Peptides and compounds capable to bond to a receptor of thrombopoetin

The present invention relates to a compound that binds to the thrombopoietin receptor, a pharmaceutical composition and the use of a compound that binds to the thrombopoietin receptor.

The present invention provides peptides and compounds that bind and activate the thrombopoietin receptor (c-mpl or TPO-R), or otherwise act as TPO agonists. The invention finds application in the field of biochemistry and medical chemistry, and in particular provides TPO agonists for use in the treatment of human diseases.

Megakaryocytes are cells derived from bone marrow that are responsible for the production of platelets in the circulation. Although in most species they constitute less than Q, 25% of bone marrow cells, their volume is> 1Q times that of typical bone marrow cells. See Kuter et al. Natl. Acad. Sci. USA 91: 111Q4-111Q8 (1994). Megakaryocytes undergo a process known as endomitosis, as a result of which their nuclei divide, but there is no cell division and therefore the number of polyploid cells increases. In response to a reduced platelet count, the rate of endomitosis increases, megakaryocytes with higher ploidy are formed, and the number of megakaryocytes may increase 3-fold. See Harker J. Clin. Invest. 47-458-465 (1968). In turn, in response to elevated platelets, the rate of endomitosis is reduced, megakaryocytes with lower ploidality are formed, and the number of megakaryocytes may decrease by 5Q%.

The exact physiological feedback mechanism by which the mass of circulating platelets regulates the rate of endomitosis and the number of bone marrow megakaryocytes is unknown. It is currently believed that thrombopoietic factor in the circulation involved in this feedback loop is thrombopoietin (TPO). More specifically, TPO has been shown to be the main humoral regulator in situations involving thrombocytopenia. See, e.g., Mietcalf Nature 369: 519-520. Several studies have shown that TPO increases platelet counts, increases platelet size, and increases the isotope incorporation in the platelets of recipient animals. In particular, TPO is thought to affect megakaryocytogenesis in several ways: (1) it increases the size and number of megakaryocytes; (2) causes an increase in DNA content, in the form of polyploidy, in megakaryocytes; (3) increases megakaryocyte endomitosis; (4) causes increased maturation of megakaryocytes; and (5) causes an increase in the percentage of precursor cells in the form of small acetylcholinesterase positive cells in the bone marrow.

Because platelets (thrombocytes) are necessary for blood clotting, and when their numbers are very low, the patient is at serious risk of dying from a catastrophic hemorrhage, TPO has the potential to be useful in both the diagnosis and treatment of various hematological disorders, for example diseases mainly caused by platelet defects. Clinical trials conducted with TPO indicated that TPO could be safely administered to patients. In addition, recent studies have laid the foundation for planning the effectiveness of TPO therapy in the treatment of thrombocytopenia, and especially thrombocytopenia arising from chemotherapy, radiotherapy or bone marrow transplantation as a treatment for cancer or lymphoma. See, e.g., McDonald (1992) Am. J. Ped. Hematology / Oncology 14: 8-21 (1992).

The gene encoding TPO was cloned and characterized. See Kuter et al. 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 of at least two forms, with apparent molecular weights of 25 kDa and 31 kDa and a common N-terminal amino acid sequence. See Bartley et al. Cell 77: 1117-1124 (1994). Thrombopoietin has been shown to have two distinct regions separated by a potential ArgArg cleavage site. The amino-terminal region is highly conserved in man and mouse and has some homology to erythropoietin and interferon a and interferon b. The carboxy-terminal region exhibits wide species diversity.

DNA sequences and sequences of encoded human TPO-R peptide (also known as c-mpl) are described. See Vigon et al. Natl. Acad, Sci. USA 89: 5640-5644 (1992). TPO-R is a member of the hematopoietin growth factor receptor family, characterized by a common structural pattern of the extracellular domain, including four conserved C residues at the N-terminal part 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 hemopoiesis includes observations that its expression is limited to the spleen, bone marrow or fetal liver in mice (see Souyri et al. Cell 63: 1137-1147 (1990)) and to megakaryocytes, platelets and CD34 + cells in humans (see Methia et al. Blood 82: 1395-1401 (1993)). In addition, exposure of CD34 + cells to synthetic mpl 20 RNA antisense oligonucleotides significantly inhibits megakaryocyte colony formation without affecting erythroid or myeloid colony formation. Some researchers postulate that the receptor acts as a homodimer, as is the case with G-CSF and erythropoietin receptors.

The availability of cloned TPO-R genes facilitates the agonist studies of this important receptor. The availability of the recombinant receptor protein enables the study of receptor-ligand interactions using various random and semi-random systems for the production of various peptides.

These systems include the "peptides on plasmids" system described in US Patent Nos. 5, 270 170 and 5,338,665; the "peptides on phage" system described in US Patent Application No. 07/718 577, filed June 20, 1991, in US Patent Application No. 07/541 108, filed June 20, 1990 and in Cwirla et al., Natl. Acad. Sci. USA 87: 6378-6382 (1990); the "polysomes" system described in United States Patent Application No. 08/300 262, filed September 2, 1994, which is a continuation in part of the application based on United States Patent Application No. 08/144 775 filed October 29, 1993 . and in PCT International Patent No. 95/11992; the "encoded synthetic library" system described in U.S. Patent Application Nos. 08/146 886, filed November 12, 1993, 07/946 239, filed September 16, 1992. and 07/762 522, filed September 18, 1991, and the "very large scale synthesis of immobilized polymer" system described in U.S. Patent No. 5,143,854; PCT Patent Publication No. 90/15070, published December 13, 1990; U.S. Patent Application No. 07/624 120, filed December 6, 1990; Fodor et al.

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Science 251: 767-773 (2/1991); Dower and Fodor Ann. Rep. Med. Chem. 26: 271-180 (1991) and U.S. Patent Application No. 07/805 727, filed December 6, 1991; each of the above patent applications and publications are incorporated herein by reference.

The slow recovery of platelet levels in patients suffering from thrombocytopenia is a serious problem and prompts the diligent search for a blood growth factor agonist capable of accelerating platelet regeneration. The present invention provides just such an agonist.

The present invention relates to a compound that binds to the thrombopoietin receptor, characterized in that the compound has a molecular weight m ^^^ e: ^; less than about 8000, binding affinity to the thrombopoietin receptor, expressed as an IC50 of not more than about 100 μM; and in that the compound includes amino acid sequences:

X, X2 X3 X4 X5 X6 X7 where Χ1 is C, L, M, P, Q, V; Χ2 is F, K, L, N, Q, R, S, T or V; Χ3 is C,

F, I, L, M, R, S, V or W; Χ4 means any of 20 genetically coded L-amino acids; Χ5 is A, D, E, G, K, M, Q, R, S, T, V or Y; X6 is C, F, G, L, M, S, V, W or Y; and Χ7 is C, G, I, K, L, M, N, R or V. In a preferred embodiment, the amino acid sequence may be cyclic. In another preferred embodiment, the sequence may be dimeric.

In a further preferred embodiment, the compound of the invention comprises the amino acid sequence:

C Χ2 Χ3 Χ4 Χ5 X6 X7 where Χ2 is K, L, N, Q, R, S, T or V; Χ3 is C, F, I, L, M, R, S or V; Χ4 means any of 20 genetically coded L-amino acids; Χ5 is A, D, E, G, S,

V or Y; X6 is C, F, G, L, M, S, V, W or Y; and Χ7 is C, G, I, K, L, M, N, R or V.

In a more preferred version of the invention, the compound comprises a sequence that contains Χ4, which is A, E, G, H, K, L, M, P, Q, R, S, T or W, and in the most preferred embodiment the compound is characterized by that Χ2 is S or T; Χ3 is L or R; X6 is R; Χ5 is D, E or G; X6 is F, L or W; and Χ7 is I, K, L, R or V.

In another preferred version, the compound of the invention comprises the amino acid sequence:

X 8 C X 2 X 3 X X 5 X 6 Χ7 in which Χ2 is F, K, L, N, Q, R, S, T or V; Χ3 is C, F, I, L, M, R, S, V or W; Χ4 means any of 20 genetically coded L-amino acids; Χ5 is A, D, E, G, K, M, Q, R, S, T, V or Y; X6 is C, F, G, L, M, S, V, W or Y; Χ7 is C,

G, I, K, L, M, N, R or V; and X8 is any of the 20 genetically coded amino acids.

Preferably the compound of the invention comprises the sequence in which X₈ means G, S,

Y or R.

Even more preferably, the compound comprises the amino acid sequence: GGCTLRE WLHGGFCGG.

Still another preferred embodiment of the invention is possible, wherein the compound of the invention comprises an amino acid sequence:

X₈ GX, X₂ Χ3 Χ4 X₅ WX₇ where Χ1 is L, M, P, Q, or V; Χ2 is F, R, S or T; Χ3 is F, L, V or W; Χ4 is A, K, L, M, R, S, V or T; Χ5 is A, E, G, K, M, Q, R, S or T; Χ7 is C, I, K, L, M, or V; and X₈ means any of 20 genetically coded L-amino acids.

More preferably the compound of the invention is characterized in that oznacza1 is P; Χ2 is T; Χ3 is L; Χ4 is R; Χ5 is E or Q; Χ7 is I or L. Also preferably, the compound of the invention is characterized in that it comprises an amino acid sequence:

188 795

X9 X8 GX, X2 X₃ X X5 W X7 is X8 is A, C, D, E, K, L, Q, R, S, T or V; and X9 is A, C, E, G, I, L, M, P, R, Q, S, T or V. ...

In the compound of the invention, in the amino acid sequence X8 is preferably D, E or K; and Xa is A or I.

Preferably the compound of the invention is characterized in that the compound is selected from the group consisting ofGGCADGPTLREWISFCGG; GNADGPTLRQWLE GRRPKN; GGCADGPTLREWISFCGGK; TIKGPTLRQWLKSREHTS; SIEGPTLREWLTSRTPHS; LAIEGPTLRQWLHGNGRDT; CADGPT LREWISFC and IEGPTLRQWLAARA.

The subject of the invention is also a compound that binds to the thrombopoietin receptor, characterized in that the compound is selected from the group consisting of:

CADGPTLREWISFC [Acc] - CADGPTLREWISFC - [amide] = CADGPTLREWISFC-Hl2; and

And 1

AND

CH₂-----------------------£

IEGPTLRQWLAARA

AND

IEGPTLRQWLAARA (Pala) - K [NH₂]

A further object of the invention is a pharmaceutical composition characterized in that as active substance it contains a compound that binds to the thrombopoietin receptor, having a molecular weight of less than about 8000, binding affinity for the thrombopoietin receptor, expressed as IC50, not greater than about 100 μΜ and comprising amino acid sequence:

Xl X₂ Χ3 Χ4 Χ5 X6 Χ7 where Xi is C, L, M, P, Q, V; X₂ is F, K, L, N, Q, R, S, T or V; X3 is C, F, I, L, M, R, S, V or W; X4 is any of the 20 genetically coded L-amino acids; X5 is A, D, E, G, K, M, Q, R, S, T, V or Y; X (, is C, F, G, L, M, S, V, W or Y; and X7 is C, G, I, K, L, M, N, R or V, and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutical composition is characterized in that said amino acid sequence is cyclic, or optionally, in another preferred embodiment, the amino acid sequence may be dimeric.

In a preferred embodiment of the pharmaceutical composition according to the invention, said sequence comprises the amino acid sequence:

CX₂ Χ3 Χ4 Χ5 X₆ Χ7 where X2 is K, L, N, Q, R, S, T or V; X3 is C, F, I, L, M, R, S or V; X4 is any of 20 genetically coded L-amino acids; X5 is A, D, E, G, S, V or Y; X₆ is C, F, G, L, M, S, V, W or Y; and X7 is C, G, I, K, L, M, N, R or V.

Also preferably, the pharmaceutical composition of the invention comprises a compound comprising the amino acid sequence in which X4 is A, E, G, H, K, L, M, P, Q, R, S, T

188 795 or W. More preferably X₂ can be S or T; X₃ can be L or R; X₃ may be R; X5 may be D, E or G; X6 can be F, L or W; and X7 can be I, K, L, R or V.

In another preferred embodiment, the pharmaceutical composition of the invention comprises an amino acid sequence:

X₈ CX₂ X₃ X4 X5 X6 X7 where X₂ is F, K, L, N, Q, R, S, T or V; X₃ is C, F, I, L, M, R, S, V or W; X, 4 is any of the 20 genetically coded L-amino acids; X5 is A, D, E, G, K, M, Q, R, S, T, V or Y; X6 is C, F, G, L, M, S, V, W or Y; X7 is C, G, I, K, L, M, N, R or V; and Xg is any of the 20 genetically coded amino acids.

More preferably, Xg is G, S, Y or R, and most preferably the compound comprises the amino acid sequence: GGC TLRE WL HG GF CG G.

In another preferred embodiment, the pharmaceutical composition of the invention comprises an amino acid sequence:

X₈ GX, X2X3 X4X5 in X7 where Xt is L, M, P, Q, or V; X₂ is F, R, S or T; X3 is F, L, V or W; X is A, K, L, M, R, S, V or T; X5 is A, E, G, K, M, Q, R, S or T; X7 is C, I, K, L, M, or V; and X₈ means any of 20 genetically coded L-amino acids. More preferably, the pharmaceutical composition of the invention comprises a compound comprising the amino acid sequence in which Xj is P; X₂ is T; X3 is L; X is R; X5 is E or Q; X7 is I or L; and most preferably, the compound comprises an amino acid sequence:

Xq X8G X! X₂X3 X4 X5W X7 where X8 is A, C, D, E, K, L, Q, R, S, T or V; and Xq is A, C, E, G, I, L, M, P, R, Q, S, T or V. Also very preferably X8 is D, E or K; and X9 is A or I.

In the most preferred embodiment, the active compound compound of the invention may be selected from the group consisting of: GGCADGPT LR EWISF CGG; GNADGPTLRQWLEGRRPKN; GGCADGPTLREWISFCGG K; TIKGPTLRQWLKSREHTS; SIEGPTLREWLTSHGLRD LA; CADGPTLREWISFC and IEGPTLRCQWL A ARA.

The invention also relates to a pharmaceutical composition containing an active substance which is a compound that binds to the thrombopoietin receptor selected from the group consisting of:

CADGPTLREWISFC

<img file="PL188795B1_D0001.tif" />

[Ac] - CADGPTLREWISFC - [amide]

O = CADGPTLREWISFC-NH2; and 1 1 and 1 ch₂----------------------- S

IEGPTLRQWLAARA

AND

IEGPTLRQWLAARA (Pala) - K [NH2]

Another object of the invention is the use of a compound that binds to the thrombopoietin receptor, which compound as mentioned previously has a molecular weight

188 795 less than about 8000, binding affinity to the thrombopoietin receptor, expressed as IC50, not greater than about 100 pM; and which compound comprises amino acid sequences:

X1 X2 X3 X4 X5 X6 X7 where X: is C, L, M, P, Q, V; X2 is F, K, L, N, Q, R, S, T or V; X3 is C, F, I, L,. M, R, S, V or W; X4 is any of the 20 genetically coded L-amino acids; X5 is A, D, E, G, K, M, Q, R, S, T, V or Y; X6 is C, F, G, L, M, S, V, W. or Y; and X7 is C, G, .I, K, L, M, N, R or V for the manufacture of a medicament for the treatment of patients suffering from hematological diseases that are sensitive to treatment with a thrombopoietin agonist. This compound in all its preferred and described above embodiments can be used for the preparation of medicaments for the treatment of hematological diseases sensitive to treatment with thrombopoietin agonist.

The invention also relates to the use of a compound that binds to the trembopeetin receptor, which compound is selected from the group consisting of:

CADGPTLREWISFC

<img file="PL188795B1_D0002.tif" />

[Ac] - CADGPTLREWISFC - [amide]

O = CADGPTLREWISFC - NH2

CH2

IEGPTLRQWLAARA

IEGPTLRQWLAARA (Pala) - K [NH2] for the manufacture of a medicament for the treatment of patients suffering from hematological diseases that are sensitive to treatment with a thrombopoietin agonist.

The invention is based on the unexpected discovery that certain low molecular weight peptides and peptide mimetics have the property of strongly binding to TPO-R and can activate TPO-R. Thus, such peptides and peptide mimetics are useful for therapeutic purposes in the treatment of TPO dependent conditions (e.g. thrombocytopenia arising as a result of chemotherapy, radiotherapy or bone marrow transfusion), as well as for diagnostic purposes in studying the mechanisms of hemopoiesis and in vitro multiplication of megaarocytes and harvested precursor cells.

Peptides and peptide mimetics suitable for therapeutic and / or therapeutic purposes have an IC 50 of about 2 mM 10 or lower as determined by binding affinity assay as shown in Example 3 below, in which the lower IC 50 is correlated with stronger binding affinity for TPO-R. For pharmaceutical purposes, peptides and peptidomimetics preferably have an IC 50 not higher than about 100 pM,^,higher than 500 nM. In a preferred embodiment, the peptide molecular weight or peptide mimetic is from about 250 to about 8000.

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

188 795

Figures 1A-B illustrate the results of a functional assay in the presence of various peptides; the assay is described in example 2. Figure 1A is a graphical representation of the results of TPO-R transfected Ba / F3 cell proliferation assay for selected peptides of the invention:

§ means results for GGCADGPTLREWISFCGGK (biotin);

X means results for GGCADGPTLREWISFCGG;

Δ is the results for LAIEGPTLRQWLHGNGRDT;

O is the result for GN AD GPTLRQ W LEG RR P KN; and + denotes the results for TIKGPTLRQWLKSREHTS.

Figure IB is a graphical representation of results obtained with the same peptides and parent cell line.

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

Figures 3A-G depict 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 the corresponding parental line or line EPO dependent cells. Figure 3A shows the results for TPO in a cell proliferation assay using TPO-R transfected Ba / F3 cell line and its corresponding parental line. Figure 3B shows the results for EPO in a cell proliferation assay using TPO-R transfected Ba / F3 cell line and its corresponding parental line. Figure 3C shows 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 from SA) in the TPO-R transfected Ba / F3 cell line. The results for the corresponding parent cell line are shown in Figure 3D. Figure 3E shows the results for TPO in a cell proliferation assay using an EPO dependent cell line. Figure 3F shows the results for EPO in a cell proliferation assay using an EPO dependent cell line. Figure 3G shows the results for the complexed biotinylated peptide (AF 12885 with streptavidin (SA)) and the complexed form of the biotinylated EPO-R binding peptide (AF 11505 from SA) in the EPO dependent cell line.

