HRP960256A2 - Peptides and compounds that bind to a receptor - Google Patents

Description

This application is a partial continuation of the U.S. Patent Application Serial Nos. 08/484,090, filed June 7, 1995; 08/485,301, filed June 7, 1995; 08/473,604, filed June 7, 1995; 08/472,371, filed June 7, 1995; 08/478,128, filed June 7, 1995; and 08/476,168, filed Jun. 7, 1995, each of which is incorporated herein by reference in their entirety for all purposes.

Background of the invention

This invention provides peptides and compounds that bind to and activate the thrombopoietin receptor (c-mpl or TPO-R) or otherwise act as a TPO agonist. The invention has application in the fields of biochemistry and medicinal chemistry, and in particular it determines TPO agonists for use in the treatment of human diseases.

Megakaryocytes are cells that originate in the bone marrow and are responsible for the production of circulating blood platelets. Although they make up <0.25% of bone marrow cells in most species, they have > 10 times the volume of a typical bone marrow cell. See Kuter et al. Proc. Natl. Acad. Sci. USA 91:11104-11108 (1994).

Megakaryocytes undergo a process known as endomitosis where they replicate their nuclei but fail to divide, thus causing the formation of polyploid cells. In response to the reduced number of platelets, the endomitotic rate increases, more ploidy megakaryocytes are formed, and the number of megakaryocytes can increase up to 3-fold.

See Harker J. Clin. Invest. 47:458-465 (1968). Conversely, as an agreement to the increased number of platelets, the endomitotic rate decreases, megakaryocytes of lower ploidy are formed, and the number of megakaryocytes can be reduced by 50%.

The exact physiological feedback mechanism by which the mass of circulating platelets regulates the endomitotic rate is not known. Thrombopoietin tai (TPO) is not thought to be a circulating thrombopoietic factor involved in mediating this feedback loop. More specifically, TPO has been shown to be the main humoral regulator in situations that cause thrombocytopenia. See, eg, Metcalf Nature 369:519-520 (1994). TPO has been shown in various studies to increase platelet count, increase platelet size, and increase isotope incorporation into platelets in recipient animals. In particular, TPO is thought to act on megakaryocytopoiesis in different ways: (1) it produces an increase in the size and number of megakaryocytes; (2) produces an increase in the amount of DNA, in the form of polyploidy, in megakaryocytes; (3) increases endomitosis of megakaryocytes; (4) produces increased senescence of megakaryocytes; and (5) produces an increase in the percentage of precursor cells, in the form of small acetylcholinesterase-positive cells in the bone marrow.

Since platelets (platelets) are necessary for blood clotting and when their number is low the patient is in serious mortal danger of catastrophic bleeding, TPO has a potentially useful application in the diagnosis as well as in the treatment of various hematological disorders, for example, diseases primarily caused by platelet defects. Ongoing clinical trials with TPO have shown that TPO can be taken safely by the patient. Furthermore, recent studies have determined the basis for projecting the efficacy of TPO therapy in the treatment of thrombocytopenia, particularly thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transplantation as a treatment for cancer or lymphoma. See, eg, McDonald (1992) Am. J. Ped. Hematology/Oncology 14:8-21 (1992).

The gene encoding TPO was cloned and characterized. See Kuter et al. Proc. Natl. Acad. Sci USA 91:11104-11108 (1994), Barley et al. Cell 77:1117-1124 (1994); Kaushansky et al. Nature 369:568:571 (1994); Wendling et al. Nature 369:571:574 (1994); and Sauvage et al. Nature 369:533:538 (1994). 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 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 human and mouse, and has some homologies with erythropoietin and interferon-a and interferon-b. The carboxy-terminal region shows a wide range of species divergence.

The DNA sequences and coding peptide sequences for human TPO-R (also known as c-mpl) are described. See Vigon et al. Proc. Natl. Acad. Sci. USA 89:5640-5644 (1992). TPO-R is a member of the hematopoietic growth factor receptor family, a family characterized by a common structural design of the extracellular region, including four conserved C residues in the N-terminus and a WSXWS motif near the transmembrane region. See Bazan Proc. Natl. Acad. Sci. USA 87:6934-6938 (1990). Evidence that this receptor plays a functional role in hematopoiesis includes observations that its expression is restricted to the spleen, bone marrow, or fetal liver in the mouse (see Souyri et al. Cell 63:1137-1147 (1990)) and to megakaryocytes, platelets, and CD34+ cells in man (see Methia et al. Blood 82:1395-1401 (1993)). Furthermore, exposure of CD34+ cells to synthetic oligonucleotides antisensitive to mpl RNA significantly inhibits the appearance of megakaryotic colonies without affecting the formation of erythroid and myeloid colonies. Some workers postulate that the receptor functions as a homodimer, similar to the situation with the receptor for G-CSF and erythropoietin.

The availability of cloned genes for TPO-R facilitates the search for agonists of this important receptor. The availability of recombinant receptor protein enables the study of receptor-ligand interactions in different random and semi-random systems of generating different peptides. These systems include "peptides on plasmids", a system described in U.S. Pat. Patent Nos. 5,270,170 and 5,338,665; "peptides on phage", a system described in the U.S. Patent Application Serial No. 07/718,577, filed Jun. 20, 1991, U.S. Pat. to Patent Application Serial No. 07/541,108, filed June 20, 1990 and in Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990); "polysomes", a system described in the U.S. Patent Application Serial no. 08/300,262, filed Sep. 2, 1994, which application is based on a continuation-in-part of U.S. Pat. Patent Applications Serial No. 08/144,775, filed October 29, 1993 and PCT WO 95/11992; "encoded synthetic library", the system described in U.S. Pat. Patent Application Serial Nos. 08/146,886, filed November 12, 1993, 07/946,239, filed September 16, 1992, and 07/762,522, filed September 18, 1991; and "very large-scale immobilized polymer synthesis," a system described in U.S. Pat. Patent No. 5,143,854; PCT Patent Publication No. 90/15070, published on December 13, 1990; LOUSE. Patent Application Serial Number 07/624,120, filed on December 6, 1990; Fodor et al. Science 251:767-773 (2/1991); Dower and Fodor Ann. Tail. Honey. Chem.. 26:271-180 (1991); and the U.S. Patent Application Serial Number 07/805,727, filed December 6, 1991; each of the aforementioned patent applications and publications is incorporated herein by reference.

The slow recovery of platelet levels in patients suffering from thrombocytopenia is a serious problem, and has caused an urgency to investigate blood growth factor agonists capable of accelerating platelet regeneration. The present invention provides such an agonist.

Summary of the invention

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 TPO-R and can activate TPO-R. Therefore, such peptides and peptide mimetics are useful for therapeutic purposes in the treatment of TPO-mediated conditions (eg, thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transfusion) as well as for diagnostic purposes in studying the mechanisms of hematopoiesis and for in vitro expansion megakaryocytes and engaged progenitor cells.

Peptides and peptide mimetics suitable for therapeutic and/or diagnostic purposes have an IC50 of about 2 mM or less, as determined by the binding affinity assay described below in Example 3, while a lower IC50 is correlated with stronger binding affinity to TPO-R. For pharmaceutical purposes, the peptides and peptidomimetics preferably have an IC50 of no greater than about 100 μM, and more preferably, no greater than 500 nM. In a preferred embodiment, the molecular weight of the peptide or peptide mimetic is from about 250 to about 8,000 daltons.

When used for diagnostic purposes, peptides and peptide mimetics are preferably labeled with detectable labels and, therefore, peptides and peptide mimetics without such a label serve as intermediates in the preparation of labeled peptides and peptide mimetics.

Peptides that meet defined criteria for molecular weight and TPO-R binding affinity contain 9 or more amino acids where these amino acids are naturally occurring or synthetic (not naturally occurring) amino acids. Peptide mimetics include peptides that have one or more of the following modifications:

peptides such that one or more peptidyl [-C(O)NR-] linkages are replaced by non-peptide linkages such as a -CH2-carbamate linkage [-CH2-OC(O)NR-]; with -CH2-sulfonamide [-CH2-S(O)2NR-] bond; with urea [-NHC(O)NR-] bond; with a -CH2-secondary amine bond; or with an alkylated peptidyl bond [-C(O)NR6- where R6 is lower alkyl];

peptides such that the N-terminus is derivatized at the -NRR1 group; - NRC(O)R group; -NRC(O)OR group; -NRS(O)2R group; -NHC(O)NHR group where R and R1 are hydrogen or lower alkyl provided that R and R1 are not both hydrogen; succinimide group; benzyloxycarbonyl-NH- (CBZ-NH-) group; or 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; or

peptides wherein the C terminus is derivatized to -C(O)R 2 wherein R 2 is selected from the group consisting of lower alkoxy, and NR 3 R 4 wherein R 3 and R 4 are independently selected from the group consisting of hydrogen and lower alkyl.

Accordingly, preferred peptides and peptide mimetics contain an ingredient having:

(1) a molecular weight of less than about 5000 daltons,

and

(2) binding affinity to TPO-R as expressed by an IC50 of no more than about 100 μm,

wherein zero to all -C(O)NH- bonds of the peptide are replaced by bonds selected from the group consisting of -CH2OC(O)NR- bonds; phosphonate bonds; -CH2S(O)2NR-bonds; -CH2NR-bonds; and -C(O)NR6-bond; and -NHC(O)NH- bond where R is hydrogen or lower alkyl and R6 is lower alkyl.

Furthermore, where the N-terminus of said peptide or peptide mimetic is selected from the group consisting of -NRR1 group; -NRC(O)R groups; -NRC(O)OR groups; -NRS(O)2R groups; -NHC(O)NHR groups; succinimide groups; benzyloxycarbonyl-NH- groups; and benzyloxycarbonyl-NH- groups 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 R1 are independently selected from the group consisting of hydrogen and lower alkyl,

and even further, where the C-terminus of said peptide or peptide mimetic has the formula -C(O)R2 where R2 is selected from the group consisting of hydroxy, lower alkoxy, and -NR3R4 where R3 and R4 are independently selected from the group consisting of consist of hydrogen and lower alkyl and where the nitrogen atom from the -NR3R4 group can optionally be the amine group of the N-terminus of the peptide so as to form a cyclic peptide, and their physiologically acceptable salts.

In a related embodiment, the invention is directed to a labeled peptide or peptide mimetic comprising the peptide or peptide mimetic described above having a detectable label covalently attached thereto.

In some embodiments of the invention, preferred peptides for use include peptides having a core structure consisting of the amino acid sequence:

X1 X2 X3 X4 X5 X6 X7

where X1 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; X4 is any of the 20 genetically encoded L-amino acids; X5 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; and X7 is C, G, I, K, L, M, N, R or V.

In a preferred embodiment, the central peptide consists of the amino acid sequence:

X8 G X1 X2 X3 X4 X5 W X7

where 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; X4 is A, K, L, M, R, S, V, or T; X5 is A, E, G, K, M, Q, R, S, or T; X 7 is C, I, K, L, M, or V; and each X8 residue is independently selected from any of the 20 genetically encoded L-amino acids, their stereoisomeric D-amino acids; and non-natural amino acids. Preferably, each X8 residue is independently selected from any of the genetically encoded L-amino acids and their stereoisomeric D-amino acids. In a preferred embodiment, X1 is P; X2 is T; X3 is L; X4 is R; X5 is E or Q; and X7 is I or L.

Even more preferably, the central peptide consists of the amino acid sequence:

X9 X8 G X1 X2 X3 X4 X5 W X7

where X9 is A, C, E, G, I, L, M, P, R, Q, S, T, or V; and X8 is A, C, D, E, K, L, Q, S, T, or V. More preferably, X9 is A or I; and X8 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 O 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 ; I I E G P T L R Q W L A A R A.

In further embodiments of the invention, preferred peptides for use in the present invention include peptides having a core structure consisting of the amino acid sequence:

C X2 X3 X4 X5 X6 X7

where X 2 is K, L, N, Q, R, S, T or V; X 3 is C, F, I, L, M, R, S or V; X4 is any of the 20 genetically encoded L-amino acids; X5 is A, D, E, G, S, V or Y; X 6 is C, F, G, L, M, S, V, W or Y; and X7 is C, G, I, K, L, M, N, R or V. In even more preferred embodiments, X4 is A, E, G, H, K, L, M, P Q R, S T or W. In further in embodiments, X 2 is S or T; X 3 is L or R; X4 is R; X5 is D, E, or G; X6 is F, L, or W; and X7 is I, K, L, R or V. Particularly preferred peptides include: G G C T L R E W L H G G F C G G.

