Novel peptides that bind to the erythropoietin receptor

CROSS REFERENCES FOR RELATED STATEMENTS

Priority requirements are given in Provisional Applications for the United States under serial numbers 60 / 469,993 and 60 / 470,244 filed May 12, 2003. The contents of these provisional applications are incorporated herein by reference and in their entirety.

POWER ENGINEERING

The present invention relates to peptide compounds that are erythropoietin receptor agonists (EPO-R). The invention also relates to therapeutic methods using such peptide compounds for the treatment of diseases which are associated with insufficient or defective production of red blood cells. Pharmaceutical compositions comprising the peptide compounds of the invention are also provided.

STAN.IETEHNIKE

Erythropoietin (EPO) is a glycoprotein hormone with 165 amino acids, with a molecular weight of about 34 kilodaltons (kD) and a priority site for glycosylation at amino acid positions 24, 38, 83 and 126. It is initially produced as a precursor protein with a signal peptide 23 amine . EPO can occur in three forms: α, β and asialo. The α and β forms differ slightly in their carbohydrate components but have the same potency, biological activity, and molecular weight. The asialo form is the ct or β form with the terminal carbohydrate (sialic acid) removed. DNA sequences encoding EPO were recorded [US Pat. Br. 4,703,008 for Lin].

EPO stimulates mitotic division and differentiation of erythrocyte precursor cells and thus ensures erythrocyte production. It is produced in the kidneys when hypoxic conditions prevail. During EPO-induced differentiation of erythrocyte precursor cells, depth synthesis is induced; heme complex synthesis is stimulated; and the number of ferritin receptors increases. These changes allow the cell to use more iron and synthesize functional hemoglobin, which binds oxygen in mature erythrocytes. Thus, erythrocytes and their hemoglobin play a key role in supplying the body with oxygen. These changes begin with the interaction of EPO with the corresponding receptor on the cell surface of erythrocyte precursor cells [See e.g._t Graber and Krantz (1978) Ann. Rev. Med. 29.51-66],

51130 Β

ΕΡΟ is found in very low plasma concentrations when the body is in a healthy state in which the tissue receives enough oxygen from the existing number of erythrocytes. This normally low concentration is sufficient to stimulate the replacement of red blood cells that are normally lost during aging.

The amount of EPO in the circulation increases in conditions of hypoxia when the transport of oxygen with 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 due to excessive exposure to radiation, reduced oxygen intake due to high altitude or prolonged fainting or various forms of anemia. In response to such hypoxic stresses, elevated EPO levels increase red blood cell production by stimulating erythroid progenitor cell proliferation. When the number of red blood cells in the circulation is higher than the normal oxygen needs of the tissue, the EPO level in the circulation decreases.

Because EPO is essential in the process of red blood cell formation, this hormone has potentially useful applications in both the diagnosis and treatment of blood diseases characterized by low or defective red blood cell production. Recent research has provided the basis for projecting the efficacy of EPO therapy for a variety of disease states, disorders, and conditions of hematologic abnormalities, including: beta-thalassemia [see Vedovato, / sar (1984) Acta. Haematol. 71: 211-213]; cystic fibrosis [see Vichinsky, et al. (1984) J. Pediatric 105: 15–21]; menstrual and diseases in pregnancy [see Cotes, et al (193) Brit. J. Ostet. Gyneacol. 90: 304-311]; early anemia of premature infants [see Haga, isar (1983) Acta Pediatr. Scand. 72; 827 831]; spinal cord injuries [see Claus-Walker, et al (1984) Arch. Phys. Med. Rehabilitate. 65: 370-374]; space flights [see Dunn, / 53 / (1984) Eur. J. Appl. Physiol. 52; 178 - 182]; acute blood loss [see Miller, isar (1982) Brit. J. Haematol. 52: 545-590]; aging [see Udupa, isar (1984) J. Lab. Clin. Med. 103: 574-580 and 581-588 and Lipschitz, / sar (1983) Blood 63: 502-509]; various neoplastic disease states 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. eZa / (1987) N. Eng. J. Med. 316: 73 - 78],

Purified, homogeneous EPO was characterized by [U.S. Pat. Br. 4, 677,195 to Hewick], DNA sequences encoding EPO were purified, cloned and expressed to produce recombinant polypeptides with the same biochemical and immunological properties as native EPO. , isar (1987) J. Biol. Cherh. 262: 12059-12076],

The biological effect of EPO appears to be mediated, in part, through interactions with the cell membrane boundary receptor. Initial studies, using immature erythroid cells isolated from mouse spleen, suggest that EPO-binding proteins on the cell surface contain two polypeptides having approximately molecular weights of 85,000 Daltons and 100,000 Daltons, respectively [Sawyer, et al. (1987) Proc. Natl. Acađ. Sci. USA 84: 3690 - 3694], The EPO3 number was calculated

51130 Β binding sites on average from 800 to 1000 per cell surface. Of these binding sites, approximately 300 bind EPO with approximately 90 pM (picomoles), while the remaining bind EPO with a reduced affinity of approximately 570 pM [Sawyer, et al (1987) J, Biol. Chem. 262: 5554 - 5562], an independent study suggests that EPO-sensitive splenic erythroblasts, obtained from mice injected with the anemic species (FVA) Friend leukemia virus, have a total of approximately 400 high and low affinity EPO binding sites with Kj values ​​of approximately 100 pM and 800 pM, respectively [Landschulz, et al (1989) blood 73: 1476-1486],

The following work indicates that two forms of EPO receptors (EPO-R) are encoded by a single gene. This gene was cloned [See larg., Jones, et al. (1990) Blood 76, 31-35; Noguchi, isar (1991) Blood 78: 2548–2556; Maouche, isar. (1991) Blood 78: 2557–2563]. For example, DNA sequences and encoded peptide sequences for murine and human EPO-R proteins are described in PCT Pub. Br. WO 90/08822 by D'Andrea, isar. Current models suggest that binding of EPO to EPO-R leads to dimerization and activation of two EPO-R molecules, leading to subsequent steps in signal transduction [See, e.g., Watowich, isar (1992) Proc. Natl. Acad. Sci. USA 89: 2140–2444].

The availability of cloned EPO-R genes facilitates the search for agonists and antagonists of these important receptors. The availability of recombinant receptor protein allows the investigation of receptor-ligand interactions in a variety of random and semi-random peptide different generation systems. These systems include a peptide system on plasmids [described in U.S. Pat. Br. 6,270,170]; phage peptide system [described in U.S. Pat. Br. 5,432,018 and Cwirla, / sar (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382]; coded synthesized library (ESL) system [described in U.S. Pat. Br. 946,239, filed Sept. 16. 1992]; and a very large scale system for the synthesis of immobilized polymers [described in U.S. Pat. Br. 5,143,854; PCT Pub. Br. 90/15070; Fodor, isar (1991) Science 251: 767–773; Dower and Fodor (1991) Ann. Rep. Med. Chem. 26: 271 -180; and U.S. Pat. Br. 5,424,186].

Peptides that react, at least to some extent, with EPO-R have been identified and described, for example, in U.S. Pat. Br. 5,773,569; 5,830,851; and 5,986,047 by Wrighton, et al. PCT Pub. Br. WO 96/40749 or Wrighton, U.S. Pat. Br. 5,767,78 and PCT Pub. Br. 96/40772 by Johnson and Zivin; PCT Pub. Br. WO 01/38342 by Balu; and WO 01/91780 by SmithSwintosky 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. Also, the peptides identified to date that contain the motif stimulate EPO-dependent cell proliferation in vitro with EC50 values ​​of about 20 nanomoles (nM) to about 250 nM. Thus, a peptide concentration of 20 nM to 250 nM is required to stimulate 50% of the maximum cell proliferation stimulated with EPO.

51130Β

Given the enormous potential of EPO-R agonists, both for investigations of important biological activities mediated by this receptor and for the treatment of disease, there is still a need to identify peptide EPO-R agonists with increased potency and activity. The present invention provides such compounds.

The citations and / or discussions of the references cited in this section and to the end of the specification are provided solely to clarify the description of the present invention and are not intended to claim that any of these references is prior experience.¹¹ for this invention.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides novel peptide compounds, which are EPO-R agonists for dramatically increased potency 1 activity. These peptide compounds are homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEKID NO: 1) or homodimers of peptide monomers having the amino acid sequence (AcG) GLYPLMKPK BR: 2), homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) (SEKID BR: 3); wherein each amino acid is designated by a standard one-letter abbreviation, (AcG) is Nacetylglycine, (1-nal) is 1-naphthylalanine, and (MeG) is N-methylglycine, also known as sarcosine. Each peptide monomer of the peptide dimer contains an intramolecular disulfide bond between the cysteine ​​monomer residues.

Peptide monomers can dimerize by covalent binding to a branched tertiary amide linker. A tertiary amide linker can be described as:

- ^ O-SNuH-SN ^ Where: X is NCO-CC ^ rN'H-; C¹ the linker forms an amide bond with an ε-amino group from the C-terminal lysine residue of the first peptide monomer; C² the linker forms an amide bond with an ε-amino grab from the C-terminal lysine residue of the second peptide monomer; and N¹ from X is linked via a carbamate bond or an amide bond to the 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 weigh more, some less, of the stated molecular weight).

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

51130Β

<img file="RS51130B_D0001.tif" />

When each homodimer monomer has the amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEKID NO: 1) and N¹ the linker is linked via an amide bond to the activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="RS51130B_D0002.tif" />

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

51130 Β

<img file="RS51130B_D0003.tif" />

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (l'nal) VCQPLR (MeG) K (SEKID NO: 2) and N¹ the linker is linked via an amide bond to the activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="RS51130B_D0004.tif" />

The peptide monomers can also be dimerized by covalent attachment to a tertiary branched amide linker. The tertiary amide linker can be described as:

-S ^ -SNg-H-SNg- ^ Ogde; X is NCO- (CH2) 2-NH-C³O-; C¹ the linker forms an amide bond with an ε-amino group from the Cterminal lysine residue of the first peptide monomer; go² the linker forms an amide bond with the ε-amino group from the C-terminal lysine residue of the second peptide monomer. The peptide dimers of the invention further comprise a spacer with a structure:

51130 Β

-ΝΉ-ίΟΗ ^ Η-ΝΉgde: C⁴ the spacer is covalently bound to C³ ίζΧ; N¹ the spacer is covalently linked via a carbamate or amide bond to the activated polyethylene glycol (PEG) moiety; and N² the spacer is covalently linked 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 (the term about indicates that in PEG preparations, some molecules will weigh more, some less than the stated molecular weight ). Each PEG portion may be, individually, 10,000 Daltons (10 kD), 20 kD, 30 kD, 40 kD, or 50 kD.

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEKID NO: 1) and both N¹ and № spacers are covalently linked via a carbamate bond to the activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="RS51130B_D0005.tif" />

In preferred embodiments, the C-tenninal lysine from the two peptide monomers is Llysine. Also, one skilled in the art will understand from the above chemical structures that the two lines of the PEG moiety are fused to lysine (e.g., as mPEGrLiz-NHS or as mPEG ^ Lysinol-NPC), which is also preferred that L-lysine and which gives the following stereochemistry.

51130Β (AcOGLVACHMGPITa-naDvĆoPL-NH ^ -NH ;,

<img file="RS51130B_D0006.tif" />

Alternatively, one or more lysine residues may be D-lysine, providing an alternative stereochemistry that will be more readily recognizable to one skilled in the art.

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VC0PLRK (SEKID NO: 1) and both N¹ and N² spacers are covalently linked via an amide bond to the activated PEG moiety, novel peptide compounds of the invention can be represented as follows:

<img file="RS51130B_D0007.tif" />

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

51130 Β

I ------------------------------ 1 U (AcG) GLYACHMGPITa-Nal) VCQPLR- NH NH₂ (AcG) GLYACHMGPIT (l-Nal) VCQ

<img file="RS51130B_D0008.tif" />

<img file="RS51130B_D0009.tif" />

<img file="RS51130B_D0010.tif" />

Alternatively, one or more lysine residues may be D-lysine, which provides an alternative stereochemistry that will be readily recognizable to one skilled in the art.

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEK1Đ BR: 2) and both N¹ and № spacers are covalently linked via a carbamate bond to the activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="RS51130B_D0011.tif" />

(AcG) GLYACHMGPrr (knal) V (j: QPLR (MeG) -

<img file="RS51130B_D0012.tif" />

nh₂

Preferably, the lysine residues associated with the peptide monomer and linear PEG moieties in this molecule are all E-lysine, giving the following stereochemistry:

51130Β (AcGJGLVAĆHMGPITa-NaDvioPLRCMeGl-NH ^ zA-NHj

<img file="RS51130B_D0013.tif" />

(AcG) GLYACHMGPIT (l-Nal) VCQPLR (MeG) -NH

<img file="RS51130B_D0014.tif" />

Alternatively, one or more lysine residues may be D-lysine, giving an alternative stereochemistry that will be readily recognizable to those of ordinary skill in the art.

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEKID NO: 2) and both N¹ and № spacers are covalently linked via an amide bond to the activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="RS51130B_D0015.tif" />

V

PEG₁₀._50K

Preferably, the lysine residues attached to the peptide monomer and linear PEG moieties of this molecule are all b-lysine, giving the following stereochemistry.

51130 Ι ι ---------------------------- 1 Ac (AcG) GLYACHMGPrra-Nal) VCQPLR (MeG) -NHY-NH₂

<img file="RS51130B_D0016.tif" />

<img file="RS51130B_D0017.tif" />

10-50Κ

PFG ^{rC, V}W50K

In other embodiments, one or more lysine residues may be D-lysine, giving an alternative stereochemistry that will be readily recognizable to a person of ordinary skill in the art.

The peptide monomers can also be dimerized by binding to a lysine linker, wherein one peptide monomer is attached by its C-terminus to the lysine ε-amino group and the other peptide monomer is attached by its C-terminus to the lysine α-amino group.

The peptide dimers of the invention further comprise a spacer portion (spacer) with the following structure:

^^ - (ODg-O-SSN ^^ O- ^ NgJg- ^ NNat one end, N¹ the spacer is connected via an amide bond to the carbonyl carbon of the lysine linker. At the opposite end, N² the spacer is linked via a carbamate bond or an amide bond to the 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 weigh more, some less of the stated molecular weight).

When the spacer is connected via a carbamate bond to the activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention may be presented as follows:

Oh

NH θ^—'^ G_{2 (M}ok (AcG) GLYA0HMGPrrQ-nal) VCQPLR (MeG) -NH

<img file="RS51130B_D0018.tif" />

(AcG) GLYACHMGPlT (l-nal) V ^ QPLR (MeG) - NH _NH_^

<img file="RS51130B_D0019.tif" />

When the spacer is linked via an amide bond to the activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention may be represented as follows:

51130Β

II (AcG) GLYACHMGPITO-nal) VCQPLR (MeG) -ΝΗ

<img file="RS51130B_D0020.tif" />

(AcG) GLYACHMGPrT (l-naI) VCQPLR (MeG) -ΝΗ

I____________________________________________________________

The invention further provides pharmaceutical compositions comprising such peptide compounds and methods for treating various medical conditions in which such peptide compounds are used,

DETAILED DESCRIPTION OF THE INVENTION

Definitions ·.

Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Fen or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Tr 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 Liz or K; Aspartic acid is Asp or D; Glutamic acid is Glu or E; Cysteine ​​is Cis or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gli or G. Unconventional amino acids in the peptides are abbreviated 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.

As used herein, the term polypeptide or protein refers to a polymer of amino acid monomers that are alpha amino acids joined together via amide bonds. The polypeptides are therefore at least two amino acid residues in length and are usually longer. In general, the term peptide refers to a polypeptide having a length of only a few amino acid residues. The novel EPO-R agonist peptides of the present invention are preferably of a length of no more than about 50 amino acid residues. More preferably, the lengths are from about 17 to about 40 amino acid residues. A polypeptide, unlike a peptide, can contain any number of amino acid residues. Thus, the term polypeptide includes peptides as well as longer amino acid sequences.

As used herein, the phrase pharmaceutically acceptable refers to molecular entities and compositions that are generally considered safe, e.g., that are physiologically tolerable and do not normally cause allergic or similar adverse reactions, such as gastric disturbance, dizziness, and the like, when are applied to humans. Preferably, as used herein, the term pharmaceutically acceptable means entities that are approved by the regulatory agencies of the Federal or state administrations or that are listed in the US Pharmacopoeia or in other generally accepted

51130 Β pharmacopoeias for use in animals and especially in humans. The term carrier refers to the diluent, adjuvant, excipient or carrier to which the compound is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanuts. oil, soybean oil, mineral oil, sesame oil and the like. Lead or aqueous solutions, saline and aqueous solutions of dextrose and glycerol solutions are preferably used as carriers, especially for injectable solutions. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin.

As used herein, the term agonist refers to a biologically active ligand that binds to its complementary biologically active receptor and activates it either to elicit a biological response at the receptor or to enhance existing biological receptor activity.

New peptides that are EPO-R agonists

The present invention provides novel peptide compounds, which are EPO-R agonists for dramatically enhancing potency and activity. These peptide compounds are homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEKID BR: 1) or homodimers of peptide monomers having the amino acid sequence (AcG) SEK (1G) MeKYKL1KYQRP BR: 2); where each amino acid is labeled with a standard one-letter abbreviation, (AcG) is N-acetylglycine, (1-nal) is 1-naphthylalanine. and (MeG) is Nmethylglycine, also known as sarcosine. Each peptide monomer of the peptide dimer contains an intramolecular disulfide bond between the cysteine ​​monomer residues. Such monomers can be represented schematically as follows:

(AcG) GLYA (!: HMGPITG-nal) VCQPLRK (AcG) GLYA (^ HMGPITQ-naI) V ^ QPLRK (AcG) GLYA (!; HMGPITa-nal) VCQPLR (MeG) K _iH &lt;^AcG)^{GLYA (}₁^{: HMGPIT}(¹-^cash)^{V (}|^:Q^pLR&lt;^MeG)^K

These monomeric peptides were dimerized to give peptide dimers with enhanced EPO-R agonist activity. The linker (Lk) portion is a tertiary branched amide, which connects the C-terminals of the two peptide monomers, by simultaneously binding to the C-terminal lysine residue of each monomer. The tertiary amide linker can be described as:

-S ^ -SNuH-SN ^ Where: X is C¹ the linker forms an amide bond with an ε-amino group from the C-terminal lysine residue of the first peptide monomer; C² the linker forms an amide bond with an ε-amino group from the C-terminal lysine residue of the second peptide monomer; and N^l from X is linked via a carbamate bond or an amide bond to the activated polyethylene glycol (PEG) moiety, where PEG has

51130 Β a molecular weight of about 20,000 to about 40,000 Daltons (the term about indicates that in PEG preparations, some molecules will weigh more, some less than the stated molecular weight). The tertiary amide linker can also be represented as:

- ^ O-SNg-H-SN ^ Where: X is NCO- (CH2) 2-NH-C³O-; C¹ the linker forms an amide bond with an ε-amino group from the Cterminal lysine residue of the first peptide monomer; go² the linker forms an amide bond with the εamino group from the C-terminal lysine residue of the second peptide monomer. The peptide dimers of the invention further comprise a spacer (spacer) having the following structure:

-NWH2)₄-C⁴HN²Where: C⁴ the spacer is covalently bound to C³ from X; N¹ the spacer is covalently linked via a carbamate or amide bond to the activated PEG moiety; and N² the spacer is covalently linked 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 (the term about indicates that in PEG preparations, some molecules will weigh more, some less than the stated molecular weight ).

Thus, the novel peptides of the invention may also contain a PEG moiety, which is covalently linked via a carbamate bond or an amide bond to the tertiary amide linker from the peptide dimer. PEG is a water-soluble polymer that is pharmaceutically acceptable. The PEG for use in the present invention may be a linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20 K) to about 60 K (the term about indicates that in PEG preparations, some molecules will weigh more, some less than said molecular weight). Most preferably, the PEG has a molecular weight of about 30 K to about 40 K. One skilled in the art will be able to select the desired polymer size based on such considerations as the desired dose; circulation time; resistance to proteolysis; effects, if any, on biological activity; ease of use; degree or absence of antigenicity; and other known effects of PEG on therapeutic peptides.

