Patent EA-003998-B1 (EA)
Title: Dimeric thrombopoietin peptide mimetics binding to mp1 receptor and having thrombopoietic activity
Inventor: LIU CHUAN-FA
IPC: C12N15/09
Filed: 1999-10-22
Abstract: 1. A compound that binds to an mpl receptor comprising the structure: TMP1-(L1)n-TMP2 wherein TMP1 and TMP2 are each independently selected from the group of core compounds comprising the structure: X2-X3-X4-X5-X6-X7-X8-X9-X10, wherein, X2 is selected from the group consisting of Glu, Asp, Lys, and Val; X3 is selected from the group consisting of Gly and Ala; X4 is Pro; X5 is selected from the group consisting of Thr and Ser; X6 is selected from the group consisting of Leu, Ile, Val, Ala, and Phe; X7 is selected from the group consisting of Arg and Lys; X8 is selected from the group consisting of Gln, Asn, and Glu; X9 is selected from the group consisting of Trp, Tyr, and Phe; X10 is selected from the group consisting of Leu, Ile, Val, Ala, Phe, Met, and L1 is a linker; and n is 0 or 1; and physiologically acceptable salts thereof. 2. The compound according to Claim 1 wherein said TMP1 and TMP2 are independently selected form the group consisting of: X2-X3-X4-X5-X6-X7-X8-X9-X10-X11; X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12; X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13; X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14; X1- X2-X3-X4-X5-X6-X7-X8-X9-X10; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12; X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13; and X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein X2 - X10 are as defined; X1 is selected from the group consisting of Ile, Ala, Val, Leu, Ser, and Arg; X11 is selected from the group consisting of Ala, Ile, Val, Leu, Phe, Ser, Thr, Lys, His, and Glu; X12 is selected from the group consisting of Ala, Ile, Val, Leu, Phe, Gly, Ser, and Gln; X13 is selected from the group consisting of Arg, Lys, Thr, Val, Asn, Gln, and Gly; and X14 is selected from the group consisting of Ala, Ile, Val, Leu, Phe, Thr, Arg, Glu, and Gly. 3. The compound according to Claim 1 wherein said TMP1 and/or TMP2 are derivatized as set forth in one or more of the following: one or more of the peptidyl [-C(O)NR-] linkages (bonds) have been replaced by a non-peptidyl linkage such as a -CH2-carbamate linkage [-CH2-OC(O)NR-]; a phosphonate linkage; a -CH2-sulfonamide [-CH2-S(O)2NR-] linkage; a urea [NHC(O)NH-] linkage; a -CH2-secondary amine linkage; or an alkylated peptidyl linkage [-C(O)NR6- where R<6> is lower alkyl]; the N-terminus is a -NRR<1> group; to a -NRC(O)R group; to a -NRC(O)OR group; to a -NRS(O)2R group; to a -NHC(O)NHR group where R and R<1> are hydrogen and lower alkyl with the proviso that R and R<1> are not both hydrogen; to a succinimide group; to a benzyloxycarbonyl-NH- (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, chloro, and bromo; the C terminus is -C(O)R<2> where R<2> is selected from the group consisting of lower alkoxy and -NR<3>R<4> where R<3> and R<4> are independently selected from the group consisting of hydrogen and lower alkyl. 4. The compound according to Claim 1 wherein all of the amino acids have a D configuration. 5. The compound according to Claim 1 wherein at least one of the amino acids has a D configuration. 6. The compound according to Claim 1 which is cyclic. 7. The compound according to Claim 1 wherein TMP1 and TMP2 are each Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala. (SEQ ID NO: 1). 8. The compound according to Claim 1 wherein L1 comprises a peptide. 9. The compound according to Claim 8 wherein L1 comprises Yn, wherein Y is a naturally-occurring amino acid or a stereoisomer thereof and n is 1 through 20. 10. The compound according to Claim 8 wherein L1 comprises (Gly)n, wherein n is 1 through 20, and when n is greater than 1, up to half of the Gly residues may be substituted by another amino acid selected from the remaining 19 natural amino acids or a stereoisomer thereof. 11. The compound according to Claim 8 wherein L, is selected from the group consisting of (Gly)3Lys(Gly)4 (SEQ ID NO: 6); (Gly)3AsnGlySer(Gly)2 (SEQ ID NO: 7); (Gly)3Cys(Gly)4 (SEQ ID NO: 8); and GlyProAsnGly (SEQ ID NO: 9). 12. The compound according to Claim 8 wherein L1 comprises a Cys residue. 13. The compound according to Claim 1 wherein L1 comprises (CH2)n, wherein n is 1 through 20. 