WO1996040189A1 - Peptides et composes se fixant a un recepteur - Google Patents

Peptides et composes se fixant a un recepteur Download PDF

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Publication number
WO1996040189A1
WO1996040189A1 PCT/US1996/008998 US9608998W WO9640189A1 WO 1996040189 A1 WO1996040189 A1 WO 1996040189A1 US 9608998 W US9608998 W US 9608998W WO 9640189 A1 WO9640189 A1 WO 9640189A1
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WIPO (PCT)
Prior art keywords
group
peptide
compound
amino acids
peptides
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Application number
PCT/US1996/008998
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English (en)
Inventor
William J. Dower
Ronald W. Barrett
Steven E. Cwirla
David J. Duffin
Christian M. Gates
Sherril S. Johnson
Larry C. Mattheakis
Peter J. Schatz
Christopher R. Wagstrom
Nicholas C. Wrighton
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Glaxo Group Limited
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Application filed by Glaxo Group Limited filed Critical Glaxo Group Limited
Priority to AU60466/96A priority Critical patent/AU6046696A/en
Publication of WO1996040189A1 publication Critical patent/WO1996040189A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • 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 provides peptides and compounds that bind to and activate the thrombopoietin
  • TPO receptor c-mpl or TPO-R
  • the invention has application in the fields of biochemistry and medicinal chemistry and particularly provides TPO agonists for use in the treatment of human disease.
  • Megakaryocytes are bone marrow-derived cells, which are responsible for producing circulating blood platelets. Although comprising ⁇ 0.25% of the bone marrow cells in most species, they have >10 times the volume of typical marrow cells. See Kuter et. al. Proc. Natl. Acad. Sci. USA
  • Megakaryocytes undergo a process known as endomitosis whereby they replicate their nuclei but fail to undergo cell division and thereby give rise to polyploid cells.
  • endomitotic rate increases, higher ploidy megakaryocytes are formed, and the number of megakaryocytes may increase up to 3-fold.
  • the endomitotic rate decreases, lower ploidy megakaryocytes are formed, and the number of megakaryocytes may decrease by 50%.
  • TPO thrombopoietin
  • TPO is thought to affect megakaryocytopoiesis in several ways: (1) it produces increases in megakaryocyte size and number; (2) it produces an increase in DNA content, in the form of
  • acetylcholinesterase-positive cells in the bone marrow.
  • TPO has potential useful application in both the diagnosis and the treatment of various hematological disorders, for example, diseases primarily due to platelet defects. Ongoing clinical trials with TPO have indicated that TPO can be administered safely to patients. In addition, recent studies have provided a basis for the projection of efficacy of TPO therapy in the treatment of thrombocytopenia, and particularly
  • thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transplantation as treatment for cancer or lymphoma. See, e.g., McDonald (1992) Am. J. Ped. Hematology/Oncology 14:8-21 (1992).
  • Thrombopoietin is a glycoprotein with at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, with a common N-terminal amino acid sequence. See, Bartley et al. Cell 77:1117-1124 (1994). Thrombopoietin appears to have two distinct regions separated by a potential Arg-Arg cleavage site. The amino-terminal region is highly conserved in man and mouse, and has some homology with
  • carboxy-terminal region shows wide species divergence.
  • TPO-R human TPO-R
  • haematopoietin growth factor receptor family a family characterized by a common structural design of the extracellular domain, including four conserved C residues in the N-terminal portion and a WSXWS motif close to the transmembrane region. See Bazan Proc. Natl. Acad. Sci. USA 87:6934-6938 (1990).
  • mpl RNA significantly inhibits the appearance of megakaryocyte colonies without affecting erythroid or myeloid colony formation.
  • TPO-R The availability of cloned genes for TPO-R facilitates the search for agonists of this important
  • invention provides such an agonist.
  • This invention is directed, in part, to the novel and unexpected discovery that defined low molecular weight peptides and peptide mimetics have strong binding properties to the TPO-R and can activate the TPO-R. Accordingly, such peptides and peptide mimetics are useful for therapeutic purposes in treating conditions mediated by TPO (e.g., thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transfusions) as well as for TPO (e.g., thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transfusions) as well as for
  • Peptides and peptide mimetics suitable for therapeutic and/or diagnostic purposes have an IC 50 of about 2 mM or less, as determined by the binding affinity assay set forth in Example 3 below wherein a lower IC 50 correlates to a stronger binding affinity to TPO-R.
  • the peptides and peptidomimetics preferably have an IC 50 of no more than about 100 ⁇ m, more preferably, no more than 500 nM.
  • the molecular weight of the peptide or peptide mimetic is from about 250 to about 8000 daltons.
  • the peptides and peptide mimetics When used for diagnostic purposes, the peptides and peptide mimetics preferably are labeled with a detectable label and, accordingly, the peptides and peptide mimetics without such a label serve as intermediates in the preparation of labeled peptides and peptide mimetics.
  • Peptides meeting the defined criteria for molecular weight and binding affinity for TPO-R comprise 9 or more amino acids wherein the amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids.
  • Peptide mimetics include peptides having one or more of the following modifications :
  • peptides wherein one or more of the peptidyl [-C(O)NR-] linkages (bonds) have been replaced by a non-peptidyl linkage such as a -CH 2 -carbamate linkage [-CH 2 -OC(O)NR-]; a
  • N-terminus is derivatized to a -NRR 1 group; to a -NRC(O)R group; to a -NRC(O)OR group; to a
  • benzyloxycarbonyl-NH- group having from 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chlor ⁇ , and bromo; or
  • peptides wherein the C terminus is derivatized to -C(O)R 2 where 2 is selected from the group consisting of lower alkoxy, and -NR 3 R 4 where R 3 and R 4 are independently selected from the group consisting of hydrogen and lower alkyl.
  • preferred peptides and peptide mimetics comprise a compound having:
  • N-terminus of said peptide or peptide mimetic is selected from the group consisting of a -NRR 1 group; a -NRC(O)R group; a -NRC(O)OR group; a -NRS(O) 2 R group; a -NHC(O)NHR group; a succinimide group; a
  • benzyloxycarbonyl-NH- group and a benzyloxycarbonyl-NH- group having from 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chloro, and bromo, where R and R 1 are independently selected from the group consisting of hydrogen and lower alkyl,
  • the C-terminus of said peptide or peptide mimetic has the formula -C(O)R 2 where R 2 is selected from the group consisting of hydroxy, lower alkoxy, and -NR 3 R 4 where R 3 and R 4 are independently selected from the group consisting of hydrogen and lower alkyl and where the nitrogen atom of the -NR 3 R 4 group can optionally be the amine group of the N-terminus of the peptide so as to form a cyclic peptide,
  • the invention is directed to a labeled peptide or peptide mimetic comprising a peptide or peptide mimetic described as above having covalently attached thereto a label capable of detection.
  • preferred peptides for use include peptides having a core structure comprising a sequence of amino acids:
  • X 1 is C, L, M, P, Q, V
  • X 2 is F, K, L, N, Q, R, S, T or V
  • X 3 is C, F, I, L, M, R, S, V or W
  • X 4 is any of the 20 genetically coded L-amino acids
  • X 5 is A, D, E, G, K, M, Q, R, S, T, V or Y
  • X 6 is C, F, G, L, M, S, V, W or Y
  • X 7 is C, G, I, K, L, M, N, R or V.
  • X 1 is L, M, P, Q, or V
  • X 2 is F, R, S, or T
  • X 3 is F, L, V, or W
  • X 4 is A, K, L, M, R, S, V, or T
  • X 5 is A, E, G, K, M, Q, R, S, or T
  • X 7 is C, I, K, L, M or V
  • each X 8 residue is independently selected from any of the 20 genetically coded L-amino acids, their stereoisomeric D-amino acids
  • each X 8 residue is independently selected from any of the 20 genetically coded L-amino acids and their stereoisomeric D-amino acids.
  • X 1 is P; X 2 is T; X 3 is L; X 4 is R; X 5 is E or Q; and X 7 is I or L.
