Peptides and compounds that bind to a thrombopoietin receptor.

PCT WORLDINTELLECTUAL PROPERTY ORGANLZATION * ^x Intenutioail Bureau -

INTERNATIONAL APPUCATION PUBLISHED UNDER THE PATENT COOPERATED TREATY (PCT)

<td>(51) Interaatlanal Patent OasriBcatlon ®: C07K 2/7, 7/06, 7/08, 7/50, 7 / 54,14 / 52</td><td>To the</td><td colspan="2">(11) International Publication Number: WO 96/40750 (43) International Publication Date: 19 Decanber 1996 (19.12.96)</td>

<td colspan="3">(21) International AppUcatfon Number: PCT / US96W623 (22) International FUing Date: 7 June 1996 (07.06.96) (30) Priority Data: 08 / 485,301 7 June 1995 (07Ό6.95) US 08 / 478,128 7 June 1995 (07.06.95) US (60) Parent Applfcatkms ar Granb (63) Related by Cofltinuation US 08 / 478,128 (CIP) Filed on 7 June 1995 (0706.95) US 08 / 485,301 (CIP) Filed «i 7 June 1995 (07.06.95) (71) Applicant (/ iw all designated Statrs except US) i GLAXO OROUP LIMTTED [GB / GBJ; Glaxo Wellcome House, Berkeley Avenue, Greenfond, Middlesex UB6 0NN (GB). (72) Inventor; and (75) Inventor / Applicants (for US otdy): DOWER, William, J. [US / USJ; 2307 Branner Avenue, Menlo Partí, CA 94025 (US). BARRET, RonaM, W. [US / USJ; 12900 Arroyo de ArqueUo, Saratoga, CA 95070 (US). CWIRLA, Steven, E. [US / USJ; 111 Hedge Road, Menlo Park. CA 94025 (US).</td><td>DUFFIN, David, J. [US / US]; 1735 Woodland Avenue # 13, East Polo Alto, CA 94303 (US). GATES, Christian, M. [US / USJ; 5770 A Croy Road, Morpn HUI, CA 95037 (US). JOHNSON, Sherril, S. [US / USJ; 27 Eaatridge Orive, Santa Cruz, CA 95060 (US). MATTHEAK1S, Lany, C. [US / USJ; 20612 Sunrise Drive, Cupertino, CA 95014 (US). SCHATZ, Peter, J. [US / USJ; 2080 Marich Way # 15, Mountain Vfew, CA 94040 (US). WAGSTROM, Christopher, R. [US / US]; 1909 Paredón Avenue, Loe Altos, CA 94024 (US). WRTGHTON, Nlcholas, C. [GB / USJ; 947 Standord Avenue. Palo Alto, CA 94306 (US). (74) Agent; FITTS, Renee, A. et al .; Townsend and Townsend and Crew LLP., 8th floor, Two Embarcadero Ccnter, San Francisco, CA 94111 -3834 (US). (81) Derignated States: AL, AM, AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SL SK, TI, TM, TR, ΊΤ, UA, UO, US, UZ, VN, ARIPO patent (KE, LS, MW. SD, SZ, UG), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, 17, TM), European patent (AT, BE, CH, DE, DK, ES, Π, FR, GB, GR, IE, IT, LU, MC, NI, PT, SE), OAPI patent (BF, BJ, CE, CO. CI. CM, GA, GN, ML, MR, NE, SN, TD, TG). Publbhed WítA Íntemoffónd rearch nport</td>

<td colspan="4">(54) Tille: PEPTIDES AND COMPOUNDS THAT BIND TO A THROMBOPOIETIN RECEIVER (57) Ahrtract Receptor are peptides and peptide mimetic that bind to and activate the thrombopoictin receptor. Such peptides and peptide mimetics are useftjl in methoda for treating hetnatologica! disoideri and particularty, thrombocytopenia resultíng from chcmothcrapy, radiation therapy, or bone marrow tnnsñisions as well as in diagnostic methoda employing labeled peptides and peptide mimetics.</td>

PEPTIDES AND COMPOUNDS THAT JOIN A RECEIVER OF

THROMBOPOYETINE BACKGROUND OF THE INVENTION

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

Megakaryocytes are cells derived from the bone marrow, which are responsible for producing circulating blood platelets. Although they comprise &lt;0.25% of the spinal cord cells in most species, they are&gt; 10 times the volume of typical marrow cells. See Kuter et. to the. Proc. Nati. Acad. Sci. USA 91: 11104-11108 (1994). Megakaryocytes undergo a process known as endomitosis by means of which it replicates in its nucleus but they fail to undergo cell division, and therefore they give rise to polyploid cells. In response to a decreased platelet count, the endomitotic rate increases, more polypolyid megakaryocytes form, and the number of megakaryocytes can increase up to three times. See Harker J. Clin. Invest. 47: 458-465 (1968). In contrast, in response to a high platelet count, endomitotic velocity decreases, fewer chloid megakaryocytes form, the number of megakaryocytes may decrease by 50%.

The exact physiological feedback mechanism by which circulating platelet mass regulates endomitotic velocity and the number of megakaryocytes in the bone marrow is unknown. The circulating thrombopoietic factor involved in mediating this feedback loop is now thought to be thrombopoietin (TPO). More specifically, TPO has been shown to be the primary humoral regulator in situations involving thrombocytopenia. See, for example, Metcalf Nature 369: 519520 (1994). TPO has been shown in several studies to increase platelet counts, increase platelet size, and increase isotope incorporation into platelets from recipient animals. Specifically, TPO is thought to affect megakaryocytopoiesis in several ways: (1) it produces increases in the size and number of megakaryocytes; (2) produces an increase in DNA content, in the form of polyploidy, in megakaryocytes; (3) increases megakaryocytic endomitosis; (4) produces increased maturation of megakaryocytes; and (5) produces an increase in the percentage of precursor cells, in the form of small actylcholinesterase positive cells, in the bone marrow.

Because platelets (thrombocytes) are necessary for both coagulation and when their numbers are very low, a patient is at serious risk of death from catastrophic bleeding, OPT has a useful potential application in both diagnosis and treatment of various hematological diseases, for example diseases due primarily to platelet defects. Ongoing clinical trials with TPO have indicated that TPO can be safely administered to patients. Furthermore, recent studies have provided a basis for projecting the efficacy of TPO therapy in the treatment of thrombocytopenia, and particularly thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transplantation as a treatment for cancer or lymphoma. . See, for example, McDonald (1992) Am. J. Ped. Hematology / Oncology 14: 8-21 (1992).

The gene that codes for TPO has been cloned and characterized. See Kuter et al. Proc. Nati. 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: 533-536 (1994). Thrombopoietin is a glycoprotein with at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, with the common N-terminal amino acid sequence. See, Bartley et al. Cell

77: 1117-1124 (1994). Thrombopoietin appears to have two distinct regions separated by a potential Arg-Arg hydrolysis site. The amino-terminal region is highly conserved in humans and mice, and has some homology to erythropoietin and interferon-a and interferon-b. The carboxy terminal region shows wide divergence in species.

DNA sequences and peptide sequences encoded for human TPO-R (also known as cmpl) have been described. See Vigon et al. Proc. Nati. Acad. Sci. USA 89: 5640-5644 (1992). TPO-R is a member of the hematopoietin growth factor receptor family, a family characterized by a common structural design of the extracellular domain, which includes four conserved C residues in the N-terminal portion and a WSXWS motif close to the transmembrane region. See Bazan Proc. Nati. Acad. Sci. USA 87: 6934-6938 (1990). Evidence that this receptor plays a functional role in hematopoiesis includes observations that its expression is restricted to the spleen, bone marrow, or fetal liver in mice (see Souyri et al. Cell 63: 1137-1147 (1990)) and megakaryocytes. , platelets and CD34 cells⁺ in humans (see Methia et al. Blood 82: 1395-1401 (1993)). Additionally, exposure of CD34 cells⁺ to synthetic antisense oligonucleotides to mpl RNA inhibits

<img file="MX9709315A_D0001.tif" />

significantly the appearance of megakaryocyte colonies, without affecting the formation of erythroid or myeloid colonies. Some researchers postulate that the receptor functions as a homodimer, similar to the situation with G-CSF and erythropoietin receptors.

The availability of cloned genes for TPO-R facilitates research for agonists of this important receptor. The availability of the recombinant receptor protein allows the study of receptor-ligand interaction in a variety of random and semi-random peptide rate generation systems. These systems include the peptide-on-plasmid systems described in US Patent Nos. 5,270,170 and 5,338,665; the phage peptide system described in US Patent Application Serial No. 07 / 718,577, filed June 20, 1991, US Patent Application No. 07 / 541,108, filed June 20, 1990, and in Cwirla et al., Proc. Nati. Acad. Sci. USA 87: 6378-6382 (1990); the polysome system described in North American Patent Application with No. Serial No. 08 / 300,262, filed September 2, 1994, which is a continuation-in-part application based on North American Patent Application Serial No. 08 / 144,775, filed October 29, 1993 and PCT WO 95/11992; the encoded synthetic library system described in the Patent Application

North American with Serial No.

08 / 146,886, filed on November 1993,

07 / 946,239, filed on September 1992, and

07 / 762,522, filed on September 1991; and the very large scale immobilized polymer synthesis system described in US Patent No. 5,143,854; the

PCT Patent Publication No. 90/15070, published December 13, 1990; US Patent Application Serial No. 07 / 624,120, filed on December 6, 1990; Fodor et al. Science 251: 767-773 (2/1991); Dower and Fodor Ann. Rep. Med. Chem. 26: 271-180 (1991); and the North American Patent Application with No. of Series 07 / 805,727, filed on December 6, 1991; each of the foregoing patent applications and publications is incorporated herein by reference.

The slow recovery of platelet concentrations in patients suffering from thrombocytopenia is a serious problem, and has urged the investigation of a blood growth factor agonist capable of accelerating platelet regeneration. The present invention provides such an agonist.

BRIEF DESCRIPTION OF THE INVENTION

This invention is directed, in part, to the novel and unexpected discovery that defined low molecular weight mimic peptides and peptides have strong TPO-R binding properties and can activate TPO-R. Accordingly / such peptides and mimic peptides are useful for therapeutic purposes in the treatment of conditions mediated by TPO (eg, thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transfusions) as well as for diagnostic purposes in the study of the mechanism of hematopoiesis, and for the in vitro expansion of megakaryocytes and registered progenitor cells.

Peptides and mimic peptides suitable for therapeutic and / or diagnostic purposes have an IC₅₀ of about 2 mM or less, determined by the binding affinity assay set forth in Example 3 below, where an IC₅or less correlates to stronger binding affinity for TPO-R. For pharmaceutical purposes, the mimic peptides and peptides preferably have an IC₅₀ not more than about 100 pm, more preferably, not more than 500 nM. In a preferred embodiment, the molecular weight of the peptide or mimic peptide is from about 250 to about 8000 Daltons.

When used for diagnostic purposes, mimic peptides and peptides are preferably labeled with a detectable marker, and accordingly mimic peptides and peptides without such a marker serve as intermediates in the preparation of labeled mimic peptides and peptides.

Peptides meeting the defined criteria for molecular weight and binding affinity for TPO-R comprise 9 or more amino acids, where the amino acids are naturally occurring or synthetic (not naturally occurring) amino acids. Mimic peptides include

<td>peptides that</td><td>have one or</td><td>plus</td><td>of</td><td>the</td><td colspan="2">following</td>

<td>modifications:</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>peptides</td><td>where one or</td><td>plus</td><td>of</td><td>the</td><td>unions</td><td>of</td>

<td colspan="2">peptidyl [-C (O) NR ~] (links)</td><td>have</td><td>been</td><td colspan="2">replaced</td><td>by</td>

a non-peptidyl bond, such as a -CH bond₂-carbamate [-CH₂-OC (O) NR-]; a phosphonate bond; a -CH link₂-sulfonamide [-CH₂-S (O)₂NR-J; a urea bond [-NHC (O) NH-]; a union of -CH₂-secondary amine; or an alkylated peptidyl linkage [-C (O) NR⁶-] where R⁶ it is lower alkyl];

peptides where the N-terminus is derivatized to a -NRR group¹; to a group -NRC (O) R; to a group -NRC (O) OR; to a group -NRS (O)₂R; to a group -NHC (O) NHR where R and R¹are hydrogen or lower alkyl, with the proviso that R and R¹ they are not both hydrogen; to a succinim group; to a benzyloxycarbonyl-ΝΉ- (CBZ-NH-) group; or to a benzyloxycarbonyl-NH- group having from 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chlorine, and bromine; or peptides where the C-terminus is derivatized to -C (O) R², where R² is selected from the group consisting of lower alkoxy, and -NR³R⁴ where R³ and R⁴ they are independently selected from the group consisting of hydrogen and lower alkyl.

Accordingly, the preferred mimic peptides and peptides comprise a compound having:

&lt;1} a molecular weight of less than about 5000 Daltons, and (2) a TPO-R binding affinity expressed by an IC₅or not more than about 100 pm, where from zero to all the -C (O) NH- junctions of the peptide have been replaced by a bond selected from the group consisting of a -CH junction₂OC (0) NR-; a phosphonate bond; a -CH link₂S (O) ₂NR-; a -CH link₂NR-; and a -C (O) NR bond⁶-; and a bond -NHC (O) NH- where R is hydrogen or lower alkyl and R⁶ it is lower alkyl;

further wherein the N-terminus of the peptide or mimic peptide is selected from the group consisting of a group -NRR¹; a -NRC (O) R group; a -NRC (O) OR group; a group -NRS (O)₂R; a -NHC (O) NHR group; a succinimide group; a benzyloxycarbonyl-NH- group; and a group

-1010 benzyloxycarbonyl-NH having 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chlorine, and bromine, where R and R¹ are independently selected from the group consisting of hydrogen and lower alkyl, and still further wherein the C-terminus of the peptide or mimic peptide has the formula -C (O) R², where R² is selected from the group consisting of hydroxy, lower alkoxy, and -NR³R⁴, where R³ and R⁴ are independently selected from the group consisting of hydrogen and lower alkyl, and wherein the nitrogen atom of the group -NR³R⁴ it may optionally be the amino group of the N-terminus of the peptide, such that it forms a cyclic peptide, and the physiologically acceptable salts thereof.

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

In some embodiments of the invention, preferred peptides to be used include peptides having a core structure comprising an amino acid sequence:

-1111

Χ1 Χ₂ Χ₃ Χ₄ x₅ χ₆ Χν

<td colspan="4">where Xi is C, L, M,</td><td colspan="2">P, Q, V; X₂ is</td><td>F, K, L, N, Q, R,</td><td>yes</td>

<td>T 0</td><td>V; X₃ is</td><td>C,</td><td>F, I, L,</td><td>, M, R, S, V</td><td colspan="2">or W; X₄ is either</td><td>of</td>

<td>the</td><td colspan="2">20 L-amino</td><td>acids</td><td>coded</td><td colspan="2">genetically; X₅ is</td><td>TO,</td>

<td>D,</td><td>E, G, K,</td><td>M,</td><td>Q, R, S</td><td>, T, V or Y;</td><td>X₆</td><td>is C, F, G, L, M,</td><td>yes</td>

<td>v,</td><td>W or Y; and</td><td>X7</td><td>is C, G,</td><td>I, K, L, M,</td><td>N,</td><td>R or V.</td><td></td>

<td></td><td>In a</td><td colspan="2">modality</td><td>preferred,</td><td>the</td><td colspan="2">nucleus peptide</td>

it comprises an amino acid sequence:

<td colspan="2"></td><td colspan="6">X₀ G Xi X₂ X₃ X₄ X₅ WX₇</td>

<td>in</td><td colspan="2">where Xi is L, Μ, P, Q, or</td><td>V;</td><td>X₂</td><td>is</td><td>F, R, S, or T; X₃ is</td><td>F,</td>

<td>L,</td><td>V,</td><td>or W; X₄ is A, K, L, M,</td><td>R,</td><td>Yes,</td><td>v,</td><td>0 T; Xs is A, E, G,</td><td>K /</td>

<td>M,</td><td>Qr</td><td>R, S or T; X₇ is C, I,</td><td>K,</td><td>L,</td><td>M</td><td>or V; and each residue</td><td>of</td>

Χθ is independently selected from any of the 20 genetically encoded L-amino acids, its stereoisomeric D-amino acids; and unnatural amino acids. Preferably, each residue of X₈ it is independently selected from any of the 20 genetically encoded L-amino acids and their stereoisomeric D-amino acids. In a preferred embodiment, Xi is P; X₂its T; X₃ is L; X₄ is R; X $ is E or Q; and X₇ is I or L.