Figures 4A-C illustrate the construction of peptide-plasmid libraries in the pJS142 vector. Figure 4A shows the restriction map and gene positions. The plasmid in which the library was created includes the rrnB transcription terminator, the bla gene to allow selection on ampicillin, the Ml3 phage intigenic region (Ml3 IG) to allow single-stranded DNA release, the origin of plasmid (ori) replication, two lacO sequences_and and the araC gene to allow positive and negative regulation of the expression directing araB promoter, the lac fusion gene. Figure 4B shows the sequence of the cloning region at the 3 'end of the lac 1 gene, eternally with the Sfil and Eagl sites used during library construction. Figure 4C shows the ligation of the linked oligonucleotides of the library, ON-829 and ON-830, at the Sfil sites of pJS142 to create a library. Single gaps in the sequence indicate ligation sites.

Figures 5Λ-Β illustrate cloning in MBP vectors pELM3 and pELM15. Figure 5A shows the 3 'end sequence of the malE fusion gene, including the MBP coding sequence, polyaspartic linker, protease cleavage site, factor Xa and available cloning sites. Other parts of the vector are from pMALc2 (pELM3) and pMALp2 (pELM15), available from New England Biolabs. Figure 5B shows the sequence of the vectors after transferring a fragment of the BspEII-Scal library to pELM3 / pELM15 digested with Agel-Scad. The transferred sequences include the GGG sequence encoding the peptide linker from the pJS142 library.

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Figure 6A shows the restriction map and gene positions for construction of the "helmet" domain dimer library in the pCMG14 vector. The library plasmid includes: rrnB transcription terminator, bla gene to allow selection on ampicillin, M13 phage intigenic region (M13 IG) to allow single-stranded DNA release, plasmid origin (orz), one lacO sequence_£ and the araC gene to allow positive and negative regulation of the araB promoter directing expression of the "helmet" domain dimer fusion gene. Figure 6b shows the sequence of the cloning region at the 3 'end of the helmet domain dimer gene', including the Sfil and Eagl sites used in constructing the library. Figure 6C shows the ligation of the combined oligonucleotides ON-1679, ON-829 and ON-830 at the Sfil sites of pCMG14 to create a library. Single gaps in the sequence indicate ligation sites.

Figures 7 and 9 show the results of further determinations determining the activity of the peptides and peptide mimetics according to the invention. Thrombocytopenia was induced in this assay using carboplatin. Figure 7 shows typical results obtained when Balb / C mice are treated with carboplatin (125 mg / kg, ip) on day 0. Dashed lines represent untreated animals from three experiments. The solid line represents carboplatin treated groups in three experiments. Thick solid lines represent historical data. Figure 8 shows the effect of carboplatin titration on platelet counts in mice treated with the indicated amounts of carboplatin (in mg / kg, intraperitoneal (ip) on day 0). Figure 9 shows improvement of carboplatin-induced thrombocytopenia on day 10 under the influence of peptide AF12513 (513). Carboplatin (CBP; 50-125 mg / kg, ip) was administered on day 0. AF12513 (1 mg / kg, ip) was given on days 1-9.

The following definitions are given to illustrate and define the meaning and scope of the various terms used herein to describe the invention.

"Agonist" refers to a biologically active ligand that binds to a biologically active receptor complementary to it and activates it either by causing a biological response of the receptor, or enhances pre-existing receptor activity.

"Pharmaceutically acceptable salts" refer to non-toxic alkali, alkaline earth and ammonium metal 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 science. The term also includes non-toxic acid addition salts, which are generally prepared by reacting the compounds of this invention with an appropriate organic or inorganic acid. Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate and the like.

"Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness and properties of the free bases, and which are not biologically or otherwise undesirable, formed from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, 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, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. For a description of the pharmaceutically acceptable acid addition salts as prodrugs, see Bundgaard, H., supra.

"Pharmaceutically acceptable ester" refers to those esters that 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 prodrugs, see Bundgaard, H., ed., Design of Prodrugs, Elsevier Science Publishers, Amsterdam (1985). These esters are usually formed from the corresponding carboxylic acid and alcohol. In general, ester formation can be carried out using conventional synthetic techniques. (See, e.g., March Advanced Organic Chemistry, 3rd Edition, John Wiley &amp; Sons, New York (1985), p. 1157,

188 795 and references cited therein, and Mark et al. Encyclopedia of Chemical Technology, John Wiley &amp; Sons, New York (1980)). The alcohol component of the ester will generally include (i) a C2-C12 aliphatic alcohol that may or may not contain one or more double bonds and may or may not contain branched carbon chains, and (ii) aromatic or heteroaromatic alcohols containing from 7 up to 12 carbon atoms. This invention also contemplates the use of those compositions that are both esters as described herein, and at the same time, their pharmaceutically acceptable acid addition salts.

"Pharmaceutically acceptable amide" refers to those amides that 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 the pharmaceutically acceptable amides as prodrugs, see Bundgaard, H., ed., Design of Prodrugs. Elsevier Science Publishers, Amsterdam (1985). These amides are usually formed from the corresponding carboxylic acid and amine. In general, amide formation can be carried out using conventional synthetic techniques. (See, e.g., March Advanced Organic Chemistry, 3rd ed., John Wiley &amp; Sons, New York (1985), p. 1152, and Mark et al. Encyclopedia of Chemical Technology,. John Wiley &amp; Sons, New York (1980)) ). This invention also contemplates the use of those compositions that are both amides as described herein and at the same time as their pharmaceutically acceptable acid addition salts.

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

"Stereoisomer" refers to a chemical compound having the same molecular weight, chemical composition and structure as another compound, but with differently distributed atoms. That is, some identical chemical residues have different orientations in space and, therefore, in their pure state have the ability to twist the plane of polarized light. However, some pure stereoisomers may have an optical rotation so low that it is undetectable by existing apparatus. The compounds of this invention may have one or more asymmetric carbon atoms, and therefore include various stereoisomers. All stereoisomers are within the scope of the invention.

A "therapeutically or pharmaceutically effective amount" in relation to the composition of this invention refers to the amount of the composition sufficient to induce the desired biological result. The result may be relief of signs, symptoms or causes of the disease, any other desired change in the biological system. In the present invention, the result will usually include an immune and / or inflammatory response to infection or tissue damage.

Amino acid residues in peptides are shortened as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Cheese or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine ​​is Cys or C; tryptophan is Trp or W; Arginine is Arg or R and glycine is Gly or G. In addition, Bu is a butoxy group, Bzl is a benzyl group, CHA is a cyclohexylamine, Ac is an acetyl group, Me is a methyl group, Pen is penicillamine, Aib is aminoisobutyric acid, Nva is norvaline , Abu is aminobutyric acid, Thi is thienylalanine, Obn is O-benzyl and hyp is hydroxyproline.

In addition to peptides consisting only of naturally occurring amino acids, peptidomimetics or peptide analogues are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the reference peptide. These types of non-peptide compounds are referred to as "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 into this description by reference in the literature). Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce equivalent or enhanced therapeutic or prophylactic effects. Generally, peptidomimetics are structurally similar to a reference polypeptide (i.e. a polypeptide that has biological or pharmacological activity), such as a naturally occurring receptor binding polypeptide but has one or more peptide bonds freely replaced by a bond selected from the group consisting of -CH2NH-, -CH₂S-, -CH₂CH₂-, -CH = CH- (cis or trans), -COCH₂-, -CH (OH) CH₂- their₂SO- methods known in science and further described in the following references: Spatola, AF in: Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, ed., Marcel Dekker, New York, p. 267 (1983); Spatola, AF, Vega Data (March 1983), volume 1, number 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm. Sci. (1980) pp. 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); Almquist et al. J. Med. Chem. 23: 1392-1398 (1980) (-COCH₂-); Jennings-White et al. Tetrahedron Lett. 23: 2533 (1982) (-COCH₂-); Szelka et al. European Patent Application Number 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) (-CH₂S-), each of which has been incorporated herein by reference. A particularly preferred non-peptide bond is -CH₂NH-. Such peptide mimetics may have significant advantages over solutions with polypeptides, including, for example, more economical production, greater chemical stability, enhanced pharmacological properties (half life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad range of biological activities), reduced antigenicity and others. Labeling of peptidomimetics typically involves the covalent attachment of one or more labels, directly or via a spacer (e.g., an amide group) in the non-interfering position (s) of peptidomimetics, which is predicted based on quantitative structure / activity data and / or molecular modeling. Such non-disruptive positions are generally positions that do not create direct sites of interaction with the macromolecule (s) (e.g. immunoglobulin superfamily molecules) to which the peptidomimetic binds to produce a therapeutic effect. Derivatives (e.g., labeling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetics. Generally, receptor-binding peptidomimetics bind to the receptor with high affinity and possess detectable biological activity (i.e. are agonists or antagonists of one or more receptor-dependent phenotypic changes).

Systematic substitution of one or more amino acids of a D-amino acid reference sequence with the same type (e.g. D-lysine instead of L-lysine) can be used to form more stable peptides. In addition, forced sequence peptides containing a reference sequence or substantially identical reference sequence variations can be created by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61: 387 (1992), incorporated herein by reference), for example, by the addition of an internal cysteine ​​residue capable of forming intramolecular disulfide bridges that convert the peptide into a cyclic form.

Synthetic or non-naturally occurring amino acids refer to amino acids that do not occur naturally in vzvo, but which nevertheless can be incorporated into the peptide structures described herein. Preferred synthetic amino acids are Da-amino acids of naturally occurring La-amino acids, as well as non-naturally occurring D- and La-amino acids of formula H₂NCHR5COOH, where R '⁵ is 1) a lower alkyl group. 2) a cycloalkyl group containing from 3 to 7 carbon atoms, 3) a heterocyclic group containing 1 ^ 404. from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen atoms, 4) aromatic residue containing from 6 to 10 carbon atoms, optionally having from 1 to 3 substituents in the aromatic part selected from the group consisting of from the hydroxyl, lower alkoxy, amino and carboxyl groups, 5) -alkylene-Y group, where the alkylene group is an alkylene group containing from 1 to 7 carbon atoms, and Y is selected from the group consisting of (a) hydroxyl group, (b) amino group, (c) cycloalkyl and cycloalkenyl group containing from 3 to 7 carbon atoms, (d) aryl group containing from 6 to 10 carbon atoms, optionally having

188 795 from 1 to 3 substituents in the aromatic part selected from the group consisting of hydroxyl, lower alkoxy, amino and carboxyl groups, (e) heterocyclic group containing from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen atoms , sulfur and nitrogen, (f) -C (O) R2, where R2 is selected from the group consisting of hydrogen, hydroxyl, lower alkyl, lower alkoxy and -NRk, wherein R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl, (g) -S (O) n R6, where n is an integer from 1 to 2, and r6 is a lower, alkyl, and that R5 does not specify the side chain of a naturally occurring amino acid.

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

Particularly preferred synthetic amino acids include amino acids, for example, D-amino acids of naturally occurring L-amino acids, L-1-naphthyl-alanine, L-2-naphthyl-alanine, L-cyclohexylalanine, L-2-aminoisobutyric acid, and sulfoxide and sulfonic acid methionine derivatives (i.e. HO0c- (H2NCH) CH2CH2-S (O) "R⁶), where n and R are the same as defined above, as well as methionine derivatives with lower alkoxy groups (i.e. HOOC- (H2NCH) CH2CH2-OR6, where R6 is as defined above).

"Detectable label" refers to materials that, if covalently linked to the peptides and peptide mimetics of this invention, allow detection of the peptide and peptide mimetics in vivo in a patient to which the peptide or peptide mimetic has been administered. Suitable detectable labels are well known in the art and include, for example, radioactive isotopes, fluorescent labels (e.g., fluorescein) and the like. The specific type of detectable label is not critical and is selected in terms of the degree of labeling used, as well as the toxicity of the label for the degree of labeling used.

The choice of marker in terms of such factors remains within the skill of a specialist in this field.

The covalent association of the detectable label with the peptide or peptide mimetic is carried out by conventional methods well known in the art. For example, if radioactive isotope 1 is used as the detectable tracer^2SJ, covalent binding of 125j to a peptide or peptide mimetic can be achieved by introducing into the peptide or peptide mimetic the amino acid tyrosine followed by iodination of the peptide. If tyrosine is not present in the peptide or peptide mimetic, the introduction of tyrosine to the N or C terminus of the peptide or peptide mimetics can be obtained by well-known chemical methods. Similarly, 3²P can be introduced into the peptide or peptide mimetics as a phosphate group by, for example, a peptide hydroxyl group or peptide mimetics using conventional chemical methods.

The present invention provides compounds that bind to and active TPO-R or otherwise behave like a TPO agonist. These compounds include a "lead" peptide compound and "derivatives" constructed to have the same or similar molecular structure or shape as the lead compounds, but which differ from the lead compounds either in terms of susceptibility to hydrolysis or proteolysis and / or in terms of other biological properties, such as increased receptor affinity. The present invention also provides compositions comprising an effective amount of agonists, and more particularly a compound that is useful for treating hematological disorders, and especially thrombocytopenia associated with chemotherapy, radiotherapy or bone marrow transfusion.

Peptides having binding affinity for TPO-R can be easily identified using randomized systems for the production of various peptides treated with an affinity enrichment process.

Specifically, random systems for the production of a variety of peptides include the "peptides on plasmids" system described in US Patent Nos. 5, 270 170 and 5,338,665; the "peptides on phage" arrangement described in United States Patent Application Serial No. 07/718 577, filed June 20, 1991, which is a continuation in part of United States Patent Application Serial Number 07/541 108, filed June 20, 1990. and in Cwirla et al., Proc. Natl. Acad. Sci. USA 87:

188 795

6378-6382 (1990); "Polysomal system" described in United States Patent Application Serial No. 08/300 262, filed September 2, 1994, which is a continuation in part of the application based on United States Patent Application Serial Number 08/144 775, filed October 29, 1993 . and PCT World Patent No. 95/11992; the "coded synthetic library (ESL)" system described in United States Patent Application Serial No. 08/146 886, filed November 12, 1993, which is the continuation of part of the United States Patent Application Serial Number 07/946 239, filed September 16, 1992 which is a continuation in part of the United States patent application serial number 07/762 522, filed on September 18, 1991, and a "very large scale" synthesis of immobilized polymer "described in U.S. Patent No. 5,143,854; PCT Patent Publication No. 90/15070, published December 13, 1990; United States Patent Application Serial No. 07/624 120, filed December 6, 1990; Fodor et al. Science 251: 767-773 (2/1991); Dower and Fodor Ann. Rep. Med. Chem. 26: 271-180 (1991) and United States Patent Application Serial Number 07/805 727, filed December 6, 1991.

Using the procedures described above, random peptides with a specific number of amino acid residues (e.g. 12) were generally generated. To create a set of oligonucleotides encoding random peptides, a codon motif (NKK) x was used, where N is nucleotide A, C, G or T (equimolar; depending on the methodology used, other nucleotides can be used), K is G or T (equimolarly ), ax is an integer corresponding to the number of amino acids in the peptide (e.g. 12) to determine all of the 32 possible codons that can arise from the NNK motif: 1 for each of the 12 amino acids, 2 for each of the 5 amino acids, 3 for each of the three amino acids and only one for the three stop codons. Thus, the NNK motif encodes all amino acids, encodes only one stop codon, and reduces codon diversity.

In the systems used, random peptides were presented either on the surface of the phage particle, as part of the fusion protein comprising the pili envelope protein or pVIII of the phage fd derivative (peptides on the phage), or as a fusion protein with the Lacl-peptide fused protein plasmid (peptides on plasmids) .

Phage or peptides, including peptide encoding DNA, were identified and isolated by an affinity enrichment process using immobilized TPO-R. The affinity enrichment process, sometimes referred to as "leaching", involves multiple rounds of incubation of phage, plasmids or polysomes with an immobilized receptor, collection of phage, plasmids or polysomes that bind the receptor (along with associated DNA or mRNA), and the production of more collected phages or plasmids (together with the Lacl-peptide fusion protein). During enrichment, an extracellular domain (ECD) was typically used.

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

By comparing test wells with control wells (no receptor), it can be determined whether the fusion proteins bind specifically to the receptor. Phage populations found to be bound to TPO-R were screened using a colony analysis procedure using a radiolabeled receptor as a monovalent probe. This probe can be created using protein kinase to phosphorylate the ceptidic sequence linked to the C-terminus of the soluble receptor. The "engineered" form of the TPO receptor is then expressed in host cells, typically CHO cells. After the PI-PCL has collected the receptors, the receptor is tested for binding

188 795 from TPO or TPO-R specific phage clones. The receptor is then labeled 32p to obtain high specific activity for use as a monovalent probe for identifying high affinity ligands using colony analysis.

Peptides that were found to bind specifically to the receptor were then synthesized as free peptides (e.g., without phage) and tested in a blocking assay. The blocking assay was carried out in a similar manner as an ELISA except that TPO or reference peptide was added to the wells before the fusion protein (control wells were of two types: (1) no receptor, and (2) without TPO or reference peptide). Fusion proteins whose binding to the receptor was blocked by TpO or a reference peptide contain peptides which are preferred compounds of the invention in a random peptide part.

TPO-R, as well as its extracellular domain, was produced in recombinant host cells. One useful form of TPO-R is constructed using standard methods by expressing the protein as a soluble protein in baculovirus-transformed host cells; another useful form is constructed with a protein secretion signal peptide and a membrane glycophospholipid anchoring site. This form of anchoring point is called the "PIG end". See Caras and Wendell Science 243: 11.96-1198 (1989) and Lin et al. Science 249: 677-679 (1990).

Using the PIG end system, phospholipase C can cleave the receptor from the surface of its expressed cells (e.g., transformed CHO cells selected using a cell sorter for high receptor expression). The cleaved receptor still has a carboxy-terminal amino acid sequence, nazaz, the "HPAP end", from the signal protein for membrane anchoring, and can be immobilized without further purification. The recombinant receptor protein can be immobilized by coating the wells of microtiter plates with an antibody directed against the HPAP end (Ab 179 or Mab 179), blocking non-specific binding of bovine serum albumin (BSA) in PBS, and then binding of the cleaved recombinant receptor to the antibody. Using this procedure, one can perform an immobilization reaction at different receptor concentrations to determine the optimal amount for a given preparation, because different recombinant protein preparations often contain different amounts of the desired protein. In addition, it must be ensured that the immobilizing antibody is completely blocked (using TPO or some other blocking compound) during the affinity enrichment process. Otherwise, an unblocked antibody may bind unwanted phage during the affinity enrichment procedure. To avoid this problem, peptides that bind to the immobilizing antibody can be used to block sites that remain unblocked after receptor immobilization, or the receptor can simply be immobilized directly in the wells of microtiter plates without the help of an immobilizing antibody. See United States Patent Application Serial No. 07/947 399, filed September 18, 1992, incorporated by reference.