The peptides, peptide dimers and other peptide molecules of the invention can be bound to water-soluble polymers (e.g., PEG) using any of a variety of chemical reactions to bind water-soluble polymers to the receptor-binding portion of the molecules (e.g., a peptide). + spacer). Typically, the solution involves the coupling of a single bond to a covalent coupling site, in a water-soluble polymer, for a receptor-binding portion, however, in alternative solutions multiple bonding may be used, including further variations in which different species of water-soluble polymers are used.

51130Β are coupled to a receptor-binding moiety at various binding compounds, which may include a covalent binding coupler to the spacer ί and / or a common or both peptide chains. In some embodiments, the higher order dimer or multimer will comprise different types of peptide chain (e.g., a heterodimer or other heteromultimer). By way of example and without limitation, the dimer may comprise a first peptide chain having a PEG binding compound and the second peptide chain may be without a PEG binding compound or use other coupling reactions relative to the first peptide chain and in some variations the spacer may contain or be without a PEG binding compound and said spacer, if PEGylated, may use a coupling reaction other than the first and / or second peptide chain, An alternative solution includes PEG attached to the spacer portion of the receptor-binding portion and a different water-soluble polymer (e.g., a carbohydrate) conjugated to the outer chain of one of the amino acids from the peptide portion of the molecule.

A very wide range of polyethylene glycol (PEG) species can be used for PEGylation of the receptor-binding portion (peptides + spacer). Basically, any suitable reactive PEG reagent can be used. In a preferred embodiment, the reactive PEG reagent will result in the formation of a carbamate or amide bond after conjugation to the receptor-binding moiety. Suitable reactive PEG species include, but are not limited to, those available for sale in the NOF Corporation (2003) Catalog of Drug Supply Systems (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome, Shibuya-ku, Tokuo 150 - 6019 ) and the Catalog of Molecular Engineering (2003) from Nectar Therapeutics (490 Discoveoma Drive, Huntsville, Alabama 35806). For example, and without limitation, the following PEG reagents are often prioritized in different solutions: mPEG2-NHS, rnPEG2-ALD, multi-Arm PEG, mPEG (MAL) 2, mPEG2 (MAL), mPEG-NH2, mPEG-SPA, mPEG-SBA, mPEG-thioesters, mPEGdouble esters, mPEG-BTC, mPEG-ButirALD , mPEG-ACET, heterofunctional PEG (NH2-PEGCOOH, Bok-PEG-NHS, Fmok-PEG-NHS, NHS-PEG-VS, NHS-PEG-MAL), PEG acrylates (ACRL · PEG-NHS), PEG-phospholipids (e.g. mPEG-DSPE), multiple PEGs from the SUNBRITE series including GL series of Glycerin PEGs activated by a reaction selected by one skilled in the art, any of SUNBRITE activated PEGs (including but not limited to carboxyl-PEG, p-NP-PEG, Tresyl-PEG, aldehyde PEG, acetal-PEG, amino-PEG, thiol-PEG, maleimido-PEG, hydroxyl-PEG-amines, amino-PEG-COOH, hydroxyl-PEG-aldehydes, PEG-carboxylic anhydride type, functionalized PEG-phospholipids and other similar and / or suitable reactive PEGs to be selected by one skilled in the art for their particular application and use.

The novel peptides of the invention may also contain two PEG moieties that are covalently linked via a carbamate or amide bond to a portion of the spacer, wherein the space moiety is covalently linked to the tertiary amide linker from the peptide dimer. Each of the two PEG moieties used in such embodiments of the present invention may be linear and may be linked together at a single binding point. Preferably, each PEG moiety has a molecular weight of about 10 kilodaltons (10 K) to about

51130 Β

Κ (the term "eye" indicates that in preparations with PEG, some molecules will weigh more, some less than the stated molecular weight). Lineami PEG parts are particularly preferred. More preferably, each of the two PEG moieties has a molecular weight of about 20 K to about 40 K, and more preferably between about 20 K and about 40 K. More preferably, each of the two PEG moieties has a molecular weight of about 20 K. One skilled in the art will be able to select the desired polymer size based on such considerations as the desired dose; circulation time; resistance to proteolysis; effects, if any, on biological activity; ease of handling; degree or absence of antigenicity; and other known effects of PEG on therapeutic peptides.

The present invention also encompasses peptide agonists that are homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) (SEKID BR: 3), wherein each amino acid is labeled with the standard one-letter Ac abbreviation. -acetylglycine, (1-nal) is 1-naphthylalanine and (MeG) is N-methylglycine, also known as sarcosine. Each peptide monomer from the peptide dimer contains an intramolecular disulfide bond between the cysteine ​​monomer residues. Such monomers can be represented schematically as follows:

II (AcG) GLYACHMGPlT (l-nal) VCQPLR (MeG)

<img file="RS51130B_D0021.tif" />

Formula II

These monomeric peptides were dimerized to give peptide dimers with enhanced EPO-R agonist activity. The linker (Lk) portion is a lysine residue, which binds the C-terminus of two peptide monomers, by simultaneously binding to the C-terminal amino acid in each monomer. One peptide monomer is attached by its C-terminus to the lysine ε-amino group and the other peptide monomer is attached by its C-terminus to the lysine α-amino group. For example, the dimer can be illustrated structurally as shown in Formula I and summarized as shown in Formula P:

Formula I

Monomerl— n²H

I fH₂ sn „I

Mononier2 — ΝΉ— CH—_g\ .

Spacer-PEG_{2 (M0K}

In Formula I and in Formula II, N² represents a nitrogen atom from the lysine ε-amino group and N¹ represents a nitrogen atom from the lysine α-amino group.

The peptide dimers of the invention further comprise a spacer (spacer) having the following structure:

Monomerl — N²H

<img file="RS51130B_D0022.tif" />

Spacer-PEG.

51130 Β

-M ^ -SSNgJg-O-dag-O-SODg- ^ NAt one end, N¹ the spacer is connected via an amide bond to the carbonyl carbon of the lysine linker. At the opposite end, N² the spacer is linked via a carbamate bond or an amide bond to the activated polyethylene glycol (PEG) moiety, where the PEG has a molecular weight of about 10,000 to about 60,000 Daltons (the term about indicates that in PEG preparations, some molecules will weigh more, some less of the stated molecular weight). More preferably, the PEG has a molecular weight of about 20,000 to 40,000 Daltons.

Thus, the novel peptides of the invention also contain a PEG moiety, which is covalently attached to the peptide dimer. PEG is a water-soluble polymer that is pharmaceutically acceptable. The PEG for use in the present invention may be a linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20 K) to about 60 K (the term about indicates that in PEG preparations, some molecules will weigh more, some less than said molecular weight). Most preferably, the PEG has a molecular weight of about 20 K to about 40 K and more preferably a molecular weight of about 30 K to about 40 K. One skilled in the art will be able to select the desired polymer size based on such considerations as are the desired dose; circulation time; resistance to proteolysis; effects, if any, on biological activity; ease of handling; degree iE absence of antigenicity; and other known effects of PEG on therapeutic peptides.

When each homodimer monomer has the amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEKID NO: 1) and N¹ the linker is linked via a carbamate bond to the activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="RS51130B_D0023.tif" />

When each homodimer monomer has the amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEKID NO: 1) and N¹ the linker is connected via an amide bond to activated polyethylene

51130 Β the glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

<img file="RS51130B_D0024.tif" />

<img file="RS51130B_D0025.tif" />

ΡΕθ2 (Μ0Κ

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEKID NO: 2) and N¹ linker is linked via a carbamate bond to the activated polyethylene glycol (PEG) moiety, novel peptide compounds of the invention can be introduced

<img file="RS51130B_D0026.tif" />

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEKID NO: 2) and N¹ the linker is linked via an amide bond to the activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

51130Β

<img file="RS51130B_D0027.tif" />

Ρ ^ θ20-40Κ

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

<img file="RS51130B_D0028.tif" />

51130 Β

<img file="RS51130B_D0029.tif" />

51130Β

<img file="RS51130B_D0030.tif" />

51130 Β

<img file="RS51130B_D0031.tif" />

51130 Β

<img file="RS51130B_D0032.tif" />

4ΟΚ

<img file="RS51130B_D0033.tif" />

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

<img file="RS51130B_D0034.tif" />

<img file="RS51130B_D0035.tif" />

-50K

10-50Κ

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (lnal) VCQPLRK (SEKID NO: 1) and both N¹ and N² spacers are covalently linked via an amide bond to the activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

51130 Β

<img file="RS51130B_D0036.tif" />

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEKID NO: 2) and both N¹ and N² spacers are covalently linked via a carbamate bond to the activated PEG moiety, the novel peptide compounds of the invention can be represented as follows:

(AcG) GLYAĆHMGPrrO-nal) VĆQPLR (MeG) - ΝΗ ^ Λ-ΝΗ_Ζ

HN o _{0 0}

ΌγΌ PEG_{! |&gt; mk}NH Ac (AcG) GLYACHMGPITd-nal) V_i: qPLR (MeG) -NH^/V ~^NH2

When each homodimer monomer has an amino acid sequence, (AcG) GLYACHMGPIT (1-nal) VCQPLRK (SEKID NO: 2) and both N¹ and N² the spacers are covalently linked via an amide bond to the activated PEG moiety, the novel peptide compounds of the invention may be represented as follows;

51130Β

<img file="RS51130B_D0037.tif" />

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

<img file="RS51130B_D0038.tif" />

51130 Β

<img file="RS51130B_D0039.tif" />

51130 Β

<img file="RS51130B_D0040.tif" />

51130 Β

<img file="RS51130B_D0041.tif" />

51130Β

<img file="RS51130B_D0042.tif" />

51130Β

<img file="RS51130B_D0043.tif" />

51130 Β

<img file="RS51130B_D0044.tif" />

51130 Β

When the spacer is connected via a carbamate bond to the activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

(AcG) GLYACHMGPTT (1-naI) VCQPLR (MeG) -NH

<img file="RS51130B_D0045.tif" />

<img file="RS51130B_D0046.tif" />

When the spacer is connected via an amide bond to the activated polyethylene glycol (PEG) moiety, the novel peptide compounds of the invention can be represented as follows:

| I (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) -NH

<img file="RS51130B_D0047.tif" />

(AcG) GLYApiMGPIT (l-nal) V (pQPLR (MeG)

<img file="RS51130B_D0048.tif" />

These smoke structures can be written [Ac-peptide, disulfide] 2Liz-spacer-PEG₂(M0K to denote the N-terminal acetylated peptide bond for both the α and ε amino groups on lysine with each peptide containing an intramolecular disulfide loop and a spacer molecule forming a covalent bond between the C-terminus on lysine and the PEG moiety, where PEG has a molecular weight of about 20,000 to about 40,000 Daltons.

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

<img file="RS51130B_D0049.tif" />

51130 Β

<img file="RS51130B_D0050.tif" />

Stereoisomers (e.g., D-amino acids) of twenty conventional amino acids, naturally occurring amino acids such as α, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acid components may also be suitable. Examples of unconventional amino acids include, but are not limited to: β-alanine, 3-pridylalanine, 4-hydroxyproline, O-phosphoserine, Nmethylglycine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, norleucine, and others similar amino acids and imino acids. Other modifications are also possible, including amino terminus modifications, carboxy terminus modifications, replacement of one or more natural genetically encoded amino acids with unconventional amino acids, side chain modifications of one or more amino acid residues, peptide phosphorylation, and the like.

51130Β

The peptide sequences of the present invention may be present alone or in conjunction with the N-terminal and / or C-thermal extension of the peptide chain. Such extensions may be naturally encoded peptide sequences optionally with or substantially free of naturally occurring sequences; extensions may include any addition, deletion, point mutation, or other modification of the sequence or combination depending on the need of one skilled in the art. For example, and without limitation, natural sequences may be full-length or partial-length and may include amino acid substitution to provide binding sites for carbohydrates, PEG, other polymers, or the like, via side chain conjugation. In variations, amino acid substitution leads to humanization of the sequence to make it compatible with the human immune system. Fusion proteins of all types are provided, including immunoglobulin sequences adjacent to or in close proximity to the EPO-R activating sequences of the present invention with or without a non-immunoglobulin spacer sequence. One type of solution is an immunoglobulin chain having EPO-R activating sequences in place of a variable (V) region on a dense and / or simple chain,

Preparation of peptide compounds of the invention:

Peptide synthesis

The peptides of the invention can be prepared by conventional methods known in the art. These standard methods include methods of exclusively solid-phase synthesis, partial solid-phase synthesis, condensation of fragments, classical solution synthesis, and recombinant DNA technologies [See, e.g., Merrifield J. Am. Chem. Soc. 1963 85: 2149].

In one embodiment, the peptide monomers from the peptide dimer are synthesized individually and then dimerized for synthesis.

In another embodiment, the peptide monomers from the dimer are linked via their C-terminus by a branched tertiary aroid linker Lk moiety having two functional groups that can serve as initial sites for peptide synthesis and a third functional group (e.g., a carboxyl group or amino group) that allows binding to another molecular moiety (e.g._F which may be present on the surface of the solid support). In this case, the two peptide monomers can be synthesized directly into two reactive nitrogen groups from the linker Lk moiety with variations in solid-phase synthesis techniques. Such syntheses can be sequential or simultaneous.

In another embodiment, the two peptide monomers can be synthesized directly into two reactive nitrogen groups from the linker Lk moiety with variations in solid-phase synthesis techniques. Such syntheses can be sequential or simultaneous. This solution uses a lysine linker (Lr) moiety that has two amino groups that can serve as initial sites for synthesis. peptide and a third functional group (e.g., a carboxyl group from lysine; or an amino group from lysine amide, a lysine residue in which

51130 Β the carboxyl group converts to the amide moiety -CONH2) which allows binding to another molecular moiety (e.g., which may be present on the surface of the solid support).

When performed in a series of syntheses of peptide chains of dimers on the linker, two amino functional groups on the linker molecule are protected (protected) with two different orthogonal removable amine protecting groups. The protected linker is coupled to the solid support via the linker's third functional group. The first amine protecting group is removed and the first dimer peptide is synthesized on the first deprotected amine moiety. The second amine protecting group is then removed and the second peptide from the dimer is synthesized on the second deprotected amine moiety. For example, the first amine portion of the linker may be protected with Alok and the second with Fmok. In this case, the Fmok group (but not the Alok group) can be removed by treatment with a weak base [e.g., 20% piperidine in dimethyl formamide (DMF)] and the first peptide chain synthesized. Thereafter, the Alok group can be removed using a suitable reagent [e.g., Pd (PPh3) / 4-methyl morpholine and chloroform] and another peptide chain synthesized. It should be noted that when different thiol-protecting groups for cysteine ​​are used, in order to control the formation of a disulfide bond (such as the discussed jole) this technique must be applied even when the final amino acid sequences of the peptide chains are identical.

When simultaneous synthesis of dimer peptide chains on a linker is performed, the two amine functional groups of the linker molecules are protected with the same removing amine protecting groups. The protected linker is coupled to the solid support via the linker's third functional group. In this case, two protected functional groups of linker molecules are simultaneously deprotected and the two peptide chains are simultaneously synthesized on deprotected amines. Note that using this technique, the sequences of the peptide chains in the dimer will be identical and the thiol-protecting groups for cysteine ​​residues are all the same.

The priority method for peptide synthesis is solid-phase synthesis. Methods for solid-phase peptide synthesis are well known in the art [see, e.g., Stewart Solid Phase Peptide Svntheses (Freeman and Co .: San Francisco) 1969; 2002/2003 General Catalog of Novabiochem Corp., San Diego, USA; Goodman Synthesis of Peptides and Peptidomimetics (Houben-Weil, Stuttgart) 2002], In solid-phase synthesis, synthesis is usually started from the C-terminal end of the peptide using an amino-protected resin. A suitable starting material can be obtained, for example, by coupling the desired α-amino acid to a chloromethylated resin, a hydroxymethyl resin, a polystyrene resin, a benzhydrylamine resin or the like. One such chloromethylated resin is sold under the commercial name BIO-BEADS SX-1 and is produced by Bio Rad Laboratories (Richmond, CA). The preparation of hydroxymethyl resin is described in [Bodonszky, isar. (1966) Chem. Ind. London 38: 1597]. Benhydrylamine (BHA) resin is described in [Pietta and Marshall (1970) Chem. Commun. 650] and the 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 the aid of

51130 Β catalysis with cesium bicarbonate, according to the method described by Gisin (1973) Helv. Chim, Acta 56: 1467.

After initial coupling, the α-amino protecting group is removed, for example, using trifluoroacetic acid (TFA) or hydrochloric acid (HCl) dissolved in organic solvents at room temperature. Thereafter, the α-arnino-protected amino acid is successively coupled to a growing, carrier-bound peptide chain. α-amino protecting groups are those known to be useful in the field of step peptide synthesis, including: protecting groups of the acyl type (e.g., formyl, trifluoroacetyl, acetyl), protecting groups of the aromatic urethane type [e.g., benzyloxycarboyl (Cbz) and substituted Cbz], protecting groups of aliphatic urethanes [e.g., t-butyloxycarbonyl (Bok) , isopropyloxycarbonyl, cyclohexyloxycarbonyl] and alkyl-type protecting groups (e.g. benzyl, triphenylmethyl), fluorenylmethyl oxycarbonyl (Fmok), allyloxycarbonyl (Alok) and 1- (4,4-dimethyl-2,6-dioxocydehex-1-yl) ).

The side chain of the protecting group (usually ethers, esters, trityl, PMC and the like) remains intact during coupling and does not cleave during deprotection of amino-terminal protecting groups or during coupling. The side chain of the protecting group must be removable upon completion of the synthesis of the final peptide and under reaction conditions that will not affect the modification of the target peptide. The side chain of the protecting group for Tire includes tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz and 2,5-dichlorobenzyl. The side chain protecting group for Asp includes benzyl, 2,6-dichlorobenzyl, methyl, ethyl and cyclohexyl. The protecting side chain for Tr and Ser includes acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl and Cbz. The side chain protecting grape for Arg includes nitro, Tozil (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), 4-methoxy-2,3,6 -trimethyl-benzenesulfonyl (Mtr) or Bok. The side chain of the Liz protecting group includes Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-Br-Cbz), Tosili Bok.

After removal of the α-amino protecting group, the remaining protected amino acids are coupled in steps in the desired order. Each protected amino acid is generally reacted in about 3-fold excess, using an appropriate carboxyl group activator such as 2- (1H-benzotriazol-1-yl) 1,1,3,3 tetramethyluronium hexafluorophosphate (HBTU) or dicyclohexylcarbodimide (DCC) in solution , for example, in methylene chloride (CH2Cl2), N-methyl pyrrolidone, dimethyl formamide (DMF) or mixtures thereof.

Once the desired amino acid sequence is completed, the desired peptide is decoupled from the resin carrier 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 the remaining groups. side chain. When chloromethylated resins are used, treatment with hydrogen fluoride leads to the formation of free peptide acids. When benzhydrylamine resins are used, treatment with

51130Β with hydrogen fluoride leads directly to the formation of free peptide amides. Alternatively, when chloromethylated resins are used, the protected side chain peptide can be decoupled by treating the peptide resin with ammonia to give the desired protected amide side chain or with an alkylamine to obtain a protected alkylaniide or dialkylamide side chain. The side chain protection is then removed in the usual manner by treatment with hydrogen fluoride to give free amides, alkylamides or dialkylamides.

To prepare the esters of the invention, the resins used to prepare the peptide acids are used and the side chain of the protected peptide is cleaved with a base and a suitable alcohol (e.g., methanol). The side chain of the protecting group is then removed in the usual manner by treatment with hydrogen fluoride to give the desired ester.

