. Peptide monomers can be illustrated as follows:

(AcGJOLYACHMGPIT (l-_thanO) VC &lt;3PLftK, NH₁ (_{SEQ ID NO:} [0206] Step 2 - Synthesis of the trifunctional linker: To a solution of diethyl iminoacetate (10.0 g, 52.8 mmol) and Boc-beta-alanine (10.0 g, 52.8 mmol) in 100 mL DCM was added diisopropylcarbodiimide (8 , 0 mL, 51.1 mmol) for 10 minutes at room temperature. During the addition, the reaction mixture warmed to ~ 10 degrees, then cooled back to room temperature for 20 minutes. The reaction mixture was stirred overnight and the precipitated diisopropyl urea was filtered off. The solvent was removed under reduced pressure to give a gum, and the residue was dissolved in ethyl acetate and filtered again to remove additional precipitated urea. The organic phase was placed in a separatory funnel, washed (sat. NaHCO3, brine, 0.5 N HCl, brine), dried (MgSO4), filtered and concentrated in vacuo to give the diester product as a colorless oil. The diester was dissolved in a 1: 1 MeOH: THF (100 mL) mixture and water (25 mL) was added thereto followed by NaOH (5 g, 125 mmol). The measured pH was&gt; 10. The reaction mixture was stirred at room temperature for 2 h and then acidified to pH 1 with 6 N HCl. The aqueous phase was saturated with NaCl and extracted 4 times with ethyl acetate. The combined organic phases were washed (brine), dried (MgSO₄), and concentrated under reduced pressure to obtain a white semi-solid. The solid was dissolved in 50 mL DCM and 300 mL hexane was added thereto to obtain a white suspension. The solvent was removed under reduced pressure to give the diacid as a white solid (14.7 g, 91.5% yield for 2 steps). To a solution of diacid (1 g, 3.29 mmol) in 20 ml DMF was added N-hydroxy succinimide (770 mg, 6.69 mmol) and diisopropylcarbodiimide (1.00 mL, 6.38 mmol) and 4-dimethylaminopyridine (3 mg, 0.02 mmol). The reaction mixture was stirred overnight and the solvent removed under reduced pressure. The residue was dissolved in ethyl acetate and filtered to remove the precipitated urea. The organic phase was placed in a separatory funnel, washed (sat. NaHCO₃, brine, 0.5 N HCl, brine), dried (MgSO₄), filtered and concentrated under reduced pressure to give the di-NHS ester product as a white solid (1.12 g, 68% yield).

<img file="PL1629007T3_D0051.tif" />

[0207] Step 3 - Coupling of the trifunctional linker with peptide monomers: For coupling with the linker, 2 equivalents of the peptide are mixed with 1 equivalent of the 3-functional linker in dry DMF to form a clear solution, after 5 minutes 5 equivalents of DIEA are added. The mixture is stirred at ambient temperature for 14 h. The solvent is removed under reduced pressure and crude

The product is dissolved in 80% TFA in DCM for 30 min to remove the Boc group, and then purified using reverse phase C18 HPLC. The dimer structure is confirmed by mass spectrometry in electrospray mode. This coupling reaction attaches the linker to the nitrogen atom of the ε-amino group of the lysine residue of each monomer. The method for SEK is shown below. ID NO .: 1.

<img file="PL1629007T3_D0052.tif" />

[0208] Step 4 - PEGylation of the peptide dimer:

PEGylation via carbamate bond: The peptide dimer is mixed with an equal amount (molar) of the activated PEG type (mPEG-NPC from NOF Corp., Japan) in dry DMF to give a clear solution. After 5 minutes, 4 equivalents of DIEA are added to the above solution. The mixture is stirred at ambient temperature for 14 h and then purified using reverse-C18 HPLC

EP 1629007 B1 in phases. The structure of PEGylated peptide is confirmed by the MALDI mass method. The purified peptide was also subjected to cation ion exchange chromatography, as outlined below. The following shows PEGylation of MPEG-NPC using SEK. ID NO .: 1.

<img file="PL1629007T3_D0053.tif" />

PEGylation via an amide bond: The peptide dimer is mixed with an equal amount (molar) of the activated PEG type (PEG-SPA-NHS from Shearwater Corp, USA) in dry DMF to obtain a clear solution. After 5 minutes, 10 equivalents of DIEA are added to the above solution. The mixture is stirred at ambient temperature for 2 h and then purified by reverse phase HPLC C18. The structure of PEGylated peptide was confirmed by the MALDI mass method.

The purified peptide was also subjected to cation ion exchange chromatography, as outlined below. PEGylation of PEG-SPA-NHS using SEK is shown below. ID NO .: 1.

EP 1629007 B1

<img file="PL1629007T3_D0054.tif" />

[0209] Step 5: - Ion Exchange Purification of Peptides: For several types of media their ability to separate the above PEG-PEG conjugate from unreacted (or hydrolyzed) PEGs was checked, and in addition their ability to retain parent dimeric peptides. Ion exchange resin (2-3 g) was added to a 1 cm column and then converted to the sodium form (0.2 N

NaOH was added to the column until the eluent had a pH of 14, about 5 column volumes), and then into the hydrogen form (eluted with either 0.1 N HCl al or 0.1 M HOAc, until the eluent had a pH corresponding to the input, approx. 5 column volumes), and then washed with 25% ACN / water up to pH 6. Either the peptide before coupling or the peptide-PEG conjugate was dissolved in 25% ACN / water (10 mg / mL) and pH adjusted to &lt;3 using TFA, then loaded onto the column. After washing with 2-3 column volumes of 25% ACN / water and collecting 5 ml fractions, the peptide was released from the column by elution with 0.1 M NH₄OAc in 25% ACN / water, again collecting 5 ml fractions. HPLC analysis revealed which fractions contained the desired peptide. Analysis using a laser evaporation scattered light detector (ELSD) showed that when the peptide was retained on the column and eluted with NH solution₄OAc (generally between fractions 4 and 10), no unconjugated PEG was observed as impurity. When the peptide was eluted with the initial washing buffer (generally the first 2 fractions), no separation of the desired PEG conjugate and excess PEG was observed.