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

X₉ X₈ G Xi X₂ X₃ X &lt;Xs WX₇

-1212 where Xg is A, C, E, G, 1 / L, Μ, P, R, Q, S, T, or V; and Xg is A, C, D; E, K, L, Q, R, S, T, or V. More preferably, Xg is A or I; and Xs is D, E, or K.

Particularly preferred peptides include: GG

<td> 5</td><td>C</td><td>TO</td><td>D</td><td>G</td><td>P</td><td>TLR</td><td>AND</td><td>W</td><td>I</td><td>S</td><td>F</td><td>CGG;</td><td>GNADGP</td><td>TL</td><td>R</td><td>Q</td><td>W</td><td>L</td><td>AND</td>

<td></td><td>G</td><td>R</td><td>R</td><td>P</td><td>K</td><td>N; GG</td><td>C</td><td>TO</td><td>D</td><td>G</td><td>P</td><td>TLR</td><td>EWISFC</td><td colspan="2">GGK;</td><td>T</td><td>I</td><td>K</td><td>G</td>

<td></td><td>P</td><td>T</td><td>L</td><td>R</td><td>Q</td><td>WLK</td><td>S</td><td>R</td><td>AND</td><td>H</td><td>T</td><td>S; YES</td><td>EGPTLR</td><td>EW</td><td>L</td><td>T</td><td>s</td><td>R</td><td>T</td>

<td></td><td>P</td><td>H</td><td>S;</td><td>I</td><td></td><td>k IEG</td><td>P</td><td>T</td><td>L</td><td>R</td><td>Q</td><td>WLH</td><td>GNGRDT;</td><td>AC</td><td>D</td><td>G</td><td>P</td><td>T</td><td>L</td>

<td></td><td>R</td><td>AND</td><td>w</td><td>I</td><td>s</td><td>FC; and</td><td>I</td><td>AND</td><td>G</td><td>P</td><td>T</td><td>LRQ</td><td>WLAAR A.</td><td></td><td></td><td></td><td></td><td></td><td></td>

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

CX₂ X₃ X₄ X₅ X_&amp; X7 where X₂ it is K, L, N, Q, R, S, T or V; X₃ is C, F, I, L, M,

R, S or V; X₄ it is any of the 20 genetically encoded L-amino acids; X₅ it is A, D, E, G, S, V or Y; X₆ is

C, F, G, L, M, S, V, W or Y; X-? is C, G, I, K, L, Μ, N, R or

V. In a more preferred embodiment, X₄ is A, E, G, Η, K, L,

Μ, P, Q, R, S, T, or W. In an additional embodiment, X₂ is S or T; X₃ is L or R; X₄ is R; X $ is D, E, or G; X₆ is F, L, or W; and X₇ it is I, K, L, R, or V. Particularly preferred peptides include: GGCTLREWLHGGFCGG.

-1313

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

<td>Xa CX₂ X₃ X₄ Xs X₆ X?</td>

<td>where X₂ is F, K, L, N, Q, R, S, T, or V; X₃ is C, F, I, L,</td>

<td>M, R, S, V or W; X₄ is any of the 20 L-amino acids</td>

<td>genetically encoded; X₅ is A, D, E, G, K, M, Q, R, S,</td>

<td>T, V or Y; X₆ is C, F, G, L, M, S, V, W or Y; X₇ is C, G, I,</td>

K, L, Μ, N, R or V; and Χθ is any of the 20 genetically encoded L-amino acids. In some embodiments, Xa is preferably G, S, Y, or R.

The compounds described herein are useful for the prevention and treatment of TPO mediated diseases, and particularly for treating hematologic disorders, including, but not limited to, thrombocytopenia resulting from chemotherapy, radiation therapy, or bone marrow transfusions. Thus, the present invention also provides a method of treatment, wherein a patient having a disorder that is amenable to treatment with a TPO agonist receives, or is administered, a therapeutically effective dose or amount of a compound of the present invention. .

The present invention also provides pharmaceutical compositions comprising one or more of the compounds described herein, and a carrier.

-1414 Physiologically acceptable. These pharmaceutical compositions can be in a variety of forms including oral dosage forms, as well as powders and inhalable solutions, and solutions for injection and that can be administered by infusion.

BRIEF DESCRIPTION OF THE FIGURES

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 the proliferation assay in TPO-R transfected Ba / F3 cells for selected peptides of the invention:

designates results for GGCADGPTL REWISFCGGK (biotin);

X designates the results for GG.CADGPTLR EWISFCGG;

^TO designates results for LAIEGPTLR QWLHGNGRDT;

or designates the results for GNADGPTLRQ WLEGRRPKN; and + designates the results for TIKGPTLRQW LKSREHTS.

<td>The figure</td><td>IB</td><td>is a</td><td>representation</td><td>graph</td><td>of the</td>

<td>results with</td><td>the</td><td>themselves</td><td>peptides and the</td><td>line of</td><td>cells</td>

father.

-1515

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

Figures 3A-G show the results of a series of control experiments showing the activity of TPO, the peptides of the present invention, EPO, and peptides that bind to EPO-R in a cell proliferation assay using either the Ba / F3 cell line transfected with TPOR and its corresponding parent line, or an EPO-dependent cell line. Figure 3A depicts the results for TPO in a cell proliferation assay using the TPO transfected Ba / F3 cell line and its corresponding parent line. Figure 3B depicts the results for EPO in a cell proliferation assay using the TPO-R transfected Ba / F3 cell line and its corresponding parent line. Figure 3C represents the results for the complexed biotinylated peptide (AF 12285

-1616 with streptavidin (SA)) and a complexed form of the biotinylated EPO-R binding peptide (AF 11505 with SA) in the Ba / F3 cell line transfected with TPO-R. The results for the corresponding parent cell line are shown in Figure 3D. Figure 3E depicts the results for TPO in the cell proliferation assay using the EPO dependent cell line. Figure 3F depicts the results for EPO in the cell proliferation assay using the EPO dependent cell line. Figure 3G depicts the results for the complexed biotinylated peptide (AF 12885 with streptavidin (SA)) and the complexed form of a biotinylated EPO-R binding peptide (AF 11505 with SA) in the EPO-dependent cell line.

Figures 4A-C illustrate the construction of peptide-over-plasmid libraries in vector pJS142. Figure 4A shows a restriction map and the position of the genes. The library plasmid includes the rrnB transcriptional terminator, the bla gene to allow selection over ampicillin, the M13 phage intragenic region (M13 IG) to allow rescue of single-stranded DNA, an origin of plasmid replication (ori ), two lacO sequences_to, and the araC gene to allow up and down regulation of the araB promoter that handles the expression of the lac fusion gene. Figure 4B shows the sequence of the cloning region in

-1717 the 3 'end of the lac I gene, which includes the Sfil and EagI sites used during library construction. Figure 4C shows the binding of library ringed oligonucleotides, ON-829 and ON-830 to Sfil sites of pJS142 to produce a library. The individual spaces in the sequence indicate binding sites.

Figures 5A-B illustrate cloning into pELM3 and pELM15 MBP vectors. Figure 5A shows the sequence at the 3 'end of the malE fusion gene, including the MBP coding sequence, the poly-aspargine linker, the factor Xa protease hydrolysis site, and available cloning sites. The remaining portions of the vectors are derived from pMALc2 (pELM3) and pMALp2 (pELM15), available from New England Biolabs. Figure 5B shows the sequence of the vectors after the transfer of the BspEII-Scal library fragment into pELM3 / pELM15 digested with Agel-Scal. The transferred sequences include the sequence encoding the GGG peptide linker from the pJS142 library.

Figure 6A depicts a restriction map and position of genes for construction of libraries of the main part dimer in vector pCMG14. The library plasmid includes: the rrnB transcriptional terminator, the bla gene to allow selection over ampicillin, the intragenic region of phage M13 (M13

-1818

IG) to allow rescue of single-stranded DNA, an origin of replication of the plasmid (ori), a lacO sequence_sf and the araC gene to allow up and down regulation of the araB promoter that handles the expression of the main part dimer fusion gene. Figure 6B depicts the sequence of the cloning region at the 3 'end of the main dimer gene, which includes the Sfil and EagI sites used during library construction. Figure 6C shows the binding of the ringed ON-1679, ON-829 and ON-830 to Sfil sites of pCMG14 to produce a library. The individual spaces in the sequence indicate binding sites.

Figures 7 to 9 show the results of additional tests evaluating the activity of the peptide and the mimic peptides of the invention. In this assay, mice are made thrombocytopenic with carboplatin. Figure 7 depicts typical results when Balb / C mice are treated with carboplatin (125 mg / kg intraperitoneally) on Day 0. The solid line represents untreated animals from three experiments. The solid line represents carboplatin-treated groups in three experiments. Thick solid lines represent historical data. Figure 8 depicts the effect of carboplatin titration on platelet counts in mice treated with the indicated amounts of

-19carboplatin (in mg / kg, intraperitoneally (ip) on Day 0) Figure 9 depicts the improvement in carboplatin-induced thrombocytopenia on Day 10 by the AF12513 peptide (513). Carboplatin (CBP; 50-125 mg / kg, intraperitoneally) was administered on Day 0. AF12513 (1 mg / kg, ip) was given on Days 1-9.

DESCRIPTION OF SPECIFIC MODALITIES

I. DEFINITIONS AND GENERAL PARAMETERS

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

Agonist refers to a biologically active ligand that binds to its complementary biologically active receptor and activates the latter, either to cause a biological response in the receptor, or to enhance the receptor's pre-existing biological activity.

Pharmaceutically Acceptable Salts refers to the non-toxic alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry, including the sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine salts of zinc, which are prepared by methods well known in the art. The term also includes non-toxic acid addition salts, which are generally prepared by reacting the compounds of this invention with a suitable organic or inorganic acid. Representative salts include hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsilate, and the like.

Pharmaceutically Acidic Acid Addition Salts refers to those salts that retain the biological effectiveness and properties of free bases, and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, acid nitric, 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 pharmaceutically acceptable acid addition salts as prodrugs, see Bundgaard, H., supra.

Pharmaceutically acceptable ester refers to those esters that retain, with the hydrolysis of the ester bond, the biological effectiveness and properties of carboxylic acid or alcohol, and that are not undesirable

-2121 biologically or otherwise. For a description of pharmaceutically acceptable esters as prodrugs, see Bundgaard, H., editor, Design of Prodrugs, Elsevier Science Publishers, Amsterdam (1985). These esters are typically formed from the corresponding carboxylic acid and an alcohol. Generally, ester formation can be accomplished via conventional synthetic techniques. (See, eg, March Advanced Organic Chemistry, 3rd Ed., John Wiley &amp; Sons, New York (1985) page 1157 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 comprise (i) an aliphatic alcohol of 2 to 12 carbon atoms that may or may not contain one or more double bonds, and may or may not contain branched carbons, or (ii) aromatic or heteroaromatic alcohols from 7 to 12 carbon atoms. This invention also contemplates the use of these compositions, which are both esters as described herein and at the same time are the pharmaceutically acceptable acid addition salts thereof.

Pharmaceutically acceptable amide refers to those amides that retain, with the hydrolysis of the amide bond, the biological effectiveness and properties of the carboxylic acid or amine, and that are not biologically or otherwise undesirable. For a description of

-2222 pharmaceutically acceptable amides as prodrugs, see Bundgaard, H., editor, Design of Prodrugs, Elsevier Science Publishers, Amsterdam (1985). These amides are typically formed from the corresponding carboxylic acid and an amine. Generally, amide formation can be accomplished via conventional synthetic techniques. (See, for example, March, Advanced Organic Chemistry, 3rd Ed._z John Wiley &amp; Sons_z New York (1985) page 1152 and Mark et al. Encyclopedia of Chemical Technology, John Wiley &amp; Sons, New York (1980)). This invention also contemplates the use of these compositions, which are both amides as described herein and at the same time are the pharmaceutically acceptable acid addition salts thereof.

Carrier pharmaceutically or therapeutically

<td colspan="5">acceptable refers to a carrier medium that does not interfere</td>

<td>with effectiveness</td><td>of</td><td>the</td><td colspan="2">biological activity of</td>

<td>active ingredients,</td><td>and</td><td>than</td><td>not</td><td>is toxic to the host or</td>

<td>patient.</td><td></td><td></td><td></td><td></td>

<td>Stereoisomer</td><td>I know</td><td colspan="2">refers</td><td>to a chemical compound that</td>

it has the same molecular weight, chemical composition, and constitution as another, but with the atoms grouped differently. That is, certain identical chemical portions are in different orientations in space, and therefore, when pure, they have the ability to rotate the plane of polarized light. However, some

-2323 pure stereoisomers may have an optical orientation that is so small that it is not detectable with the instrumentation present. The compounds of the present invention can have one or more asymmetric carbon atoms, and therefore include several stereoisomers. All stereoisomers are included within the scope of the invention.

Therapeutically or pharmaceutically effective amount applied to the compositions of the present invention, refers to the amount of composition sufficient to induce a desired biological result. This result may be the improvement of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system . In the present invention, the result will typically involve a decrease in the immune and / or inflammatory responses to infection or tissue injury.

The amino acid residues in the peptides are abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is lie or I; Methionine is Met or M; Valine is Val or V; Serina is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanina 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

-2424

<td colspan="2">is Trp or W; Arginine is Arg or</td><td>R; and</td><td>Wisteria is</td><td>Gly o</td><td>G.</td>

<td>Additionally, Bu is</td><td>Butoxi,</td><td>Bzl</td><td>is benzyl,</td><td>Cha</td><td>is</td>

<td>cyclohexylamine, Ac is</td><td>acetyl</td><td>, I</td><td>it is methyl.</td><td>Pen</td><td>is</td>

<td>penicillamine, Aib is</td><td colspan="2">amino acid</td><td>isobutyric,</td><td>Nva</td><td>is</td>

<td>Norvalina, Abu is</td><td>acid</td><td colspan="2">aminobutyric,</td><td>Thi</td><td>is</td>

<td>Thienylalanine, Obn is 0-</td><td>benzyl,</td><td>and hyp</td><td colspan="2">it is hydroxyproline.</td><td></td>

In addition to peptides consisting only of naturally occurring amino acids, mimic peptides or peptide analogs are also provided. Peptide analogues are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are called mimic peptides or mimic peptides (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS ​​page 392 (1985); and Evans et al. J. Med. Chem. 30: 1229 (1987), which are incorporated herein by reference). Mimic peptides that are structurally similar to therapeutically useful peptides can be used to produce an improved therapeutic or prophylactic or equivalent effect. In general, mimic peptides are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), such as a naturally occurring receptor-binding polypeptide, but have one or more

-2525 peptide junctions optionally replaced by a junction selected from the group consisting of: -CH₂NH-, -CH₂S-,

-CH2-CH2-, -CH = CH- (cis and trans), -COCH₂-, -CH (OH) CH₂-, and -CH₂SO-, by methods known in the art and further described in the following references: Spatola, AF in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, editors, Marcel Dekker, New York, page 267 (1983) ; Spatola, AF, Vega Bata (March

1983), Vol. 1, Number 3, Peptide Backbone Modifications (overhaul); Morley, Trends Pharm. Sci. (1980) pages 463-468 (general review); Hudson, D. et al., Int J Pept Prot Res L4: 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); Szelke et al.

European Application EP 45665 CA ¢ 1982): 97: 39405 (1982) (-CH (OH) CH₂-); Holladay et al. Tetrahedron Lett 24: 44014404 (1983) (-C (OH) CH₂-); and Hruby Life Sci. 31: 189-199 (1982) (-CH₂-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide junction is -CH₂NH-. Such mimic peptides may have significant advantage over polypeptides of the embodiments, including, for example:

-2626 cheaper production, increased chemical stability, improved pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (eg, a broader spectrum of biological activities), reduced antigenicity, and others.