In another embodiment, preferred peptides for use in the present invention include peptides having a structure consisting of the amino acid sequence:

X8 C X2 X3 X4 X5 X6 X7

where 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; X4 is any of the 20 genetically encoded L-amino acids; X5 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; and X8 is any of the 20 genetically encoded L-amino acids. In some embodiments, X8 is preferably G, S, Y, or R.

The compounds described herein are useful for the prevention and treatment of TPO-mediated diseases, and particularly for the treatment of hematologic disorders, including, but not limited to, thrombocytopenia as a result of chemotherapy, radiation therapy, or bone marrow transfusion. Thus, the present invention also provides a method of treatment wherein a patient having a disorder responsive to treatment with a TPO agonist receives, or is treated with, a therapeutically effective dose or amount of an ingredient of the present invention.

The invention also provides pharmaceutical compositions containing one or more of the ingredients described herein and their physiologically acceptable carriers. These pharmaceutical compositions can be in a variety of forms including oral dosage forms as well as powders and solutions for inhalation and solutions for injections and infusions.

Short description of the pictures

Figures 1A-B illustrate the results of functional experiments in the presence of different peptides; The experiment is described in Example 2. Figure 1A is a graphical description of the results of the proliferation experiment of Ba/F3 cells transfected with TPO-R, for selected peptides of the invention:

¦ indicates results for G G C A D G P T L R E W I S F C G G K (biotin);

X indicates results for G G C A D G P T L R E W I S F C G G;

^ indicates results for L A I E G P T L R Q W L H G N G R D T;

Ο denotes results for G N A D G P T L R Q W L E G R R P K N; and

+ indicates results for T I K G P T L R Q W L K S R E H T S.

Figure 1B is a graphical description of the results with the same peptides and parental cell line.

Figures 2A-C show the results of peptide oligomerization using a proliferation assay of Ba/F3 cells transfected with TPO-R. Figure 2A shows the results of experiments for complexed biotinylated peptide (AF 12285 with streptavidin (SA) for transfected and parental cell lines. Figure 2B shows the results of experiments for free biotinylated peptide (AF 12285) for transfected and parental cell lines. Figure 2C shows the results of streptavidin alone experiments for transfected as well as parental cell lines.

Figures 3A-G show the results of a series of control experiments showing the activity of TPO, 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 respective parental line, or the EPO- dependent line of the station. Figure 3A describes the result for TPO in a cell proliferation experiment using the TPO-R transfected Ba/F3 cell line and its respective parental line. Figure 3B describes the results for EPO in a cell proliferation experiment using the TPO-R transfected Ba/F3 cell line and its respective parental line. Figure 3C describes the results for complexed biotinylated peptides (AF 12285 with streptavidin (SA)) and the complexed form of biotinylated EPO-R binding peptide (AF 11505 with SA) in the TPO-R transfected Ba/F3 cell line. The results for the respective parental cell line are shown in Figure 3D. Figure 3E describes the results for TPO in a cell proliferation experiment using an EPO-dependent cell line. Figure 3F describes the results for EPO in a cell proliferation experiment using an EPO-dependent cell line. Figure 3G describes the results for complexed biotinylated peptides (AF 12885 with streptavidin (SA)) and complexed form of biotinylated EPO-R binding peptide (AF 11505 with SA) in an EPO-dependent cell line.

Figures 4A-C illustrate the construction of a peptide-on-plasmid library in the pJS142 vector. Figure 4A shows the restriction map and gene position. The plasmid library includes the rrnB transcriptional terminator, and the bla gene to allow selection on ampicillin, the M13 phage intragenic region (M13 IG) to allow single-stranded DNA rescue, the plasmid origin of replication (ori), two lacO3 sequences, and the araC gene to allow positive and negative regulation of the araB promoter driving the 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 SfiI and EagI sites used during library construction. Figure 4C shows the binding of annealed library oligonucleotides, ON-829 and ON-830, to the SfiI site of pJS142 to produce a library. The positions themselves in the sequence indicate the binding sites.

Figures 5A-B illustrate cloning into the pELM3 and pELMI5 MBP vectors. Figure 5A shows the sequence at the 3' end of the malEgen binding site, including the MBP coding sequence, poly asparagine linker, factor Xa protease cleavage site, and available cloning sites. The remaining parts of the vector were obtained from pMALc2 (pELM3) and pMALp2 (pELMI5), available from New England Biolabs. Figure 5B shows the vector sequence after transfer of the BspEII-Scal library fragment into AgeI-ScaI digested pELM3/pELM15. The transferred sequence includes the sequence encoding the GGG peptide linker from the pJS142 library.

Figure 6A describes the restriction map and gene position for the construction of the main piece of the dimer library in the pCMG14 vector. The plasmid library includes: an rrnB transcriptional terminator, a bla gene to allow selection on ampicillin, an M13 phage intragenetic region (M13 IG) to allow rescue of single-stranded DNA, a plasmid origin of replication (ori), a single laco3 sequence, and an araC gene to allow positive and negative regulation of the araB promoter that controls the expression of the main dimer of the fusion gene. Figure 6B depicts the sequence of the cloning region at the 3' end of the major dimer gene, including the SfiI and EagI sites used during library construction. Figure 6C shows the binding of quenched ON-1679, ON-829, and ON-830 to the SfiI site of pCMG14 to produce a library. The positions themselves in the sequence indicate the binding sites.

Figures 7 to 9 show the results of further experiments evaluating the activity of the peptides and peptide mimetics of the invention. In this experiment, mice were rendered thrombocytopenic with carboplatin. Figure 7 describes typical results when Balb/C mice were treated with carboplatin (125 mg/kg intraperitoneally) on Day 0. The dashed line represents untreated animals from three experiments. The solid line represents the carboplatin-treated groups in three experiments. The thick solid line represents historical data. Figure 8 describes the effect of carboplatin titration on platelet counts in mice treated with the indicated amount of carboplatin (in mg/kg, intraperitoneally (ip) on Day 0). Figure 9 describes the amelioration of carboplatin-induced thrombocytopenia on Day 10 by peptide AF12513 (513). Carboplatin (CBP; 50-125 mg/kg, i.p.) was given on Day 0. AF12513 (1 mg/kg, ip) was given on Days 1-9.

Descriptions of specific achievements

I. Definitions and general parameters

The following definitions are set forth to illustrate and define the meaning and scope of various terms used in describing this invention.

"Agonist" refers to a biologically active ligand that binds to its complementary biologically active receptor and activates the latter either to cause a biological response in the receptor or to increase preexisting biological activity of the receptor.

"Pharmaceutically acceptable salts" refer to non-toxic alkali metals, alkaline earth metals, and ammonium salts commonly used in the pharmaceutical industry including sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and zinc protamine salts, which are prepared by methods well known in the profession. The term also includes non-toxic acid addition salts, which are generally prepared by reacting the ingredients of this invention with suitable organic or inorganic acids. 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 added acid salts" refer to those salts which retain biological effectiveness and free base properties and are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and 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 , mandalic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

For a description of pharmaceutically acceptable salts of the added acid as drugs, see Bundgaard, H., supra.

"Pharmaceutically acceptable ester" refers to those esters which retain, after hydrolysis of the ester bond, the biological effectiveness and properties of the carboxylic acid or alcohol and are not biologically or otherwise undesirable. For a description of pharmaceutically acceptable esters as drugs, see Bundgaard, H. ed., Design of Prodrugs, Elsevier Science Publishers, Amsterdam (1985). These esters are typically formed from the corresponding carboxylic acid and an alcohol. In general, ester formation can be achieved via conventional synthetic techniques. (See, e.g., March Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York (1985) p. 1157 and references cited therein, and Mark et al. Encyclopedia of Chemical Technology, John Wiley & Sons, New York ( 1980)). The alcohol component of the ester will generally contain (i) a C2-C12 aliphatic alcohol which may or may not contain one or more double bonds and may or may not contain branched carbons or (ii) C7-C12 aromatic or heteroaromatic alcohols. This invention also contemplates the use of those ingredients which are both esters as described herein and at the same time pharmaceutically acceptable added acid salts thereof.

"Pharmaceutically acceptable amide" refers to those amides which retain, after hydrolysis of the amide bond, the biological effectiveness and properties of the carboxylic acid or amine and are not biologically or otherwise undesirable. For a description of pharmaceutically acceptable amides as drugs, see Bundgaard, H., ed., Design of Prodrugs, Elsevier Science Publishers, Amsterdam (1985). These amides are typically formed from the corresponding carboxylic acid and an amine. In general, 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 ingredients which are both amides as described herein and at the same time pharmaceutically acceptable acid addition salts thereof.

"Pharmaceutically or therapeutically acceptable carrier" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredients and is not toxic to the host or patient.

"Stereoisomer" refers to a chemical compound that has the same molecular weight, chemical composition, and constitution as another, but with the atoms grouped differently. This means that certain identical chemical parts are in different orientations in space, and therefore, when pure, has the ability to rotate the plane of polarized light. However, some pure stereoisomers may have an optical rotation that is so slight that it cannot be detected with current instrumentation. The compounds of this invention may have one or more asymmetric carbon atoms and thus include different stereoisomers. All stereoisomers are included within the scope of this invention.

"Therapeutically or pharmaceutically effective amount" as applied to the ingredients of this invention refers to an amount of the ingredient sufficient to induce the desired biological result. The result may be an alleviation of the signs, symptoms or cause of the disease, or any other change in the biological system. In the present invention, the result will typically be a reduction in immune and/or inflammatory responses to infection or tissue injury.

Amino acid residues in peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Valin 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 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. Additionally, 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 is O-benzyl, and hyp is hydroxyproline.

In addition to peptides consisting only of naturally occurring amino acids, there are also peptidomimetics or peptide analogs. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the matrix peptide. These types of non-peptide compounds are called "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 can be used to produce an equivalent or increased therapeutic or prophylactic effect. In general, peptidomimetics are structurally similar to paradigm polypeptides (ie, a polypeptide having biological or pharmacological activity), such as naturally occurring receptor-binding polypeptides, but having one or more peptide bonds optionally replaced with a bond selected from the group consisting of : -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, by the method known in the field and further described in the following references: Spatola, A.F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A.F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morlev, Trends Pharm Sci (1980) p. 463-468 (general review); Hudson, D. et al., Int. J. Pept. prot. Res 14:177-185 (1979) (-CH2NH-, CH2CH2-); Spatola et al. Life Sci 38:1243-1249 (1986) (-CH2-S); Hann J. Chem. Soc. Perkin Trans. I 307-314 (1982) (-CH-CH-, cis and trans); Almqusit et al. J. Med. Chem. 23:1392-1398 (1980) (-COCH2-); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (-COCH2-); Szelke et al. European Appl. EP 45665 CA (1982): 97:39405 (1982) (-CH(OH)CH2-); Holladay et al. Tetrahedron Lett 24:4401-4404 (1983) (-C(OH)CH2-); and Hruby Life Sci 31:189-199 (1982) (-CH2-S-); each of which is incorporated herein by reference.

A particularly preferred non-peptide bond is -CH2NH-. Such peptide mimetics can have significant advantages over polypeptide realizations, including, for example: higher economic production, greater chemical stability, increased pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (eg, a broad spectrum of biological activities) , reduced antigenicity, and others. Labeling of a peptidomimetic typically involves the covalent attachment of one or more labels, either directly or through a spacer (eg, amide group), to non-interfering site(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling. . Such non-interfering sites are generally sites that do not make direct contacts with the macromolecule(s) (eg, molecules of the immunoglobulin superfamily) to which the peptidomimetic binds to produce a therapeutic effect. Derivatization (eg, labeling) of the peptidomimetic must not significantly interfere with the desired biological or pharmacological activity of the peptidomimetic. In general, receptor-binding peptide peptidomimetics bind to the receptor with high affinity and possess appreciable biological activity (ie, they are agonistic or antagonistic to one or more receptor-promoted phenotypic changes).

Systematic replacement of one or more amino acids of the consensus sequence with a D-amino acid of the same type (eg, D-lysine instead of L-lysine) can be used to generate more stable peptides. Furthermore, restricted peptides comprising the consensus sequence or a variant of a substantially identical consensus sequence can be generated by methods known in the art (Rizo and Gieraqsch Ann. Rev, Biochem. 61:387 (1992), incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize peptides.