  • the core peptide comprises a sequence of amino acids :
  • X 9 is A, C, E, G, I, L , M, P, R, Q, S, T, or V; and X 8 is A, C, D, E, K, L, Q, R, S, T, or V. More preferably, X 9 is A or I; and X 8 is D, E, or K.
  • Particularly preferred peptides include: G G C A D G P T L R E W I S F C G G; G N A D G P T L R Q W L E G R R P K N; G G C A D G P T L R E W I S F C G G K; T I K G P T L R Q W L K S R E H T S; S I E G P T L R E W L T S R T P H S; L A I E G P T L R Q W L H G N G R D T; C A D G P T L R E W I S F C; and I E G P T L R Q W L A A R A.
  • preferred peptides for use in this invention include peptides having a core structure comprising a sequence of amino acids: C X 2 X 3 X 4 X 5 X 6 X 7 where X 2 is K, L, N, Q, R, S, T or V; X 3 is C, F, I, L, M, R,
  • X 4 is any of the 20 genetically coded L-amino acids
  • X 5 is A, D, E, G, S, V or Y; X 6 is C, F, G, L, M, S, V, W or Y; and X 7 is C, G, I, K, L, M, N, R or V.
  • X 4 is A, E, G, H, K, L, M, P, Q, R, S, T, or W.
  • X 2 is S or T;
  • X 3 is L or R;
  • X 4 is R;
  • X 5 is D, E, or G
  • X 6 is F, L, or W
  • X 7 is I, K, L, R, or
  • Particularly preferred peptides include: G G C T L R E W L H G G F C G G.
  • preferred peptides for use in this invention include peptides having a structure
  • X 2 is F, K, L, N, Q, R, S, T or V
  • X 3 is C, F, I, L, M, R, S, V or W
  • X 4 is any of the 20 genetically coded L-amino acids
  • X 5 is A, D, E, G, K, M, Q, R, S, T, V or Y
  • X 6 is C, F, G, L, M, S, V, W or Y
  • X 7 is C, G, I, K, L, M, N, R or V
  • X 8 is any of the 20 genetically coded L-amino acids.
  • X 8 is preferably G, S, Y, or R.
  • the compounds described herein are useful for the prevention and treatment of diseases mediated by TPO, and particularly for treating hematological disorders, including but not limited to, thrombocytopenia resulting from
  • the present invention also provides a method for treating wherein a patient having a disorder that is
  • TPO agonist susceptible to treatment with a TPO agonist receives, or is administered, a therapeutically effective dose or amount of a compound of the present invention.
  • compositions comprising one or more of the compounds described herein and a physiologically acceptable carrier.
  • These pharmaceutical compositions can be in a variety of forms including oral dosage forms, as well as inhalable powders and solutions and injectable and infusible solutions.
  • Figures 1A-B illustrates the results of a functional assay in the presence of various peptides; the assay is described in Example 2.
  • Figure 1A is a graphical depiction of the results of the TPO-R transfected Ba/F3 cell proliferation assay for selected peptides of the invention:
  • Figure 1B is a graphical depiction of the results with the same peptides and the parental cell line.
  • Figure 2A-C show the results of peptide
  • Figure 2A shows the results of the assay for the complexed biotinylated peptide (AF 12285 with
  • FIG. 2B shows the results of the assay for the free biotinylated peptide (AF 12285) for both the transfected and parental cell lines.
  • Figure 2C shows the results of the assay for streptavidin alone for both the transfected and parental cell lines.
  • Figures 3A-G show the results of a series of control experiments showing the activity of TPO, the peptides of the present invention, EPO, and EPO-R binding peptides in a cell proliferation assay using either the TPO-R transfected Ba/F3 cell line and its corresponding parental line, or an
  • FIG. 3A depicts the results for TPO in the cell proliferation assay using the TPO-R
  • FIG. 3B depicts the results for EPO in the cell proliferation assay using the TPO-R transfected Ba/F3 cell line and its corresponding parental line.
  • Figure 3C depicts the results for complexed biotinylated peptide (AF 12285 with streptavidin (SA)) and a complexed form of a biotinylated EPO-R binding peptide (AF 11505 with SA) in the TPO-R
  • FIG. 3E depicts the results for TPO in the cell
  • FIG. 3F depicts the results for EPO in the cell proliferation assay using the EPO-dependent cell line.
  • Figure 3G depicts the results for complexed biotinylated peptide (AF 12885 with streptavidin (SA)) and the complexed form of a biotinylated EPO-R binding peptide (AF 11505 with SA) in the EPO-dependent cell line.
  • SA streptavidin
  • Figures 4A-C illustrates the construction of peptides-on-plasmids libraries in vector pJS142.
  • Figure 4A shows a restriction map and position of the genes.
  • the library plasmid includes the rrnB transcriptional terminator, the bla gene to permit selection on ampicillin, the M13 phage intragenic region (M23 IG) to permit rescue of single-stranded DNA, a plasmid replication origin (ori), two lacO s sequences, and the araC gene to permit positive and negative regulation of the araB promoter driving expression of the lac fusion gene.
  • Figure 4B shows the sequence of the cloning region at the 3' end of the lac I gene, including the Sfil and EagI sites used during library construction.
  • Figure 4C shows the ligation of annealed library oligonucleotides, ON-829 and ON-830, to Sfil sites of pJS142 to produce a library. Single spaces in the sequence indicate sites of ligation.
  • Figures 5A-B illustrate cloning into the pELM3 and pELM15 MBP vectors .
  • Figure 5A shows the sequence at the 3' end of the malE fusion gene, including the MBP coding sequence, the poly asparagine linker, the factor Xa protease cleavagge site, and the available cloning sites.
  • pMALc2 pELM3
  • pMALp2 pELM15
  • Figure 5B shows the sequence of the vectors after transfer of the BspEII-Scal library fragment into Agel-Scal digested pELM3/pELM15.
  • the transferred sequence includes the sequence encoding the GGG peptide linker from the pJS142 library.
  • Figure 6A depicts a restriction map and position of the genes for the construction of headpiece dimer libraries in vector pCMG14.
  • the library plasmid includes: the rmB transcriptional terminator, the bla gene to permit selection on ampicillin, the M13 phage intragenic region (M13 IG) to permit rescue of single-stranded DNA, a plasmid replication origin (ori), one lacO s ssequence, and the araC gene to permit positive and negative regulation of the araB promoter driving expression of the headpiece dimer fusion gene.
  • Figure 6B depicts the sequence of the cloning region at the 3' end of the headpiece dimer gene, including the Sfil and EagI sites used during library construction.
  • Figure 6C shows the
  • Figures 7 to 9 show the results of further assays evaluating activity of the peptides and peptide mimETICS of the invention. In this assay mice are made thrombocytopenic with carboplatin. Figure 7 depicts typical results when
  • FIG. 8 depicts the effect of carboplatin titration on platelet counts in mice treated with the indicated amounts of carboplatin (in mg/kg, intraperitoneally (ip) on Day 0).
  • Figure 9 depicts amelioration of carboplatin-induced thrombocytopenia on Day 10 by peptide AF12513 (513).
  • Carboplatin (CBP; 50-125 mg/kg, intraperitoneally) was administered on Day 0.
  • AF12513 (1 mg/kg, ip) was given on Days 1-9.
  • Antist 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.
  • “Pharmaceutically acceptable salts” refer to the non-toxic alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry including the sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art. The term also includes
  • non-toxic acid addition salts which are generally prepared by reacting the compounds of this invention with a suitable organic or inorganic acid.
  • Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate, and the like.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, menthanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • acid addition salts as prodrugs, see Bundgaard, H., supra.
  • “Pharmaceutically acceptable ester” refers to those esters which retain, upon hydrolysis of the ester bond, the biological effectiveness and properties of the carboxylic acid or alcohol and are not biologically or otherwise undesirable.