These methods can also be used to synthesize peptides that contain other amino acids relative to 20 natural, genetically encoded amino acids that are substituted at one, two or more of the possible positions on the compounds of the invention. Synthetic amino acids that may be substituted in the peptides of the present invention include, but are not limited to, N-methyl, L · hydroxypropyl, Iz3,4-dihydroxyphenylalanyl, b amino acids such as L · b-hydroxylysyl and D-bmethylalanyl, Lra-methylalanyl , β amino acids and isoquinolyl. Naturally non-occurring D-amino acids and synthetic amino acids can also be incorporated into the peptides of this invention.

Peptide modifications

It is also possible to modify the amino and / or carboxy terminuses of the peptide compounds of the invention to produce other compounds of the invention. For example, the amino terminus may be acetylated with acetic acid or a halogenated derivative thereof (such as α-chloroacetic acid, α-bromoacetic acid or α-iodoacetic acid).

It is possible to replace the natural side chain of 20 genetically encoded amino acids (or stereoisomeric D amino acids) with other side chains, for example with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl , amide, lower alkyl amide, di (lower alkyl) amide, lower alkoxy, hydroxy, carboxy and their lower ester derivatives and with 4-, 5-, 6-, to 7-membered heterocycles. In particular, proline analogs can be used in which the ring size of the proline residue is altered from

5th member to 4.6 or 7th member. Cyclic groups may be saturated or unsaturated and, if unsaturated, may be aromatic or non-aromatic. Preferably, the heterocyclic groups contain one or more nitrogen, oxygen and / or sulfur heteroatoms. Examples of such groups include furazanil, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g. 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thionyl thionyl, triazolyl.

51130Β

These heterocyclic groups may be substituted or unsubstituted. When the group is substituted, the substituent may be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

Peptides can also be easily modified by phosphorylation and other methods [e.g., described in Hruby, isar. (1990) Biochem J. 268: 249-262],

The peptide compounds of the invention also serve as model structures for non-peptide compounds with similar biological activity. One skilled in the art will appreciate that various techniques are available to construct a compound with the same or similar desired biological activity as the major peptide compound, but with more favorable activity than the major in terms of solubility, stability and susceptibility to hydrolysis and proteolysis [See Morgan and Gainor (1989). Ann. Rep. Med. Chem, 24: 243 - 252], These techniques involve replacing the peptide support with a support consisting of phosphonates, amidates, carbamates, sulfonamides, secondary amines and N-methylamino acids.

Formation of disulfide bonds

The compounds of this invention contain two intramolecular disuffide bonds. Such disulfide bonds can be formed by the oxidation of cysteine ​​residues on each peptide monomer.

In one embodiment, the control of cysteine ​​bond formation is applied by selecting the type of oxidizing agent and its concentration that is effective to optimize the formation of the desired isomer. For example, the oxidation of a peptide dimer to form two intramolecular disulfide bonds (one on each peptide chain) is preferably achieved (relative to the formation of an intermolecular disulfide bond) when the oxidizing agents are DMSO or iodine (I₂).

In other embodiments, the formation of cysteine ​​bonds is controlled by the selective use of a thiol-protecting group during peptide synthesis. For example, when a dimer with two intramolecular disulfide bonds is desired, the first monomeric peptide chain is synthesized with two cysteine ​​residues from the nucleus of the sequence protected by the first thiol protecting group [e.g., trityl (Trt), allyloxycarbonyl (Alok) or " (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) or the like], then a second peptide monomer is synthesized with two cysteine ​​residues from the nucleus of the sequence which is protected by a second thiol protecting group other than the first thiol protecting group [e.g., acetamidomethyl (Acm), t-butyl (tBu) or the like]. Thereafter, the first thiol protecting group is removed to give the bisulfide cyclization of the first monomer and then the second thiol protecting group is removed to give the bisulfide cyclization of the second monomer.

Other solutions of the present invention provide analogs of these disulfide derivatives in which one of the sulfur is replaced by CH₂ group or other sulfur isotherm. These analogs can be obtained from the compounds of the present invention, in which each peptide monomer contains at least one C or homocysteine ​​residue and α-amino-v-biiternic acid instead of the other C residue, via

51130 Β intramolecular or intermolecular displacement, using methods known in the art [See e.g., Barker, isar (1992) J. Med. Chem. 35: 2040 - 2048 and Or, isar (1991) J. Org. Chem. 56: 3146 - 3149], One skilled in the art will readily recognize that these displacements also occur using other homologues of α-amino-7-butyric acid and homocysteine.

In addition to the above cycEzation strategy, other non-disulfide peptide cyclization strategies may be used. Such alternative cyclization strategies include, for example, amide cyclization strategies as well as those involving the formation of thioether bonds. Thus, the compounds of the present invention may exist in cyclic form with either an intramolecular amide bond or an intramolecular thioether bond. For example, peptides can be synthesized when one cysteine ​​of the core sequence is replaced with lysine and the other cysteine ​​is replaced with glutamic acid. Thereafter, the cyclic monomer can be formed via an amide bond between the side chains of these two residues. Alternatively, the peptide may be synthesized when one cysteine ​​of the core sequence is replaced with lysine (or serine). The cyclic monomer can then be formed via a thioether bond between the side chains of a lysine (or serine) residue and another cysteine ​​residue of the core sequence. As such, in addition to the disulfide cyclization strategy, the amide cyclization strategy and the thioether cyclization strategy can be readily applied to the cyclization of the compounds of this invention. Alternatively, the amino-terminus of the peptide may be captured with α-substituted acetic acid, wherein the α-substituent is a leaving group, such as α-haloacetic acid, for example, α-chloroacetic acid, ct'bromoacetic acid or α-iodoacetic acid.

Addition of a branched tertiary amide linker

Peptide monomers can be dimerized using a branched tertiary amide linker moiety. In one embodiment, the linker is incorporated into the peptide during peptide synthesis. For example, when the linker Lk moiety contains two functional groups that serve as initial sites for peptide synthesis and one or more other functional groups (e.g., a carboxyl group or an amino group) that may bind to one or more other molecular moieties, Lyricer can be conjugated to a solid support. Thereafter, the two peptide monomers can be synthesized directly into the two reactive nitrogen groups of the linker Lk moiety with variations in solid-phase synthesis techniques.

In an alternative embodiment, the linker can be conjugated to two peptide monomers of the peptide dimer after peptide synthesis. Such conjugation can be achieved by methods well known in the art. In one embodiment, the linker comprises two functional groups suitable for binding to the target functional groups of the synthesized peptide monomers. For example, an ink containing two functional groups, either pre-activated or in the presence of suitable coupling reagents, can be reacted with a targeted lysine side chain of an amino group from each of the two peptide monomers.

51130 Β

For example, peptide moomers can be chemically coupled to tertiary amide linkers,

ACH ^ O-SN? -H-SN? -S²O-V * wherein: X is NCO- (CH2) 2-NH-Y and Y is a suitable protecting group, such as t-butyloxycarbonyl (Bok) protecting group; A * is a suitable functional group, such as N-oxy succinimide, used for C conjugation¹ a ε-amino group linker from the Gterminal lysine residue of the first peptide monomer; and B * is an affected functional group, such as N-oxy succinimide, used to conjugate C² a linker for an ε-amino group from the C-terminal lysine residue of another peptide monomer.

In addition, for example, peptide monomers may be chemically coupled to a tertiary amide linker,

ACHMSN ^ H-SNg-SCJ-V * where: X is NCO- (CH₂) 2-NH-C³O ·; A * is a suitable functional group, such as N-oxy succinimide, used for C conjugation¹ a linker for the ε-amino group from the C-thermal lysine residue of the first peptide monomer; and B * is a suitable functional group, such as N-oxy succinimide, used to conjugate C² a linker for the ε-amino group from the C-terminal lysine residue of the expensive peptide monomer; and the tertiary amide linker is chemically linked to the spacer,

Y-NH- (CH2)₄-C⁴H-NH-Y where: C³ from X is covalently bound to C⁴ spacers; and Y is a suitable protecting group, such as a tbutyloxycarbonyl (Boc) protecting group.

Addition of lysine linker

Peptide monomers can be dimerized using the lysine linker Lk moiety. In one embodiment, the lysine linker is incorporated into the peptide during peptide synthesis. For example, when the lysine linker Lk moiety contains two functional groups that may serve as initial sites for peptide synthesis and a third functional group (e.g., a carboxyl group or amino group) that may bind to another molecular moiety, the linker may be conjugated to a solid support. Thereafter, the two peptide monomers can be synthesized directly into the two reactive nitrogen groups of the lysine linker Lk moiety with variations in solid-phase synthesis techniques.

In alternative embodiments, when the peptide dimer is dimerized using a lysine linker Lr moiety, said linker can be conjugated to two peptide monomers of the peptide dimer after peptide synthesis. Such conjugation can be achieved by methods well known in the art. In one embodiment, the Inker comprises at least two functional groups suitable for binding to the target functional groups of the synthesized peptide monomer.

51130 Β

For example, lysine two free amino groups can react with the C-terminal carboxyl group of each of the two peptide monomers.

Adding a spacer

The peptide compounds of the invention further comprise a spacer. In one embodiment, the spacer may be incorporated into the peptide during peptide synthesis. For example, when the spacer contains a free amino group and another functional group (e.g., a carboxyl group or an amino group) that may bind to another molecular moiety, the spacer may be conjugated to a solid support.

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 Lr moiety that has two functional groups that can serve as initial sites for peptide synthesis and a third functional group (e.g., a carboxyl group or an amino group) that can bind to another molecular moiety is conjugated to the spacer via spacers of the second functional group and lmker of the third functional group. Thereafter, the two peptide monomers can be synthesized directly into two reactive nitrogen groups of the linker Lr moiety with variations in solid-phase synthesis techniques. For example, a spacer coupled on a solid support with a free amino group can react with the Iisin linker via a linker free carboxyl group.

In alternative embodiments, the spacer may be conjugated to a peptide dimer after peptide synthesis. Such conjugation can be achieved by methods well known in the art. In one embodiment, the linker comprises at least one functional group suitable for binding to a target functional group of the synthesized peptide. For example, a spacer with a free amino group can be reacted with a peptide C-terminal carboxyl group. In a preferred example, the linker with a free carboxyl group can be reacted with a free amino group from lysine amide.

Polyethylene glycol (PEG) binding

In recent years, water-soluble polymers, such as polyethylene glycol (PEG), have been used for covalent peptide modifications of therapeutic and diagnostic significance. Binding of such polymers is performed due to the increase of biological activity, prolongation of blood circulation time, decrease of immunogenicity, increase of water solubility and increase of resistance to protease digestion. For example, it has been observed that covalent binding of PEG to therapeutic polypeptides such as interleukins [Knauf, isar. (1988) J. Biol. Chem. 263; 15064; Tsutsumi, isar. (1995) J. Controlled Release 33: 447), interferons (Kita, isar (1990) DrugDes. Delivery 6: 157), catalase (Abuchowski, / sar (1977) J. BioLChem. 252: 582), superoxide dismutase (Beauchamp , 7 ^ ag (1983) Anal. Biochem. 131: 25) and adenosine deaminase (Chen, 7sa / - (1981) Biochim. Biophy. Acta 660: 293), prolongs their half-life in vivo, and / or reduces their immunogenicity and antigenicity.

51130 Β

The peptide compounds of the invention may comprise a polyethylene glycol (PEG) moiety, which is covalently attached to a tertiary branched amide linker or spacer from the peptide dimer via a carbamate bond or via an amide bond. One example of the PEG used in the present invention is lineami, unbranched PEG having a molecular weight of about 20 kiloDaltons (20 K) to about 40 K (the term about indicates that in PEG preparations, some molecules will weigh more, some less than the stated molecular weight). Preferably, the PEG has a molecular weight of from about 30 K to about 40 K.

Another example of a PEG used in the present invention is a lineami PEG having a molecular weight of from about 10 K to about 60 K (the term about '¹ indicates that in preparations with PEG, some molecules will weigh more, some less than the stated molecular weight). Preferably, the PEG has a molecular weight of from about 20 K to about 40 K. More preferably, the PEG has a molecular weight of about 20 K.

Examples of methods for covalently binding PEG (PEGylation) are described below. These illustrative descriptions are given but without limitation. One of ordinary skill in the art will appreciate that different methods for covalently binding a wide range of different PEGs are well known in the art. As such, the peptide compounds to which PEG is to bind by any of a number of binding methods known in the art are encompassed by the present invention.

For example, PEG may be covalently linked to a linker via a reactive group to which the activated PEG molecule may be attached (e.g., a free amino group or a carboxyl group). PEG molecules can be attached to amino groups using methoxylated PEG (mPEG) having various reactive moieties. Such polymers include mPEG-succinimidyl succinate, mPEGsuccinimidyl carbonate, mPEG-imidate, mPEG-4-hydrotrophenyl carbonate, and m. Similarly, PEG molecules can be attached to carboxyl groups using methoxylated PEG with a free amino group (mPEG-NH?).

In some embodiments, the linker or spacer comprises a terminal amino group (i.e., positioned at the spacer terminus). This terminal amino group can be reacted with a suitably 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 suitably activated PEG molecule, such as mPEG-succinimidyl butyrate (mPEGSBA) or mPEG-succinimidyl propionate (mPEG-SPA) containing a reactive N-hydroxy-succinimide (NHS) group, to obtained a stable covalent carbamate bond. In other embodiments, the linker reactive group comprises a carboxyl group that can be activated to form a covalent bond with an amine-containing PEG molecule under suitable reaction conditions. Suitable PEG molecules include mPEG-NH2 and suitable reaction conditions include the formation of amides via carbodiimide or the like.

Analysis of EPO-R agonist activity:

51130 Vit ϊη in vitro functional analysis

In vitro competitive binding assays quantify the ability of the tested peptide to compete with EPO for binding to EPO-R. For example (see, e.g., as described in U.S. Patent 5,773,569), the extracellular region of human EPO-R (EPO binding protein, EBP) can be recombinantly produced in E. coli and the recombinant protein is coupled to a solid. a carrier, such as a microtiter vessel or synthetic microspheres [e.g., Sulfolink beads manufactured by Pierce Chemical Co. (Rockford, IL)]. Immobilized EBP is then incubated with labeled recombinant EPO or with labeled recombinant EPO and peptide tested. Serial dilutions of the tested peptide are used for such experiments. Analytical points without added peptide tested define total EPO binding to EBP. For reactions containing the tested peptide, the amount of bound EPO is quantified and expressed as a percentage of control (total = 100%) binding. These values ​​are plotted against peptide concentrations. The IC50 value is defined as the concentration of test peptide that reduces EPO binding to EBP by 50% (i.e., 50% inhibition of EPO binding).

Various in vitro competitive binding assays measure the weak signal generated as a function of the proximity of two microspheres: one EPO-conjugate microsphere and one EPO-R-conjugate microsphere. The proximity of the microspheres was formed by the binding of EPO to EPO-R. The tested peptide that competes with EPO for binding to EPO-R will prevent that binding, leading to a reduction in light emission. The concentration of test peptide leading to a 50% reduction in light emission is defined as the IC50 value.

The peptides of the present invention are in very effective competition with EPO in binding to EPO-R. This enhanced function is represented by their ability to inhibit EPO binding at significantly lower peptide concentrations (i.e., they have very low IC50 values),

The biological activity and potency of the monomeric and dimeric peptides of the EPO-R agonists of the invention, which bind specifically to the EPO receptor, can be measured using in vitro cell-based functional assays.

One assay is based on a pre'B-cell line of mice expressing human EPO-R and further transfected with a phosgene construct containing a promoter-guided luciferase reporter. Upon exposure to EPO or another EPO-R agonist, the cell reacts to synthesize luciferase. Luciferase causes light to be emitted after the addition of its luciferin substrate. Thus, the level of EPO R activation in such cells can be quantified by measuring luciferase activity. The activity of the test peptide is measured by adding serial dilutions of the test peptide to the cells, which are incubated for 4 hours. After incubation, luciferin substrate is added to the cells and light emission is measured. The concentration of test peptide leading to half of the maximum light emission is recorded as EC50.

The peptides of the present invention show a dramatically increased ability to support EPOR signaling-dependent luciferase expression in this assay. This enhanced function represents their ability to lead to half of maximal luciferase activity at significantly lower levels

51130Β peptide concentrations (i.e., they have very low EC50 values). This assay is the preferred method for determining the potency and activity of the EPO-R agonist peptide of the invention.

A second assay can be performed using FDC-Pl / ER cells [Dexter, isar. (1980) J, Ehr. Med. 152: 1036 -1047], well-characterized untransformed mouse bone marrow cell lines, in which EPO-R was transiently transfected. These cells show EPO-dependent proliferation.

In one such assay, cells grow to half stationary density in the presence of necessary growth factors (see, e.g., as described in U.S. Patent 5,773,569). The cells were then washed in PBS and fasted for 16-24 hours in complete growth factor-free medium. After determining the viability of the blanket (e.g., by trypan blue staining), stock solutions (in complete medium without growth factor) are prepared to give about 10⁵ cells at 50 pL. Serial dilutions of peptide EPO-R agonist compounds (usually free, dissolved peptides as opposed to phage-linked or otherwise bound or immobilized peptides) to be tested are prepared in 96 wells on a tissue cell culture plate in a final volume of 50 μL per well. Cells (50 μL) were added to each well and the cells were incubated for 24-48 hours, during which time the negative controls should die or be at rest. Cell proliferation is then measured using techniques known in the art, such as MTT analysis that measures H³-thymidine incorporation as an indication for cell proliferation [see Mosmann (1983) J. Imunol. Methods 65: 55 - 63], Peptides were tested on both the EPO-R-expressing cell line and the parental non-expressing cell line. The concentration of test peptide required to result in one-half of maximal cell proliferation is recorded as the EC50.

The peptides of the present invention show a dramatically increased ability to support EPO-dependent cell growth in this assay. This enhanced function is represented by their ability to lead to half of the maximum stimulatory activity for cell proliferation at significantly lower peptide concentrations (i.e., they have very low EC50 values). This assay is the preferred method for determining the potency and activity of the EPO-R agonist peptide of the invention.

In another assay, cells are grown to a stationary phase in EPO-supplied medium, harvested and then cultured for an additional 18 h in EPO-free medium. Cells were divided into three groups with the same cell density: one group without added factors (negative control), the EPO group (positive control) and the experimental group with the tested peptide. The cultured cells are then collected at various time intervals, and stained with DNA-binding fluorescent dye (e.g., propidium iodide or Hoechst dye, 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 with CellFIT software (Becton Dickinson). Cells treated with EPO or active peptidors will show a higher proportion of cells in the S phase (which is determined by increased fluorescence as an indicator of an increase in DNA content) compared to the negative control group.

Similar tests can be performed using FDCP-1 [see eg “Dexter isar. (1980).

51130 Β

Ehr. Med. 152: 1036 * 1047] or TF-1 [Kitamura, isar. (1989) Blood 73: 375–380] cell lines. FDCP-1 is a multi-potential primitive growth factor-dependent hematopoietic progenitor cell line from mice that can multiply but not differentiate when WEHI-3-conditioned medium (IL-3-containing medium) is added. , ATCC number TIB-68). For such experiments, the FDCP-1 cell line is transfected with human or murine EPO-R to produce FDCP-1-hEPO-R or FDCP-1-mEPO-R cell lines, respectively, that can replicate but cannot differentiate, in the presence of EPO. TF-1, a single EPO-dependent cell line, can also be used to measure the effects of EPO-R agonist peptides on cell proliferation.

In another analysis, the procedure set out in Krystal (1983) Ehr. Hematol 11: 649 - 660 for X-based microanalysis³-thymidine incorporation in spleen cells can be used to test the ability of compounds of the present invention to serve as EPO agonists. Briefly, B6C3Fj mice are injected, daily for two days, with phenylhydrazine (60 mg / kg). On the third day, spleen cells are removed and their ability to multiply over a period of 24 hours is examined using ΜΊΤ analysis.