If random peptide production systems are used that enable. multivalent ligand-receptor interaction, one must be aware that the density of immobilized receptor is an important factor in determining the affinity of ligands that I can bind to an immobilized receptor. At higher receptor densities (e.g. if each well coated with anti-receptor antibody is treated with 0.25 to 0.5 mg of receptor), multivalent binding is more likely to occur than at lower receptor densities (e.g., if each well coated with anti-receptor antibody is treated with 0.5 to 0.5 mg of receptor) And receptor). If there is multivalent binding, it will be more likely to isolate ligands with a relatively low affinity, unless high immobilized receptor densities are used to identify lead compounds and lower receptor densities are used to isolate derivatives with higher affinity.

To distinguish between higher affinity peptides, a monovalent receptor probe is often used. This probe can be generated using protein kinase A to phosphorylate the ceptidic sequence linked to the C-terminus of the soluble receptor. Next

188 795, "engineered" forms of the TPO receptor are expressed in host cells, typically CHO cells. After collecting the receptors by PI-PLC, the receptor is tested for binding to TPO or TPO-R specific phage clones. The receptor is then labeled²P for high specific activity for use as a monovalent probe to identify high affinity ligands using colony analysis.

Preferred screening methods to facilitate the identification of peptides that bind to TPO-R include first the identification of lead peptides that bind to the extracellular domain of the receptor, followed by the preparation of other peptides that lead to ^ p ^ and ^ peptides. Specifically, using pII or pVIII based peptides in a phage system, a random library can be screened to discover a phage that displays TPO-R binding peptide. The phage DNA is sequenced to determine the sequence of peptides displayed on the surface of the phages.

Clones capable of specifically binding TPO-R were identified in the pVIII random library of 10-amino acid linear peptides and the random pYIII library of 10- and 12-amino acid cyclic peptides. The sequences of these peptides serve as the basis for constructing other peptide libraries designed to contain with high frequency derivatives of pre-identified peptides. These libraries can be synthesized to promote the production of peptides that differ from the binding peptide with only a few residues. This approach involves the synthesis of an oligonucleotide with a coding sequence for the binding peptide, with the exception that rather than using pure preparations of each of the four nucleoside triphosphates, mixtures thereof are used (i.e. one preferred mixture for this purpose is 55% "proper" nucleotide and 15% each of the other three nucleotides, and another preferred mixture ;, for this purpose there is 70% "proper" nucleotide and 10% each of the three remaining nucleotides) so that derivatives of the binding peptide coding sequence are formed.

A variety of strategies have been used to obtain derivative lead peptides by preparing "motif mutagenesis" libraries. These included the creation of a mutated pVIII phagemid library based on a standard sequence mutagenized with a frequency of 70: 10: 10: 10 and extended from each end with random residues to form clones that encode the sequence ΧΧΧΧ (C, S, P or R) TLREWL ΧΧΧΧΧΧ (C or S). A similar elongated mutagenized library was constructed using a peptide-plasmid system to form clones that encode the TL (C, S, P or R) TLREWL XXXXXXX sequence. Additional elongated mutagenized. the library, XxXx (S, S, P or R) TLREWL ΧΧΧΧΧΧ (C or S), was constructed using a polysomal presentation system. All three libraries were screened by eluting peptides and using a radiolabeled monovalent receptor as a probe.

"Peptide plasmid" techniques have also been used for screening and research by mutagenesis and are described in more detail in U.S. Patent No. 5,338,665, which has been dragged into the following description, for all purposes, by reference. According to this approach, random peptides are linked to the LacI C terminus by expression from a plasmid vector containing the fusion gene. The binding of Lacl-plasmid fusion to the DNA encoding them occurs via lacO plasmid sequences, forming a stable peptide-LacI-plasmid complex that can be screened by affinity purification (enrichment) on the immobilized receptor. The plasmids thus isolated can then be reintroduced into E. coli by electroporation to amplify the selected population to perform additional rounds of screening or single-clone studies.

In addition, random peptide screening and mutagenesis studies were performed using a LacI modified C-terminal presentation system in which the presentation value was reduced ("helmet domain dimer" presentation system). The libraries were screened and the resulting DNA inserts were cloned as a population in the maltose binding protein (MBP) vector enabling their expression as the C-terminus of the fusion protein. The crude cell lysates from randomly picked individual MBP fusion clones were then assayed for TPO-R binding using the ELISA procedure as discussed above.

188 795

Studies using peptide mutagenesis were also carried out using a polysomal presentation system as described in the U.S. Patent Application Serial Number 08/300 262 filed September 2, 1994, which is a continuation in part of the U.S. Patent Application America serial number 08/144 775, filed October 29, 1993. and PCT World Patent No. 95/11992, each of which is incorporated herein, for all purposes, by reference in the literature. A mutagenized library based on sequence XXXX (C, P, R or S) t 1 re 1 XXXXXX (C or S) was constructed, where X is a random NNK codon and lower case letters are codons of frequency mutagenized amino acids. 70: 10: 10: 10 in positions 1 and 2 and having K (G or T) in position 3 of the codon. The library was enriched for 5 turns at the TPO receptor, which was immobilized on magnetic beads. After the fifth round, the population amplified by PGR was cloned into pAFF6 and the ELISA positive clones were sequenced. The sequences were subcloned in the MBP vector and their binding affinities were determined by MBP ELISA.

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

Searching the various libraries described above resulted in TPO receptor binding peptides as shown in Tables 1 and 2 below, as well as others not listed here.

Table 1

<td>peptide</td>

<td> 1</td>

<td>REGPTLRQ WM</td>

<td>REGPTLRQ WM</td>

<td>SRGMTLREWL</td>

<td>EGPTLRGWLA</td>

<td>REGQTLKEWL</td>

<td>ERGPFWAKAC</td>

<td>REG PRC VM WM</td>

<td>CSGLTLREWLVC</td>

<td>CLTGPFVTQ WLYEC</td>

<td>CGEGLTLTQWLEHC</td>

<td>CRAGPTLLEWLTLC</td>

<td>CRAGPTLLEWLTLC</td>

<td>CRQGPTLTAWLLEC</td>

<td>CADGPTLREWISFC</td>

<td>CELVGPSLMS WLTC</td>

<td>CGTEGPTLSTWLDC</td>

<td>CDQLGVTLSRWLEC</td>

188 795 cont. table 1

<td> 1</td>

<td>SGTGLTLREWLGSFSLLS</td>

<td>CPEGPTLLQWLKRGYSSC</td>

<td>RGDGPTLSQWLYSLMIMC</td>

<td>MVAGPTLREFIASLPIHC</td>

<td>SMQGPTFREWVSMMKVLC</td>

<td>SVQCGPTLRQWLAARNHLS</td>

<td>GNADGPTLRQWLEGRRPKN</td>

<td>SVRCGPTLRQWLAARTHLS</td>

<td>LAIEGPTLRQWLHGNGRDT</td>

<td>HGRVGPTLREWKTQVATKK</td>

<td>C ADGPTLREWISFC</td>

<td>ISDGPTLKEWLSVTRGAS</td>

<td>SIEGPTLREWLTSRTPHS</td>

<td>TIKGPTLRQWLKSREHTS</td>

<td>GNADGPTLRQWLEGRRPKN</td>

<td>SIEGPTLREWLTSRTPHS</td>

<td>ISDGPTLKEWLSYTRGAS</td>

Table 2

<td>peptide</td>

<td> 1</td>

<td>CSLEDLRKRC</td>

<td>CRRSELLERC</td>

<td>CTFKQFLDGC</td>

<td>CTRGEWLRCC</td>

<td>CTLRQWLQGC</td>

<td>CTLEELRACC</td>

<td>CTREELMRLC</td>

<td>CQRADLINFC</td>

<td>CNRNDLLLFC</td>

<td>CTRTEWLHGC</td>

<td>CTLEFMNGC</td>

<td>CSLGELRRLC</td>

<td>CNINQLRSIC</td>

<td>CTMRQFLVCC</td>

188 795 cont. table 2

<td> 1</td>

<td>CTRSEWLERC</td>

<td>CTLHEYLSGC</td>

<td>CTREELLRQC</td>

<td>CTFREFVNGC</td>

<td>CSRADFLAAC</td>

<td>CSCAQVVQCC</td>

<td>CTLRQWILLGMC</td>

<td>CTLREWLHGGFC</td>

<td>CTLRAWLMSETC</td>

<td>CTLRAWLMESCC</td>

<td>CTFQVWKLARNC</td>

<td>CLLREWLDXRTC</td>

<td>CVLREWLLXXSC</td>

<td>CLLSEFLAGQQC</td>

<td>CSLRQYLDFGLGSC</td>

<td>CTLQELKQSSLYEC</td>

<td>CDLSELKTHGYAYC</td>

<td>CKLSDWLMNGVAAC</td>

<td>CSLQEFLSHGGYVC</td>

<td>CSLKEFLHSGLMQC</td>

<td>CTFRQLLEYGVSSC</td>

<td>CTMREFLVASGVAC</td>

<td>CTLAEFLASGVEQC</td>

<td>CTLAEFLASGVEQC</td>

<td>CTLKEWLVSHEVWC</td>

<td>CTLREFLSLGMNAC</td>

<td>CTLREFLDPTTAVC</td>

<td>CSLLEFLALGVALC</td>

<td>GGGRGCTLKQWKQGDCGRS</td>

<td>CNRSQLLAAC</td>

<td>CTLQQWLSGC</td>

<td>CTLREFKAGC</td>

<td>CTRAQFLKGC</td>

<td>CTLREFNRGC</td>

<td>CTLSDFKRGC</td>

188 795 cont. table 2

<td> 1</td>

<td>CTFRQWKEAC</td>

<td>CTLSEFRGGC</td>

<td>CTLQEFLEGC</td>

<td>CTLQQWKDGC</td>

<td>CTRSQWLEGC</td>

<td>CSLQEFKHGC</td>

<td>CTLGEWKRGC</td>

<td>CTLWGCGKRGC</td>

<td>CTLQEWRGGC</td>

<td>CTRLSGCWLC</td>

<td>CTRTQWLLDC</td>

<td>CTLAEFRRGC</td>

<td>CTSTQWLLAC</td>

<td>CSRSQFLRSC</td>

<td>CTLREWLEGC</td>

<td>CTLREFLMMGAC</td>

<td>CTLKEWLL WSSC</td>

<td>CTLLEWLRNPVC</td>

<td>CTLRQ WLGDA WC</td>

<td>CTLGQ WLQMGMC</td>

<td>CTLREWVFAGLC</td>

<td>CLLLEFLSGADC</td>

<td>CTLGEFLAGHLC</td>

<td>CRLREFLVDLTC</td>

<td>CSFRSWLVDQTC</td>

<td>CTLREWLEDLGC</td>

<td>CTLQD WL VS WTC</td>

<td>CTLSEWLSELSC</td>

<td>CTLMQWLGGWPC</td>

<td>CTLREWLSYGYC</td>

<td>CTLQEWLSGGLC</td>

<td>GSHGCTLREWLCMKIVPC</td>

<td>QWQGCTLRDC1LRGVFWS</td>

<td>SVNSCTLREFLTGCRVFC</td>

<td>SYDGCTLRHWLMDIYGDC</td>

188 795 cont. table 2

<td> 1</td>

<td>QRSGCTLRDWFLLNCLAS</td>

<td>NYRGCTLSQWFSEQIVGC</td>

<td>GRSGCTLREYLGGMCYLS</td>

<td>ASWYCTVPELMEMQLPEC</td>

<td>GSTGCTLREXLHMLGLDC</td>

<td>ACEGCTLRQWLEYVRVGC</td>

<td>AQRGCTLQYFVSYGXDMC</td>

<td>GVCGCTLREFLA1PHTSC</td>

<td>SEGGCTLREWVASSLANC</td>

<td>SNSRCTLREWIIQGCDFS</td>

<td>SNSRCTLREWIIQGCDFS</td>

<td>CLGCTLSQWRKRTRCDTH</td>

<td>YRGCSRAQLLGGECRKK</td>

<td>GRGCTLKQWKQGDCGRS</td>

<td>VRGGCALRDWVAGECFDWT</td>

<td>LWRGCTLNGFKSRHCGSPP</td>

<td>CTLRSWKHRGCAP</td>

<td>GRGCTRAQWLAGCTTGH</td>

<td>RAGCTLREFRKGCLAL</td>

<td>KRGCTLAEMIRGCCNRSN</td>

<td>GRGCTLKQWKQGDCGRS</td>

<td>RWRGCSLAKLKKGAACGRG</td>

<td>RGGCTLREWRRVRVIN</td>

<td>GRGCTLKQWKQGDCGRS</td>

<td>RYGCTRHQWLVGTCVRH</td>

The IC50 values ​​of some additional representative peptides are given in the table below. Various methods can be used to assess the IC50 value. For example, an equilibrium binding ELISA using either MBP-TPO or lacl-peptide as indicator, was used to determine whether peptides inhibit TPO binding to the extracellular domain of the TPO receptor. Usually IC50 values ​​were determined using free peptide. The IC50 value can be determined using a free peptide, which may optionally have a C-terminated moiety, or it may be prepared as an ester or other carboxamide.

To accurately reproduce phage sequences, one or two glycine residues are often inserted after the N-terminal and C-terminal amino acids of a synthetic peptide. These glycines are not considered necessary for binding or activity. Similarly, to accurately mimic the polysomal sequences, the MAS sequence is often inserted after the C-terminal amino acids of synthetic peptides. Also in this case, this sequence is not considered necessary for binding or activity.

188 795

The IC50 value is indicated symbolically by the symbols "-," + ", and" ++ ". For example, those peptides that had IC50 values ​​above 200 pM were indicated as Those peptides that had IC50 values ​​less than or equal to 200 µM were designated "+", while those that had IC50 values ​​of 500 nm or lower were indicated by These peptides that showed IC50 values ​​equal to or near the limit point for a given symbol are indicated by a mixed designation, e.g. Those peptides for which an IC50 value was not determined are listed as "NO". The IC50 value for peptides having the GGCTL ReWlHGGFCGG structure was 50 nM or less. (It should be noted that two glycine residues are inserted after the N-terminal and C-terminal amino acids to reproduce the exact sequence displayed by phage. These glycines are not considered necessary for binding or activity.)

Table 3

<td>peptide</td><td>Affinity</td>

<td> 1</td><td> 2</td>

<td>GGCADGPTLREWISFCGG</td><td> ++</td>

<td>GNADGPTLRQWLEGRRPKN</td><td> ++</td>

<td>GGCADGPTLREWISFCGGK</td><td> ++</td>

<td>TIKGPTLRQWLKSREHTS</td><td> ++</td>

<td>GPTLRQWL</td><td> -</td>

<td>LAIEGPTLRQWLHGNGRDT</td><td>-H-</td>

<td>SIEGPTLREWLTSRTPHS</td><td> ++</td>

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

X, X2 Χ3 Xx Χ5 X6 Χ7 where X1 is C, L, M, P, Q, V; X2 is F, K, L, N, Q, R, S, T or V; X3 is C, F, I, Ł, M, R, S, V or W; X4 is any of the 20 genetically coded L-amino acids; X.5 is A, D, E, G, K, M, Q, R, S. T, V or Y; X6 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 comprises an amino acid sequence:

X₈ GX, X2 X3 X, X5 WX₇ where X1 is L, M, P, Q, or V; X2 is F, R, S or T; X3 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; and X₇ is C, I, K, L, M, or V; and each X8 residue is independently selected from any of the 20 genetically coded L-amino acids, their stereoisomeric D-amino acids, and unnatural amino acids. Preferably, each residue X₈ are independently selected from any of the 20 genetically coded 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.

More preferably, the peptide core comprises an amino acid sequence:

X9 X8 GX, X₂ X3 X4 X5 WX₇ 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: GGCADGPTLREWISFCGG; GN ADGPTLRQWLEGRRPKN; GGCADGPTLREWISFCGGK; TIKGP TLRQWLKSREHTS; SIEGPTLREWLTSRTPHS; LAIEGPTLRQW LHGNGRDT; CADGPTLREWISFC and IEGPTLRQWLA ^ I ^? V.

188 795

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

C X2 X3 X x 5 X6 X7 where Χ2 is K, L, N, Q, R, S, T or V; Χ3 is C, F, I, L, M, R, S or V; X * is any of 20 genetically coded L-amino acids; Χ5 is A, D, E, G, S, V or Y; X₆ is C, F, G, L, M, S, V, W or Y; and Χ7 is C, G, I, K, L, M, N, R or V. In a more preferred embodiment, Χ4 is A, E, G, H, K, L, M, P, Q, R, S, T or W. In a further embodiment, Χ2 is S or T; Χ3 is L or R; X is R; Xs is D, E or G; X &lt;, is F, L or W; and Χ7 is I, K, L, R or V. Particularly preferred peptides include: GGCTLREWLHGGFCGG.

In a further embodiment, preferred peptides for use in this invention include peptides having a structure comprising an amino acid sequence:

X8 C X2 X3 XX 5 X6 X 7 where Χ2 is F, K, L, N, Q, R, S, T or V; Χ3 is C, F, I, L, M, R, S, V or W; Χ4 is any of the 20 genetically coded L-amino acids; Xs is A, D, E, G, K, M, Q, R, S, T, V or Y; Xe is C, F, G, L, M, S, V, W or Y; Χ7 is C, G, I, K, L, M, N, R or V; and Xg is any of the 20 genetically coded amino acids. In some embodiments, Xg is preferably G, S, Y or R.

Peptides and peptide mimetics having an IC 50 higher than about 100 mM do not exhibit sufficient binding to allow use in diagnostic or therapeutic aspects of this invention. Preferably, for diagnostic purposes, the peptides and pepticetics have an IC50 of about 2 mM or lower, and for pharmaceutical purposes, the peptides and peptide mimetics have an IC50 of about 100 μΜ or lower.