  • pharmaceutically acceptable esters as prodrugs, see Bundgaard, H., ed., Design of Prodrugs,
  • esters are typically formed from the corresponding carboxylic acid and an alcohol. Generally, ester formation can be
  • the alcohol component of the ester will generally comprise (i) a C 2 -C 12 aliphatic alcohol that can or can not contain one or more double bonds and can or can not contain branched carbons or (ii) a C 7 -C 12 aromatic or heteroaromatic alcohols.
  • This invention also contemplates the use of those compositions which are both esters as
  • “Pharmaceutically acceptable amide” refers to those amides which retain, upon hydrolysis of the amide bond, the biological effectiveness and properties of the carboxylic acid or amine and are not biologically or otherwise undesirable.
  • pharmaceutically acceptable amides as prodrugs, see Bundgaard, H., ed., Design of Prodrugs,
  • amides are typically formed from the corresponding carboxylic acid and an amine. Generally, amide formation can be accomplished via conventional synthetic techniques. (See, e.g., March Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York (1985) p. 1152 and Mark et al. Encyclopedia of Chemical Technology, John Wiley & Sons, New York (1980)). This invention also contemplates the use of those compositions which are both amides as described herein and at the same time are the pharmaceutically acceptable acid addition salts thereof.
  • “Pharmaceutically or therapeutically acceptable carrier” refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.
  • Stepoisomer refers to a chemical compound having the same molecular weight, chemical composition, and
  • the compounds of the instant invention may have one or more asymmetrical carbon atoms and therefore include various stereoisomers. All stereoisomers are included within the scope of the invention.
  • composition sufficient to induce a desired biological result. That result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In the present invention, the result will typically involve a
  • Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L;
  • Isoleucine is lie. or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin 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.
  • Bu is Butoxy
  • Bzl is benzyl
  • CHA is cyclohexylamine
  • Ac is acetyl
  • Me is methyl
  • Pen is penicillamine
  • Aib is amino isobutyric acid
  • Nva is norvaline
  • Abu is amino butyric acid
  • Thi is thienylalanine
  • OBn O-benzyl
  • hyp is
  • peptidomimetics or peptide analogs are also provided.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics” or “peptidomimetics” (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference). Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect.
  • peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a paradigm polypeptide (i.e., a polypeptide that has a paradigm polypeptide (i.e., a polypeptide that has a paradigm polypeptide (i.e., a polypeptide that has a paradigm polypeptide (i.e., a polypeptide that has a paradigm polypeptide (i.e., a polypeptide that has a
  • polypeptide embodiments including, for example: more
  • Such non-interfering positions generally are positions that do not form direct contacts with the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s) (e.g., immunoglobulin superfamily molecules) to which the macromolecules (s)
  • peptidomimetic binds to produce the therapeutic effect.
  • Derivitization e.g., labeling
  • peptidomimetics should not substantially interfere with the desired biological or
  • peptidomimetics of receptor-binding peptides bind to the receptor with high affinity and possess detectable biological activity (i.e., are agonistic or antagonistic to one or more receptor-mediated phenotypic changes).
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type may be used to generate more stable peptides.
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein by reference) ; for example, by adding internal cysteine residues capable of forming
  • Synthetic or non-naturally occuring amino acids refer to amino acids which do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein.
  • Preferred synthetic amino acids are the D-a-amino acids of naturally occurring L-a-amino acid as well as non-naturally occurring D- and L-a-amino acids represented by the formula H 2 NCHR 5 COOH where R 5 is 1) a lower alkyl group, 2) a cycloalkyl group of from 3 to 7 carbon atoms, 3) a heterocycle of from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen, 4) an aromatic residue of from 6 to 10 carbon atoms optionally having from 1 to 3 substituents on the aromatic nucleus selected from the group consisting of
  • alkylene is an alkylene group of from 1 to 7 carbon atoms and Y is selected from the group consisting of (a) hydroxy, (b) amino, (c) cycloalkyl and cycloalkenyl of from 3 to 7 carbon atoms, (d) aryl of from 6 to 10 carbon atoms optionally having from 1 to 3 substituents on the aromatic nucleus selected from the group consisting of hydroxyl, lower alkoxy, amino and carboxyl, (e) heterocyclic of from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen, (f) -C(O)R 2 where R 2 is selected from the group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, and -NR 3 R 4 where R 3 and R 4 are independently selected from the group consisting of hydrogen and lower alkyl, (g) -S(
  • amino acids wherein the amino group is separated from the carboxyl group by more than one carbon atom such as b-alanine,
  • D-amino acids of naturally occurring L-amino acids include, by way of example, the D-amino acids of naturally occurring L-amino acids, L-1-napthyl-alanine,
  • methionine i.e., HOOC- (H 2 NCH)CH 2 CH 2 -S(O) n R 6
  • n and R 6 are as defined above as well as the lower alkoxy derivative of methionine (i.e., HOOC- (H 2 NCH) CH 2 CH 2 -0R 6 where R 6 is as defined above) .
  • Detectable label refers to materials, which when covalently attached to the peptides and peptide mimetics of this invention, permit detection of the peptide and peptide mimetics in vivo in the patient to whom the peptide or peptide mimetic has been administered.
  • Suitable detectable labels are well known in the art and include, by way of example,
  • radioisotopes e.g., fluorescein
  • fluorescent labels e.g., fluorescein
  • the particular detectable label employed is not
  • Covalent attachment of the detectable label to the peptide or peptide mimetic is accomplished by conventional methods well known in the art.
  • covalent attachment of 125 I to the peptide or the peptide mimetic can be achieved by incorporating the amino acid tyrosine into the peptide or peptide mimetic and then iodating the peptide. If tyrosine is not present in the peptide or peptide mimetic, incorporation of tyrosine to the N or C terminus of the peptide or peptide mimetic can be achieved by well known chemistry.
  • 32 P can be incorporated onto the peptide or peptide mimetic as a phosphate moiety through, for example, a hydroxyl group on the peptide or peptide mimetic using conventional chemistry.
  • the present invention provides compounds that bind to and activate the TPO-R or otherwise behave as a TPO
  • These compounds include “lead” peptide compounds and “derivative” compounds constructed so as to have the same or similar molecular structure or shape as the lead compounds but that differ from the lead compounds either with respect to susceptibility to hydrolysis or proteolysis and/or with respect to other biological properties, such as increased affinity for the receptor.
  • compositions comprising an effective amount of a TPO agonist, and more particularly a compound, that is useful for treating hematological disorders, and particularly,
  • thrombocytopenia associated with chemotherapy, radiation therapy, or bone marrow transfusions are associated with chemotherapy, radiation therapy, or bone marrow transfusions.
  • Peptides having a binding affinity to TPO-R can be readily identified by random peptide diversity generating systems coupled with an affinity enrichment process.
  • random peptide diversity generating systems include the "peptides on plasmids" system described in U.S. Patent Nos. 5,270,170 and 5,338,665; the "peptides on phage” system described in U.S. Patent Application Serial No. 07/718,577, filed June 20, 1991 which is a continuation in part application of U.S. Patent Application Serial No.
  • random peptides were generally designed to have a defined number of amino acid residues in length (e.g., 12).
  • the codon motif (NNK) x where N is nucleotide A, C, G, or T (equimolar; depending on the methodology employed, other nucleotides can be employed) , K is G or T (equimolar) , and x is an integer corresponding to the number of amino acids in the peptide (e.g., 12) was used to specify any one of the 32 possible codons resulting from the NNK motif: 1 for each of 12 amino acids, 2 for each of 5 amino acids, 3 for each of 3 amino acids, and only one of the three stop codons.
  • the NNK motif encodes all of the amino acids, encodes only one stop codon, and reduces codon bias.
  • the random peptides were presented either on the surface of a phage particle, as part of a fusion protein comprising either the piII or the pVIII coat protein of a phage fd derivative (peptides on phage) or as a fusion protein with the Lad peptide fusion protein bound to a plasmid (peptides on plasmids) .
  • the phage or plasmids including the DNA encoding the peptides, were identified and isolated by an affinity enrichment process using immobilized TPO-R.
