WO2009025957A1 - Formulations de peptide de récepteur d'érythropoïétine et utilisations - Google Patents

Formulations de peptide de récepteur d'érythropoïétine et utilisations Download PDF

Info

Publication number
WO2009025957A1
WO2009025957A1 PCT/US2008/070926 US2008070926W WO2009025957A1 WO 2009025957 A1 WO2009025957 A1 WO 2009025957A1 US 2008070926 W US2008070926 W US 2008070926W WO 2009025957 A1 WO2009025957 A1 WO 2009025957A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
peg
patient
moiety
epo
Prior art date
Application number
PCT/US2008/070926
Other languages
English (en)
Inventor
Richard Stead
Anne-Marie Duliege
Robert Hong Choon Leong
Julia Iwashita
William Lang
Mark A. Weinberg
Original Assignee
Takeda Pharmaceuticals Company Limited
Affymax
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Pharmaceuticals Company Limited, Affymax filed Critical Takeda Pharmaceuticals Company Limited
Priority to JP2010521912A priority Critical patent/JP2010536857A/ja
Priority to US12/195,832 priority patent/US20090221510A1/en
Publication of WO2009025957A1 publication Critical patent/WO2009025957A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptide compounds that are agonists of the erythropoietin receptor (EPO-R).
  • EPO-R erythropoietin receptor
  • the invention also relates to therapeutic methods using such peptide compounds to treat disorders associated with insufficient or defective red blood cell production, including chemotherapy induced anemia.
  • Pharmaceutical compositions that comprise the peptide compounds of the invention are also provided.
  • EPO Erythropoietin
  • EPO can occur in three forms: ⁇ , ⁇ , and asialo.
  • the ⁇ and ⁇ forms differ slightly in their carbohydrate components, but have the same potency, biological activity, and molecular weight.
  • the asialo form is an ⁇ or ⁇ form with the terminal carbohydrate (sialic acid) removed.
  • the DNA sequences encoding EPO have been reported [U.S. Pat. No. 4,703,008 to Lin].
  • EPO stimulates mitotic division and differentiation of erythrocyte precursor cells, and thus ensures the production of erythrocytes. It is produced in the kidney when hypoxic conditions prevail.
  • EPO-induced differentiation of erythrocyte precursor cells globin synthesis is induced; heme complex synthesis is stimulated; and the number of ferritin receptors increases. These changes allow the cell to take on more iron and synthesize functional hemoglobin, which in mature erythrocytes binds oxygen.
  • erythrocytes and their hemoglobin play a key role in supplying the body with oxygen.
  • EPO is present in very low concentrations in plasma when the body is in a healthy state wherein tissues receive sufficient oxygenation from the existing number of erythrocytes. This normal low concentration is sufficient to stimulate replacement of red blood cells which are lost normally through aging.
  • the amount of EPO in the circulation is increased under conditions of hypoxia when oxygen transport by blood cells in the circulation is reduced.
  • Hypoxia may be caused, for example, by substantial blood loss through hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen intake due to high altitude or prolonged unconsciousness, or various forms of anemia.
  • elevated EPO levels increase red blood cell production by stimulating the proliferation of erythroid progenitor cells.
  • EPO levels in circulation are decreased.
  • EPO-binding cell surface proteins comprise two polypeptides having approximate molecular weights of 85,000 Daltons and 100,000 Daltons, respectively [Sawyer, et al. (1987) Proc. Natl. Acad. ScL USA 84:3690-3694].
  • the number of EPO-binding sites was calculated to average from 800 to 1000 per cell surface. Of these binding sites, approximately 300 bound EPO with a Kd of approximately 90 pM (picomolar), while the remaining bound EPO with a reduced affinity of approximately 570 pM [Sawyer, et al. (1987) J. Biol. Chem. 262:5554-5562].
  • EPO- responsive splenic erythroblasts prepared from mice injected with the anemic strain (FVA) of the Friend leukemia virus, possess at total of approximately 400 high and a low affinity EPO binding sites with K d values of approximately 100 pM and 800 pM, respectively [Landschulz, et al. ( 1989) Blood 73 : 1476- 1486] .
  • FVA anemic strain
  • K d values approximately 100 pM and 800 pM
  • EPO-R The availability of cloned genes for EPO-R facilitates the search for agonists and antagonists of this important receptor.
  • the availability of the recombinant receptor protein allows the study of receptor-ligand interaction in a variety of random and semi-random peptide diversity generation systems. These systems include the "peptides on plasmids" system [described in U.S. Pat. No. 6,270,170]; the "peptides on phage” system [described in U.S. Pat. No. 5,432,018 and Cwirla, et al (1990) Proc. Natl. Acad. ScL USA 87:6378-6382]; the "encoded synthetic library” (ESL) system [described in U.S.
  • a group of peptides containing a peptide motif has been identified, members of which bind to EPO-R and stimulate EPO- dependent cell proliferation.
  • peptides identified to date that contain the motif stimulate EPO-dependent cell proliferation in vitro with EC50 values of about 20 nanomolar (nM) to about 250 nM.
  • nM nanomolar
  • peptide concentrations of 20 nM to 250 nM are required to stimulate 50% of the maximal cell proliferation stimulated by EPO.
  • Still other peptides and constructs thereof that bind to the EPO receptor have been described in U.S. application nos. 60/470,244, 60/470,245, and 60/469,993, all filed on May 12, 2003; U.S. application nos.
  • EPO-R agonists Despite the immense potential of EPO-R agonists, there still remains a need for identifying their applicability to the treatment of specific diseases, for example, chemotherapy-induced anemia. Anemia is a frequent complication of cancer chemotherapy. Within such a broad category, there also remains the need to identify sub-types of cancer for which specific EPO-R agonists are promising targets for treating chemotherapy induced anemia. The present invention provides such methods.
  • the present invention provides novel methods involving peptide compounds for the treatment of chemotherapy induced anemia, such as can occur during the treatment of breast, prostate or lung cancer.
  • the peptide compounds used by the methods of this inventions include homodimers of peptide monomers having the amino acid sequence (AcG)GLYACHMGPIT(l -nal)VCQPLRK (SEQ ID NO: 1), homodimers of peptide monomers having the amino acid sequence (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 2), and homodimers of peptide monomers having the amino acid sequence (ACG)GLYACHMGPIT( I -nal)VCQPLR(MeG) (SEQ ID NO: 3); where each amino acid is indicated by standard one letter abbreviation, "(AcG)” is JV-acetylglycine, "(1-nal)” is 1- naphthylalanine, and "
  • the invention provides a method of treating a disorder characterized by deficiency of erythropoietin or a low or defective red blood cell population in a patient who is undergoing, has undergone, or will undergo, chemotherapy for lung, breast or prostate cancer, comprising administering to the patient a therapeutically effective amount of a compound comprising: (a) a first peptide monomer comprising the amino acid sequence (AcG)GLYACHMGPIT(l-nal)VCQPLR (SEQ ID NO: 14); (b) a second peptide monomer comprising the amino acid sequence ( AcG)GLY ACHMGPIT( 1-nal) VCQPLR (SEQ ID NO: 14); (c) a linker moiety covalently bonding the first peptide monomer to the second peptide monomer; and (d) a spacer moiety covalently joining the linker moiety and a poly(ethylene glycol) (PEG) moiety, said PEG moiety comprising: (a) a
  • the amino acid sequence additionally comprises (MeG), K, or (MeG)K at the C-terminus.
  • the amino acid sequence is (AcG)GLYACHMGPIT( l -nal)VCQPLRK (SEQ ID NO: 1).
  • the amino acid sequence is (AcG)GLYACHMGPIT(l -nal)VCQPLR(MeG) (SEQ ID NO: 3).
  • the amino acid sequence is (AcG)GLYACHMGPIT(l- nal)VCQPLR(MeG)K (SEQ ID NO: 2).
  • the two cysteine residues of the first peptide monomer are bonded together through a disulfide bridge, and wherein two cysteine residues of the second peptide monomer are bonded together through a disulfide bridge.
  • the linker moiety comprises an amide derivative of a lysine residue.
  • the lysine residue is covalently bonded through the nitrogen atom of its side chain.
  • the linker moiety is defined by the formula:
  • the spacer moiety is defined by the formula:
  • the compound is further defined by the formula:
  • the compound can be further defined as: or pharmaceutically acceptable salts thereof, and substantially free from other optical isomers thereof.
  • the PEG moiety has a molecular weight from about 10,000 Dalton to about 50,000 Daltons, In some embodiments, the PEG moiety has a molecular weight from about 20,000 Daltons to about 40,000 Daltons.
  • the disorder is anemia, for example, chemotherapy-induced anemia.
  • the patient has cancer of the bladder, blood, bone, brain, breast, central nervous system, colon, endometrium, esophagus, genitourinary tract, head, larynx, liver, lung, neck, ovary, pancreas, prostate, spleen, small intestine, large intestine, stomach, or testicle.
  • the patient is undergoing, has undergone, or will undergo, chemotherapy for lung cancer, for example, non-small cell lung cancer.
  • lung cancer for example, non-small cell lung cancer.
  • the patient is undergoing, has undergone, or will undergo, chemotherapy for prostate cancer.
  • the patient is undergoing, has undergone, or will undergo, chemotherapy for breast cancer.
  • the patient is a primate, for example, a human.
  • the method further comprises identifying a patient in need of treatment.
  • the compound is administered locally. [0033] In some embodiments, wherein the compound is administered systemically. [0034] In some embodiments, the compound is administered intravenously, intra- arterially, intramuscularly, intraperitoneally, subcutaneously or orally.
  • the therapeutically effective amount is a dosage from about 0.01 milligram to about 1,000 milligram of the compound per kilogram of body weight of the patient. In some embodiments, the therapeutically effective amount is a dosage from about 0.025 milligram to about 0.5 milligram of the compound per kilogram of body weight of the patient. In some embodiments, the therapeutically effective amount is a dosage from about 0.025 milligram to about 0.2 milligram of the compound per kilogram of body weight of the patient. In some embodiments, the therapeutically effective amount is a dosage from about 0.05 milligram to about 0.1 milligram of the compound per kilogram of body weight of the patient.
  • the therapeutically effective amount is administered in a single dose per day. In some embodiments, the therapeutically effective amount is administered in two or more doses per day. In some embodiments, the therapeutically effective amount is administered once every 3 to 4 weeks.
  • the invention further provides pharmaceutical compositions comprised of such peptide compounds.
  • the invention provides a pharmaceutical composition for preventing or treating a disorder characterized by deficiency of erythropoietin or a low or defective red blood cell population in a patient who is undergoing, has undergone, or will undergo, chemotherapy for lung, breast or prostate cancer, which comprises a compound comprising:
  • the pharmaceutical composition comprises any one of the above compounds and a pharmaceutically acceptable carrier.