[0210] The following columns efficiently retained both the peptide and the PEG-PEG conjugate, and effectively purified the Peptide-PEG conjugate from unconjugated peptide:

Table 3: Ion exchange resins

<td>fulfillment</td><td>Source</td>

<td>Column ready, powerful Mono S HR 5/5 cation exchanger</td><td>Amersham Biosciences</td>

<td>Filling made of SE53 microfine cellulose, strong cation exchanger</td><td>Whatman</td>

<td>SP Sepharose Fast Flow filling, a strong cation exchanger</td><td>Amersham Biosciences</td>

Example 6: Synthesis of EPO-R agonist peptide homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2) [0211] EPO-R agonist peptide peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2) is synthesized as described in Example 1, except that the peptide monomers synthesized in Step 1 are:

(AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2) [0212] When PEG is attached to the linker via a carbamate bond, the final product of this synthesis using SEQ ID NO: 2. ID NO: 2 can be structurally illustrated as follows:

<img file="PL1629007T3_D0055.tif" />

[0213] When PEG is attached to the linker via an amide bond, the final product of this synthesis using SEK. ID NO: 2 can be structurally illustrated as follows:

<img file="PL1629007T3_D0056.tif" />

EP 1629007 B1

Example 7: In vitro activity assays [0214] This example describes various in vitro assays that are useful for assessing the activity and potency of EPO-R agonist peptides of the invention. The results for these tests show that the new peptides of the present invention bind to EPO-R and activate EPO-R signal transduction. In addition, the results for these tests show that the new peptide compositions exhibit an unexpected increase in binding affinity for EPO-R and biological activity compared to EPO mimetic peptides, which were previously described.

[0215] EPO-R agonist peptide monomers and dimers are prepared according to the methods given in Example 1 or Example 2. The potency of these peptide dimers is assessed using a number of in vitro activity assays, including: reporter assay, proliferation assay, competitive binding assay, and C / BFU-e test. These four tests are described in more detail below.

[0216] The results of these in vitro activity tests are summarized in Table 4.

1. Reporter assay [0217] This assay is based on Baf3 / EpoR / GCSFR fos / lux reporter cells derived from mouse pre-B cell lines. This reporter cell line expresses a chimeric receptor comprising the extracellular portion of the human EPO receptor with the intracellular portion of the human GCSF receptor. This cell line is further transfected with a luciferase reporter gene construct driven by the phosp promoter. Activation of this chimeric receptor by the addition of an erythropoietic factor results in the expression of a luciferase reporter gene, and thus the production of light by the addition of luciferase substrate luciferase. Thus, the level of EPO-R activation in such cells can be quantified by measuring luciferase activity.

[0218] Baf3 / EpoR / GCSFR fos / lux cells are cultured in DMEM / F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3 supernatant (WEHI-3 cell culture supernatant, ATCC) # TIB-68), and penicillin / streptomycin. Approximately 18 h before the test, cells are starved by transferring them to DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day of the test, cells are washed once with DMEM / F12 medium supplemented with 10% FBS (without WEHI-3 supernatant), then 1 x 10⁶ cells / ml are cultured in the presence of known concentration of test peptide, or from EPO (R&amp;D Systems Inc., Minneapolis, MN) as a positive comparison, in DMEM / F12 medium supplemented with 10% FBS (without WEHI-3 supernatant). In this test, subsequent dilutions of the test peptide are tested simultaneously. Test plates are incubated for 4 h at 37 ° C in an atmosphere containing 5% CO₂and luciferin (Steady-Glo; Promega, Madison, WI) is added to each well. After a five-minute incubation, light emission is measured on a Packard Topcount luminometer (Packard Instrument Co., Downers Grove, IL). Light measurements are plotted versus test peptide concentration and analyzed using the Graph Pad program. The concentration of test peptide that produces half the maximum light emission is recorded as EC50

2. Proliferation assay [0219] This assay is based on the Baf3 line of mouse pre-B cells transfected to express human EPOR. The proliferation of the resulting cell line, BaF3 / Gal4 / Elk / EPOR, is dependent on EPO-R activation. The degree of cell proliferation is quantified using MTT, where the signal in the MTT test is proportional to the number of viable cells.

EP 1629007 B1 [0220] BaF3 / Gal4 / Elk / EPOR cells are grown in culture vessels with stirrers in DMEM / F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3 supernatant (ATCC # TIB-68) . The cultured cells are starved overnight in a stirring culture vessel at a cell density of 1 x 10⁶ cells / ml, in DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant. The starved cells are then washed twice with Dulbecco's PBS (Gibco), and resuspended to a density of 1x10⁶ cells / ml in DMEM / F12 supplemented with 10% FBS (without WEHI3 supernatant). Aliquots of 50 pL (~ 50,000 cells) are then placed in three uniform embodiments on 96-well assay plates. Aliquots of 50 pL of subsequent dilutions of EPO mimetic peptide test, or 50 pL EPO (R&amp;D Systems Inc., Minneapolis, MN) or Aranesp ™ (darbapoetin alfa, a commercially available EPO-R agonist from Amgen) in DMEM / F12 medium supplemented with 10% FBS (without supernatant I WEHI-3) is added to 96-well assay plates (final well volume 100 pL). For example, 12 different dilutions can be tested, where the final concentration of test peptide (or comparative EPO peptide) ranges from 810 pM to 0.0045 pM. The cells on the plates are then incubated for 48 h at 37 ° C. Then 10 pL MTT (Roche Diagnostics) is added to each well of the culture plate and left to incubate for 4 h. Then stop the reaction by adding 10% SDS + 0.01 N HCl. The plates are then incubated overnight at 37 ° C. The absorbance of each well is then measured spectrophotometrically at 595 nm. Plots of absorbance readings versus test peptide concentration are created and the EC50 is calculated using the Graph Pad program. The concentration of test peptide that gives half the maximum absorbance is recorded as the EC50.