Synthetic or non-naturally occurring amino acids refer to amino acids that do not occur naturally in vivo, but which can nevertheless be incorporated into the peptide structure described herein. Preferred synthetic amino acids from D-a-amino acids naturally occurring L-a-amino acids as well as unnaturally occurring D- and L-a-amino acids represented by the formula H2NCHR5COOH where R5 is 1) a lower alkyl group, 2) a cycloalkyl group of 3 to 7 carbon atoms, 3) a heterocycle of the form of 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 6 to 10 carbon atoms that optionally has 1 to 3 substituents on the aromatic nucleus selected from a group consisting of hydroxyl, lower alkoxy, amino, and carboxyl, 5) -alkylene-Y where alkylene is an alkylene group of 1 to 7 carbon atoms and Y is selected from the group consisting of (a) hydroxy, (b) amino, (c) cycloalkyl and cycloalkylene of 3 to 7 carbon atoms, (d) aryl of 6 to 10 carbon atoms which optionally has from 1 to 3 substituents on the aromatic ring selected from the group consisting of hydroxyl, lower alkoxy, amino and carboxyl, (e) heterocycle and from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen, (f) -C(O)R2 where R2 is selected from the group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, and -NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl, (g) -S(O)nR6 where n is an integer from 1 to 2 and R6 is lower alkyl and provided that R5 does not define a naturally occurring amino acid side chain.

Other preferred synthetic amino acids include amino acids where the amino group is separated from the carboxyl group by one or more carbon atoms such as b-alanine, g-aminobutyric acid, and the like.

Particularly preferred synthetic amino acids include, by way of example, D-amino acids, naturally occurring L-amino acids, L-1-napthyl-alanine, L-2-napthylalanine, L-cyclohexylalanine, L-2-amino isobutyric acid, sulfoxide and sulfonic acid. methionine derivatives (ie, HOOC-(H 2 NCH)CH 2 CH 2 -S(O)nR 6 where n and R 6 are as defined above as well as a lower alkoxy derivative of methionine (ie, HOOC-(H 2 NCH)CH 2 CH 2 -OR 6 where R 6 is as defined up).

"Detectable label" refers to materials which, when covalently bound to the peptides and peptide mimetics of the present invention, permit the detection of the peptide and peptide mimetics in vivo in a patient to whom the peptide or peptide mimetic is administered as a drug. Suitable detectable labels are well known in the art and include, by way of example, radioisotopes, fluorescent labels (eg, fluorescein), and the like. The particular detectable label used is not critical and is chosen relative to the amount of label used as well as the toxicity of the label to the amount of label used. Selection of markers relative to such factors is well within the skill of the art.

Covalent attachment of a detectable label to a peptide or peptide mimetic is accomplished by conventional methods well known in the art. For example, when using 125I radioisotope as a detectable label, covalent attachment of 125I to a peptide or peptide mimetic can be achieved by incorporating the amino acid tyrosine into the peptide or peptide mimetic and then iodizing the peptide. If tyrosine is not present in the peptide or peptide mimetic, incorporation of tyrosine at the N or C terminus of the peptide or peptide mimetic can be achieved by known chemistry. Likewise, 32 P can be incorporated onto a peptide or peptide mimetic as a phosphate moiety through, for example, a hydroxyl group onto the peptide or peptide mimetic using conventional chemistry.

Review

This invention provides compounds that bind to and activate the TPO-R or otherwise act as a TPO agonist. These ingredients include "lead" peptide ingredients and "derivative" ingredients engineered to have the same or similar molecular structure or shape as the lead ingredients but to differ from the lead ingredients either in susceptibility to hydrolysis or proteolysis and/or in other biological properties. , such as increased affinity for the receptor. The present invention also provides compositions containing an effective amount of a TPO agonist, and more specifically, a composition useful for treating hematological disorders, and in particular, thrombocytopenia associated with chemotherapy, radiation therapy, or bone marrow transfusion.

Identification of tpo-agonists

Peptides having TPO-R binding affinity can be readily identified by random peptide diversity generation systems coupled to an affinity enrichment procedure.

Specifically, random peptide diversity generation systems include the "peptides on plasmids" system described in U.S. Pat. Patents no. 5,270,170 and 5,338,665; the "peptides on phage" system, described in U.S. Pat. Patent Application Serial No. 07/718,577, filed June 20, 1991, which is a continuation-in-part of U.S. Appl. Patent Application Serial No. 07/541,108, filed June 20, 1990, and in Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1980); "polysomic system" described in the U.S. Patent Application Serial No. 08/300,262, filed Sep. 2, 1994, which application is a continuation-in-part based on U.S. Pat. Patent Application Serial No. 08/144,775, filed October 29, 1993 and PCT WO 95/11992; the "encoded synthetic library (ESL)" system described in the U.S. Patent Application Serial No. 08/146,886, filed Nov. 12, 1993, which is a continuation-in-part of U.S. Appl. Patent Application Serial No. 07/946,239, filed Sep. 16, 1992, which is a continuation-in-part of U.S. App. Patent Applications Serial No. 07/762,522, filed September 18, 1991; and the "very large scale immobilized polymer synthesis" system described in U.S. Patent No. 5,143,854; PCT Patent Publication No. 90/15070, published Dec. 13, 1990; U.S. Patent Application Serial No. 07/624,120, filed Dec. 6, 1990; Fodor et al. Science 251:767-773 (2/1991); Dower and Fodor Ann. Rep. Med. Chem. 26:271-180 (1991); and U.S. Patent Application Serial No. 805,727, filed Dec. 6, 1991.

Using the procedures described above, random peptides are generally designed to have a defined number of amino acid residues in length (eg, 12). To generate a collection of oligonucleotides encoding random peptides, the codon motif (NNK)x was used, where N is the nucleotide A, C, G or T (equimolar; depending on the methodology used, other nucleotides can be used), K is G or T ( equimolar), and x is an integer corresponding to the number of amino acids in the peptide (e.g. 12), to specify any 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 the three amino acids, and only one of the three stop codons. Thus, the NNK motif encodes all amino acids, encodes only one stop codon and reduces codon bias.

In the system used, random peptides are presented either on the surface of the phage particle, as part of a binding protein consisting of either the pIII or pVIII coat protein of the phage derivative fd (peptides on phage) or as a binding protein with a LacI peptide binding protein linked to a plasmid (peptides on plasmids).

Phage or plasmids, including DNA encoding peptides, were identified and isolated by an affinity enrichment procedure using immobilized TPO-R. The affinity enrichment process, sometimes called "panning", requires multiple rounds of incubating the phage, plasmid, or polysome with an immobilized receptor, collecting the phage, plasmid, or polysome that bind to the receptor (along with the accompanying DNA or mRNA ), and the production of multiple assembled phages or pasmids (together with the associated LacI-peptide for protein fusion). The extracellular domain (ECD) of TPO-R is typically used during "washout".

After several rounds of affinity enrichment, the phages or plasmids and associated peptides were assayed by ELISA to determine whether the peptides bind specifically to TPO-R. This experiment was performed similarly to the procedures used in the affinity enrichment procedure, except that after removal of unbound phages, the wells were typically treated with rabbit anti-phage antibodies, followed by alkaline phosphatase (AP)-conjugated goat anti-rabbit antibody. The amount of alkaline phosphatase in each well was determined by standard methods. A similar ELISA procedure for use in peptide-on-plasmid systems is described in detail below.

By comparing experimental wells with control wells (no receptor), it can be determined whether the binding proteins bind specifically to the receptor. Pools of phages found to bind to TPO-R were screened in a "colony lift" assay format using radiolabeled monovalent receptors. This probe can be produced using protein kinase A that phosphorylates a kemptid sequence fused to the C-terminus of the soluble receptor. the so-called "engineered" form of the TPO receptor is then expressed in host cells, typically CHO cells. Following PI-PLC yield of the receptor, the receptor was tested for binding to TPO or TPO-R specific phage clones. The receptor was then labeled for high-specificity activity with 33P for use as a monovalent probe to identify high-specificity ligands using colony lifts.

Peptides found to bind specifically to the receptor were then synthesized as free peptides (eg, phage-free) and tested in a batch experiment. The batch experiment was performed similarly to the ELISA, except that TPO or reference peptide was added to the wells before the fusion protein (there were two types of control wells: (1) no receptor; and (2) no TPO or reference peptide). Binding proteins for which binding to the receptor is blocked by TPO or the reference peptide contains peptides in random peptide portions that are preferred ingredients of the invention.

TPO-R, as well as its extracellular domain, are produced in recombining host cells. A useful form of TPO-R was constructed by expressing the protein as a soluble protein in baculovirus-transformed host cells using standard methods; another useful form is engineered with only one protein for protein secretion and for glycophospholipid anchoring of the membrane. 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).

Using a "PIG-tailing" system, one can cleave the receptor from the surface of cells expressing the receptor (eg, transformed CHO cells selected for high receptor expression with a cell sorter) with C phospholipase. The separated receptor still contains the carboxy terminal amino acid sequence, called the "HPAP tail", from the membrane anchoring signaling protein and can be immobilized without further purification. Recombinant receptor protein can be immobilized by coating wells of microtiter plates with a single anti-HPAP rep antibody (Ab 179 or Mab 179), blocking non-specific binding with bovine serum albumin (BSA) in PSA, and then binding the separated recombinant receptor to the antibody. Using this procedure, the immobilization reaction can be performed by varying the receptor concentration to determine the optimal amount for a given preparation, since different recombinant protein preparations often contain different amounts of the desired protein. Furthermore, it must be ensured that the immobilizing antibody is completely blocked (with TPO or another blocking agent) during the affinity enrichment procedure. Otherwise, the unblocked antibody may bind the unwanted phage during the affinity enrichment procedure. Peptides that bind to the immobilizing antibody can be used to block unbound sites that remain after immobilizing the receptor to avoid this problem, or one can simply directly immobilize the receptor on the wells of microtiter plates, without the help of an immobilizing antibody. See U.S. Patent Application Serial no. 07/947,339, filed Sep. 18, 1992, incorporated herein by reference.

When using a random peptide generation system that allows for multivalent ligand-receptor interaction, it must be recognized that the density of the immobilized receptor is an important factor in determining the affinity of the ligands that can bind to the immobilized receptor. At higher receptor densities (eg, 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 (eg, each anti-receptor antibody-coated well well treated with 0.5 to 1 ng receptor). If multivalent binding occurs, then ligands with relatively low activity are more likely to be isolated, unless high densities of immobilized receptors are used to identify lead compounds and lower receptor densities are used to isolate higher affinity derivative compounds.

A monovalent receptor probe is often used to discriminate between higher affinity peptides. This probe can be produced using 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 yield of the receptor, the receptor was tested for binding to TPO or TPO-R specific phage clones. The receptor is then labeled for highly specific activity with 33P for use as a monovalent probe, using "colony lifts" to identify high affinity ligands.

Preferred assay methods that facilitate the identification of peptides that bind to the TPO-R include first identifying lead peptides that bind to the extracellular region of the receptor and then generating other peptides that resemble the lead peptides. Specifically, using a pIII or pVIII-based peptide phage system, a random library can be screened to discover a phage presenting a peptide that binds to the TPO-R. DNA phages were sequenced to determine the sequences of peptides displayed on the phage surface.

Clones capable of binding specifically to TPO-R were identified from a random linear 10-mer pVIII library and random cyclic 10-mer and 12-mer pVIII libraries. The sequences of these peptides serve as a basis for the construction of other peptide libraries designed so that contain a high frequency of derivatives at the beginning of the identified peptides. These libraries can be synthesized by favoring peptides that differ from the binding peptide by only a few residues. This approach involves the synthesis of oligonucleotides with the binding peptide coding sequence, except that rather than using pure preparations of each of the four nucleoside triphosphates in the synthesis, mixtures of the four nucleoside triphosphates (ie, 55% of the "correct" nucleotide, and 15% of of each of the other three nucleotides is one preferred mixture for this purpose and 70% of the "correct" nucleotide and 10% of each of the other three nucleotides is another preferred mixture for this application) so as to generate derivatives of the binding peptide coding sequences.

Different strategies were used to derivatize the lead peptides by performing "topical mutagenesis" of the library. These included a pVIII phagemid mutagenesis library based on the consensus sequence mutagenized at a frequency of 70:10:10:10 and extended at each end with random residues to produce clones encoding the sequence XXXX (C, S, P, or R) TLREWL XXXXXX (C or S). A similar expanded/mutagenized library was constructed using a peptide-on-plasmid system to generate clones encoding the sequence XXXXX (C, S, P, or R) TLREWL XXXXXXX. An additional expanded/mutagenized library, XXXX (C, S, P, or R) TLREWL XXXXXX (C or S), was constructed using a polysome display system. All three libraries were screened with peptide elution and probed with radiolabeled monovalent receptor.