14. The compound according to Claim 1, which is selected from the group consisting of IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (SEQ. ID NO: 9) IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (cyclic) (SEQ. ID NO: 10) IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (linear) (SEQ. ID NO: 11) IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA (SEQ. ID NO: 12) IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA (SEQ.ID NO: 13) IEGPTLRQWLAARA-GGGK(BrAc)GGGG-IEGPTLRQWLAARA (SEQ.ID NO: 14) IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (SEQ.ID NO: 15) IEGPTLRQWLAARA-GGGK(PEG)GGGG-IEGPTLRQWLAARA (SEQ.ID NO: 16) IEGPTLRQWLAARA-GGGC(PEG)GGGG-IEGPTLRQWLAARA (SEQ.ID NO: 17) IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA (SEQ.ID NO: 18) IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ.ID NO: 20). 15. The compound according to Claim 1 or 2, which has the formula (Fc)m-(L2)q-TMP1-(L1)n-TMPn-(L3)r(Fc)p, wherein L1, L2 and L3 are linker groups which are each independently selected from the linker groups consisting of Yn, wherein Y is a naturally-occurring amino acid or a stereoisomer thereof and n is 1 through 20; (Gly)n, wherein n is 1 through 20, and when n is greater than 1, up to half of the Gly residues may be substituted by another amino acid selected from the remaining 19 natural amino acids or a stereoisomer thereof; (Gly)3Lys(Gly)4 (SEQ ID NO: 6); (Gly)3AsnGlySer(Gly)2 (SEQ ID NO: 7); (Gly)3Cys(Gly)4 (SEQ ID NO: 8); GlyProAsnGly (SEQ ID NO: 9); a Cys residue; and (CH2)n, wherein n is 1 through 20; Fc is an Fc-region of an immunoglobulin; m, p, q and r are each independently selected from the group consisting of 0 and 1, wherein at least one of m or p is 1, and further wherein if m is 0 then q is 0, and if p is 0, then r is 0; and physiologically acceptable salts thereof. 16. The compound according to Claim 15 wherein L1, L2 and L3 are each independently selected from the group consisting of Yn, wherein Y is selected a naturally-occurring amino acid or a stereoisomer thereof and n is 1 through 20. 17. The compound according to Claim 16 wherein L, comprises (Gly)n, wherein n is 1 through 20, and when n is greater than 1, up to half of the Gly residues may be substituted by another amino acid selected from the remaining 19 natural amino acids or a stereoisomer thereof. 18. The compound according to Claim 17 wherein L1, L2 and L3 are independently selected from the group consisting of: (Gly)3Lys(Gly)4 (SEQ ID NO: 6); (Gly)3AsnGlySer(Gly)2(SEQ ID NO: 7); (Gly)3Cys(Gly)4 (SEQ ID NO: 8); and GlyProAsnGly (SEQ ID NO: 9). 19. The compound according to Claim 15 wherein L1, L2, or L3 comprises a Cys residue. 20. The compound according to Claim 15 wherein L1, L2 or L3 comprises (CH2)n, wherein n is 1 through 20. 21. The compound according to Claim 1, which is selected from the group consisting of: Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (SEQ. ID NO: 21) Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA-Fc (SEQ. ID NO: 22) IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA-Fc (SEQ. ID NO: 23) Fc-GG-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (SEQ. ID NO: 24) Fc-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 25) Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (cyclic) (SEQ. ID NO: 26) Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (linear) (SEQ. ID NO: 27) Fc-IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA (SEQ. ID NO: 28) Fc-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 29) Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 30) Fc-IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA (SEQ. ID NO: 31) Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 32) Fc-GGGGG-IEGPTLRQWLA,ARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 33). 22. A dimer of the compound according to Claim 12. 23. The dimer according to claim13 which is TMP1-Gly3-Cys-Gly4-TMP2 TMP1-Gly3-Cys-Gly4-TMP2. 24. A dimer of the compound according to Claim 19. 25. A method of increasing megakaryocytes or platelets in a patient in need thereof, which comprises administering to said patient an effective amount of a compound according to Claim 1. 26. The method according to Claim 25, wherein said amount is from 1 pg/kg to 100 mg/kg. 27. A pharmaceutical composition comprising a compound according to Claim 1 in admixture with a pharmaceutically acceptable carrier thereof. 28. A polynucleotide that encodes a compound according to claim 8. 29. A polynucleotide that encodes a compound according to claim 16. 30. A polynucleotide that encodes a compound according to claim 22. 31. A polynucleotide that encodes a compound according to claim 24. 32. A vector that comprises a polynucleotide according to any of claims 28-31. 33. A host cell that comprises a vector according to claim 32. 34. A method of producing a compound according to claims 8, 16, 22 or 24, which comprises growing a host cell according to claim 33 in a suitable nutrient medium and isolating said compound from said cell or nutrient medium.