  • the present invention is intended to encompass all pharmaceutically acceptable ionized forms (e.g., salts) and solvates (e.g., hydrates) of the compounds, regardless of whether such ionized forms and solvates are specified since it is well known in the art to administer pharmaceutical agents in an ionized or solvated form. It is also noted that unless a particular stereochemistry is specified, recitation of a compound is intended to encompass all possible stereoisomers (e.g., enantiomers or diastereomers depending on the number of chiral centers), independent of whether the compound is present as an individual isomer or a mixture of isomers.
  • pharmaceutically acceptable ionized forms e.g., salts
  • solvates e.g., hydrates
  • FIG. 1 Correction of Anemia with Peptide I Treatment by Cancer Patient Group. The percentage of patient responders by cancer group upon treatment with Peptide I is shown. Response was defined by an increase in hemoglobin (Hgb) concentration of greater than or equal to 1 g/dL by Week 9 of treatment (see FIG. 3).
  • Hgb hemoglobin
  • NSCLC non-small cell lung cancer.
  • FIG. 2 Mean Effective Peptide I Dose by Cancer Patient Group.
  • the dose (Y axis) is given in mg of Peptide I per kg of body weight.
  • the bars indicate the minimum dose and the maximum dose administered to a patient of the indicated group.
  • NSCLC is non-small cell lung cancer.
  • the group mean dose was calculated from the last dose administered to each patient in that group.
  • FIG. 3. Hemoglobin Curves as a Function of Peptide I Treatment by Cancer Patient Group. Blood hemoglobin (Hgb) levels in g/dL were monitored over a four dose multi-week study. The three series of curves correspond to three groups of patients, each undergoing treatment for different types of cancer. [0043] FIG. 4. Hemoglobin Curve as a Function of Peptide I Treatment for Breast Cancer Patient Group. Blood hemoglobin (Hgb) levels in g/dL were monitored over a four dose multi-week study. Error bars were assigned to each value.
  • FIG. 5 Hemoglobin Curve as a Function of Peptide I Treatment for NSCLC Cancer Patient Group. Blood hemoglobin (Hgb) levels in g/dL were monitored over a four dose multi-week study. Error bars were assigned to each value.
  • Hgb Blood hemoglobin
  • FIG. 6 Hemoglobin Curve as a Function of Peptide I Treatment for Prostate Cancer Patient Group. Blood hemoglobin (Hgb) levels in g/dL were monitored over a four dose multi-week study. Error bars were assigned to each value.
  • Hgb Blood hemoglobin
  • Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is De or I; Methionine is Met or M; Valine is VaI or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
  • 1-naphthylalanine is 1 -nal or Np
  • N- methylglycine also known as sarcosine
  • MeG or Sc methylglycine
  • acetylated glycine N- acetylglycine
  • polypeptide or "protein” refers to a polymer of amino acid monomers that are alpha amino acids joined together through amide bonds. Polypeptides are therefore at least two amino acid residues in length, and are usually longer. Generally, the term “peptide” refers to a polypeptide that is only a few amino acid residues in length.
  • the novel EPO-R agonist peptides of the present invention are preferably no more than about 50 amino acid residues in length. They are more preferably of about 17 to about 40 amino acid residues in length.
  • a polypeptide, in contrast with a peptide, may comprise any number of amino acid residues.
  • polypeptide included peptides as well as longer sequences of amino acids.
  • pharmaceutically acceptable refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
  • agonist refers to a biologically active ligand which binds to its complementary biologically active receptor and activates the latter either to cause a biological response in the receptor, or to enhance preexisting biological activity of the receptor.
  • the present invention provides novel uses of peptide compounds, which are EPO- R agonists of dramatically enhanced potency and activity.
  • These peptide compounds are homodimers of peptide monomers having the amino acid sequence (AcG)GLYACHMGPIT(l-nal)VCQPLRK (SEQ ID NO: 1), or homodimers of peptide monomers having the amino acid sequence (AcG)GLY ACHMGPIT(I -nal) VCQPLR(MeG)K (SEQ ED NO: 2); where each amino acid is indicated by standard one letter abbreviation, "(AcG)" is N-acetylglycine, "(1-nal)" is 1-naphthylalanine, and "(MeG)” is "(MeG)” is N- methylglycine, also known as sarcosine.
  • Each peptide monomer of a peptide dimer contains an intramolecular disulfide bond between the
  • the linker (L K ) moiety is a branched tertiary amide, which bridges the C-termini of two peptide monomers, by simultaneous attachment to the C- terminal lysine residue of each monomer.
  • the tertiary amide linker can be depicted as:
  • X is NCO-(CH 2 ) 2 -N 1 H-; C 1 of the linker forms an amide bond with the ⁇ -amino group of the C-terminal lysine residue of the first peptide monomer; C 2 of the linker forms an amide bond with the ⁇ -amino group of the C-terminal lysine residue of the second peptide monomer; and N 1 of X is attached via a carbamate linkage or an amide linkage to an activated polyethylene glycol (PEG) moiety, where the PEG has a molecular weight from about 20,000 to about 40,000 Daltons (the term "about” indicating that in preparations of PEG, some molecules will weigh more, some less, than the stated molecular weight).
  • PEG polyethylene glycol
  • the tertiary amide linker may also be depicted as: -C 1 O-CH 2 -X-CH 2 -C 2 O- where: X is NCO-(CH 2 ) 2 -NH-C 3 O-; C 1 of the linker forms an amide bond with the ⁇ -amino group of the C-terminal lysine residue of the first peptide monomer; and C' of the linker forms an amide bond with the ⁇ -amino group of the C-terminal lysine residue of the second peptide monomer.
  • the peptide dimers of the invention further comprise a spacer moiety of the following structure:
  • C 4 of the spacer is covalently bonded to C 3 of X; N 1 of the spacer is covalently attached via a carbamate or an amide linkage to an activated PEG moiety; and N 2 of the spacer is covalently attached via a carbamate or an amide linkage to an activated PEG moiety, where PEG has a molecular weight of about 10,000 to about 60,000 Daltons (the term "about" indicating that in preparations of PEG, some molecules will weigh more, some less, than the stated molecular weight).
  • the peptides used in this invention can also contain a PEG moiety, which is covalently attached via a carbamate linkage or an amide linkage to the tertiary amide linker of the peptide dimer.
  • PEG is a water soluble polymer that is pharmaceutically acceptable.
  • PEG for use in the present invention may be linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20K) to about 6OK (the term "about” indicating that in preparations of PEG, some molecules will weigh more, some less, than the stated molecular weight). Most preferably, the PEG has a molecular weight of about 30K to about 4OK.
  • One skilled in the art will be able to select the desired polymer size based on such considerations as the desired dosage; circulation time; resistance to proteolysis; effects, if any, on biological activity; ease in handling; degree or lack of antigenicity; and other known effects of PEG on a therapeutic peptide.
  • Peptides, peptide dimers and other peptide-based molecules of the invention can be attached to water-soluble polymers (e.g., PEG) using any of a variety of chemistries to link the water-soluble polymer(s) to the receptor-binding portion of the molecule (e.g., peptide+spacer).
  • a typical embodiment employs a single attachment junction for covalent attachment of the water soluble polymer(s) to the receptor-binding portion, however in alternative embodiments multiple attachment junctions may be used, including further variations wherein different species of water-soluble polymer are attached to the receptor- binding portion at distinct attachment junctions, which may include covalent attachment junction(s) to the spacer and/or to one or both peptide chains.
  • the dimer or higher order multimer will comprise distinct species of peptide chain (i.e., a heterodimer or other heteromultimer).
  • a dimer may comprise a first peptide chain having a PEG attachment junction and the second peptide chain may either lack a PEG attachment junction or utilize a different linkage chemistry than the first peptide chain and in some variations the spacer may contain or lack a PEG attachment junction and said spacer, if PEGylated, may utilize a linkage chemistry different than that of the first and/or second peptide chains.
  • An alternative embodiment employs a PEG attached to the spacer portion of the receptor-binding portion and a different water-soluble polymer (e.g., a carbohydrate) conjugated to a side chain of one of the amino acids of the peptide portion of the molecule.
  • a PEG attached to the spacer portion of the receptor-binding portion and a different water-soluble polymer (e.g., a carbohydrate) conjugated to a side chain of one of the amino acids of the peptide portion of the molecule.
  • PEG polyethylene glycol
  • Suitable reactive PEG species include, but jure not limited to, those which are available for sale in the Drug Delivery Systems catalog (2003) of NOF Corporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome, Shibuya-ku, Tokyo 150-6019) and the Molecular Engineering catalog (2003) of Nektar Therapeutics (490 Discovery Drive, Huntsville, Ala. 35806).
  • PEG reagents are often preferred in various embodiments: mPEG2-NHS, mPEG2-ALD, multi-Arm PEG, mPEG(MAL)2, mPEG2(MAL), mPEG-NH2, mPEG-SPA, mPEG-SBA, mPEG-thioesters, mPEG-Double Esters, mPEG-BTC, mPEG-ButyrALD, mPEG-ACET, heterofunctional PEGs (NH2-PEG- COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-VS, NHS-PEG-MAL), PEG acrylates (ACRL-PEG-NHS), PEG-phospholipids (e.g., mPEG-DSPE), multiarmed PEGs of the SUNBRITE series including the GL series of glycerine-based PEGs activated by a chemistry chosen by those skilled in
  • the novel peptides of the invention can also contain two PEG moieties that are covalently attached via a carbamate or an amide linkage to a spacer moiety, wherein the spacer moiety is covalently bonded to the tertiary amide linker of 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 point of attachment.
  • Each PEG moiety preferably has a molecular weight of about 10 kilodaltons (10K) to about 6OK (the term "about” indicating that in preparations of PEG, some molecules will weigh more, some less, than the stated molecular weight). Linear PEG moieties are particularly preferred.
  • each of the two PEG moieties has a molecular weight of about 2OK to about 4OK, and still more preferably between about 20K and about 4OK. Still more preferably, each of the two PEG moieties has a molecular weight of about 2OK.
  • One skilled in the art will be able to select the desired polymer size based on such considerations as the desired dosage; circulation time; resistance to proteolysis; effects, if any, on biological activity; ease in handling; degree or lack of antigenicity; and other known effects of PEG on a therapeutic peptide.
  • the present invention also comprises methods and formulations of peptide agonists that are homodimers of peptide monomers having the amino acid sequence (AcG)GLYACHMGPITO-nal)VCQPLR(MeG) (SEQ ID NO: 3), where each amino acid is indicated by standard one letter abbreviation, "(AcG)” is N-acetylglycine, “(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 residues of the monomer.
  • Such monomers may be represented schematically as follows:
  • These monomelic peptides are dimerized to provide peptide dimers of enhanced EPO-R agonist activity.