  • the affinity enrichment process sometimes called “panning,” involves multiple rounds of incubating the phage, plasmids, or
  • polysomes with the immobilized receptor collecting the phage, plasmids, or polysomes that bind to the receptor (along with the accompanying DNA or mRNA) , and producing more of the phage or plasmids (along with the accompanying Lad-peptide fusion protein) collected.
  • the extracellular domain (ECD) of the TPO-R typically was used during panning.
  • the phage or plasmids and accompanying peptides were examined by ELISA to determine if the peptides bind specifically to TPO-R.
  • This assay was carried out similarly to the procedures used in the affinity enrichment process, except that after removing unbound phage, the wells were typically treated with rabbit anti-phage antibody, then with alkaline phosphatase
  • test wells By comparing test wells with control wells (no receptor), one can determine whether the fusion proteins bind to the receptor specifically.
  • the phage pools found to bind to TPO-R were screened in a colony lift probing format using radiolabelled monovalent receptor. This probe can be produced using protein kinase A to phosphorylate a kemptide sequence fused to the C-terminus of the soluble receptor.
  • TPO receptor is then expressed in host cells, typically CHO cells. Following PI-PLC harvest of the receptors, the receptor was tested for binding to TPO or TPO-R specific phage clones. The receptor is then labeled to high specific activity with 33 P for use as a monovalent probe to identify high affinity ligands using colony lifts.
  • Peptides found to bind specifically to the receptor were then synthesized as the free peptide (e.g., no phage) and tested in a blocking assay.
  • the blocking assay was carried out in similar fashion to the ELISA , except that TPO or a reference peptide was added to the wells before the fusion protein (the control wells were of two types: (1) no
  • Fusion proteins for which the binding to the receptor was blocked by TPO or the reference peptide contain peptides in the random peptide portion that are preferred compounds of the invention.
  • TPO-R as well as its extracellular domain, were produced in recombinant host cells.
  • One useful form of TPO-R is constructed by expressing the protein as a soluble protein in baculovirus transformed host cells using standard methods; another useful form is constructed with a signal peptide for protein secretion and for glycophospholipid membrane anchor attachment. This form of anchor attachment is called "PIG-tailing". See Caras and Wendell Science 243:1196-1198 (1989) and Lin et al. Science 249:677-679 (1990).
  • the receptor e.g., transformed CHO cells selected for high level
  • the cleaved receptor still comprises a carboxy terminal sequence of amino acids, called the "HPAP tail", from the signal protein for membrane attachment and can be immobilized without further purification.
  • the recombinant receptor protein can be immobilized by coating the wells of microtiter plates with an anti-HPAP tail antibody (Ab 179 or MAb 179), blocking non-specific binding with bovine serum albumin (BSA) in PBS, and then binding cleaved recombinant receptor to the antibody. Using this procedure, one should perform the immobilization reaction in varying concentrations of receptor to determine the optimum amount for a given preparation, because different preparations of recombinant protein often contain different amounts of the desired protein.
  • BSA bovine serum albumin
  • the density of the immobilized receptor is an important factor in determining the affinity of the ligands that can bind to the immobilized receptor.
  • receptor densities e.g., each anti-receptor antibody-coated well treated with 0.25 to 0.5 mg of receptor
  • multivalent binding is more likely to occur than at lower receptor densities (e.g., eacn anti-receptor antibody-coated well created witn 0.5 to 1 ng of the receptor) . If multivalent binding is occurring, then one will be more likely to isolate ligands with relatively lower affinity, unless one uses high densities of immobilized receptor to identify lead compounds and uses lower receptor densities to isolate higher affinity derivative compounds .
  • a monovalent receptor probe frequently is used.
  • This probe can be produced using protein kinase A to phosphorylate a kemptide sequence fused to the C-terminus of the soluble receptor.
  • the "engineered" form of the TPO receptor is then expressed in host cells, typically CHO cells. Following PI-PLC harvest of the receptors, the receptor was tested for binding to TPO or TPO-R specific phage clones. The receptor is then labeled to high specific activity with 33 P for use as a monovalent probe to identify high affinity ligands using colony lifts.
  • Preferred screening methods to facilitate identification of peptides which bind TPO-R involve first identifying lead peptides which bind to the extracellular domain of the receptor and then making other peptides which resemble the lead peptides. Specifically, using a pill or pVIII-based peptides on phage system, a random library can be screened to discover a phage that presents a peptide that binds to TPO-R. The phage DNAs are sequenced to determine the sequences of the peptides displayed on the surface of the phages .
  • Clones capable of specific binding to the TPO-R were identified from a random linear l ⁇ -mer pVIII library and a random cyclic 10-mer and 12-mer pVIII libraries.
  • a mutagenesis library was constructed based on the sequence X X X X X (C,P,R,or S) t l r e f l X X X X X X (C or S), in which X represents a random NNK codon, and the lower case letters represent amino acid codons containing
  • the library was panned for 5 rounds against TPO receptor which had been immobilzed on magnetic beads. After the fifth round, the PCR amplified pool was cloned into pAFF6 and the ELISA positive clones were
  • sequences were subcloned into an MBP vector and their binding affinities were determined by an MBP ELISA.
  • IC 50 values for some additional representative peptides are given in the table below.
  • a variety of methods can be used to evaluate IC 50 values. For example, an equilibrium binding ELISA assay, using either MBP-TPO or lad-peptide tracer, was used to determine whether the peptides inhibit the binding of TPO to the extracellular domain of the TPO receptor.
  • the IC 50 value were determined using the free peptide.
  • the IC 50 value can be determined using the free peptide, which optionally can be C-terminally amidated, or can be prepared as an ester or other carboxy amide.
  • the N-terminal and C-terminal amino acids of the synthetic peptides are often preceded by one or two glycine residues. These glycines are not believed to be necessary for binding or activity.
  • the C-terminal amino acids of the synthetic peptides are often preceded by the sequence M A S. Again, this sequence is not believed to be necessary for binding or activity.
  • IC 50 values are indicated symbolically by the symbols "-", “+”, and "++". For examples, those peptides which showed IC 50 values in excess of 200 ⁇ M are indicated with a " - ". Those peptides which gave IC 50 values of less than or equal to 200 ⁇ M are given a "+”, while those which gave IC 50 values of 500 nm or less are indicated with a "++”. Those peptides which gave IC 50 values at or near the cutoff point for a particular symbol are indicated with a hybrid designator, e.g., "+/-”. Those peptides for which IC 50 values were not determined are listed as "N.D.”.
  • the IC 50 value for peptides having the structure: G G C T L R E W L H G G F C G G was 500 nm or less. (Note the N-terminal and C-terminal amino acids were preceded by two glycines to recreate the exact sequence displayed by the phage. These glycines are not believed to be necessary for binding or activity.)
  • X 1 is C, L, M, P, Q, V
  • X 2 is F, K, L, N, Q, R, S, T or V
  • X 3 is C, F, I, L, M, R, S, V or W
  • X 4 is any of the 20 genetically coded L-amino acids
  • X 5 is A, D, E, G, K, M, Q, R, S, T, V or Y
  • X 6 is C, F, G, L, M, S, V, W or Y
  • X 7 is C, G, I, K, L, M, N, R or V.
  • X 1 is L, M, P, Q, or V
  • X 2 is F, R, S, or T
  • X 3 is F, L, V, or W
  • X 4 is A, K, L, M, R, S, V, or T
  • X 5 is A, E, G, K, M, Q, R, S, or T
  • X 7 is C, I, K, L, M or V
  • each X 8 residue is independently selected from any of the 20 genetically coded L-amino acids, their stereoisomeric D-amino acids
  • each X 8 residue is independently selected from any of the 20 genetically coded L-amino acids and their stereoisomeric D-amino acids.
  • X 1 is P; X 2 is T; X 3 is L; X 4 is R; X 5 is E or Q; and X 7 is I or L.