Binding of EPO to EPO-R in an erythropoietin-sensitive cell line induces tyrosine phosphorylation of both receptors and a number of intracellular proteins, including Shc, vav, and JAK2 kinases. Thus, another in vitro assay measures the ability of the peptides of the invention to induce tyrosine phosphorylation of EPO-R and further affect intracellular signal transducem proteins. The active peptides, identified by the binding and proliferation assays described above, exhibit a phosphorylation pattern almost identical to that of EPO in erythropoietin-sensitive cells. For these assays, FDC-Pl / ER cells [Dexter, isar (1980) J Ehr Med 152: 1036 - 47] were maintained in EPO-supplied medium where they were grown to the stationary phase. These cells were then cultured in EPO-free medium for 24 h. A defined number of such cells are then incubated with the test peptide for approximately 10 min at 37 ° C. A control sample of EPO cells is also introduced into each assay. The treated cells were then harvested by centrifugation, resuspended in SDS lysis buffer, and subjected to SDS polyacrylamide gel electrophoresis. Electrophoresis proteins in the gel are converted to nitrocellulose and proteins containing phosphotyrosine to blot which are examined using standard immunological techniques. For example, the blot can be tested with anti-phosphotyrosine antibodies (e.g., anti-phosphotyrosine IgG from mice, manufactured by Upstate Biotechnology, Inc.), washed, and then tested with secondary antibodies [e.g., peroxidase-labeled anti-mouse IgG from goats by Kirkegaard &amp; Reggu Laboratories, Inc. (Washington, DC)]. Thereafter, phosphotyrosine-containing proteins can be visualized using standard techniques including colorimetric, chemiluminescent, or fluorescent assays. For example, chemiluminescent analysis can be performed using the ECL West Blotting System manufactured by Amersham.

Another in vitro cell-based assay that can be used to assess the peptide activity of the present invention is colony analysis, which is performed using bone marrow cells from mice or human peripheral blood cells. Mouse bone marrow can be obtained from the femur of mice while being sampled

51130 Β Human blood peripherals can be obtained from healthy donors. In the case of peripheral blood, mononuclear cells are first isolated from the blood, for example, by centrifugation over a FicollHypaque gradient [Stem Cells Technologies, Inc. (Vancouver, Canada)]. For this analysis, a nucleated cell count is performed to determine the number and concentration of nucleated cells in the original sample. The defined number of cells is coated with methyl cellulose according to the manufacturer's instructions [Stem Cells Technologies, Inc. (Vancouver, Canada)]. One experimental group was treated with the peptide tested, the positive control group was treated with EPO and the negative control group was not treated. The number of growing colonies for each group is then recorded after defined incubation periods, generally after 10 days and 18 days. If active, the peptide will promote colony formation.

Other m virobiological assays that can be used to test the activity of a compound of the present invention are described in Greenberger, isar (1983) Proc. Natl. Acad. Sci. USA 80: 2931 - 2935 (EPO-dependent hematopoietic progenitor 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 (Tyrosine phosphorylation of EPO receptors in human EPOsensitive cells); Quelle, isar (1992) J. Biol. Chem. 267: 17055-17060 (Tyrosine phosphorylation of cytosolic proteins, p. 100, in FDC-ERcells); Wortington, isar. (1987) Ehr. Hematol. 15: 85 - 92 (colorimetric analysis for hemoglobin); Kaiho and Miuno (1985) Anal. Biochem. 149: 117 -120 (detection of hemoglobin with 2,7-diaminofluorene); Patel, et al (1992) J. Biol. Chem. 267: 21300 - 21302 (c-mib expression); Witthuhn, et al (1993) Cell 74: 227-236 (association and tyrosine phosphorylation JAK2); Leonard, 7sar (1993) Blood 82: 1071–1079 (expression of GATA transcription factors); and Ando, ​​et al (1993) Proc. Natl. Acad. Sci. USA 90: 9571 - 9575 (regulation of Gi transition by cycling D2 and D3).

An instrument designed by Molecular Devices Corp., known as a microphysiometer, has been used successfully to measure the effects of agonists and antagonists on various receptors. The basis of this apparatus is the measurement of changes in the acidification degree of the extracellular medium in response to receptor activation.

In vivo functional tests

One in wVofunctional assay that can be used to test the potency of a tested peptide is a bioassay of polycythemic exhypoxic mice. For this analysis, mice are subjected to an alternately conditioned cycle for several days. In this cycle, mice are alternated between periods of hypobaric conditions and ambient pressure conditions. After that, the mice are maintained at ambient pressure for 2-3 days before the application of the tested samples. The tested peptide samples, or EPO standards in the case of mice from the positive control group, are injected subcutaneously into conditioned mice. Radiolabeled iron (eg Fe⁵⁹) is applied 2 days later and blood samples are taken two days after the application of radiolabeled iron. Hematocrit and radioactivity measurements are then determined for each blood sample using standard techniques.

51130Β

Blood samples from mice injected with the active test peptides showed higher radioactivity (due to Fe binding⁵⁹ for hemoglobin from erythrocytes) than mice that did not receive tested peptides or EPO.

Another in vivo functional assay that can be used to determine the potency of a tested peptide is reticulocyte assay. For this analysis, 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 were collected from mice. The percentage (%) of reticulocytes in the blood is determined with the help of thiazole orange staining and flow cytometric analysis (reticcount program). In addition, hematocrits are determined manually. The percentage of corrected reticulocytes is determined using the following formula:% RETIK_{K0R [G0V}an! h '% RFTIK_{Ђ (}) b [je \ ih X (Hematocrit_NDI\_4DUALNi / Hematocrit_N0RMAL) Active tested compounds show an increase in the levels of% RETIC corrected ^u compared to mice that did not receive tested peptides or EPO.

Use of an EPO-R peptide agonist of the invention

The peptide compounds of the invention are useful in vitroaridm for understanding the biological role of EPO, including the assessment of many factors thought to affect and that influence EPO production and EPO binding to EPO-R (e.g., EPO mechanism). / ERO-R signal transduction / receptor activation). The peptides of the invention are also useful for the development of other EPO-R-binding compounds, as the compounds of the invention provide important information about the structure-activity relationship that facilitates this development.

In addition, based on their ability to bind to EPO-R, the peptides of the present invention can be used as reagents for the detection of EPO-R in living cells; fixed cells; in biological fluids; in tissue homogenates; in purified, natural biological materials; etc. For example, by labeling such peptides, cells that have EPO-R on their surfaces can be identified. In addition, based on their ability to bind to EPO-R, the peptides of the present invention can be used in in situ staining, FACS (separation by fluorescence-activated cells) assays, Western blotting, ELISA (enzyme-linked immunosorbent assays), and td. Additionally, based on their ability to bind to EPO-R, the peptides of the present invention can be used in the purification of receptors or in the purification of cells that express EPO-R on the cell surface (or within permeabilized cells).

The peptides of the invention may also be used as commercial reagents for various medical research and diagnostic purposes. Such uses may include, but are not limited to; (1) use as a calibration standard to quantify the activity of possible EPO-R agonists in various functional assays; (2) use as blocking reagents in random peptide screening, i.e., in the search for new families of EPO-R peptide ligands, peptides can be used to block the release of EPO peptides from the present invention; (3) use in

51130 Β co-crystallization with EPO-R, i.e., EPO-R-bound peptide crystals of the invention can be formed, allowing determination of the receptor / peptide structure by X-ray crystallography; (4) use in measuring the capacity of erythrocyte precursor cells to induce depth synthesis and heme complex synthesis and to increase the number of ferritin receptors, by initiating differentiation; (5) use in maintaining the proliferation and growth of EPO-dependent cell lines, such as FDCP-1-mEPO-Ri TF-1 cell lines; (6) using related peptides of the invention with radioactive chromophores; and (7) other research applications in which it is preferred that the EPO-R is activated or such activation is typically calibrated to a known amount of EPO-R agonist and the like.

In further aspects of the present invention, methods for the treatment and manufacture of medicaments are provided. The peptide compounds of the invention may be administered to warm-blooded animals, including humans, to stimulate EPO binding to EPO-R in vivo. Thus, the present invention encompasses methods for the therapeutic treatment of diseases associated with EPO deficiency, wherein the methods comprise administering the peptides of the invention in amounts sufficient to stimulate EPO-R and thereby alleviate the symptoms resulting from 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 failure / dialysis; anemia associated with AIDS; anemia associated with chronic inflammatory diseases (e.g., rheumatoid arthritis and chronic intestinal inflammation) and autoimmune diseases; and to increase the number of red blood cells in patients before surgery. Other disease states, disorders and conditions of haematological abnormalities that can be treated using the peptides of the present invention include: beta-thalassemia; cystic fibrosis; diseases of pregnancy and menstrual diseases; early anemia in preterm infants; spinal cord injuries; space flights; acute blood loss; aging; stroke, ischemia (both CNS and cardiac); and various neoplastic disease states associated with abnormal erythropoiesis.

In other embodiments, the peptide compounds of the invention may be used to treat diseases not characterized by low levels or deficiency of red blood cells, for example as in the treatment prior to transfusions. Additionally, administration of a compound of the present invention may result in a reduction in bleeding time and thus, may find use in patients prior to surgery or when there are indications that bleeding is expected to occur. In addition, the compounds of the present invention may find use in the activation of megakaryocytes.

Since EPO has been shown to have a mitogenic and chemotactic effect on vascular endothelial cells as well as an effect on central cholinergic neurons [see e.g., Amagnostou, isar (1990) Proc. Natl. Acad, Sci. USA 87: 5978 - 5982 and Konishi, isar (1993) Brain Res. 609: 29-35], the compounds of the present invention may also find use in the treatment of various vascular diseases, such as: improving wound healing; improving the growth of lateral coronary blood vessels (such as those that may occur after myocardial infarction); injury treatment; and post-vascular graft treatment. The compounds of the present invention may also find use in the treatment of various neurological

51130 Β disorders, which are generally characterized by low absolute levels of acetyl choline or low relative levels of acetyl choline compared to other neuroactive substances e.g., neurotransmitters.

Pharmaceutical compositions

In another aspect of the present invention, pharmaceutical compositions of the aforementioned EPO-R agonist peptide compounds are provided. Conditions that are alleviated or modulated by the use of these compositions include those listed previously. Such pharmaceutical compositions may be for administration by oral, parenteral, intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passive or using iontophoresis or electroporation), transmucosal (nasal, vaginal, rectal or sublingual) use. inserts and can be formulated in conjunction with forms suitable for each mode of application. Generally within the scope of the invention are pharmaceutical compositions comprising effective amounts of EPO-R agonist peptides or derivative products of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions include diluents with various buffers (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; additives such as detergents and solubilizing agents (eg Tween 20, Tween 80, Polysorbate 80), anti-oxidants (eg ascorbic acid, sodium metabisulphite), preservatives (eg Timerzol, benzyl alcohol) and fillers (e.g., lactose, mannitol); the incorporation of the material into particles of polymeric compounds such as polylactic acid, polyglycolic acid, etc. can be performed. or in liposomes. Hyaluric acid can also be used. Such compositions may affect the physical state, stability, degree of release, and degree of clearance of proteases and derivatives of the present invention. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA18042) pages 1435 1712 which are incorporated herein by reference. The compositions may be in liquid form or may be in the form of dry powders (eg lyophilized).

Oral administration

The use of oral solid dosage forms, which are described in the generabio in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton PA18042) in Chapter 89, which is incorporated herein by reference. Solid dosage forms include tablets, capsules, pills, trochees or lozenges, flakes, pellets, powders or granules. Also, liposomal or proteinoid encapsulation can be used to formulate a composition of the present invention (such as, for example, the proteinoid microspheres disclosed in U.S. Patent No. 4,925,673). Liposomal encapsulation can be used and liposomes can be derivatized with

51130 Β various polymers (e.g., U.S. Patent No. 5,013,556). A description of possible solid dosage forms for therapeutics is given by Marshall, K. in: Modem Phaiznaceutics№ \ te &amp; by GS Banker and CT Rhodes Chapter 10,1979, which is incorporated herein by reference. In general, the formulation will include EPOR agonist peptides (or their chemically modified forms) and inert ingredients that provide protection from environmental conditions in the stomach and the release of biologically active materials in the intestinal tract.

It is also contemplated herein to use liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions and syrups, which may contain other components including inert diluents; adjuvants such as wetting agents, emulsifiers and suspending agents; and sweeteners, flavors and fragrances.

Peptides can be chemically modified so that oral administration of the derivative is effective. In general, a chemical modification in question is the binding of at least one moiety to a component of the molecule itself, wherein said moiety allows (a) inhibition of proteolysis; and (b) introducing blood into the bloodstream from the stomach or intestine. It is also desirable to increase the overall stability of the component or components and to speed up the circulation time in the body. As noted earlier, PEGylation is a preferred chemical modification for pharmaceutical use. Other possible uses include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-thioxocane [see e.g. , Abuchovsky and Davis (1981) Soluble Polymer-Enzyme Adducts, in Enzymes asDrugs. Hozenberg and Roberts, eds. (Wiley-Interscience: New York, NY) p. 367 - 383; and Newmark, isar. (1982) J. Appl. Biochem. 4: 185 -189].

For oral formulations, the sites of release may be the stomach, small intestine (duodenum, jejunum or ileum) or colon. Someone with experience in the field knows which formulations will not dissolve in the stomach but will release material in the duodenum or elsewhere in the intestines. Preferably, the release is protected from the adverse effects of the gastric environment, either by protecting the peptide (or derivative) or by releasing the peptide (or derivative) after the gastric medium, such as e.g. in the intestines.

To ensure complete gastric resistance for at least pH 5.0, an impermeable coating is essential. Examples of more common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Cetazole Aquateric , Eudragit S and Selak. These coatings can be used as mixed films.

The coating or coating mixtures can also be used on tablets, which do not have the role of protection against the stomach. These may include sugar coatings or coatings that make the tablets easier to swallow. Capsules may consist of a hard shell (such as gelatin) for dry administration

51130 Β Therapeutics (i.e., powders), and soft gelatin coatings can be used for liquid forms. The flake wrapping material may be a starch coating or other edible paper. For pills, lozenges, molded tablets or crushed tablets, the wet mixing technique can be used.

The peptide (or derivative) can be included in the formulation as tiny multiparticulates in the form of granules or pellets with a particle size of about 1 pip. The formulation material for administration in the form of capsules may also be powder, lightly compressed pieces or even tablets. These therapeutics can be produced by compression.

Colorants and / or flavoring agents may also be included. For example, a peptide (or derivative) may be formulated (e.g., as a liposome or by encapsulation microspheres) and then housed within an edible product, such as chilled beverages containing color and flavoring agents.

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

Disintegrants may be included in the formulation of solid dosage forms. Materials used as disintegrants include, but are not limited to, starch, including the commercial starch-based disintegrant, Explotab. Starch sodium glycolate, Amberlite, sodium carboxymethyl cellulose, ultramilopectin, sodium alginate, gelatin, orange peel, acidic carboxymethyl cellulose, natural sponge and bentonite can also be used. Disintegrating agents may also be insoluble cation exchange resins. Powdered rubbers can be used as disintegrating agents and as binders (binders) and may include powdered rubbers such as agar, Karaja Tragacanth. Alginic acid and its sodium salts are also useful as a disintegrant.

Binders can be used to hold peptides (or derivatives) and agents together to form hard tablets and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) can be used in alcoholic solutions to granulate peptides (or derivatives).

Anti-friction agents may be included in the formulation of the peptide (or derivative) to prevent sticking during the formulation process. Lubricants may be used as a layer between the peptide (or derivative) and the mold wall and they 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 can also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Karbovax 4000 and 6000.

51130 Β

Sliding agents (glidants), which can improve the flow properties of the drug during formulation and to help rearrange during compression, can be added. Glidants may include starch, talc, pyrogenic silicon, and hydrated silicoaluminate.

Surfactants (surfactants) can be added as a wetting agent to aid in the dissolution of the peptide (or derivative) in the aqueous medium. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents may also be used and may include benzalkonium chloride or benzetomy chloride. The list of potential non-ionic detergents that can be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10.50 and 60, glycerol monostearate, polysorbate 20,40,60,65 and 80, sucrose methyl fatty acid esters cellulose and carboxymethyl cellulose. These surfactants may be present in the formulation of proteins or derivatives either individually or as a mixture with different ratios.

Additives that potentially enhance the yield of peptides (or derivatives) are, for example, fatty acids, oleic acid, linolenic acid and linoleic acid.

Controlled release of formulations may be desirable. The peptide (or derivative) can be incorporated into an inert matrix that allows release either by diffusion or by wetting mechanisms, e.g., into gums. Slow-decomposing matrices can also be incorporated into the formulation. Some enteric coatings also delay the release effect. Another form of controlled release is using a method based on the Oros therapeutic system (Alza Corp.), that is, the drug is surrounded by a semipermeable membrane that allows water to enter and push the drug through a small opening, using osmotic effects.

Other coatings can be used for formulation. These include various sugars that can be applied to the coating pan. The peptide (or derivative) can also be administered in the form of a film-coated tablet and the materials used in this case are divided into 2 groups. The first group are non-enteric materials and includes methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxymethyl cellulose, providon and polyethylene glycols. The second group consists of enteric materials which are usually phthalic acid esters.

Mixtures of materials can be used to obtain optimal film coatings. Film-coated coatings can be made in coating vessels or in a fluidized bed III using compressed coating.

Parenteral administration

Compositions of the present invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or carriers 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

51130Β Preservatives, wetting, emulsifying and dispersing agents. They can be sterilized by, for example, filtration through bacterial filters, by inserting sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be produced using sterile water or other sterile injectable media, immediately before use.

Rectal or vaginal administration

Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or suppository wax. Compositions for nasal or sublingual administration are also produced with standard excipients well known in the art.

Pulmonary application

Also provided herein is the administration of EPO-R agonist peptides (or derivatives thereof) to pulmonaries. The peptide (or derivative) is used for pulmonary inhalation in mammals and passage through the pulmonary epithelium into the bloodstream [see, e.g., Adjei, isar (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 (sup 5): 143-146 (endothelin-1); Hubbard, isar (1989) Annals of Intemal Medicine, Vol. III, p. 206-212 (al-antitrypsin); Smith, 7sar (1989) J. Clin. Invest. 84: 11451146 (α-1-proteinase); Oswein, isar (1990) Aerosolization of Proteins, Proceedings of Symposium on Respiratory Drug Delivery II Keystone, Colorado (recombinant human growth hormone); Debs, isar (1988) J. Imunol. 140: 3482 - 3488 (interferon-γ and tumor necrotic factor a); and U.S. Pat. Br. No. 5,284,656 to Platz, isar (granulocyte colony stimulating factor). A method and composition for the pulmonary administration of drugs for systemic effects is described in U.S. Pat. Br. No. 5,451,569 to Wong, et al.

Intended for use in the present invention are many mechanical devices for the pulmonary administration of therapeutic products, including but not limited to nebulizers, meme dose inhalers, and powder inhalers, all known to those skilled in the art. Some specific examples of commercially available devices suitable for use according to the present invention are the Ultravent sprayer (Mallinckrodt Inc., St. Louis, MO); Akom II Atomizer (Marquest Medical Product s, Englewood, CO); Ventolin meme dose inhaler (Glaxo Inc., Research Triangle Park, NC); and Spinhaler powder inhaler (Fizons Corp., Bedford, MA).

All of these devices require the use of formulations that are suitable for the distribution of peptides (or derivatives). Typically, each formulation is specific to the type of device used and may involve the use of appropriate propellant materials, in addition to conventional diluents, adjuvants and / or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes or other types of carriers is contemplated. Chemically modified peptide can also be obtained in different formulations depending on the type of chemical modification or the type of device

51130 Β which applies.

A formulation suitable for administration with a nebulizer, whether jet or ultrasonic, typically contains a peptide (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include buffer and individual sugar (e.g., for protein stabilization and osmotic pressure regulation). The spray formulation may also contain a surfactant, to reduce or prevent surface-induced aggregation of the peptide (or derivative) resulting in atomization of the solution in the form of an aerosol.