3. Competitive binding test [0221] Competitive binding calculations are performed using a test in which the light signal generated depends on the proximity of two beads: streptavidin donor beads having a biotinylated EPO-R binding peptide as an indicator, and the acceptor beads to which it is associated EPO-R. Light is created by the radiationless transfer of energy, during which under the influence of lighting from the first pearl singlet oxygen is released, and contact with the released singlet oxygen causes that the second pearl emits light. These bead sets are commercially available (Packard). The proximity of the beads is caused by the binding of an indicator that is an EPO-R binding peptide to EPO-R alone. A test peptide that competes with the indicator for binding to EPO-R will prevent this binding, resulting in a decrease in light emission.

[0222] More specifically, the method is as follows: 4 pL of serial dilutions of the EPO-R agonist test peptide, or positive or negative comparative assays are added to the wells of the 384 well plate. Then 2 pL / well of receptor / pearl cocktail are added. The receptor / pearl cocktail consists of: 15 pL streptavidin donor beads (5 mg / ml; Packard), 15 pL monoclonal antibody ab 179 (5 mg / ml; this antibody recognizes a portion of human alkaline phosphatase protein contained in recombinant EPO-R) , acceptor beads coated with protein A (protein A will bind to antibody ab 179; Packard), 112.5 pL of a 1: 6.6 dilution of recombinant EPO-R (produced in Chinese hamster ovary cells as a fusion protein with part of the human alkaline phosphatase protein that contains the ab 179 target epitope) and 607.5 pL of Alphaquest buffer ( 40 mM HEPES, pH 7.4; 1 mM MgCl2; 0.1% BSA, 0.05% Tween 20). To mix, tap carefully. 2 pL / well of biotinylated indicator being EPO-R binding peptide (30 nM final concentration) is added.

EP 1629007 B1

According to the methods described in Example 1, a peptide indicator being an EPO-R binding peptide is prepared using SEQ ID NO: 1. ID NO .: 4.

Peptide indicator

Bio1ywa-00LYAięHMQPrrWVCQFLRG

K-NHi [0223] Mix on a planetary mixer for 1 min. The plate is sealed with Packard Top Seal and wrapped in foil. Incubate overnight at room temperature. After 18 hours, the light emission is read using an AlphaQuest reader (Packard). Light emission is plotted versus peptide concentration and analyzed by Graph Pad or Excel.

[0224] The concentration of test peptide that results in a 50% decrease in light emission, relative to the emission observed without the test peptide, is recorded as IC50.

4. Test C / BFU-e [0225] Signal transduction by EPO-R stimulates the differentiation of bone marrow stem cells into proliferating red blood cell precursors. This test measures the ability of test peptides to stimulate the proliferation and differentiation of red blood cell precursors from primary human bone marrow stem cells with the ability of multidirectional differentiation.

[0226] For this assay, further dilutions of assay peptide are prepared in IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These subsequent dilutions, or a positive comparison with EPO peptide, are then added to methyl cellulose to give a final volume of 1.5 ml. Then the methylcellulose and peptide mixture is thoroughly mixed by vortexing. Portions (100,000 cells / ml) of CD34 + cells derived from human bone marrow (Poietics / Cambrex) are thawed. Thawed cells are added gently to 0.1 ml DNAse (1 mg / ml; Stem Cells) in a 50 ml tube. Then 40-50 ml IMDM medium is gently added to the cells: the first 10 ml medium is added dropwise along the wall of the 50 mL tube, after which the remaining volume of medium is slowly dispensed down the wall of the tube. The cells are then centrifuged at 900 rpm for 20 min, and the medium is carefully removed by gentle aspiration. The cells are resuspended in 1 ml IMDM medium and the cell density per ml is counted on the hemocytometer slide (10 µl aliquot of cell suspension on the hemocytometer slide, and the cell density expressed in cells / ml is the average count result x 10,000). The cells are then diluted in IMDM medium to a cell density of 15,000 cells / ml. Then for each 1.5 ml methyl cellulose plus peptide samples, 100 pL of diluted cells are added (final cell concentration in the test medium is 1000 cells / ml), and the mixture is vortexed. After the disappearance of bubbles in the mixture, 1 ml is aspirated using a blunt needle. 0.25 ml of aspirated mixture is added to each of 4 wells of a 24-well plate (Falcon brand) from each sample. The plate mixtures are incubated in a humid incubator at 37 ° C in an atmosphere with 5% CO₂ for 14 days. Using a phase microscope (5X-10X lens, final magnification of 100X) the presence of erythroid colonies is scored in points. The concentration of test peptide at which the number of colonies formed is 90% relative to the maximum number observed for the positive EPO control assay is recorded as EC90 [See Table 4: EC90 C / BFU-e].