"Peptide-on-plasmid" techniques have also been used for peptide screening and mutagenesis studies and are described in more detail in U.S. Pat. Patent no. 5,338,665, which is incorporated herein by reference for all purposes. According to this approach, random peptides are fused to the C-terminus of LacI through expression from a plasmid vector carrying the splicing gene. Binding of the LacI-peptide bond to its coding DNA takes place through the lacO sequence on the plasmid, forming a stable peptide-Lacl-plasmid complex that can be tested by affinity purification (panning) on the immobilized receptor. Plasmids thus isolated can then be reintroduced into E. coli by electroporation to amplify the selected population for an additional round of testing, or to test individual clones.

Furthermore, random peptide screening and mutagenesis studies were performed using a modified C-terminus Lac-I display system in which the displayed valence was reduced ("master dimer" display system). Libraries were screened and the resulting DNA inserts were cloned as a pool into a maltose-binding protein (MBP) vector allowing their expression as a C-terminal junction protein. Crude cell lysates from randomly selected individual MBP clones for fusion were then assayed for TPO-R binding in an ELISA format, as discussed above.

Peptide mutagenesis studies were also performed, using a polysome display system, as described in the accompanying application of U.S. Pat. Patent Applications Serial no. 08/300,262, filed Sep. 2, 1994, which is a continuation-in-part of U.S.-based App. Patent Application Serial no. 08/144,775, filed Oct. 29, 1993, and PCT WO 95/11992, each of which is incorporated herein by reference for all purposes. A mutagenesis library was constructed based on the sequence X X X X (c, P, R, or S) t l r e f l X X X X X X (C or S), where X represents a random NNK codon and lowercase letters represent amino acid codons containing 70:10:10 :10 mutagenesis at positions 1 and 2 and K (G or T) at position 3 of the codon. The library was panned through 5 rounds against TPO receptors that were immobilized on magnetic beads. After the fifth round, the PCR amplified pool was cloned into pAFF6, and ELISA positive clones were sequenced. The sequences were subcloned into the MBP vector, and their binding affinities were determined by MBP ELISA.

To immobilize TPO-R for polysome assay, Ab 179 was first chemically conjugated to tosyl-activated magnetic beads (available from Dynal Corporation) as described by the manufacturer. Beads were incubated with antibodies in 0.5 M borate buffer (pH 9.5) overnight at room temperature. The beads were washed and combined with TPO-R containing "HPAP" tail. Antibody- and receptor-coated beads were incubated for 1 hour at 4°C, and the beads were washed again before addition of the polysome library.

Screening of the various libraries described above led to the TPO receptor-binding peptides shown in Tables 1 and 2 below, as well as others not listed here.

[image]

[image]

[image]

[image]

[image]

IC50 values for some additional representative peptides are given in the table below. Various methods can be used to determine IC50 values. For example, an equilibrium binding ELISA experiment, using either MBP-TPO or a lacI-peptide label, was used to determine whether the peptides would inhibit TPO binding to the extracellular region of the TPO receptor. Typically, IC50 values are determined using free peptides. The IC50 value can be determined using the free peptide, which can optionally be amidated at the C-terminus, or can be prepared as an ester or other carboxy amide.

To recreate the exact sequence displayed on the phage, the amino acids at the N-terminus and C-terminus of the synthetic peptides are often preceded by one or two glycine residues. These glycines are not believed to be essential for binding or activity. Also, to mimic the exact sequence of peptides displayed on polysomes, the amino acid C-termini of synthetic peptides are often preceded by the sequence M A S. Again, this sequence is not believed to be essential for binding or activity.

IC50 values are indicated symbolically by the symbols "-", "+", and "++". For example, those peptides that showed IC50 values in excess of 200 μM are marked with "-". Those peptides that gave IC50 values less than or equal to 200 μM were given a "+" sign, while those that gave IC50 values of 500 nm or less were marked with "++". Those peptides that gave IC50 values at or near the breakpoint for a particular symbol are indicated with the hybrid sign npt., "+/-". Those peptides for which IC50 values were not determined are marked as "N.D.". The IC50 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 that the N-terminal and C-terminal amino acids are preceded by two glycines to recreate the exact sequence displayed by the phage. These glycines are not believed to be required for binding or activity.)

[image]

The above tables, especially Table 3, illustrate that the preferred core peptide contains the amino acid sequence:

X1 X2 X3 X4 X5 X6 X7

where X1 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; X4 is any of the 20 genetically encoded L-amino acids; X5 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; and X7 is C, G, I, K, L, M, N, R or V.

In a preferred embodiment, the peptide core contains the amino acid sequence:

X8 G X1 X2 X3 X4 X5 W X7

where 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; X4 is A, K, L, M, R, S, V, or T; X5 is A, E, G, K, M, Q, R, S, or T; X 7 is C, I, K, L, M, or V; and each X8 residue is independently selected from any of the 20 genetically encoded L-amino acids, their stereoisomeric D-amino acids; and non-natural amino acids. In a preferred embodiment, X1 is P; X2 is T; X3 is L; X4 is R; X5 is E or Q; and X7 is I or L.

Even more preferably, the peptide core contains the amino acid sequence:

X9 X8 G X1 X2 X3 X4 X5 W X7

where X9 is A, C, E, G, I, L, M, P, R, Q, S, T, or V; and X8 is A, C, D, E, K, L, Q, R, S, T, or V. More preferably, X9 is A or I; and X8 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.

In the following embodiments of the invention, preferred peptides for use in the present invention include peptides having a core structure comprising the amino acid sequence:

C X2 X3 X4 X5 X6 X7

where X 2 is K, L, N, Q, R, S, T or V; X 3 is C, F, I, L, M, R, S or V; X4 is any of the 20 genetically encoded L-amino acids; X5 is A, D, E, G, S, V or Y; X 6 is C, F, G, L, M, S, V, W or Y; and X7 is C, G, I, K, L, M, N, R or V. In a more preferred embodiment, X4 is A, E, G, H, K, L, M, P, Q, R, S, T, or W. In another embodiment, X 2 is S or T; X 3 is L or R; X4 is R; X5 is D, E, or G; X 6 is P, L, or W; and X7 is I, K, L, R, or V. Particularly preferred peptides include: G G C T L R E W L H G G P C G G.

Peptides and peptidomimetics having an IC50 greater than about 100 mM lack sufficient binding to permit use in either the diagnostic or therapeutic aspects of this invention. Preferably, for diagnostic purposes, the peptides and peptidomimetics have an IC50 of about 2 mM or less, and for pharmaceutical purposes, the peptides and peptidomimetics have an IC50 of about 100 μM or less.

The binding peptide sequence also provides a means to determine the smallest measure of the TPOR binding agent of the invention. Using an "encoded synthetic library" (ESL) system or a "very large-scale immobilized polymer synthesis" system, one can not only determine the smallest measure of peptides with such activity, but also make all peptides that form a group of peptides that differ from the preferred motif ( or of the smallest measure for that motif) in one, two, or more residues. This collection of peptides can then be tested for their ability to bind to the TPO-receptor. These immobilized polymer synthesis systems or methods of synthesizing other peptides can also be used to synthesize truncated analogs, deletion analogs, substitution analogs, and combinations thereof of all peptide constituents of the invention.

Peptides and peptide mimetics of the present invention were also evaluated in a thrombopoietin-dependent cell proliferation assay, as described in more detail in Example 2 below. Cell proliferation is measured by techniques known in the art, such as the MTT assay which correlates with 3H-thymidine incorporation as an indication of cell proliferation (see Mossmann J. Immunol. Methods 65:55 (1993)). The tested peptides stimulated the 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 1B.

Figures 7 to 9 show the results of further experiments evaluating the activity of the peptides and peptide mimetics of the invention. In this experiment, mice were made thrombocytopenic with carboplatin. Figure 7 shows typical results where Balb/C mice were treated with carboplatin (125 mg/kg intraperitoneally) on day 0. Dashed lines represent untreated animals from three experiments. The solid line represents the carboplatin-treated groups in three experiments. Thick solid lines represent historical data. Figure 8 describes the effect of carboplatin titration on platelet counts in mice treated with the indicated amounts of carboplatin (in mg/kg, intraperitorally (ip) on day 0). Figure 9 shows amelioration of carboplatin-induced thrombocytopenia at day 10 by peptide AF12513 (513). Carboplatin (CBP; 50-125 mg/kg, intraperitoneal) was given on day 0. AF12513 (1 mg/kg, ip) was given on days 1-9. These results demonstrate that the peptides of the invention can improve thrombocytopenia in a mouse model.

Furthermore, certain peptides of this invention can be dimerized or oligomerized, thereby increasing the affinity and/or activity of the ingredients. In order to examine the effect that peptide dimerization/oligomerization has on the potency of the TPO mimetic in the cell proliferation experiment, a biotinylated analog of the peptide G G C A D G P T L R E W I S F C G G (G G C A D G P T L R E W I S F C G G K (Biotin)) was synthesized at the C-end. The peptide was preincubated with streptavidin in serum-free RPMI buffered with HEPES in a molar ratio of 4:1. The complex was tested for stimulation of cell proliferation of TPO-R transfected Ba/F3 cells, as above, along with free biotinylated peptide and non-biotinylated parent peptide. Figure 2A shows the results of experiments for complexed biotinylated peptide (AF 12885 with streptavidin (SA)) for both transfected and parental cell lines. Figure 2B shows the results of experiments for free biotinylated peptide (AF 12285) for both transfected and parental cell lines. Figure 2C shows the results of streptavidin alone experiments for both transfected and parental cell lines. All images show that the previously formed complex is approximately 10 times stronger than the free peptide.

The binding specificity and activity of the peptides of the present invention were also investigated by studying the cross-reactivity of the peptide erythropoietin receptors (EPO-R). EPO-R is also a member of the hematopoietin growth factor receptor family, such as TPO-R. The peptides of the invention, as well as TPO, EPO, and the known EPO-binding peptide, were tested in a cell proliferation assay using an EPO-dependent cell line. This experiment used FDCP-1, a murine multi-potential primitive, growth factor-dependent hematopoietic 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 cannot differentiate when supplied with WEHI-3-conditioned media (media containing IL-3, ATCC number T1B68). The parental cell line was transfected with human or murine EPO-R to generate the FDCP-1-EPO-R cell line.

These transfected cell lines can proliferate but not differentiate in the presence of human or murine EPO.

Cells were grown to half stationary density in the presence of essential growth factors. Cells were then washed in PBS and starved for 16-24 hours in complete medium without growth factors. After determining the viability of the cells, stock solutions were made (in the entire medium without growth factors) such that they yield approximately 105 cells per 50 microliters. Serial dilutions of constituents (typically, peptide from the free solution phase as opposed to phage-bound or otherwise immobilized peptide) to be tested were made in 96-well tissue culture plates, for a final volume of 50 microliters per well. Cells (50 microliters) were added to each well and then incubated for 24-48 hours, at which point negative controls should be dead or quiescent. Cell proliferation is then measured by techniques known in the art, such as the MTT assay.

Figures 3A-G show the results of a series of control experiments showing the activity of TPO, 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 respective parental line, or the EPO- dependent cell line and its corresponding parent line. Figure 3A shows the results for TPO in a cell proliferation experiment using the TPO-R transfected Ba/F3 cell line and its respective parental line. Figure 3B shows the results for EPO in a cell proliferation experiment using the TPO-R transfected Ba/F3 cell line and its corresponding parental line. Figure 3C describes the results for the complexed biotinylated peptide (AF 12285 with streptavidin (SA)) and the complexed form of the biotinylated EPO-R binding peptide (AF 11505 with SA) in the TPO-R transfected Ba/F3 cell line. The results for the respective parental cell line are shown in Figure 3D. Figure 3E shows the results for TPO in a cell proliferation experiment using an EPO-dependent cell line. Figure 3F shows the results for EPO in a cell proliferation experiment using an EPO-dependent cell line. Figure 3G describes the results for complexed biotinylated peptides (AF 12285 with streptavidin (SA)) and complexed form of biotinylated RPO-R binding peptide (AF 11505 with SA) in an EPO-dependent cell line. These results demonstrate that the peptides of the present invention bind and activate TPO-R with a high degree of specificity.