Formulations for use with meme dosage inhalers generally comprise a finely divided powder containing a peptide (or derivative) suspended in a propellant by a surfactant. The propellant may be any conventional material for use for these purposes, such as chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon or hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane or a combination thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid can also be used as a surfactant,

Formulations for distribution from powder inhaler devices comprise a finely divided dry powder containing a peptide (or derivative) and may also include excipients such as lactose, sorbitol, sucrose or mannitol in amounts that facilitate dispersion of the powder from the device, e.g., 50 to 90% by weight of the formulation. The peptide (or derivative) must be suitably prepared in a special form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm for the most effective application to the distal lungs.

Nasal application

Nasal administration of an EPO-R peptide agonist (or derivative) is also contemplated. Nasal administration allows the peptide to pass into the bloodstream immediately after administration of the therapeutic manufacturer in the nose, without the need for the product to penetrate the lungs. Formulations for nasal administration include those with dextran or cyclodextran.

Doses

For all peptide compounds, as further studies are conducted, information will emerge in relation to appropriate dose levels for the treatment of different conditions in different patients and a worker with usual experience, considering the therapeutic context, age and general health of the recipient, will be able to establish appropriate dosage. . The selected dose depends on the desired therapeutic effect, the method of administration and the duration of the desired treatment. General dose levels from 0.001 to 10

51130Β mg / kg body weight are administered daily in mammals. Generally, for intravenous injections or infusions, the doses may be lower. The dosage schedule may vary, depending on the half-life of the circulation and the formulation used.

The peptides of the present invention (or derivatives thereof) may be administered together with one or more additional active ingredients or pharmaceutical compositions.

Examples

The present invention is further described by the following examples. However, the use of these other examples anywhere in the specification is illustrative only and in no way limits the field and meaning of the invention or any of the above forms. Likewise, the invention is not limited to any particular priority solution described herein. In fact, many modifications and variations of the invention may be apparent to one skilled in the art upon reading these specifications and may be made without departing from the scope and scope of the invention. The invention is therefore limited only by the conditions of the appended claims, together with the full fields of equivalents for which the claims are made.

Example 1: Synthesis of EPO-R agonist peptide dimers by solid-phase synthesis

Step -Synthesis of Cbz-TAP: A solution containing commercially available diamine (TAP manufactured by Aldrich Chemical Co.) (10g, 67.47mmol) in anhydrous DCM (100 mL) was cooled to 0 ° C. A solution of benzyl chloroformate (4.82ml, 33.7mmol) in anhydrous DCM (50ml) was added slowly through a dropping funnel over a period of 6-7 h, maintaining the temperature of the reaction mixture at 0 ° C until complete, then allowing to warm to room temperature (~ 25 ° C). After a further 16 h, the DCM was removed in vacuo and the residue was partitioned between 3N HCl and ether. The aqueous layers were collected and neutralized with 50% aq. NaOH to pH 8-9 and extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous Na₂SO4, then concentrated in vacuo to give crude mono-Cbz-TAP (5g, about 50% yield). This compound was used for the next reaction without any further purification.

NHCbz

<img file="RS51130B_D0051.tif" />

Cbz-Cl, DCM

Kogak 2 - Synthesis of Cbz-TAP-Bok: To a vigorously mixed suspension with Cbz-TAP (5g, 17.7mmol) in hexane (25ml) was added Bok₂O (3.86g, 17.7mmol) and stirring was continued at RT overnight.

51130 Β

The reaction mixture was diluted with DCM (25ml) and washed with 10% aq. citric acid (2X), water (2X) and saline. The organic layer was dried over anhydrous Na2SO₄ and concentrated in vacuo. The crude product (yield 5g) was used directly in the next reaction.

NHCbz

()

Hi₂0, Hexane

NHCbz nh₂

<img file="RS51130B_D0052.tif" />

NHBok

KogakZ-Sin thesis Bok-TAP: The crude product from the previous reaction was dissolved in methanol (25 ml) and hydrogenated in the presence of 5% Pd on Carbon (5% w / w) under balloon pressure for 16 h. The mixture was filtered, washed with methanol and the filtrate was concentrated in vacuo to give the crude H-TAP-Boc product (yield 3.7g). The total approximate yield of Bok-TAP after Steps 1-3 was 44% (calculated based on the amounts of Cbz-Cl used).

NHCbz

<img file="RS51130B_D0053.tif" />

NHBok

Pd-C, H₂, MeOH

--&gt;►

<img file="RS51130B_D0054.tif" />

Step 4- Synthesis of TentaGel-IJnker: TenteGe [bromide (2.5 g, 0.48 mmol / g, Rapp Polymers, Germany), phenolic linker (5 equivalents) and K₂SO 2 (5 equivalents) was heated in 20 mL of 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-colored resin.

<img file="RS51130B_D0055.tif" />

<img file="RS51130B_D0056.tif" />

Step 5-Synthesis of TentaGel-linker-TAP (Boc): 2, [&gt; g of the above resin and H-TAP-Boc (1.5 g, 5 eq.) And glacial AcOH (34 μΐ, 5 eq.) Were added in a 1: 1 mixture of MeOH-THF and stirred overnight. A 1M solution of sodium cyanoborohydride (5 eq.) In THF was added to this and everything was stirred for a further 7 h. The resin was washed by filtration (DMF, THF, 0.1 N HCl, water, MeOH) and dried. A small amount of resin was benzolated with Bz-Cl and DIEA in DCM and separated with 70% TFA-DCM and verified by LCMS and HPLC.

51130 Β

<img file="RS51130B_D0057.tif" />

<img file="RS51130B_D0058.tif" />

Step 6- Synthesis of TentaGel-linker-TAF-IJz: The resin obtained above was treated with activated solution of Fmok-Liz (Fmok) -OH (obtained from 5 eq. Of amino acid and 5 eq. Of HA.TU dissolved to 0.5 M in DMF , after which 10 eq of DIEA was added) and left to stir gently for 14 h. The resin was washed (DMF, THF, DCM, MeOH) and dried to give a protected resin. Residual amino groups were captured by treating the resin with a solution of 10% acetic anhydride, 20% pyridine in DCM for 20 minutes, after which it was washed as above. Fmok groups were removed by gently stirring the resin in 30% piperidine in DMF for 20 minutes, after which it was washed (DMF, THF, DCM, MeOH) and dried.

<img file="RS51130B_D0059.tif" />

1) Fmok-Liz (Fmok) -OH DIC, HOBt

2) Piperidine -------&gt;

<img file="RS51130B_D0060.tif" />

<img file="RS51130B_D0061.tif" />

Step 7-Synthesis. TentaGel-Linker-TAP-IIz (Peptide) 2: The resin obtained above was subjected to repeated cycles of Fmok-amino acid coupling with HBTU / HOBt activation and Fmok removal with piperidine to build both peptide chains simultaneously. This is typically performed on an ABI433 automated peptide synthesizer available from Applied Biosystems, Inc. After final Fmok removal, the terminal amino groups were acylated with acetic anhydride (10 eq.) And DIEA (20 eq.) In DMF over 20 minutes, followed by washing as above.

<img file="RS51130B_D0062.tif" />

Step 8 - Separation from the resin: The resin obtained above was suspended in solution with TFA (82.5%), phenol (5%), ethanedithiol (2.5%), water (5%) and thioanisole (5%) in progress. 3 h at room temperature. An alternative separation mixture, such as TFA (95%), water (2.5%) and triisopropylsilane

51130 Β (2.5%) can also be used. The TFA solution was cooled to 5 ° C and placed in Et₂The peptide would precipitate. Filtration and drying under reduced pressure gave the desired peptide. Purification by preparative HPLC with a C18 column gave pure peptide.

<img file="RS51130B_D0063.tif" />

Step 9- Oxidation of the peptide to form an intramolecular disulfide cut / The peptide dimer was dissolved in 20% DMSO / water (1 mg dry weight of the peptide / mL) and allowed to stand at room temperature for 36 h. The peptide was purified by introducing the reaction mixture into a C18 HPLC column (Waters Delta-Pak C18, particle size 5 microns, pore size 300 angstroms, 40 mm x 200 mm length), followed by ACN / water / 0.01% TFA lines. gradient from 5 to 95% ACN over 40 minutes. Lyophilization of the fractions containing the desired peptide gave the product as a fluffy white solid.

HS

SH

Ac-XCYCZ— NH

<img file="RS51130B_D0064.tif" />

DMSO / H 2 O

Ac-XCYCZ - NH

<img file="RS51130B_D0065.tif" />

Dimemi peptide (() containing reduced cysteine ​​residues

Dimemic peptide (() containing oxidized disulfide bonds

Step 10- PEGylation of the -NH2 groups:

PEGylation through precarbamate cuts. The peptide dimer was mixed with 1.5 eq. (molar basis) of activated PEG species (mPEG-NPC manufactured by NOF Corp. Japan) in dry DMF to give a clear solution. After 5 minutes 4 eq. DIEA was added to the stock solution. The mixture was stirred at room temperature for 14 h, after which it was purified by C18 reverse phase HPLC. The structure of the PEGylated peptide was confirmed by MALDI mass. The purified peptide was also subjected to purification by cation exchange chromatography as shown below.

51130 Ι ι ------------------------------------------------ ----------- 1

<img file="RS51130B_D0066.tif" />

PEGylation via amide care / Peptidium dimer was mixed with 1.5 eq. (molar basis) of 1 eq. activated PEG species (PEG-SPA-NHS manufactured by Shearwater Corp, USA) in dry DMF to obtain a clear solution. After 5 minutes 10 eq. DIEA was added to the stock solution. The mixture was stirred at room temperature for 2 h, after which it was purified by C18 reverse phase HPLC.

The structure of the PEGylated peptide was confirmed by MALDI mass. The purified peptide was also subjected to purification by cation exchange chromatography as shown below.

(AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) “NH

<img file="RS51130B_D0067.tif" />

(AcG) GLYACHMGPIT (1-nal) V (j: QPLR (MeG) -NH

<img file="RS51130B_D0068.tif" />

V (AcG) GLYA (!: HMGPIT (l-nal) VCQPLR (MeG) -

PEG-SPA-NHS DIEA DMF

<img file="RS51130B_D0069.tif" />

20K (AcG) GLYAfHMGPIT (1-nal) VfQPLR (MeG) -NH

Step 11 - Ion exchange purification: For several exchange carriers, their ability to separate upper peptide-PEG conjugates from unreacted (or hydrolyzed) PEG, in addition to their ability to retain starting dimetic peptides, was tested. Ion exchange resins (2-3 g) were inserted into a 1 cm column, after which the conversion was performed

51130 Β in the sodium form (0.2 N NaOH was added to the column until the eluent reached pH 14, ca, 5 column volumes) and then in the hydrogenated form (eluted with either 0.1 N HCl or 0.1 M HOAc until the eluent reaches the pH of the charge, about 5 column volumes), which is washed with 25% ACN / water mixture until pH 6. Either the peptide before conjugation or the peptide-PEG conjugate is dissolved in 25% ACN / water mixture (10 mg / mL ) and the pH was adjusted to &lt;3 saTFA, then the column was loaded. After washing with 2-3 column volumes with 25% ACN / leader mixture and collecting 5 mL fractions, the peptide was discharged from the column eluting with 0.1 M NH₄OAc in 25% ACN / water mixture, recovered 5 mL fraction. HPLC analyzes show which fraction contains the desired peptide. Analyzes with the Evaporative Light-scattering Detector (ELSD) show that when the peptide remains on the column and the eluate is eluted with NH solution₄OAc (generally between fractions 4 and 10), no non-conjugated PEGs present as contaminants. When the peptide is eluted in the initial wash buffer (generally the first 2 fractions), no separation of the desired PEG-conjugates is present and an excess of PEG is observed.

The following columns successfully retain both the peptide and peptide-PEG conjugate and successfully purify the peptide-PEG conjugate from unconjugated peptides:

Table 1: Ion exchange resins

<td>Carrier</td><td>Source</td>

<td>Mono S HR 5/5 highly cation exchange pre-filled column</td><td>Amersham Biosciences</td>

<td>SE53 Cellulose, microgranules strongly cation exchange carrier</td><td>Whatman</td>

<td>SP Sepharose fast-flowing high cation exchange carrier</td><td>Amersham Biosciences</td>

Example 2: Synthesis of EPO-R agonist peptide dimers by condensation of fragments

Step - Synthesis (Cbz) z-IJz: Lysine is reacted under standard conditions with a solution of benzyl chloroformate to obtain lysine protected on its two amino groups with the Cbz group.

<img file="RS51130B_D0070.tif" />

Step 2- Synthesis Bok-TARBok-TAP was synthesized as described in Steps 1 to 3 of

Example 1.

51130 Β

Step 3 - Coupling (Cbz) 2-Lizi Bok-TAP: (Cbz) 2-Liz and Bok-TAP were coupled under standard coupling conditions to obtain (Cbz) 2-Liz-TAP-Bok.

<img file="RS51130B_D0071.tif" />

<img file="RS51130B_D0072.tif" />

NHBok

<img file="RS51130B_D0073.tif" />

Step 4- Πζ-ΤΑΡ-Wok: The solid product from the previous reaction was dissolved in methanol (25ml) and hydrogenated in the presence of 5% Pd on carbon (5% w / w) under balloon pressure for 16 h. The mixture was filtered, washed with methanol and the filtrate was concentrated in vacuo to give the crude Liz-TAPBok product.

<img file="RS51130B_D0074.tif" />

Step 5- Synthesis of peptide monomers by fragment condensation: Four peptide fragments of peptide monomer sequences were synthesized using standard techniques. These partially protected fragments were then exposed to two independent coupling series. In the first series, the N-terminal half of the monomer is formed by coupling two peptide fragments, while the C-thermal half of the monomer is formed by coupling the other two peptide fragments. In the second coupling series, the N-terminal and C-terminal halves are coupled to form a fully protected monomer. The monomer was then OBn-deprotected (deprotected) using standard techniques.

51130Β {AcG) -GLYA-OH

I

OtBu

S (Acm)

I

Fmok-GII-MG-OH

I

Trt

S (Acm)

Fmok-PIT- (1-nal) -VCQP-OH

II

OtBu Trt

H2N-LR- (MeG) -OBn k coupling

In coupling

V

S (Acm) i

(AcG) -GLYACHMG-OH II OtBu Trt

S (Acm) i

Ktok-R4-T-O-pa1) -U-S-e-R-1 ^ - (MeS ^ V III

OtBu Trt Pbf Coupling

V

S (Acm)

S (Acra) (AsSg) -Sg-1, u-A-ON-M-St-r-1-T-a-pa1) -U-O0-R-1 ^ K- (MeS) -0Vp I ¹ &gt; II

OtBu Trt OtBu Trt Pbf deprotection OBn

V

S (Acm) S (Acm) 'I (ΑοΟ-ο-υγ-Α-σΗ-Μ-Θ-ρ-ι-τ-α-η ^ -ν-σο-ρ-υκ-ίΜεΟ-οΗ ¹¹ I ¹ L

OtBu Trt OtBu Trt Pbf

Step 6 - Oxidation of peptide monomers to form intramolecular disulfide bonds: OB-deprotected fused peptide monomers are then oxidized under iodine to form intramolecular disulfide bonds between Acm-protected cysteine ​​monomer residues.

S (Acm) S (Acm) (AsO) -S-E-U-A-S-N-M-S-R-1-T- (1-pa1) -U-S - () - R-1 ^ k- (MeS) -ON

II II I

OtBu Trt OtBu Trt PM

I₂ oxidation c

ss

II (AsO) -S-V-U-A-S-N-M- &amp; R-RT- (1-pa1) -U-S- (J-R-1J-E- (Me6) -0N

II III

OtBu Trt OtBu T_rt Pbf

Step 7- Coupling of Liz-TAP-Box oxidized OBn-deprotected monomer to form a peptide dimer: Liz-TAP-Box was coupled for twice the excess oxidized OB deprotected monomer under standard conditions until peptide dimer formation. The peptide dimer was then deprotected under standard conditions.

51130Β

<img file="RS51130B_D0075.tif" />

S ----------------- S

II (AsO) -O-1 ^ rA-S-N-M - &amp; - R-1-T-a-pa1) -U-S-CR-kN- (MeS) -ON OtBu Trt

<img file="RS51130B_D0076.tif" />

(Αεβ) -6-ΥτΥ-Α-σΗ-Μ-04^&gt;-Ι-Τ-α-ηα1) -ν-ϋ-0-Ρ-ΙζΚ- (ΜβΟ — (

<img file="RS51130B_D0077.tif" />

The 8-PEGylation of the deprotected dimer ^ &amp; pr ^ k ^ dsd peptide dimer was then PEGylated as described in Step 10 of Example 1.

Step 9-Ion exchange purification: The PEGylated peptide dimer was then purified as described in Step 11 of Example 1.

Example 3: In vitro activity assays

This example describes various in vitro assays that are useful in assessing the activity and potency of the EPO-R agonist peptide of the invention. The results of these assays indicate that the novel peptides of the present invention bind to EPO-R and activate EPO-R signaling. In addition, the results from these studies indicate that the new peptide compositions show a surprising increase in EPO-R binding affinity and biological activity compared to the EPO mimic peptides described previously.

51130Β

EPO-R agonist peptide dimers were obtained according to the methods shown in Example 1 or Example 2. The potency of these peptide dimers was assessed using a series of in vitro activity assays, including: reporter assay, proliferation assay, competitive binding assay, and C / BFU-e assay. These four analyzes are described in more detail below.

The results of these in vitro activity assays are shown in Table 2.

1. Reporter analysis

This assay is based on reporter cells derived from pre-B cell lines from mice, Baf3 / EpoR / GCSFRfos / lux. These reporter cell lines express chimeric receptors that include the extra-cellular portion of the human EPO receptor for the intra-cellular portion of the human GCSF receptor. These cell lines were further transfected with a phosgene construct containing a promoter-guided luciferase reporter. Activation of this chyme receptor by the addition of an erythropoietic agent leads to the expression of the luciferase reporter gene and thus the production of light afterwards. addition of luciferin substrate luciferin. Thus the level of EPO-R activation in such cells can be quantified by measuring luciferase activity.

Baf3 / EpoR / GCSFR fos / lux cells were cultured in DMEM / F12 medium (Gibco) enriched with 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3 supernatant (WEHI-3 cell culture supernatant, ATCC # TIB-68) and a mixture of penicillin / streptomycin. Approximately 18 h before analysis, cells were starved by transferring them to DMEM / F12 medium enriched with 10% FBS and 0.1% WEHi-3 supernatant. On the day of analysis, cells were washed once with DMEM / F12 medium enriched in 10% FBS (without WEHI-3 supernatant), then 1X10⁶cells / mL cultured in the presence of a known concentration of test peptide or with EPO (R&amp;D Systems Inc., Minneapolis, MN) as a positive control, in DMEM / F12 medium enriched with 10% FBS (without WEHI-3 supernatant). Serial dilutions of the tested peptide were tested simultaneously in this assay. Analytical plates were incubated for 4 h at 37 ° C in a 5% CO2 atmosphere, after which luciferin (Steady-Glo; Promega, Madison, Wi) was added to each well. After a 5-minute incubation, light emission was measured on a PackardTopcount Luminometer (Packard Instrument Co., Downers Grove, HI.). Light values ​​were plotted against the peptide concentrations tested and analyzed using Graph Pad software. The concentration of test peptide leading to half of the maximum light emission is recorded as EC50 [See Table 2: Reporter EC50].

2. Proliferation analysis

This assay is based on a pre-B cell line from mice, Baf3, transfected to express human EPO-R. The proliferation of the resulting cell lines, BaF3 / Gal4 / Elk / EPOR, is dependent on EPO-R activation. The degree of cell proliferation was quantified using MTT, where the signal in the Μ1Τ analysis is proportional to the number of viable cells.