EP 1629007 B1

Table 4: In vitro activity tests for peptide dimers

<td>Mark Relationship</td><td>Peptide dimer</td><td>EC50 reporter (PM)</td><td>EC50 proliferation (PM)</td><td>AQ IC50 (MM)</td><td>EC90 C / BFU-e (NM)</td>

<td>Peptide IV (SEQ ID NO: 1)</td><td>1-1 Fr. (AtGKJLYACHMOWTCl-MDyCęPLRrNH ^ L-NHL v_s ° Ϋ from</td><td></td><td></td><td></td><td> 6,2</td>

<td></td><td>(AciOGLYAęHMGWTOMijyęępLR-ms ^ y-NLA</td><td></td><td></td><td></td><td></td>

Example 8: In vivo activity assays [0227] This example describes various in vivo assays that are useful for assessing the activity and potency of EPO-R agonist peptides of the invention. Peptide monomers and dimers that are EPO-R agonists are prepared according to the methods given in Example 1 or Example 2. The in vivo activity of these peptide monomers and dimers is assessed using a number of tests, including bioassay in hypoxic polycythemia mice and reticulocyte test. These two tests are described in more detail below.

1. Bioassay in polycythemia mice after hypoxia [0228] The activity of test peptides in vivo is tested in bioassay mice with hypoxia polycythemia adapted from the method described by Cotes and Bangham (1961), Nature 191: 1065-1067. This test examines the ability of the test peptide to act as an EPO mimetic, i.e., to activate EPO-R and induce the synthesis of new red blood cells. Red blood cell synthesis is quantified by incorporating radiolabeled iron into the hemoglobin of synthesized red blood cells.

[0229] BDF1 mice are accustomed to ambient conditions for 7-10 days. Body weight is determined for all animals and low weight animals (&lt;15 grams) are not used. Mice undergo subsequent conditioning cycles in a hypobaric chamber for a total of 14 days. Each 24-hour cycle consists of 18 hours at 0.40 ± 0.02% atmospheric pressure and 6 hours at ambient pressure. After conditioning, the mice are kept under ambient pressure for an additional 72 h before dosing.

[0230] Test peptides or recombinant human EPO standards are diluted in PBS + 0.1% BSA (PBS / BSA) medium. Stock solutions of peptide monomers are first dissolved in dimethyl sulfoxide (DMSO). Negative comparison groups include one group of mice injected with PBS / BSA alone, and one group of mice injected with 1% DMSO. Each dose group contains 10 mice. Mice are injected subcutaneously (in the neck fold) with 0.5 ml of the appropriate sample.

[0231] Forty-eight hours after injection of the sample, mice are given an intraperitoneal injection of 0.2 ml Fe⁵⁹ (Dupont, NEN), to obtain a dose of approximately 0.75 pCi / mouse. 24 hours after the administration of Fe⁵⁹ the body weight of the mice is determined and 48 h after Fe administration⁵⁹ mice are sacrificed. From

In each animal, blood is collected by cardiac puncture and hematocrit (heparin used as anticoagulant) is determined. Each blood sample (0.2 ml) is analyzed for Fe incorporation⁵⁹ using a Packard gamma counter. Non-responding mice (i.e., those mice with less radioactivity than those in the negative control group) are eliminated from the appropriate data set. Mice that have hematocrit values ​​less than 53% of the negative control group are also eliminated.

[0232] The results are from sets of 10 animals for each experimental dose. The average amount of radioactivity [counts per minute (CPM)] included in the blood samples from each group is calculated.

2. Reticulocyte assay [0233] Normal BDF1 mice are dosed (0.5 mL, subcutaneous injection) for three consecutive days with either EPO control or test peptide. On the third day, mice are also dosed (0.1 ml, intraperitoneal injection) with iron dextran (100 mg / ml). On the fifth day, mice are anesthetized with CO₂ and bleeds by heart puncture. The percentage (%) reticulocytes for each blood sample is determined by thiazole orange staining and flow cytometric analysis (retic-count program). Hematocrites are determined by hand. The corrected reticulocyte percentage is determined using the following formula:

% ^RheticusCorrected. =%^RhaetianMEASURE. ^{x (Hematocr}Y^tINDIVIDUAL^{/ Hematokr}s^tNORMAL⁾

3. Hematology test [0234] Normal CD1 mice are dosed with four weekly high dose intravenous injections of either a positive EPO control test, or test peptide or vehicle. Dose ranges of the positive control and test peptide, expressed as mg / kg, are tested by varying the concentration of active compound in the formulation. The injected volumes are 5 ml / kg. The vehicle comparison group consists of twelve animals, while each of the other dose groups contains 8 animals. Life is recorded daily and body weight is weekly.

[0235] After dosing, the mice are starved and then anesthetized with inhaled isoflurane, and final blood samples are collected by cardiac or abdominal aortic puncture on day 1 (for mice from vehicle control group) and on days 15 and 29 (4 mice / group / day). Blood is transferred to Vacutainer® brand tubes. The preferred anticoagulant is ethylenediaminetetraacetic acid (EDTA).

[0236] The synthesis and physiology of red blood cells is assessed by measuring the end parameters of blood samples such as hematocrit (Hct), hemoglobin (Hgb) and red blood cell count in mm³ (RBC), using known automated clinical analyzers (e.g., from Coulter, Inc.).

Example 9: Synthesis of EPO-R agonist peptide homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEQ ID NO: 1) [0237] Step 1 - Synthesis of peptide monomers: Peptide monomers are synthesized by using normal Fmoc chemistry on the ABI 431A peptide synthesizer using TG-RAM resin (0.18 mmol / g Rapp Polymere, Germany). In the synthesis of peptide monomers with an amidated carboxy terminus, the fully completed peptide is cleaved from the resin using 82.5% TFA, 5% water, 6.25% anisole, 6.25% ethanedithiol. The deprotected product is filtered off with resin and precipitated with diethyl ether. After thorough drying, the product is purified by C18 reverse phase high performance liquid chromatography using an acetonitrile / water gradient in 0.1% trifluoroacetic acid. structure

The peptide is confirmed by electrospray mass spectrometry. Peptide monomers can be illustrated as follows:

(AoG) GLYACHMGPIT (1-nal) VCQPLRK-NH₂ (SEQ ID NO: 1)

Stage 2 - Synthesis of the three-functional linker:

[0238] To a solution of diethyl iminoacetate (10.0 g, 52.8 mmol) and Boc-beta-alanine (10.0 g, 52.8 mmol) in 100 mL of DCM was added diisopropylcarbodiimide (8.0 mL, 51.1 mmol) for 10 minutes at room temperature. During the addition, the reaction mixture warmed to ~ 10 degrees, then cooled back to room temperature for 20 minutes. The reaction mixture was stirred overnight and the precipitated diisopropyl urea was filtered off. The solvent was removed under reduced pressure to give a gum, and the residue was dissolved in ethyl acetate and filtered again to remove additional precipitated urea. The organic phase was placed in a separatory funnel, washed (sat. NaHCO₃, brine, 0.5 N HCl, brine), dried (MgSO₄), filtered and concentrated under reduced pressure to give the diester product as a colorless oil. The diester was dissolved in a 1: 1 MeOH: THF (100 mL) mixture and water (25 mL) was added thereto followed by NaOH (5 g, 125 mmol). The measured pH was&gt; 10. The reaction mixture was stirred at room temperature for 2 h and then acidified to pH 1 with 6 N HCl. The aqueous phase was saturated with NaCl and extracted 4 times with ethyl acetate. The combined organic phases were washed (brine), dried (MgSO₄), and concentrated under reduced pressure to obtain a white semi-solid. The solid was dissolved in 50 mL DCM and 300 mL hexane was added thereto to obtain a white suspension. The solvent was removed under reduced pressure to give the diacid as a white solid (14.7 g, 91.5% yield for 2 steps). To a solution of diacid (1 g, 3.29 mmol) in 20 ml DMF was added N-hydroxy succinimide (770 mg, 6.69 mmol) and diisopropylcarbodiimide (1.00 mL, 6.38 mmol) and 4-dimethylaminopyridine (3 mg, 0.02 mmol). The reaction mixture was stirred overnight and the solvent removed under reduced pressure. The residue was dissolved in ethyl acetate and filtered to remove the precipitated urea. The organic phase was placed in a separatory funnel, washed (sat. NaHCO₃, brine, 0.5 N HCl, brine), dried (MgSO₄), filtered and concentrated under reduced pressure to give the di-NHS ester product as a white solid (1.12 g, 68% yield).

<img file="PL1629007T3_D0057.tif" />

Stage 3 - Coupling of the three-functional linker with peptide monomers:

[0239] For conjugation to the linker, 2 equivalents of the peptide are mixed with 1 equivalent of the trifunctional linker in dry DMF to obtain a clear solution, and after 2 minutes 5 equivalents of DIEA are added. The mixture is stirred at ambient temperature for 14 h. The solvent is removed under reduced pressure and the crude product is dissolved in 80% TFA in DCM for 30 min to remove the Boc group, followed by purification using reverse phase C18 HPLC. The dimer structure is confirmed by mass spectrometry in electrospray mode. This coupling coupling reaction

EP 1629007 B1 linker to the nitrogen atom of the ε-amino group of the lysine residue of each monomer. Coupling using SEK. ID NO .: 1 is shown below.

<img file="PL1629007T3_D0058.tif" />

Step 4 - Synthesis of PEG moiety containing two linear PEGS chains linked by mPEG2-Lysinol-NPC lysine [0240] Lysinol, which can be obtained commercially, is treated with an excess of mPEG2-NPC to give MPEG2-lysinol, which is then reacted with NPC to give mPEG2 -lizynol-NPC.

mPEG2-Lys-NHS [0241] This product can be obtained commercially, for example, from the Molecular Engineering (2003) catalog from Nektar Therapeutics (490 Discovery Drive, Huntsville, Alabama 35806), cat. number 2Z3XOT01.

Step 5 - PEGylation of the peptide dimer:

PEGylation via a carbamate bond:

[0242] The peptide dimer and type of PEG (mPEG2-Lysinol-NPC) are mixed in a 1: 2 molar ratio in dry DMF to obtain a clear solution. After 5 minutes, 4 equivalents of DIEA are added to the above solution. The mixture is stirred at ambient temperature for 14 h and then purified by reverse phase HPLC C18. The structure of PEGylated peptide is confirmed by the MALDI mass method. The purified peptide was also subjected to cation ion exchange chromatography, as outlined below. PEGylation using mPEG-Lysinol-NPC is shown below using SEQ. ID NO .: 1.

EP 1629007 B1

<img file="PL1629007T3_D0059.tif" />

PEGylation via an amide bond:

[0243] The peptide dimer and type of PEG (mPEG2-Lys-NHS from Shearwater Corp, USA) are mixed in a 1: 2 molar ratio in dry DMF to obtain a clear solution. After 5 minutes, 10 equivalents of DIEA are added to the above solution. The mixture is stirred at ambient temperature for 2 h and then purified by reverse phase HPLC C18. The structure of PEGylated peptide was confirmed by the MALDI mass method. The purified peptide was also subjected to cation ion exchange chromatography, as outlined below. PEGylation using mPEG2-LysNHS is shown below using SEQ. ID NO .: 1.