  • the linker (L K ) moiety is a lysine residue, which bridges the C- termini of two peptide monomers, by simultaneous attachment to the C-terminal amino acid of each monomer.
  • One peptide monomer is attached at its C-terminus to the lysine's ⁇ -amino group and the second peptide monomer is attached at its C-terminus to the lysine's ⁇ -amino group.
  • the dimer may be illustrated structurally as shown in Formula I, and summarized as shown in Formula II:
  • N represents the nitrogen atom of lysine's ⁇ -amino group
  • N 1 represents the nitrogen atom of lysine's ⁇ -amino group.
  • the peptide dimers of the invention further comprise a spacer moiety of the following structure:
  • N 1 of the spacer is attached via an amide linkage to a carbonyl carbon of the lysine linker.
  • N 2 of the spacer is attached via a carbamate linkage or an amide linkage to an activated polyethylene glycol (PEG) moiety, where the PEG has a molecular weight of about 10,000 to about 60,000 Daltons (the term "about” indicating that in preparations of PEG, some molecules will weigh more, some less, than the stated molecular weight). More preferably, the PEG has a molecular weight of about 20,000 to
  • 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.
  • PEG for use in the present invention may be linear, unbranched PEG having a molecular weight of about 20 kilodaltons (20K) to about 6OK (the term "about” indicating that in preparations of PEG, some molecules will weigh more, some less, than the stated molecular weight). Most preferably, the PEG has a molecular weight of about 2OK to about 4OK, and still more preferably a molecular weight of about 30K to about 4OK.
  • each monomer of the homodimer has the amino acid sequence, (AcG)GLYACHMGPIT( l -nal)VCQPLRK (SEQ ED NO: 1) and N 1 of the linker is attached via an amide linkage to an activated polyethylene glycol (PEG) moiety
  • the peptide compounds used in this invention may be represented as follows:
  • each monomer of the homodimer has the amino acid sequence, (ACG)GLYACHMGPIT( I -nal) VCQPLR(MeG)K (SEQ ID NO: 2) and N 1 of the linker is attached via a carbamate linkage to an activated polyethylene glycol (PEG) moiety
  • the peptide compounds used in this invention may be represented as follows:
  • each monomer of the homodimer has the amino acid sequence, (AcG)GLYACHMGPIT(l-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and N 1 of the linker is attached via an amide linkage to an activated polyethylene glycol (PEG) moiety
  • the peptide compounds used in this invention may be represented as follows:
  • peptide dimers that may be used according to the present invention include, but are not limited to:
  • each monomer of the homodimer has the amino acid sequence, (AcG)GLYACHMGPIT( l -nal) VCQPLRK (SEQ ID NO: 1 ) and both N 1 and N 2 of the spacer are covalently attached via a carbamate linkage to an activated PEG moiety, the peptide compounds used in this invention may be represented as follows:
  • each monomer of the homodimer has the amino acid sequence, (AcG)GLYACHMGPIT(l-nal) VCQPLRK (SEQ ID NO: 1 ) and both N 1 and N 2 of the spacer are covalently attached via an amide linkage to an activated PEG moiety, the peptide compounds used in this invention may be represented as follows:
  • each monomer of the homodimer has the amino acid sequence, (AcG)GLYACHMGPIT(l -nal)VCQPLR(MeG)K (SEQ ID NO: 2) and both N 1 and N 2 of the spacer are covalently attached via a carbamate linkage to an activated PEG moiety
  • the peptide compounds used in this invention may be represented as follows:
  • each monomer of the homodimer has the amino acid sequence, (AcG)GLYACHMGPIT( l-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and both N 1 and N 2 of the spacer are covalently attached via an amide linkage to an activated PEG moiety, the peptide compounds used in this invention may be represented as follows:
  • peptide dimers that may be used with the present invention include, but are not limited to:
  • the peptide compounds used in this invention may be represented as follows:
  • the peptide compounds used in this invention may be represented as follows:
  • This dimeric structure can be written [Ac-peptide, disulf ⁇ de] 2 Lys-spacer-PEG 2 ⁇ - 40K to denote an N-terminally acetylated peptide bound to both the ⁇ and ⁇ amino groups of lysine with each peptide containing an intramolecular disulfide loop and a spacer molecule forming a covalent linkage between the C-terminus of lysine and a PEG moiety, where the PEG has a molecular weight of about 20,000 to about 40,000 Daltons.
  • Stereoisomers e.g., D-amino acids of the twenty conventional amino acids, unnatural amino acids such as a,a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for compounds of the present invention.
  • unconventional amino acids include, but are not limited to: ⁇ - alanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine, N-methylglycine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, nor-leucine, and other similar amino acids and imino acids.
  • peptide sequences of the present invention and be present alone or in conjunction with N-terminal and/or C-terminal extensions of the peptide chain.
  • extensions may be naturally encoded peptide sequences optionally with or substantially without non-naturally occurring sequences; the extensions may include any additions, deletions, point mutations, or other sequence modifications or combinations as desired by those skilled in the art.
  • naturally-occurring sequences may be full-length or partial length and may include amino acid substitutions to provide a site for attachment of carbohydrate, PEG, other polymer, or the like via side chain conjugation.
  • the amino acid substitution results in humanization of a sequence to make in compatible with the human immune system.
  • Fusion proteins of all types are provided, including immunoglobulin sequences adjacent to or in near proximity to the EPO-R activating sequences of the present invention with or without a non-immunoglobulin spacer sequence.
  • One type of embodiment is an immunoglobulin chain having the EPO-R activating sequence in place of the variable (V) region of the heavy and/or light chain.
  • the peptides used in the invention may be prepared by classical methods known in the art. These standard methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis, and recombinant DNA technology [See, e.g., Merrifield J. Am. Chem. Soc. 1963 85:2149]. [0078] In one embodiment, the peptide monomers of a peptide dimer are synthesized individually and dimerized subsequent to synthesis. The synthesis of the specific peptides in this application is present in US Patent Publication US 2007/0104704, which is incorporated by reference herein in its entirety.
  • the peptide monomers of a dimer are linked via their C- termini by a branched tertiary amide linker L K moiety having two functional groups capable of serving as initiation sites for peptide synthesis and a third functional group ⁇ e.g., a carboxyl group or an amino group) that enables binding to another molecular moiety (e.g. ., as may be present on the surface of a solid support).
  • the two peptide monomers may be synthesized directly onto two reactive nitrogen groups of the linker L K moiety in a variation of the solid phase synthesis technique. Such synthesis may be sequential or simultaneous.
  • the two peptide monomers may be synthesized directly onto two reactive nitrogen groups of the linker L K moiety in a variation of the solid phase synthesis technique. Such synthesis may be sequential or simultaneous.
  • a lysine linker (L K ) moiety having two amino groups capable of serving as initiation sites for peptide synthesis and a third functional group e.g., the carboxyl group of a lysine; or the amino group of a lysine amide, a lysine residue wherein the carboxyl group has been converted to an amide moiety -CONH 2
  • another molecular moiety e.g., as may be present on the surface of a solid support
  • two amine functional groups on the linker molecule are protected with two different orthogonally removable amine protecting groups.
  • the protected linker is coupled to a solid support via the linker's third functional group.
  • the first amine protecting group is removed, and the first peptide of the dimer is synthesized on the first deprotected amine moiety.
  • the second amine protecting group is removed, and the second peptide of the dimer is synthesized on the second deprotected amine moiety.
  • the first amino moiety of the linker may be protected with Alloc, and the second with Fmoc.
  • the Fmoc group (but not the Alloc group) may be removed by treatment with a mild base [e.g., 20% piperidine in dimethyl formamide (DMF)], and the first peptide chain synthesized. Thereafter the Alloc group may be removed with a suitable reagent [e.g., Pd(PPh 3 )/4-methyl morpholine and chloroform], and the second peptide chain synthesized.
  • a mild base e.g. 20% piperidine in dimethyl formamide (DMF)
  • a suitable reagent e.g., Pd(PPh 3 )/4-methyl morpholine and chloroform
  • Solid phase peptide synthesis procedures are well-known in the art [see, e.g., Stewart Solid Phase Peptide Syntheses (Freeman and Co.: San Francisco) 1969; 2002/2003 General Catalog from Novabiochem Corp, San Diego, USA; Goodman Synthesis of Peptides and Peptidomimetics (Houben-Weyl, Stuttgart) 2002].
  • synthesis is typically commenced from the C-terminal end of the peptide using an ⁇ -amino protected resin.
  • a suitable starting material can be prepared, for instance, by attaching the required ⁇ -amino acid to a chloromethylated resin, a hydroxymethyl resin, a polystyrene resin, a benzhydrylamine resin, or the like.
  • the ⁇ -amino protecting group is removed, for example, using trifluoroacetic acid (TFA) or hydrochloric acid (HCI) solutions in organic solvents at room temperature. Thereafter, ⁇ -amino protected amino acids are successively coupled to a growing support-bound peptide chain.
  • TFA trifluoroacetic acid
  • HCI hydrochloric acid
  • the ⁇ -amino protecting groups are those known to be useful in the art of stepwise synthesis of peptides, including: acyl-type protecting groups (e.g., formyl, trifluoroacetyl, acetyl), aromatic urethane-type protecting groups [e.g., benzyloxycarboyl (Cbz) and substituted Cbz], aliphatic urethane protecting groups [e.g., t- butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl], and alkyl type protecting groups (e.g., benzyl, triphenylmethyl), fluorenylmethyl oxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), and l-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde).
  • acyl-type protecting groups e
  • the side chain protecting groups (typically ethers, esters, trityl, PMC, and the like) remain intact during coupling and are not split off during the deprotection of the amino- terminus protecting group or during coupling.
  • the side chain protecting group must be removable upon the completion of the synthesis of the final peptide and under reaction conditions that will not alter the target peptide.
  • the side chain protecting groups for Tyr include tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl.
  • the side chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl, and cyclohexyl.
  • the side chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and Cbz.
  • the side chain protecting groups for Arg include nitro, Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf).
  • the side chain protecting groups for Lys include Cbz, 2- chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-Br-Cbz), Tos, or Boc. [0086] After removal of the ⁇ -amino protecting group, the remaining protected amino acids are coupled stepwise in the desired order.