51130 Β

BaF3 / Gal4 / Elk / EPOR cells were cultured in rotating bottles in DMEM / F12 medium (Gibco) enriched with 10% FBS (Hyclone) and 2% WEHI-3 supernatant (ATCC # TIB-68). Cultured cells were fasted overnight, in rotating bottles at a cell density of 1 x 10⁶cell / ml, in DMEM / F12 medium enriched in 10% FBS and 0.1% WEHI-3 supernatant. The smoothed cells were then washed twice with Dulbecco's PBS (Gibco) and resuspended at a density of 1 x 10⁶ ceHja / ml in DMEM / F12 enriched in 10% FBS (without WEHI-3 supernatant). Aliquots of 50 pL (-50,000 cells) of the cell suspension were then inserted, in triplicate, into a 96-well assay plate. Aliquots of 50 pL serial dilutions of tested EPO mimetic peptides or 50 pL EPO (R&amp;D Systems Inc., Minneapolis, MN) or Aranesp® (darbpoeitin alpha, EPOR agonist commercially available from Amgen) in DMEM / F12 medium enriched with 10% FBS (without WEH1-3 supernatant I) was added to 96-well assay plates (final well volume of 100 μL). For example, 12 different dilutions can be tested where the final concentration of test peptide (or control EPO peptide) ranges between 810 pM to 0.0045 pM. The cells in the plates were then incubated for 48 h at 37 ° C. Thereafter, 10 μL MTT (Roche Diagnostics) was added to each culture well and then left in an incubator for 4 h. The reaction was then quenched by the addition of 10% SDS + 0.01 N HCl. The plates were then incubated overnight at 37 ° C. The absorbance at a wavelength of 595 nM, for each well was then measured on a spectrophotometer. Diagrams of absorbance values ​​according to the concentrations of the tested peptide were constructed and the EC50 value was calculated using Graph Pad software. The concentration of the tested peptide leading to half of the maximum absorbance is recorded as EC50 [See Table 2: Proliferation of EC50].

3. Competitive binding analysis

Competitive binding calculations were performed using an analysis in which a light signal is generated as a function of the proximity of two microspheres: the streptavidin donor microsphere carrying the biotinylated EPO-R-binding peptide tracer and the acceptor microsphere to which EPO-R binds. Light is generated by the transfer of non-radiative energy, during which one oxygen is released from the first microsphere during illumination and comes into contact with the released oxygen, causing the second microsphere to emit light. The sets of these microspheres are commercially available (Packard). The proximity of the microspheres is created by binding the EPO-R-binding peptide tracer to EPO-R. The tested peptide that competes with the EPO-R-binding peptide tracer in binding to EPO-R will prevent this binding, leading to a reduction in light emission.

A more detailed presentation of the method is as follows: 4 pL of serial dilutions of the tested EPO-R peptide agonist or positive or negative control are added to wells on a 384 well plate. Thereafter, 2 μL / well of the receptor / microsphere mixture was added. The mixture of receptors and microspheres consists of: 15 pL of 5mg / ml streptavidin donor microspheres (Packard), 15 pL of 5mg / ml monoclonal antibodies abl79 (these antibodies recognize part of the human placental alkaline protein

51130Β phosphatase found in recombinant EPO-R), acceptor microspheres coated with protein A (protein A binds to abl79 antibodies; Packard), 112.5 pL of recombinant EPO-R at a dilution of 1: 6.6 (which was produced in Chinese hamster ovarian cells as a fusion protein in a portion of human placental alkaline phosphatase protein containing abl79 target epitopes) and 607.5 pLAlfaquest buffer (40 mM HEPES, pH 7.4; 1 mM MgCl 3; 0.1% BSA, 0.05 % Tween 20). Everything comes together to mix. 2 pL / well of biotinylated EPO-R-binding peptide tracer, AF33068 (30 nM final concentration) was added. AF33068, an EPO-Binding peptide (see Table 3 Reporter EC50 (pM)), was obtained according to the methods described in Example 1.

AF3306Ž

I ---------------------------------------- 1

Biotin-GGLYACHMGPITWVCQPLRG: k-nh,

Biotui-GGLYACHMGPITWVCQPLRG

Centrifuge for 1 min to mix. 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 data are plotted against peptide concentration and analyzed with Graph Pad or Excel.

The concentration of the tested peptide leading to a 50% reduction in light emission, compared to that obtained without the tested peptide, is recorded as IC50 [See Table 2: AQIC50].

4. C / BFU-e analysis

EPO-R signaling stimulates the differentiation of cell lineage from bone marrow into proliferative precursors of red blood cells.

For this analysis, serial dilutions of the tested peptide were prepared in IMDM medium (Gibco) enriched with 10% FBS (Hyclone). These serial dilutions, or positive control of the EPO peptide, were then added to methylcellulose to give a final volume of 1.5 mL. The mixture of methylcellulose and peptide was then vortexed vigorously. Aliquots (100,000 cells / mL) of human bone marrow CD34 + cells (Poietics / Cambrex) were dissolved. Dissolved cells were slowly iodinated in 0.1 mL of 1 mg / ml DNAse (cell line) in 50 mL tubes. Then, 40-50 mL of IMDM medium was slowly added to the cells: the medium was added dropwise along the side walls of the 50 mL tubes for the first 10 mL and then the remaining volume of medium was slowly drained along the side walls of the tube. The cells were then centrifuged at 900 rpm for 20 min and the medium was carefully removed by gentle aspiration. Cells were resuspended in 1 ml of IMDM medium and cell density per mL was determined on a hemacytometer slide (an aliquot of 10 μL of cell suspension on slide and cell density was an average of X 10,000 cells / ml). The cells were then diluted in IMDM medium to cell density

51130Β of 15,000 cells / mL. 100 μL of diluted cells were then added to each 1.5 mL of methyl cellulose and peptide sample (final cell concentration in analytical medium was 1000 cells / mL) and the mixture was vortexed. Allow the bubbles in the mixture to disappear and then aspirate 1 mL using a blunt-tipped needle. 0.25 mL of aspirated mixture from each sample was added to each of the 4 wells on a 24-well plate (Falcon brand). The plate mixtures were incubated at 37 ° C under an atmosphere of 5% CO2 in a humidified incubator for 14 days. The presence of erythroid colonies was assessed using a phase microscope (5Χ-10Χ lens, final magnification of 100Χ). The concentration of test peptide at which the number of colonies formed is 90% of the maximum, relative to the number obtained with the EPO positive control, is recorded as EC90 [See Table 2: C / BFUs of EC90].

5. Competitive radioligand binding analysis

An alternative radioligand binding binding assay can also be used to measure the IC50 values ​​of the peptides of the present invention. This analysis measures binding¹²⁵EPO for EPOr. Preferably, the analysis is performed according to the following procedure given as an example:

A. Materials

<td>Recombinant Human EPO R / Fc chimera</td><td>• Identification: Recombinant Human EPO R / Fc chimera • Manufacturer: R&amp;D systems (Minneapolis, MN, US) • Catalog number: 963-ER • Lot number: ΕΟΚ033071 • Storage: 4 ° C</td>

<td>Iodized recombinant human Erythropoietin</td><td>• Identification: (3 [¹²⁵I] iodotyrosyl) Erythropoietin, human recombinant, high specific activity, 370 kBq, 10 pCi • Manufacturer; Amersham Biosciences (Piscataway, NJ, US) • Catalog number: 1Μ219-10 pCi • Lot number: • Storage: 4 ° C</td>

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

<td>Analytical 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. Determination of appropriate receptor concentration

One bottle of 50 pg of lyophilized recombinant EPOr extracellular region fused to the Fc portion of human IgG1 was reconstituted in 1 mL of assay buffer. To determine the required amount of receptor for use in the assay, 100 pL of serial dilutions of this receptor preparation is

51130 Β combined with approximately 20,000 cpm in 200 pL of iodinated recombinant human erythropoietin (¹²⁵I-EPO) in 12 x 75 mm polypropylene test tubes. The tubes were capped and gently stirred at 4 ° C overnight on a LabQuake rotary shaker.

The next day, 50 μL of a 50% suspension of Protein-G Sepharose was added to each tube. The tubes were then incubated for 2 hours at 4 ° C, with gentle stirring. The tubes were then centrifuged for 15 min at 4000 RPM (3297 h G) to precipitate protein-G sepharose. The supernatants were carefully removed and discarded. After washing 3 times with 1 mL of assay buffer at 4 ° C, the precipitate was counted in a Wallac Wizard gamma counter. The results were then analyzed and the dilution required to reach 50% of the maximum binding value was calculated.

C. Determination of IC₅$ for the peptide

To determine the IC50 for AF37702, serial dilutions of 100 pL of peptide were combined with 100 pL of recombinant erythropoietin receptor (100 pg / tube) in 12 x 75 mm polypropylene test tubes, then 100 pL of iodinated recombinant human erythropoietin was added.¹²⁵ΙΈΡΟ) into each tube and tubes were capped and gently stirred at 4 ° C overnight.

The next day, tied up ¹²⁵I-EPO was quantified as described above. The results were analyzed and IC50 values ​​were calculated using Graphpad Prism version 4.0, manufactured by GraphPad Software, Inc. (San Diego, CA). The assay was repeated two or more times for each peptide tested, in a total of 3 or more replicates of IC50 determination.

Table 2: 1p in vitro analysis of peptide dimer activity

<img file="RS51130B_D0078.tif" />

51130Β

Example 4: In vivo activity assays

This example describes various in vivo assays useful for assessing the activity and potency of the EPO-R agonist peptide of the invention. EPO-R agonist peptide dimers were prepared according to the methods given in Example 1 or Example 2. In vivo activity of these peptide monomers and dimers were assessed using a series of assays, including bioassays in polycythemic exhypoxic mice and reticulocyte analyzes. These two tests are described in more detail below.

1. Bioanalyzed by polycythemic and exhypoxic mice

Bioanalysis examined the in vivo activity of the tested peptides in polycythemic exhypoxic mice adopted from the method described by Cotes and Bangham (1961), Nature 191: 1065-1010. This assay examines the ability of the test peptide to function as an EPO mimic: that is, to activate EPO-R and induce the synthesis of new red blood cells. The synthesis of red blood cells was quantified on the basis of the incorporation of radiolabeled iron into the hemoglobin of synthesized red blood cells.

BDFl mice were allowed to acclimatize to ambient conditions for 7-10 days. Body weights were determined for all animals and animals with low weight (&lt;15 grams) were not used. Mice were subjected to successive conditioned cycles in the hypobamic chamber for a total of 14 days. Each 24-hour cycle consists of 18 h at 0.40 ± 0.02% atmospheric pressure and 6 h at ambient pressure. After conditioning, mice were maintained at ambient pressure for an additional 72 h before dosing.

Test peptides, or recombinant human EPO standards, were diluted in PBS + 0.1% BSA vehicle (PBS / BSA). Stock solutions of peptide monomers were first dissolved in dimethyl sulfoxide (DMSO). Negative control groups included one group of mice injected with PBS / BSA alone and one group injected with 1% DMSO. The group for each dose contained 10 mice. Mice were injected subcutaneously (into the nape of the neck) with 0.5 mL of the appropriate sample.

Forty-eight hours after injection of the sample, mice were given an intraperitoneal injection of 0.2 ml of Fe⁵⁹ (Dupont, NEN), for a dose of approximately 0.75 pKiria / per mouse. Mice body weights were determined 24 h after Fe⁵⁹ applications and mice were sacrificed 48 h after Fe⁵⁹ applications. Blood was collected from each animal by cardiac puncture and hematocrits were determined (heparin was used as an anticoagulant). Each blood sample (0.2 ml) was analyzed for Fe⁵⁹ incorporation using Packard gamma counters. Insensitive mice (i.e., those mice in which the radioactive incorporation is less than the negative control grape) were eliminated from the appropriate data set. Mice in which hematocrit values ​​were less than 53% of the negative control group were also eliminated.

51130Β

Results were obtained from sets of 10 animals for each experimental dose. The average amount of radioactivity incorporated [amount per minute (CPM)] in blood samples from each group was calculated.

2. Reticulocyte analysis

Normal BDF1 mice were dosed (0.5 mL, injected subcutaneously) for three consecutive days with either EPO control or peptide tested. On the third day, mice were also dosed (0.1 mL, injected intraperitoneally) with iron dextran (100 mg / ml). On day 5, mice were anesthetized with SO₂ and blood samples were taken using cardiac puncture. The percentage (%) of reticulocytes for each blood sample was determined by thiazole orange staining and flow cytometry analysis (retic-count program). Hematocrits were determined manually. The corrected reticulocyte percentage was determined using the following formula:

% RETIKcorrected =% RETIKobtained X (HematocritNDiviDUAL / Hematocrit_NORMALAN)

3. Haematological analysis

Normal CD1 mice were dosed with four weeks of bolus intravenous injection with either an EPO positive control, a tested peptide, or a vehicle. The range of positive control doses and test peptides, expressed as mg / kg, was tested by varying the concentration of active compound in the formulation. The injected volumes are 5 ml / kg. the vehicle control group included twelve animals, while 8 animals were in each of the remaining dosing groups. Daily viability and weekly body weights were recorded.

Dosing mice were fasted and then anesthetized by inhalation of isoflurane and terminal blood samples were collected by cardiac or abdominal aortic puncture, days 1 (for vehicle control mice) and days 15 and 29 (4 mice / group / day). Blood was transferred to a Vacutainer® branch tube, the preferred anticoagulant being ethylenediaminetetraacetic acid (EDTA).

Blood samples were evaluated for endpoints at which red blood cell synthesis and physiology such as hematocrit (Hct), hemoglobin (Hgb), and total erythrocyte count (RBC) were measured using automated clinical analyzers known in the art (e.g., those from the manufacturer). Coulter, Inc.).

Example 5: Synthesis of EPO-R agonist peptide homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPITO-nal) VCQPLRK (SEKID NO: 1)

Step 1 - Synthesis of peptide monomers: Peyhdm monomers were synthesized using a staidard Fmok chemical reaction on an ABI431A peptide synthesizer, using TG-RAM resin (0.18 mmol / g Rapp Polymere, Germany). For the synthesis of peptide monomers with amidated

51130Β carboxy terminus, the fully assembled peptide was isolated from the resin with 82.5% TFA, 5% water, 6.25% anisole, 6.25% ethanedithiol. The deprotected product was filtered from the resin and precipitated with diethyl ether. After complete drying, the product was purified by high performance C18 reverse phase liquid chromatography with an acetonitrile / water gradient in 0.1% trifluoroacetic acid. The structure of the peptide was confirmed by electrospray mass spectrometry. The peptide was dissolved in a 1: 1 solution of DMSO: water at a concentration of 1 mg / mL to support disulfide formation. The product was purified by high performance C18 reverse phase liquid chromatography with an acetonitrile / water gradient in 0.1% trifluoroacetic acid. Peptide monomers can be illustrated as follows:

I ₍ (AcG) GLYACHMGPIT (1-nal) VCQPLRK-NH₂

Step 2- Synthesis of triAinkdonInoglinker: To a solution with diethyl iminoacetate (10.0 g, 52.8 mmol) and Bok-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. The reaction mixture was heated to -10 degrees during the addition, then cooled back to room temperature over 20 minutes. The reaction mixture was allowed to stir overnight and the precipitated diisopropylurea 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 further precipitated urea. The organic phase was placed in a separatory funnel, washed (sat. NaHCO 3a, brine, 0.5 N HCl, brine), dried (MgSO 4), filtered and concentrated under reduced pressure to give the diester product as a colorless oil. . The diester was added to a 1: 1 mixture of MeOH: THF (100 mL) and water (25 mL) was added thereto followed by NaOH (5g, 125 mmol), the pH was measured to be&gt; 10. The reaction mixture was stirred at room temperature for 2 h and then acidified to pH 1 with 6N HCl. The aqueous phase was saturated with NaCl and extracted 4 times with ethyl acetate. The combined organic phase was washed (brine), dried₄) and concentrated under reduced pressure to give a white semi-solid. The solid was dissolved in 50 mL of DCM and 300 mL of hexane was added to this to give a white suspension. The solvent was removed under reduced pressure to give the diacid as a white solid (14.7 g, 91.5% yield in 2 steps). To a solution of diacid (1 g, 3.29 mmol) in 20 mL DMF was added N-hydroxysuccinimide (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 was removed under reduced pressure. The residue was taken up in ethyl acetate and filtered to remove precipitated urea. The organic phase was placed in a separatory funnel, washed (sat. NaHCO.3, brine, 0.5 N HCl, brine), dried (MgSO 4).₄), filtered and concentrated under reduced pressure to give the di-NHS ester product as a white solid (1.12g, 68% yield).

51130Β

<img file="RS51130B_D0079.tif" />

CO₂Et

BokHN

<img file="RS51130B_D0080.tif" />

Ο

<img file="RS51130B_D0081.tif" />

&gt;

BokHN

<img file="RS51130B_D0082.tif" />

<img file="RS51130B_D0083.tif" />

KogakZ-Coupling of trifunctional linker peptide monomers: Z'A coupling for linker, 2 eq. peptide was mixed with 1 eq. of a trifunctional linker in dry DMF to give a clear solution, 5 eq. DIEA was added after 2 minutes. The mixture was stirred at room temperature for 14 h. The solvent was removed under reduced pressure and the crude product was dissolved in 80% TFA in DCM for 30 min to remove the Boc group, followed by purification by C18 reverse phase HPLC. The dimer structure was confirmed by electrospray mass spectrometry. This coupling reaction binds a linker to a nitrogen atom from the ε-amino group from the lysine residue of each monomer.

51130 Β

<img file="RS51130B_D0084.tif" />

TFA

<img file="RS51130B_D0085.tif" />

51130 Β

Step 4 - PEGylation of the peptide dimer:

PEGylation via a carbamate bond. The teptide dimer is mixed. with an equal amount (molar base) of activated PEG species (mPEG-NPC manufactured by NOF Corp. Japan) in dry DMF to obtain a clear solution. After 5 minutes 4 eq. DIEA was added to the stock solution. The mixture was stirred at room temperature for 14 h, followed by purification by C18 reverse phase HPLC. The structure of the PEGylated peptide was confirmed by MALDI mass. The purified peptide was also subjected to purification by cation exchange chromatography as shown below.

<img file="RS51130B_D0086.tif" />

<img file="RS51130B_D0087.tif" />

PEGylation via an amide bond: The peptide dimer was mixed with an equal amount (molar base) of activated PEG species (PEG-SPA-NHS manufactured by Shearwater Corp, USA) in dry DMF to

51130Β đobijanja clear solution. After 5 minutes of 10 eq, DIEA was added to the stock solution. The mixture was stirred at room temperature for 2 h, followed by purification by C18 reverse phase HPLC. The structure of the PEGylated peptide was confirmed by MALDI mass. The purified peptide was also subjected to purification by cation exchange chromatography as shown below.

<img file="RS51130B_D0088.tif" />

Step 5: - Ion exchange peptide purification: The ability to separate the above-obtained pepdd-PEG conjugates from unreacted (or hydrolyzed) PEG, in addition to their ability to retain the starting smoke peptides, was tested on several exchange carriers. Ion exchange resins (2-3g) were placed in a 1 cm column, after which conversion to sodium form (0.2 N NaOH charged to the column was performed until the eluent reached pH 14, ca. 5 column volume) and then in hydrogenated form (eluted or with 0.1 N HCl

51130 Β or with 0.1 Μ HOAc until the eluent reaches the filling pH, approx. 5 column volumes), then washed with 25% ACN / water mixture until pH 6. Either the peptide before conjugation or the peptide-PEG conjugate was dissolved in 25% ACN / water mixture (10 mg / mL) and the pH was adjusted to &lt;3 with TFA, then placed in a column. After washing with 2-3 column volumes with 25% ACN / water mixture and collecting 5 mL fractions, the peptides were discharged from the column eluting with 0.1 M NH₄OAc in 25% ACN / water mixture, recovered fractions of 5 mL. Analyzes by HPLC show which fraction contains the desired peptide. Analyzes with the Evaporation Light-Scattering Detector (ELSD) indicate that when the peptides are retained on the column when eluted with NH4OAC solution (generally between fractions 4il0), no unconjugated PEG as contaminants are present. When the peptides were eluted in the initial wash buffer (generally the first 2 fractions), no separation of the desired PEG-conjugate and excess PEG was observed.