  • the core peptide comprises a sequence of amino acids :
  • X 9 is A, C, E, G, I, L , M, P, R, Q, S, T, or V; and X 8 is A, C, D, E, K, L, Q, R, S, T, or V. More preferably, X 9 is A or I; and X 8 is D, E, or K.
  • Particularly preferred peptides include: G G C A D G P T L R E W I S F C G G; G N A D G P T L R Q W L E G R R P K N; G G C A D G P T L R E W I S F C G G K; T I K G P T L R Q W L K S R E H T S; S I E G P T L R E W L T S R T P H S; L A I E G P T L R Q W L H G N G R D T; C A D G P T L R E W I S F C; and I E G P T L R Q W L A A R A.
  • preferred peptides for use in this invention include peptides having a core structure comprising sequence of amino acids:
  • X 2 is K, L, N, Q, R, S, T or V
  • X 3 is C, F, I, L, M, R,
  • X 4 is any of the 20 genetically coded L-amino acids
  • X 5 is A, D, E, G, S, V or Y;
  • X 6 is C, F, G, L, M, S, V, W or
  • Y; and X 7 is C, G, I, K, L, M, N, R or V.
  • X 4 is A, E, G, H, K, L, M, P, Q, R, S, T, or W.
  • X 2 is S or T;
  • X 3 is L or R;
  • X 4 is R;
  • X 5 is D, E, or G
  • X 6 is F, L, or W
  • X 7 is I, K, L, R, or
  • Particularly preferred peptides include: G G C T L R E W
  • preferred peptides for use in this invention include peptides having a structure
  • X 2 is F, K, L, N, Q, R, S, T or V
  • X 3 is C, F, I, L, M
  • X 4 is any of the 20 genetically coded L-amino acids
  • X 5 is A, D, E, G, K, M, Q, R, S, T, V or Y
  • X 6 is C, F,
  • X 7 is C, G, I, K, L, M, N, R or V;
  • X 8 is any of the 20 genetically coded L-amino acids. In some embodiments, X 8 is preferably G, S, Y, or R.
  • Peptides and peptidomimetics having an IC 50 of greater than about 100 mM lack sufficient binding to permit use in either the diagnostic or therapeutic aspects of this invention.
  • the peptides and peptidomimetics have an IC 50 of about 2 mM or less and, for pharmaceutical purposes, the peptides and peptidomimetics have an IC 50 of about 100 ⁇ M or less.
  • the binding peptide sequence also provides a means to determine the minimum size of a TPOR binding compound of the invention.
  • ESL encoded synthetic library
  • very large scale immobilized polymer synthesis one can not only determine the minimum size of a peptide with such activity, but one can also make all of the peptides that form the group of peptides that differ from the preferred motif (or the minimum size of that motif) in one, two, or more residues. This collection of peptides can then be screened for ability to bind to TPO-receptor.
  • These immobilized polymers synthesis systems or other peptide synthesis methods can also be used to synthesize truncation analogs, deletion analogs, substitution analogs, and
  • peptides and peptide mimetics of the present invention were also evaluated in a thrombopoietin dependent cell proliferation assay, as described in greater detail in Example 2 below.
  • Cell proliferation is measured by techniques known in the art, such as an MTT assay which correlates with 3 H-thymidine incorporation as an indication of cell
  • the peptides tested stimulated proliferation of TPO-R transfected Ba/F3 cells in a dose dependent manner as shown in Figure 1A. These peptides have no effect on the parental cell line as shown in Figure IB.
  • Figures 7 to 9 show the results of a further assay evaluating activity of the peptides and peptide mimetics of the invention.
  • mice are made thrombocytopenic with carboplatin.
  • Figure 7 depicts typical results when
  • FIG. 8 depicts the effect of carboplatin titration on platelet counts in mice treated with the indicated amounts of carboplatin (in mg/kg, intraperitoneally (ip) on Day 0).
  • Figure 9 depicts amelioration of carboplatin-induced thrombocytopenia on Day 10 by peptide AF12513 (513).
  • Carboplatin (CBP; 50-125 mg/kg, intraperitoneally) was administered on Day 0.
  • AF12513 (1 mg/kg, ip) was given on Days 1-9.
  • FIG. 2B shows the results of the assay for the free biotinylated peptide (AF 12285) for both the transfected and parental cell lines.
  • Figure 2C shows the results of the assay for streptavidin alone for both the transfected and parental cell lines.
  • the specificity of the binding and activity of the peptides of the invention was also examined by studying the cross reactivity of the peptides for the erythropoieitin receptor (EPO-R).
  • EPO-R erythropoieitin receptor
  • TPO-R haematopoietin growth factor receptor family
  • This assay utilized FDCP-1, a growth factor dependent murine multi-potential primitive haematopoietic progenitor cell line (see, e.g., Dexter et al. J. Exp. Med. 152:1036-1047 (1981)) as the parental cell line.
  • This cell line can proliferate, but not differentiate when supplemented with
  • WEHI-3-conditioned media (a medium that contains IL-3, ATCC number T1B68) .
  • the parental cell line is transfected with human or murine EPO-R to produce the FDCP-1-EPO-R cell line. These transfected cell lines can proliferate, but not
  • the cells were grown to half stationary density in the presence of the necessary growth factors. The cells are then washed in PBS and starved for 16-24 hours in whole media without the growth factors. After determining the viability of the cells, stock solutions (in whole media without the growth factors) are made to give about 10 5 cells per 50 microliters. Serial dilutions of the compounds (typically, the free solution phase peptide as opposed to a phage-bound or other bound or immobilized peptide) to be tested are made in 96-well tissue culture plates for a final volume of 50
  • Figures 3A-G show the results of a series of control experiments showing the activity of TPO, the peptides of the present invention, EPO, and EPO-R binding peptides in a cell proliferation assay using either the TPO-R transfected Ba/F3 cell line and its corresponding parental line, or an
  • FIG. 3A depicts the results for TPO in the cell
  • FIG. 3B depicts the results for EPO in the cell proliferation assay using the TPO-R transfected Ba/F3 cell line and its corresponding parental line.
  • Figure 3C depicts the results for complexed biotinylated peptide (AF 12285 with streptavidin (SA) ) and a complexed form of a biotinylated EPO-R binding peptide (AF 11505 with SA) in the TPO-R transfected Ba/F3 cell line.
  • SA streptavidin
  • FIG. 3E depicts the results for TPO in the cell proliferation assay using the EPO-dependent cell line.
  • Figure 3F depicts the results for EPO in the cell proliferation assay using the EPO-dependent cell line.
  • Figure 3G depicts the results for complexed biotinylated peptide (AF 12285 with streptavidin (SA) ) and the complexed form of a biotinylated EPO-R binding peptide (AF 11505 with SA) in the EPO-dependent cell line.
  • SA streptavidin
  • the peptides of the invention can be prepared by classical methods known in the art, for example, by using standard solid phase techniques.
  • the standard methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis, and even by recombinant DNA technology. See, e.g., Merrifield J. Am. Chem. Soc. 85:2149 (1963), incorporated herein by reference.
  • the synthesis is typically
  • a suitable starting material can be prepared, for instance, by attaching the required
  • chloromethylated resin a chloromethylated resin, a hydroxymethyl resin, or a benzhydrylamine resin.
  • chloromethylated resin is sold under the tradename BIO-BEADS SX-1 by Bio Rad Laboratories, Richmond, CA, and the preparation of the
  • the compounds of the invention can be prepared by coupling an alpha-amino protected amino acid to the
  • the alpha-amino protecting group is removed by a choice of reagents including trifiuoroacetic acid (TFA) or hydrochloric acid (HCl) solutions in organic solvents at room temperature.
  • TFA trifiuoroacetic acid
  • HCl hydrochloric acid
  • 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)
  • alkyl type protecting groups e.g. benzyl, triphenylmethyl.
  • Boc and Fmoc are preferred protecting groups.
  • the side chain protecting group remains intact during coupling and is not split off during the deprotection of the amino-terminus protecting group or during coupling.