  • Each protected amino acid is generally reacted in about a 3-fold excess using an appropriate carboxyl group activator such as 2-(1H- benzotriazol-1-yl)-l,1,3,3 tetramethyluronium hexafluorophosphate (HBTU) or dicyclohexylcarbodimide (DCC) in solution, for example, in methylene chloride (CH 2 Cl 2 ), N- methyl pyrrolidone, dimethyl formamide (DMF), or mixtures thereof.
  • an appropriate carboxyl group activator such as 2-(1H- benzotriazol-1-yl)-l,1,3,3 tetramethyluronium hexafluorophosphate (HBTU) or dicyclohexylcarbodimide (DCC) in solution, for example, in methylene chloride (CH 2 Cl 2 ), N- methyl pyrrolidone, dimethyl formamide (DMF), or mixtures thereof.
  • the desired peptide is decoupled from the resin support by treatment with a reagent, such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF), which not only cleaves the peptide from the resin, but also cleaves all remaining side chain protecting groups.
  • a reagent such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF)
  • TFA trifluoroacetic acid
  • HF hydrogen fluoride
  • the side chain protected peptide can be decoupled by treatment of the peptide resin with ammonia to give the desired side chain protected amide or with an alkylamine to give a side chain protected alkylamide or dialkylamide. Side chain protection is then removed in the usual fashion by treatment with hydrogen fluoride to give the free amides, alkylamides, or dialkylamides.
  • the resins used to prepare the peptide acids are employed, and the side chain protected peptide is cleaved with base and the appropriate alcohol (e.g., methanol). Side chain protecting groups are then removed in the usual fashion by treatment with hydrogen fluoride to obtain the desired ester.
  • Synthetic amino acids that can be substituted into the peptides of the present invention include, but are not limited to, N- methyl, L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, ⁇ amino acids such as L- ⁇ - hydroxylysyl and D- ⁇ -methylalanyl, L- ⁇ -methylalanyl, ⁇ amino acids, and isoquinolyl.
  • D- amino acids and non-naturally occurring synthetic amino acids can also be incorporated into the peptides used in the present invention.
  • amino and/or carboxy termini of the peptide compounds of the invention can also modify the amino and/or carboxy termini of the peptide compounds of the invention to produce other compounds of the invention.
  • the amino terminus may be acetylated with acetic acid or a halogenated derivative thereof such as ⁇ - chloroacetic acid, ⁇ -bromoacetic acid, or ⁇ -iodoacetic acid).
  • proline analogues in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed.
  • Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazoliny), 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., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl.
  • piperazinyl e.g., 1 -piperazinyl
  • piperidyl e.g., 1 -piperidyl, piperidino
  • pyranyl pyrazinyl, pyrazolidiny
  • heterocyclic groups can be substituted or unsubstituted.
  • the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.
  • the peptide compounds of the invention also serve as structural models for non- peptidic compounds with similar biological activity.
  • Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as the lead peptide compound, but with more favorable activity than the lead with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis [See, Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24:243-252]. These techniques include replacing the peptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, and iV-methylamino acids.
  • the compounds used in the present invention contain two intramolecular disulfide bonds. Such disulfide bonds may be formed by oxidation of the cysteine residues of each peptide monomer.
  • the control of cysteine bond formation is exercised by choosing an oxidizing agent of the type and concentration effective to optimize formation of the desired isomer. For example, oxidation of a peptide dimer to form two intramolecular disulfide bonds (one on each peptide chain) is preferentially achieved (over formation of intermolecular disulfide bonds) when the oxidizing agent is DMSO or iodine (I 2 ).
  • the formation of cysteine bonds is controlled by the selective use of thiol-protecting groups during peptide synthesis.
  • the first monomer peptide chain is synthesized with the two cysteine residues of the core sequence protected with a first thiol protecting group [e.g., trityl(Trt), allyloxycarbonyl (Alloc), and l -(4,4-dimethyl-2,6- dioxocyclohex-1-ylidene)ethyl (Dde) or the like]
  • the second monomer peptide is synthesized the two cysteine residues of the core sequence protected with a second thiol protecting group different from the first thiol protecting group [e.g., acetamidomethyl (Acm), t-butyl (fBu), or the like].
  • the first thiol protecting groups are removed
  • analogues of these disulfide derivatives in which one of the sulfurs has been replaced by a CH 2 group or other isotere for sulfur.
  • These analogues can be prepared from the compounds of the present invention, wherein each peptide monomer contains at least one C or homocysteine residue and an ⁇ - amino- ⁇ -butyric acid in place of the second C residue, via an intramolecular or intermolecular displacement, using methods known in the art [See, e.g., Barker, et al. (1992) J. Med. Chem. 35:2040-2048 and Or, et al. (1991) J. Org. Chem. 56:3146-3149].
  • Barker, et al. (1992) J. Med. Chem. 35:2040-2048 and Or, et al. (1991) J. Org. Chem. 56:3146-3149 One of skill in the art will readily appreciate that this displacement can also occur using other homologs of ⁇ -amino- ⁇
  • cyclization strategies include, for example, amide-cyclization strategies as well as those involving the formation of thio-ether bonds.
  • the compounds of the present invention can exist in a cyclized form with either an intramolecular amide bond or an intramolecular thio-ether bond.
  • a peptide may be synthesized wherein one cysteine of the core sequence is replaced with lysine and the second cysteine is replaced with glutamic acid. Thereafter a cyclic monomer may be formed through an amide bond between the side chains of these two residues.
  • a peptide may be synthesized wherein one cysteine of the core sequence is replaced with lysine (or serine).
  • a cyclic monomer may then be formed through a thio-ether linkage between the side chains of the lysine (or serine) residue and the second cysteine residue of the core sequence.
  • amide-cyclization strategies and thio- ether cyclization strategies can both be readily used to cyclize the compounds of the present invention.
  • the amino-terminus of the peptide can be capped with an ⁇ - substituted acetic acid, wherein the ⁇ -substituent is a leaving group, such as an ⁇ -haloacetic acid, for example, ⁇ -chloroacetic acid, ⁇ -bromoacetic acid, or ⁇ -iodoacetic acid.
  • an ⁇ -haloacetic acid for example, ⁇ -chloroacetic acid, ⁇ -bromoacetic acid, or ⁇ -iodoacetic acid.
  • the peptide monomers may be dimerized by a branched tertiary amide linker moiety.
  • the linker is incorporated into the peptide during peptide synthesis.
  • a linker L K moiety contains two functional groups capable of serving as initiation sites for peptide synthesis and one or more other functional groups (e.g., a carboxyl group or an amino group) that enables binding to one or more other molecular moieties
  • the linker may be conjugated to a solid support.
  • two peptide monomers may be synthesized directly onto the two reactive nitrogen groups of the linker L K moiety in a variation of the solid phase synthesis technique.
  • the linker may be conjugated to the two peptide monomers of a peptide dimer after peptide synthesis. Such conjugation may be achieved by methods well established in the art.
  • the linker contains two functional groups suitable for attachment to the target functional groups of the synthesized peptide monomers.
  • a linker containing two carboxyl groups either preactivated or in the presence of a suitable coupling reagent, may be reacted with the target lysine side chain amine groups of each of two peptide monomers.
  • the peptide monomers may be chemically coupled to the tertiary amide linker,
  • a *-C 1 O-CH 2 -X-CH 2 -C 2 O-B* where: X is NCO-(CH 2 ) 2 -NH-Y and Y is a suitable protecting group, such as a t-butyloxycarbonyl (Boc) protecting group; A* is a suitable functional group, such as N-oxy succinimide, used to conjugate C 1 of the linker to the ⁇ -amino group of the C-terminal lysine residue of the first peptide monomer; and B* is a suitable functional group, such as /V-oxy succinimide, used to conjugate C 2 of the linker to the ⁇ -amino group of the C-terminal lysine residue of the second peptide monomer.
  • X is NCO-(CH 2 ) 2 -NH-Y and Y is a suitable protecting group, such as a t-butyloxycarbonyl (Boc) protecting group
  • A* is a suitable functional group,
  • the peptide monomers may be chemically coupled to the tertiary amide linker, A*-C 1 O-CH 2 -X-CH 2 -C 2 O-B* where: X is NCO-(CH 2 ) 2 -NH-C 3 O-; A* is a suitable functional group, such as N-oxy succinimide, used to conjugate C 1 of the linker to the ⁇ -amino group of the C-terminal lysine residue of the first peptide monomer; and B* is a suitable functional group, such as N-oxy succinimide, used to conjugate C 2 of the linker to the ⁇ -amino group of the C-terminal lysine residue of the second peptide monomer; and the tertiary amide linker is chemically bonded to the spacer moiety, Y-NH-(CH 2 ) 4 -C 4 H-NH-Y where: C 3 of X is covalently bonded to C 4 of
  • the peptide monomers may be dimerized by a lysine linker L K moiety.
  • the lysine linker in incorporated into the peptide during peptide synthesis.
  • a lysine linker LK moiety contains two functional groups capable of serving as initiation sites for peptide synthesis and a third functional group (e.g., a carboxyl group or an amino group) that enables binding to another molecular moiety
  • the linker may be conjugated to a solid support.
  • two peptide monomers may be synthesized directly onto the two reactive nitrogen groups of the lysine linker L K moiety in a variation of the solid phase synthesis technique.
  • a peptide dimer is dimerized by a lysine linker L K moiety
  • said linker may be conjugated to the two peptide monomers of a peptide dimer after peptide synthesis. Such conjugation may be achieved by methods well established in the art.
  • the linker contains at least two functional groups suitable for attachment to the target functional groups of the synthesized peptide monomers. For example, the lysine's two free amine groups may be reacted with the C-terminal carboxyl groups of each of two peptide monomers.
  • the peptide compounds used in the invention further comprise a spacer moiety.
  • the spacer may be incorporated into the peptide during peptide synthesis.
  • a spacer contains a free amino group and a second functional group (e.g., a carboxyl group or an amino group) that enables binding to another molecular moiety
  • the spacer may be conjugated to the solid support.
  • a spacer containing two functional groups is first coupled to the solid support via a first functional group.
  • the lysine linker L K moiety having two functional groups capable of serving as initiation sites for peptide synthesis and a third functional group (e.g., a carboxyl group or an amino group) that enables binding to another molecular moiety is conjugated to the spacer via the spacer's second functional group and the linker's third functional group.