The labeling of the mimic peptides generally involves the covalent attachment of one or more labels, directly or through a spacer (eg, an amide group), to position (s) of non-interference on the mimic peptide that are predicted by quantitative structure-activity and / or molecular modeling data. Such non-interference positions are generally positions that do not directly contact the macromolecules (s) (eg, molecules of the immunoglobulin superfamily) to which the mimic peptides bind to produce the therapeutic effect. Formation of derivatives (eg, labeling) of mimic peptides should not substantially interfere with the desired biological or pharmacological activity of the mimic peptide. Receptor-binding peptide-mimicking peptides generally bind to the receptor with high affinity, and possess detectable biological activity (i.e., are agonists or antagonists of one or more receptor-mediated phenotypic changes).

-2727

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

Synthetic or naturally occurring amino acids refer to amino acids that do not naturally exist in vivo, but which, however, can be incorporated into the peptide structures described herein. Preferred synthetic amino acids are naturally occurring Da-amino acids from naturally occurring La-amino acids as well as D- and non-naturally occurring La-amino acids represented by the formula HINCHR⁵COOH where R⁵ is 1) a lower alkyl group, 2) a cycloalkyl group from 3 to 7 carbon atoms, 3) a heterocycle with from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen, 4) an aromatic residue from

-2828 to 10 carbon atoms, optionally having from 1 to 3 substituents on the aromatic nucleus selected from the group consisting of hydroxyl, lower alkoxy, amino, and carboxyl, 5) -alkylene-Y where alkylene is an alkylene group from 1 to 7 carbon atoms and Y is selected from the group consisting of (a) hydroxy, (b) amino, (c) cycloalkyl and cycloalkenyl from 3 to 7 carbon atoms, (d) aryl from 6 to 10 carbon atoms , optionally having from 1 to 3 substituents on the aromatic nucleus, selected from the group consisting of hydroxyl, lower alkoxy, amino and carboxyl, (e) heterocycle from 3 to 7 carbon atoms and 1 to 2 heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen, (f) -C (O) R² where R² is selected from the group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy, and -NR³R⁴ where R³ and R⁴are independently selected from the group consisting of hydrogen and lower alkyl, (g) -S (O)_nR⁶ where n is an integer from 1 to 2 and R⁶ is lower alkyl, and with the proviso that R⁵ do not define a side chain of a naturally occurring amino acid.

Other preferred synthetic amino acids include amino acids where 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.

-2929

Particularly preferred synthetic amino acids include, by way of example, naturally occurring L-amino acids D-amino acids, Ll-naphthyl-alanine, L-2-naphthylalanine, L-cyclohexylalanine, L-2-aminoisobutyric acid, sulfoxide derivatives and methionine sulfone (i.e. HOOC- (H₂NCH) CH₂CH₂-S (O) _nR⁶) where ny R⁶ are as defined above, as well as the lower alkoxy derivative of methionine (i.e. HOOC (H₂NCH) CH₂CH₂-OR⁶ where R⁶ is as defined above).

Detectable marker refers to materials, which when covalently linked to the mimic peptides and peptides of this invention, allow detection of the peptide and mimic peptides in vivo in the patient to whom the mimic peptide or peptide has been administered. Suitable detectable labels are well known in the art and include, for example, radioisotopes, fluorescent labels (eg, fluorescein), and the like.

The particular detectable marker employed is not critical, and is selected relative to the amount of marker to be employed, as well as the toxicity of the marker to the amount of marker employed. Selection of the marker relative to such factors is within the skill of the art.

Covalent binding of the detectable marker to the peptide or mimic peptide is accomplished by very conventional methods.

-3030 known in the art. For example, when the radioisotope is used¹²⁵I as the detectable marker, the oval union of ¹²⁵I to the mimic peptide or peptide can be accomplished by incorporating the amino acid tyrosine into the mimic peptide or peptide, and then iodizing the peptide. If tyrosine is not present in the mimic peptide or peptide, incorporation of tyrosine into the N or C terminus of the mimic peptide or peptide can be accomplished by well known chemistry. Also, the³²P can be incorporated into the mimic peptide or peptide as a phosphate moiety through, for example, a hydroxyl group on the mimic peptide or peptide using conventional chemistry.

II. REVISION

The present invention provides compounds that bind to and activate TPO-R or otherwise behave like a TPO agonist. These compounds include main peptide compounds and derived compounds constructed to have the same or similar molecular structure or shape as the main compounds, but which differ from the main compounds either with respect to susceptibility to hydrolysis or proteolysis and / or with with respect to other biological properties, such as increased affinity for the receptor. The present invention also provides compositions comprising an effective amount of a TPO agonist, and more.

-3131 particularly a compound, which is useful for treating hematologic disorders, and particularly, thrombocytopenia associated with chemotherapy, radiation therapy, or bone marrow transfusions.

III. IDENTIFICATION OF THE TPO AGONISTS

Peptides that have a TPO-R binding affinity can be easily identified by generation systems from a variety of random peptides coupled with an affinity enrichment process.

Specifically, systems for generating a variety of random peptides include the peptide-on-plasmid system described in US Patent Nos. 5,270,170 and 5,338,665; the phage peptide system described in US Patent Application Serial No. 07 / 718,577, filed June 20, 1991 which is a continuation application in part of US Patent Application Serial No. of Series 07 / 541,108, filed on June 20, 1990, and in Cwirla et al-, Proc. Nati. Acad. Sci. USA 87: 6378-6382 (1980); the polysome system described in North American Patent Application Serial No. 08 / 300,262, filed on September 2, 1994, which is a continuation application in part based on North American Patent Application Serial No. 08 / 144,775, filed October 29, 1993 and PCT WO

-3232

95/11992; the encoded synthetic library (ESL) system described in North American Patent Application Serial No. 08 / 146,886, filed on November 12, 1993, which is a continuation application in part of North American Patent Application Serial No. Series 07 / 946,239, filed on September 16, 1992, which is a continuation application in part of the North American Patent Application with No. of Series 07 / 762,522, filed on September 18, 1991; and the very large scale immobilized polymer synthesis system described in US Patent No. 5,143,854; PCT Patent Publication No. 90/15070, published December 13, 1990; US Patent Application Serial No. 07 / 624,120, filed on December 6, 1990; Fodor et al. Science 251: 767-773 (2/1991); Dower and Fodor Ann. Rep. Med. Chem. 26: 271-180 (1991); and North American Patent Application with Serial No. 805,727, filed on December 6, 1991.

Using the procedures described above, random peptides were generally designed to have a defined number of residues and amino acids in length (eg, 12). To generate the collection of oligonucleotides encoding the random peptides, the codon motif (NNK) x, where N is nucleotide A, C, G, or T (equimolar; depending on the methodology used,

-3333 (other nucleotides may be used), K is G or T (equimolar), and x is an integer corresponding to the number of amino acids in the peptide (eg 12) was used to specify any of the 32 possible codons resulting from the motif NNK: 1 each of 12 amino acids, 2 each of 5 amino acids, 3 each of 3 amino acids; and only one of the three stop codons. Thus, the NNK motif encodes all amino acids, encodes only one stop codon, and reduces codon polarization.

In the systems employed, the random peptides were presented either on the surface of a phage particle, as part of a fusion protein comprising either the pIII coat protein or the pVIII of a phage fd derivative (peptides on phage ) or as a fusion protein with the plasmid-linked peptide LacI fusion protein (peptides on plasmids).

The phage or plasmids, including the DNA encoding the peptides, were identified and isolated by an affinity enrichment process using immobilized TPO-R. The affinity enrichment process, sometimes called panning, involves multiple incubation sets of the phage, plasmids, or polysomes with the immobilized receptor, collection of the phage, plasmids, or polysomes that bind to the receptor (along with the DNA or mRNA that the

-3434 accompanies), and the production of more of the phages or plasmids (along with the accompanying LacI peptide fusion protein) collected. The TPOR extracellular domain (ECD) was typically used during panning.

After several rounds of affinity enrichment, the phages or plasmids and accompanying peptides were examined by ELISA to determine if the peptides specifically bound to TPO-R. This assay was carried out similarly to the procedures used in the affinity enrichment process, except that after removing the unbound phage, the wells were typically treated with rabbit anti-phage antibody, then with goat anti-rabbit antibody. 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 the peptide-on-plasmid system is described in detail below.

By comparing the test wells with the control wells (without receptor), one can determine if the fusion proteins specifically bind to the receptor. Phage concentrations found to bind to TPO-R were investigated in a colony-supported probe format, using a radioactively labeled monovalent receptor. This probe can be produced using protein kinase A to phosphorylate a

-3535 Kemptido sequence fused to the C-terminus of a soluble receptor. The engineered form of the TPO receptor is then expressed in host cells, typically CHO cells. Following PI-PLC harvesting of the receptors, the receptor was tested for binding to TPO or TPO-R specific phage clones. The receptor was then marked for high specific activity with³³P to be used as a monovalent probe to identify high affinity ligands using colony lift.

Peptides that were found to specifically bind to the receptor were then synthesized as the free peptide (eg, without phage) and tested in a blocking assay. The blocking assay was carried out in a similar manner to that of the ELISA, except that TPO or a reference peptide was added to the wells before the fusion protein (the control wells were of two types): (1) without receiver; and (2) without TPO or reference peptide). The fusion proteins for which receptor binding was blocked by TPO or the reference peptide contain peptides in the portion of the random peptide that are the preferred compounds of the invention.

TPO-R, as well as its extracellular domain, were produced in recombinant host cells. A useful form of TPO-R was constructed by expressing the protein as a

-3636 soluble protein in baculovirus transformed host cells using standard methods; another useful form is constructed with a signal peptide for protein secretion and for glycophospholipid membrane anchor binding. This form of anchor attachment is called the PIG tail extension. See Caras and Wendell Science 243: 1196-1198 (1989) and Lin et al. Science 249: 677-679 (1990).

Using PIG's tail extension system, one can hydrolyze the receptor from the surface of receptor-expressing cells (eg, transformed CHO cells selected for their high level of receptor expression with a cell distributor or classifier) ​​with phospholipase C. The hydrolyzed receptor is still

<td>comprises a</td><td>sequence</td><td colspan="2">carboxy</td><td>terminal</td><td>from amino</td><td>acids,</td>

<td colspan="2">called the HPAP queue,</td><td>of</td><td>the</td><td>protein</td><td>signal</td><td>for the</td>

<td>union to the</td><td>membrane,</td><td>and</td><td colspan="2">can be</td><td colspan="2">immobilized without</td>

<td>purification</td><td>additional</td><td></td><td>The</td><td>protein</td><td>of the</td><td>receiver</td>

Recombinant can be immobilized by coating the microtiter plate wells with an anti-HPAP tail antibody (Ab 179 or Mab 179), blocking non-specific binding with bovine serum albumin (BSA) in PBS, and then binding the recombinant receptor hydrolyzed to the antibody. Using this procedure, one must perform the immobilization reaction at varying concentrations of the

-3737 receptor, to determine the optimal amount for a given preparation, because different recombinant protein preparations frequently contain different amounts of the desired protein. Furthermore, one must ensure that the immobilizing antibody is completely blocked (blocking) during affinity.

Otherwise, the unblocked antibody can bind to unwanted phages during the affinity enrichment procedure. One can use peptides that bind to the immobilizing antibody to block unbound sites that remain after receptor immobilization, to avoid this problem, or one can simply immobilize the receptor directly to the walls of the microtiter plates, without the help of an immobilizing antibody. View Application

North American patent with

No.

of Series 07 / 947,339, filed on September 18,

1992, incorporated herein by reference.

When using random peptide generation systems that allow multivalent receptor-1gando interaction, one must recognize that the density of the immobilized receptor is an important factor in determining the affinity to bind to the immobilized receptor.

the ligands that can

At higher densities of

-3838 receptor (for example, each well coated with anti-receptor antibody treated with 0.25 to 0.5 mg receptor), multivalent binding is more likely to occur than at lower receptor densities (for example, each well coated with anti-receptor antibody). receptor treated with 0.5 to 1 ng of receptor). If multivalent binding occurs, then one will be more likely to isolate ligands with relatively lower affinity, unless one uses high densities of immobilized receptor to identify major compounds and uses lower receptor densities to isolate higher affinity derived compounds.

To discriminate between high affinity peptides, a monovalent receptor probe is frequently used. This probe can be produced using protein kinase A to phosphorylate a kemptido sequence fused to the C-terminus of the soluble receptor. The engineered form of the TPO receptor is then expressed in host cells, typically CHO cells. Following PI-PLC harvesting of these receptors, the receptor was tested for binding to TPO or TPO-R specific phage clones. The receptor was then marked for high specific activity with ³³P to be used as a monovalent probe to identify high affinity ligands using colony lift.

Preferred research methods to facilitate the identification of peptides that bind to TPO-R

-3939 involve first identifying major peptides that bind to the receptor's extracellular domain, and then making other peptides that resemble the major peptides. Specifically, using a pl 11 or pVI 11 based phage-on-phage system, a random library can be screened to discover a phage displaying a peptide that binds to TPO-R. Phage DNAs are sequenced to determine the sequences of the peptides displayed on the surface of the phages.

Clones capable of specifically binding to TPO-R were identified from a randomized linear 10-mer pVIII library and a random cyclic 10-mer and 12mer pVIII libraries. The sequences of these peptides serve as the basis for the construction of other peptide libraries designed to contain a high frequency of derivatives of the initially identified peptides. These libraries can be synthesized to promote the production of peptides that differ from binding peptides by only a few residues. This approach involves the synthesis of an oligonucleotide with the binding sequence of the binding peptide, except that rather than using pure preparations of each of the four nucleoside triphosphates in the synthesis, one uses mixtures of the four nucleoside triphosphates (i.e. 55% of the correct nucleotide, and

-4040

15% of each of the other three nucleotides is a preferred mixture for that purpose, and 70% of correct nucleotide and 10% of each of the three nucleotides is another preferred mixture for this purpose) in order to generate sequence derivatives coding for the binding peptide.

A variety of strategies were used to form derivatives of the major peptides, making mutagenesis libraries on one subject. These include a library of pVIII phagemid mutagenesis based on the consensus sequence mutagenized at 70: 10: 10: 10 frequency and spread over each term with random residues to produce clones encoding sequence XXXX (C, S, P , or R) TLREWL XXXXXX (C or S). A similar extended / mutagenized library was constructed using the peptide-over-plasmid system to produce clones encoding the sequence XXXXX (C, S, P, or R) TLREWL XXXXXXX. An additional extended / mutagenized library, XXXX (C, S, P, or R) TLREWL XXXXXX (C or S), was constructed using the polysome display system. All three libraries were screened with elution for peptides, and probed with radiovalently labeled monovalent receptor.

Peptide on plasmid techniques were also used to investigate peptides and mutagenesis studies,

-4141 and are described in greater detail in US Patent No. 5,338, 665, which is incorporated herein by reference for all purposes. According to this approach, random peptides are fused at the C-terminus of LacI through the expression of a plasmid vector carrying the fusion gene. The binding of the fusion Lacl-peptide to its decoded DNA occurs via the LacO sequences on the plasmid, forming a stable peptide-LacI-plasmid complex that can be investigated by affinity purification (panning) on ​​an immobilized receptor. Plasmids isolated in this way can then be reintroduced into E. coli by electroporation, to amplify the selected population for additional research cycles, or for the examination of individual clones.

In addition, random peptide and mutagenesis research studies were performed, using a modified C-terminal LacI display system, in which the display valency was reduced (main part dimer display system). Libraries were investigated, and the resulting DNA inserts were cloned as a combination into a maltose binding protein vector (MBP), allowing expression as a protein.

-4242 C-terminal fusion. Cell raw materials from randomly selected individual MBP fusion clones were then assayed for TPO-R binding in an ELISA format, as discussed above.