  • the side chain protecting group remains intact during coupling and is not split off during the deprotection of the amino-terminus protecting group or during coupling.
  • the side chain protecting group remains intact during coupling and is not split off during the deprotection of the amino-termin
  • 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 group for Thr and Ser is benzyl.
  • the side chain protecting groups for Arg include nitro, Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), or Boc.
  • the side chain protecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl
  • each protected amino acid is coupled stepwise in the desired order.
  • An excess of each protected amino acid is generally used with an appropriate carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH 2 Cl 2 ), dimethyl formamide (DMF) mixtures.
  • DCC dicyclohexylcarbodiimide
  • CH 2 Cl 2 methylene chloride
  • DMF dimethyl formamide
  • the desired peptide is decoupled from the resin support by treatment with a reagent such as trifiuoroacetic acid or hydrogen fluoride (HF), which not only cleaves the pepci ⁇ e rrom the resin, but also cleaves all remaining side chain protecting groups.
  • a reagent such as trifiuoroacetic acid or hydrogen fluoride (HF)
  • 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.
  • synthesis methods can also be used to synthesize truncation analogs and deletion analogs and combinationc of truncation and deletion analogs of all of the peptide compounds of the invention.
  • D amino acids and non-naturally occurring synthetic amino acids can also be incorporated into the peptides of the present invention.
  • proline analogs in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be
  • Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic.
  • 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 sulphur heteroatoms. Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g. 1-piperazinyl), piperidyl (e.g.
  • the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.
  • peptide compounds of the invention can also readily modify the peptides of the instant invention by phosphorylation, and other methods for making peptide derivatives of the compounds of the present invention are described in Hruby et al. 42
  • the peptide compounds of the invention also serve as a basis to prepare peptide mimetics with similar biological activity.
  • peptide compounds of the invention can be covalently modified to one or more of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkenes, in the manner set forth in U.S. Patent No. 4,640,835; U.S. Patent No.
  • modifications can be coupled in one peptide mimetic structure (e.g., modification at the C-terminal carboxyl group and inclusion of a -CH 2 -carbamate linkage between two amino acids in the peptide).
  • the peptides typically are synthesized as the free acid but, as noted above, could be readily prepared as the amide or ester.
  • Amino terminus modifications include methylating (i.e., -NHCH 3 or -NH(CH 3 ) 2 ), acetylating, adding a carbobenzoyl group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO- , where R is selected from the group
  • Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints.
  • Amino terminus modifications are as recited above and include alkylating, acetylating, adding a carbobenzoyl group, forming a succinimide group, etc. Specifically, the N-terminal amino group can then be reacted as follows:
  • reaction can be conducted by contacting about equimolar or excess amounts (e.g., about 5 equivalents) of an acid halide to the peptide in an inert diluent (e.g., dichloromethane) preferably containing an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge the acid generated during reaction.
  • Reaction conditions are otherwise conventional (e.g., room temperature for 30
  • succinimide group by reaction with succinic anhydride.
  • an approximately equimolar amount or an excess of succinic anhydride e.g., about 5 equivalents
  • succinic anhydride e.g., about 5 equivalents
  • an excess e.g., ten equivalents
  • a tertiary amine such as diisopropylethylamine in a suitable inert solvent (e.g., dichloromethane).
  • alkyl substituents are prepared by reaction of a lower olefin
  • Wollenberg, et al., supra, and -SR substituents are prepared by reaction of RSH with maleic anhydride where R is as defined above;
  • benzyloxycarbonyl chloride or a substituted CBZ-Cl in a suitable inert diluent (e.g., dichloromethane) preferably containing a tertiary amine to scavenge the acid generated during the reaction;
  • a suitable inert diluent e.g., dichloromethane
  • the inert diluent contains excess tertiary amine (e.g., ten equivalents) such as
  • reaction conditions are otherwise conventional (e.g., room temperature for 30 minutes);
  • the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge any acid generated during reaction.
  • Reaction conditions are otherwise conventional (e.g., room temperature for 30
  • a suitable inert diluent e.g., dichloromethane
  • the inert diluent contains an excess (e.g., about 10 equivalents) of a tertiary amine, such as diisopropylethylamine.
  • Reaction conditions are otherwise conventional (e.g., room temperature for about 30 minutes).
  • benzhydrylamine resin is used as the solid support for peptide synthesis. Upon completion of the synthesis, hydrogen
  • C-terminus is -C(O)NH 2 ).
  • C-terminus is -C(O)NRR 1 where R and R 1 are as defined above).
  • Side chain protection is then removed in the usual fashion by treatment with hydrogen fluoride to give the free amides, alkylamides, or dialkylamides.
  • the C-terminal carboxyl group or a C-terminal ester can be induced to cyclize by internal displacement of the -OH or the ester (-OR) of the carboxyl group or ester respectively with the N-terminal amino group to form a cyclic peptide.
  • the free acid is converted to an activated ester by an appropriate carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH 2 Cl 2 ),
  • DCC dicyclohexylcarbodiimide
  • DMF dimethyl formamide
  • 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 Ann. Rep. Med. Chem. 24:243-252 (1989), incorporated herein by reference.
  • [-C(O)NR 6 - where R 6 is lower alkyl] are prepared during conventional peptide synthesis by merely substituting a suitably protected amino acid analogue for the amino acid reagent at the appropriate point during synthesis.
  • Suitable reagents include, for example, amino acid analogues wherein the carboxyl group of the amino acid has been replaced with a moiety suitable for forming one of the above linkages. For example, if one desires to replace a -C(O)NR- linkage in the peptide with a -CH 2 -carbamate linkage (-CH 2 OC(O)NR-), then the carboxyl (-COOH) group of a suitably protected amino acid is first reduced to the -CH 2 OH group which is then converted by conventional methods to a -OC.(O) Cl functionality or a para-nitrocarbonate -OC(O)O-C 6 H 4 -p-NO 2 functionality.
  • Replacement of an amido linkage in the peptide with a -CH 2 -sulfonamide linkage can be achieved by reducing the carboxyl (-COOH) group of a suitably protected amino acid to the -CH 2 OH group and the hydroxyl group is then converted to a suitable leaving group such as a tosyl group by conventional methods. Reaction of the tosylated derivative with, for example, thioacetic acid followed by hydrolysis and oxidative chlorination will provide for the -CH 2 -S(O) 2 Cl functional group which replaces the carboxyl group of the otherwise suitably protected amino acid. Use of this suitably
  • protected amino acid analogue in peptide synthesis provides for inclusion of an -CH 2 S(O) 2 NR- linkage which replaces the amido linkage in the peptide thereby providing a peptide mimetic.
  • a -CH 2 S(O) 2 Cl group For a more complete description on the conversion of the carboxyl group of the amino acid to a -CH 2 S(O) 2 Cl group, see, for example, Weinstein, Boris Chemistry & Biochemistry of Amino Acids. Peptides and Proteins Vol. 7, pp. 267-357, Marcel Dekker, Inc., New York (1983) which is incorporated herein by reference.
  • Secondary amine linkages wherein a -CH 2 NH- linkage replaces the amido linkage in the peptide can be prepared by employing, for example, a suitably protected dipeptide
  • analogue wherein the carbonyl bond of the amido linkage has been reduced to a CH 2 group by conventional methods For example, in the case of diglycine, reduction of the amide to the amine will yield after deprotection H 2 NCH 2 CH 2 NHCH 2 COOH which is then used in N-protected form in the next coupling reaction.
  • the preparation of such analogues by reduction of the carbonyl group of the amido linkage in the dipeptide is well known in the art.
  • the suitably protected amino acid analogue is employed in the conventional peptide synthesis in the same manner as would the corresponding amino acid.
  • typically about 3 equivalents of the protected amino acid analogue are employed in this reaction.
  • An inert organic diluent such as methylene chloride or DMF is employed and, when an acid is generated as a reaction by-product, the reaction solvent will typically contain an excess amount of a tertiary amine to scavenge the acid generated during the reaction.