  • two peptide monomers may be synthesized directly onto the two reactive nitrogen groups of the linker L K moiety in a variation of the solid phase synthesis technique.
  • a solid support coupled spacer with a free amine group may be reacted with a lysine linker via the linker's free carboxyl group.
  • the spacer may be conjugated to the peptide dimer after peptide synthesis. Such conjugation may be achieved by methods well established in the art.
  • the linker contains at least one functional group suitable for attachment to the target functional group of the synthesized peptide.
  • a spacer with a free amine group may be reacted with a peptide's C-terminal carboxyl group.
  • a linker with a free carboxyl group may be reacted with the free amine group of a lysine amide.
  • PEG Polyethylene Glycol
  • PEG polyethylene glycol
  • covalent attachment of PEG to therapeutic polypeptides such as interleukins [Knauf, et al. ( 1988) J. Biol. Chem. 263; 15064; Tsutsumi, et al. ( 1995) J. Controlled Release 33:447), interferons (Kita, et al. (1990) Drug Des.
  • the peptide compounds of the invention may comprise a polyethylene glycol (PEG) moiety, which is covalently attached to the branched tertiary amide linker or the spacer of the peptide dimer via a carbamate linkage or via an amide linkage.
  • PEG polyethylene glycol
  • An example of PEG used in the present invention is linear, unbranched PEG having a molecular weight of about 20 kiloDaltons (20K) to about 4OK (the term "about” indicating that in preparations of PEG, some molecules will weigh more, some less, than the stated molecular weight).
  • the PEG has a molecular weight of about 3OK to about 40K.
  • PEG used in the present invention is linear PEG having a molecular weight of about 1OK to about 60K (the term "about” indicating that in preparations of PEG, some molecules will weigh more, some less, than the stated molecular weight).
  • the PEG has a molecular weight of about 2OK to about 4OK. More preferably, the PEG has a molecular weight of about 2OK.
  • Examples of methods for covalent attachment of PEG (PEGylation) are described below. These illustrative descriptions are not intended to be limiting. One of ordinary skill in the art will appreciate that a variety of methods for covalent attachment of a broad range of
  • PEG is well established in the art. As such, peptide compounds to which PEG has been attached by any of a number of attachment methods known in the art are encompassed by the present invention.
  • PEG may be covalently bound to the linker via a reactive group to which an activated PEG molecule may be bound (e.g., a free amino group or carboxyl group).
  • PEG molecules may be attached to amino groups using methoxylated PEG ("mPEG") having different reactive moieties.
  • mPEG methoxylated PEG
  • Such polymers include mPEG-succinimidyl succinate, mPEG- succinimidyl carbonate, mPEG-imidate, mPEG-4-nitrophenyl carbonate, and mPEG-cyanuric chloride.
  • PEG molecules may be attached to carboxyl groups using methoxylated PEG with a free amine group (HiPEG-NH 2 ).
  • the linker or spacer contains a terminal amino group (i.e., positioned at the terminus of the spacer). This terminal amino group may be reacted with a suitably activated PEG molecule, such as mPEG-para-nitrophenylcarbonate (mPEG-NPC), to make a stable covalent carbamate bond.
  • a suitably activated PEG molecule such as mPEG-para-nitrophenylcarbonate (mPEG-NPC)
  • this terminal amino group may be reacted with a suitably activated PEG molecule, such as an mPEG-succinimidyl butyrate (mPEG-SBA) or mPEG-succinimidyl propionate (mPEG-SPA) containing a reactive N- hydroxl-succinimide (NHS) group, to make a stable covalent carbamate bond.
  • a suitably activated PEG molecule such as an mPEG-succinimidyl butyrate (mPEG-SBA) or mPEG-succinimidyl propionate (mPEG-SPA) containing a reactive N- hydroxl-succinimide (NHS) group
  • the linker reactive group contains a carboxyl group capable of being activated to form a covalent bond with an amine-containing PEG molecule under suitable reaction conditions.
  • Suitable PEG molecules include mPEG-NH 2 and suitable reaction conditions include carbodiimide-mediated
  • EPO binding protein EBP
  • EBP extracellular domain of the human EPO-R
  • EBP EPO binding protein
  • a different in vitro competitive binding assay measures the light signal generated as a function of the proximity of two beads: an EPO-conjugated bead and an EPO-R- conjugated bead. Bead proximity is generated by the binding of EPO to EPO-R. A test peptide that competes with EPO for binding to EPO-R will prevent this binding, causing a decrease in light emission. The concentration of test peptide that results in a 50% decrease in light emission is defined as the IC50 value.
  • the peptides of the present invention compete very efficiently with EPO for binding to the EPO-R. This enhanced function is represented by their ability to inhibit the binding of EPO at substantially lower concentrations of peptide (i.e., they have very low IC50 values).
  • EPO-R agonists of the invention which bind specifically to the EPO-receptor
  • One assay is based upon a murine pre-B-cell line expressing human EPO-R and further transfected with a fos promoter-driven luciferase reporter gene construct.
  • EPO or another EPO-R agonist Upon exposure to EPO or another EPO-R agonist, such cells respond by synthesizing luciferase. Luciferase causes the emission of light upon addition of its substrate luciferin.
  • the level of EPO-R activation in such cells may be quantitated via measurement of luciferase activity.
  • test peptide The activity of a test peptide is measured by adding serial dilutions of the test peptide to the cells, which are then incubated for 4 hours. After incubation, luciferin substrate is added to the cells, and light emission is measured. The concentration of test peptide that results in a half-maximal emission of light is recorded as the EC50.
  • the peptides of the present invention show dramatically enhanced ability to promote EPO-R signaling-dependent luciferase expression in this assay. This enhanced function is represented by their ability to yield half of the maximal luciferase activity at substantially lower concentrations of peptide (i.e., they have very low EC50 values).
  • This assay is a preferred method for estimating the potency and activity of an EPO-R agonist peptide of the invention.
  • Another assay may be performed using FDC-P1/ER cells [Dexter, et al. (1980) J. Exp. Med. 152: 1036-1047], a well characterized nontransformed murine bone marrow derived cell line into which EPO-R has been stably transfected. These cells exhibit EPO- dependent proliferation.
  • FDC-P1/ER cells Dexter, et al. (1980) J. Exp. Med. 152: 1036-1047
  • these cells exhibit EPO- dependent proliferation.
  • the cells are grown to half stationary density in the presence of the necessary growth factors (see, e.g., as described in U.S. Pat. No. 5,773,569). The cells are then washed in PBS and starved for 16-24 hours in whole media without the growth factors.
  • the cells are grown to stationary phase in EPO-supplemented medium, collected, and then cultured for an additional 18 hr in medium without EPO.
  • the cells are divided into three groups of equal cell density: one group with no added factor (negative control), a group with EPO (positive control), and an experimental group with the test peptide.
  • the cultured cells are then collected at various time points, fixed, and stained with a DNA-binding fluorescent dye (e.g., propidium iodide or Hoechst dye, both available from Sigma). Fluorescence is then measured, for example, using a FACS Scan Flow cytometer.
  • a DNA-binding fluorescent dye e.g., propidium iodide or Hoechst dye, both 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 may then be determined, for example, using the SOBR model of CeIIFIT software (Becton Dickinson). Cells treated with EPO or an active peptide will show a greater proportion of cells in S phase (as determined by increased fluorescence as an indicator of increased DNA content) relative to the negative control group.
  • Similar assays may be performed using FDCP-I [see, e.g., Dexter et al. (1980) J. Exp. Med. 152: 1036-1047] or TF- I [Kitamura, et al. (1989) Blood 73:375-380] cell lines.
  • FDCP-I is a growth factor dependent murine multi-potential primitive hematopoietic progenitor cell line that can proliferate, but not differentiate, when supplemented with WEHI- 3-conditioned media (a medium that contains IL-3, ATCC number TIB-68).
  • WEHI- 3-conditioned media a medium that contains IL-3, ATCC number TIB-68.
  • the FDCP-I cell line is transfected with the human or murine EPO-R to produce FDCP- 1-hEPO-R or FDCP- 1-mEPO-R cell lines, respectively, that can proliferate, but not differentiate, in the presence of EPO.
  • TF-I an EPO-dependent cell line, may also be used to measure the effects of peptide EPO-R agonists on cellular proliferation.
  • the blot may be probed with an anti-phosphotyrosine antibody (e.g., mouse anti-phosphotyrosine IgG from Upstate Biotechnology, Inc.), washed, and then probed with a secondary antibody [e.g., peroxidase labeled goat anti-mouse IgG from Kirkegaard & Perry Laboratories, Inc. (Washington, D. C.)].
  • an anti-phosphotyrosine antibody e.g., mouse anti-phosphotyrosine IgG from Upstate Biotechnology, Inc.
  • a secondary antibody e.g., peroxidase labeled goat anti-mouse IgG from Kirkegaard & Perry Laboratories, Inc. (Washington, D. C.)
  • phosphotyrosine-containing proteins may be visualized by standard techniques including colorimetric, chemiluminescent, or fluorescent assays.
  • a chemiluminescent assay may be performed using the ECL Western Blotting System from Amers
  • Another cell-based in vitro assay that may be used to assess the activity of the peptides of the present invention is a colony assay, using murine bone marrow or human peripheral blood cells.
  • Murine bone marrow may be obtained from the femurs of mice, while a sample of human peripheral blood may be obtained from a healthy donor.
  • mononuclear cells are first isolated from the blood, for example, by centrifugation through a Ficoll-Hypaque gradient [Stem Cell Technologies, Inc. (Vancouver, Canada)]. For this assay a nucleated cell count is performed to establish the number and concentration of nucleated cells in the original sample.
  • a defined number of cells is plated on methyl cellulose as per manufacturer's instructions [Stem Cell Technologies, Inc. (Vancouver, Canada)].
  • An experimental group is treated with a test peptide, a positive control group is treated with EPO, and a negative control group receives no treatment.
  • the number of growing colonies for each group is then scored after defined periods of incubation, generally 10 days and 18 days. An active peptide will promote colony formation.
  • Other in vitro biological assays that can be used to demonstrate the activity of the compounds of the present invention are disclosed in Greenberger, et al. (1983) Proc. Natl. Acad.
  • One in vivo functional assay that may be used to assess the potency of a test peptide is the polycythemic exhypoxic mouse bioassay.
  • mice are subjected to an alternating conditioning cycle for several days. In this cycle, the mice alternate between periods of hypobaric conditions and ambient pressure conditions. Thereafter, the mice are maintained at ambient pressure for 2-3 days prior to administration of test samples.
  • Test peptide samples, or EPO standard in the case positive control mice are injected subcutaneously into the conditioned mice.
  • Radiolabeled iron e.g., 59 Fe