Peptide mutagenesis studies were also conducted using the polysome display system, as described in co-pending application US Patent Application Serial No. 08 / 300,262, filed September 2, 1994, which is a continued in part based on North American Patent Application No. Series 08 / 144,775, filed October 29, 1993, and PCT WO 95/11992, each of which is incorporated herein by reference for all purposes. A mutagenesis library based on the sequence XXXX (C, P, R, or S) t IreflXXXXXX (CoS) was constructed, in which X represents a random NNK codon, and the letters in the lower box represent amino acid codons that they contain 70: 10: 10: 10 mutagenesis at positions 1 and 2 and K (G or T) at position 3 of the codon. The library was panned for 5 series against the TPO receptor, which had been immobilized on magnetic beads. After the fifth series, the PCR amplified pool was cloned into pAFF6, and the sequence was determined by

-4343 ELISA positive clones. The sequences were subcloned into an MBP vector, and their binding affinities were determined by an MBP ELISA.

To immobilize TPO-R for polysome investigation, Ab 179 was first chemically conjugated to tosyl activated magnetic beads (available from Dynal Corporation) as described by the manufacturer. The beads were incubated with antibody in 0.5M borate buffer (pH 9.5) overnight at room temperature.

The beads were washed and combined with TPO-R containing the HPAP glue. Antibody-coated beads and receptor were incubated for 1 hour at 4^or C, and the beads were washed again before adding the polysome library.

Investigation of the various libraries described above provided the TPO receptor binding peptides shown in Tables 1 and 2 below, as well as others not listed herein.

-4444

Table 1

Pep-tida

RQ w

<img file="MX9709315A_D0002.tif" />

RQW

<td>AND</td><td>G</td><td>P</td><td>T</td><td>L</td><td>R</td><td>G</td><td>W</td><td>L</td><td>A 1</td>

<td>R</td><td>AND</td><td>G</td><td>Q</td><td>T</td><td>L</td><td>K</td><td> £</td><td>W</td><td>ly</td>

<td>AND</td><td>R</td><td>G</td><td>P</td><td>F</td><td>W</td><td>TO</td><td> :&lt;</td><td>TO</td><td>c II</td>

<td> 1 ^c</td><td>• i</td><td>Q</td><td>G</td><td>P</td><td>T</td><td> 1»</td><td>T</td><td>TO</td><td>w</td><td>L »</td><td>L</td><td>c.</td><td>G</td>

<td>c</td><td>TO</td><td>D</td><td>G</td><td>P</td><td>T</td><td>L</td><td>R</td><td>AND</td><td>w</td><td>I</td><td>s</td><td>F</td><td>C</td>

<td>c</td><td> £</td><td>L</td><td>V</td><td>G</td><td>P</td><td>S</td><td>L</td><td>M</td><td>s</td><td>w</td><td>L</td><td>T</td><td>c</td>

-4545

<img file="MX9709315A_D0003.tif" />

<td>s</td><td colspan="2">: e</td><td>G</td><td>P</td><td>T</td><td>L</td><td>R</td><td>AND</td><td>W</td><td>L</td><td>T</td><td>s</td><td>R</td><td>T</td><td>P</td><td>H</td><td>s 1</td>

<td>T</td><td> -</td><td>K</td><td>G</td><td>P</td><td>T</td><td>L</td><td>R</td><td>Q</td><td>w</td><td>L</td><td>K</td><td>s</td><td>R</td><td>AND</td><td>H</td><td>T</td><td>s |</td>

<td>i G</td><td></td><td>TO</td><td>D</td><td>G</td><td>P</td><td>T</td><td>L</td><td>R</td><td> 0</td><td>W</td><td>L</td><td>AND</td><td>G</td><td>R</td><td>R</td><td>P</td><td>KN |</td>

<td>1 s</td><td></td><td>AND</td><td>G</td><td>P</td><td>T</td><td>L</td><td>R</td><td>AND</td><td>w</td><td>L</td><td>T</td><td>s</td><td>R</td><td>T</td><td>P</td><td>H</td><td>S |</td>

<td>1 I</td><td></td><td>D</td><td>G</td><td>P</td><td>T</td><td>L</td><td>K</td><td>AND</td><td>w</td><td>L</td><td>s</td><td>V</td><td>T</td><td>R</td><td>G</td><td>TO</td><td>s N</td>

<img file="MX9709315A_D0004.tif" />

CSLEDLRKRC

CRRSELLERC

CTRGEWLRCC

CTLEELRACC

CTREELMRLC

CQRADLIN'FC

CTLRQWLQGC

CTFKQFLDGC

CNRNDLLLFC

CTLEFMNGC

CSLGELRRLC

CNINQLRSIC

CTMRQFLVCC

CTRSEWLERC

CTLHEYLSGC

CTRSELLRQC

CTFREFVNGC

CSCAQVVQCC

CTLRQWILLGMC

TABLE 2 nesnaEsog

Peptide

<img file="MX9709315A_D0005.tif" />

-4646

ICTLREWLHGGFC_______

CTLRAWLMSETC_______

CTLRAWLMESCC_______

CTFQVWKLARNC_____ '

CLLREWLDXRTC_______

CVLREWLLXXSC_______

CLLSEFLAGQQC_______

CSLRQYLDFGLGSC_____

CTLQELKQSSLYEC_____

CDLSELKTHGYAYC_____

CKLSDWLMMGVAAC_____

CSLQEFLSHGGYVC_____

CSLKEFLHSGLMQC_____

CTFRQLLEYGVSSC_____

CTMREFLVASGVAC_____

CTLAEFLASGVEQC_____

CTLAEFIiASGVEQC_____

CTLKEWLVSHEVWC_____

CTLREFLSLGMNAC_____

ICTLREFLDPTTAVC_____

CSLLEFLALGVALC

GGGRGCTLKQWKQGDCGRS CNRSQLLAAC_________

CTLQQWLSGC_________

CTLREFKAGC_________

IcTRAQFLKGC _______ CTLREFNRGC_________

CTLSDFKRGC_________

ICTFRQWKEAC__________

CTLSEFRGGC_________

CTLQEFLEGC_________

CTLQQWKDGC

-4747

<img file="MX9709315A_D0006.tif" />

<img file="MX9709315A_D0007.tif" />

<img file="MX9709315A_D0008.tif" />

-4848

<td>NYRGCTLSQWVSEQ IV GC</td>

<td>GRSGCTLREYLGGMCYLS</td>

<td>ASWYCTVPELMEMQLPEC</td>

<td>gstgctlrexlhmlgldc</td>

<td>ACEGCTLRQWLEYVRVGC</td>

<td>AQRGCTLQYFVSYGXDMC</td>

<td>GVCGCTLREFLAIPHTSC</td>

<td>SZGGCTLREWVASSLANC</td>

<td>SNSRCTLREWIIQGCDFS</td>

<td>SNSRCTLREWIIQGCDFS</td>

<td>CLGCTLSQWRKRT RCDTH</td>

<td>YRGCSRAQLLGGECRKK</td>

<td>GRGCTLKQWKQGDCGRS</td>

<td>VRGGCALRDWVAGECFDWT</td>

<td>LWRGCTLNGFKSRHCGSPE</td>

<td>CTLRSWKHRGCAP</td>

<td>GRGCTRAQWLAGCCTGH</td>

<td>RAGCTLREFRKGCLAL</td>

<td>KRGCTLAEMIRGCNRSN</td>

<td>GRGCTLKQWKQGDCGRS</td>

<td>RWRGCSLAKLKKGAACGRG</td>

<td>RGGCTLREWRRVRVIN</td>

<td>GRGCTLKQWKQGDCGRS |</td>

<td>RYGCTRHQWLVGTCVRH |</td>

IC values₅or for some additional representative peptides are given in the table below. A variety of methods can be used to evaluate IC values.₅or. For example, an equilibrium binding ELISA assay, using either MBP-TPO or lacl-peptide tracer,

-4949 was used to determine whether the peptides inhibit TPO binding to the TPO receptor extracellular domain. Typically IC values₅₀ were determined using the free peptide. The IC50 value can be determined using the free peptide, which can optionally be C-terminally amidated, or can be prepared as an ester or other carboxy amide.

To recreate the exact sequence displayed on the phage, the N-terminal and C-terminal amino acids of the synthetic peptides are frequently preceded by one or two glycine residues. These glycins are not believed to be necessary for binding or activity. Likewise, to mimic the exact peptide sequence displayed on the polysomes, the C-terminal amino acids of the synthetic peptides are frequently preceded by the M AS sequence. Again, this sequence is not believed to be necessary for binding or activity.

IC50 values ​​are symbolically indicated by the symbols and For example, those peptides that showed IC values₅₀ Greater than 200 μΜ are indicated with a Those peptides that gave IC50 values ​​less than or equal to 200 μΜ are given with a while those that gave IC values₅₀ 500 nm or less are indicated with a Those peptides

-5050 that gave IC values₅or at or near the limit point for a particular symbol are indicated with a hybrid indicator, for example, those peptides for which IC values ​​were not determined₅or are listed as ND :. IC value₅or for peptides having the structure: GGCTLREWLHGGFCGG is 500 nm or less. (Note that the N-terminal and C-terminal amino acids were preceded by two glycins, to recreate the exact sequence exhibited by the phage. These glycins are not believed to be necessary for binding or activity.

TABLE 3

<td>1 Pejid</td><td>Affinity '</td>

<td>| ggcadgptlrewisfcgg</td><td></td>

<td>Gnadgptlrqwlegrrpkn</td><td> ++</td>

<td>JGGCADGPTLREWISFCGGK</td><td> +4-</td>

<td>| ti kgptlrqwlksrehts</td><td> 4·+</td>

<td>Jgptlrqwl</td><td> -</td>

<td>| laxegpti.rq Ȓi, hgngrdt</td><td> ** 1</td>

<td>IsiEGPTLREWLTSRTPHS</td><td> ++ |</td>

The tables above, especially Table 3, illustrate that a preferred core peptide comprises an amino acid sequence:

-5151

Χι χ₂ χ₃ χ₄ χ₅ x_and X?

where Χι is C, L, Μ, 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 it is any of the 20 genetically encoded L-amino acids; X₅ that,

D, E, G, K, M, Q, R, S, T, V or Y; X_and is C, F, G, L, M, S,

V, W or Y; and X₇ is C, G, I, K, L, Μ, N, R or V.

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

<td></td><td colspan="10">X_and G Xi X₂ X₃ X₄ Xs WX₇</td>

<td>in</td><td colspan="2">where xi</td><td>is</td><td>L, Μ, P, Q,</td><td>or</td><td>V;</td><td>X₂ is</td><td>F, R,</td><td>S, or T; X₃ is</td><td>F,</td>

<td>L,</td><td>V,</td><td>or W;</td><td>X₄</td><td>is A, K, L,</td><td>M,</td><td>R,</td><td>S, V,</td><td>or T;</td><td>X₅ is A, E, G,</td><td>K,</td>

<td>M,</td><td>Q,</td><td>R, S,</td><td>or</td><td>T; X₇ is C,</td><td>To go</td><td>K,</td><td>L, M</td><td>or V;</td><td>and each residue</td><td>X₍</td>

is independently selected from any of the 20 genetically encoded amino acids, their stereoisomeric D-amino acids; and unnatural amino acids. Preferably each residue X_g it is independently selected from any of the 20 genetically encoded L-amino acids and their stereoisomeric D-amino acids. In a preferred embodiment, X_: is P; X₂its T; X₃ is L; X₄ is R; X₅ is E or Q; and X? is I or L.

More preferably, the core peptides comprise an amino acid sequence:

X₉ X_and G Xi X₂ X₃ X₄ X₅ WX₇

-5252 where Xg is A, C, E, G, I, L, Μ, P, R, Q, S, T, or V; and Χθ is A, C, D, E, K, L, Q, R, S, T, or V. More preferably, Xg is A or I; and X₈ is D, E, or K.

Particularly preferred peptides include: GG CADGPTLREWISFCGG; GNADGPTLRQWLE GRRPKN; GGCADGPTLREWISFCGGK; TIKG PTLRQWLKSREHTS; SIEGPTLREWLTSRT PHS; LAIEGPTLRQWLHGNGRDT; CADGPTL REWISFC; ylEGPTLRQWLAARA.

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

CX₂ X₃ x &lt;x₅ x₆ Xi where X₂ is K_z L, N, Q, R, S, T or V; X₃ is C, F, I, L, M, R, S or V; X₄ it is any of the 20 genetically encoded L-amino acids; X₅ it 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, Μ, N, R or V. In a more preferred embodiment, X₄ is A, E, G, Η, K, L, Μ, P, Q, R, S, T, or W. In a further embodiment, X₂ is S or T; X₃ is L or R; X₄ is R; X₅ is D, E, or G; X₆ is F, L, or W; and X₇ it is I, K, L, R, or V. Particularly preferred peptides include: GGCTLREWLHGGFCGG.

-5353

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

Χ_θ CX₂ X₃ X₄ X₅ x₆ X?

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; X₄ it is any of the 20 genetically encoded L-amino acids; X₃ it 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, I, K,

L, Μ, N, R or V; and X₈ it is any of the 20 genetically encoded L-amino acids. In some modalities, X_s is G, S, Y or R.

Peptides and mimic peptides that have an IC₅or greater than about 100 mM lack sufficient binding to allow its use in either diagnostic or therapeutic aspects of this invention. Preferably, for diagnostic purposes, the mimic peptides and peptides have an IC₅₀ about 2 mM or less and, for pharmaceutical purposes, the mimic peptides and peptides have an IC_&amp;or about 100 μΜ or less.

The binding peptide sequence also provides a means of determining the minimum size of a TPO-R binding compound of the invention. Using the encoded synthetic library (ESL) system or the very large scale immobilized polymer synthesis system,

-5454 one can not only determine the minimum size of a peptide with such activity, but can also make all of the peptides that form the group of peptides that differ from the preferred motif (or the minimum size of that motif) by one, two or more waste. This collection of peptides can

<td>so</td><td colspan="5">be investigated to determine your</td><td>capacity</td><td>of</td>

<td>union to</td><td>receiver of</td><td>TPO</td><td>. These</td><td>systems</td><td>of</td><td>synthesis</td><td>of</td>

<td>polymer</td><td>immobilized</td><td>or</td><td>others</td><td>methods</td><td>of</td><td>synthesis</td><td>of</td>

Peptides can also be used to synthesize truncation analogs, deletion assays, substitution analogues, and combinations thereof of all the peptide compounds of the invention.

The peptides and mimic peptides of the present invention were also evaluated in a thrombopoietin-dependent cell proliferation assay, described in greater detail in Example 2 below. Cell proliferation is measured by techniques known in the art, such as the MTT assay, which correlates with the incorporation of³H-thymidine as an indication of cell proliferation (see Mossman, J. Immunol. Methods 65:55 (1983)). The tested peptides stimulated the proliferation of Ba / F3 cells transfected with TPO-R, in a dose-dependent manner, as shown in

-5555

Figure 1A. These peptides have no effect on the parent cell line, as shown in Figure IB.

Figures 7 to 9 show the results of one test

<td>additional</td><td colspan="2">which evaluates</td><td>activity</td><td>of</td><td>the</td><td>peptides</td><td>and</td>

<td>peptides</td><td>imitators</td><td>of</td><td>the invention.</td><td>In</td><td>this</td><td>test,</td><td>the</td>

<td colspan="2">mice turn</td><td colspan="2">thrombocytopenic</td><td>with</td><td colspan="2">carboplatin.</td><td>The</td>

Figure 7 illustrates the typical results when Balb / C mice are treated with carboplatin (125 mg / kg intraperitoneally) on Day 0. The non-continuous 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 illustrates the effect of carboplatin titration on platelet counts in mice treated with the indicated amounts of carboplatin (in mg / kg, intraperitoneally (ip) on Day 0). Figure 9 illustrates the improvement in carboplatin-induced thrombocytopenia on day 10 by the AF12513 peptide (513). Carboplatin (CBP; 50-125 mg / kg, intraperitoneally) was administered on Day 0. AF12513 (1 mg / kg, ip) was given on Days 1-9. These results show that the peptides of the invention can improve thrombocytopenia in a mouse model.