  • One particularly preferred tertiary amine is diisopropylethylamine which is typically employed in about 10 fold excess.
  • the reaction results in incorporation into the peptide mimetic of an amino acid analogue having a non-peptidyl linkage. Such substitution can be repeated as desired such that from zero to all of the amido bonds in the peptide have been replaced by non-amido bonds.
  • cyclized compounds are provided in Tables 4, 5, 6, 8 , and 9.
  • the compounds of the present invention may exist in a cyclized form with an intramolecular disulfide bond between the thiol groups of the cysteines .
  • an intramolecular disulfide bond between the thiol groups of the cysteines .
  • cysteine residues may also be substituted with a homocysteine.
  • intramolecular or intermolecular disulfide derivatives can be represented schematically as shown below:
  • n and n are independently 1 or 2.
  • the amino-terminus of the peptide can be capped with an alpha-substituted acetic acid, wherein the alpha substituent is a leaving group, such as an a-haloacetic acid, for example, a-chloroacetic acid, a-bromoacetic acid, or a-iodoacetic acid.
  • the compounds of the present invention can be cyclized or dimerized via displacement of the leaving group by the sulfur of the cysteine or homocysteine residue. See, e.g., Barker et al. J. Med. Chem. 35:2040-2048 (1992) and Or et al. J. Org. Chem. 56:3146-3149 (1991), each of which is incorporated herein by reference. Examples of dimerized compounds are provided in Tables 7, 9, and 10.
  • V. UTILITY The compounds of the invention are useful in vn tro as unique tools. for understanding the biological role of TPO, including the evaluation of the many factors thought to influence, and be influenced by, the production of TPO and the receptor binding process.
  • the present compounds are also useful in the development of other compounds that bind to and activate the TPO-R, because the present compounds provide important information on the relationship between structure and activity that should facilitate such development.
  • the compounds are also useful as competitive binders in assays to screen for new TPO receptor agonists.
  • the compounds of the invention can be used without modification or can be modified in a variety of ways; for example, by labeling, such as covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.
  • the materials thereto can be labeled either directly or indirectly.
  • Possibilities for direct labeling include label groups such as: radiolabels such as 125 I, enzymes (US Patent 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Patent Np. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or
  • labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups.
  • the compounds may also include spacers or linkers in cases where the compounds are to be attached to a solid support.
  • the peptides of the present invention can be used as reagents for detecting TPO receptors on living cells, fixed cells, in biological fluids, in tissue homogenates, in
  • the peptides of the present invention can be used in in si tu staining, FACS (fluorescence-activated cell sorting), Western blotting, ELISA, etc.
  • the peptides of the present invention can be used in receptor purification, or in purifying cells expressing TPO receptors on the cell surface (or inside permeabilized cells).
  • the compounds of the present invention can also be utilized as commercial reagents for various medical research and diagnostic uses. Such uses include but are not limited to: (1) use as a calibration standard for quantitating the activities of candidate TPO agonists in a variety of
  • transduction/receptor activation (5) other research and diagnostic applications wherein the TPO-receptor is preferably activated or such activation is conveniently calibrated against a known quantity of a TPO agonist, and the like.
  • the compounds of the present invention can be used for the in vi tro expansion of megakaryocytes and their
  • thrombocytopenia by killing the rapidly dividing, more mature population of megakaryocytes.
  • these therapeutic treatments can also reduce the number and viability of the immature, less mitotically active megakaryocyte precursor cells.
  • amelioration of the thrombocytopenia by TPO or the compounds of the present invention can be hastened by infusing patients post chemotherapy or radiation therapy with a population of his or her own cells enriched for
  • the compounds of the invention can also be administered to warm blooded animals, including humans, to activate the TPO-R in vivo .
  • the present invention encompasses methods for therapeutic treatment of TPO related disorders that comprise administering a compound of the invention in amounts sufficient to mimic the effect of TPO on TPO-R in vivo.
  • the peptides and compounds of the invention can be administered to treat a variety of
  • hematological disorders including but not limited to platelet disorders and thrombocytopenia, particularly when associated with bone marrow transfusions, radiation therapy, and
  • TPO antagonists are preferably first administered to patients undergoing chemotherapy or radiation therapy, followed by administration of the tpo agonists of the invention.
  • compositions of the present invention are useful for treating thrombocytopenia associated with bone marrow transfusions, radiation therapy, or chemotherapy.
  • the compounds typically will be administered prophylactically prior to chemotherapy, radiation therapy, or bone marrow transplant or after such exposure.
  • compositions comprising, as an active
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.
  • Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, with the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
  • aqueous or non-aqueous solutions examples include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • non-aqueous solvents or vehicles examples include 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 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 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 preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.
  • compositions containing the compounds can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as
  • therapeutically effective dose Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient.
  • compositions of the- invention can also be microencapsulated by, for example, the method of Tice and Bibi (in Treatise on Controlled Drug Delivery, ed. A. Kydonieus, Marcel Dekker, N.Y. (1992), pp. 315-339).
  • compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a "prophylactically effective dose”. In this use, the precise amounts again depend on the patient's state of health and weight.
  • TPO agonist necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vi tro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for
  • N-oxtrisdimethylaminophosphonium hexafluorophosphate N-oxtrisdimethylaminophosphonium hexafluorophosphate
  • HOBt 1-hydroxybenzotriazole
  • the resin used was HMP resin or PAL
  • the Fmoc group was used for amino protection during the coupling procedure.
  • Primary amine protection on amino acids was achieved with Fmoc and side chain protection groups were t-butyl for serine, tyrosine, asparagine, glutamic acid, and threonine; trityl for glutamine; Pmc
  • Bioactivity of the peptides can be measured using a thrombopoietin dependent cell proliferation assay.
  • Murine IL-3 dependent Ba/F3 cells were transfected with full length human TPO-R. In the absence of IL-3 (WEHI-3 conditioned media), these cells are dependent on TPO for proliferation. The parental, untransfected cell line does not respond to human TPO, but remains IL-3 dependent.
  • Bioassays have been performed on both of the above cell lines using synthetic peptides derived from library screening.
  • the cells were grown in complete RPMI-10 media, containing 10% WEHI-3 conditioned media, then washed twice in PBS, resuspended in media which lacked WEHI-3 conditioned media, and added to wells containing dilutions of peptide or TPO at 2 x 10 4 cells/well.
  • the cells were incubated for 48 hours at 37°C in a humidified 5% CO 2 atmosphere and metabolic activity was assayed by the reduction of MTT to formazan, with absorbance at 570 nM measured on an ELISA plate reader.
  • the peptides tested stimulated proliferation of TPO-R transfected Ba/F3 cells in a dose dependent manner as shown in Figure 1. These peptides have no effect on the parental cell line.
  • peptides for TPO-R were measured in a competition binding assay.
  • the wells of a microtiter plate were coated with 1 mg streptavidin, blocked with PBS/1% BSA, followed by 50 ng of biotinylated anti-receptor immobilizing antibody (Abl79).
  • the wells were then treated with a 1:10 dilution of soluble TPO-R harvest.
  • Various concentrations of peptide or peptide mimetic were mixed with a constant amount of a truncated form of TPO consisting of residues 1-156 fused to the C-terminus of maltose binding protein (MBP-TPO 156 ).
  • MBP-TPO 156 maltose binding protein
  • MBP-TPO 156 that was bound at equilibrium was measured by adding a rabbit anti-sera directed against MBP, followed by alkaline phosphatase conjugated goat anti-rabbit IgG. The amount of alkaline phosphatase in each well was then
  • the assay is conducted over a range of peptide concentrations and the results are graphed such that the y axis represents the amount of bound MBP-TPO 156 and the x axis represents the concentration of peptide or peptide mimetic.
  • the dissociation constant (Kd) for the peptide should be similar to the measured IC 50 using the assay conditions described above.
  • the pJS142 vector is used for library construction and is shown in Figure 4.