  • Plasma samples taken two days after administration of radiolabeled iron. Hematocrits and radioactivity measurements are then determined for each blood sample by standard techniques. Blood samples from mice injected with active test peptides will show greater radioactivity (due to binding of Fe 59 by erythrocyte hemoglobin) than mice that did not receive test peptides or EPO.
  • reticulocyte assay Another in vivo functional assay that may be used to assess the potency of a test peptide is the reticulocyte assay.
  • normal untreated mice are subcutaneously injected on three consecutive days with either EPO or test peptide. On the third day, the mice are also intraperitoneally injected with iron dextran. At day five, blood samples are collected from the mice. The percent (%) of reticulocytes in the blood is determined by thiazole orange staining and flow cytometer analysis (retic-count program). In addition, hematocrits are manually determined. The percent of corrected reticulocytes is determined using the following formula:
  • % RETIC CORRECTED % RETIC OBSERVED X(Hematocrit INDIVIDUAL /Hematocrit NORMAL ) Active test compounds will show an increased % RETIC CORRECTED level relative to mice that did not receive test peptides or EPO.
  • compositions of the above EPO-R agonist peptide compounds are provided. Conditions alleviated or modulated by the administration of such compositions include those indicated above.
  • Such pharmaceutical compositions may be for administration by oral, parenteral (intramuscular, intraperitoneal, intravenous (FV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.
  • compositions comprising effective amounts of an EPO-R agonist peptide, or derivative products, of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 20, Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulf ⁇ te), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used.
  • buffer content e.g., Tris-HCl, acetate, phosphate
  • additives e.g., Tween 20, Tween 80, Polysorbate 80
  • anti-oxidants e.g., ascorbic acid, sodium metabisulf ⁇ t
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference.
  • the compositions may be prepared in liquid form, or may be in dried powder (e.g., lyophilized) form.
  • compositions designed to administer the compounds of the present invention by parenteral administration generally characterized by subcutaneous, intramuscular or intravenous injection.
  • injectables may be prepared in any conventional form, for example as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • excipients examples include, but are not limited to water, saline, dextrose, glycerol or ethanol.
  • the injectable compositions may also optionally comprise minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.
  • Implantation of a slow- release or sustained-release system, such that a constant level of dosage is maintained see, e.g., U.S. Pat. No.
  • Parenteral administration of the formulations includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as the lyophilized powders described herein, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.
  • suitable carriers include, but are not limited to physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • pharmaceutically acceptable carriers that may optionally be used in parenteral preparations include, but are not limited to aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.
  • aqueous vehicles that may optionally be used include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection.
  • nonaqueous parenteral vehicles examples include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.
  • Antimicrobial agents in bacteriostatic or fungistatic concentrations may be added to parenteral preparations, particularly when the preparations are packaged in multiple-dose containers and thus designed to be stored and multiple aliquots to be removed. Examples of antimicrobial agents that may be used include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Examples of isotonic agents that may be used include sodium chloride and dextrose.
  • Examples of buffers that may be used include phosphate and citrate.
  • antioxidants that may be used include sodium bisulfate.
  • Examples of local anesthetics that may be used include procaine hydrochloride.
  • Examples of suspending and dispersing agents that may be used include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone.
  • Examples of emulsifying agents that may be used include Polysorbate 80 (TWEEN 80).
  • a sequestering or chelating agent of metal ions includes EDTA.
  • Pharmaceutical carriers may also optionally include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
  • concentration of an inhibitor in the parenteral formulation may be adjusted so that an injection administers a pharmaceutically effective amount sufficient to produce the desired pharmacological effect.
  • concentration of an inhibitor and/or dosage to be used will ultimately depend on the age, weight and condition of the patient or animal as is known in the art.
  • Unit-dose parenteral preparations may be packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile, as is known and practiced in the art.
  • Injectables may be designed for local and systemic administration.
  • a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1 % w/w up to about 90% w/w or more, for example, more than 1 % w/w of a EPO-R agonist peptide compound to the treated tissue(s).
  • the inhibitor may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment will be a function of the location of where the composition is parenterally administered, the carrier and other variables that may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data.
  • concentrations and dosage values may also vary with the age of the individual treated. It is to be further understood that for any particular subject, specific dosage regimens may need to be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. Hence, the concentration ranges set forth herein are intended to be exemplary and are not intended to limit the scope or practice of the claimed formulations.
  • the EPO-R agonist peptide compound may optionally be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the disease state and may be empirically determined.
  • Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules.
  • liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673).
  • Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No.
  • the formulation will include the EPO-R agonist peptides (or chemically modified forms thereof) and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
  • 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, emulsifying and suspending agents; and sweetening, flavoring, and perfuming agents.
  • the peptides may be chemically modified so that oral delivery of the derivative is efficacious.
  • the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine.
  • the increase in overall stability of the component or components and increase in circulation time in the body is also desired.
  • PEGylation is a preferred chemical modification for pharmaceutical usage.
  • moieties that may be used include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly- l ,3-dioxolane and poly- 1 ,3,6- tioxocane [see, e.g., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, N. Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem.
  • the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine.
  • the release will avoid the deleterious effects of the stomach environment, either by protection of the peptide (or derivative) or by release of the peptide (or derivative) beyond the stomach environment, such as in the intestine.
  • a coating impermeable to at least pH 5.0 is essential.
  • examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.
  • a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow.
  • Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (i.e. powder), for liquid forms a soft gelatin shell may be used.
  • the shell material of cachets could be thick starch or other edible paper.
  • moist massing techniques can be used.
  • the peptide (or derivative) can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs, or even as tablets. These therapeutics could be prepared by compression.
  • Colorants and/or flavoring agents may also be included.
  • the peptide (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
  • these diluents could include carbohydrates, especially mannitol, ⁇ -lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
  • Certain inorganic salts may be 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 Avicell.
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab.
  • Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
  • the disintegrants may also be insoluble cation ⁇ c exchange resins.
  • Powdered gums may be used as disintegrants and as binders and can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salf are also useful as disintegrants.
  • Binders may be used to hold the peptide (or derivative) agent together to form a hard tablet 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) could both be used in alcoholic solutions to granulate the peptide (or derivative).
  • An antifrictional agent 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 die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. [00145] Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • surfactant might be added as a wetting agent.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
  • nonionic detergents that could 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 fatty acid ester, methyl cellulose and carboxymethyl cellulose.
  • surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.
  • Additives which potentially enhance uptake of the peptide (or derivative) are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
  • Controlled release oral formulations may be desirable.
  • the peptide (or derivative) could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums.
  • Slowly degenerating matrices may also be incorporated into the formulation.
  • Some enteric coatings also have a delayed release effect.
  • Another form of a controlled release is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects.
  • Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan.
  • the peptide (or derivative) could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups.
  • the first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl -methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols.
  • the second group consists of the enteric materials that are commonly esters of phthalic acid.
  • a mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.
  • Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • non- aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.
  • compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.
  • compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.
  • Pulmonary Delivery also contemplated herein is pulmonary delivery of the EPO-R agonist peptides (or derivatives thereof).
  • the peptide (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream [see, e.g., Adjei, et al. (1990) Pharmaceutical Research 7:565-569; Adjei, et al. (1990) Int. J. Pharmaceutics 63: 135-144 (leuprolide acetate); Braquet, et al. (1989) J. Cardiovascular Pharmacology 13(sup5): 143-146 (endothelin-1); Hubbard, et al.
  • Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
  • Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N. C); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.).
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual 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 peptides may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.