The binding peptide sequence also provides a method for determining the minimum size of a TPOR binding compound of the invention. Using the "coded synthetic library" (ESL) system or the "large scale immobilized polymer synthesis" system, you can not only determine the minimum size of the peptide with such activity, but also prepare all of the peptides that make up a group of peptides that differ from the preferred motif (or motif size) with one or more residues. This set of peptides can then be screened for TPO receptor binding capacity. This synthesis system of immobilized polymers, or other peptide synthesis method can also be used to synthesize truncated analogs, deletion analogs, substitution analogs and combinations thereof for all of the peptide compounds of the invention.

The peptides and peptide mimetics of the present invention were also evaluated in a thrombopoietin-dependent cell proliferation assay, described in more detail in Example 2 below. Cell proliferation is measured by the 3H-thymidine incorporation technique as an indicator of cell proliferation (see Mossmann J. Immunol. Methods 65: 55 (1983)). Test peptides stimulated the proliferation of TPO-R-transformed Ba / F3 cells in a dose-dependent manner as shown in Figure 1A. These peptides had no effect on the parent cell line as shown in Figure IB.

Figures 7 to 9 show the results of a further assay in which the activity of the peptides and peptide mimetics according to the invention is assessed. In this assay, thrombocytopenia is induced in mice using carboplatin. Figure 7 shows typical results for treatment of Balb / C mice with carboplatin (125 mg / kg, intraperitoneally) on day 0. Dashed lines correspond to untreated animals from three experiments. The solid line corresponds to the carboplatin treated group in three experiments. Thick solid lines correspond to historical data. Figure 8 shows the effect of carboplatin titration on platelet counts in mice treated with the indicated amounts of carboplatin (in mg / kg, intraperitoneal (ip) on day 0). Figure 9 shows improvement of carboplatin-induced thrombocytopenia on day 10 under the influence of peptide AF12513 (513). Carboplatin (CBP; 50-125 mg / kg, ip) was administered on day 0. AF125513 (1 mg / kg, ip) was given on days 1-9. These results show that the peptides of the invention can lead to improved thrombocytopenia in a mouse model.

188 795

In addition, some peptides of the invention can be dimerized or oligomerized, thereby increasing the affinity and / or activity of the compounds. To investigate the effect that dimerization / oligomerization of peptides exerts on TPO mimicking potency in cell proliferation assays, the peptide analog GGCADGPT LREWISFCGG with biotinylated C-terminus (GGCADGPTLREWISFCGG K (biotin)) was synthesized. The peptide was pre-incubated with streptavidin in serum-free RPMI buffered HEPES in a 4: 1 molar ratio. The complex was tested for stimulation of TPO-R transformed Ba / F3 cell proliferation as described above in parallel with free biotinylated peptide and unbiotinylated parent peptide. Figure 2A shows the results of the assay for the complexed biotinylated peptide (AF 12885 with streptavidin. (SA) for both transformed and parental cell lines. Figure 2B shows the results of the free biotinylated peptide (AF 12285) assay for both transformed and parental cell lines. Figure 2C shows the results of the assay for streptavidin alone for both the transformed and the parent cell line. These figures show that the pre-formed complex was approximately 10 times stronger than the free peptide.

The binding specificity and activity of the peptides of the invention can also be checked by testing the cross-reactivity of the erythropoietin receptor (EPO-R) peptides. EPO-R is also a member of the growth factor receptor family, as is 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. In this assay, FDCP-1, a growth factor-dependent mouse murine multipotent primary precursor hemopoietic cell line, is used as the parent cell line (see, e.g., Dexter et al. J. Exp. Med. 152-1036-1047 (1981)). This cell line may proliferate but not differentiate if placed in WEHI-3 conditioned medium (medium that contains IL-3, ATCC number T1B68). The parent cell line is transfected with human or mouse EPO-R to produce the FDCP-1-EPO-R cell line. This transfected cell line may proliferate but not differentiate in the presence of human or mouse EPO.

Cells were grown to half the stationary phase density in the presence of essential growth factors. .

The cells were then washed with PBS and starved for 16-24 hours in complete medium without growth factors. After determining cell viability, stock solutions (in complete media without growth factors) were prepared with a density of about 10⁵ cells per 50 microliters. Subsequent dilutions of test compounds (usually, the peptide in the free solution phase, in contrast to the phage-bound peptide or other bound peptide or immobilized peptide) are made in 96-well tissue culture plates in a final volume of 50 microliters per well. Cells (50 microliters) are added to each well and incubated for 24-48 hours, after which time the negative controls should be dead or at rest. Cell proliferation is then measured by techniques known in the art, such as an MTT assay.

Figures 3A-3G 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 or the corresponding line parent. Figure 3A shows the results for TPO in a cell proliferation assay using the TPO-R transfected Ba / F3 cell line and the corresponding parent line. Figure 3B shows the results for EPO in a cell proliferation assay using the TPO-R transfected Ba / F3 cell line and the corresponding parent line. Figure 3C shows the results for the complexed biotinylated peptide (AF 12285 with streptavidin (SA)) and the complexed form of the biotinylated EPO-R binding peptide (Af 11501 from SA) in the TPO-R transfected Ba / F3 cell line. The results for the corresponding parent cell line are shown in Figure 3D. Figure 3E shows the results for TPO in a cell proliferation assay using an EPO dependent cell line. Figure 3F shows the results for

188 795

EPO in a cell proliferation assay using an EPO dependent cell line. Figure 3G shows the results for the complicated biotinylated peptide (AF 12285 with streptavidin (SA) and the complexed form of the biotinylated EPO-R binding peptide (AF 11501 from SA) in the EPO dependent cell line. These results show that the peptides of the invention bind and activate TPO -R with a high degree of specificity.

The peptides of the invention can be prepared by conventional methods known in the art, for example using standard solid phase synthesis techniques. Standard methods include solid phase synthesis only, partially solid phase synthesis, fragment condensation, classical solution synthesis, and even recombinant DNA technology. See, e.g., Mer ^ field J. Am. Chem. Soc. 85: 2149 (1963), dragged to this description by reference. Solid phase synthesis is usually started from the C-terminus of the peptide using a blocked alpha-amino resin. A suitable starting material can be prepared, for example, by attaching the required alpha-amino acid to a chloromethylated resin, hydroxymethyl resin or benzohydrylamine resin. One such chloroformated resin is sold under the factory name BIO-BEADS SX-1 by Bio Rad Laboratories, Richmond, CA, and the preparation of hydroxymethyl resin is described by Bodonszky et al. Chem. Indium. (London) 38: 1597 (1966). The bronzehydramamme resin (BHA) was described by Pietta and Marshall Chero. Commn. 650 (1970) and is commercially available for Rockman Instnimcnts, Inc., Palo Alto, CA, as the hydrochloride salt.

Thus, the compounds of the invention can be prepared by coupling an alpha-amino blocked amino acid to a chloroformate. resin with, for example, cesium bicarbonate catalyst according to the method described by Gisin, Helv. Chim. Acta 56: 1467 (1973). After initial coupling, the alpha-amino blocking groups are removed by choosing from reagents including solutions of trifluoroacetic acid (TFA) or hydrochloric acid in organic solvents at room temperature.

Alpha-amino blocking groups are those known to be useful in the field of stepwise peptide synthesis. These include acyl type blocking groups (e.g., trifluoroacetyl, acetyl), aromatic urethane blocking groups (e.g., benkylxycarbene (Cbz) or substituted Cbz), aliphatic urethane blocking groups (e.g., t-butyloxycarbonyl) (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type blocking groups (e.g. benzyl, triphenylmethyl). The preferred blocking groups are Boc and Fmoc. The side chain blocking group remains intact during coupling and is not cleaved during unblocking of the amino terminus blocking or during coupling. The side chain blocking group must be removable after completion of the final peptide synthesis and under reaction conditions that will not change the target peptide.

Tyr side chain blocking groups include tetrahydropyranyl, tert-butyl, trityl, benzyl, Cb2, O-Br-Cbz and 2,5-dichlorobenzyl. Asp side chain blocking groups include benzyl, 2,6-dichlorobenzzyl, methyl ethyl, and cyanide. Thr and Ser side chain blocking groups include acetyl, benzoyl, trityite tetrahydropyranic benzyl. 2,6-dichlorobenzyl and Cbz. The block group side chain Thr and Ser is a benzyl group. Arg side chain blocking groups include nitro, tosyl (Tos), Cbz, adamantyloxycarbonylmethylsilylsulfonyl (Mts) or Boc. Lys side chain blocking groups include Cbz, 2-chlororebenzyxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbenyl (2-BrCbz), Tos or Boc.

After removal of the alpha-amino blocking group, the remaining blocked amino acids are coupled in stages in the desired order. Generally, an excess of each blocked amino acid with a suitable carboxyl group activator, such as dicyclohexylcarbediimide (DDC) in solution, is used, for example, in mixtures of methylene chloride (CH2Cl2) and dimethylformamide (DMF).

After completing the desired amino acid sequence, the desired peptide is cleaved from the resin support 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

188 795 all remaining side chain blocking groups. If a chloromethylated resin is used, the result of the treatment with hydrogen fluoride is the formation of free peptide acids. If a benzohydrylamine resin is used, the result of the treatment with hydrogen fluoride is the formation of free peptide amides. Alternatively, if a chloromethylated resin is used, the blocked side chain can be cleaved by treating the resin with the bound peptide ammonia to obtain the desired blocked side chain amide, or an alkylamine to give an alkylamide or blocked side chain dialkylamide. The side chain blocking groups are then removed in the usual manner by treatment with hydrogen fluoride to obtain free amides, alkylamides or dialkylamides.

Solid phase peptide synthesis procedures are well known in the art and are further described in Stewart Solid Phase Peptide Syntheses (Freeman and Co., San Francisco, (1969)).

Using "large-scale" encoded synthetic library "or" synthesis of immobilized polymer "systems described in U.S. Patent Application Nos. 07/492 462, filed March 7, 1990; 07/624 120, filed December 6, 1990. and 07/805 727, filed December 6, 1991, you can not only determine the minimum size of a peptide with such activity, but also prepare all of the peptides forming a group of peptides that differ from the preferred motif (or the minimum size of that motif) by one, two or more residues. This set of peptides can then be screened for TPO-R binding capacity. This immobilized polymer synthesis system or other peptide synthesis methods can also be used to synthesize truncated and deletion analogs and combinations of truncated and deletion analogues of all peptide compounds of the invention.

These procedures can also be used to synthesize peptides in which amino acids other than naturally occurring, genetically coded amino acids are substituted at one, two or more positions of each of the compounds of the invention. For example, naphthylalanine can be substituted with trypophan, facilitating synthesis. Other synthetic amino acids that can be substituted for the peptides of the present invention include L-hydroxypropyl, L-3,4-dihydroxy-phenylalanyl, d-amino acids such as Ld-hydroxylyzyl and Dd-methylalanyl, La-methylalanyl, b amino acids, and isoquinolyl. D amino acids and non-naturally occurring synthetic amino acids can also be introduced into the peptides of the present invention.

The naturally occurring side chains of 20 genetically encoded amino acids (or D amino acids) can be replaced with other side chains, for example, groups such as alkyl, lower alkyl, 4-, 5-, 6- to 7-membered cycloalkyl, amide, an amide group substituted with a lower alkyl group, an amide group substituted with two lower alkyl groups, a lower alkoxy group, a hydroxyl group, a carboxyl group and their derivatives with lower esters and 4-, 5-, A 6- to 7-membered heterocyclic group. In particular, proline analogs in which the ring size of the proline residue changes from 5 membered to 4, 6 or 7 membered can be used. Cyclic groups can be saturated or unsaturated, and if they are unsaturated, they can be aromatic or non-aromatic.

Cyclic groups can be saturated or unsaturated, and if they are unsaturated, they can. be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen and / or sulfur heteroatoms. Examples of such groups include furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl groups. (e.g. morpholine), oxazolyl, piperazinyl (e.g. 1-piperazine), piperidinium (e.g. 1-piperidinium, piperidine), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl. (e.g. 1-pyrrolidinyl), pyrrolyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl and (e.g. thiomorpholine) and triazolyl. These heterocyclic groups can be substituted or unsubstituted. If the group is substituted, the substituent may be an alkyl, alkoxy group, halogen, oxygen atom or a substituted or unsubstituted phenyl group.

188 795

The peptides of this invention can be easily modified by phosphorylation, and other methods for preparing peptide derivatives of compounds of the present invention are described in Hruby et al. ⁴⁵. Thus, the peptide compounds of the invention also serve as a basis for preparing peptide mimetics with similar biological activity.

The peptide compounds of the invention, including peptide-mimetics, can be modified covalently to obtain one or more different non-protein polymers, e.g. polyethylene glycol, polypropylene glycol or polyoxyalkenes as described in U.S. Patent No. 4,640,835, U.S. Patent No. 4,496,689, U.S. Patent No. 4,301,144, U.S. Patent No. 4,670,417 , U.S. Patent No. 4,791,192 or U.S. Patent No. 4,179,337, all of which have been fully incorporated into the following description by reference.

Those skilled in the art will know that various techniques are available for constructing peptide mimetics with the same or similar biological activity as the corresponding peptide compound, but with better activity than the peptide in terms of solubility, stability and susceptibility to hydrolysis and proteolysis. See, for example, Morgan and Gainor Ann. Rep. Med. Chem. 24: 243-252 (1989). Methods for preparing peptide mimetics with a modified N-terminal amino group, a C-terminal carboxyl group, and / or one or more peptide amide bond changed to a nonamide bond are described below. It is understood that two or more of these modifications can be combined in the structure of one peptide mimetic (e.g., modification of the C-terminal carboxyl group and introduction between amino acids in the -CHA-carbaminia bond peptide).

Peptides are usually synthesized as the free acid, but as noted above, they can easily be prepared as an amide or ester. The amino and / or carboxy terminus of the peptide compound of the invention may also be modified to form other compounds of the invention. Modifications to the amino terminus include methylation (i.e. -NHCH3 or -NH (CH₂)₂ acetylation, attachment of a carbobenzoyl group or blocking of the amino terminus with any blocking group containing a carboxylate functional group defined as RCOO-, wherein R is selected from the group consisting of naphthyl, acridinyl, steroidyl and similar groups. Modifications to the carboxy terminus include substitution of the free acid carboxamide group or formation of a cyclic lactam at the carboxyl terminus to introduce structural restrictions.

Modifications of the amino terminus are as described above and include alkylation, acetylation, addition of a carbobenzoyl group, formation of a succinimide group, etc. In detail, the Nk. terminal amino group can then be subjected to the following reactions:

(a) forming an amide group of formula RC (O) NH-, where R is as defined above, by reaction with an acid halide [e.g. RC (O) Cl] or acid anhydride. Typically, the reaction can be carried out by allowing approximately equimolar or excess amounts (e.g., about 5 equivalents) of the acid halide to contact the peptide in an inert diluent (e.g. dichloromethane), preferably containing excess (e.g. about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to remove the acid formed during the reaction. In other respects, the reaction conditions are conventional (e.g. room temperature within 30 minutes). Alkylation of the amino terminus to provide N-terminal substitution with a lower alkyl group followed by reaction with the acid halide as described above results in the N-terminal alkylamide group of formula RC (O) NR-;

(b) forming a succinimide group by reaction with succinic anhydride. As before, an approximately equimolar amount or excess of succinic anhydride (e.g., about 5 equivalents) can be used and the amino group is converted to a succinimide group by methods well known in the art, including the use of excess (e.g., ten equivalents) of a tertiary amine such as diisopropylethylamine , in a suitable inert solvent (e.g. 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 an amber group can be substituted, for example, with a C2-C6 alkyl group or with -SR substituents, which are prepared in a conventional manner to obtain a substituted succinimide group at the N-terminus of the peptide. Such alkyl substituents are prepared by reacting lower olefins (C2-C6) with maleic anhydride as described by Wollenberg et al., Supra, and the -SR substituents are prepared by reacting RSH with maleic anhydride, where R is as defined above;

(c) forming a benzyloxycarbonyl-NH- group or a substituted benzyloxycarbonyl-NH- group by reaction with an approximately equivalent amount or excess of CBZ-C1 (i.e., benzyloxycarbonyl chloride) or substituted CBZ-C1 in a suitable inert diluent (e.g., dichloromethane), preferably containing tertiary amine to remove the acid formed during the reaction;

(d) forming a sulfonamide group by reaction with an equivalent amount or excess (e.g., 5 equivalents) of RS (O) 2Cl in a suitable inert diluent (dichloromethane) to convert the terminal amino group into a sulfonamide group, where R is as defined above. Preferably, the inert diluent contains an excess of tertiary amine (e.g., ten equivalents) such as diisopropylethylamine to remove the acid formed during the reaction. In other respects, the reaction conditions are conventional (e.g. room temperature within 3Q minutes);

(e) the formation of a carbamate group by reaction with an equivalent amount or excess (e.g., 5 equivalents) of R-OC (O) Cl or R-OC (O) OC₍, H4-pNO2 in a suitable inert diluent (e.g., dichloromethane) to convert the terminal amino group into a carbamate group, where R is as defined above. Preferably, the inert diluent contains an excess (e.g., ten equivalents) of a tertiary amine, such as diisopropylethylamine, to remove the acid formed during the reaction. In other respects, the reaction conditions are conventional (e.g. room temperature within 3Q minutes); and (f) the formation of a urea group by reaction with an equivalent amount or excess (e.g. 5 equivalents) of RN = C = O in a suitable inert diluent (e.g. dichloromethane) to convert the terminal amino group into a urea group (i.e. RNHC (O) NH-), where R is as defined above. Preferably, the inert diluent contains an excess (e.g., about ten equivalents) of a tertiary amine, such as diisopropylethylamine. In other respects, the reaction conditions are conventional (e.g., room temperature in about 3Q minutes).

When preparing peptide mimetics in which the C-terminal carboxyl group is replaced with an ester (i.e. -C (O) OR, where R is as defined above), the resins used to prepare the peptide acids, and the side chain blocked peptide are used the base is cleaved in a suitable alcohol, e.g. methanol. Side chain blocking groups are then removed in the usual manner by treatment with hydrogen fluoride to obtain the desired ester.