EP 1629007 B1

<img file="PL1629007T3_D0060.tif" />

[0244] Step 6: - Ion exchange purification of peptides: For several types of media their ability to separate the above PEG-PEG conjugate from unreacted (or hydrolyzed) PEGs was checked, and in addition their ability to retain parent dimeric peptides. Ion exchange resin (2-3 g) was added to a 1 cm column and then converted to the sodium form (0.2 N

NaOH was added to the column until the eluent had a pH of 14, about 5 column volumes), and then into the hydrogen form (eluted with either 0.1 N HCl or 0.1 M HOAc until the eluent had a pH corresponding to the input, approx. 5 column volumes), and then washed with 25% ACN / water up to pH 6. Either the peptide prior to coupling or the peptide-PEG conjugate was dissolved in 25% ACN / water (10 mg / mL) and The pH was adjusted to &lt;3 using TFA, then loaded onto the column. After washing with 2-3 column volumes of 25% ACN / water and collecting 5 ml fractions, the peptide was released from the column by elution with 0.1 M NH₄OAc in 25% ACN / water, again collecting 5 ml fractions. HPLC analysis revealed which fractions contained the desired peptide. Analysis using a laser evaporation scattered light detector (ELSD) showed that when the peptide was retained on the column and eluted with NH solution₄OAc (generally between fractions 4 and 10), no unconjugated PEG was observed as impurity. When the peptide eluted with the initial one

With washing buffer (generally the first 2 fractions), no separation of the desired PEG conjugate and excess PEG was observed.

[0245] The following columns efficiently retained both the peptide and the PEG-PEG conjugate, and effectively purified the Peptide-PEG conjugate from the unconjugated peptide:

Table 5: Ion exchange resins

<td>fulfillment</td><td>Source</td>

<td>Column ready, powerful Mono S HR 5/5 cation exchanger</td><td>Amersham Biosciences</td>

<td>Filling made of SE53 microfine cellulose, strong cation exchanger</td><td>Whatman</td>

<td>SP Sepharose Fast Flow filling, a strong cation exchanger</td><td>Amersham Biosciences</td>

Example 10: Synthesis of EPO-R agonist peptide homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2) [0246] EPO-R agonist peptide monomers peptide having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2) is synthesized as described in Example 1, except that the peptide monomers synthesized in Step 1 are:

(AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEQ ID NO: 2) [0247] When PEG is attached to the spacer via carbamate bonds, the final product of this synthesis using SEQ ID NO: 2. ID NO: 2 can be structurally illustrated as follows:

<img file="PL1629007T3_D0061.tif" />

[0248] When PEG is attached to the spacer via amide bonds, the final product of this synthesis using SEQ ID NO: 1. ID NO: 2 can be structurally illustrated as follows:

EP 1629007 B1

<img file="PL1629007T3_D0062.tif" />

Example 11: In vitro activity assays [0249] This example describes various in vitro assays that are useful for assessing the activity and potency of EPO-R agonist peptides of the invention. The results for these tests show that the new peptides of the present invention bind to EPO-R and activate EPO-R signal transduction. In addition, the results for these tests show that the new peptide compositions exhibit an unexpected increase in binding affinity for EPO-R and biological activity compared to EPO mimetic peptides, which were previously described.

[0250] EPO-R agonist peptide monomers and dimers are prepared according to the methods given in Example 1 or Example 2. The potency of these peptide dimers is assessed using a number of in vitro activity tests, including: reporter test, proliferation test, competitive binding assay, and C / BFU-e test. These four tests are described in more detail below.

[0251] The results of these in vitro activity tests are summarized in Table 6.

1. Reporter assay [0252] This assay is based on Baf3 / EpoR / GCSFR fos / lux reporter cells derived from mouse pre-B cell lines. This reporter cell line expresses a chimeric receptor comprising the extracellular portion of the human EPO receptor with the intracellular portion of the human GCSF receptor. This cell line is further transfected with a luciferase reporter gene construct driven by the phosp promoter. Activation of this chimeric receptor by the addition of an erythropoietic factor results in the expression of a luciferase reporter gene, and thus the production of light by the addition of luciferase substrate luciferase. Thus, the level of EPO-R activation in such cells can be quantified by measuring luciferase activity.

[0253] Baf3 / EpoR / GCSFR fos / lux cells are cultured in DMEM / F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3 supernatant (WEHI-3 cell culture supernatant, ATCC) # TIB-68), and penicillin / streptomycin. Approximately 18 h before the test, cells are starved by transferring them to DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day of the test, cells are washed once with DMEM / F12 medium supplemented with 10% FBS (without WEHI-3 supernatant), then 1 x 10⁶ cells / ml are cultured in the presence of known concentration of test peptide, or from EPO (R&amp;D Systems Inc., Minneapolis, MN) as a positive comparison, in DMEM / F12 medium supplemented with 10% FBS (without WEHI-3 supernatant). In this test, subsequent peptide dilutions are tested simultaneously

EP 1629007 B1 test. Test plates are incubated for 4 h at 37 ° C in an atmosphere containing 5% CO₂and luciferin (Steady-Glo; Promega, Madison, WI) is added to each well. After a five-minute incubation, light emission is measured on a Packard Topcount luminometer (Packard Instrument Co., Downers Grove, IL). Light measurements are plotted versus test peptide concentration and analyzed using the Graph Pad program. The concentration of test peptide that produces half the maximum light emission is recorded as EC50

2. Proliferation assay [0254] This assay is based on the Baf3 line of mouse pre-B cells transfected to express human EPOR. The proliferation of the resulting cell line, BaF3 / Gal4 / Elk / EPOR, is dependent on EPO-R activation. The degree of cell proliferation is quantified using MTT, where the signal in the MTT test is proportional to the number of viable cells.