  • Formulations suitable for use with a nebulizer will typically comprise 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 a buffer and a simple sugar ⁇ e.g., for protein stabilization and regulation of osmotic pressure).
  • the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the peptide (or derivative) caused by atomization of the solution in forming the aerosol.
  • Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the peptide (or derivative) suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof.
  • Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
  • Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing peptide (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
  • the peptide (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most effective delivery to the distal lung.
  • Nasal delivery of the EPO-R agonist peptides is also contemplated. Nasal delivery allows the passage of the peptide to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. [00160] Other penetration-enhancers used to facilitate nasal delivery are also contemplated for use with the peptides of the present invention (such as described in International Patent Publication No. WO 2004056314, filed Dec. 17, 2003, incorporated herein by reference in its entirety).
  • any one of the peptides of the present invention may be used to treat individuals with renal failure prior to dialysis or during dialysis (pre-dialysis or dialysis patients).
  • the therapeutic dose range in this embodiment can be 0.025 to 0.2 milligrams (mg) of compound per 1 kilogram (kg) of body weight of the individual (0.025- 0.2 mg/kg). More particularly, the dose range of 0.05-0.1 mg/kg would be preferred. Furthermore, a physician may initially use escalating dosages, starting at 0.025 mg/kg, and then titrate the dosage at approximately 0.025 mg/kg increments for each individual being treated based on their individual hemoglobin responses. Thus, the physician may titrate the dosage for each individual until an adequate hemoglobin response is achieved.
  • the adequate hemoglobin response would be to approximately attain normal hemoglobin levels (14-15 g/dL) or another hemoglobin level as determined by the physician.
  • the pharmacologically active dose (PAD) for each individual pre-dialysis or dialysis patient is expected to be 0.067-0.075 mg/kg.
  • An advantage of this embodiment is expected to be a lower dosing frequency of once every three to four weeks for each individual patient instead of weekly as is the case for other current erythropoiesis stimulating agents (ESAs).
  • ESAs erythropoiesis stimulating agents
  • Many routes of administration may be used (oral, IV, etc. as described above).
  • a preferred route of administration for dialysis patients would be intravenously.
  • a preferred route of administration for pre-dialysis patients would be subcutaneously.
  • one of the compounds described above may be used to treat individuals with anemia associated with malignancies (oncology patients).
  • the therapeutic dose range in this embodiment is expected to be three to five times the range for pre-dialysis or dialysis patients (i.e., 0.075-0.5 mg/kg). More particularly, the dose range of 0.2-0.4 mg/kg would be preferred.
  • the physician treating the oncology patients may titrate the dosage, starting at 0.075 mg/kg, and increasing at 0.075 mg/kg increments until an adequate hemoglobin response is attained.
  • the PAD for each individual oncology patient is expected to be approximately 0.25 mg/kg.
  • the dosage may be administered prior to chemotherapy (for example, 3-5 days beforehand) or co-administered with chemotherapy to prevent the decline in hemoglobin during the lag phase between reticulocyte stimulation and hemoglobin rise.
  • Many routes of administration may be used (oral, IV, etc. as described above).
  • Subcutaneous administration would be a preferred route of administration for oncology patients. Examples of compounds for use in treating oncology patients, include those shown below.
  • Amide linkage, with sarcosine, and range of PEG weights (here showing SEQ ED NO: 2): [00170] Amide linkage, sarcosine, and the preferred PEG weights (here showing SEQ ID NO: 2):
  • the peptides of the present invention may be administered in conjunction with one or more additional active ingredients or pharmaceutical compositions.
  • the compounds used in the methods of the present invention may be synthesized according to the procedures disclosed in U.S. patent application publications 2007/0104704 and 2008/0081783. These methods can be further modified and optimized using the principles and techniques of organic chemistry and/or biotechnology as applied by a person skilled in the art.
  • the biological activity of the peptide dimers that may be used in the context of the present invention has been tested.
  • the tests have included both in vitro and in vivo assays to evaluate the activity and potency of EPO-R agonist peptides.
  • the in vivo assays include polycythemic exhypoxic mouse bioassays, reticulocyte assasys and hematological assays.
  • Examples of in vitro assays conducted on the EPO-R agonist peptides include reporter assay, proliferation assays, competitive binding assays and C/BFU-e assays. Further, pharmacokinetic studies in rats, dogs monkeys and humans have been conducted on Peptide I.
  • Peptide I appeared safe and well tolerated after single IV doses of 0.025, 0.05, or 0.1 mg/kg, with a safety profile similar to placebo.
  • the pharmacokinetic results showed a halflife ranging from approximately 15 to 33 hours, with a mean of 23.5 hours at the 0.1 mg/kg dose. The median was comparable for all doses, occurring 15 minutes after the start of the infusion.
  • C 1113x appeared to have a linear relationship with dose; AUC(o.,nfin ⁇ ty) appeared to be nonlinear at the 0.1 mg/kg dose.
  • First order kinetics were observed at lower drug concentrations only, suggesting saturation of metabolic/elimination processes at plasma concentrations >400 ng/mL.
  • Peptide I showed pharmacological activity for reticulocytes at all doses evaluated; generally the responses were dose-dependent with greater and longer responses with increasing dose.
  • the 0.1 mg/kg dose group was defined as the PAD in NHV as it was associated with a clinically and statistically significant increase in hemoglobin from baseline, with an average maximum increase from baseline of 1.36+0.39 g/dL among the 10 Peptide I recipients. Changes in other pharmacodynamic parameters (increased red cell count and hematocrit, transient decreases in ferritin and reticulocyte hemoglobin content, transient increase in soluble transferrin receptor protein, and transient decrease in EPO) were consistent with stimulation of erythropoiesis. [00174] Incorporated herein by reference in its entirety is the poster and abstract #0470 presented at the European Hematology Association 10th Annual Congress in Sweden on Jun. 4, 2005.
  • a highly effective method of birth control is defined as one that results in a low failure rate (i.e., less than 1% per year) when used consistently and correctly such as implants, injectables, combined oral contraceptives, some IUDs, sexual abstinence (only acceptable if practiced as a life-style and not acceptable if one who is sexually active practices abstinence only for the duration of study) or vasectomized partner; (3) Patients with histologically confirmed solid tumor malignancy or lymphoma who are scheduled to receive at least 9 weeks of cyclic myelosuppressive chemotherapy while on study; (4) a hemoglobin value of > 8 and ⁇ 1 1 g/dL within 1 week prior to administration of study drug; (5) an ECOG Performance Status of 0-2; (6) one reticulocyte hemoglobin content (CHr) > 29 pg within 4 weeks prior to study drug administration; (7) one transferrin saturation > 15% within 4 weeks prior to study drug administration; (8) one serum or red cell folate level above the lower limit of normal within
  • the patient was considered by the investigator as iron-deficient and IV iron supplementation was required, the patient was re-screened weekly (no sooner than 7 days after iron administration) until hemoglobin has not increased more than 0.5 g/dL from the previous week.
  • Patient exclusion criteria included: (1 ) treatment with any erythropoiesis stimulating agent (ESA) in the past 90 days; (2) history of failure to respond to ESA treatment; (3) known antibodies to other ESAs or history of pure red cell aplasia (PRCA); (4) acute or chronic leukemia, myelodysplastic syndrome (MDS), or multiple myeloma; (5) any previous or planned radiotherapy to more than 50% of either the pelvis or spine; (6) known intolerance to parenteral iron supplementation; (7) red-blood cell (RBC) transfusion within 4 weeks prior to study drug administration; (8) known hemoglobinopathy (e.g., homozygous sickle-cell disease, thalassemia of all types, etc.); (9) known hemolysis; (10) history of pulmonary embolism or DVT in the previous 2 years or current therapeutic doses of anticoagulants; (1 1 ) known blood loss as a cause of anemia; (12) uncontrolled, or symptomatic inflammatory disease (e.g.,
  • Tables 2-4 provide clinical results by cancer group.
  • the data includes hemoglobin concentrations (the top portion of each table) and dosing information (bottom portion of each table) for Peptide I.
  • the abbreviations used in these tables are as follows: “Hgb” refers to Hgb concentration in g/dL; “n” indicates the number of observations; “std” refers to standard deviation.
  • “Taxane” indicates that the patient was being treated with a taxane-based chemotherapeutic agent.
  • Non-Taxane indicates that the patient was being treated with a non-taxane based chemotherapeutic agent. Empty cells indicate that no data was obtained or that no dose was given.
  • Hgb values are censored due to transfusion, as was planned for in the protocol.
  • Censoring is a statistical technique that ignores data that might have been confounded by something other that what is being studied. Censoring of some data is typically described ahead of the analysis either in the protocol or in the statistical analysis plan. In this case, when study patients receive a blood transfusion, their Hgb data is confounded for a period of time after the transfusion. The protocol specified that pharmacodynamic data, such as Hgb, would be censored for 4 weeks following a transfusion. The marked values are those that occurred in the 4-week period after a transfusion and were thus "ignored" in the analysis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Diabetes (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne de nouvelles utilisations de composés peptidiques qui sont des agonistes du récepteur d'érythropoïétine (EPO-R). L'invention concerne également des procédés d'utilisation de tels composés peptidiques pour traiter des troubles associés à une production insuffisante ou défectueuse de globules rouges, y compris une anémie provoquée par une chimiothérapie. Des compositions pharmaceutiques, qui comportent les composés peptidiques de l'invention, sont également proposées. Des kits et des articles manufacturés comportant de tels composés sont également proposés.
PCT/US2008/070926 2005-06-03 2008-07-23 Formulations de peptide de récepteur d'érythropoïétine et utilisations WO2009025957A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2010521912A JP2010536857A (ja) 2007-08-22 2008-07-23 エリスロポエチン受容体ペプチドの製剤及び使用
US12/195,832 US20090221510A1 (en) 2005-06-03 2008-08-21 Erythropoietin receptor peptide formulations and uses