When preparing peptide mimetics in which the C-terminal carboxyl group is replaced with the -C (O) NRk amide, a benzohydrylamine resin is used as the solid support for peptide synthesis. At the end of the synthesis, the treatment with hydrogen fluoride to release the peptide from the support results directly in free peptide amide (i.e. the end is -C (O) NH2). Alternatively, the use of chloromethylated resin together with the reaction during peptide synthesis with ammonia to cleave peptide with blocked side chains from the support gives free peptide amide and after reaction with the alkylamine. or a dialkylamine to give an alkylamide or dialkylamide with blocked side chains (i.e. the C-terminus is -C (O) NRR^!where R and R^! are as defined above). The side chain blocking groups are then removed in the usual manner by treatment with hydrogen fluoride to obtain free amides, alkylamides or dialkylamides.

In another alternative embodiment, cyclization of the C-terminal carboxyl group of the C-terminal ester can be induced by internal replacement of the -OH or ester (-OR) of the carboxyl group or ester with an N-terminal amino group, respectively, to form a cyclic peptide. For example, after synthesis and cleavage to obtain a peptide acid, the free acid is converted into an activated ester with a suitable kar32 group activator

188 795 bauxite, such as dicyclohexylcarbodiimide (DCC) in solution, for example in mixtures of methylene chloride (CH2Cl2), dimethylformamide (DMF). The cyclic peptide is then formed by internal replacement of the activated ester with an N-terminal amino group. Internal cyclization, as opposed to polymerization, can be enhanced by using very dilute solutions. Such methods are well known in the art.

It is also possible to cyclize the peptides of the invention, or to introduce a deamine or descarboxyl residue at the peptide ends such that there is no amino or carboxy terminal group to reduce protease sensitivity or to reduce peptide conformation. The C-terminal functional groups of the compounds of this invention include an amide group, an amide group, substituted with: an alkyl group, an amide group substituted with two lower alkyl groups, a lower alkoxy group, a hydroxyl and carboxyl group, and derivatives thereof with lower esters and their pharmaceutically acceptable salts.

Other methods for preparing peptide derivatives of compounds of the invention are described in Hruby et al. Biochem J. 268 (2): 249-262 (1990), incorporated herein by reference. Thus, the peptide compounds of the invention also serve as structural models of non-peptide compounds with similar biological activity. Those skilled in the art know that various techniques are available for constructing compounds with the same or similar desired biological activity as the lead peptide compound, but with more favorable activity than solubility, stability or susceptibility to hydrolysis or proteolysis. See Morgan and Gainor Ann. Rep. Med. Chem. 24: 243-252 (1989), incorporated herein by reference. These techniques include replacing the peptide backbone with a backbone consisting of phosphonates, amidates, carbamates, sulfonamides, secondary amines and N-methylamino acids.

Peptide mimetics in which one or more [-C (O) BH-] peptide bonds have been replaced with such bonds as -CHh-carbamate bond, phosphonium bond, -CH2-sulfonamide bond, urea bond, secondary amine bond (-CH2NH- ) and the alkylated peptide bond [-C (O) NR6-, wherein R⁶ is a lower alkyl group], prepared during conventional peptide synthesis by merely replacing, at the appropriate synthesis point, the amino acid reagent with a suitably blocked amino acid analog.

Suitable reagents include, for example, amino acid analogs in which the carboxy group has been replaced with a residue suitable to form one of the above bonds. For example, if it is desired to replace the -C (O) NR-bond in the peptide with a -CH2Carbamate (-CH2OC (O) NR-) bond, then the carboxyl group (-COOH) of the appropriately blocked amino acid is first reduced to the -CH2OH group, which is then is transformed by conventional methods into the -OC (O) C1 functional group or the para-nitrocarbonate -OC (O) O-C6H4-p-NO2 functional group. The reaction of either of these functional groups with a free amine or an alkylated amine at the N-terminus of the partially formed solid-supported peptide leads to the formation of the -CH2OC (O) NR bond. For a more detailed description of the formation of such -Cfk-carbamate bonds, see Cho et al. Science 261: 1303-1305 (1993).

Similarly, replacement of an amide bond in a peptide with a phosphonate bond can be obtained as described in United States Patent Applications Serial Numbers 07/943 805, 08/081 577 and 08/119 700, the disclosures of which are incorporated in full by reference herein.

Replacement of the amide bond in the peptide with a -CIT-sulfonamide bond can be achieved by reducing the carboxyl group (-COOH) of the appropriately blocked amino acid to the -CH2OH group, and then the hydroxyl group is converted by conventional methods into a suitable leaving group, such as a tosyl group. The reaction of the tosylated derivative with, for example, thioacetic acid, followed by hydrolysis and oxidative chlorination will provide a functional group. -CH2-S (O) 2Cl, which replaces the carboxyl group of the otherwise blocked amino acid. The use of this appropriately blocked amino acid analog in peptide synthesis ensures the introduction of the -CH2S (O) 2NR- bond, which replaces the amide bond in the peptide, thereby providing a peptide mimetic. For a more complete description of the conversion of the carboxyl group of the amino acid into the -CH2S (O) 2Cl group, see, for example, Weinstein. Boris Chemistry &amp; Biochemistry of Amino Acids. Peptides and Proteins. Vol. 7, pp. 267-357, Marcel Dekker, Inc., New York (1983), which has been incorporated into this description by reference.

Replacement of a peptide bond in a peptide with a urea bond can be obtained as shown in United States Patent Application Serial Number 08/147 805, which application is incorporated in its entirety by reference.

Bonds through a secondary amine, where the -CH2NH- bond replaces the amide bond in the peptide, can be prepared by using, for example, a suitably blocked dipeptide analog in which the carbonyl bond of the peptide bond has been reduced by conventional methods to the CH2 group. For example, in the case of diglycin, the result of amide reduction to amine will be after deprotection of H2NCH2CH2NHCH2COOH, which is then used in a blocked N form in the subsequent coupling reaction. The preparation of such analogs by reduction of the carbonyl group of an amide bond in a dipeptide is well known in the art.

A properly blocked amino acid analog is used in conventional peptide synthesis in the same way as if it were a corresponding amino acid. For example, typically about 3 equivalents of a blocked amino acid analog is used in this reaction. An inert organic diluent such as methylene chloride or DMF is used, and if acid is formed as a byproduct, the reaction solvent will usually contain an excess of tertiary amine to remove the acid produced during the reaction.

One particularly preferred tertiary amine is diisopropylethylamine, which is usually used in a 10-fold excess. The reaction results in the introduction into the peptide mimetic of an amino acid analog having a non-peptide bond. If desired, such substitution can be repeated, also from none to all of the amide bonds in the peptide are replaced with non-amide bonds.

It is also possible to cyclize the peptides according to the invention, or to introduce a deamine or decarboxylic residue at the peptide ends, so as not to reduce protease sensitivity or to limit peptide conformation, such that no amino or carboxy terminal group is present. The C-terminal functional groups of the compounds of the present invention include an amide group, an amide group substituted with a lower alkyl group, an amide group substituted with two lower alkyl groups, a lower alkoxy group, a hydroxyl and carboxyl group, and their derivatives with lower esters and their pharmaceutically acceptable salts. Examples of cyclic compounds are given in Tables 4, 5, 6, 8 and 9.

The compounds of the present invention may exist in a cyclic form with an intramolecular disulfide bond between thiol groups of cysteine ​​residues. Alternatively, an intermolecular disulfide bond may be formed between thiol groups of the cysteine ​​residues to obtain a dimeric compound (or higher order oligomer). One or more cysteine ​​residues can also be substituted with a homocysteine ​​residue. These intramolecular and intermolecular disulfide derivatives can be schematically represented as shown below:

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Ρ^Η2&gt; π where min are 1 or 2 independently of each other.

Other embodiments of this invention provide analogs of those disulfide derivatives in which one of the sulfur atoms has been replaced by a CH group₂ or other sulfur isostere. These analogs can be prepared by intramolecular or intermolecular substitution using methods known in the art as shown below:

<img file="PL188795B1_D0003.tif" />

<img file="PL188795B1_D0004.tif" />

where p is 1 or 2. A person skilled in the art will readily understand that this replacement can also be performed using other homologues of the α-amino-g-butyric acid derivative described above and homocysteine.

Alternatively, the amino terminus of the peptide may be terminated with acetic acid substituted in the alpha position, where the substituent in the alpha position is a leaving group! such as α-halogenoacetic acid, e.g., α-chloroacetic acid, α-bromoacetic acid or α-iodoacetic acid. The compounds of the present invention can be cyclized or dimerized by replacing the leaving group with a sulfur atom for a cysteine ​​or homocysteine ​​residue. See, e.g., Barker et al. J. Med. Chem. 35: 2040-2048 (1992) and Or et al. J. Org. Chem. 56: 3146-3149 (1991), each of which is incorporated herein by reference. Examples of dimerized compounds are given in Tables 7, 9 and 10.

The compounds of the invention are useful in vitro as unique tools for understanding the biological role of TPO, including the assessment of many factors considered to be influencing and influencing TPO production and receptor binding. The present compounds are also useful in the development of other compounds that bind and activate TPO-R, because they provide important information about the relationships between structure and activity that should facilitate such development.

Compounds p. Also useful as competitive binders in assays designed to screen for new TPO receptor agons. In such solutions

188 In connection with the assay, compounds of the invention may be used without modification, or may be modified in a variety of ways; for example by labeling, such as covalent or non-covalent attachment of a residue, which directly or indirectly provides a detectable signal. In each of these markings, the materials used can be marked either directly or indirectly. Direct tagging capabilities include tag groups such as radioactive tags such as ^ J, enzymes (U.S. Patent No. 3,645,090), such as peroxidase and alkaline phosphatase, and fluorescent tags (U.S. Patent No. 3,940,475) to enable monitoring the change in fluorescence intensity, maximum wavelength shift or fluorescence polarization. Indirect labeling options include biotinylation of one component followed by avidin binding coupled to one of the foregoing tag groups. In cases where the compounds are to be attached to a solid support, they may also include spacers or linkers.

In addition, based on their ability to bind to the TPO receptor, the peptides of the present invention can be used as reagents for detecting receptors on living cells, fixed cells, in biological fluids, in tissue homogenates, in purified, natural biological materials, etc., for example, by labeling such peptides can identify cells having TPO-R on the surface. In addition, based on their TPO receptor binding capacity, the peptides of the present invention can be used in staining in situ, FACS (fluorescence activated cell sorting), Western blotting, ELISA, etc. In addition, based on their TPO receptor binding capacity, the peptides of the present invention can be used to purify the receptor or purify cells that express TPO receptors on the surface (or inside permyalyzed cells).

The compounds of the present invention can also be used as commercial reagents for various medical research and diagnostic applications. Such applications include, but are not limited to: (1) use as a calibration standard for quantifying the activity of potential agonists in TPO with a variety of functional assays; (2) use for maintaining the proliferation and growth of TPO-dependent cell lines; (3) use in structural analysis of the TPO receptor by co-crystallization; (4) use for testing the mechanism of TPO signal transduction / receptor activation; and (5) other research and diagnostic applications in which activation of the TPO receptor is beneficial or such activation is conveniently calibrated against a known amount of TPO agon, and the like.

The compounds of the present invention can be used to propagate from n-megakaryocytes and their harvested precursor cells, both together with additional cytokines, and alone. See, e.g., DiGiusto et al. PCT publication number 95/05843, which has been incorporated herein by reference. Chemotherapy and radiation treatments cause thrombocytopenia by killing the rapidly dividing, more mature megahariocyte population. However, these therapeutic effects can also reduce the amount and viability of immature megaRariocyte precursor cells with less mitotic activity. Thus, the improvement of thrombocytopenia under the influence of TPO or the compounds of the present invention can be accelerated by infusing patients after chemotherapy or radiation therapy of a population of his or her own cells enriched by in v / tro culture with megakaryocytes and immature precursors.

The compounds of the present invention can also be administered to warm-blooded animals, including humans, to activate TPO-R in vivo. Thus, the present invention includes methods of therapeutic action on TPO-dependent disorders that comprise administering a compound of the invention in amounts sufficient to mimic the effect of TPO on TPO-R in vivo. For example, the peptides and compounds of the invention may be administered to treat various hematological disorders, including but not limited to platelet and thrombocytopenia disorders, especially if they involve bone marrow transfusions, radiation therapy. and chemotherapy.

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In some embodiments of the invention, patients undergoing chemotherapy or radiation therapy are preferably administered antagonists first, followed by administration of the TPO agonists of the invention.

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

According to one embodiment, the compositions of the present invention are useful for the treatment of thrombocytopenia associated with bone marrow transfusions, radiotherapy or chemotherapy. The compounds will usually be given prophylactically before or after chemotherapy, radiotherapy or bone marrow transplantation.

Thus, the present invention also provides pharmaceutical compositions as an active ingredient comprising at least one of the peptides or peptide mimetics of the invention, together with a pharmaceutical carrier or diluent. The compounds of this invention can be administered by oral, pulmonary, parenteral routes (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), by inhalation (in the form of a fine powder), transdermal, intranasal, vaginal, rectal or sublingual injection. dosage form suitable for each route of administration. See, e.g., Bernstein et al. PCT Patent Publication Number WO 93/25221, Pitt et al. PCT Patent Publication Number WO 94/17784 and Pitt et al. European Patent Application No. 613 683, each of which is incorporated herein by reference.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable carrier, such as sucrose, lactose or starch. Such dosage forms may also include, in accordance with normal practice, additional substances other than inert diluents, e.g. glidants such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills may also be prepared with enteric coatings.

Liquid dosage forms for oral administration 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 compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and flavoring agents.

The formulations of this invention for parenteral administration include sterile, aqueous and non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. They can be sterilized by, for example, filtration through a bacterial-retaining filter, by incorporating sterilizing agents into the composition, by irradiating the composition or by heating the composition. They can also be created using sterile water or some other injection medium immediately before use.

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

Compositions containing compounds may be administered for prophylactic and / or therapeutic purposes. In therapeutic applications, the compositions are administered to a patient already suffering from a disease as described above in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is termed "therapeutically effective dose." The amounts effective in this application will depend on the severity of the disease and the weight and general condition of the patient.

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The compositions of the invention may also be encapsulated, for example, the Tice and Bibi methods (in: Treatise on Controlled Drug Delivery, ed. A. Kydonieus, Marcel Dekker, NY (1992), pp. 315-339).

For prophylactic applications, compositions containing compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease. This amount is referred to as "prophylactically effective dose". Also with such use, the exact amounts depend on the patient's state of health and weight.

The amounts of TPO agonist necessary for successful treatment will depend on a variety of factors, including the mode of administration, destination, physiological status of the patient, and other medications administered. Therefore, therapeutic doses should be gradually increased to optimize safety and efficacy. Typically, in vitro doses may provide useful guidance on the amounts useful in in situ administration of these factors. Animal testing of effective doses for the treatment of a particular disorder will provide further guidance on human doses. Various considerations on this subject are described e.g. in Gilman et al. (ed.) Goodman and Gilman's: The Pharmacological Basis of Therapeutics, ed. 8, Pergamon Press (199Q); and Remington's Pharmaceutical Sciences, ed. 7, Mack Publishing Co., Easton, Pennsylvania (1985); each of which is hereby incorporated by reference.

The peptides and peptide mimetics of this invention are effective in treating TPO-dependent conditions when administered in a dose range of from about Q, QQ1 mg to about 1Q mg / kg body weight per day. The specific dose used depends on the particular condition being treated, the route of administration as well as the opinion of the treating physician depending on such factors 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 described above, it should be understood that modifications and variations of the invention are possible without departing from the spirit and intended scope of the invention.

Example 1

Solid phase peptide synthesis

Using Memfield's solid phase synthesis technique (see Steward and Young, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical, Rockford, IL (1984) and Merrifield J. Am. Chem. Soc. 85: 2149 (1963), various peptides were synthesized according to of the invention using an automated Milligen / Biosearch 96QQ apparatus or the Applied Biosystems Inc. Model 431 A peptide synthesizer. The peptides were synthesized using standard protocols of Applied Biosystems Inc. System Software, version 1.Q1. Each coupling was carried out for one to two hours with BOP (benzotriazolyl-N-oxytrisdimethylaminophosphonium hexafluorophosphate) and HOBt (1-hydroxybenzotriazole).

The resin used was HMP or PAL resin (Milligen / Biosearch), which is a cross-linked polystyrene resin with 5- (4'-Fmoc-aminomethyl-3,5'-dimethoxyphenoxy) valeric acid as the linker. The result of using PAL resin is to obtain a carboxy-terminal amide functional group after cleavage of the peptide from the resin. After cleavage, the HMP resin forms a carboxylic acid residue at the C-terminus of the final product. Most reagents, resins and blocked amino acids (free or on resin) were purchased from Millipore or Applied Biosystems Inc.

Fmoc was used to block amino groups during the coupling procedure. Blocking of the primary amino group of amino acids was achieved using Fmoc, and the side chain blocking groups were the t-butyl group for serine, tyrosine, asparagine, glutamic acid and threonine; trityl group for glutamine, Pmc (2,2,5,7,8-pentamethylchroma sulfonate) for arginine; Nt-butyloxycarbonyl for tryptophan; N-trityl for histidine and glutamine and S-trityl for cysteine.

Removal of peptides from the resin and simultaneous deprotection of side chain functional groups was achieved by treatment with K reagent or minor modifications thereof. Alternatively, in the synthesis of these carboxy-terminated peptides, the fully synthesized peptide was cleaved with a mixture of 9Q% trifluoroacetic acid, 5% ethanedithiol and 5% water, first at 4 ° C and then raising the temperature to room temperature. Unblocked peptides were precipitated with diethyl ether. In all38

188 In 95 cases, purification was performed by preparative high performance liquid chromatography, reversed phase on a silica gel column associated with a C18 with an acetone-yl / water gradient in 0.1% trifluoroacetic acid. Homogeneous peptides were characterized by mass atomic bombardment or fast-atomic bombarding and mass spectrometry as well as analysis of the amino acid composition if it could be used.

Example 2

Biological markings

The biological activity of the peptides can be measured using a thrombopoietin-dependent cell proliferation assay. IL-3 dependent Ba / F3 mouse cells were transfected with full-length human TPO-R. In the absence of IL-3 (WEHI-3 conditioned media), the proliferation of these cells is dependent on TPO. The parent, non-transfected cell line does not respond to human TPO, but remains dependent on IL-3.

Biological assays were performed using both of the above cell lines using synthetic peptides derived from library screening. Cells were cultured in complete RPMI-10 media containing 10% conditioned WEHI-3 medium, and added to wells containing 2 × 104 peptide or TPO dilutions with well / well. Cells were incubated for 48 hours at 37 ° C in a humidified 5% CO2 atmosphere and metabolic activity was determined by reducing MTT to formazan, measuring absorbance at 570 nM measured using an ELISA plate reader. Test peptides stimulated 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 parental cell line.