[0255] BaF3 / Gal4 / Elk / EPOR cells are grown in culture vessels with stirrers in DMEM / F12 medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3 supernatant (ATCC # TIB-68). The cultured cells are starved overnight in a stirring culture vessel at a cell density of 1 x 10⁶ cells / ml, in DMEM / F12 medium supplemented with 10% FBS and 0.1% WEHI-3 supernatant. The starved cells are then washed twice with Dulbecco's PBS (Gibco), and resuspended to a density of 1x10⁶ cells / ml in DMEM / F12 supplemented with 10% FBS (without WEHI3 supernatant). Aliquots of 50 pL (~ 50,000 cells) are then placed in three uniform embodiments on 96-well assay plates. Aliquots of 50 pL of subsequent dilutions of EPO mimetic peptide test, or 50 pL EPO (R&amp;D Systems Inc., Minneapolis, MN) or Aranesp ™ (darbapoetin alfa, a commercially available EPO-R agonist from Amgen) in DMEM / F12 medium supplemented with 10% FBS (without supernatant I WEHI-3) is added to 96-well assay plates (final well volume 100 pL). For example, 12 different dilutions can be tested, where the final concentration of test peptide (or comparative EPO peptide) ranges from 810 pM to 0.0045 pM. The cells on the plates are then incubated for 48 h at 37 ° C. Then 10 pL MTT (Roche Diagnostics) is added to each well of the culture plate and left to incubate for 4 h. Then stop the reaction by adding 10% SDS + 0.01 N HCl. The plates are then incubated overnight at 37 ° C. The absorbance of each well is then measured spectrophotometrically at 595 nm. Plots of absorbance readings versus test peptide concentration are created and the EC50 is calculated using the Graph Pad program. The concentration of test peptide that gives half the maximum absorbance is recorded as the EC50.

3. Competitive binding test [0256] Competitive binding calculations are performed using a test in which the light signal produced depends on the proximity of two beads: streptavidin donor beads having a biotinylated EPO-R binding peptide as an indicator, and the acceptor beads to which it is associated EPO-R. Light is created by the radiationless transfer of energy, during which under the influence of lighting from the first pearl singlet oxygen is released, and contact with the released singlet oxygen causes that the second pearl emits light. These bead sets are commercially available (Packard). The proximity of the beads is caused by the binding of an indicator that is an EPO-R binding peptide to EPO-R alone. A test peptide that competes with the indicator for binding to EPO-R will prevent this binding, resulting in a decrease in light emission.

[0257] More specifically, the method is as follows: 4 pL of subsequent dilutions of the EPO-R agonist test peptide, or positive or negative comparative assays, are added to the wells of the 384 well plate. Then 2 pL / well of receptor / pearl cocktail are added. The receptor / pearl cocktail consists of: 15 pL streptavidin donor beads (5 mg / ml; Packard), 15 pL monoclonal antibody ab 179 (5 mg / ml; this antibody recognizes a portion of human alkaline phosphatase protein contained in recombinant EPO-R) , acceptor beads coated with protein A (protein A will bind to antibody ab 179; Packard), 112.5 pL of a 1: 6.6 dilution of recombinant EPO-R (produced in Chinese hamster ovary cells as a fusion protein with part of the human alkaline phosphatase protein that contains the ab 179 target epitope) and 607.5 pL of Alphaquest buffer ( 40 mM HEPES, pH 7.4; 1 mM MgCl2; 0.1% BSA, 0.05% Tween 20). To mix, tap carefully. 2 pL / well of biotinylated indicator being EPO-R binding peptide (30 nM final concentration) is added. According to the methods described in Example 1, a peptide indicator being an EPO-R binding peptide is prepared using SEQ ID NO: 1. ID NO .: 4.

Peptide indicator

Biogna-GQLYAÓHMGFnWTjQi&gt; LRGx

JK-on

Biotjna-OGLYACHMGPmtycąPŁRG / [0258] Mix on a planetary mixer for 1 min. The plate is sealed with Packard Top Seal and wrapped in foil. Incubate overnight at room temperature. After 18 hours, the light emission is read using an AlphaQuest reader (Packard). Light emission is plotted versus peptide concentration and analyzed by Graph Pad or Excel.

[0259] The concentration of test peptide that causes a 50% decrease in light emission, relative to the emission observed without the test peptide, is recorded as IC50.

4. Test C / BFU-e [0260] Signal transduction by EPO-R stimulates the differentiation of bone marrow stem cells into proliferating red blood cell precursors. This test measures the ability of test peptides to stimulate the proliferation and differentiation of red blood cell precursors from primary human bone marrow stem cells with the ability of multidirectional differentiation.

[0261] For this assay, further dilutions of assay peptide are prepared in IMDM medium (Gibco) supplemented with 10% FBS (Hyclone). These subsequent dilutions, or a positive comparison with EPO peptide, are then added to methyl cellulose to give a final volume of 1.5 ml. Then the methylcellulose and peptide mixture is thoroughly mixed by vortexing. Portions (100,000 cells / ml) of CD34 + cells derived from human bone marrow (Poietics / Cambrex) are thawed. Thawed cells are added gently to 0.1 ml DNAse (1 mg / ml; Stem Cells) in a 50 ml tube. Then 40-50 ml IMDM medium is gently added to the cells: the first 10 ml medium is added dropwise along the wall of the 50 mL tube, after which the remaining volume of medium is slowly dispensed down the wall of the tube. The cells are then centrifuged at 900 rpm for 20 min, and the medium is carefully removed by gentle aspiration. The cells are resuspended in 1 ml IMDM medium and the cell density per ml is counted on the hemocytometer slide (10 µl aliquot of cell suspension on the hemocytometer slide, and the cell density expressed in cells / ml is the average count result x 10,000). 77

EP 1629007 B1

The cells are then diluted in IMDM medium to a cell density of 15,000 cells / ml. Then for each 1.5 ml methyl cellulose plus peptide samples, 100 pL of diluted cells are added (final cell concentration in the test medium is 1000 cells / ml), and the mixture is vortexed. After the disappearance of bubbles in the mixture, 1 ml is aspirated using a blunt needle. 0.25 ml of aspirated mixture is added to each of 4 wells of a 24-well plate (Falcon brand) from each sample. The plate mixtures are incubated in a humid incubator at 37 ° C in an atmosphere with 5% CO₂ for 14 days. Using a phase microscope (5X-10X lens, final magnification of 100X) the presence of erythroid colonies is scored in points. The concentration of test peptide at which the number of colonies formed is 90% relative to the maximum number observed for the EPO positive control is recorded as EC90 [See Table 2: EC90 C / BFU-e].