-5656

Furthermore, certain peptides of the present invention can be dimerized or oligomerized, thereby increasing the affinity and / or activity of the compounds. To investigate the effect that peptide dimerization / oligomerization has on TPO-mimicking potency in cell proliferation assays, a C-terminally biotinylated analog of the peptide GGCADG PTLREWISFCGG (GGCADGPTLREWISFCG GK (Biotin)) was synthesized. The peptide was previously incubated with streptavidin in RPMI buffered with serum-free HEPES in a 4: 1 molar ratio. The complex was tested to determine the stimulation of cell proliferation of Ba / F3 cells transfected with TPO-R, as above, together with free biotinylated peptide and non-biotinylated parent peptide. Figure 2A shows the results of the assay for the complexed biotinylated peptide (AF 12885 with streptavidin (SA)) for both cell lines, the transfected and the parent. Figure 2B shows the results of the assay for the free biotinylated peptide (AF 12885) for both cell lines, the transfected and the parent. Figure 2C shows the results of the assay for streptavidin alone, for both the transfected and the parent cell lines. These figures illustrate that the

-5757 previously formed complex was approximately 10 times as potent as the free peptide.

The binding specificity and activity of the peptides of the invention was also examined by studying the cross-reactivity of the peptides for the erythropoietin receptor (EPO-R). The

EPO-R is also a member of the hematopoietin growth factor receptor family, such as

TPO-R. The peptides of the invention, as well as

TPO, EPO, and a known EPO-binding peptide, were examined in a cell proliferation assay, using an EPO-dependent cell line. This trial used FDCP-1, a multi-potential primitive hematopoietic progenitor cell line

<td>murine</td><td colspan="2">dependent</td><td>of the</td><td>factor</td><td>of</td><td colspan="2">growth (see, for</td>

<td>example,</td><td>Dexter</td><td>et</td><td>al ·,</td><td>J. Exp.</td><td>Med.</td><td> 152:1036-1047 (</td><td> 1981))</td>

<td>as the</td><td>line</td><td colspan="2">mobile</td><td>father.</td><td>This</td><td>Cellphone line</td><td>can</td>

proliferate, but not differentiate when supplemented with WEHI-3 conditioned medium (a medium containing IL-3, ATCC number T1B68). The parent cell line is transfected with human or murine EPO-R, to produce the FDCP-l-EPO-R cell line.

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

-5858

Cells were cultured at half stationary density, in the presence of the necessary growth factors. Cells were then washed in PBS, and deprived for 16-24 hours in complete medium without growth factors. After determining the viability of the cells, standard solutions were made (in complete medium without growth factors), to give about 10⁵ cells per 50 microliters. Serial dilutions of the compounds (typically the free-phase peptide in solution, as opposed to the phage-linked peptide or other bound or immobilized peptide) were made to be tested, in 96-well tissue culture plates, for a final volume of 50 microliters per well. Cells (50 microliters) were added to each well, and the cells were incubated for 24-48 hours, and at this point the negative controls had to die or be quiescent. Cell proliferation was then measured by techniques known in the art, such as an MTT assay.

Figures 3A-G show the results of a series of control experiments showing the activity of TPO, the peptides of the present invention, EPO, and peptides that bind to EPO-R in a cell proliferation assay, using either the Ba / F3 cell line transfected with TPO and

-5959 its corresponding parent line, or an EPO dependent cell line and its corresponding parent line.

Figure 3A illustrates the results for TPO in the cell proliferation assay, using the Ba / F3 cell line transfected with TPO-R and its corresponding parent line. Figure 3B illustrates the results for EPO in the cell proliferation assay, using the TPO-R transfected Ba / F3 cell line and its corresponding parent line. Figure 3C illustrates the results for the complexed biotinylated peptide (AF 12285 with streptavidin (SA)) and a complexed form of a biotinylated EPO-R binding peptide (AF 11505 with SA) in the Ba / F3 cell line transfected with TPO -R. The results for the corresponding parent cell line are shown in Figure 3D. Figure 3E illustrates the results for TPO in the cell proliferation assay, using the EPO dependent cell line. Figure 3F illustrates the results for EPO in the cell proliferation assay, using the EPO dependent cell line. Figure 3G illustrates the results for the complexed biotinylated peptide (AF 12285 with streptavidin (SA)) and the complex form of a biotinylated EPO-R binding peptide (AF 11505 with SA) in the EPO-dependent cell line. This results

-6060 show that the peptides of the invention bind and activate TPO-R with a high degree of specificity.

IV. PREPARATION OF PEPTIDES AND IMITATING PEPTIDES

A. SOLID PHASE SYNTHESIS

The peptides of the invention can be prepared by classical methods known in the art, for example, using standard solid phase techniques. Standard methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis, and even by recombinant DNA technology. See, eg, Merrifield, J. Am. Chem. Soc., 85: 2149 (1963), incorporated herein by reference. In solid phase, synthesis typically starts from the extreme

C-terminus of the peptide, using a protected alpha-amino resin

A suitable starting material can be prepared, for example, by bonding the required alpha-amino acid to a chloromethylated resin, a hydroxymethyl resin, or a benzhydrylamine resin. Such chloromethylated resin is sold under the trademark BIO-BEADS SX-1 by Bio Rad Laboratories, Richmond, CA, and the preparation of the hydroxymethyl resin is described by Bodonszky et al., Chem. Ind. (London) 38: 1597. (1966). Benzhydrylamine resin (BHA) has been described by Pietta and

-6161 Marshall, Chem. Coima. 650 (1970), and is commercially available from Beckman Instrumente, Inc., Palo Alto, CA, in the hydrochloride form.

Thus, the compounds of the invention can be prepared by coupling a protected alpha-amino amino acid to the chloromethylated resin with the help of, for example, a cesium bicarbonate catalyst, according to the method described by Gisin, Helv. Chim. Acta, 56: 1467 (1973). After initial coupling, the alpha-amino protecting group is removed by a selection of reagents that include solutions of trifluoroacetic acid (TFA) or hydrochloric acid (HC1) in organic solvents at room temperature.

Protecting alpha-amino groups are those known to be useful in the peptide step synthesis technique. Included are acyl-type protecting groups (eg formyl, trifluoroacetyl, acetyl), aromatic urethane-type protecting groups (eg benzyloxycarbonyl (Cbz), and substituted Cbz), aliphatic urethane protecting groups (eg t-butyloxycarbonyl (Boc), isopropyloxycarbonyl , cyclohexyloxycarbonyl), and alkyl-type protecting groups (eg benzyl, triphenylmethyl). Preferred protecting groups are Boc and Fmoc. The protecting group

Side chain -6262 remains intact during coupling, and does not hydrolyze during amino-terminal protecting group deprotection, or during coupling. The side chain protecting group must be capable of being removed upon termination of the final peptide synthesis, and under reaction conditions that will not alter the target peptide.

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

After removal of the alpha-amino protecting group, the remaining protected amino acids are coupled step by step in the desired order. Usually a

-6363 excess of each amino acid protected with an appropriate activator of the carboxyl group, such as dicyclohexylcarbodiimide (DCC) in solution, for example, in mixtures of methylene chloride (CH2CI2) and dimethyl formamide (DMF).

After the desired amino acid sequence has been completed, the desired peptide is decoupled from the resin support, by treatment with a reagent such as trifluoroacetic acid or hydrogen fluoride (HF), which not only hydrolyzes the peptide from the resin , but also hydrolyzes all remaining side chain protecting groups. When the chloromethlated resin is used, treatment with hydrogen fluoride results in the formation of the free acids of the peptide. When benzhydrylamine resin is used, treatment with hydrogen fluoride results directly in the free amide of the peptide. Alternatively, when the chloromethylated resin is employed, the side chain protected peptide can be decoupled by treating the peptide resin with ammonia, to give the desired side chain protected amide, or with an alkylamine, to give a protected alkylamide or dialkylamide side chain. The side chain protection is then removed in the usual way,

-64 by treatment with hydrogen fluoride, to give alkylamide amides, or free dialkylamides.

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

Using the encoded synthetic library system or the very large scale immobilized polymer synthesis system, described in North American Patent Applications Serial Nos. 07 / 492,462, filed March 7, 1990; 07 / 624,120, filed on 6

December 1990; and 07 / 805,727, filed on

December 1991; one can not only determine the minimum size of a peptide with such activity, one can also make all of the peptides that form the group of peptides that differ from the preferred motif (or the minimum size of that motif) by one, two, or more waste. This collection of peptides can then be investigated to determine their ability to bind to TPO-R. This immobilized polymer synthesis system, or other methods of peptide synthesis can also be used to synthesize truncation analogs and deletion analogs, and combinations of truncation analogs and deletion analogs of all the peptide compounds of the invention.

B. AMINO SYNTHETIC ACIDS

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

One can replace the naturally occurring side chains of the 20 genetically encoded amino acids (or D amino acids) with other side chains, for example with groups such as alkyl, lower alkyl, 4-, 5-, 6- or 7-membered cyclic alkyl, amide, amide-lower alkyl, amide-di (lower alkyl), lower alkoxy, hydroxy, carboxy and the ester derivatives

-6666 lower thereof, and with heterocyclic groups of 4, 5, 6,. to 7 members. In particular, proline analogs may be employed in which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members. Cyclic groups can be saturated or unsaturated and if unsaturated, they can be aromatic or non-aromatic.

Cyclic groups can be saturated or unsaturated, and if 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 furazanil, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (for example morpholin), oxazolyl, piperazinyl (for example 1 piperazinyl), piperidyl (for example

1-piperidyl, piperidino), pyranium, pyrazinyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (eg 1pyrrolidinyl), pyrrolinyl, pyrrolyl, thiazololyl, thienyl, thienyl, thienyl, thienyl, thienyl, thyrolyl, thienyl, thyrolyl, thienyl, thyrolyl, thienyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl, thyrolyl triazolyl. These heterocyclic groups can be substituted or unsubstituted. When a group is substituted, the substituent can be alkyl,

-6767 alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

One can also easily modify the peptides of the present invention by phosphorylation, and other methods for making peptide derivatives of the compounds of the present invention are described in Hruby et al.⁴² Thus, the peptide compounds of the invention also serve as a base for preparing mimic peptides with similar biological activity.

The peptide compounds of the invention, including the mimic peptides, can be covalently modified to one or more of a variety of non-proteinaceous polymers, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkenes, in the manner set forth in US Patent No. 4,640,835; US Patent No. 4,496,689; US Patent No. 4,301,144; US Patent No. 4,670,417; North American Patent No. 4,791,192; North American Patent No. 4,179,337; all of which are incorporated by reference in their entirety herein.

C. TERMINAL MODIFICATIONS

Those skilled in the art will recognize that a variety of techniques are available to construct mimic peptides with the same or similar biological activity.

-6868 desired than the corresponding peptide compound, but with more favorable activity than the peptide with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis. See, for example, Morgan and Gainor Ann. Rep. Med. Chem. 24: 243-252 (1989). The following describes methods for preparing modified mimic peptides at the N-terminal amino group, the C-terminal carboxyl group, and / or changing one or more of the amido bonds in the peptide to a non-amido bond. It is understood that two or more of such modifications may be coupled into a 'mimic peptide' structure (eg, modification at the C-terminal carboxyl group, and the inclusion of a -CH bond₂-carbamate between two amino acids in the peptide).

one. N-TERMINAL MODIFICATIONS

The peptides are typically synthesized as the free acid but, as noted above, they could easily be prepared as the amide or the ester. One can also modify the amino and / or carboxy terminus of the peptide compounds of the invention, to produce other compounds of the invention. Modifications to the amino term include methylar (i.e., -NHCH₃ or -NH (CH₃)₂), acetylate, add a carbobenzyloxy group, or block the amino terminus with a blocking group containing a carboxylate functionality defined by RCOO-, where R is

-6969 selects from the group consisting of naphthyl, acridinyl, steroidyl, and the like. Modifications of the carboxy terminus include replacing the free acid with a carboxyamide group, or forming a cyclic lactam at the carboxy terminus, to introduce structural constraints.

Modifications to the amino term are as discussed above, and include renting, acetylating, adding a carbobenzoyl group, forming a succinimido group, etc. Specifically, the N-terminal amino group can then be reacted as follows:

(a) to form an amide group of the formula RC (0) NH-, where R is as defined above, by reaction with an acid halide [eg RC (O) C1] or acid anhydride. Typically, the reaction can be conducted by contacting about equimolar or excess amounts (eg, about 5 equivalents) of an acid halide to the peptide, in an inert diluent (eg, dichloromethane), which preferably contains an excess ( for example, about 10 equivalents) of a tertiary amine, such as diisopropylethylamine, to capture the acid generated during the reaction. Reaction conditions are otherwise conventional (eg, room temperature for 30 minutes). The alkylation of the terminal amino to

-7070 providing a lower alkyl N-substitution, followed by reaction with an acid halide as described above, will provide an N-alkyl amide group of

<td colspan="6">the formula RC (O) NR-;</td>

<td colspan="4">(b) to form a succinimido group, for</td><td colspan="2">reaction with</td>

<td>anhydride</td><td>succinic. How</td><td>before you can</td><td>to be</td><td>employee</td><td>a</td>

<td>quantity</td><td>approximately</td><td>equimolar, or</td><td>a</td><td>excess</td><td>of</td>

succinic anhydride (eg, about 5 equivalents), and the amino group is converted to succinimide by methods well known in the art, including the use of an excess (eg, ten equivalents) of a tertiary amine such as diisopropylethylamine, in a suitable inert solvent (for example, dichloromethane). See, eg, Wollemberg et al., US Patent No. 4,612,132, which is incorporated herein by reference in its entirety. It is understood that the succinic group can be substituted with, for example, alkyl substituents of 2 to 6 carbon atoms or -SR, which are prepared in a conventional manner, to provide the N-terminus substituted succinimide of the peptide. Such alkyl substituents are prepared by reacting a lower definition (2 to 6 carbon atoms) with maleic anhydride, as described by Wollemberg et al., Supra., And the

-7171 -SR substituents are prepared by the reaction of RSH with maleic anhydride, where R is as defined above;

(c) to form a benzyloxycarbonyl-NH- group or a substituted benzyloxycarbonyl-NH group, by reaction with approximately an equivalent amount or an excess of CBz-Cl (i.e., benzyloxycarbonyl chloride) or a CBzCl substituted in a suitable inert diluent (for example, dichloromethane), which preferably contains an amine

<td>tertiary, to capture</td><td>the</td><td>acid generated</td><td>during</td><td>the</td>

<td>reaction;</td><td></td><td></td><td></td><td></td>

<td colspan="2">(d) to form a group</td><td>sulfonamide, by</td><td>reaction</td><td>with</td>

<td>an equivalent amount</td><td>or</td><td>an excess (for</td><td>example,</td><td> 5</td>

equivalent) of RS (O)₂C1 in a suitable inert diluent (dichloromethane), to convert the terminal amine to a sulfonamide, where R is as defined above. Preferably, the inert diluent contains an excess of tertiary amine (eg, ten equivalents), such as diisoproylethylamine, to capture the acid generated during the reaction. Reaction conditions are otherwise

<td>conventional</td><td>(for example,</td><td>temperature</td><td>environment by</td><td> 30</td>

<td>minutes);</td><td></td><td></td><td></td><td></td>

<td>(e) to</td><td>form a group</td><td>carbamate,</td><td>by reaction</td><td>with</td>

<td>an amount</td><td>equivalent or</td><td>An excess</td><td>(for example,</td><td> 5</td>

<td>equivalents)</td><td>from R-OC (O) C1</td><td colspan="2">o R-OC (0) OCeH.-p-NOj in</td><td>a</td>

-7272 suitable inert diluent (eg dichloromethane) to convert the terminal amine to a carbamate, where R is as defined above. Preferably, the inert diluent contains an excess of tertiary amine (eg, about 10 equivalents), such as diisoproylethylamine, to capture the acid generated during the reaction. Reaction conditions are otherwise conventional (eg, room temperature for 30 minutes); and (f) to form a urea group, by reaction with an equivalent amount or excess (eg 5 equivalents) of RN = C = O in a suitable inert diluent (eg dichloromethane) to convert the terminal amine to a group urea (ie RNHC (O) NH-), where R is as defined above. Preferably, the inert diluent contains an excess of tertiary amine (eg, about 10 equivalents), such as diisoproylethylamine. Reaction conditions are otherwise conventional (eg, room temperature for 30 minutes).