Preparation of peptides and peptide mimetics

A. Solid phase synthesis

Peptides of the present invention can be prepared by conventional methods known in the art, for example, using standard solid phase techniques. Standard methods include exclusive solid phase synthesis, partial solid phase synthesis methods, phagemental condensation, classical solution synthesis, and even recombinant DNA technology. See, e.g., Merrifield J. Am. Chem. Soc. 85:2149 (1963), incorporated herein by reference. On solid phase, synthesis typically starts from the C-terminus of the peptide using an alpha-amino protected resin. A suitable starting material can be prepared, for example, by attaching the desired alpha-amino acid chloromethylated resin to a hydroxymethyl resin or to a benzhydrylamine resin. One such chloromethylated resin is sold under the tradename BIO-BEADS SX-1 from Bio Rad laboratories, Richmond, CA, and a preparation of the hydroxymethyl resin is described by Bodonszky et al. Chem. Ind. (London) 38:1597 (1966). Benzhydrylamine (BHA) resin is described by Pietta and Marshall Chem. Commn. 650 (1970) and is commercially available from Beckman Instruments, Inc., Palo Alto, CA, in the hydrochloride form.

Thus, the compounds of the invention can be prepared by attaching an alpha-amino protected amino acid to a chloromethylated resin with the aid of, for example, a cesium bicarbonate catalyst, according to the method described by Giesin Helv. Chem. Acta 56:1467 (1973). After initial attachment, the alpha-amino protecting group is removed by a choice of reagents including solutions of trifluoroacetic acid (TFA) or hydrochloric acid (HCl) in organic solvents at room temperature.

Alpha-amino protecting groups are those known to be useful in the art for the stepwise synthesis of peptides. Included are the type of acyl protecting groups (eg, formyl, trifluoroacetyl, acetyl), the type of aromatic urethane protecting groups (eg, benzyloxycarbyl (Cbz) and substituted Cbz), aliphatic urethane protecting groups (eg, t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl) and the type of alkyl protecting group (eg, benzyl, triphenylmethyl). Boc and Fmoc are preferred protecting groups. The terminal chain of the protecting group remains intact during binding and is not cleaved during deprotection of the amino-terminus of the protecting group or during binding. The end chain of the protecting group must be able to be removed after the completion of the synthesis of the final peptide and under the reaction conditions this will not change the target peptide.

End chain protecting groups for Tyr include tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl. Chain-end protecting groups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl, and cyclohexyl. End chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl, and Cbz. The end chain protecting group for Thr and Ser is benzyl. End chain protecting groups for Arg include nitro, Tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), or Boc. End chain protecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-BrCbz), Tos, or Boc.

After removing the alpha-amino protecting group, the remaining protected amino acids are attached step by step in the desired order. An excess of each protected amino acid is generally used with a suitable carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH 2 Cl 2 ), a dimethyl formamide (DMF) mixture.

Once the desired amino acid sequence is complete, the desired peptide is delinked from the support resin by treatment with a reagent such as trifluoroacetic acid or hydrogen fluoride (HF), which not only cleaves the peptide from the resin, but also cleaves any remaining side chain protecting groups. When a chloromethylated resin is used, treatment with hydrogen fluoride results in the formation of free peptide acids. When a benzhydrylamine resin is used, treatment with hydrogen fluoride results directly in the free peptide amide. Alternatively, when a chloromethylated resin is used, the side chain protecting group can be removed by treating the peptide resin with ammonia to give the desired side chain protected amide or with an alkylamine to give the side chain protected alkylamide or dialkylamide. The side chain is then deprotected in the usual manner by treatment with hydrogen fluoride to give the free amides, alkylamides or dialkylamides.

Procedures for the synthesis of solid phase peptides are well known in the art and are further described in Stewart Solid Phase Peptide Syntheses (Freeman and Co., San Francisco, (1969)).

Using the "encoded synthetic library" or "large-scale immobilized polymer synthesis" system described in U.S. Pat. Patent Applications Serial Nos. 07/492,462, filed March 7, 1990; 07/624,120, filed Dec. 6, 1990; and 07/805,727, filed Dec. 6, 1991; not only can the smallest measure of a peptide with such activity be determined, but all peptides that form a group of peptides that differ from the preferred motif (or the smallest measure of that motif) by one, two or more residues can be made. This collection of peptides can then be tested for their ability to bind to TPO-R. This immobilized polymer synthesis system or other peptide synthesis method can also be used to synthesize truncated analogs, deletion analogs, and combinations of truncated and deleted analogs from all peptide constituents of the invention.

Synthetic amino acids

These procedures can also be used to synthesize peptides in which amino acids other than the naturally occurring 20, which are genetically encoded amino acids, are substituted at one, two, or more positions than any of the compounds of the invention. For example, naphthylalanine can be substituted for tryptophan, thereby facilitating synthesis. Other synthetic amino acids that can be substituted into the peptides of this invention include L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, d amino acids such as L-d-hydroxylysyl and D-d-methylalanyl, L-a-methylalanyl, b amino acids, and isoquinolyl. D amino acids and synthetic amino acids that do not occur naturally may also be incorporated into the peptides of this invention.

The naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) can be replaced with other side chains, for example with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, carboxy and their lower ester derivatives, and with 4-, 5-, 6-, to 7-membered heterocyclics. In particular, proline analogs in which the ring size of the proline residue varies from 5-membered to 4-, 6-, or 7-membered can be used. Cyclic groups may be saturated or unsaturated, and if unsaturated, may be aromatic or non-aromatic.

Cyclic groups may be saturated or unsaturated, and if unsaturated, may be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (eg, morpholino), oxazolyl, piperazinyl (eg, 1-piperazinyl), piperidyl (eg, 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (eg, 1-pyrrolidinyl), pyrrolinyl, pyrlyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (eg, thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. Where the group is substituted, the substituent may be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

One can also readily modify the peptides of the present invention by phosphorylation, and other methods for making peptide derivatives of the ingredients of the present invention described in Hruby et al. 42 therefore, the peptide components of this invention also serve as a base for the preparation of peptide mimetics with similar biological activities.

Peptide compounds of the invention, including peptidomimetics, may be covalently modified into one or more non-proteinized polymer variants, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkenes, in the manner established in U.S. Pat. Patent no. 4,640,835; LOUSE. Patent no. 4,496,689; LOUSE. Patent no. 4,301,144; LOUSE. Patent no. 4,670,417; LOUSE. Patent no. 4,791,192; or U.S. Patent no. 4,179,337, all of which are incorporated herein by reference in their entirety.

End modifications

Those skilled in the art recognize that a variety of techniques are available for constructing peptide mimetics with the same or similar desired biological activity as the corresponding peptide constituent, but with more favorable activity than the peptide in terms of solubility, stability, and susceptibility to hydrolysis and proteolysis. See, for example, Morgan and Gainor Ann. Tail. Honey. Chem. 24:243-252 (1989). The following are descriptions of methods for the preparation of peptide mimetics modified at the N-terminus of the amino group, the C-terminus of the carboxyl group, and/or by changing one or more amido bonds in the peptide to non-amido bonds. It is understood that two or more such modifications can be linked into a single peptide mimetic structure (eg, a modification at the C-terminus of the carboxyl group and the inclusion of a -CH 2 -carbamate bond between two amino acids in the peptide).

1. Modification of the N-terminus

Peptides are typically synthesized as the free acid but, as noted above, can be readily prepared as an amide or ester. One may also modify the amino and/or carboxy terminus of peptide compounds of the invention to produce other compounds of the invention. Modification of the amino terminus includes methylation (eg, -NHCH3 or -NH(CH3)2), acetylation, addition of a carbobenzoyl group, or blocking of the amino terminus with any blocking group consisting of a carboxylate functionally defined by RCOO-, where R is chosen from the group consisting of naphthyl, acridinyl, steroidyl, and similar groups. Modifications of the carboxy terminus include replacement of the free acid with a carboxamide group or formation of a cyclic lactam at the carboxy terminus to introduce structural constraints.

Modifications to the amino terminus are as recited above and include alylation, acetylation, addition of a carbobenzoyl group, formation of a succinimide group, etc. Specifically, the N-terminus of the amino group can then be reacted as follows:

(a) so as to form an amide group of the formula RC(O)NH- where R is as defined above by reaction with an acid halide [eg, RC(O)Cl] or an acid anhydride. Typically, the reaction can be performed by contracting approximately equimolar or excess amounts (e.g., about 5 equivalents) of the 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 created during the reaction. The reaction conditions are otherwise conventional (eg, room temperature for 30 minutes). Alkylation of the amino terminus to effect N-substitution of the lower alkyl, followed by reaction with an acid halide as described above will remove the N-alkyl group of the formula RC(O)NR-;

(b) by reacting with succinic anhydride to form a succinamide group. As before, an approximately equimolar amount or excess of succinic anhydride (e.g., about 5 equivalents) can be used and the amino group converted to succinimide by methods well known in the art including using an excess (e.g., ten equivalents) of a tertiary amine such as diisopropylethylamine in suitable inert solvent (eg, dichloromethane). See, for example, Wollenberg, et al., U.S. Patent no. 4,612,132 which is incorporated herein by reference in its entirety. It is understood that the succinide group can be substituted with, for example, C2-C6 alkyl or -SR substituents prepared in a conventional manner to replace the substituted succinamide at the N-terminus of the peptide. Such alkyl substituents are prepared by reacting a lower olefin (C2-C6) with maleic anhydride as described by Wollenberg, et al., supra and -SR substituents are prepared by reacting RSH with maleic anhydride where R is as defined above;

(c) by forming a benzyloxycarbonyl-NH- or substituted benzyloxycarbonyl-NH- group by reaction with an approximately equivalent amount or excess of CBZ-C1 (ie, benzyloxycarbonyl chloride) or substituted CBZ-C1 in an appropriate inert solvent (eg, dichloromethane) per the ability of the tertiary amine to scavenge the acid generated during the reaction;

(d) by forming a sulfonamide group by reaction with an equivalent amount or excess (eg, 5 equivalents) of R-S(O)2Cl in a suitable inert diluent (dichloromethane) to convert the terminal amine to a sulfonamide where R is as defined above. Preferably, the inert diluent contains an excess of a tertiary amine (eg, ten equivalents) such as diisopropylethylamine, to scavenge the acid generated during the reaction. The reaction conditions are otherwise conventional (eg, room temperature for 30 minutes);

(e) so as to form a carbamate group by reaction with an equivalent amount or excess (eg, 5 equivalents) of R-OC(O)Cl or R-OC(O)OC6H4-p-NO2 in a suitable inert diluent (eg, dichloromethane) to convert the terminal amine to the carbamate where R is as described above. Preferably, the inert diluent contains an excess (eg, about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to scavenge any acid generated during the reaction. The reaction conditions are otherwise conventional (eg, room temperature for 30 minutes); and

(f) such that it forms a urea group by reaction with an equivalent amount or excess (eg, 5 equivalents) of R-N=C=O in a suitable inert diluent (eg, dichloromethane) to convert the amine terminus to urea (ie, RNHC(O )NH-) group where R is as defined above. Preferably, the inert diluent contains an excess (eg, about 10 equivalents) of a tertiary amine, such as diisopropylethylamine. The reaction conditions are otherwise conventional (eg, room temperature for about 30 minutes).

2. Modifications of the C-terminus

In the preparation of peptide mimetics where the C-terminus carboxyl group is replaced by an ester (ie -C(O)OR where R is as defined above), resins used for the preparation of peptide acids are used, and the side chain protected peptide is attached to a base and with a suitable alcohol, eg methanol. The side chain protecting groups were then deprotected in the usual manner by treatment with hydrogen fluoride to give the desired ester.

In the preparation of peptide mimetics where the carboxyl group at the C-end is replaced by amide -C(O)NR3R4, benzhydrylamine resin is used as a coarse support for peptide synthesis. After completion of the synthesis, treatment with hydrogen fluoride to release the peptide from the support directly results in the free peptide amide (ie, the C-terminus is -C(O)NH2). Alternatively, the use of a chloromethylated resin during peptide synthesis coupled with reaction with ammonia to cleave the side chain protected peptide from the substrate leads to a free peptide amide and reaction with an alkylamine or dialkylamine leads to a side chain protected alkylamide or dialkylamide (ie the C-terminus is -C (O)NRR1 where R and R1 are as defined above). The side chain is then deprotected in the usual manner by treatment with hydrogen fluoride to give free amides, alkylamides, or dialkylamides.

In another alternative embodiment, the C-terminal carboxyl group or C-terminal ester can be induced to cyclize by internal displacement of the -OH or ester (-OR) carboxyl group with the N-terminal amino group to form a cyclic peptide. For example, after synthesis and cleavage to give the peptide acid, the free acid is converted to the activated ester using a suitable carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example, in methylene chloride (CH2Cl2), dimethyl formamide (DMF) mixtures . A cyclic peptide is then formed by internal displacement of the activated ester with the N-terminal amine. Internal cyclization as opposed to polymerization can be enhanced by using a very dilute solution. Such methods are well known in the art.