Example 3

Binding affinity

The binding affinities of chemically synthesized peptides to TPO-R were measured in a competitive binding assay. The wells of the microtiter plate were lowered with 1 mg streptavidin, blocked with PBS / 1% BSA, followed by 50 ng biotinylated immobilizing anti-receptor antibody (Abl79). The wells were then treated with harvested soluble TPO-R diluted 1:10. Different concentrations of peptide or peptide mimetics were mixed with a solid, shortened form of TPO, consisting of residues 1-156 linked to the C-terminus of maltose binding protein (MBP-TPO156). Mixtures with MBP-TPO156 peptide were added to wells coated with TPO-R, incubated for 2 hours at 4 ° C, followed by washing with PBS. The amount of MBP-TPO156 that was bound at equilibrium was measured by the addition of anti-MBP rabbit antiserum followed by goat anti-rabbit IgG conjugated with alkaline phosphatase. The amount of alkaline phosphatase in each well was then determined using standard methods.

The assay was carried out over a range of different peptide concentrations and the results are graphically represented such that the y-axis represents the amount of bound MBP-TPO156 and the x-axis represents the peptide concentration or peptide mimetic. Then, the concentration at which the peptide or peptide mimetic will reduce by 50% (IC50) the amount of MBP-TPO156 bound to TPO-R can be determined. Using the assay conditions described above, the dissociation constant (Kd) for the peptide should be similar to the measured IC50.

Example 4 "Peptides on plasmids"

The pJS142 vector is used to construct the library and it is shown in Figure 4. Three oligonucleotide sequences are necessary to construct the library: ON-829 (5 'ACC ACC TCC GG); ON-830 (5 'TTA CTT AGT TA) and library specific oligonucleotide of interest (5' GA GGT GgT {NNK} n TAA CTA AGT AAA GC, where (NNK)_n means a random region of the desired length and sequence. Oligenucleotides can be chemically phosphorylated at the 5 'end during synthesis or after purification using a polynucleotide kinase. They are then combined in a 1: 1: 1 molar ratio and ligated with the vector.

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The E. coli strain, which is preferably used for enrichment, has the genotype: A / sril-recA) endA1 nup (/ lon-11 sulAlAsAR // Δ (ompy-ff7? CC2ó6 AclpA319: .kan ólacl lac ZU.//8 can prepare from E. coli strain from E. coli Genetic Stock Center at Yale University (E coli b / r, Stock Center CGSC: 6573) with lon-11 suAl genotype. The above E. coli strain is prepared for use in electroporation as described Dower et al. Nucleic Acids Res. 16: 6127 (1988), except that 10% glycerol is used at all washing stages. Cells are tested for efficiency using 1 pg of Bluescript plasmid (Stratagene). These cells are used to propagate the original library and to amplify the enriched population after each enrichment round.

Peptides on plasmids are released for enrichment from cells by gentle enzymatic digestion of cell walls using lysozyme. After deposition of cellular debris, the crude lysate can be directly used for most receptors. If some additional purification of plasmid complexes is needed, a molecular filtration column can be used to remove many of the low molecular weight impurities present in the crude lysate.

Enrichment is carried out in buffer (HEKL) with a lower salt concentration than in most physiological buffers. Enrichment can be carried out in microtiter wells with a receptor immobilized on a non-blocking monoclonal antibody (Mab) or by enrichment on beads or columns. More specifically, 24 wells may be used in the first round of enrichment, each coated with a receptor. The second round usually uses six wells coated with the receptor (PAN sample) and six wells without the receptor (NC sample). Comparison of the number of plasmids in these two samples can provide an indication of whether receptor-specific clones are enriched using the procedure. "Enrichment" is defined as the ratio of PAN transformants to those obtained from the NC sample. A 10-fold enrichment is usually an indication that receptor specific clones are present.

In later enrichment rounds, it is useful to reduce the initial amount of lysate in the wells to reduce nonspecific background binding of plasmid complexes. In round 2, 100 pl lysate per well is usually used. In round 3, 100 pl of lysate diluted 1/10 in HEKL / BSA is used. Later rounds of enrichment typically use a starting amount of plasmid transforming units at least 1000 times the estimated remaining diversity.

The binding properties of peptides encoded by individual clones are usually checked after 3, 4 or 5 rounds of enrichment, depending on the values ​​observed. Typically, an ELISA assay is used in which receptor specific binding of Lacl-peptide fusion proteins is detected. Normally LacI is a tetramer and the smallest functional form of DNA binding is a dimer. Thus, the peptides are presented on the fusion protein in a multivalent manner. Assuming that the receptor can be immobilized in wells with sufficient density, LacI-linked peptides will bind to the surface in a cooperative, multivalent manner. This cooperative binding allows detection of binding cases with low intrinsic affinity. The sensitivity of this assay is an advantage in that preliminary binding cases with low affinity can be easily identified, but there is a disadvantage in that the ELISA signal is not correlated with intrinsic peptide affinity. The fusion of peptides with maltose binding protein (MBP), as described below, enables testing in an ELISA procedure where signal strength is better correlated with affinity. See figure 5A-B.

DNA from the clones of interest can be prepared in double-stranded form using any standard mini-preparation procedure. The coding sequences of the individual clones of interest or clone populations can be transferred to vectors that combine these sequences in one reading frame with the MBP coding gene, a protein that is generally in the form of a monomer in solution. Cloning the library in pJS142 creates a BspEI restriction site near the beginning of the random coding region of the library. Digestion with BspEI and Scal restriction enzymes allows the purification of a 900 bp DNA fragment that can be cloned into one of two vectors, pELM3 (cytoplasmic) or pELM15 (periplasmic), which are simple modifications

188 795 vectors pMALc2 and pMALp2 commercially available in New England BioLabs. See figure 5A-B. Digestion of pELM3 and pELM15 with Agel and Scal restriction enzymes allows efficient cloning of the Bspl-Scal fragment from the pJS142 library. The BspEI and Agel ends are compatible for ligation. In addition, the correct ligation of Scal sites is essential for the reconstitution of a functioning bla gene (Amp resistance), which allows a reduction in the level of background clones resulting from unwanted ligation. The expression of the MBP-peptide fusion driven by the tac promoter can then be induced with IPTG.

Lacl or MBP ELISA lysates are prepared from individual clones using lysozyme and removing insoluble cell debris by centrifugation. Lysates are then added to wells containing the immobilized receptor and to control wells without the receptor. Binding of LacI or MBP fusion to peptide is detected by incubation with rabbit anti-LacI or MBP anti-LacI polyclonal antiserum followed by incubation with alkaline phosphatase-labeled goat anti-rabbit immunoglobulins. Bound alkaline phosphatase is detected using a chromogenic p-nitrophenyl phosphate substrate.

Example 5

"Helmet domain dimer" system

A variant of the Lacl-peptide-technique on plasmids uses a DNA binding protein called "helmet domain dimer". DNA binding by the E. coli lac repressor occurs through a "helmet" domain of approximately 60 amino acids. The "helmet" domain dimer, which binds the lac operator, is normally formed by the interaction of a much larger C-terminal domain of about 300 amino acids. The "helmet domain dimer" system uses "helmet" domain dimer molecules containing two "helmet" domains connected by a short peptide linker. These elm proteins DNA stable enough to allow binding of the peptide epitope presented at the C-terminus of the "helmet domain dimer" to the plasmid encoding that peptide.

Random peptides bind to the C-terminus of the "helmet" domain dimer, which binds to the plasmid encoding them, forming a peptide-dimer complex of "helmet" domains - plasmid; this complex can be searched by enrichment. The dimer system of "helmet domains peptides on plasmids" allows greater selectivity for high affinity ligands than the LacI system. Thus, the "helmet" domain dimer system is useful for preparing mutagenic libraries based on initially low binding affinity and selecting variants with higher affinity for these initial sequences.

Libraries are constructed as for peptides on plasmids using the pCMG14 vector encoding the "helmet" domain dimer (see Figure 6A-c). The presence of the lac operator is not required for binding the plasmid to the "helmet" dimer protein. The libraries were introduced into the E. coli strain (lon-11 sw / A h.soR / 7 (ompT-fCjpC) AclpA319: .kan ΔΙαοΙ lac ZU118 Δ (srl-recA) 306 :: Tn10 and amplified under basic conditions (A) promoter induction. Enrichment of "helmet" domain dimers is carried out according to procedures similar to those used for Lac libraries, except for the use of HEK buffer instead of HEKL buffer and elution of plasmids from wells run with aqueous phenol instead of IPTG. Sequences obtained as a result of enrichment of "helmet" domain dimer libraries are often characterized after transfer to the MBP vector, so that they can be tested in the affinity-sensitive MBP ELISA, and that clone populations can also be tested by colony analysis using a labeled receptor.

Example 6

In this example, cyclic compounds were subjected to three determinations. First, IC 50 values ​​were obtained as described above. In addition, MTT cell proliferation assay was performed to calculate EC50 values. Finally, the assay was performed using a micrograph (Molecular Devices Corp.). In general, the acidification rate of extracellular medium in response to stimulation of the TPO receptor with compounds of the invention was determined in this assay. The EC50 ranges are indicated symbolically as for the IC50 described above. The results are summarized in Table 4.

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Table 4

Structure ^EC50 (^nH) EC (ⁿM ftolife- Physiation microration

IC₅₀ (nM) [H] - (Pen) ADGPTLREWISF (Cys) - [NH₂ ]

SS ++ ++ ++ [O = C-NH] -ADGPTLREWISF (Cys) - [NHj] ++ ++ ++

CH₂ [H] - (Homocys) ADGPTLREWISF (Cys) - [NH₂ ] ++ ++

NO [O = C-NH] -ADGPTLREWISF (Cys) - [NH₂ ] + ++ CH₃ [H] - (D-Cys) ADGPTLREWISF (D-Cys) - [NH₂ ] + + NO [H] - (Cys) ADGPTLREWISF (D-Cys) - [NH₂ ]

Cys + - + ++

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Structure

EC50 (nM) EC0 (nH)

Prlife- Micro IC55 ration physioTOtr [H] - (D-Pen) ADGPTLREWISF (D-Cys) - [NH₂ ]

SS + + ++ [H] - (HoBocys) ADGPTLREWISF (Hoeocys) - [NH_and ] + + ++ [O = C-NH] -ADGPTLREWISF (Hoiocys) - [NH₂ ] + ++ ch₂ [O = C-NH] -ADGPTLREWISF (Pen) - [NH₂ ]

I + ch₂-s + - + [O = C-NH] -ADGPTLREWISF (Cys) - [NH₂ ] | | ++ +- ++

Ph-CH-S [H] -KADGPTLREWISFE- [NH₂ ]

II + - + - NO

NH-C = O

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Structure [H] -EADGPTLREWISFK- [NH₂ ]

And '

O = C-NHEC_5o (n «) EC5 (nH)

Prooife- Mikro IC5 (nH) ration physiometer + NO [O = C-NH] -ADGPTLREWISF (Cys) - [NH_and ] ++ + NO [O = C-NH] -ADGPTLREWISF (Cys) - [NH₂ ] ++ + - NO [HN] -ADGPTLREWISFE- [NH₂ ] 'And

-c = o + - + - + [H] - (Pen) ADGPTLREWISF (Pen) - [NH₂ ]

NO + - + Example 7

In this example, the amino acid substitution products at the D, E, I, S or F positions of the cyclic compound

CADGPTLREWISFC was determined for EC50 and IC50 values ​​as described above. The results of the tests with the use of a microsizometer are given in brackets. The results are summarized in Table 5 below.

Table 5

CADGPTLREWISFC

<td>Substitution</td><td>EC50 (nM) cell proliferation</td><td>IC50 (nM)</td>

<td> 1</td><td> 2</td><td> 3</td>

<td>E - Q</td><td> ++(+)</td><td> ++</td>

<td>DA</td><td> + (+)</td><td>-H-</td>

188 795 cd table 5

<td> 1</td><td> 2</td><td> 3</td>

<td>IA</td><td> +-(+)</td><td> +</td>

<td>ARE</td><td> ++(++)</td><td> ++</td>

<td>S - D-Ala</td><td> +</td><td> +</td>

<td>S - Sar</td><td> +-</td><td> ++</td>

<td>S-Aib</td><td> ++ (++)</td><td> ++</td>

<td>S - D-Ser</td><td> ++</td><td> ++</td>

<td>S-Nva</td><td> ++(++)</td><td> ++</td>

<td>S - Abu</td><td> ++</td><td> ++</td>

<td>S - (N-Me-Ala)</td><td> +</td><td> +</td>

<td>S - (N-Me-Val)</td><td> +</td><td> +-</td>

<td>S - (N-Me-Ala) *</td><td> +</td><td> +-</td>

<td>S - (Nor-Leu)</td><td> ++</td><td> ++</td>

<td>S - (t-Bu-Gly)</td><td> +</td><td> ++</td>

<td>S - [N-MeSerOBzl)]</td><td></td><td> +</td>

<td>S - (Homoser)</td><td>WELL</td><td>WELL</td>

<td>S - (N + Mj + Lju)</td><td> +</td><td>WELL</td>

<td>FA</td><td> +-(+)</td><td> ++</td>

<td>F - D-Ala</td><td> +</td><td> ++</td>

<td>F - D-Phe</td><td> +</td><td> ++</td>

<td>F - Homo-Phe</td><td> ++(++)</td><td> ++</td>

<td>F-CHA</td><td> ++(++)</td><td> ++</td>

<td>F-Thi</td><td> ++</td><td> ++</td>

<td>F - (SwCBd))</td><td> ++</td><td> ++</td>

<td>F - (N-Me-Ala)</td><td> +-</td><td> +-</td>

<td>F - (phenylgly)</td><td> ++(++)</td><td> ++</td>

<td>F - (pyridylal)</td><td> ++</td><td> ++</td>

<td>F - (p-nitrepha)</td><td> ++(++)</td><td> ++</td>

<td>F - (3,4-di-Cl-Phe)</td><td> ++(+)</td><td> ++</td>

<td>F - (p-Cl-Phe)</td><td> ++</td><td> ++</td>

<td>F - (2-Nal)</td><td> ++(++)</td><td> ++</td>

<td>F - (1-Nal)</td><td> ++</td><td> ++</td>

<td>F - (DiPh-Ala)</td><td> ++</td><td> ++</td>

<td>F - (N-Me-Phe)</td><td> ++</td><td>WELL</td>

<td>S, F - Ava (thioether)</td><td> +-</td><td>-H-</td>

<td>S, F - Ava (cys-cys)</td><td> +</td><td> ++</td>

188 795 cont. table 5

<td> 1</td><td> 2</td><td> 3</td>

<td>S, F - Ava</td><td> +-</td><td> ++</td>

<td>AD - deletion</td><td> +-(+)</td><td>WELL</td>

<td>ADG - deletion</td><td> (+)</td><td> +</td>

Ava = H.₂NXA^AND.COOH

Example 8

In this example, the amino acid substitutions in the compound [O = C ~ NH] -ADGPTLREWISF (CYS) were evaluated.

II

CH₂-s in positions D, S or F as indicated in table 6 below. ECsq and ICsq values ​​were calculated as described above. The results of the tests with the use of a microsizometer are given in brackets.

Table 6 [O = C - NH] -ADGPTLREWISF (CYS)

CH₂-S

<td>Substitution</td><td>EC50 (nM) cell proliferation</td><td>IC50 (nM)</td>

<td>DE</td><td> (+)</td><td>WELL</td>

<td>Free acid form</td><td> ++(+)</td><td>WELL</td>

<td>Gly addition finally C</td><td> ++</td><td> ++</td>

<td>S - Abu</td><td> ++(++)</td><td>WELL</td>

<td>F - DiPh-Ala</td><td> (++)</td><td> ++</td>

<td>S, F - Abu, DiPh-Ala</td><td> +-(+)</td><td> ++</td>

188 795

Table 7

ECgo (nM) ECjg (nM)

Micro Prolife- IC₅0 physiator right

ABOUT

II [Br + C-NH] -ADGPTLREWISFC- [NH_and ]

O ++ ++ ++

II [Br + C-NH] -ADGPTLREWISFC- [NH_FROM] [H] -IEGPTLRQWLAARA ++ ++ ++ [H] -IEGPTLRQWLAARA (B-AlaK- [NH_andj [H] -CIEGPTLRQWLAARA- [NH_and ] ++ ++ ++ [H] -CIEGPTLRQWLAARA- [NH₂ ] [H] -CADGPTLREWISF- [NH₂ ] ++ ++ ++ [H] -CADGPTLREWISF- [NH₂ ] [H] -SVQCADGPTLRQWLAARNHLS- [NH_and ] ++ ++ ++ [H] -SVQCADGPTLRQWLAARNHLS- [NH_and ] [H] -MVGPTLRSGC- [NH₂ ] NO + - + [H] -MYGPTLRSGC- [NH_and 3

188 795

<td></td><td>EC50 (nM) micro- fizjoietr</td><td>EC₅₀ (NK) proliferation right</td><td>ICf (nK)</td>

<td>CADGPTLREWISFC L 1</td><td> ++</td><td> ++</td><td> ++</td>

<td>[Ac] -ADGPTLREWISFC 1 [AC] "ADGPTLREWISFC</td><td> ++</td><td></td><td> ++</td>

<td>ADGPTLREWISFC 1 ADGPTLREWISFC</td><td> ++</td><td></td><td> ++</td>

<td>[Ac] -DGPTLREWISFC 1 [Ac] -DGPTLREWISFC</td><td> ++</td><td> ++</td><td> ++</td>

<td>[Ac] -GPTLREWISFC 1 [Ac] -GPTLREWISFC</td><td>WELL</td><td> ++</td><td> ++</td>

<td>GPTLREWISFC 1 GPTLREWISFC</td><td> ++</td><td> ++</td><td> +</td>

<td>[Ac] -PTLREWISFC 1 [Ac] -PTLREWISFC</td><td>WELL</td><td> ++</td><td> ++</td>

<td>PTLREWISFC 1 PTLREWISFC</td><td> ++</td><td> ++</td><td> +-</td>

<td>[Ac] -TLREWISFC 1 [Ac] -TLREWISFC</td><td> ++</td><td> +-</td><td></td>

<td>TLREWISFC 1</td><td> ++</td><td> +-</td><td> +-</td>

TLREWISFC

188 795

Table 8

<img file="PL188795B1_D0005.tif" />

[Η] -CLLSEFLAGQQC · - [NH₂ ]

I_I [H] -CTFQVWKLARNC "[NH_and) l_i [H] -CTLGQWLQMGMC- [NH₂ ]

I_1 [H] -CLTGPFVTQWLYEC- [NH_and ] I_I [H] "CTLREFLDPTTAVC- [NH₂ ] I_I [H] -CGTEGPTLSTWLDC- [NH₂ ] I_I [H] -CELVGPSLMSWLTC- [NH_and ] I_I [H 3-CSLKEFLHSGLMQC- [NH₂ ] [H] -CTLAEFLASGVEQC- [NH₂ ] I_ | [H] -CTLKEWLVSHEVWC- [NH₂ ]

I_I [H] -CIEGPTLRQWLAARAC- [NH₂ ] I_I [H] "REGPTLRQWM- [NH₂ ] [H] -REGPTLRQWLMSRS- [NH₂ ]

188 795

Example 9

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

CADGPTLREWISFC enabled for comparison.