2. C-TERMINAL MODIFICATIONS

In the preparation of mimic peptides where the C-terminal carboxyl group is replaced by an ester (i.e., -C (O) OR, where R is as defined above, the resins used to prepare the peptides are employed.

-7373

<td>acids,</td><td>and the</td><td colspan="2">protected peptide</td><td>of</td><td>side chain it</td>

<td>hydrolyzes</td><td>with</td><td>base and</td><td>the alcohol</td><td colspan="2">appropriate for example</td>

<td>methanol.</td><td>The</td><td>groups</td><td>protectors</td><td>of</td><td>side chain it</td>

<td>eliminate</td><td>then</td><td>of the</td><td colspan="2">usual way,</td><td>by treatment with</td>

<td>fluoride</td><td colspan="2">hydrogen,</td><td>to get</td><td>the</td><td>desired ester.</td>

In the preparation of mimic peptides where the C-terminal carboxyl group is replaced by the amide -C (0) NR³R⁴, a benzhydrylamine resin is used as the solid support for the synthesis of the peptide. Upon completion of the synthesis, treatment with hydrogen fluoride to release the peptide from the support results directly in the free acid amide (i.e., the C-term is -C (0) NH₂). Alternatively, use of the chloromethylated resin during peptide synthesis, coupled with the reaction with ammonia to hydrolyze the side chain protected peptide from the support provides the amide of the free peptide, and reaction with an alkylamine or dialkylamine provides an alkylamide. or side chain protected dialkylamide (i.e. the term C20 is -CÍOJNRR¹, where R and R¹ are as defined above). The side chain protection is then removed in the usual manner, by treatment with hydrogen fluoride to give the free amides, alkylamides, or dialkylamides.

-7474

In another alternative embodiment, the C-terminal carboxyl group or a C-terminal ester can be induced to cyclize by internal displacement of the -OH or the (-OR) ester of the carboxyl or ester group respectively with the N-terminal amino group, to form a cyclic peptide. For example, after synthesis and hydrolysis to give the acid peptide, the free acid is converted to an ester activated by an appropriate carboxyl group activator, such as dicyclohexylcarbodiimide (DCC) in solution, for example, in mixtures of methylene chloride (CH₂C1₂) and dimethyl formamide (DMF). The cyclic peptide is then formed by internal displacement of the N-terminal amine activated ester. Internal cyclization, as opposed to polymerization, can be improved by using very dilute solutions. Such methods are well known in the art.

One can also cyclize the peptides of the invention, or incorporate a deamino or decarboxy residue at the terminus of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases, or to restrict the conformation of the peptide. . The C-terminal functional groups of the compounds of the present invention include amide, amide-lower alkyl, amide-di (lower alkyl), alkoxy

-7575 lower, hydroxy, and carboxy, and the lower ester derivatives of misinos, and the pharmaceutically acceptable salts thereof.

D. MODIFICATIONS TO THE MAIN CHAIN

Other methods of making peptide derivatives of the compounds of the present invention are described by 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 for non-peptide compounds with similar biological activity. Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as the primary peptide compound, but with more favorable activity than the primary with respect to solubility, stability, and susceptibility to hydrolysis. and proteolysis. See Morgan and Gainor, Ann. Rep. Med. Chem. 24: 243-252 (1989), incorporated herein by reference. These techniques include replacing the peptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, and N-methylamino acids.

-7676

Mimicrying peptides where one or more of the peptidyl [-C (O) NH-] linkages have been replaced by linkages such as a -CH linkage₂-carbamate, a phosphonate bond, a -CH bond₂-sulfonamide, a urea bond, a secondary amine bond (-CH₂NH-), and an alkylated peptidyl bond [-C (O) NR⁶-, where R⁶ is lower alkyl] are prepared during conventional peptide synthesis, merely by substituting a suitably protected amino acid analog for the reactive amino acid at the appropriate point during synthesis.

Suitable reagents include, for example, amino acid analogs where the carboxyl group of the amino acid has been replaced with a suitable portion to form one of the above bonds. For example, if one wants to replace a -C (O) NR- bond in the peptide with a -CH bond₂-carbamate (-CH₂0C (0) NR-), then the carboxyl group (-COOH) of a suitably protected amino acid is first reduced to the -CH group₂0H, which is then converted by conventional methods to a -OC (O) Cl functionality, or a para-nitrocarbonate functionality -OC (0) OCeHí-p-NOi. Reaction of any such functional group with the free amine or an alkylated amine on the N-terminus of the partially manufactured peptide on the solid support results in formation

-7777 from a -CH link₂OC (O) NR-. For a more detailed description of the formation of such -CH bonds₂-carbamate, see Cho et al., Science, 261: 1303-1305 (1993).

Similarly, the replacement of an amido bond in the peptide with a phosphonate bond can be accomplished in the manner set forth in US Patent Applications Serial Nos. 07 / 943,805, 08 / 081,577, and 08 / 119,700, the disclosures of which are incorporated herein by reference in their entirety.

Replacement of the amido bond in the peptide with a -CH bond₂-sulfonamido can be achieved by reducing the carboxyl group (-COOH) of a suitably protected amino acid to the -CH group₂OH, and the hydroxyl group is then converted to a suitable leaving group such as a tosyl group by conventional methods. Reaction of the tosylated derivative with eg thioacetic acid followed by hydrolysis and oxidative chlorination will provide the functional group -CH₂-S (O) ₂C1, which replaces the otherwise adequately protected carboxyl group of the amino acid. The use of this suitably protected amino acid analog in the synthesis of peptides provides for the inclusion of a -CH bond₂S (O) ₂NR-, which replaces the amido bond in the peptide, thereby providing an mimic peptide. For a more complete description of group conversion

-7878 carboxyl of the amino acid to a -CH group₂SW)₂C1, see for example, Weinstein, Boris, Chemistry &amp; Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pages 267357, Marcel Dekker, Inc., New York (1983), which is incorporated herein by reference .

The replacement of an amido bond in the peptide with a urea bond can be accomplished in the manner set forth in US Patent Application Serial No. 08 / 147,805, the application is hereby incorporated by reference in its entirety.

The secondary amine bonds where a bond

-CH₂NH- replaces the amido bond in the peptide can be prepared using, for example, a suitably protected dipeptide analog, where the carbonyl of the amido bond has been reduced from binding to a group

CH₂ by conventional methods.

For example, in the case of diglycine, reduction of the amide to the amine will provide after deprotection the

H₂NCH₂CH₂NHCH₂COOH, which is then used in the form

Nprotected in the next coupling reaction.

The preparation of such carbonyl analogues of the dipeptide group reduction amido bond is well known in the art.

-7979

The suitably protected amino acid analog is employed in conventional peptide synthesis in the same manner as the corresponding amino acid. For example, about 3 equivalents of the protected amino acid analog are typically employed in this reaction. An inert organic diluent such as methylene chloride or DMF is employed and, when an acid is generated as a by-product of the reaction, the reaction solvent will typically contain an excess amount of a tertiary amine, to capture the generated acid during the reaction. A particularly preferred tertiary amine is diisoproylethylamine, which is typically used in about a 10-fold excess. The reaction results in the incorporation into the mimic peptide of an amino acid analog having a non-peptidyl bond. Such a substitution can be repeated as desired, such that from scratch all amide linkages in the peptide have been replaced by non-amido linkages.

One can also cyclize the peptides of the invention, or incorporate a deamino or decarboxy residue at the terminus of the peptide, so that there is no amino or carboxy terminal group, to decrease susceptibility to proteases, or to restrict the conformation of the peptide. .

-8080

The C-terminal functional groups of the compounds of the present invention include amide, amide-lower alkyl, amide-di- (lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof. Examples of cyclized compounds are provided in Tables 4, 5, 6, 8, and 9.

E. FORMATION OF THE DISULFIDE LINK

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

s—

-8181 where m and n are independently 1 or 2.

Other embodiments of this invention provide analogs of these disulfide derivatives, in which one of the sulfurs has been replaced by a CH group.₂ or another isostere for sulfur. These analogs can be made via an intramolecular or intermolecular shift, using methods known in the art, as shown below:

H where p is 1 or 2. One skilled in the art will readily appreciate that this shift can also occur using other homologs of the a-amino-gbutyric acid derivative shown above and homocysteine.

Alternatively, the amino terminus of the peptide can be coated with an alpha-substituted acetic acid, in

-8282 where the alpha substituent is a leaving group, such as an α-haloacetic acid, for example α-chloroacetic acid, α-bromoacetic acid, or α-iodoacetic acid. The compounds of the present invention can be cyclized or dimerized via the displacement of the leaving group by the sulfur of the cysteine ​​or homocysteine ​​residue. See, for example, Barker et al-, J. Med. Chem., 35: 2040-2048 (1992) and Or et al., J. Org. Chem., 56: 3146-3149 (1991), each of which is incorporated herein by reference. Examples of dimerized compounds are provided in Tables 7, 9, and 10.

V. PROFIT

The compounds of the present invention are useful in vitro as useful tools for understanding the biological role of TPO, including evaluating the many factors thought to influence and are influenced by TPO production and the receptor binding process. . The compounds present are also useful in the development of other compounds that bind and activate TPO-R, because the compounds present provide important information about the relationship between structure and activity that such development should facilitate.

The compounds are also useful as competitive adherents in assays to investigate new agonists.

-8383 from the TPO receptor. In such test modalities, the compounds of the invention can be used without modification, or can be modified in a variety of ways, for example, by labeling, such as covalently or non-covalently linking a portion that directly or indirectly provides a detectable signal. In any of these tests, the materials therefor can be labeled either directly or indirectly. Possibilities for direct marking include marking groups such as: radioactive markings such as ¹²⁵I, enzymes (US Patent No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (US Patent No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or polarization of fluorescence. Possibilities of indirect labeling include biotinylation of a constituent, followed by binding to avidin coupled to one of the labeling groups above. The compounds may also include spacers or linkers, in cases where the compounds are to be attached to a solid support.

On the other hand, based on their ability to bind to the TPO receptor, the peptides of the present invention can be used as reagents to detect receptors.

-8484 of TPO on living cells, fixed cells, in biological fluids, in homogenized tissue materials, in purified, natural materials, etc. For example, by marking such peptides, one can identify cells that have TPO-R on their surfaces. Furthermore, based on their ability to bind the TPO receptor, the peptides of the present invention can be used in in situ staining, FACS (fluorescence activated cell sorting), Western staining, ELISA, etc. Furthermore, based on their ability to bind to the TPO receptor, the peptides of the present invention can be used in receptor purification, or in purifying cells that express TPO receptors on the cell surface (or within permeabilized cells).

The compounds of the present invention can also be used as commercial reagents for various diagnostic and medical research uses. Such uses include, but are not limited to: (1) use as a calibration standard, to quantify the activities of candidate TPO agonists, in a variety of functional assays; (2) use to maintain the proliferation and growth of TPO-dependent cell lines; (3) use in structural analysis of the TPO receptor through co-crystallization; (4) use to investigate the mechanism

-8585 transducer / receptor signal activation; and (5) other diagnostic and research applications, wherein the TPO receptor is preferably activated, or such activation is conveniently calibrated against a known amount of a TPO agonist, and the like.

The compounds of the present invention can be used for the in vitro expansion of megakaryocytes and their registered progenitors, both in conjunction with additional cytokines, or independently. See, eg, DiGiusto et al, PCT Publication No. 95/05843, which is incorporated herein by reference. Chemotherapy and radiation therapies cause thrombocytopenia by killing the rapidly maturing population of megakaryocytes. However, these therapeutic treatments can also reduce the number and viability of less mitotically active, immature megakaryocyte precursor cells. Thus, improvement of thrombocytopenia by TPO or the compounds of the present invention can be accelerated administered to patients by infusion post chemotherapy or radiation therapy with a population of their own cells enriched in megakaryocytes and immature precursors by in vitro culture.

-8686

The compounds of the invention can also be administered to warm-blooded animals, including humans, to activate TPO-R in vivo. Thus, the present invention includes methods for the therapeutic treatment of TPO-related disorders, which comprises administering a compound of the invention in an amount sufficient to mimic the effect of TPO on TPO-R in vivo. For example, the peptides and compounds of the invention can be administered ^ to treat a variety of hematologic disorders, including but not limited to platelet disorders and thrombocytopenia, particularly when associated with bone marrow transfusions, radiation therapy, and chemotherapy.

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

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

<img file="MX9709315A_D0009.tif" />

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

Accordingly, the present invention also provides pharmaceutical compositions comprising, as an active ingredient, at least one of the mimic peptides or peptides of the invention, in association with a pharmaceutical carrier or diluent. The compounds of this invention can be administered by the oral, pulmonary, parenteral routes of administration (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (via a fine powder formulation), transdermal, nasal, vaginal, rectal, or sublingual, and can be formulated in appropriate dosage forms for each route of administration. See, for example, Bernstein et al., PCT Patent Publication No. WO 93/25221; Pitt et al., PCT Patent Publication No. WO 94/17784; and Pitt et al., European Patent Application No. 613, 683, each of which is incorporated herein by reference.

<img file="MX9709315A_D0010.tif" />

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 pharmaceutically acceptable inert carrier, such as sucrose, lactose, or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, for example, lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, and elixirs contain inert diluents commonly used in the art, such as water. Beside such inert diluents, the compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and flavoring agents.

Preparations according to this invention for parenteral administration include sterile aqueous or nonaqueous 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 can also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. They can be sterilized for example by filtration through a bacteria-retaining filter by incorporating sterilizing agents into the compositions, irradiating the compositions, or heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.

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

The compositions containing the compounds can be administered for prophylactic and / or therapeutic treatments. In therapeutic applications,

-9090 compositions are administered to the patient who is already suffering from a disease, as described above, in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. An adequate amount to achieve this is defined as the therapeutically effective dose. The effective amounts for this use will depend on the severity of the disease and the weight and general condition of the patient.

The compositions of the invention can also be microencapsulated, for example by the method of Tice and Bibi (in Treatise on Controlled Drug Delivery, ed. Kydonieus, Marcel Dekker, NY (1992), pages 315-339).

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

The amounts of TPO agonist needed for effective therapy will depend on many different factors, including means of administration, target site, patient's physiological status, and other medications.

-9191 administered. Thus, treatment dosages should be assessed to optimize safety and efficacy. Typically, the dosages used in vitro can provide useful guidance in the amounts useful for in si tu administration of these reagents. Animal testing of the effective doses for the treatment of particular disorders will provide a further predictive indication of dosage in humans. Various considerations are described, for example, in Gilman et al. (eds.) Goodman and Gilman's: The Pharmacological Basis of Therapeutica, 8th ed., Pergamon Press (1990); and in Remington's Pharmaceutical Sciences, 7th ed., Mack Publishing Co., Easton, Penn. (1985); each of which is incorporated herein by reference.

The peptides and mimic peptides of this invention are effective in treating conditions mediated by TPO when administered in a dosage range from about 0.001 mg to about 10 mg / kg of body weight per day. The specific dose employed is regulated by the particular condition being treated, the route of administration, as well as the judgment of the attending physician, depending on factors such as the severity of the condition, the age, and the general condition of the patient. , and the like.

-9292

Although only preferred embodiments of the invention are specifically described above, it will be appreciated that modifications and variations of the invention are possible without departing from the spirit and scope of the invention.

EXAMPLE 1

SYNTHESIS OF PEPTIDES IN SOLID PHASE

Various peptides of the invention were synthesized using Merrifield's solid phase synthesis techniques (see Steward and Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical, Rockford, IL (1984) and Merrifield, J. Am. Chem. Soc. 85: 2149 (1963)) on a Milligen / Biosearch 9600 automated instrument or a Model 431A peptide synthesizer from Applied Biosystems Inc. The peptides were assembled using standard protocols of version 1.01 of System Software from Applied Biosystems Inc. Each coupling was performed for one to two hours with BOP (benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate) and HOBt (1hydroxybenzotriazole).