One can also cyclize the peptides of this invention, or incorporate a desamino or decarboxy residue at the ends of the peptide, so that there is no terminal amino or carboxyl group, to reduce susceptibility to proteases or to limit the conformation of the peptide. The C-terminal functional groups of the compounds of this invention include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and lower ester derivatives thereof, and pharmaceutically acceptable salts thereof.

B Modifications of the backbone

Other methods for making peptide derivatives of the compounds of this invention are described in Hruby et al. Biochem J. 268(2):249-262 (1990), incorporated herein by reference. Accordingly, the peptide compounds of the invention also serve as structural models for non-peptide compounds with similar biological activity. Those skilled in the art recognize that various techniques are available for constructing ingredients with the same or similar desired biological activity as the lead peptide ingredient but with more favorable activity than lead in terms of solubility, stability, and susceptibility to hydrolysis and proteolysis. See Morgan and Gainor Ann. Tail. Honey. Chem. 24:243-252 (1989), which is incorporated herein by reference. These techniques involve replacing the peptide backbone with a backbone composed of phosphonates, amidates, carabamates, sulfonamides, secondary amines, and N-methylamino acids.

Peptide mimetics where one or more peptidyl linkages [-C(O)NH-] are replaced by linkages such as a -CH2-carbamate linkage, a phosphonate linkage, a -CH2-sulfonamide linkage, a urea linkage, a secondary amine (-CH2NH-) linkage, and alkylated peptidyl bond [-C(O)NR6- where R6 is lower alkyl] are prepared during conventional peptide synthesis by merely substituting the corresponding protected amino acid analog for the amino acid reagent at the appropriate site during synthesis.

Suitable reagents include, for example, amino acid analogues where the carboxyl group of the amino acid is replaced with a moiety suitable for forming one of the above linkages. For example, if it is desired to replace the -C(O)NR- bond in a peptide with a -CH2-carbamate bond (-CH2OC(O)NR-), then the carboxyl (-COOH) group of a suitably protected amino acid is first reduced to -CH2OH group which is then converted by conventional methods into -OC(O)Cl functionality or para-nitrocarbonate -OC(O)-C6H4-p-NO2 functionality. Reaction of any such functional group with a free amine or an alkylated amine at the N-terminus of a partially fabricated peptide found on a solid support leads to the formation of a -CH2OC(O)NR- bond. For a more detailed description of the formation of such -CH2-carbamate bonds, see Cho et al. Science. 261:1303-1305 (1993).

Similarly, replacement of an amido bond in a peptide with a phosphonate bond can be accomplished in a manner established in U.S. Pat. Patent Application Nos. 07/943,805, 08/081,577, and 09/119,700, the disclosures of which are incorporated herein by reference in their entirety.

Replacement of an amido bond in a peptide with a -CH2-sulfonamide bond can be achieved by reducing the carboxyl (-COOH) group of a suitable protecting amino acid to a -CH2OH group and the hydroxyl group is then converted to an appropriate leaving group, such as a tosyl group by conventional methods. Reaction of tosylated derivatives with, for example, thioacetic acid followed by hydrolysis and oxidative chlorination will produce a -CH 2 -S(O) 2 Cl functional group that replaces the carboxyl group of an otherwise appropriately protected amino acid. The use of this suitable amino acid analogue in the synthesis of peptides leads to the inclusion of the -CH2S(O)2NR- bond, which replaces the amido bond in the peptide, thereby obtaining a peptide mimetic. For a more complete description of the conversion of the carboxyl group of an 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, p. 267-357, Marcel Dekker, Inc., New York (1983) which is incorporated herein by reference.

Replacement of the amido bond in the peptide with a urea bond can be accomplished in the manner set forth in U.S. Pat. Patent Application No. 08/147,805, and that application is incorporated herein by reference in its entirety.

Secondary amine bonds where the -CH2NH- bond replaces the amido bond in the peptide can be prepared using, for example, the corresponding protected dipeptide analog where the carbonyl bond of the amido bond is reduced to a CH2 group by conventional methods. For example, in the case of diglycine, the reduction of the amide to the amine will yield after deprotection H2NCH2CH2NHCH2COOH which is then used in the N-protected form, in the following coupling reaction. Preparation of such analogs by reduction of the carbonyl group of the amido compound in the dipeptide is well known in the art.

An appropriately protected amino acid analog is used in conventional peptide synthesis in the same way as the corresponding amino acid would be used. For example, typically about 3 equivalents of the protected amino acid analog are used in this reaction. An inert organic solvent such as methylene chloride or DMF is used, and when acid is generated as a reaction by-product, the reaction solvent will typically contain a tertiary amine in excess to remove the acid generated during the reaction. One particularly preferred tertiary amine is diisopropylethylamine, which is typically used in approximately tenfold excess. The reaction results in the incorporation into the peptide mimetic of an amino acid analog having a non-peptidyl linkage. Such substitution can be repeated as desired by replacing zero to all amido linkages in the peptide with non-amido linkages.

One can also cyclize the peptides of this invention, or incorporate a desamino or decarboxy residue at the ends of the peptide, so that it does not end with an amino or carboxyl group, to reduce susceptibility to proteases or to limit peptide formation. C-terminus functional groups of the compounds of this invention include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and lower ester derivatives thereof, and pharmaceutically acceptable salts thereof. Examples of cyclized compounds are shown in Tables 4, 5, 6, 8, and 9.

E. Disulfide bond formation

The compounds of this invention may exist in a cyclized form with an intramolecular disulfide bond between thiol groups of cysteine. Alternatively, an intermolecular disulfide bond can be produced between the thiol groups of cysteine to give a dimeric (or higher oligomeric) constituent. One or more cysteine residues may also be substituted with homocysteine. These intramolecular or intermolecular disulfide derivatives can be represented schematically as shown below:

[image]

where m and n are independently 1 or 2.

Other embodiments of this invention provide analogs of these disulfide derivatives in which one of the sulfurs is replaced by a CH2 group or some other isostere for sulfur. These analogs can be made via intramolecular or intermolecular displacement, using methods known in the art, as shown below:

[image]

where p is 1 or 2. One skilled in the art will readily appreciate that this shift can also be achieved using homologues of the α-amino-g-butyric derivatives shown above as homocysteine.

Alternatively, the amino-terminus of the peptide may be terminated with one alpha-substituted acetic acid, where the alpha substituent is a leaving group, such as one α-haloacetic acid, for example, α-chloroacetic acid, α-bromoacetic acid, or α- iodoacetic acid. The compounds of this invention can be cyclized or dimerized by displacement of the sulfur leaving group of a cysteine or homocysteine residue. See, eg, Barker et al. J. Med. Chem. 35:2040-2048 91992) and Or et al. J. Org. Chem. 56:3146-3149 91991), each of which is incorporated herein by reference. Examples of dimerized ingredients are given in Tables 7, 9 and 10.

Utility

The compounds of the invention are useful in vitro as a unique tool for understanding the biological role of TPO, including the evaluation of many factors thought to influence, and are affected by, the production of TPO and the receptor binding process. The disclosed compounds are also useful for the development of other compounds that bind to and activate the TPO-R, as the disclosed compounds provide important structure-activity relationship information that may facilitate such development.

The compounds are also useful as competitive binders in an attempt to test new TPO receptor agonists. In such experimental embodiments, the ingredients of the invention may be used without modification or may be modified in various ways; for example, by labeling, by covalently or non-covalently attaching a moiety that directly or indirectly produces a detectable signal. In any of these experiments, the materials can be labeled either directly or indirectly. Direct labeling options include label groups such as: radiolabels such as 125I, enzymes (US Patent 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Patent No. 3,940,475) capable of monitoring changes in fluorescence intensity, wavelength shift, or fluorescence polarization. Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin attached to one of the above-labeled moieties. The ingredients may also include spacers or bonders in cases where the ingredients need to adhere to a rigid support.

Furthermore, based on their ability to bind to the TPO receptor, the peptides of the present invention can be used as reagents for the detection of TPO receptors on living cells, fixed cells, in biological fluids, in tissue homogenates, in purified natural biological materials, etc. for example, by labeling such peptides, one can identify cells that have TPO-R on their surface. Furthermore, based on their ability to bind the TPO receptor, the peptides of the present invention can be used in in situ staining, FACS (Fluorescence-activated cell sorting), Western blotting, ELISA, etc. Furthermore, based on their ability to bind the TPO receptor , the peptides of the present invention can be used in receptor purification, or in the purification of cells displaying TPO receptors on the cell surface (or within permeabilized cells).

The compounds of this invention may also be used as commercial reagents for various medical research and diagnostic purposes. Such purposes include, but are not limited to: (1) use as a calibration standard to quantify the activity of candidate TPO agonists in various functional assays; (2) use to sustain the proliferation and growth of TPO-dependent cell lines; (3) use in structural analyzes of the TPO-receptor through co-crystallization; (4) use to investigate the activation mechanism of TPO signal transduction/reception; and (5) other research and diagnostic applications where the TPO receptor is preferably activated or such activation is conventionally calibrated against a known amount of TPO agonist, and the like.

The compositions of the present invention can be used for the in vitro expansion of megamyocytes and their committed progenitors, either in conjunction with additional cytokinesis or alone. See, eg, DiGiusto et al. PCT Publication No. 95/05843, which is incorporated herein by reference. Chemotherapy and radiation therapies cause thrombocytopenia by killing and rapidly dividing the senescent population of megakaryocytes. However, these therapeutic treatments may also reduce the number and durability of immature, less miotically active megakaryocytic precursor cells. Thus, amelioration of thrombocytopenia by TPO or an ingredient of the present invention can be enhanced by infusing a post-chemotherapy or radiation therapy patient with his or her own cell population enriched in megakaryocytes and immature precursors by in vitro culture.

The compounds of the present invention can also be administered to warm-blooded animals, including humans, to activate TPO-R in vivo. Accordingly, the present invention encompasses methods for the therapeutic treatment of TPO-related disorders that consist in administering an ingredient of the invention in amounts sufficient to mimic the effect of TPO on the TPO-R in vivo. For example, the peptides and compounds of the invention can be administered to treat various hematological disorders, including but not limited to platelet disorders and thrombocytopenia, particularly when associated with bone marrow transfusions, radiation therapy, and chemotherapy.

In some embodiments of the invention, TPO antagonists are first administered to a patient undergoing chemotherapy or radiation therapy, followed by TPO agonists of the present invention.

The activity of the compounds of this invention can be evaluated either in vitro or in vivo in one of the numerous models described in McDonald Am. J. of Pediatric Hematology/Oncology 14:8-21 (1992), which is incorporated herein by reference.

According to one embodiment, the compounds of this invention are useful for treating thrombocytopenia associated with bone marrow transfusions, radiation therapy, or chemotherapy. The ingredients will typically be administered prophylactically before or after chemotherapy, radiation therapy or bone marrow transplantation.

Accordingly, the present invention also provides pharmaceutical compositions containing, as an active ingredient, at least one of the peptides or peptide mimetics of the invention together with pharmaceutical carriers or diluents. The compounds of this invention can be administered by oral, pulmonary, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (by forming a fine powder), transdermal, nasal, vaginal, rectal, or sublingual routes of administration and can be formulated in dosage forms suitable for each of the routes of administration. See, eg, Bernstein et al. PCT Patent Publication No. WO 94/17784, and Pitt et al. European Patent Application 613,683, each of which is incorporated herein by reference.

Solid dosage forms for oral administration of the drug include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms may also contain, as in normal practice, additional substances other than the inert diluent, eg, lubricating substances such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also contain buffering substances. Tablets and pills can be additionally prepared with enteric coatings.

Liquid dosage forms for oral administration of the drug include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, with elixirs containing inert diluents commonly used in the art, such as water. In addition to such inert diluents, the ingredients may also include auxiliaries, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, as well as fragrance agents.

Formulations of the present invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or agents 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 aids such as protective, moisturizing, emulsifying and dispersing substances. They can be sterilized, for example, by filtering through a bacteria-retaining filter, adding a sterilizing agent to the compositions, irradiating the compositions, or heating the compositions. They may also be made using sterile water, or some other sterile injectable medium, immediately prior to use.

Compositions for rectal or vaginal administration of the drug are preferably suppositories which can contain, in addition to the active substance, an excipient such as cocoa butter or wax for suppositories. Compositions for nasal or sublingual drug administration are also prepared with standard excipients well known in the art.

Compositions containing the ingredients can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, the compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to treat or at least partially stop the symptoms of the disease and its complications. An amount adequate to achieve this is defined as a "therapeutically effective dose". The amount effective for this use will depend on the severity of the disease as well as the weight and general condition of the patient.