The following columns successfully retain both peptide and peptide-PEG conjugates and successfully purify the peptide-PEG conjugate from unconjugated peptides:

Table 3: Ion exchange resins

<td>Carrier</td><td>Source</td>

<td>Mono S HR 5/5 highly cation exchange pre-filled column</td><td>Amersham Biosciences</td>

<td>SE53 Cellulose, microgranule strongly cation exchange carrier</td><td>Whatman</td>

<td>SP Sepharose fast-flowing high cation exchange carrier</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) VCQPLRK (SEKID NO: 1)

EPO-R agonist peptide homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEKID BR: 2) were synthesized as described in Example 1, except that in Step 1 they were synthesized pep :

(AcG) GLYACHMGPrr (l-nal) VCQPLR (MeG) K

Where PEG is attached to the linker via a carbamate bond, the final product of this synthesis can be illustrated structurally as follows:

51130Β

<img file="RS51130B_D0089.tif" />

Where PEG is attached to the linker via an amide bond, the final product of this synthesis can be illustrated structurally as follows:

<img file="RS51130B_D0090.tif" />

Example 7: In vitro activity assays

This example describes various in vitro assays that are useful in assessing the activity and potency of the EPO-R agonist peptide of the invention. The results of these assays indicate that the new peptides of the present invention bind to EPO-R and activate EPO-R signaling. In addition, the results of these assays indicate that the new peptide compositions show a surprising increase in EPO-R binding affinity and biological activity compared to the EPO mimetic peptides described previously.

EPO-R agonist peptide monomers and dimers were obtained according to the methods given in Example 1 or Example 2. The potency of these peptide dimers was assessed using a series of in vitro activity assays, including: reporter assay, proliferation assay, competitive binding assay, and C / BFU -e analysis. These four analyzes are described in more detail below.

The results of these in vitro activity assays are shown in Table 2.

51130Β

1. Reporter analysis

This assay is based on reporter cells derived from pre-B cell lines from mice, Baf3 / EpoR / GCSFR phos / lux. These reporter cell lines express chimeric receptors that include the extra-cellular portion of the human EPO receptor for the intra-cellular portion of the human GCSF receptor. These cell lines were further transfected with a phosgene construct containing a promoter-guided luciferase reporter. Activation of this chyme receptor by the addition of an erythropoietic agent leads to the expression of the luciferase reporter gene and thus light production upon administration of the luciferase substrate luciferin. Thus, the level of EPO-R activation in such cells can be quantified by measuring luciferase activity.

Baf3 / EpoR / GCSFR fos / lux cells were cultured in DMEM / F12 medium (Gibco) enriched with 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3 supernatant (WEHI-3 cell culture supernatant, ATCC # TIB-68) and a mixture of penicillin / streptomycin. Approximately 18 h before analysis, cells were starved by transferring them to DMEM / F12 medium enriched with 10% FBS and 0.1% WEHI-3 supernatant. On the day of analysis, cells were washed once with DMEM / F12 medium enriched in 10% FBS (without WEHI-3 supernatant), then 1X10⁶cells / mL cultured in the presence of a known concentration of test peptide or with EPO (R&amp;D Systems Inc., Minneapolis, MN) as a positive control, in DMEM / F12 medium enriched with 10% FBS (without WEHI-3 supernatant). Serial dilutions of the tested peptide were tested simultaneously in this assay. Analytical plates were incubated for 4 h at 37 ° C in an atmosphere of 5% CO2, after which luciferin (Steady-Glo; Promega, Madison, Wi) was added to each well. After a 5-minute incubation, light emission was measured on a Packard Topcount Luminometer (Packard Instrument Co., Downers Grove, HI.). Light values ​​were plotted against the peptide concentrations tested and analyzed using Graph Pad software. The concentration of test peptide leading to half of the maximum light emission is recorded as EC50.

2, Proliferation analysis

This assay is based on a pre-B cell line from mice, Baf3, transfected to express human EPO-R. The proliferation of the resulting cell lines, BaF3 / GaI4 / Elk / EPOR, is dependent on EPO-R activation. The degree of cell proliferation was quantified using MTT, where the signal in MTT analysis is proportional to the number of viable cells.

BaF3 / GaI4 / Elk / EPOR cells were cultured in rotating bottles in DMEM / F12 medium (Gibco) enriched with 10% FBS (Hyclone) and 2% WEHI-3 supernatant (ATCC # TIB-68). Cultured cells were smoothed overnight, in rotating bottles at a cell density of 1 x 10⁶cell / ml, in DMEM / F12 medium enriched in 10% FBS and 0.1% WEHI-3 supernatant. Starved cells were then washed twice with Dulbecco's PBS (Gibco) and resuspended at a density of 1 x 10⁶ cell / ml in DMEM / F12 enriched in 10% FBS (without WEHI-3 supernatant). Aliquots of 50 μL (~ 50,000 cells) of the cell suspension were then inserted, in triplicate, into a 96-well assay plate. Aliquots of 50 pL serial dilutions of tested EPO mimics

51130 Β peptide or 50 pL EPO (R&amp;D Systems Inc., Minneapolis, MN) or Aranesp ™ (darbpoeitin alpha, EPOR agonist commercially available from Amgen) in DMEM / F12 medium enriched with 10% FBS (without WEHI-3 supernatant I) were added to analytical wells with 96 wells (final well volume of 100 μΕ). For example, 12 different dilutions can be tested where the final concentration of test peptide (or control EPO peptide) ranges between 810 pM to 0.0045 pM. The cells in the plates were then incubated for 48 h at 37 ° C. Thereafter, 10 μL MTT (Roche Diagnostics) was added to each culture well and then left in an incubator for 4 h. The reaction was then quenched by the addition of 10% SDS + 0.01 N HCl. The plates were then incubated overnight at 37 ° C. The absorbance at a wavelength of 595 nM, for each well was then measured on a spectrophotometer. Diagrams of absorbance values ​​according to the concentrations of the tested peptide were constructed and the EC50 value was calculated using Graph Pad software. The concentration of test peptide leading to half of the maximum absorbance is recorded as the EC50.

3. Analysis of competitive binding

Competitive binding calculations were performed using an analysis in which a light signal is generated as a function of the proximity of two microspheres: the streptavidin donor microsphere carrying the biotinylated EPO-R-binding peptide tracer and the acceptor microsphere to which EPO-R binds. Light is generated by the transfer of non-radiative energy, during which one oxygen is released from the first microsphere during illumination and comes into contact with the release of one oxygen causing the other microsphere to emit light. The sets of these microspheres are commercially available (Packard). The proximity of the microspheres is created by binding the EPO-R-binding peptide tracer to EPO-R. The tested peptide that competes with the EPO-R-binding peptide tracer in binding to EPO-R will prevent this binding, leading to a reduction in light emission.

A more detailed presentation of the method is as follows: 4 pL of serial dilutions of the tested EPO-R peptide agonist or positive or negative control are added to wells on a 384 well plate. Thereafter, 2 [mu] L / well of the receptor / microsphere mixture was obtained. The mixture of receptors and microspheres consists of: 15 μΕ of 5mg / ml streptavidin donor microspheres (Packard), 15 pL of 5mg / ml monoclonal antibodies abl79 (these antibodies recognize part of the human placental alkaline phosphatase protein found in recombinant EPO-R), acceptor microspheres coated with protein A (protein A binds to abl79 antibodies; Packard), 112.5 pL of recombinant EPO-R at a dilution of 1: 6.6 (produced in Chinese hamster ovarian cells as a fusion protein in a portion of human placental alkaline phosphatase protein containing abl79 target epitopes) and 607.5 pLAlfaquest buffer ( 40 mM HEPES, pH 7.4, 1 mM MgCl 3, 0.1% BSA, 0.05% Tween 20). Everything comes together to mix. Add 2 μΕ / ρο well to a biotinylated EPO-R-binding peptide tracer, AF33068 (30 nM final concentration). AF33068, an EPO-R binding peptide (see Table 3 Reporter EC50 (pM)), was obtained according to the methods described in Example 1.

51130Β

AF33068

Biotin-GGLYACHMGPnWVCQPLRG

<img file="RS51130B_D0091.tif" />

k-nh₂

Biotin-GGLYACHMGPITWVCQPLRG /

Centrifuge for 1 min to mix. 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). The light emission data are plotted against the peptide concentration and analyzed with Graph Pad or Excel,

The concentration of the tested peptide that leads to a 50% reduction in light emission, compared to that obtained without the tested peptide, is recorded as IC50.

4. C / BFU-e analysis

EPO-R signaling stimulates the differentiation of cell lineage from bone marrow into proliferative precursors of red blood cells. This assay measures the ability of the tested peptides to stimulate the proliferation and differentiation of precursors of red blood cells from human multipotent bone marrow cell lines.

For this analysis, serial dilutions of the tested peptide were prepared in IMDM medium (Gibco) enriched in 10% FBS (Hyclone). These serial dilutions, or positive control of EPO peptides, were then added to methylcellulose to give a final volume of 1.5 mL. The mixture of methylcellulose and peptide was then vortexed vigorously. Aliquots (100,000 cells / mL) of human bone marrow CD34 + cells (Poietics / Cambrex) were dissolved. Dissolved cells were slowly added to 0.1 mL of 1 mg / ml DNAse (cell line) in 50 mL tubes. Then, 40-50 mL of IMDM medium was slowly added to the cells: the medium was added dropwise along the side walls of the 50 mL tubes for the first 10 mL and then the remaining volume of medium was slowly drained along the side walls of the tube. The cells were then centrifuged at 900 rpm for 20 min. and the medium is carefully removed by gentle aspiration. Cells were resuspended in 1 ml of IMDM medium and cell density per mL was determined on a hemacytometer slide (an aliquot of 10 gL of cell suspension on slide and cell density was an average of X 10,000 cells / ml). Cells were then diluted in IMDM medium to a cell density of 15,000 cells / mL 100 pL of diluted cells were then added to each 1.5 mL of methyl cellulose and peptide sample (final cell concentration in analytical medium was 1000 cells / mL) and the mixture was vortexed. . Allow the bubbles in the mixture to disappear and then aspirate with 1 mL using a blunt-tipped needle. 0.25 mL of aspirated mixture from each sample was added to each of the 4 wells on a 24-well plate (Falcon brand). The plate mixtures were incubated at 37 ° C under an atmosphere of 5% CO₂ in a humid incubator for 14 days. The presence of erythroid colonies was assessed using a phase microscope (5Χ-10Χ lens, final magnification of 100Χ). The concentration of test peptide at which the number of colonies formed is 90% of the maximum, relative to the number obtained with the EPO positive control, is recorded as EC90 [See Table 2: C / BFUs of EC90J.

Table 4: In vitro analysis of peptide dimer activity

<td>C / BFUs EC90 (nM) 1</td><td>6.2 1 i</td>

<td>AQ IC50 (nM)</td><td>1 i</td>

<td>Proliferation EC50 (pM)</td><td>1 1 l</td>

<td>Reporter EC50 (pM)</td><td> 1 1</td>

<td>Peptide dimer</td><td>* os? (9 CL o = G . § A 3 σ fi, Oi σ&gt; g-o θ ' 2; a • z d * &amp; s &amp; ij —Ϋ ·&gt; rn o and § § z s</td>

<td>Label of the compound</td><td>AF36205 1</td>

51130Β

Example 8: Tests of vivo activity

This example describes various in wV &lt;9 assays that are useful for assessing the activity and potency of the EPO-R agonist peptide of the invention. EPO-R agonist peptide dimers were obtained according to the methods described in Example 1. The in vivo activity of these peptide monomers and dimers was assessed using a series of assays, including bioassays in polycythemic exhypoxic mice and reticulocyte analyzes. These two analyzes are described in more detail below.

1. Bioanalysis in polydemic exhypoxic mice

Bioanalysis examined the in vivo activity of the tested peptides in polycythemic exhypoxic mice adopted from the method described by Cotes and Bangham (1961), Nature 191: 1065-1010. This assay examines the ability of the test peptide to function as an EPO mimic: that is, to activate EPO-R and induce the synthesis of new red blood cells. The synthesis of red blood cells was quantified based on the incorporation of radiolabeled iron into hemoglobin synthesized red blood cells.

BDFl mice were allowed to acclimatize to ambient conditions for 7-10 days. Body weights were determined for all animals and animals with low weight (&lt;15 grams) were not used. Mice were subjected to successive conditioned cycles in the hypobamic chamber for a total of 14 days. Each 24-hour cycle consists of 18 h at 0.40 ± 0.02% atmospheric pressure and 6 h at ambient pressure. After conditioning, mice were maintained at ambient pressure for an additional 72 h before dosing.

Test peptides, or recombinant human EPO standards, were diluted in PBS + 0.1% BSA vehicle (PBS / BSA). Stock solutions of peptide monomers were first dissolved in dimethyl sulfoxide (DMSO). Negative control groups included one group of mice injected with PBS / BSA alone and one group injected with 1% DMSO. The group for each dose contained 10 mice. Mice were injected subcutaneously (into the nape of the neck) with 0.5 mL of the appropriate sample.

Forty-eight hours after injection of the sample, mice were given an intraperitoneal injection of 0.2 ml of Fe⁵⁹ (Dupont, NEN), a dose of approximately 0.75 pKiria / per mouse. Mice body weights were determined 24 h after Fe⁵⁹ applications and mice were sacrificed 48 h after Fe⁵⁹ applications. Blood was collected from each animal by cardiac puncture and hematocrits were determined (heparin was used as an anticoagulant). Each blood sample (0.2 ml) was analyzed for Fe⁵⁹ incorporation using Packard gamma counters. Insensitive mice (i.e., those mice in which the radioactive incorporation is less than the negative control group) were eliminated from the appropriate data set. Mice in which hematocrit values ​​were less than 53% of the negative control group were also eliminated.

Results were obtained from sets of 10 animals for each experimental dose. The average amount of radioactivity incorporated [amount per minute (CPM)] in blood samples from each group was calculated.

51130 Β

2. Reticulocyte analysis

Normal BDF1 mice were dosed (0.5 mL, injected subcutaneously) for three consecutive days with either EPO control or peptide test. On the third day, mice were also dosed (0.1 mL, injected intraperitoneally) with iron dextran (100 mg / ml). On the fifth day, the mice were anesthetized with CO? and blood samples were taken using cardiac puncture. The percentage (%) of reticulocytes for each blood sample was determined by thiazole orange staining and flow cytometry analysis (retic-count program). Hematocrits were determined manually. The corrected percentage of reticulocytes was determined using the following formula:

% RETIKrogirovani =% RETIKdobueno X (HematocritNDiviDUAL / HematocritNORMALAN)

3. Haematological analysis

Normal CD1 mice were dosed with four weeks of bolus intravenous injection with either an EPO positive control, a tested peptide, or a vehicle. The range of positive control doses and test peptides, expressed as mg / kg, was tested by varying the concentration of active compound in the formulation. The injected volumes are 5 ml / kg. the vehicle control group included twelve animals, while 8 animals were in each of the remaining dosing groups. Daily viability and weekly body weights were recorded.

Dosing mice were fasted and then anesthetized by inhalation of isoflurane and terminal blood samples were collected by cardiac or abdominal aortic puncture on day 1 (for vehicle control mice) and days 15 and 29 (4 mice / group / day). Blood transferred to Vacutainer® brand tubes. The preferred anticoagulant is ethylenediaminetetraacetic acid (EDTA).

Blood samples were evaluated for endpoints at which red blood cell synthesis and physiology such as hematocrit (Hct), hemoglobin (Hgb), and total erythrocyte count (RBC) were measured using automated clinical analyzers known in the art (e.g., those of manufactured by Coulter, Inc.).

Example 9: Synthesis of EPO-R aganist peptide homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPrT (1-nal) VCQPLRK (SEKID NO: 1)

Step 1 - Synthesis of peptide monomers: Peptide monomers were synthesized using a standard Fmok chemical reaction on an ABI431Α peptide synthesizer, using TG-RAM resin (0.18 mmol / g Rapp Polymere, Germany). For the synthesis of peptide monomers with an amidated carboxy terminus, the fully assembled peptide was isolated from the resin with 82.5% TFA, 5% water, 6.25% anisole, 6.25% ethanedithiol. The deprotected product was filtered from the resin and precipitated with diethyl ether. After complete drying, the product was purified by C18 reverse phase liquid chromatography

51130 Β High performance with acetonitrile / water gradient in 0.1% trifluoroacetic acid. The structure of the peptide was confirmed by electrospray mass spectrometry. Peptide monomers can be illustrated as follows:

(AcG) GLYACHMGPIT (1-nal) YCQPLRK-NH2

Step 2- Synthesis of a trifunctional linker:

To a solution of diethyl iminoacetate (10.0 g, 52.8 mmol) and Bok-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. The reaction mixture was heated to -10 degrees during the addition, then cooled back to room temperature over 20 minutes. The reaction mixture was allowed to stir overnight and the precipitated diisopropylurea was filtered off. The solvent was removed under reduced pressure to rub the gum and the residue was dissolved in ethyl acetate and filtered again to remove further precipitated urea. The organic phase was placed in a separatory funnel, washed (sat. NaHCO 3)₃, brine, 0.5 N HCl, brine), dried₄), filtered and concentrated under reduced pressure to give the diester product as a colorless oil. The diester was added to a 1: 1 mixture of MeOH: THF (100 mL) and water (25 mL) was added thereto followed by NaOH (5g, 125 mmol). The pH was measured to be&gt; 10. The reaction mixture was stirred at room temperature for 2 h and then acidified to pH 1 with 6N HCl. The aqueous phase was saturated with NaCl and extracted 4 times with ethyl acetate. The combined organic phase was washed (brine), dried₄) and concentrated under reduced pressure to give a white semi-solid. The solid was dissolved in 50 mL of DCM and 300 mL of hexane was added to this to give a white suspension. The solvent was removed under reduced pressure to give the diacid as a white solid (14.7 g, 91.5% yield in 2 steps). To a solution of the diacid (1 g, 3.29 mmol) in 20 mL of DMF was added N-hydroxysuccinimide (770 mg, 6.69 mmol) and diisopropylcarbodiimide (1.00 mL, 6.38 mmol) and 4-dimethylamino- pyridine (3 mg, 0.02 mmol). The reaction mixture was stirred overnight and the solvent was removed under reduced pressure. The residue was taken up in ethyl acetate and filtered to remove precipitated urea. The organic phase was taken up in levax separation, washed (sat. HaNSO 2, brine, 0.5 N HCl, brine), dried (MgSO 4).₄), filtered and concentrated under reduced pressure to give the diNHS ester product as a white solid (1.2 g, 68% yield).

HN

<img file="RS51130B_D0092.tif" />

Z C.

N

CO₂Et

51130 Β

Step 3 - Coupling of trtfunkdonal linker for peptide monomers: For coupling for linker, 2 eq. peptide was mixed with 1 eq. of a trifunctional linker in dry DMF to give a clear solution, 5 eq. DIEA was added after 2 minutes. The mixture was stirred at room temperature for 14 h. The solvent was removed under reduced pressure and the crude product was dissolved in 80% TFA in DCM for 30 min to remove the Boc group, followed by purification by 018 reverse phase HPLC. The dimer structure was confirmed by electrospray mass spectrometry. This coupling reaction binds a linker to a nitrogen atom from the ε-amino group from the lysine residue of each monomer.

g

<img file="RS51130B_D0093.tif" />

TFA

<img file="RS51130B_D0094.tif" />

Step 4 - Synthesis of a FEG moiety involving two PEGS chain linkages linked to Ldzin mPEG2-tizinol-NPC

Commercially available lysinol was treated with excess mPEG2-NPC to give MPEG2-lysinol, which then reacted with NPC to form mPEG2-Lysinol-NPC.

51130Β mPEG2-Liz-NHS

This product can be obtained commercially, for example, from the Molecular Engineering catalog (2003) by NektarTherapeutics (490 Discovery Drive, Huntsville, Alabama 35806), item no. 2Ζ3ΧΟΤ01.