Table 6: In vitro activity tests for peptide dimers

<td>Mark Relationship</td><td>Peptide dimer</td><td>EC50 reporter (PM)</td><td>EC50 proliferation (PM)</td><td>IC50 radioligand (PM)</td><td>EC90 C / BFU-e (Nm)</td>

<td>Peptide I</td><td>^{1 1} ii</td><td> 195</td><td> 165</td><td> 111</td><td> 3</td>

<td>(SEQ ID NO</td><td>(AflG) GLY ACIttiK ί ΡΠ (l «nl) VCQTIi&gt; - fu | AT</td><td></td><td></td><td></td><td></td>

<td>ID .: 2)</td><td>Λ Ύ OO about</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td>

Example 12: In vivo activity assays [0262] This example describes various in vivo assays that are useful for assessing the activity and potency of EPO-R agonist peptides of the invention. Peptide monomers and dimers that are EPO-R agonists are prepared according to the methods given in Example 1. The in vivo activity of these peptide monomers and dimers is assessed using a number of tests, including bioassay in hypoxic hypoxia mice and reticulocyte test. These two tests are described in more detail below.

1. Bioassay in Hypoxia Post Hypoxia Mice [0263] The activity of test peptides in vivo is tested in a bioassay test on hypoxia mice with hypoxia adapted from a method described by Cotes and Bangham (1961), Nature 191: 1065-1067. This test examines the ability of the test peptide to act as an EPO mimetic, i.e., to activate EPO-R and induce the synthesis of new red blood cells. Red blood cell synthesis is quantified by incorporating radiolabeled iron into the hemoglobin of synthesized red blood cells.

[0264] BDF1 mice are accustomed to ambient conditions for 7-10 days. Body weight is determined for all animals and low weight animals (&lt;15 grams) are not used. Mice undergo subsequent conditioning cycles in a hypobaric chamber for a total of 14 days. Each 24-hour cycle consists of 18 hours

EP 1629007 B1 at 0.40 ± 0.02% atmospheric pressure and 6 h at ambient pressure. After conditioning, the mice are kept under ambient pressure for an additional 72 h before dosing.

[0265] Test peptides or recombinant human EPO standards are diluted in PBS + 0.1% BSA (PBS / BSA) medium. Stock solutions of peptide monomers are first dissolved in dimethyl sulfoxide (DMSO). Negative comparison groups include one group of mice injected with PBS / BSA alone, and one group of mice injected with 1% DMSO. Each dose group contains 10 mice. Mice are injected subcutaneously (in the neck fold) with 0.5 ml of the appropriate sample.

[0266] Forty-eight hours after injection of the sample, mice are given an intraperitoneal injection of 0.2 ml Fe⁵⁹ (Dupont, NEN), to obtain a dose of approximately 0.75 pCi / mouse. 24 hours after the administration of Fe⁵⁹ the body weight of the mice is determined and 48 h after Fe administration⁵⁹ mice are sacrificed. Blood is collected from each animal by cardiac puncture and hematocrit (heparin used as anticoagulant) is determined. Each blood sample (0.2 ml) is analyzed for Fe incorporation⁵⁹ using a Packard gamma counter. Non-responding mice (i.e., those mice with less radioactivity than those in the negative control group) are eliminated from the appropriate data set. Mice that have hematocrit values ​​less than 53% of the negative control group are also eliminated.

[0267] The results are derived from sets of 10 animals for each experimental dose. The average amount of radioactivity [counts per minute (CPM)] included in the blood samples from each group is calculated.

2. Reticulocyte assay [0268] Normal BDF1 mice are dosed (three mL, subcutaneous injection) for three consecutive days with either EPO control or test peptide. On the third day, mice are also dosed (0.1 ml, intraperitoneal injection) with iron dextran (100 mg / ml). On the fifth day, mice are anesthetized with CO₂ and bleeds by heart puncture. The percentage (%) reticulocytes for each blood sample is determined by thiazole orange staining and flow cytometric analysis (retic-count program). Hematocrites are determined by hand. The corrected reticulocyte percentage is determined using the following formula:

% ^RheticusMEASURE. ^{x (Hematocrit}INDIVIDUAL^{/ Hematocrit}NORMAL⁾

3. Hematology test [0269] Normal CD1 mice are dosed with four weekly high dose intravenous injections of either a positive EPO control test, or test peptide or vehicle. Dose ranges of the positive control and test peptide, expressed as mg / kg, are tested by varying the concentration of active compound in the formulation. The injected volumes are 5 ml / kg. The vehicle comparison group consists of twelve animals, while each of the other dose groups contains 8 animals. Life is recorded daily and body weight is weekly.

[0270] After dosing, the mice are starved and then anesthetized with inhaled isoflurane, and final blood samples are collected by cardiac or abdominal aortic puncture on day 1 (for mice from vehicle control group) and on days 15 and 29 (4 mice / group / day). Blood is transferred to Vacutainer® brand tubes. The preferred anticoagulant is ethylenediaminetetraacetic acid (EDTA).

[0271] The synthesis and physiology of red blood cells is assessed by measuring the final parameters of blood samples, such as hematocrit (Hct), hemoglobin (Hgb) and red blood cell count in mm³ (RBC), using known automated clinical analyzers (e.g., from Coulter, Inc.).

[0272] The present invention should not be limited in its scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0273] Hereinafter, it is to be understood that all numerical values ​​are approximate and that they are given to describe the invention.

[0274] Numerous references are cited and discussed in the description of the present invention, including patents, patent applications, and various publications. Citation and / or discussion of such references is provided for the sole purpose of explaining the description of the present invention and does not imply that any such references constitute "prior art" for the present invention.