The compounds in Table 8 were inactive at a maximum test concentration of 10 pm.

Table 9 compares the EC50 and IC50 values ​​determined as described above for cyclic or dimerized IE GPTLRQWLAAR A variants.

Table 10 compares the truncated (H) - IEGPTLRQWLAAR dimer forms

I (H) -IEGPTLRQWLAARA (Bala) K - (NH₂)

EC50 and IC50 values ​​were calculated as described above. The results of the tests with the use of a microsizometer are given in brackets.

Table 9

<td></td><td>EC₅₀ (NH) Hikro- fizjosetr</td><td>EC₅₀ (NH) Prolife- IC50 (nH) right</td>

<td>[H 3-1EGPTLRQWLAARA- [NH₂ ]</td><td>WELL</td><td> ++ ++</td>

[H] -CIEGPTLRQWLAARAC "[NH,] 1_I

<td>ł, 1 ",, 1</td><td>WELL</td><td> ++</td><td> ++</td>

<td>[H] -IEGPTLRQWLAARA- [NH₂ ]</td><td></td><td></td><td></td>

<td> \</td><td> ++</td><td> ++</td><td> ++</td>

<td>[H 3-IEGPTLRQWLAARA (β-Ala) K- [NH₂ ]</td><td></td><td></td><td></td>

<td>[H 3-CIEGPTLRQWLAARA- [NH₂ 3</td><td> ++</td><td> ++</td><td> ++</td>

[H] -CIEGPTLRQWLAARA- [NH₂ j

188 795

Table 10 (H) ~ IEGPTLRQWLAARA (H) -IEGPTLRQWLAARA (B-Ala) K- (NH₂ )

<td>Sequence</td><td>EC50 (nM) cell proliferation</td><td>IC50 (NM)</td>

<td>(Ac) -IEGPTLRQWLAARA 1 (Ac) -IEGPTLRQWLAARA-BA-K (NH₂)</td><td> ++</td><td>WELL</td>

<td>(H) -IEGPTLRQWLAAR 1 (H) -IEGPTLRQWLAAR-BA-C (NH₂)</td><td> ++</td><td>WELL</td>

<td>(H) -IEGPTLRQWLAA 1 (H) -IEGPTLRQWLAA-BA-C (NH₂)</td><td> ++ (++)</td><td>WELL</td>

<td>(Ac) -EGPTLRQWLAARA 1 (Ac) -EGPTLRQWLAARA-BA-C (NH₂ )</td><td>WELL</td><td>WELL</td>

<td>(H) -EGPTLRQWLAARA 1 (H) -EGPTLRQWLAARA-BA-K- (NH₂)</td><td> ++</td><td>WELL</td>

<td>(H) -EGPTLRQWLAAR 1 (H) -EGPTLRQWLAAR-BA-C (NH₂)</td><td> ++ (++)</td><td>WELL</td>

<td>(Ac) -EGPTLRQWLAA 1 (Ac) -EGPTLRQWLAA-BA-C (NH₂)</td><td> ++</td><td>WELL</td>

<td>(H) -EGPTLRQWLAA 1 (H) -EGPTLRQWLAA-BA-C (NH₂)</td><td> ++</td><td>WELL</td>

188 795

Example 1Q

This example introduces various substitutions at the G, P and W positions in the cyclic compound [H] - CADGPTLREWISFC - [NH₂]

I___1

Table 11 lists examples of substituted compounds that exhibit TPO agon activity. The substitutions given in the abbreviations in the table are as follows:

Table 11

<td colspan="3">[H] - CADGPTLREWISFC - [NH2]</td>

<td>G</td><td>P</td><td>IN</td>

<td>Sar</td><td>Hyp (OBn)</td><td>Nal</td>

<td>Sar</td><td>Hyp (OBn)</td><td>Nal</td>

<td>Gly</td><td>Pro</td><td>Trp</td>

<td>Gly</td><td>Pro</td><td>Trp</td>

<td>Sar</td><td>Hyp (OBn)</td><td>Nal</td>

<td>Gaba</td><td>Pro</td><td>^trp</td>

<td>Cpr-Gly</td><td>Pro</td><td>^trp</td>

<td>Sar</td><td>Hyp (OBn)</td><td>Nal</td>

<td>Gly</td><td>Pro</td><td>Trp</td>

<td>Gly</td><td>Pro</td><td>Nal</td>

<td>Sar</td><td>Pro</td><td>^trp</td>

<td>Cpr-Gly</td><td>L-Tic</td><td>Nal</td>

<td>Gly</td><td>D-Tic</td><td>D-Trp</td>

<td>Cpr-Gly</td><td>D-Tic</td><td>Trp</td>

<td>Gaba</td><td>Hyp (OBn)</td><td>Trp</td>

188 795

Proline replacement residues

<img file="PL188795B1_D0006.tif" />

ζχ

COOH

<img file="PL188795B1_D0007.tif" />

H

D-Pro

COOH

<img file="PL188795B1_D0008.tif" />

COOH

L-Pro

L-4-Hyp (0BN)

<img file="PL188795B1_D0009.tif" />

COOH

<img file="PL188795B1_D0010.tif" />

'' COOH

HNK-L-pipeko Sowy D-pipeko! Inic acid

COOH

L-Azetidimicarboxylic acid

<img file="PL188795B1_D0011.tif" />

<img file="PL188795B1_D0012.tif" />

L-Tiq

188 795

Tryptophan replacement residues

<img file="PL188795B1_D0013.tif" />

COOH

<img file="PL188795B1_D0014.tif" />

COOH

<img file="PL188795B1_D0015.tif" />

<img file="PL188795B1_D0016.tif" />

COOH

<img file="PL188795B1_D0017.tif" />

COOH

NH₂

L- (Benzjoienylo) -alarina

<img file="PL188795B1_D0018.tif" />

<img file="PL188795B1_D0019.tif" />

D-2-Nal

<img file="PL188795B1_D0020.tif" />

COOH

COOH

<img file="PL188795B1_D0021.tif" />

COOH

<img file="PL188795B1_D0022.tif" />

<img file="PL188795B1_D0023.tif" />

&gt; COOH

NH

L-Tic

188 795

Glycine replacement residues

COOH

glycine

<img file="PL188795B1_D0024.tif" />

COOH

H

sarcosine

HJN

<img file="PL188795B1_D0025.tif" />

COOH

<img file="PL188795B1_D0026.tif" />

COOH

0-Alar

7-aminomastastic acid

<img file="PL188795B1_D0027.tif" />

COOH

COOH

N-penfyfoglicyna

N-cyklopropyłoglicyna

Example 11

In order to evaluate the possibility of using mice as a convenient species for testing, several in vitro experiments were performed to measure the activity of test compounds on the mouse receptor. First, bone marrow cells taken from the femur of 8- to 9-week-old Balb / C mice were incubated for 7 days in liquid culture with either rhuTPO or various concentrations of test peptides. At the end of the incubation period, the cultures were concentrated using Cytospin, stained with acetylcholinesterase (AChe, mouse megakaryocyte marker) and counted by microscopic analysis. One (1) nM rhuTPO resulted in the growth of very large (&gt; 40 pm), non-adherent cells in which AChe stained. These cells turned out to be mature megakaryocytes. An initial seeded total of 106 bone marrow cells / ml (in 50 ml cultures) developed, as estimated, 1 to 2 x 106 _meg_andand_aΓiocytów. This response to TPO has been marked as "maximum". Control cultures containing no growth factors produced very few AChe positive cells. Several of the peptide compounds at high concentration were tested in this assay and the results are summarized in Table 12. Peptide A at a concentration of 10 μΜ induced maximal bone marrow response. This was the first evidence that this family of peptides is active on the mouse receptor. In the second experiment, bone marrow cells were harvested and cultured in semi-solid medium (methyl cellulose), either containing no factors or containing 1 nM rhuTPO or 10 pm Peptide A. After 7 days of culture, large cell colonies were counted (assuming they are megakaryocytes) and they were grouped into small colonies (3-5 cells 188 795 cells) or large colonies (more than 6 cells). The results are shown in Table 13. Both TPO and peptides tested resulted in the formation of substantially more colonies of both sizes than contained in negative control cultures. This indicates that the peptides mimic TPO in its ability to stimulate the proliferation of megakaryocyte precursor cell populations.

To obtain a more quantitative comparison of the activity of test compounds on murine and human receptors, the muTPO receptor was cloned and transfected with BaF3 cells. A population of TPO-dependent cells was isolated.

Table 12

<td>peptide</td><td>Tested concentration (nM)</td><td>Reply</td>

<td>D</td><td> 100 000</td><td>lack</td>

<td>C</td><td> 40 000</td><td>maximal**</td>

<td>C + SA *</td><td> 1000</td><td>maximal**</td>

<td>only SA</td><td> 1000</td><td>lack</td>

<td>B</td><td> 100 000</td><td>minimal</td>

<td>AND</td><td> 10 000</td><td>maximal**</td>

<td>TPO (R&amp;D)</td><td> 1</td><td>"maximal":</td>

* Streptavidin complexed with 2 biotinylated peptide - putative complex concentration 1: 4 ** Compared with recombinant human TPO ** 25-30% cells with aCe staining after concentration using Cytospin Cultures without any factors - about 5% cells with AChE staining (lower number of cells)

Table 13

<td>Relationship</td><td>3-5 large cells</td><td>6-12 large cells</td>

<td>No factors 1</td><td> 2</td><td> 1</td>

<td>No factors 2</td><td> 1</td><td> 1</td>

<td>1nM TPO # 1-1</td><td> 15</td><td> 6</td>

<td>1 nM TPO # 1-2</td><td> 12</td><td> 1</td>

<td>1nM TPO # 2-1</td><td> 16</td><td> 8</td>

<td>1 nM TPO # 2-2</td><td> 13</td><td> 3</td>

<td>10 μΜ peptide # 1-1</td><td> 25</td><td> 10</td>

<td>10 μΜ peptide # 1-2</td><td> 22</td><td> 8</td>

<td>1 # || M ι ·&gt; 2 «Ή — t ΊΟ-. 1 1 \ From Ili. L</td><td> 22</td><td> 7</td>

<td>10 μΜ peptide # 2-2</td><td> 21</td><td> 10</td>

The disclosures in this application of all articles and literature sources, including patent documents, have been fully incorporated for all purposes in this specification by reference to literature.

188 795

<img file="PL188795B1_D0028.tif" />

ols αο

<img file="PL188795B1_D0029.tif" />

Log peptide concentration

FIG. 2B

188 795

FROM 570

<img file="PL188795B1_D0030.tif" />

188 795

FROM 570 FROM 570

<img file="PL188795B1_D0031.tif" />

<img file="PL188795B1_D0032.tif" />

FIG. 3B

188 795

<img file="PL188795B1_D0033.tif" />

FIG. 3C

FROM 570

<img file="PL188795B1_D0034.tif" />

FIG. 3D

188 795

FROM 570

<img file="PL188795B1_D0035.tif" />

Log TPR concentration

FIG.

<img file="PL188795B1_D0036.tif" />

<img file="PL188795B1_D0037.tif" />

OOO IA

Λ ♦ · ·

Log EPR concentration

FIG, 3F

188 795

FROM 570

<img file="PL188795B1_D0038.tif" />

FIG. 3G

188 795

<img file="PL188795B1_D0039.tif" />

2479 Sal I 2479 Hinc II 2495 Hind HI

2392 XhoI 2435 Siu I 2454 Eag I

FIG · 4A

188 795 υ σι (0 U σ * σ υ σι

4J (0 σι υ

Η &lt;

<td></td><td>σ</td><td colspan="2"> 4</td>

<td>Η</td><td></td><td>AT</td><td> 0</td>

<td></td><td></td><td>α</td><td>ο</td>

<td>AT</td><td>ο</td><td>ο</td><td>ο</td>

<td>ω</td><td></td><td>ο</td><td>ο</td>

<td>X</td><td></td><td>Α</td><td> &lt;</td>

<td></td><td></td><td>at</td><td> □</td>

υ &lt;&lt; Η e * &lt;0 0

PJS142 library vector, cloning sites at the 3 'end of the lacl gene

O (tf rM υ

(0 in&gt; 4

<td></td><td>• Η</td><td></td><td>ο</td><td>at</td>

<td></td><td>ΜΜ</td><td> 0</td><td></td><td>at</td>

<td></td><td>tn</td><td></td><td>rt</td><td>at</td>

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

<td></td><td></td><td>(ϋ</td><td>ο</td><td>about</td>

<td>Η</td><td></td><td></td><td>υ</td><td></td>

<td></td><td></td><td></td><td>ο</td><td>S</td>

<td>σι</td><td></td><td>at</td><td>ο</td><td>about</td>

<td> (0</td><td></td><td></td><td>ο</td><td>about</td>

<td>ca</td><td></td><td></td><td>ο</td><td>P</td>

<td></td><td>Μ</td><td>ζ</td><td></td><td></td>

<td></td><td></td><td></td><td> 2</td><td></td>

<td></td><td> 4</td><td></td><td></td><td></td>

<td></td><td>(X</td><td> &gt;</td><td>ε-</td><td> 2</td>

<td></td><td>X</td><td></td><td>ρ</td><td>at</td>

<td></td><td></td><td></td><td>ο</td><td>about</td>

<td></td><td></td><td></td><td>at</td><td>about</td>

ο ο ο υ ο ο υ ο ο υ ο £ Ω

Λ

<td></td><td></td><td> 01</td><td> 0</td><td>about</td>

<td>IN</td><td></td><td></td><td>H</td><td></td>

<td></td><td>IN</td><td></td><td> 0</td><td>and</td>

<td>• H</td><td></td><td> &lt;</td><td> 0</td><td> 0</td>

<td>MM</td><td> 3</td><td></td><td> 0</td><td> 0</td>

<td>ol</td><td>4J</td><td></td><td>σ</td><td>υ</td>

<td></td><td>ol</td><td>ca</td><td>RTJ</td><td>.at</td>

<td></td><td></td><td></td><td>σι</td><td>υ</td>

<td></td><td></td><td></td><td> 4-1</td><td> 4</td>

<td></td><td></td><td> 0</td><td>σι</td><td>υ</td>

<td></td><td></td><td></td><td>σι</td><td>υ</td>

<td></td><td></td><td></td><td>at</td><td> 4</td>

<td></td><td></td><td> 0</td><td>σι</td><td>υ</td>

σι σ σ »σ&gt; 4-» 4 σι σ σ »υ

Ο (0 4-1 υ σι σ »σ (0 U σ» σ σι σ σι σ σι σ 4J 4 (0 4J υ σι σι σ 4J &lt;0 σι σ σ&gt; σ

4J &lt;0 σι σ ca • Η

In • Η

C σ

• rl

Μ

4J α

• Η

0)

4J

Ο • Η

1—1 • Η

Λ σ

• Η

C

<img file="PL188795B1_D0040.tif" />

<td></td><td></td><td></td><td>at</td><td> 4</td><td></td>

<td></td><td></td><td> 0</td><td>ol</td><td>AT</td><td></td>

<td></td><td></td><td></td><td>σι</td><td>υ</td><td></td>

<td></td><td></td><td></td><td>4J</td><td> 4</td><td></td>

<td></td><td></td><td> 0</td><td>ol</td><td>AT</td><td></td>

<td></td><td></td><td></td><td>ol</td><td>υ</td><td></td>

<td></td><td></td><td></td><td> 4</td><td>u χ-</td><td></td>

<td>in</td><td></td><td> 0</td><td>ol</td><td>υ »</td><td></td>

<td>IN</td><td></td><td></td><td> 0</td><td>at ⁱⁿσι</td><td> \</td>

<td>ol</td><td></td><td></td><td> 0</td><td>ol</td><td>&lt;Μ</td>

<td>AT)</td><td></td><td> 01</td><td> 0</td><td></td><td>C0</td>

<td> «</td><td></td><td></td><td>tn</td><td></td><td> »</td>

<td></td><td>H</td><td></td><td> 0</td><td> 0</td><td> 2</td>

<td></td><td></td><td> &lt;</td><td> 0</td><td> 0</td><td>η</td>

<td></td><td> 3</td><td></td><td> 0</td><td> 0</td><td></td>

<td></td><td>4J</td><td></td><td>ol</td><td>υ</td><td></td>

<td></td><td> 0)</td><td>ca</td><td> 4</td><td> 4-1</td><td></td>

<td></td><td></td><td></td><td>0i</td><td>υ</td><td></td>

<td></td><td></td><td></td><td>4J</td><td> 4</td><td></td>

<td></td><td></td><td> 0</td><td>σι</td><td>υ</td><td></td>

<td></td><td></td><td></td><td>σι</td><td>υ</td><td></td>

<td></td><td></td><td></td><td>at</td><td> 4</td><td></td>

<td></td><td></td><td> 0</td><td>Οι</td><td>υ</td><td></td>

<td></td><td></td><td></td><td>ol</td><td>υ</td><td></td>

<td></td><td></td><td></td><td>ol</td><td>υ</td><td></td>

<td></td><td></td><td> &gt;</td><td>j</td><td> 4</td><td></td>

<td></td><td></td><td></td><td>σι</td><td>υ</td><td></td>

<td></td><td></td><td></td><td>ol</td><td>υ</td><td></td>

<td></td><td></td><td>about</td><td> 4</td><td>4J</td><td></td>

<td></td><td></td><td></td><td>υ</td><td>σι</td><td></td>

<td></td><td></td><td></td><td>ABOUT)</td><td>at</td><td></td>

<td></td><td></td><td>ca</td><td> 4</td><td>4J</td><td></td>

<td></td><td></td><td></td><td>ol</td><td>υ</td><td></td>

<td></td><td></td><td></td><td>ol</td><td>ο</td><td></td>

<td></td><td></td><td> 0</td><td>cn</td><td>υ</td><td></td>

<td></td><td></td><td></td><td> 01</td><td>υ</td><td></td>

<td></td><td></td><td></td><td> 4-1</td><td> 4</td><td></td>

<td></td><td></td><td>X</td><td> 4</td><td> 4-1</td><td></td>

<td>ο</td><td></td><td>S</td><td>«υ j</td><td> 1</td><td></td><td>ABOUT</td>

<td></td><td> 0</td><td> 0</td><td>about</td><td> 1</td><td></td><td></td>

<td></td><td> 0</td><td> 0</td><td> 3</td><td> 1</td><td></td><td></td>

<td> 0</td><td> 0</td><td> 0</td><td>μ</td><td> 1</td><td></td><td> 0</td>

<td></td><td> 0</td><td> 0</td><td>4J</td><td> 1</td><td></td><td></td>

<td></td><td> 0</td><td> 0</td><td>in</td><td> 1</td><td></td><td></td>

<td>οι</td><td> 0</td><td> 0</td><td>c</td><td> 1</td><td></td><td> 01</td>

<td></td><td></td><td> £</td><td> 0</td><td>H</td><td></td><td></td>

<td></td><td> 0</td><td> 0</td><td>X</td><td>and</td><td></td><td></td>

<td>ca</td><td></td><td>b ·</td><td></td><td> (0</td><td>H</td><td>this</td>

<td></td><td> 0</td><td> 0</td><td></td><td>rM</td><td></td><td></td>

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

<td>at</td><td></td><td> &lt;</td><td></td><td>t</td><td>AND</td><td>at</td>

□ σι σι υ U (0 σι υ σι υ U (0 σι υ υ ο ο ο ouau ο ο ο ο &lt;Ε O 0 δ Η «ο ο ο

Θ

Ο

AT,

188 795

Cloning sites of the pELM3 / pEIM15MBP vector

<td></td><td></td><td></td><td></td><td></td><td></td><td>Λ</td><td>in</td><td></td><td>Eh</td><td>rf</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 1-1</td><td></td><td>Eh</td><td> 2</td><td></td><td></td><td></td><td></td>