The compositions of the invention can also be microencapsulated, for example by the method of Tice and Bibi (in Treatise on Controlled Drug Delivery, ed. A. Kydonicus, Marcel dekker, N.Y. (1992)., pp. 315-339).

In prophylactic applications, compositions containing the ingredients of the invention are administered to a patient who is susceptible to or at risk for a particular disease. Such an amount is defined as a "prophylactically effective dose". In this use, the precise amount again depends on the patient's health and weight.

The amounts of TPO agonists necessary for effective therapy will depend on many different factors, including the route of administration, the desired condition, the patient's psychological state, and other medications the patient is taking. Therefore, treatment doses should be titrated to optimize safety and efficacy. Typically, doses used in vitro can provide useful guidance on the amount useful for in situ administration of these reagents. Animal testing of effective doses for the treatment of certain diseases will provide further predictive indications for human dosing. Various studies are described, eg, in Gilman et al. (eds), Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press (1990); and Remington's Pharmaceutical Sciences, 7th ed., Marc Publishing Co., Easton, Penn. (1985); and each is incorporated herein by reference.

The peptides and peptide mimetics of the present invention are effective in treating TPO-mediated conditions when taken in a dosage range of about 0.001 mg to about 10 mg/kg of body weight per day. The specific dose used is regulated by the particular condition being treated, through the administration of the drug, as well as by the judgment of the doctor in charge, which depends on factors such as the severity of the condition, the age and general condition of the patient, and the like.

Although only preferred embodiments of the invention have been specifically described above, it should be noted that modifications and variations of the invention are possible without departing from the spirit and intended scope of the invention.

Example 1

Synthesis of rigid peptides

The various peptides of this invention were synthesized using the Merrifield solid phase synthesis technique (see Steward and Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical, Rockford, IL (1984) and Merrifield J. Am. Chem. Soc. 85:2149 ( 1936)) on a Milligen/Biosearch 9600 automated instrument or on Applied Biosystems Inc. Model 431A peptide synthesizer. Peptides were harvested using standard Applied Biosystems Inc. protocols. system Software version 1.01. Each coupling was done for one to two hours with BOP (benzotriazolyl N-octrisdimethylaminophosphorus hexafluorophosphate) and HOBt (1-hydroxybenzotriazole).

The mass used is HMP resin or PAL (Milligen/Biosearch), which is a cross-linked polystyrene mass with 5 (4'-Fmok-aminomethyl-3,5'-dimethyloxyphenoxy) valeric acid as a bond. The use of PAL mass results obtained in the terminal carboxyl of the amide functions based on cleavage of the peptide from the mass. Based on the cleavage principle, the HMP mass produces carbonic acid at the C-terminus of the final product. Most of the reagents, stocks, and protected amino acids (free or bulk) were obtained from Millipor or Applied Biosystems Inc.

The Fmoc group was used for amino capping during the coupling procedure. Primary amino protection on amino acids is achieved with Fmoc and protection of end groups in the chain where t-butyl is for serine, tyrosine, asparagine, glutamic acid, and threonine; tritol for glutamine; Pmc (2,2,5,7,8-pentamethylchromium sulfonate) for arginine; N-t-butyloxycarbonyl for tryptophan; N-trityl for histidine and glutamine; and S-trityl for cysteine.

Removal of the peptide from the mass and successive deprotection of the end groups in the chain was achieved by treatment with reagent K or its modifications. Another possibility, in the synthesis of these peptides, is with the end amidated carbons, fully connected peptides are cleaved with a mixture of 90% trifluoroacetic acid, 5% ethanedithiol, and 5% water, initially at 4°C, and gradually increasing to room temperature. Deprotected peptides were precipitated with diethyl ether. In all cases, purification was prepared by reversed-phase, high-performance liquid chromatography on C18-bonded silica gel columns with an acetonitrile/water gradient in 0.1% trifluoroacetic acid. Homogenized peptides were characterized by Fast Atom Bombardment mass spectrometry or electrospray mass spectrophotometry and amino acid analyzes when possible.

Example 2

Bioprobe

Peptide bioactivity 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 (WEHT - 3 conditioned medium), these cells are dependent on TPO for proliferation. The underlying, untransfected cell line does not respond to human TPO, but remains dependent on IL-3. Biological tests were performed on the circumference of upper cells using synthetic derivatives of peptides from the library fine mesh. Cells were grown in confluent RPMI-10 medium containing 10% WEHI-3 conditioned medium, washed twice in PRS, resuspended in medium lacking WEHI-3 conditioned medium, and added to peptide solutions or TPO at 2 x 104 cells/ vel. Cells were incubated for 48 hours at 37°C in a humidified 5% CO2 atmosphere and metabolic activity was obtained by reducing MTT to formazan, with absorbance at 570 nM measured on an ELISA plate reader. The tested peptides stimulate the 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 basal cell line.

Example 3

Binding properties

The binding properties of chemically synthesized peptides to TPO-R were measured in competition form binding. Wells of microtiter plates were covered with PBS/1% and 50 ng of biotinylated anti-receptor immobilizing antibodies (AB179). The wells were then treated with a 1:10 solution of soluble TPO-R gain. Different concentrations of peptides or peptide mimetics were mixed with a constant amount of the truncated form of TPO consisting of residues 1-156 fused to the C-terminus of maltose-binding protein (C). Mixtures of (MBP-TPO156) peptides were added to TPO-R coated wells, incubated for 2 hours at 4°C and washed with PBS. The amount of MBP-TPO156 peptide bound at equilibrium was measured by addition of rabbit anti-serum directed against MBP, followed by alkaline phosphatase conjugated goat anti-rabbit IgG. The amount of alkaline phosphatase in each well was determined using standard methods.

This experiment was carried out in the area of peptide concentration and the results are shown in the graph so that the y axis represents the amount of bound MBP-TPO156 and the x axis represents the concentration of the peptide or peptide mimetic. The amount at which the peptides or peptide mimetic will reduce to 50% (IC50) the amount of MBP-TPO156 bound to the immobilized TPO-R can be determined. The dissociation constant (Kd) for the peptides must be similar to the IC50 measured using the assay conditions described earlier.

Example 4

"Peptides on plasmids"

The pJS142 vector was used for free construction and is shown in Figure 4. Three oligonucleotide sequences are required for free construction: ON-829 (5' ACC ACC TCC GG); ON-830 (5' TTA CTT AGT TA) and the specific free oligonucleotide of interest (5' GA GGT GGT {NNK}n TAA CTA AGT AAA GC), where {NNK}n represents a random region of desired length and sequence. Oligonucleotides can be 5' chemically phosphorylated during synthesis or after purification with polynucleotide kinase. They are then heated at a 1:1:1 molar ratio and linked into a vector.

The strain of E. coli most used for pan has the genotype: Δ (srl-recA) endA1 nupG Ion-11 sulA1 hadR17 Δ (ompT-fepC)266 ΔclpA319; kan ΔlacI lac ZU118 which is prepared from the E. coli family from the E. coli Genetic Stock Center at Yale University (E. coli b/r, stock center designation CGSC:6573) with genotype lon-11 sulA1. The above family of E. coli was prepared for use in electroporation as described in Dower et al. Nucleic Acids Res. 16:6127 (1988), except that 10% glycerol was used for all stages of washing. Cells were tested for efficiency using 1 pg of Bluescript plasmid (Stratagene). These cells are used to grow the original freedom and to expand the enriched population after each round of panic 4.

Peptides on the plasmids were released from the panning cells by mild enzymatic digestion of the cell wall using lysozyme. After creating a tablet from the broken cells, the solid lysate is used directly on most receptors. If any additional purification of the plasmid complex is required, column gel filtration is required to remove low molecular weight contaminants in the solid lysate.

Panning is carried out in a buffer (HEKL) with a lower salt concentration than usual buffers. Panning is performed in microtiter wells with the receptor immobilized on non-blocking monoclonal antibodies (Mab) or panning on layers or on a column. More specifically, in the first panning, 24 wells are used, each captured with a receptor. In the second round, six wells coated with receptors (PAN pattern) and 6 wells without receptors (NC pattern) are typically used. Comparison of the number of plasmids in the two samples gives an indication of which receptors are specific clones enriched by panning. "Enrichment is defined as the ratio of PAN transformed to those obtained from NC samples. Tenfold enrichment is usually an indication that specific receptor clones are present.

In the last panning, it is useful to reduce the input of lysate to the wells to reduce the binding of non-specific plasmid complexes to the substrate. In the second step, 100 μl of lysate is usually used per well. In the third step, 100 μl of lysate per well diluted with 1/10 HEKL/BSA is used. In further stages of panning, an input of plasmid transforming units at least 1000-fold above the specified residual divergence is typically used.

The binding properties of peptides decoded by individual clusters are typically examined after 3, 4, or 5 steps of panning, depending on the number of enrichments observed. Typically, ELISAs are used that detect specific binding of LacI-peptides by binding proteins. LacI is usually a tetramer and the smallest functional DNA-binding species are dimers. Cooperative splicing allows the detection of splicing events of low intrinsic affinity. The sensitivity of this experiment is an advantage in the easy determination of initial hits of low affinity, but the disadvantage is that the signal in ELISA is not comparable to the intrinsic affinity of the peptide. Synthesis of the peptide into maltose binding protein (MBP) as described below allows testing in an ELISA format where signal strength is better correlated with affinity. See Figure SA-B.

DNA of clones of interest can be prepared in double-stranded form using the minipred procedure. The coding parts of individual clones or populations of clones of interest can be transferred into vectors, which synthesize sequences within the decoding framework of MBP, a protein that usually exists as a monomer in solution. Cloning the librates into pJS142 creates BspEI restriction groups near the beginning of the librate random coding region. Digestion with BspEI and ScaI allows purification of 900 hp DNA fragments that can be subcloned into one or two vectors, pELM3 (cytoplasmic) or pELM15 (periplasmic), which are simple modifications of the pMALc2 and pMALp2 vectors, commercially available from New England Biolabs. See Figure 5A-B. Digestion of pELM3 and pELM15 with AgeI and ScaI allows efficient cloning of the BspEI-ScaI fragment from the pJSI42 librate. BspEi and AgeI ends are compatible for ligation. Next, it is logical to properly ligation ScaI groups to recreate functional bla (Amp resistant) genes, to reduce the level of clones in the background from unwanted ligations. Expression of tac promoter-driven MBP-peptide synthesis can be induced with IPTG.

Lysates for LacI or MBP ELISAs were prepared from individual clones by lysing cells using lysozyme and removing insoluble cell debris by centrifugation. Lysates were added to wells containing immobilized receptors and to control wells without receptors. Binding of LacI or MBP peptide syntheses was detected by incubation with rabbit polyclonal antiserum directed against LacI or MBP followed by incubation with alkaline phosphatase-labeled goat anti-rabbit second antibodies. Bound alkaline phosphatases were detected with p-nitrophenyl phosphate chromogenic substrates.

Example 5

"Headpierce dimer" system

The LacI peptide-on-plasmid technique uses a DNA binding protein called the "Headpiece dimer". The DNA-bound E. coli lac repressor is represented by approximately 60 amino acid "headpiece" domains. Dimer headpiece domains that are bound to the light operator are usually formed by joining the much larger approximately 300 amino acid C-terminal domains. The "headpiece dimer" system utilizes headpiece dimer molecules that contain two headpieces connected via short pentide linkers. That protein binds DNA with sufficient stability to allow association of the epitope peptides displayed at the C-terminus of the headpiece dimer with the plasmid encoding these peptides.

Random peptides combine with the C-terminus of headpiece dimers, which are ligated to plasmids that decode them to yield a peptide-headpiece dimer-plasmid complex that can be screened by panning. The headpiece dimer peptide-on-plasmids system allows greater selectivity for high-affinity ligands than the LacI system. Thus, the headpiece dimer system is useful for generating mutagenesis libraries based on initial low-affinity hits, and selecting high-affinity variants of initial sequences.

Libraries were constructed as with peptides on plasmids using the headpiece dimer vector pCMG14 (see Figure 6A-C). The presence of the lac operator is not required for plasmid binding by the headpiece dimer protein. Libraries were introduced into the bacterial family comprising E. coli (lon-11 sulA1 hsdR17 (compT-fepC) ΔclpA319:: kan ΔlacI lac ZU118 Δ(srl-recA) 306::Tn10, and amplified under basal conditions (A) promoter inducer. Headpiece dimer panning of libraries was performed using a procedure similar to that used for LacI libraries, except that HEK buffer was used instead of HEKL buffer and plasmid elution from the wells was performed with aqueous phenol instead of IPTG.Sequences from headpiece dimer panning were often characterized after transfer to MBP. vector so that they can be tested in prone sensitive MBP ELISAs and also that populations of clones can be framed by elevator colonies with labeled receptor.