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US95739607P 2007-08-22 2007-08-22
US60/957,396 2007-08-22
US98975807P 2007-11-21 2007-11-21
US60/989,758 2007-11-21

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US11/777,500 Division US7919461B2 (en) 2005-06-03 2007-07-13 Erythropoietin receptor peptide formulations and uses

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/195,832 Continuation US20090221510A1 (en) 2005-06-03 2008-08-21 Erythropoietin receptor peptide formulations and uses

Publications (1)

Publication Number Publication Date
WO2009025957A1 true WO2009025957A1 (fr) 2009-02-26

Family

ID=40194013

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2008/070943 WO2009025958A1 (fr) 2007-08-22 2008-07-23 Formulations de peptide de récepteur d'érythropoïétine et utilisations
PCT/US2008/070926 WO2009025957A1 (fr) 2005-06-03 2008-07-23 Formulations de peptide de récepteur d'érythropoïétine et utilisations

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2008/070943 WO2009025958A1 (fr) 2007-08-22 2008-07-23 Formulations de peptide de récepteur d'érythropoïétine et utilisations

Country Status (3)

Country Link
EP (1) EP2195002A4 (fr)
JP (2) JP2010536858A (fr)
WO (2) WO2009025958A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2709648A4 (fr) * 2011-05-19 2015-04-08 Geysen Hendrik M Composés qui se lient au récepteur d'érythropoïétine
US11013704B2 (en) * 2014-05-28 2021-05-25 The Board Of Regents Of The University Of Texas System Compounds supports hematopoietic stem cells and red blood cells

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004101606A2 (fr) * 2003-05-12 2004-11-25 Affymax, Inc. Nouveaux peptides se fixant au recepteur de l'erythropoietine
WO2006133144A2 (fr) * 2005-06-03 2006-12-14 Affymax, Inc. Preparations de peptides agonistes du recepteur de l'erythropoietine et utilisations
US20070027074A1 (en) * 2003-05-12 2007-02-01 Affymax, Inc. Novel peptides that bind to the erythropoietin receptor
US20080081783A1 (en) * 2005-06-03 2008-04-03 Affymax, Inc. Erythropoietin receptor peptide formulations and uses

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8343953B2 (en) * 2005-06-22 2013-01-01 Astex Therapeutics Limited Pharmaceutical compounds

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004101606A2 (fr) * 2003-05-12 2004-11-25 Affymax, Inc. Nouveaux peptides se fixant au recepteur de l'erythropoietine
US20070027074A1 (en) * 2003-05-12 2007-02-01 Affymax, Inc. Novel peptides that bind to the erythropoietin receptor
WO2006133144A2 (fr) * 2005-06-03 2006-12-14 Affymax, Inc. Preparations de peptides agonistes du recepteur de l'erythropoietine et utilisations
US20080081783A1 (en) * 2005-06-03 2008-04-03 Affymax, Inc. Erythropoietin receptor peptide formulations and uses

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS, BIOWORLD TODAY, vol. 19, no. 18, 28 January 2008 (2008-01-28) *
ANONYMOUS, PHARMA MARKETLETTER, 4 February 2008 (2008-02-04) *
DATABASE NLDB 28 January 2008 (2008-01-28), ANONYMOUS: "Clinic Roundup", XP002510130, retrieved from STN Database accession no. 2008:111355 *
DATABASE PHARMAML [online] 4 February 2008 (2008-02-04), ANONYMOUS: "Takeda doses first patient in Ph I CIA Hematide trial", XP002510131, retrieved from STN Database accession no. 16770024 *
FAN ET AL: "Preclinical evaluation of Hematide, a novel erythropoiesis stimulating agent, for the treatment of anemia", EXPERIMENTAL HEMATOLOGY, NEW YORK, NY, US, vol. 34, no. 10, 1 October 2006 (2006-10-01), pages 1303 - 1311, XP005656813, ISSN: 0301-472X *
GLASPY J A: "THE DEVELOPMENT OF ERYTHROPOIETIC AGENTS IN ONCOLOGY", EXPERT OPINION ON EMERGING DRUGS, ASHLEY PUBLICATIONS, GB, vol. 10, no. 3, 1 January 2005 (2005-01-01), pages 553 - 567, XP008074550, ISSN: 1472-8214 *
REDDY G K ET AL: "Preliminary results of a phase II dose-finding study of subcutaneous Hematide(TM) in patients with cancer receiving chemotherapy", SUPPORTIVE CANCER THERAPY 200701 US, vol. 4, no. 2, January 2007 (2007-01-01), pages 74 - 75, XP001539117, ISSN: 1543-2912 *
WOODBURN ET AL: "Hematide is immunologically distinct from erythropoietin and corrects anemia induced by antierythropoietin antibodies in a rat pure red cell aplasia model", EXPERIMENTAL HEMATOLOGY, NEW YORK, NY, US, vol. 35, no. 8, 26 July 2007 (2007-07-26), pages 1201 - 1208, XP022171067, ISSN: 0301-472X *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2709648A4 (fr) * 2011-05-19 2015-04-08 Geysen Hendrik M Composés qui se lient au récepteur d'érythropoïétine
US10005826B2 (en) 2011-05-19 2018-06-26 Epodose Llc Compounds that bind to the erythropoietin receptor
US11013704B2 (en) * 2014-05-28 2021-05-25 The Board Of Regents Of The University Of Texas System Compounds supports hematopoietic stem cells and red blood cells

Also Published As

Publication number Publication date
JP2010536858A (ja) 2010-12-02
WO2009025958A1 (fr) 2009-02-26
JP2010536857A (ja) 2010-12-02
EP2195002A1 (fr) 2010-06-16
EP2195002A4 (fr) 2011-09-07

Similar Documents

Publication Publication Date Title
US8304391B2 (en) Peptides that bind to the erythropoietin receptor
EP1629007B1 (fr) Nouveaux peptides se fixant au recepteur de l'erythropoietine
US7550433B2 (en) Erythropoietin receptor peptide formulations and uses
US20080108564A1 (en) Novel peptides that bind to the erythropoietin receptor
US20090221510A1 (en) Erythropoietin receptor peptide formulations and uses
US20120157660A1 (en) Novel peptides that bind to the erythropoietin receptor
WO2009025957A1 (fr) Formulations de peptide de récepteur d'érythropoïétine et utilisations
US8324159B2 (en) Erythropoietin receptor peptide formulations and uses
AU2012203812A1 (en) Erythropoietin receptor peptide formulations and uses

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08796503

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2010521912

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08796503

Country of ref document: EP

Kind code of ref document: A1