The resin used was HMP or PAL resin (Milligen / Biosearch), which is a 5- (4'-Fmoc-aminomethyl-3,5'dimethyloxyphenoxy) valeric acid crosslinked polystyrene resin as a linker. The use of

-9393 PAL resin results in terminal carboxyl amide functionality with hydrolysis of the resin peptide. With hydrolysis, the HMP resin produces a portion of carboxylic acid at the C-terminus of the final product. Most of the resins, reagents, and protected amino acids (free or on the resin) were purchased from Millipore or Applied Biosystems Inc.

The Fmoc group was used for amino protection during the coupling procedure. Protection of the primary amine over amino acids was achieved with Fmoc, and the side chain protection groups were t-butyl for serine, tyrosine, asparagine, glutamic acid, and threonine; trityl 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.

The separation of the peptides from the resin, and the simultaneous deprotection of the side chain functions were achieved by treatment with reagent K or slight modifications of it. Alternatively, in the synthesis of peptides with an amidated carboxyl terminus, the fully assembled peptide was hydrolyzed with a mixture of 90% trifluoroacetic acid, 5% ethandithiol, and 5% water, initially at 4^or C, and gradually increasing

-9494 at temperature precipitated with ambient. The unprotected peptides are diethyl ether. In all cases, purification was by preparative high-performance liquid chromatography, reversed phase, on a column of silica gel attached to Cie, with a gradient of acetonitrile / water in acid, homogeneous peptides were characterized by mass spectrometry of Rapid Atom bombardment or electrospray mass spectrometry and amino acid analysis when applicable.

EXAMPLE 2

Bioassays

The bioactivity of the peptides can be measured using a thrombopoietin dependent cell proliferation assay. Murine IL-3 dependent Ba / F3 cells were transfected with full length human TPO-R. In the absence of IL-3 (WEHI3 conditioned medium), these cells are dependent on TPO for their proliferation. The untransfected parent cell line does not respond to human TPO, but remains dependent on IL-3.

Bioassays were performed on both above cell lines, using synthetic peptides derived from library research. The cells are

-9595 were grown in complete RPMI-10 medium, containing 10% WEHI-3 conditioned medium, then washed twice in PBS, resuspended in medium lacking WEHI-3 conditioned medium, and added to wells containing dilutions of the peptide or TPO in 2 x 10⁴cells / well. Cells were incubated for 48 hours at 37 ° C in a humidified atmosphere with 5% CO2, and metabolic activity was assayed by reduction of MTT to formazan, with absorbance at 570 nm, measured in an ELISA plate reader. The tested 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 parent line of the cells.

EXAMPLE 3

UNION AFFINITY

The binding affinities of the TPO-R chemically synthesized peptides were measured in a competition binding assay. Wells from a microtiter plate were coated with 1 mg of streptavidin, blocked with PBS / 1% BSA, followed by 50 ng of immobilized anti-biotinylated receptor antibody (Abl79). Wells were then treated with a 1:10 harvest dilution of soluble TPO-R. Various concentrations of peptide were mixed or

-9696 mimic peptide with a constant amount of a truncated form of TPO consisting of residues 1-156 fused to the C-terminus of maltose binding protein (MBP-TPO156) · MBP-TPOise peptide mixtures were added to TPO-R coated wells were incubated for 2 hours at 4 ° C, and then washed with PBS. The amount of MBPTPOise that bound in equilibrium was measured by adding a rabbit antiserum directed against MBP. Followed by alkaline phosphatase conjugated goat anti-rabbit IgG. The amount of alkaline phosphatase in each well was then determined using standard methods.

The assay was conducted over a range of peptide concentrations, and the results are plotted such that the y-axis represents the amount of MBP-TPOi₅₆ bound, and the x-axis represents the concentration of the peptide or mimic peptide. One can then determine the concentration at which the mimic peptide or peptide will reduce by 50% (IC50) the amount of MBP-TPO156 bound to the immobilized TPO-R. The dissociation constant (Kd) for the peptide should be similar to IC₅₀measured using the test conditions described above.

-9797

EXAMPLE 4

PEPTIDES ON PLASMIDS

The vector pJS142 was used for the construction of libraries, and is shown in Figure 4. Three oligonucleotide sequences are necessary for the construction of

<td>the libraries: ON-829</td><td>(5 'ACC ACC TCC GG); ON-830</td><td> (5'</td><td>TTA</td>

<td>CTT AGT TA) and a</td><td>specific oligonucleotide</td><td>of</td><td>the</td>

<td>library of interest</td><td>(5 'GA GGT GGT {NNk} „TAA CTA</td><td>AGT</td><td>AAA</td>

GC), where {NNk}_n denotes a random region of the desired length and sequence. Oligonucleotides can be chemically phosphorylated 5 'during synthesis or after purification with polynucleotide kinase. Then they ring in a 1: 1: 1 molar ratio, and bind to the vector.

The E. coli strain that is preferably used for panning has the genotype: Á (srl-recA) endAl nupG Ion11 sulAl hsdR17 Δ (ompT-fecC) 266 ÁclpA319 :: kan ÁlacI lac ZU118, which can be prepared from a strain of

E.coli from the E.coli Genetic Stock Center at Yale University (E. coli b / r, CGSC pattern center designation: 6573) with the lon-11 sulAl genotype · The E. coli strain above is prepared for use in electroporation as described by Dower et al., Nucleic Acid Res. 16: 6127

-9898 ¢ 1988), except that 10% glycerol is used for all washing stages. Cells were tested for efficiency using 1 pg of a Bluescript plasmid (Stratagene). These cells were used for the culture of the

<td>original library, and</td><td>for the</td><td colspan="2">amplification</td><td>of</td><td>the</td>

<td>enriched population</td><td>then</td><td>of</td><td colspan="2">each cycle</td><td>of</td>

<td>panning.</td><td></td><td></td><td></td><td></td><td></td>

<td>Peptides on</td><td>plasmids</td><td>I know</td><td>released</td><td>of</td><td>the</td>

cells for panning by gentle enzymatic digestion of the cell wall, using a lysozyme. After pelletizing the cell debris, the raw Used material can be used directly on most receptors. If any further purification of the plasmid complexes is necessary, a gel filtration column can be used to remove many of the low molecular weight contaminants that are in the raw Used material.

Panning is performed in a buffer solution (HEKL) of a lower salt concentration than most physiological buffers. Panning can be conducted in microtiter wells, with a receptor immobilized on a non-blocking monoclonal antibody (Mab), or by panning on beads or on columns. Plus

-9999

For example, 24 wells can be used in the first pan cycle, each lined with receptor. For the second cycle, six wells coated with receptor (PAN sample) and 6 wells without receptor (sample NC) are typically used. Comparison of the number of plasmids in these two samples can give an indication of whether receptor-specific clones are being enriched by panning. Enrichment is

<td>definite</td><td>how</td><td>the</td><td>relationship</td><td>of</td><td colspan="2">transformants of PAN</td><td>to</td>

<td>those</td><td colspan="3">recovered from</td><td>the</td><td>shows</td><td>from NC.</td><td>The</td>

<td colspan="2">enrichment</td><td>of</td><td>10 times</td><td colspan="2">is usually a</td><td>indication</td><td>of</td>

that specific receptor clones are present.

In subsequent panning cycles, it is useful to reduce the ingress of lysed material into the wells, to decrease non-specific background binding of plasmid complexes. In cycle 2, 100 µΐ of lysed material is usually used per well. In cycle 3, 100 µΐ of lysed material is used per well, diluted 1/10 in HEKL / BSA. For additional panning cycles, an input of plasmid transformation units typically at least 100 times above the estimated remaining diversity is typically used.

The binding properties of the peptides encoded by individual clones are typically examined after

-100100 of 3, 4, or 5 pan cycles, depending on the observed enrichment numbers. Typically, an ELISA is used that detects specific binding to the receptor by Lacl-peptide fusion proteins. LacI is normally a tetramer, and the species that binds to minimal functional DNA is a dimer. The peptides are thus multivalently displayed on the fusion protein. Assuming that a sufficient receptor density can be immobilized in the wells, the LacI-fused peptides will bind to the surface in a cooperative, multivalent manner. This cooperative binding allows the detection of low intrinsic affinity binding events. The sensitivity of this assay is an advantage, in that low-affinity junctions can be easily identified, but it is a disadvantage in that the signal in the ELISA does not correlate with the intrinsic affinity of the peptides. The fusion of the peptides to the maltose binding protein (MBP) as described below allows testing in an ELISA format, where the signal strength is better correlated with the affinity. See Figure 5A-B.

The DNA of the clones of interest can be prepared in double strand form using any standard miniprep procedure. Clone coding sequences

-101101 individual or populations of clones of interest can be transferred to vectors that fuse those sequences

<td>framing</td><td>with</td><td>the</td><td>gene that</td><td colspan="2">encode</td><td>the MBP,</td><td>a</td><td>protein</td><td>than</td>

<td>for the</td><td colspan="2">general</td><td>exists</td><td>how</td><td>a</td><td>monomer</td><td>in</td><td>solution.</td><td>The</td>

<td>cloned</td><td>of</td><td>a</td><td colspan="2">library</td><td>in</td><td colspan="2">pJS142 creates</td><td>a place</td><td>of</td>

<td colspan="2">restriction</td><td>of</td><td colspan="2">BspEI close</td><td>of the</td><td>start</td><td>of</td><td>the region</td><td>of</td>

library random encoding. Digestion with BspEI and Scal allows the purification of a ~ 900 bp DNA fragment, which can be subcloned into one or two vectors, pELM3 (cytoplasmic) or pELMIS (periplasmic), which are simple modifications of the pMLAc vectors₂ and pMALp₂, respectively, commercially available from New England Biolabs. See Figures 5A-B. Digestion of pELM3 and pELM15 with Agel and Scal allows efficient cloning of the BspEI-Scal fragment from the pJS142 library. BspEI and Agel ends are compatible for ligation. Furthermore, correct ligation of the Scal sites is essential to recreate a functional bla (Amp resistance) gene, thus decreasing the level of background clones from unwanted ligation events. Expression of the MBP-peptide fusions managed by the tac promoter can then be induced with IPTG.

Lysates for the LacI or MBP ELISAs are prepared from individual clones, lysing cells

-102102 using lysozyme, and extracting insoluble residues from cells by centrifugation. The Used materials are then added to wells containing the immobilized receptor, and to control wells without receptor. Binding by LacI or MBP-peptide fusions is detected by incubation with a rabbit polyclonal antiserum directed against either LacI or MBP, followed by incubation with a second alkaline phosphatase labeled goat rabbit antibody. Bound alkaline phosphatase is detected with a chromagenic substrate with p-nitrophenyl phosphate.

EXAMPLE 5

MAIN PART DIMER SYSTEM

A variant of the LacI plasmid peptide technique uses a protein that binds to DNA called a main part dimer. DNA binding by the E. coli lac repressor is mediated by the principal part domain of approximately 60 amino acids. The dimer of the masterpiece domains that binds to the lac operator is normally formed by the association of the much larger Cterminal domain of about 300 amino acids. The main part dimer system uses main part dimer molecules that contain two main parts connected via a short peptide linker. These proteins bind to DNA with enough

-103103 stability to allow association of a peptide epitope displayed at the C-terminus of the main part dimer with the plasmid encoding that peptide.

Random peptides are fused to the C-terminus of the main part dimer, which binds to the plasmid that encoded it, to make a peptide-main part dimer-plasmid complex that can be investigated by panning. The peptide-over-plasmid system of the main part dimer allows greater selectivity for high affinity ligands than the LacI system. Thus, the main part dimer system is useful for making mutagenesis libraries based on low initial affinity junctions, and selecting higher affinity variants of those initial sequences.

Libraries are constructed with peptides on plasmids using the main part dimer vector (see Figure 6A-C} The presence of the lac operator is not required for binding of the plasmid by the main part dimer protein. Libraries were introduced in bacterial strains comprising E. coli (lon-11 sulAl hsdR17 (ompT-fepC) ÁclpA319 :: kan ÁlacI lac ZU118 Á (srlrecA) 306 :: Tnl0 and amplified under conditions of induction by the basal promoter (A). Panning the libraries of the main part dimer led to

-104104 performed by procedures similar to those used for the LacI libraries, except that HEK buffer was used instead of HEKL buffer, and elution of the plasmids from the wells was performed with aqueous phenol instead of IPTG . Panning dimer sequence panning sequences are often characterized after transfer to the vector of

<td>MBP so</td><td>they can</td><td>to be</td><td>proven</td><td>in the ELISA of</td><td>MBP</td>

<td>sensitive by</td><td>affinity,</td><td>and</td><td>too</td><td>so that</td><td>the</td>

<td>populations</td><td>clones</td><td colspan="2">could be</td><td>investigated</td><td>by</td>

colony support with labeled receptor.

EXAMPLE 6

In this example, the cyclized compounds were subjected to three tests. First, the IC values ​​were obtained₅or as described above. Additionally, an MTT cell proliferation assay was performed, as described above, to calculate EC values.₅or. Finally, an assay was performed on a microphysiometer (Molecular Devices Corp.). Basically, in this assay, the acidification rate of the extracellular medium was determined, in response to stimulation of the TPO receptor by the compounds of the invention. The intervals for EC₅₀ are symbolically indicated as for IC₃₀

-105105 described above. The results are summarized in the

Table 4.

-106ί

106

TABLE 4

Structure

ECSOínM) ECSOfnMi CSOínMl

Proliferate. Microoniz.

lH] -CPeniADGFTLRZWI £ F (Cys} - £ .NH21

-h I ⁺⁺⁺ H.H

ÍOC-NH) -ADCPTLREWISFfCys I - (NH 2) ch₂S (H1

<img file="MX9709315A_D0011.tif" />

++ W- NÜ

<td>(OCH) -ADGmaEilSF- (Cys) - (NH 21 CHo - '</td><td> 4</td><td>• b-</td><td> 4-</td>

<td>CH? _T</td><td></td><td></td><td></td>

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

(Hl - {D-Cys) ADGPTLBZWIHF ÍD-Cys) - {NH2 J4.

IΊ

4— Mü íHl - f Cys J ADGPTLREWISF (D-Cys I - {NH 21 + ·

-107107

Structure

ECTínMl ECSOÍnMl IC50 (nMl Prolif. Microniz.

<img file="MX9709315A_D0012.tif" />

[H] -KADGPTLEEWISIZ- (NH 7] | 1

1 ---------- NH-C-0

-108108

Structure

ECSOfaM) ECSOfaM) IGO (nM) Prolif. Microniz.

<td>(HJ -EADGPTLREWT5FK- (NH 2 i O »C-NH ---------- '</td><td> *</td><td> 4-</td><td>M £&gt;</td>

<td>tO-C-NHl -ADCTTLKHíISF fCys) - (NH 21 .1 · _ i</td><td> + *</td><td></td><td></td>

<td>/ \ z ^s</td><td></td><td></td><td></td>

<td>[OsC-HH] -ADGmEEHISFCCys) - [NH ₂J i________________________l</td><td></td><td> 4-</td><td>HO</td>

IHl

<img file="MX9709315A_D0013.tif" />

-109109

EXAMPLE 7

In this example, amino acid substitutes at the D, Ε, I, S, or F positions were tested on the cyclized compound:

CADGPTLREWISFC to determine EC values₅or IC50 as described above. The results of the microphysiometer are given in 10 parentheses. The results are summarized in Table 5 below.