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

<td> (0</td><td></td><td></td><td>about</td><td> 0</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td></td><td></td><td></td>

<td>X</td><td></td><td rowspan="2">in</td><td>rf</td><td>Eh</td><td></td><td></td><td>Ό</td><td>WHAT</td><td> 0</td><td> 0</td><td></td><td></td><td></td><td></td>

<td> 1</td><td></td><td> 0</td><td> 0</td><td></td><td></td><td>C</td><td></td><td>rf</td><td>Eh</td><td></td><td></td><td></td><td></td>

<td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>• H</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>l</td><td></td><td></td><td>about</td><td> 0</td><td></td><td></td><td>X</td><td></td><td>rf</td><td>Eh</td><td></td><td></td><td></td><td></td>

<td>l</td><td></td><td>in</td><td>Eh</td><td>rf</td><td></td><td></td><td></td><td> &lt;</td><td> 0</td><td> 0</td><td></td><td></td><td></td><td></td>

<td>V</td><td></td><td></td><td></td><td>Eh</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td></td><td></td><td></td>

<td>Λ</td><td></td><td></td><td>ABOUT</td><td> 0</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td></td><td></td><td></td>

<td> 1</td><td></td><td> 0</td><td> 0</td><td> 0</td><td></td><td></td><td></td><td>σ</td><td>rf</td><td>Eh</td><td></td><td></td><td></td><td rowspan="2">CJ CJ</td>

<td rowspan="2"> 1</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2">about</td><td rowspan="2"> 0</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2">K</td><td rowspan="2"></td><td rowspan="2"> 0</td><td rowspan="2"> 0</td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td>

<td>&lt;e- *</td>

<td></td><td></td><td></td><td>CJ</td><td> 0</td><td></td><td></td><td> 4-&gt;</td><td></td><td> 0</td><td> 0</td><td></td><td>ht</td><td></td><td>CJ CJ</td>

<td></td><td></td><td>ul</td><td>Eh</td><td>rf</td><td></td><td></td><td>IN</td><td>ie</td><td>Eh</td><td>rf</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td rowspan="2">CJ</td><td> 0</td><td></td><td></td><td>cu</td><td></td><td> 0</td><td> 0</td><td></td><td>rH</td><td></td><td>uo</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>H &lt;</td>

<td></td><td></td><td></td><td>about</td><td> 0</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td></td><td>What</td><td></td><td>OU</td>

<td></td><td></td><td>FROM</td><td>rf</td><td>Eh</td><td></td><td></td><td></td><td>Q</td><td>rf</td><td>E *</td><td></td><td></td><td></td><td rowspan="2"></td>

<td></td><td></td><td></td><td></td><td>Eh</td><td></td><td></td><td>H</td><td></td><td> 0</td><td> 0</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td>CJ</td><td> 0</td><td></td><td></td><td>r-b</td><td></td><td> 0</td><td> 0</td><td></td><td></td><td></td><td>Eh rf</td>

<td></td><td></td><td>from</td><td>rf</td><td>Eh</td><td></td><td></td><td>d</td><td> &gt;</td><td>Eh</td><td>rf</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 2</td><td>Eh</td><td></td><td></td><td>What</td><td></td><td> 0</td><td> 0</td><td></td><td></td><td></td><td>rf Eh</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>rf Eh</td>

<td></td><td></td><td></td><td>about</td><td> 0</td><td></td><td></td><td></td><td></td><td>rf</td><td>Eh</td><td></td><td></td><td></td><td> 0 0</td>

<td></td><td></td><td>from</td><td> *5</td><td>EH</td><td></td><td></td><td></td><td>X</td><td> 0</td><td> 0</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 2</td><td>Eh</td><td></td><td></td><td>H</td><td></td><td>rf</td><td>Eh</td><td></td><td>H</td><td></td><td> 0 0 0 0</td>

<td></td><td></td><td></td><td>Eh</td><td>rf</td><td></td><td></td><td> &lt;0</td><td></td><td>Eh</td><td>rf</td><td></td><td>at</td><td></td><td> 0 0</td>

<td></td><td></td><td>from</td><td>rf</td><td>EH</td><td></td><td>d</td><td>Λ</td><td>WHAT</td><td> 0</td><td> 0</td><td></td><td>WHAT</td><td></td><td></td>

<td></td><td></td><td></td><td> 2</td><td>Eh</td><td></td><td>• H</td><td>X</td><td></td><td>Eh</td><td>rf</td><td></td><td>s</td><td></td><td>Eh rf</td>

<td></td><td></td><td></td><td></td><td></td><td rowspan="2">d about</td><td>c</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0 0</td>

<td></td><td></td><td></td><td>CJ</td><td> 0</td><td> (0 £</td><td></td><td></td><td> 0</td><td> 0</td><td></td><td></td><td></td><td> 0 0</td>

<td></td><td></td><td>from</td><td>rf</td><td>Eh</td><td>ω</td><td>about</td><td>in</td><td>WHAT</td><td> 0</td><td> 0</td><td></td><td></td><td></td><td></td>

<td rowspan="2">c N</td><td></td><td></td><td> 2</td><td>Eh</td><td>Tł</td><td>c</td><td></td><td></td><td>Eh</td><td>rf</td><td>H</td><td></td><td></td><td>rf Eh</td>

<td></td><td>from</td><td>Eh rf</td><td>rf Eh</td><td colspan="2">At 0 Ή e</td><td>X £ &lt;0</td><td>about</td><td>rf 0</td><td rowspan="2">Eh 0</td><td>X 4) •AT</td><td></td><td></td><td>rf Eh rf Eh</td>

<td></td><td></td><td></td><td> 2</td><td>Eh</td><td></td><td></td><td>X</td><td></td><td> 0</td><td> 0</td><td></td><td></td><td>Eh &lt;</td>

<td>rM</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0 0</td>

<td></td><td></td><td></td><td>CJ</td><td> 0</td><td></td><td></td><td></td><td></td><td> 0</td><td rowspan="4">Eh Łk</td><td>Λ</td><td></td><td></td><td>rf Eh</td>

<td></td><td></td><td>from</td><td> &amp;</td><td>Eh</td><td></td><td></td><td>K</td><td>ω</td><td>Eh</td><td>• RJ</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 2</td><td>Eh</td><td></td><td></td><td>X</td><td></td><td>Eh</td><td> 43</td><td></td><td></td><td>rf Eh Eh rf</td>

<td>J</td><td></td><td></td><td>CJ</td><td> 0</td><td></td><td></td><td>about</td><td></td><td>rf</td><td>• Ή</td><td>Λ</td><td></td><td> 0 0</td>

<td> 1</td><td></td><td>from</td><td>rf</td><td>Eh</td><td></td><td></td><td>υ</td><td>in</td><td> 2</td><td>Eh</td><td> ·*</td><td>d</td><td></td><td></td>

<td>L</td><td></td><td></td><td> 2</td><td>Eh</td><td></td><td></td><td>ω</td><td></td><td> 0</td><td> 0</td><td> &lt;0</td><td>X</td><td></td><td>rf Eh</td>

<td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>4J</td><td>φ</td><td> ♦</td><td>2 Eh</td>

<td>t</td><td></td><td></td><td>CJ</td><td> 0</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td>CD</td><td>about</td><td></td><td>Eh rf</td>

<td> 1</td><td></td><td>from</td><td>rf</td><td>Eh</td><td></td><td></td><td>H</td><td>X</td><td> 0</td><td> 0</td><td> 5</td><td>• H</td><td></td><td></td>

<td> 1</td><td></td><td></td><td> 2</td><td>Eh</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td>g</td><td>r-b</td><td></td><td></td>

<td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td>&lt;d</td><td></td><td></td><td></td><td>• H</td><td> 43</td><td colspan="2">part</td>

<td> 1</td><td></td><td></td><td>CJ</td><td> 0</td><td></td><td></td><td> £</td><td></td><td> 0</td><td> 0</td><td>C</td><td>• H</td><td>X</td><td>zz</td>

<td> 1</td><td></td><td>from</td><td>rf</td><td>EH</td><td></td><td></td><td>WHAT</td><td> &lt;</td><td> 0</td><td> 0</td><td>d</td><td> 43</td><td></td><td>zz</td>

<td>1 J</td><td></td><td></td><td> 2</td><td>Eh</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td> 5 0</td><td>V</td><td></td><td> '— —</td>

<td> 1</td><td></td><td></td><td>CD</td><td> 0</td><td></td><td></td><td></td><td></td><td> 0</td><td> 0</td><td>c Λ</td><td>Λ</td><td></td><td>Eh rf</td>

<td> 1</td><td></td><td>in</td><td>CJ</td><td> 0</td><td></td><td></td><td></td><td> &gt;</td><td>Eh</td><td>rf</td><td> 1-4</td><td>t</td><td> 0</td><td> 0 0</td>

<td>1 AND</td><td></td><td></td><td>Eh</td><td>rf</td><td></td><td></td><td>H</td><td></td><td> 0</td><td> 0</td><td></td><td>L</td><td></td><td>0 Oh</td>

<td> 1</td><td>M</td><td></td><td>ABOUT</td><td> 0</td><td></td><td></td><td> &lt;—(</td><td></td><td> 0</td><td> 0</td><td> 3</td><td>X</td><td></td><td>Eh rf</td>

<td> 1</td><td></td><td>in</td><td> 0</td><td> 0</td><td></td><td></td><td> £</td><td>X</td><td>rf</td><td>Eh</td><td rowspan="2">N</td><td>• H</td><td> 0</td><td> 0 0</td>

<td>AND</td><td>υ</td><td></td><td>rf</td><td>Eh</td><td></td><td></td><td>X</td><td></td><td> 0</td><td> 0</td><td>.C</td><td></td><td> 0 0</td>

<td> 1</td><td> (0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 0</td><td>N</td><td></td><td></td>

<td> 1</td><td>WHAT</td><td></td><td> 0</td><td> 0</td><td></td><td></td><td></td><td></td><td>Eh</td><td>rf</td><td>X</td><td>! tf</td><td></td><td>rf Eh</td>

<td> 1</td><td></td><td>What</td><td>AT</td><td> 0</td><td></td><td></td><td></td><td>about</td><td> 0</td><td> 0</td><td rowspan="2">in ^ 4</td><td>id</td><td> 0</td><td> 0 0</td>

<td>V</td><td></td><td></td><td>Eh</td><td>rf</td><td></td><td></td><td> 1-1</td><td></td><td> 0</td><td> 0</td><td> 1 </td><td></td><td> 0 0</td>

<td>Λ</td><td></td><td></td><td>Eh</td><td>rf</td><td></td><td></td><td>Φ</td><td></td><td> 0</td><td> 0</td><td>s</td><td> 1 1</td><td></td><td> 0 0</td>

<td> 1</td><td></td><td>from</td><td>rf</td><td>EH</td><td></td><td></td><td colspan="2">CP Eh</td><td> 0</td><td> 0</td><td>in</td><td> 1</td><td>Eh</td><td> 0 0</td>

<td>1 AND</td><td></td><td></td><td>rf</td><td>Eh</td><td></td><td>V</td><td>rf</td><td></td><td>rf</td><td>Eh</td><td>X \</td><td>V</td><td></td><td>rf Eh</td>

<td rowspan="2">1 X</td><td></td><td></td><td>Eh</td><td>rf</td><td></td><td>Λ</td><td></td><td></td><td> 0</td><td> 0</td><td>ΓΌ</td><td>Λ</td><td></td><td> 0 0</td>

<td></td><td>Eh</td><td> 0</td><td> 0</td><td></td><td></td><td></td><td>X</td><td> 0</td><td> 0</td><td>Se</td><td> 1</td><td>AXIS</td><td> 0 0</td>

<td>X</td><td></td><td></td><td>rf</td><td>Eh</td><td></td><td></td><td></td><td></td><td>rf</td><td>Eh</td><td> £</td><td> 1</td><td></td><td>rf Eh</td>

<td>Σ 1</td><td></td><td rowspan="2">about</td><td> 0</td><td> 0</td><td></td><td></td><td></td><td></td><td>rf</td><td>Eh</td><td>X</td><td> 1 1</td><td></td><td>rf EH</td>

<td> 1</td><td></td><td>rf</td><td>Eh</td><td></td><td>ίθ</td><td></td><td>about</td><td> 0</td><td> 0</td><td></td><td>d</td><td> 0</td><td> 0 0</td>

<td> 1</td><td></td><td></td><td> 0</td><td> 0</td><td></td><td>X</td><td></td><td></td><td> 0</td><td> 0</td><td></td><td>X</td><td></td><td> 0 0</td>

QQ tn o

C9

lk

188 795

AIwNI 3916

Cia 1 5 Nsil 5 PpulOI

Eco47 III 4682

237 BsaB I 301 Aft II

384 EcoR V

Dra III 3348 BsaA I 3348 NgoM I 3245

Nae I 3245

<img file="PL188795B1_D0041.tif" />

2335 EcoK

Eam1105l 3000 Bsa I 2933 Gsu I 2915 Ase I 2829

Fspl 2780

Pvu I 2633 Merge 2522

670 BssH II

706 No. I 715 BspE I

914 PUM I 1074 AgeI 1077 BslE II and 1097 BsmI - 1135 Mlu 1 “1239 BamHI '1300 Nhel

FIG. 6A

1456 Ecl136 I 1456 Sac. 1 1471 Smal 1471 Xmal 1534 Rsrll

1640 Stul

1641 SfiI ·

1654 Hpal 1659 Eagl 1662 Dsal 1666 Sfi I

1674 Mscl 1684 Accl 1684 Hall

1688 BspMI

1689 Sse8337 I 1696 Sphl 1702 Hindlll

188 795

The pCMG14 library vector, the cloning site at the 3 'end of the helmet domain polymer gene

<img file="PL188795B1_D0042.tif" />

<td>tf</td><td> 0</td><td></td>

<td>H</td><td>at</td><td></td>

<td>σι υ</td><td></td><td>at</td>

<td>Λ3</td><td> 0</td><td>at</td>

<td>tf</td><td>at</td><td> 0</td>

<td>In 55</td><td></td><td>AND</td>

<td></td><td> 3</td><td></td>

<img file="PL188795B1_D0043.tif" />

C4

<img file="PL188795B1_D0044.tif" />

C5 0

ol

P &lt;

about

U 0 0 0 0 oo

about

Lu

188 795

Average platelet count (x wz / ul)

<img file="PL188795B1_D0045.tif" />

BALB / c mice received carboplatin on day 0 (125mg / kg, ip)

1. The dashed lines represent the controls from 3 experiments

2. Solid lines represent carboplatin treated groups from 3 experiments

3. Thick solid lines represent historical data from TRUrlich et al.

FIG 7 Blood 86 (3): 971-976.1995

188 795

Effect of carboplatin titration on mouse platelet count (mg / kg)

1200 _T

Average platelet count (x 10'3 / μΙ)

1000 ··

800 ·

600

400 -

200

<img file="PL188795B1_D0046.tif" />

day

Carboplatin (mg / kg, ip) given on day 0

FIG. 8

188 795

Improvement of incubated carboplatin thrombocytopenia on day 10, under the influence of AF12513:

Average platelet count (x 10'3 / ul)

Carboplatin titration

<img file="PL188795B1_D0047.tif" />

1. Carboplatin (CBP; 125-50mg / kg, ip) was given on day 0

2. AF 12513 (513; 1mg / kg; ip) was administered on days 1-9 p = &lt;0.05

188 795

FROM S70 ° · ° · ⁵⁷⁰

<img file="PL188795B1_D0048.tif" />

FIG. 1A

1.8·

1.61.41.210.80.60.40.2·

1E-8

AF1228S AF12193

AF12434 matrix 'matrix AF12359 matrix matrix AF12405: this cell line and this cell line permanent cell line this cell line and cell line ł = B = S = rrri1E-6

1E-7

Logarithm of peptide concentration

1E-S

FIG. 1B

UP Department of Publications. Circulation of 50 copies Price PLN 6.00.