Example 6

In this example, the ring joints are exposed in three trials. First, IC50 shells were obtained as described above. Subsequently, the MTT proliferated cell medium as described above was performed to calculate EC50 values. Finally, a microphysiometer (Molecular Device Corp.) was tested. Basically, in this test, the rate of acidification of the extracellular medium in response to TPO receptor stimulation was determined with the help of the test compounds. The range for EC50 is symbolically marked as for IC50 described above. The results are shown in Table 4.

[image]

[image]

[image]

Example 7

In this example, amino acid substitutions at positions D, E, I, S, or F in the ring compound

[image]

were tested for EC50 and IC50 values as described above. Microphysiometric results are given in parentheses. The results are summarized in Table 5 below.

[image]

[image]

[image]

Example 8

In this example, the amino acid substituents in the compound

[image]

are rated at sites D, S, or F as indicated in Table 6 below. EC50 and IC50 values were calculated as described above. Microphysiometric results are in parentheses.

[image]

Example 9

In this example, the EC50 and IC50 values were calculated as described above for the dimer compounds listed in Table 7 below. Ring monomer

[image]

is included for comparison.

The compounds of Table 8 were inactivated at the maximum concentration tested at 10 μm.

In Table 9, the EC50 and IC50 values determined as described above for the crystallized and dimerized variations of I E G P T L R Q W L A A R A are compared. In Table 10, dimer abbreviations

[image]

were compared. EC50 and IC50 values were calculated as described above. Microphysiometric results are given in parentheses.

[image]

[image]

[image]

[image]

[image]

[image]

Example 10

In this example, different substituents are included at the G, P, and W positions in the ring compound

[image]

Table 11 provides examples of substituted compounds exhibiting TPO activity. Abbreviated substituents are tabulated as follows:

[image]

[image]

[image]

[image]

Example 11

To test the ability of the mouse as a favorable experimental material, several in vitro experiments were made, designed to measure the activity of the test substances on mouse receptors. First, brain cells, obtained from 8- to 9-week-old mice from the femurs of one Balb/C mouse, were incubated for 7 days in liquid culture with rhuTPO or various concentrations of test peptides. At the end of the incubation period, cultures were concentrated with Cytosine, stained for acetylcholinesterase (ADhE, diagnosis of mouse megakaryocytes), and counted by microscopic analysis. One (1) nMrhuTPO produces an increase in the outgrowth of large (>40 μm) non-adherent cells stained for AChE. These cells appear to be mature megakaryocytes. From an initial seed of 10 total brain cells/ml (in 50 ml culture) a certain 1 to 2 x 106 megakaryocytes were developed. This response to TPO is labeled "maximal". Control cultures containing no growth factors produced very few AChE-positive cells. Several peptide compounds were tested at higher concentrations in this experiment and the results are summarized in Table 12. Peptides A at 10 μM produced a maximal response in the mouse brain. This finding is the first evidence that this peptide family is active on the rat receptor. In another experiment, brain cells were harvested and counted in semi-solid medium (methylcellulose) containing no factor, 1 nM rhuTPO, or 10 μM Peptide A. After seven days in culture, colonies of large cells (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. Both TPO and test peptides produced more actual colonies of both sizes than the negative control cultures. This indicates that the peptides mimic TPO in their ability to stimulate the expansion of Mk progenitor population cells.

To obtain more quantitative comparisons of the activity of the tested compounds on rat and human receptors, the muTPo receptor was cloned and transfected into BaF3 cells. A TPO-dependent population of cells was isolated.

[image]

*Streptavidin complexed into a biostimulated peptide - concentration putative 1 : 4 complex.

** Compared to recombinant human TPO

** 25 - 30% of ACE stained cells on cytospin

Without culture factor - approx. 5% AChE stained cells (less cellularity).

[image]

The disclosures in this application from all articles and references, including patent documents are incorporated herein by reference in their entirety for all purposes.

Claims (31)

1. An ingredient that binds to the thrombopoietin receptor, characterized by the fact that said ingredient has: (1) a molecular weight of less than about 8000 daltons, and (2) binding affinity to the thrombopoietin receptor as expressed by an IC50 of no more than about 100 μm, 2. The ingredient of Claim 1, characterized in that said ingredient is a peptide, i having zero to all of the -C(O)NR- bonds of the peptide replaced with bonds selected from the group consisting of -CH2-OC(O)NR- bonds; phosphonate bonds; -CH2-S(O)2NR- bonds; -CH2NR- bonds; and -C(O)NR6 bonds; and -NHC(O)NH- bond; where R is hydrogen or lower alkyl and R6 is lower alkyl, further where the N-terminus of said peptide or peptide mimetic is selected from the group consisting of -NRR1 group; -NRC(O)R groups; -NRC(O)OR groups; -NRS(O)2R groups; -NHC(O)NHR groups; succinimide groups; benzyloxycarbonyl-NH-groups; and a benzyloxycarbonyl-NH- group having from 1 to 3 substituents on the phenyl ring which is selected from the group consisting of lower alkyl, lower alkoxy, chloro, and bromo where R and R1 are independently selected from the group consisting of hydrogen and lower alkyl, and further wherein the C-terminus of said peptide or peptide mimetic has the formula -C(O)R2 wherein R2 is selected from the group consisting of hydroxy, lower alkoxy, and NR3R4 wherein R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl and where the nitrogen atom of the NR3R4 group can optionally be the amine group of the N-terminus of the peptide so as to form a cyclic peptide, and their physiologically acceptable salts. 3. Pharmaceutical composition, characterized in that it consists of the ingredient from Claim 1 in combination with a pharmaceutically acceptable carrier. 4. A method for treating a patient suffering from a disorder that is sensitive to treatment with a thrombopoietic agonist, characterized in that it consists of administering to the patient a therapeutically effective dose or amount of the composition of Claim 1. 5. The method of Claim 4, characterized in that the ingredient administered to the patient is a peptide, i wherein zero to all -C(O)NH- bonds of the peptide are replaced by bonds selected from the group consisting of -CH2OC(O)NR- bonds; phosphonate bonds; -CH2S(O)2NR- bonds; -CH2NR- bonds; and -C(O)NR6- bond; and -NHC(O)NH- bond where R is hydrogen or lower alkyl and R6 is lower alkyl, further where the N-terminus of said peptide or peptide mimetic is selected from the group consisting of -NRR1 group; -NRC(O)R groups; -NRC(O)OR groups; -NRS(O)2R groups; -NHC(O)NHR groups; succinimide groups; benzyloxycarbonyl-NH-groups; and benzyloxycarbonyl-NH- groups having from 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl. lower alkoxy, chloro, and bromo, wherein R and R1 are independently selected from the group consisting of hydrogen and lower alkyl, and even further, where the C-terminus of said peptide or peptide mimetic has the formula -C(O)R2 where R2 is selected from the group consisting of hydroxy, lower alkoxy, and -NR3R4 where R3 and R4 are independently selected from the group consisting of consist of hydrogen and lower alkyl and where the nitrogen atom from the -NR3R4 group can optionally be the amine group of the N-terminus of the peptide so as to form a cyclic peptide, and their physiologically acceptable salts. 6. The ingredient of Claim 2, characterized in that said ingredient consists of a sequence of amino acids: X1 X2 X3 X4 X5 X6 X7 where X1 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; X4 is any of the 20 genetically encoded L-amino acids; X5 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; and X7 is C, G, I, K, L, M, N, R or V. 7. The composition of claim 6, characterized in that said amino acid sequence is cyclized. 8. The composition of claim 6, characterized in that said amino acid sequence is dimerized. 9. The ingredient of claim 6, characterized in that the ingredient contains an amino acid sequence C X2 X3 X4X5 X6 X7 where X 2 is K, L, N, Q, R, S, T or V; X 3 is C, F, I, L, M, R, S or V; X4 is any of the 20 genetically encoded L-amino acids; X5 is A, D, E, G, S, V or Y; X 6 is C, F, G, L, M, S, V, W or Y; and X7 is C, G, I, K, L, M, N, R or V. 10. The compound of Claim 8, wherein X4 is A, E, G, H, K, L, M, P Q R, S, T, or W. 11. The compound of Claim 10, characterized in that X2 is S or T; X 3 is L or R; X4 is R; X5 is D, E, or G; X6 is F, L, or W; and X7 is I, K, L, R or V. 12. The ingredient of Claim 9, characterized in that said ingredient contains the sequence of amino acids: X8 C X2 X3 X4 X5 X6 X7 where X 2 is F, K, L, N, Q, R, S, T or V; X 3 is C, F, L, L, M, R, S, V or W; X4 is any of the 20 genetically encoded L-amino acids; X5 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; and X8 is any of the 20 genetically encoded amino acids. 13. The compound of Claim 12, wherein X8 is G, S, Y or R. 14. The ingredient from Claim 12, characterized in that said ingredient contains the sequence of amino acids: G G C T L R E W L H G G F C G G. 15. The ingredient from Claim 6, characterized in that said ingredient contains the sequence of amino acids: X8 G X1 X2 X3 X4X5 W X7 where 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; X4 is A, K, L, M, R, S, V, or T; X5 is A, E, G, K, M, Q, R, S, or T; X 7 is C, I, K, L, M, or V; and X8 is any of the 20 genetically encoded L-amino acids. 16. The composition of claim 15, characterized in that X1 is P; X2 is T; X3 is L; X4 is R; X5 is E or Q; and X7 is I or L. 17. The ingredient from Claim 16, characterized in that said ingredient consists of the sequence of amino acids: X9 X8 G X1 X2 X3 X4X5 W X7 where X8 is A, C, D, E, K, L, Q, S, T, or V; and X9 is A, C, E, G, I, L, M, P, R, Q, S, T, or V. 18. The compound of Claim 17, characterized in that X8 is D, E, or K; and X9 is A or I. 19. The ingredient from Claim 18, characterized in that said ingredient is selected from the group consisting of 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; I I E G P T L R Q W L A A R A. 20. The method of claim 4, characterized in that said ingredient administered to the patient contains the sequence of amino acids: C X2 X3 X4 X5 X6 X7 where X 2 is K, L, N, Q, R, S, T or V; X 3 is C, F, I, L, M, R, S or V; X4 is any of the 20 genetically encoded L-amino acids; X5 is A, D, E, G, S, V or Y; X 6 is C, F, G, L, M, S, V, W or Y; and X7 is C, G, I, K, L, M, N, R or V. 21. The method of Claim 20, wherein X4 is A, E, G, H, K, L, M, P Q R, S T or W. 22. The method of claim 21, characterized in that X2 is S or T; X 3 is L or R; X4 is R; X5 is D, E, or G; X6 is F, L, or W; and X7 is I, K, L, R or V. 23. The method of claim 22, characterized in that said ingredient administered to the patient consists of the sequence of amino acids: G G C T L R E W L H G G F C G G. 24. The method of claim 4, characterized in that the disorder sensitive to treatment with a thrombopoietin agonist is selected from the group consisting of: hematological disorders and thrombocytopenia as a result of chemotherapy, radiation therapy, or bone marrow transfusion. 25. The method of claim 4, characterized in that said ingredient administered to the patient consists of the sequence of amino acids: X8 G X1 X2 X3 X4 X5 W X7 where 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; X4 is A, K, L, M, R, S, V, or T; X5 is A, E, G, K, M, Q, R, S, or T; X 7 is C, I, K, L, M, or V; and the X8 residue is any of the 20 genetically encoded L-amino acids. 26. The method of claim 25, characterized in that X1 is P; X2 is T; X3 is L; X4 is R; X5 is E or Q; and X7 is I or L. 27. The method from Claim 26, characterized in that the said ingredient consists of the sequence of amino acids: X9 X8 G X1 X2 X3 X4 X5 W X7 where X8 is A, C, D, E, K, L, Q, S, T, or V; and X9 is A, C, E, G, I, L, M, P, R, Q, S, T, or V. 28. The method of Claim 27, wherein X8 is D, E, or K; and X9 is A or L 29. The method of Claim 28, characterized in that the ingredient administered to the patient is selected from the group consisting of 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; I I E G P T L R Q W L A A R A. 30. An ingredient that binds to the thrombopoietin receptor, characterized in that said ingredient is selected from the group consisting of [image] [image] 31. A method for treating a patient suffering from a disorder sensitive to treatment with a thrombopoietin agonist, comprising administering to the patient an ingredient selected from the group consisting of [image]