Step 5 - PEGylation of the peptide dimer:

PEGylation of rgeco carbamate bond:

The peptide dimer and PEG species (mPEGrLysinol-NPC) were mixed in a 1: 2 molar ratio in dry DMF to give a clear solution, After 5 minutes 4 eq. DIEA is added to the stock solution. The mixture was stirred at room temperature for 14 h, followed by purification by C18 reverse phase HPLC. The structure of the PEGylated peptide was confirmed by MALDI mass. The purified peptide was also subjected to purification by cation exchange chromatography as shown below.

51130Β

<img file="RS51130B_D0095.tif" />

mPEG₂-Lysinol-NPC

DIEA, DMF

<img file="RS51130B_D0096.tif" />

PEGylation via amide bond:

Peptide dimer and PEG species (mPEG-II-NHS from Shearwater Corp., USA) were mixed in a 1: 2 molar ratio in dry DMF to give a clear solution. After 5 minutes 10 eq. DIEA is added to the stock solution. The mixture was stirred at room temperature for 2 h, followed by purification by C18 reverse phase HPLC. The structure of the PEGylated peptide was confirmed by MALDI mass. The purified peptide was also subjected to purification by column exchange chromatography as shown below.

51130Β

<img file="RS51130B_D0097.tif" />

<img file="RS51130B_D0098.tif" />

Step 6: -Ion-exchange peptide purification: The ability to separate the peptide-PEG conjugate obtained above from unreacted (or hydrolyzed) PEG, in addition to their ability to retain starting dimetic peptides, was tested for several exchange carriers. Ion exchange resins (2-3g) were placed in a 1 cm column, after which conversion to sodium form was performed (0.2 N NaOH charged to the column until the eluent reached pH 14, ca. 5 column volume) and then in hydrogenated form (eluted with either 0.1 N HCl or 0.1 M HOAc until the eluent reaches the pH of the charge, approx. 5 column volume), after which they were washed with 25% ACN / water to reaching pH 6. Either the peptide before conjugation or the peptide-PEG conjugate was dissolved in 25% ACN / water mixture (10

51130Β mg / mL) and the pH was adjusted to &lt;3 saTFA, then placed in a column. After washing with 2-3 column volumes with 25% ACN / water mixture and collecting 5 mL fractions, the peptides were discharged from the column eluting with 0.1 Μ NH4OAC in 25% ACN / water mixture, re-collected 5 mL fractions. Analyzes by HPLC show which fraction contains the desired peptide. Analyzes with the Evaporation Light-Scattering Detector (ELSD) indicate that when peptides are retained on the column when eluted with NH₄With the OAc solution (generally between fractions 4 and 10), no unconjugated PEGs are present as contaminants. When the peptides were eluted in the initial wash buffer (generally the first 2 fractions), no separation of the desired PEG-conjugate and excess PEG was observed.

Table 5: Ion exchange resins

<td>Carrier</td><td>Source</td>

<td>Mono S HR 5/5 highly cation exchange pre-purine column</td><td>Amersham Biosciences</td>

<td>SE53 Cellulose, microgranules strongly cation exchange carrier</td><td>Whatman</td>

<td>SP Sepharose fast-flowing high cation exchange carrier</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 (SEKID NO: 1)

EPO-R agonist peptide homodimers of peptide monomers having the amino acid sequence (AcG) GLYACHMGPIT (1-nal) VCQPLR (MeG) K (SEKID BR: 2) were synthesized as described in Example 1, except that in Step 1 peptides were synthesized :

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

Where PEG is attached to the spacer via a carbamate bond, the final product of this synthesis can be illustrated structurally as follows:

51130 Β

<img file="RS51130B_D0099.tif" />

Where PEG is attached to the spacer via an amide bond, the final product of this synthesis can be illustrated structurally as follows:

<img file="RS51130B_D0100.tif" />

This example describes various in w &amp; Assays that are useful in assessing the activity and potency of the EPO-R agonist peptide of the invention. The results of these assays indicate that the novel peptides of the present invention bind to EPO-R and activate EPO-R signaling. In addition, the results from these studies indicate that the new peptide compositions show a surprising increase in EPO-Binding affinity and in biological activity compared to the EPO mimetic peptides described previously.

51130Β

EPO-R agonist peptide monomers and dimers were obtained according to the methods given in Example 1 or Example 2. The potency of these peptide dimers was assessed using a series of in vitro activity assays, including: reporter assay, proliferation assay, competitive binding assay, and C / BFU -e analysis. These four analyzes are described in more detail below.

The results of these m vitro activity assays are shown in Table 2.

1. Reporter analysis

This assay is based on reporter cells derived from pre-B cell lines from mice, Baf3 / EpoR / GCSFRfos / lux. These reporter cell lines express chimeric receptors that include the extra-cellular portion of the human EPO receptor for the intra-cellular portion of the human GCSF receptor. These cell lines were further transfected with a phosgene construct containing a promoter-guided luciferase reporter. Activation of this heme receptor by the administration of an erythropoietic agent leads to the expression of the luciferase reporter gene and thus the production of light after the addition of the luciferase substrate luciferin. Thus the level of EPO-R activation in such cells can be quantified by measuring luciferase activity.

Baf3 / EpoR / GCSFR fos / lux cells were cultured in DMEM / F12 medium (Gibco) enriched with 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3 supernatant (WEHI-3 cell culture supernatant, ATCC # TIB-68) and a mixture of penicillin / streptomycin. Approximately 18 h before analysis, cells were starved by transferring them to DMEM / F12 medium enriched with 10% FBS and 0.1% WEHI-3 supernatant. On the day of analysis, cells were washed once with DMEM / F12 medium enriched in 10% FBS (without WEHI-3 supernatant), then 1X10⁶cells / mL cultured in the presence of a known concentration of test peptide or with EPO (R&amp;D Systems Inc., Minneapolis, MN) as a positive control, in DMEM / F12 medium enriched with 10% FBS (without WEHI-3 supernatant). Serial dilutions of the tested peptide were tested simultaneously in this assay. Analytical plates were incubated for 4 h at 37 ° C under 5% CO₂, after which luciferin (Steady-Glo; Promega, Madison, Wi) was added to each well. After a 5-minute incubation, light emission was measured on a Packard Topcount Luminometer (Packard Instrument Co., Downers Grove, HI.). Light values ​​were plotted against the concentrations of the peptide tested and analyzed using Graph Pad software. The concentration of the tested peptide leading to half of the maximum light emission is recorded as EC50.

2. Proliferation analysis

This assay is based on a pre-B cell line from mice, Baf3, transfected to express human EPO-R. The proliferation of the resulting cell lines, BaF3 / Gal4 / Elk / EPOR, is dependent on EPO-Activation. The degree of cell proliferation was quantified using MTT, where the signal in MTT analysis is proportional to the number of viable cells.

BaF3 / Gal4 / Elk / EPOR cells were cultured in rotating bottles in DMEM / F12 medium

51130Β (Gibco) enriched with 10% FBS (Hyclone) and 2% WEHI-3 supernatant (ATCC # TIB-68). Cultured cells were fasted overnight, in rotating bottles at a cell density of 1 x 10⁶cell / ml, in DMEM / F12 medium enriched in 10% FBS and 0.1% WEHI-3 supernatant. The smoothed cells were then washed twice with Dulbecco's PBS (Gibco) and resuspended at a density of 1 x 10⁶ cell / ml in DMEM / F12 enriched in 10% FBS (without WEHI-3 supernatant). Aliquots of 50 μL (~ 50,000 cells) of the cell suspension were then inserted, in triplicate, into a 96-well assay plate. Aliquots of 50 pL of serial dilutions of tested EPO mimetic peptides or 50 pL of EPO (R&amp;D Systems Inc., Minneapolis, MN) or Aranesp® (darbpoeitin alpha, EPOR agonist commercially available from Amgen) in DMEM / F12 medium enriched with 10% FBS (without WEHI-3 supernatant I) was added to 96-well assay plates (final well volume of 100 μL). For example, 12 different dilutions can be tested where the final concentration of test peptide (or control EPO peptide) ranges between 810 pM to 0.0045 pM. The cells in the plates were then incubated for 48 h at 37 ° C. Thereafter, 10 μL MTT (Roche Diagnostics) was added to each culture well and then left in an incubator for 4 h. The reaction was then quenched by the addition of 10% SDS + 0.01 N HCl. The plates were then incubated overnight at 37 ° C. The absorbance at 595 nM was measured for each well on a spectrophotometer. Diagrams of absorbance values ​​according to the concentrations of the tested peptide were constructed and the EC50 value was calculated using Graph Pad software. The concentration of test peptide leading to half of the maximum absorbance is recorded as the EC50.

3. Cognitive binding analysis

Competitive binding calculations were performed using an analysis in which a light signal is generated as a function of the proximity of two microspheres: the streptavidin donor microsphere carrying the biotinylated EPO-R-binding peptide tracer and the acceptor microsphere to which EPO-R binds. Light is generated by the transfer of non-radiative energy, during which one oxygen is released from the first microsphere during illumination and comes into contact with the released one oxygen, causing the second microsphere to emit light. The sets of these microspheres are commercially available (Packard). The proximity of the microspheres is created by binding the EPO-R-binding peptide tracer to EPO-R. The tested peptide that competes with the EPO-R-binding peptide tracer in binding to EPO-R will prevent this binding, leading to a reduction in light emission.

A more detailed presentation of the method is as follows: 4 pL of serial dilutions of the tested EPO-R peptide agonist or positive or negative control is added to wells on a 384 well plate. After that, 2 pL / ro well of receptor / microsphere mixture is added. The mixture of receptors and microspheres consists of: 15 μL of 5 mg / ml streptavidin donor microspheres (Packard), 15 μΐ, of 5 mg / ml monoclonal antibodies abl79 (these antibodies recognize part of the human placental alkaline phosphatase protein found in recombinant EPO-R ), protein A-coated acceptor microspheres (protein A binds to abl79 antibodies; Packard), 112.5 pL of recombinant EPO-R in dilution 1

51130Β: 6.6 (produced in Chinese hamster ovarian cells as a fusion protein in a portion of human placental alkaline phosphatase protein containing abl79 target epitopes) and 607.5 pL of Alfaquest buffer (40 mM HEPES, pH 7.4; 1 mM MgClg 0.1% BSA, 0.05% Tween 20). Everything comes together to mix. 2 [mu] L / well of biotinylated EPO-R-binding peptide tracer, AF33068 (30 nM final concentration) was added. AF33068, an EPO-R binding peptide (see Table 3 Reporter EC50 (pM)), was obtained according to the methods described in Example 1.

AF33068

Biotin-GGLYACHMGPnWVCQPLRG: k-nh₂

Biotin-GGLYACHMGPnWVCQPLRG /

Centrifuge for 1 min to mix. 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 data are plotted against peptide concentration and analyzed with Graph Pad or Excel software.

The concentration of the tested peptide leading to a 50% reduction in light emission, compared to that obtained without the tested peptide, is recorded as IC50,

4. C / BFU-e analysis

EPO-R signaling stimulates the differentiation of cell lineage from bone marrow into proliferative precursors of red blood cells. This assay measures the ability of the tested peptides to stimulate the proliferation and differentiation of red blood cell precursors from primary human multipotent bone marrow cell lines.

For this analysis, serial dilutions of the tested peptide were prepared in IMDM medium (Gibco) enriched in 10% FBS (Hyclone). These serial dilutions, or positive control of the EPO peptide, were then added to methylcellulose to give a final volume of 1.5 mL. The mixture of methylcellulose and peptide was then vortexed vigorously. Aliquots (100,000 cells / mL) of human bone marrow CD34 + cells (Poietics / Cambrex) were dissolved. Dissolved cells were slowly added to 0.1 mL of 1 mg / ml DNAse (cell line) in 50 mL tubes. Then, 40-50 mL of IMDM medium was slowly added to the cells: the medium was added dropwise along the side walls of the 50 mL tubes for the first 10 mL and then the remaining volume of medium was slowly drained along the side walls of the tube. The cells were then centrifuged at 900 rpm for 20 min and the medium was carefully removed by gentle aspiration. Cells were resuspended in 1 ml of IMDM medium and cell density per mL was determined on a hemacytometer slide (an aliquot of 10 μL of cell suspension on slide and cell density was an average of X 10,000 cells / ml). The cells were then diluted in IMDM medium to a cell density of 15,000 cells / mL. 100 μL of diluted cells were then added to each 1.5 mL of methyl cellulose and peptide sample (final cell concentration in assay medium was 1000 cells / mL) and the mixture was vortexed. Allow the bubbles in the mixture to disappear and then aspirate with 1 mL using a needle with

51130Β blunt tip. 0.25 mL of the aspirated mixture from each sample was added to each of the 4 wells on a 24-well plate (Falcon brand). The plate mixtures were incubated at 37 ° C under an atmosphere of 5% CO₂ in a humid incubator for 14 days. The presence of erythroid colonies was assessed using a phase microscope (5Χ-10Χ lens, final magnification of 100Χ). The concentration of test peptide at which the number of colonies formed is 90% of the maximum, relative to the number obtained with the EPO positive control, is recorded as EC90 [See Table 2: C / BFUs of EC90].

Table 6: In vitro analysis of peptide dimer activity

<td>0) ώ PQ υ</td><td>o σ&gt; υ Μ</td><td colspan="2">(nm)</td><td colspan="3">SS</td>

<td rowspan="2">'o TZ</td><td>'ΰ</td><td>Ο</td><td>Β</td><td>ri ri</td><td></td><td></td>

<td>ω • r- *</td><td>υ ►Μ</td><td></td><td>Tl</td><td></td><td></td>

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

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

<td>&amp; b čZ</td><td>φ 1L υ</td><td>With</td><td></td><td>ιί5 Φ</td><td></td><td></td>

<td>г ~ Ч</td><td>Μ</td><td> 4/</td><td></td><td></td><td></td><td></td>

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

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

<td>£ o</td><td rowspan="2">φ Š Ο Μ</td><td> §</td><td></td><td>ΙΛ φ</td><td></td><td></td>

<td>ad</td><td></td><td></td><td>GN</td><td></td><td></td>

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

<td>(V g</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="3">ar 8 Ο Ш α. i Ρζ ⁸η \ Ο ο) Ш \ ** }&gt; Ο . _with!! V&gt; M. Υ ι 1 ι &lt;2 1 § 1 § 8L σ £ r— u £ &gt;? I &amp; t 1 a. 2 O N § o_ _o</td><td rowspan="2"></td>

<td>HJ 'S</td>

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

<td>• l</td><td></td><td></td><td></td><td></td><td>U 0 0 i</td><td></td>

<td>&lt;D</td><td></td><td></td><td></td><td></td><td> §</td><td></td>

<td> 0-1</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>ctf</td><td></td><td></td><td rowspan="2">SM φ</td><td></td><td></td>

<td>ctf</td><td>δ¹</td><td></td><td></td><td></td><td></td>

<td>c</td><td rowspan="2">&lt;υ 'Ξ * tz</td><td></td><td></td><td>the</td><td></td><td></td>

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

<td>o</td><td>&lt;υ · —Ί</td><td></td><td></td><td></td><td></td><td></td>

51130Β

Example 12: Tests and activities

This example describes various tests that are useful for assessing the activity and potency of the EPO-R agonist peptide of the invention. EPO-R agonist peptide dimers were obtained according to the methods given in Example 1. The h wactivity of these peptide monomers and dimers was assessed using a series of assays, including bioassays on polycythemic exhypoxic mice and reticulocyte analyzes. These two analyzes are described in more detail below.

1. Bioanalysis in polycythemic exhypoxic mice

Bioanalysis examined the in vivo activity of the tested peptides in polycythemic exhypoxic mice adopted from the method described by Cotes and Bangham (1961), Nature 191: 1065-1010. This assay examines the ability of the tested peptide to function as an EPO mimic: that is, to activate EPO-R and induce the synthesis of new red blood cells. The synthesis of red blood cells was quantified based on the incorporation of radiolabeled iron into the hemoglobin of synthesized red blood cells.

BDFl mice were allowed to acclimatize to ambient conditions for 7-10 days. Body weights were determined for all animals and animals with low weight (&lt;15 grams) were not used. Mice were subjected to successive conditioned cycles in the hypobamic chamber for a total of 14 days. Each 24-hour cycle consists of 18 h at 0.40 + 0.02% atmospheric pressure and 6 h at ambient pressure. After conditioning, mice were maintained at ambient pressure for an additional 72 h before dosing.

Test peptides, or recombinant human EPO standards, were diluted in PBS + 0.1% BSA vehicle (PBS / BSA). Stock solutions of peptide monomers were first dissolved in dimethyl sulfoxide (DMSO). Negative control groups include one group of TBS / BSA alone injections and one group injected with 1% DMSO. The group for each dose contained 10 mice. Mice were injected subcutaneously (into the nape of the neck) with 0.5 mL of the appropriate sample.

Forty-eight hours after injection of the sample, mice were given an intraperitoneal injection of 0.2 ml of Fe⁵⁹ (Dupont, NEN), for a dose of approximately 0.75 pKiria / per mouse. Mice body weights were determined 24 h after Fe⁵⁹ applications and mice were sacrificed 48 h after Fe⁵⁹ applications. Blood was collected from each animal by cardiac puncture and hematocrits were determined (heparin was used as an anticoagulant). Each blood sample (0.2 ml) was analyzed for Fe⁵⁹ incorporation using Packard gamma counters. Insensitive mice (i.e., those mice in which the radioactive incorporation is less than the negative control group) were eliminated from the appropriate data set. Mice in which hematocrit values ​​were less than 53% of the negative control group were also eliminated.

Results were obtained from sets of 10 animals for each experimental dose. The average amount of radioactivity incorporated [amount per minute (CPM)] in blood samples from each group was calculated.

51130Β

2. Reticulocyte analysis

Normal BDF1 mice were dosed (0.5 mL, injected subcutaneously) for three consecutive days with either EPO control or tested peptide. On the third day, mice were also iodinated (0.1 mL, injected intraperitoneally) with iron dextran (100 mg / ml). On day 5, mice were anesthetized with CO₂ and blood samples were taken using cardiac puncture. The percentage (%) of reticulocytes for each blood sample was determined by thiazole orange staining and flow cytometry analysis (retic-count program). Hematocrits were determined manually. The corrected percentage of reticulocytes was determined using the following formula:

% RETIKcorrected =% RETIK_DOBuenoX

3. Haematological analysis

Normal CD1 mice were dosed with four weeks of bolus intravenous injection with either an EPO positive control, a tested peptide, or a vehicle. The range of positive control doses and test peptides, expressed as mg / kg, was tested by varying the concentration of active compound in the formulation. The injected volumes are 5 ml / kg. the vehicle control group included twelve animals, while 8 animals were in each of the remaining dosing groups. Daily viability and weekly body weights were recorded.

Dosing mice were fasted and then anesthetized by inhalation of isoflurane and terminal blood samples were collected by cardiac or abdominal aortic puncture on day 1 (for vehicle control mice) and days 15 and 29 (4 mice / group / day). Blood was transferred to Vacutainer® brand tubes. Preferred anticoagulants are ethylenediaminetetraacetic acid (EDTA).

Blood samples were evaluated for endpoints at which red blood cell synthesis and physiology such as hematocrit (Hct), hemoglobin (Hgb), and total erythrocyte count (RBC) were measured using automated clinical analyzers known in the art (e.g., those of manufactured by Coulter, Inc.).

* * ★

The present invention is not limited in scope to the specific solutions described herein. In fact, various modifications of the invention in addition to those described herein will become apparent to one skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the attached requirements.

It should be further understood that all values ​​are approximate and are provided for description.

Numerous references, including patents, patent applications, and various publications are cited and discussed in the description of the present invention. References and / or descriptions of these references are provided only to clarify the descriptions of the present invention and it is not the recognition of such references as previous experience in the field for the present invention. All references cited and discussed in this specification are incorporated herein by reference in their entirety and to the same extent if each reference given is individually incorporated by reference.