-110110

TABLE 5

CADGPTLREWZSxC t - - - -

<td>I Substitution</td><td>EC50 (nM) Prolif. mobile</td><td>ICSD (nM) |</td>

<td>EQ</td><td>++ OO</td><td>Ί- +</td>

<td>GIVES</td><td>+ c +&gt;</td><td></td>

<td>IA</td><td>• HO)</td><td> 4</td>

<td>SA</td><td>4-4- Ú ++)</td><td>fh</td>

<td>SD-Ala</td><td> +</td><td>t-</td>

<td>S - Sar</td><td> ¥-</td><td></td>

<td>S-Aíb</td><td> 4-4-0)</td><td> +4-</td>

<td>SD-Ser</td><td> ++</td><td></td>

<td>S-Nva</td><td> 4+(*&gt;</td><td> +4</td>

<td>S-Abu</td><td> 44-</td><td> 4+</td>

<td>S - (N-Me-Ala)</td><td> 4-</td><td> 4</td>

<td>S - (N-Me-Val)</td><td>l</td><td> 4*</td>

<td>S - (N-Me-Ala)</td><td> 4-·</td><td> +’</td>

<td>S - (Nor-Len)</td><td> +4</td><td> +4</td>

<td>S - (t-Bu-Gly)</td><td> 4-</td><td></td>

<td>S - (N-Me-SeriBzDl</td><td></td><td>j ..</td>

-111111

<td rowspan="2">Substitution</td><td>ECSO (nM)</td><td>ICSO (nM)</td>

<td>Prolif. cellul</td><td></td>

<td>S- (Hoaoser)</td><td>GARLIC</td><td>NOT .</td>

<td>5 - (N-Me-Leu)</td><td> +</td><td>NOT</td>

<td>FA</td><td></td><td> + +</td>

<td>FD-Ala</td><td>X</td><td></td>

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

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

<td>F-CHA</td><td>+ 4-Í ++)</td><td> 4-4</td>

<td>F-Ihi</td><td>-H-</td><td></td>

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

<td>F * (N * Me * Ala)</td><td>TO- 4</td><td> 4-'</td>

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

<td>F- (Pyiidylala)</td><td>w ·</td><td>Ή-</td>

<td>F - (p-Nitrophe)</td><td> ++(++&gt;</td><td>W</td>

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

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

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

<td>Final)</td><td> ++</td><td>Ή ·</td>

<td>F- (KPh - Alai</td><td> ++</td><td> 44</td>

112

<td>Substitution</td><td>ECSOtaM) Prolif. cellul.</td><td>I IC50 (nM) j</td>

<td>F - (N-Me-? He)</td><td></td><td>MD</td>

<td>Sf - Ava (thioether)</td><td> +-</td><td> *&gt;+</td>

<td>SJ- Ava tcys-cys)</td><td>t-</td><td>t +</td>

<td>SJ - Ava - deletion</td><td></td><td> ++</td>

<td>AD- deletion</td><td>+ ~ (+ ύ</td><td>MD</td>

<td>adg-</td><td></td><td></td>

Ava = HjN ^/\ Z \ z COOH

-113113

EXAMPLE 8

In this example, amino acid substitutions in the compound were evaluated:

(O «C - NH] - ADGPTLREWISF (CYS)

CH₂--- in positions D, S, or F, as indicated in Table 6 below. ECso and IC values₅or were calculated as described above. The results of the microphysiometer are given in parentheses.

-114114

TABLE 6 [O = C-NH] -ADGPTLHZWISF (Cys) II

CH2 ------------------- Ξ

<td>Substitution</td><td>EC50 (nM) Prolif. cellul.</td><td>1 (30 (nM) |</td>

<td>OF</td><td>C +)</td><td>NOT</td>

<td>free acid form</td><td> *·*&lt;+)</td><td>NOT</td>

<td>C-term. ad | c. from Gly</td><td></td><td> 4-+ _t</td>

<td>S-Abu</td><td></td><td>NOT</td>

<td>F-DiPh-Ali</td><td></td><td>Ή-</td>

<td>SJ - Abu. DiPh-Ala</td><td>K + *)</td><td></td>

-115115

EXAMPLE 9

In this example, the ECso and IC50 values ​​are

<td>they calculated</td><td>as described</td><td>above</td><td>for the</td><td>compounds</td>

<td>dimerized</td><td>listed in the</td><td>Table 7</td><td>from below 0.</td><td>Monomer</td>

<td>cyclized:</td><td></td><td></td><td></td><td></td>

<td></td><td>CADGPTLRE I--</td><td>WISF</td><td>c J</td><td></td>

it is included as a comparison.

The compounds in Table 8 were inactive at the maximum tested concentration of 10 ym.

In Table 9, the EC values ​​are compared₅or IC50, determined as described above for the cyclized and dimerized variants of IEGPTLRQWLAARA.

In Table 10, the truncation of the dimer is compared:

(H) - IEGPTLRQWLAARA

I (H) - IEGPTLRQWLAARA (Shovel) K - (NH_to)

EC values₅₀ and IC50 were calculated as described above. The results of the microphysiometer are given in parentheses.

-116116

TABLE 7

EC50 (nM) ICSO (nM)

Micropnis. Prolif.

OR

ÍBr-jC-NH} -ADGFTLRSWISFC- · 1

O [Sr-K-NHl -AJDGéTLREHISTC- [8¾]

IH) -XECrTUlQMIAMa

IH] -I £ SPIIBQWIAAWP-AWK-! NH2Í! HJ -aZSFXXSQHLMKi- (NHi 1

I [H] -CHSPJLEQWIARKA- (KHj}

1H] -CAEG? HES1I5F- (* S₂1

I (Hl-aDGFHHEH £ SF- {KH₂l (H1 -SirQCGPEBQWLARRNHLS- (HH₂1

I__________ (H} -SVtX3STT3QWU¿RNELS- (l®₂) (H} -HSGPHBSGC- [NH2]

I [H1 -HVGPIXSSGC- | NH21

<td>4M-</td><td>-Ml</td><td> ¥4</td>

<td> 4-4</td><td> 4-4·</td><td>-Mr</td>

<td>-W</td><td> 4—4</td><td>-H</td>

<td>4H-</td><td> 4-4-</td><td>- W</td>

<td> 4-4</td><td>-H-</td><td>-Μ-</td>

<td></td><td> 4*</td><td>Η</td>

-117117

EC50 (ηΜ)

IC50 (πΜ)

CSDGPffiRESíISFC

I —______________

Micropnis.

++

Prolif.

(AC1-ADGP3XSEBISSC í ÍAcl-AEGPII2E «ISFC

NO r +

ADGPTLEDCSFC

I

ADGPUBEHISFC + - + (AcJ-OGPILSZHISrC (Acl-DGPHSHZSEC ++ (Ac) -GPHEZHISFC i

[AcIhSPTISZSXSFC

NOT

GPESHXSFC

GPKEEHXSFC

-b (here-phesist:

I (Acl-PUSSOSEC

NO +4 ρπιεϊεη:

___________I

PH2EHI5FC

4-4 lACI-IXSEEXSxü (Afil-USEHXSFC

4 + HJEíISrC ____________I UBENISFC

4*

-118118

TABLE 8 (Hl ^ nSMgxgSC-ltBa) (H] -CHIKQEES3Cr {KH₂] (H1JAAC- (HH₂i [H) -CrSTOTUAC- (SH₂] [H] -cggamjgc-iKg₂i (H1 -nXSEFEaGQQC- (HH₂one [H] -cmjvwmaEs :-( NH₂] (YES -giGqWLgQg- ÍSB₂] (Η1-ΟΛ55? ΝΤ0ίϋ. · ΣΕ: -ί1Η₂) [Hi-oisg¿jgraac-iHg₂] [21 - &lt;ctspjlsibip: - (νη₂ϊ [21 -eEcroegasgJC- »g₂i [21-CSIZ £ EZffiGIHQC- (lE₂l [Hl-aXaSIASGV2X-¡NH₂¡(2i- &lt;x3cSLVsaEnK: - {2s₂']

-119119 [Hl-OXSnXSQHLMBK-ÍHBz) (Hl-JHSPTLRCWM-INHjl [ΗΙ-ΚΕΠΡηΟ-ΤΜεϊδ-ίΝΗ ^ ΐ

-120120

TABLE 9

<td></td><td colspan="3">EC50 (nM) ICSO (ntó) Micropnis. Prolif.</td>

<td>(HJ -XEGnXKJWIAMA- (^ ¾)</td><td>ND</td><td></td><td> +4</td>

<td>(H) -CTEGSIXSQWLMMC- []</td><td>ND</td><td> +*</td><td>-tt-</td>

<td>[H} -XEG5TL2QHIAAIA \ í H] -IHnTLRQWIAASA (B-Ala) K- {NHj]</td><td>-H-</td><td>-M-</td><td>• H-</td>

<td>(H1-OSOTBJKÍAM &amp; - (HBjl l H] -aB5SHBq «LMUUl- (» β₂1</td><td></td><td> ++</td><td>H</td>

-121121

TABLE 10 (Η) -ΙΕϋ? Τΐ113ΛΛλ * λ

I tK) -IErJ7I3í ^ nJAKA (B-kUiK “(ΝΗ, Ι

<td colspan="4">Sequence ¹⁰⁰</td>

<td></td><td>(AcHHGFH2QHXAM * one (Acl -IEGFILXCWLAAXA-Ga-KíNHjJ</td><td> ++</td><td>MD</td>

<td></td><td>(HI-IE3TI2GWXJUUL one (HI -lEGFXLXQt'ttAAR-fiA-KÍKHjl</td><td>4-b</td><td>MD</td>

<td></td><td>(H) -nS7H30WUA 1 (HI -ΧΕ &amp; ηΧΚΟΜ-βλ-ΧΠΕ ^ Ι</td><td></td><td>MD</td>

<td></td><td>(Ae | - £ G? H3QWUa¡a 1 (AcJ -EGFIiraiLWUUl- ^ A-KtJC ^ l</td><td>MD</td><td>MD</td>

<td></td><td>(HI-EOTIIQSLWUA one (HI 'EGxmCyUAIA-SA-X n®y</td><td>4-Ψ</td><td>NOT</td>

<td></td><td>(H) -EGTZLSOWLNUt one (HJ • BSJTLKJHIAAR-flA'K (KHJ</td><td></td><td>ND</td>

<td></td><td>(AcJ-EGmXCWUA one (Ac) -EGnXSQHUU-BA-XtKS ^)</td><td> 4-4-</td><td>Nb</td>

<td></td><td>(H) -EüFH3GHLHA one (HI -ESFTLSOTXAA-ÜA-K (HHJ</td><td>+ ~ r</td><td>MD</td>

-122122

EXAMPLE 10

In this example various substitutions were introduced at the G, P, and W positions in the cyclized compound:

(H) -CADGPTLREWISFC- (NHjl.

Table 3 lists examples of the substituted compounds that show TPO agonist activity. The abbreviated substitutions in the Table are as follows:

TABLE 11

<img file="MX9709315A_D0014.tif" />

<td>G</td><td>P</td><td>W |</td>

<td>He</td><td>HypíOBnl</td><td>End 1</td>

<td>He</td><td>HypíOBnl</td><td>Nal |</td>

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

<td>Gly</td><td>Pro</td><td>^Trp 1</td>

<td>Γ ^sw</td><td>HypíOBnl</td><td>End 1</td>

<td>Gaba</td><td>Pro</td><td>Trp |</td>

<td>Cpr-Gly</td><td>Pro</td><td>Trp |</td>

<td>He</td><td>Ηγρ (ΟΒηΙ</td><td>End 1</td>

<td>L </td><td>Pro</td><td>Trp I</td>

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

<td>He</td><td>Pro</td><td>Trp</td>

<td>Cpr-Gly</td><td>L-Tic</td><td>Na! |</td>

<td>GlY</td><td>D-Tic</td><td>D-Trp I</td>

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

<td>| Gaba</td><td>HypíOBnl</td><td>Trp I)</td>

-123123

<img file="MX9709315A_D0015.tif" />

REPLACEMENTS. PROLIN

<img file="MX9709315A_D0016.tif" />

O ** COOH N

L-Pro D-Pro

<img file="MX9709315A_D0017.tif" />

L-Pipecolinic Acid

D-Pipecolinic Acid

L-azetidine carboxylic acid

<img file="MX9709315A_D0018.tif" />

<img file="MX9709315A_D0019.tif" />

L-Oic

LrTlC

D-Tk

<img file="MX9709315A_D0020.tif" />

L-Tiq

-124124

<img file="MX9709315A_D0021.tif" />

D4-Nal tryptophan replacements

<img file="MX9709315A_D0022.tif" />

<img file="MX9709315A_D0023.tif" />

<img file="MX9709315A_D0024.tif" />

<img file="MX9709315A_D0025.tif" />

D-2-N11

L- (Benzothienyl) -alanine

DL-S-Me-Tr ©

<img file="MX9709315A_D0026.tif" />

DL-Ó-Firp

<img file="MX9709315A_D0027.tif" />

DL-5-F-Trp

<img file="MX9709315A_D0028.tif" />

DL-5-Br-Trp

<img file="MX9709315A_D0029.tif" />

b-tic

-125125

GLYCINE REPLACEMENTS

<img file="MX9709315A_D0030.tif" />

Glycine

Sarcosina

Η, Ν

<img file="MX9709315A_D0031.tif" />

COOH

COOH p-alanine Γ-aminobutyric acid

<img file="MX9709315A_D0032.tif" />

COOH

<img file="MX9709315A_D0033.tif" />

N-Pentyl glycine

N-Cyclopropyl glycine

-126126

EXAMPLE 11

To determine the viability of mice as a convenient species for testing, several in vitro experiments have been done, designed to measure the activity of test compounds on the mouse receptor. First, marrow cells, harvested from the femurs of Balb / C mice for 8 to 9 weeks, were incubated for 7 days in liquid culture with either rhuTPO or various concentrations of the test peptides. At the end of the incubation period, the cultures were concentrated by Cytospin, stained for acetylcholinesterase (AChE, a diagnosis of mouse megakaryocytes), and counted by microscopic analysis. One (1) nM rhuTPO resulted in the logical consequence of very large non-adherent cells (&gt; 40 pm), staining for AChE. These cells appear to be mature megakaryocytes. From an initial planting of 10⁶ total marrow cells / ml (in 50 ml cultures) an estimate of 1 to 2 x 10 was developed⁶ megakaryocytes. This response to TPO was designated as maximum. Control cultures that did not contain added growth factors produced very few AChE positive cells. Several of the peptide compounds were tested in high concentration in this assay, and the results are summarized in Table 12. Peptide A at 10 pM produced

-127127 a maximum response from the mouse cord. This discovery was the first evidence that this family of peptides is active on the murine receptor. In a second experiment, marrow cells were harvested and cultured in semi-solid medium (methyl cellulose) containing either no factor, 1 nM rhuTPO, or 10 µΜ Peptide A. After 7 days in culture, large cell colonies (presumed to be megakaryocytes) were counted and grouped into small colonies (3-5 cells) or large colonies (more than 6 cells). Results are shown in Table 13. TPO and test peptides both produced substantially more colonies of both sizes than negative control cultures. This indicates that the peptides mimic TPO in their ability to stimulate expansion of the Mk precursor cell population.

For a more quantitative comparison of the activity of the test compounds on murine and human receptors, the muTPO receptor was cloned and transfected into BaF3 cells. A TPO-dependent cell population 20 was isolated.

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TABLE 12

<td>j Peptide</td><td>Conc. Tested (nm)</td><td>Answer</td>

<td> 1 ^D</td><td> 100,000</td><td>none</td>

<td> 1 ^c</td><td> 40,000</td><td>~ .— maximum **</td>

<td>IC + SA * alone</td><td> 1000</td><td>maximum **</td>

<td> 1 ^S-^TO'</td><td> 1000</td><td>none</td>

<td> 1 ^B</td><td> 100,000</td><td>minimal</td>

<td>| TO</td><td> 10,000</td><td>maximum **</td>

<td>II TPO (R 6 D)</td><td> 1</td><td>maximum</td>

Streptavidin supplemented to biotinylated peptide concentration of putative 1: 4 complex.

** Compared to recombinant human TPO *** 25-30% of cells that stain for AChE on cytopspin

Crops without factor - approx. 5% of cells that stain for

AChE (inferior cellularity)

TABLE 13

<td colspan="2">Compound</td><td>3-5 large cells</td><td>6-12 large cells</td>

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

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

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

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

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

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

<td>10 uM Peptide</td><td> #1-1</td><td> 25</td><td> 10 |</td>

<td>10 uM Peptide</td><td> #1-2</td><td> 22</td><td>⁸ II</td>

<td>1 10 uM Peptide</td><td> #2-1</td><td> 22</td><td> 7 1</td>

<td>I 10 uM Peptide</td><td> #2-2</td><td> 21</td><td> 10 1</td>

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

It is noted that in relation to this date, the best method known by the applicant to put the aforementioned invention into practice is&gt; that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property:

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