  • Three oligonucleotide sequences are needed for library construction: ON-829 (5' ACC ACC TCC GG) ; ON-830 (5' TTA CTT AGT TA) and a library specific
  • oligonucleotide of interest (5' GA GGT GGT ⁇ NNK ⁇ n TAA CTA AGT AAA GC), where ⁇ NNK ⁇ n denotes a random region of the desired length and sequence.
  • the oligonucleotides can be 5 1
  • the strain of E. coli which is preferably used for panning has the genotype: A (srl -recA) endAl nupG lon-11 sulAl hsdR17 A (ompT-fepC) 266 AclpA319 : :kan AlacI lac ZU118 which can be prepared from an E. coli strain from the E. coli Genetic Stock Center at Yale University (E. coli b/r, stock center designation CGSC:6573) with genotype lon-11 sulAl .
  • the above E. coli strain is prepared for use in electroporation as described by Dower et al. Nucleic Acids Res.
  • Peptides on plasmids are released from cells for panning by gentle enzymatic digestion of the cell wall using lysozyme. After pelleting of the cell debris, the crude lysate can be used directly on most receptors. If some additional purification of the plasmid complexes is needed, a gel filtration column can be used to remove many of the low molecular weight contaminants in the crude lysate.
  • Panning is carried out in a buffer (HEKL) of a lower salt concentration than most physiological buffers.
  • the panning can be conducted in microtiter wells with a receptor immobilized on a nonblocking monoclonal antibody (MAb) or by panning on beads or on columns. More specifically, in the first round of panning, 24 wells, each coated with receptor, can be used. For the second round, six wells coated with receptor (PAN sample) and 6 wells without receptor (NC sample) are typically used. Comparison of the number of plasmids in these two samples can give an indication of whether receptor specific clones are being enriched by panning. "Enrichment” is defined as the ratio of PAN transformants to those
  • binding properties of the peptides encoded by individual clones are typically examined after 3, 4, or 5 rounds of panning, depending on the enrichment numbers
  • Lad-peptide fusion proteins an ELISA that detects receptor specific binding by Lad-peptide fusion proteins.
  • Lad is normally a tetramer and the minimum functional DNA binding species is a dimer. The peptides are thus displayed
  • the peptides fused to Lad will bind to the surface in a cooperative, multivalent fashion.
  • This cooperative binding permits the detection of binding events of low intrinsic affinity.
  • the sensitivity of this assay is an advantage in that initial hits of low affinity can be easily identified, but is a disadvantage in that the signal in the ELISA is not correlated with the intrinsic affinity of the peptides.
  • MBP maltose binding protein
  • DNA from clones of interest can be prepared in double stranded form using any standard miniprep procedure.
  • populations of clones can be transferred to vectors that fuse those sequences in frame with the- gene encoding MBP, a protein that generally occurs as a monomer in solution.
  • the cloning of a library into pJS142 creates a BspEI restriction site near the beginning of the random coding region of the library.
  • Digestion with BspEI and Seal allows the purification of a "900 bp DNA fragment that can be subcloned into one of two vectors, pELM3 (cytoplasmic) or pELM15 (periplasmic) , which are simple modifications of the pMALc2 and pMALp2 vectors, respectively, available commercially from New England Biolabs. See Figure 5A-B.
  • Digestion of pELM3 and pELM15 with Agel and Seal allows efficient cloning of the BspEI-Seal fragment from the pJS142 library.
  • the BspEI and Agel ends are compatible for ligation.
  • correct ligation of the Seal sites is essential to recreate a functional bla (Amp resistance) gene, thus lowering the level of background clones from undesired ligation events. Expression of the tac
  • promoter-driven MBP-peptide fusions can then be induced with IPTG.
  • Lysates for the Lad or MBP ELISAs are prepared from individual clones by lysing cells using lysozyme and removing insoluble cell debris by centrifugation. The lysates are then added to wells containing immobilized receptor and to control wells without receptor. Binding by the Lad or MBP peptide fusions is detected by incubation with a rabbit polyclonal antiserum directed against either Lad or MBP followed by incubation with alkaline phosphatase labeled goat anti rabbit second antibody. The bound alkaline phosphatase is detected with p-nitrophenyl phosphate chromagenic substrate. EXAMPLE 5
  • a variant of the Lad peptides-on-plasmids technique utilizes a DNA binding protein called "headpiece dimer".
  • DNA binding by the E. coli lac repressor is mediated by the approximately 60 amino acid "headpiece” domain.
  • the dimer of the headpiece domains that binds to the lac operator is normally formed by association of the much larger
  • headpiece dimer utilizes headpiece dimer molecules containing two headpieces connected via short peptide linker. These proteins bind DNA with sufficient stability to allow association of a peptide epitope displayed at the C-terminus of the headpiece dimer with the plasmid encoding that peptide.
  • the random peptides are fused to the C-terminus of the headpiece dimer, which binds to the plasmid that encoded it to make a peptide-headpiece dimer-plasmid complex that can be screened by panning.
  • the headpiece dimer binds to the plasmid that encoded it to make a peptide-headpiece dimer-plasmid complex that can be screened by panning.
  • peptides-on-plasmids system allows greater selectivity for high affinity ligands than the LacI system.
  • headpiece dimer system is useful for making mutagenesis libraries based on initial low-affinity hits, and selecting higher affinity variants of those initial sequences.
  • the libraries are constructed as with peptides on plasmids using headpiece dimer vector pCMG14 (see Figure
  • the presence of the lac operator is not required for plasmid binding by the headpiece dimer protein.
  • the libraries were introduced into bacterial strain comprising E. coli
  • Sequences from headpiece dimer panning are often characterized after transfer to the MBP vector so that they can be tested in the affinity sensitive MBP ELISA and also so that populations of clones can be screened by colony lifts with labeled receptor.
  • cyclized compounds were subjected to three assays. First, IC 50 valves were obtained as described above. Additionally, an MTT cell proliferation assay as described above was performed to calculate EC 50 values.
  • amino acid substitutes at positions D, E, I, S, or F in the cyclized compound were assayed for EC 50 and IC 50 values as described above, Microphysiometer results are given in parentheses. The results are summarized in Table 5 below.
  • EC 50 and IC 50 values were calculated as described above for the dimer compounds listed in Table 7 below.
  • the cyclized monomer is included as a comparison.
  • the compounds of Table 8 were inactive at the maximum concentration tested of 10 ⁇ m.
  • Table 11 lists examples of the substituted compounds that show TPO agonist activity.
  • the substitutions abbreviated in the table are as follows:
  • mice To assess the feasibility of mice as a convenient test species, several in vitro experiments, designed to measure the activity of the test compounds on the mouse receptor, have been done.
  • marrow cells harvested from the femurs of 8 to 9 week one Balb/C mice, were incubated for 7 days in liquid culture with either rhuTPO or various
  • marrow cells were harvested and cultured in semi- solid medium (methylcellulose) containing either no factors, 1 nM rhuTPO, or 10uM Peptide A. After 7 days in culture, colonies of large cell (presumed to be megakaryocytes) were counted and grouped into small colonies (3-5 cells) or large colonies (greater than 6 cells). The results are shown in Table 13. TPO and the test peptides both produced
  • the muTPO receptor was cloned and transfected into BaF3 cells. A TPO dependent population of cells was isolated.

Abstract

La présente invention se rapporte à des peptides et à des mimétiques peptidiques qui se fixent et activent le récepteur de la thrombopoïétine. De tels peptides et mimétiques peptidiques servent dans des procédés pour le traitement de troubles hématologiques et en particulier, à la thrombocytopénie résultant de la chimiothérapie, de la radiothérapie ou des transfusions de moelle épinière, ainsi que dans des procédés diagnostiques employant des peptides et mimétiques peptidiques marqués.
PCT/US1996/008998 1995-06-07 1996-06-05 Peptides et composes se fixant a un recepteur WO1996040189A1 (fr)

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AU6046696A (en) 1996-12-30
MY113497A (en) 2002-03-30
HRP960256A2 (en) 1998-02-28
YU34196A (sh) 1999-03-04

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