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Modified natriuretic compounds and conjugates thereof are disclosed in the present invention. In particular, conjugated forms of hBNP are provided that include at least one modifying moiety attached thereto. The modified natriuretic compound conjugates retain activity for stimulating cGMP production

Modified natriuretic compounds and conjugates thereof are disclosed in the present invention. In particular, conjugated forms of hBNP are provided that include at least one modifying moiety attached thereto. The modified natriuretic compound conjugates retain activity for stimulating cGMP production, binding to NPR-A receptor, and in some embodiments an improved half-life in circulation as compared to unmodified counterpart natriuretic compounds. Oral, parenteral, subcutaneous, and intravenous forms of the compounds and conjugates may be prepared as treatments and/or therapies for heart conditions particularly congestive heart failure. Modifying moieties comprising oligomeric structures having a variety of lengths and configurations are also disclosed. Analogs of the natriuretic compound are also disclosed, having an amino acid sequence that is other than the native sequence.

대표청구항 ▼

What is claimed is: 1. A natriuretic compound conjugate comprising: (a) a biologically active natriuretic compound comprising: (i) a natriuretic molecule NPR-A binding site; and (ii) at least one modifying moiety conjugation site wherein the biologically active natriuretic compound is a brain natr

What is claimed is: 1. A natriuretic compound conjugate comprising: (a) a biologically active natriuretic compound comprising: (i) a natriuretic molecule NPR-A binding site; and (ii) at least one modifying moiety conjugation site wherein the biologically active natriuretic compound is a brain natriuretic peptide, atrial natriuretic peptide, C-type natriuretic peptide, dendroaspis natriuretic peptide or a biologically active segment thereof; and (b) a modifying moiety attached to said modifying moiety conjugation site, wherein the modifying moiety has a formula: each C is independently selected and is an alkyl moiety having m carbons and m is from 1 to 20; and each PAG is independently selected and is a polyalkylene glycol moiety having n subunits and n is from 2 to 25; each X is independently selected and is a linking moiety selected from the group consisting of —C—, —O—, —C(O)—, —NH—, —NHC(O)—, and —C(O)NH—, and o is from 1 to 15, and wherein said natriuretic compound conjugate exhibits one or more advantages selected from the group consisting of increased resistance to enzymatic degradation relative to a corresponding unconjugated natriuretic compound, increased circulating half life, increased bioavailability, and prolonged duration of effect. 2. The natriuretic compound conjugate of claim 1 further defined as retaining a therapeutically significant percentage of cGMP stimulating activity relative to the corresponding unconjugated natriuretic compound. 3. The natriuretic compound conjugate of claim 1 further defined as retaining at least 30% of the cGMP stimulating activity of the corresponding unconjugated natriuretic compound. 4. The natriuretic compound conjugate of claim 1 further defined as retaining at least 50% of the cGMP stimulating activity of the corresponding unconjugated natriuretic compound. 5. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound is hBNP. 6. The natriuretic compound conjugate of claim 1 further defined as retaining at least 90% of the cGMP stimulating activity of the corresponding unconjugated natriuretic compound. 7. The natriuretic compound conjugate of claim 1 further defined as more hydrophilic than a corresponding unconjugated natriuretic compound. 8. The natriuretic compound conjugate of claim 1 further defined as more amphiphilic than a corresponding unconjugated natriuretic compound. 9. The natriuretic compound conjugate of claim 1 further defined as more lipophilic than a corresponding unconjugated natriuretic compound. 10. The natriuretic compound conjugate of claim 1 further defined as more resistant to protease degradation than a corresponding unconjugated natriuretic compound. 11. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises a sequence: A1PX1MVQGSGCFGRX2MDRISSSSGLGCX3VLR (SEQ ID NO. 116), wherein A1 is an amino acid or series of amino acids native to a natriuretic peptide, X1, X2 and X3 are independently selected from the group consisting of Lys, Arg and Gly, and at least one of X1, X2 and X3 is a Lys. 12. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises: (a) an amino acid sequence X1-C1FGRX2MDRISSSSGLGC2-X3 (SEQ ID NO: 117) wherein X1 is optionally present and when present is an amino acid sequence having from 1-10 amino acids; X2 is Gly, Arg, or Lys; and X3 is optionally present and when present is an amino acid sequence having from 1-10 amino acids. (b) a disulfide bond between C1 and C2 to form a loop. 13. The natriuretic compound conjugate of claim 12 wherein X1 is Arg or Gly. 14. The natriuretic compound conjugate of claim 12 wherein X1 is selected from the group consisting of: (a) Lys; (b) Gly; (c) Arg; (d) SG-, GSG-, QGSG- (SEQ ID NO. 118), VQGSG- (SEQ ID NO. 119), MVQGSG- (SEQ ID NO. 120), PKMVQGSG- (SEQ ID NO. 121), and SPKMVQGSG- (SEQ ID NO. 122); (e) hBNP segments of (d) comprising a substitution selected from the group consisting of Lys-to-Gly and Lys-to-Arg; (f) hBNP segments of (d) comprising a substitution selected from the group consisting of Gly-to-Lys and Arg-to-Lys; (g) hBNP segments of(d) comprising an inserted Lys; (h) N-terminal tails and C-terminal segments of N-terminal tails of natriuretic peptides; (i) N-terminal tails and C-terminal segments of (h) comprising a substitution selected from the group consisting of Lys-to-Gly and Lys-to-Arg; (j) N-terminal tails and C-terminal segments of (h) comprising a substitution selected from the group consisting of Gly-to-Lys and Arg-to-Lys; (k) N-terminal tails and C-terminal segments of (h) comprising an inserted Lys. 15. The natriuretic compound conjugate of claim 12 wherein X3 is selected from the group consisting of: (a) Lys; (b) Gly; (c) Arg; (d) hBNP segments KV, KVL, KVLR (SEQ ID NO. 107), KVLRR (SEQ ID NO. 106), and KVLRRH (SEQ ID NO. 105); and (e) hBNP segments of (d) comprising a substitution selected from the group consisting of Lys-to-Gly and Lys-to-Arg; (f) hBNP segments of (d) comprising a substitution selected from the group consisting of Gly-to-Lys and Arg-to-Lys; (g) hBNP segments of(d) comprising an inserted Lys; (h) C-terminal tails and N-terminal segments of C-terminal tails of natriuretic peptides; (i) C-terminal tails and N-terminal segments of C-terminal tails of (h) comprising a substitution selected from the group consisting of Lys-to-Gly and Lys-to-Arg; (j) C-terminal tails and N-terminal segments of C-terminal tails of (h) comprising a substitution selected from the group consisting of Gly-to-Lys and Arg-to-Lys; (k) C-terminal tails and N-terminal segments of C-terminal tails of (h) comprising an inserted Lys. 16. The natriuretic compound conjugate of claim 12 wherein the natriuretic compound comprises a sequence selected from the group consisting of: (a) SPKMVQGSGCFGRKMDRISSSSGLGCKVL; (SEQ ID NO. 123) (b) SPKMVQGSGCFGRKMDRISSSSGLGC; (SEQ ID NO. 124) and (c) segments (a) or (b) comprising a substitution selected from the group consisting of Lys-to-Gly and Lys-to-Arg. 17. The natriuretic compound conjugate of claim 12 wherein X1 comprises a 1-9 amino acid residue sequence from the N-terminus of hBNP. 18. The natriuretic compound conjugate of claim 12 wherein X1 comprises SPX3MVQGSG (SEQ ID NO: 125), and wherein X2 comprises a modifying moiety conjugation site. 19. The natriuretic compound conjugate of claim 12 wherein X3 comprises a 1-6 amino acid residue sequence from the C-terminus of hBNP. 20. The natriuretic compound conjugate of claim 12 wherein X3 comprises KVLRRH (SEQ. ID. NO: 105), KVLRR (SEQ ID NO. 106), KVLR (SEQ ID NO. 107), KVL, KV or K. 21. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises a native hBNP sequence (SEQ ID NO. 73) having one or more mutations selected from the group consisting of Lys3Arg, Lys14Arg, Arg30Lys, Lys27Arg, and Arg31Lys. 22. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises a native hBNP sequence (SEQ ID NO. 73), having one or more insertions or deletions. 23. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises a native hBNP amino acid sequence (SEQ ID NO. 73) and a N-terminal or C-terminal Lys. 24. The natriuretic compound conjugate of claim 1 further defined as: (a) comprising a multipeptide comprising two or more amino acid sequences encoding a natriuretic compound; (b) optionally comprising a spacer sequence between each set or adjacent natriuretic compound encoding sequences; (c) optionally comprising an extension at either or both ends of the multipeptide, the extension comprising one or more amino acids. 25. The natriuretic compound conjugate of claim 24 wherein the natriuretic peptide units each comprise hBNP (SEQ ID NO. 73) or a biologically active analog, segment or segment analog thereof. 26. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound consists of a native BNP. 27. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound consists of a native hBNP. 28. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound consists of a native ANP. 29. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound consists of a canine BNP. 30. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound consists of urodilatin. 31. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound consists of DNP. 32. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises an amino acid sequence: X1MVQGSGCFGRX2MDRISSSSGLGCX3 (SEQ ID NO. 126), wherein X1, X2 and X3 are each independently selected from the group consisting of Lys, Gly and Arg, with the proviso that at least one of X1, X2 and X3 is Arg or Gly. 33. The natriuretic compound conjugate of claim 32 wherein the sequence comprises: (a) N-terminal to X1, an extension selected from the group consisting of: SPK, PK and K; and/or (b) C-terminal to X3, an extension selected from the group consisting of -VLRRH (SEQ ID NO: 19), -VLRR (SEQ ID NO: 20), -VLR, -VL, and -V. 34. The natriuretic compound conjugate of claim 32 wherein X1 is Lys, X2 is Arg and X3 is Arg. 35. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises an amino acid sequence: CFGRX1MDRISSSSGLGCX2(SEQ ID NO: 21), wherein X1 and/or X2 comprises a modifying moiety conjugation site coupled to the modifying moiety. 36. The natriuretic compound conjugate of claim 35 wherein X1 comprises Lys coupled to the modifying moiety. 37. The natriuretic compound conjugate of claim 35 wherein X2 comprises Lys coupled to the modifying moiety. 38. The natriuretic compound conjugate of claim 1 wherein the modifying moiety conjugation site comprises a moiety selected from the group consisting of natural or non-natural amino acid side chains, an N-terminus of the natriuretic compound, and a C-terminus of the natriuretic compound. 39. The natriuretic compound conjugate of claim 38 wherein the modifying moiety conjugation site is a Lys side chain. 40. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound conjugate includes only one modifying moiety. 41. The natriuretic compound conjugate of claim 1 wherein: (a) the natriuretic compound comprises a Lys3 to Cys26 segment of hBNP (SEQ ID NO. 127) and a disulfide bond coupling Cys10 of the segment to the Cys26; a single modifying moiety coupled to the natriuretic compound at the Lys3, wherein the amino acid sequence of hBNP is SEQ ID NO. 73. 42. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises a Cys10 to Cys236 segment of hBNP (SEQ ID NO. 128) and a disulfide bond coupling the Cys10 to the Cys26, wherein said natriuretic compound is a monoconjugate including a single modifying moiety coupled thereto at Lys14 of the segment. 43. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises a Cys10 to Lys27 segment of hBNP (SEQ ID NO. 129), wherein said natriuretic compound is a monoconjugate including a single modifying moiety coupled thereto at Lys27 of the segment. 44. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises a Cys10 to His32 (SEQ ID NO. 130) segment of hBNP and a disulfide bond coupling the Cys10 to Cys26 of the segment, wherein said natriuretic compound is a monoconjugate including a single modifying moiety coupled thereto at Lys27 of the segment. 45. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound comprises a Cys10 to Cys26 segment of hBNP (SEQ ID NO. 128) and a disulfide bond coupling the Cys10 to the Cys26; wherein the natriuretic compound is a monoconjugate including a single modifying moiety coupled thereto at the N-terminus of the natriuretic compound. 46. The natriuretic compound conjugate of claim 1 wherein: (a) the natriuretic compound consists of the hBNP amino acid sequence; and (b) the natriuretic compound conjugate is a diconjugate comprising: (c) a modifying moiety coupled to the natriuretic peptide at Lys3 of the hBNP amino acid sequence, wherein the amino acid sequence of hBNP is SEQ ID NO. 73, and (d) a modifying moiety coupled to the natriuretic peptide at Lys14 of the hBNP amino acid sequence, wherein the amino acid sequence of hBNP is SEQ ID NO. 73. 47. The natriuretic compound conjugate of claim 1 wherein: (a) the natriuretic compound is hBN, wherein the amino acid sequence of hBNP is SEQ ID NO. 73; and (b) the natriuretic compound conjugate is a diconjugate comprising: (i) a modifying moiety coupled to the natriuretic peptide at Lys3 of the hBNP amino acid sequence; and (ii) a modifying moiety coupled to the natriuretic peptide at Lys27 of the hBNP amino acid sequence. 48. The natriuretic compound conjugate of claim 1 wherein the natriuretic compound sequence comprises N-terminal tail and the modifying moiety is coupled to an amino acid which is positioned in the N-terminal tail. 49. The natriuretic compound conjugate of claim 48 wherein the N-terminal tail consists of a native sequence of an N-terminal tail of a natriuretic peptide or a C-terminal segment of an N-terminal tail of a natriuretic peptide. 50. The natriuretic compound conjugate of claim 1 wherein the polyalkylene glycol moiety comprises a polyethylene glycol moiety. 51. The natriuretic compound conjugate of claim 1 wherein the modifying moiety comprises a linear or branched polyalkylene glycol moiety coupled to the natriuretic compound and a linear or branched alkyl moiety coupled to the polyalkalene glycol moiety at a site which is distal relative to the natriuretic compound. 52. The natriuretic compound conjugate of claim 1 wherein the modifying moiety comprises a linear or branched alkyl moiety coupled to the natriuretic compound and a polyalkylene glycol moiety coupled to the alkyl moiety at a site which is distal relative to the natriuretic compound. 53. The natriuretic compound conjugate of claim 1 wherein the modifying moiety is coupled to the natriuretic compound by a bond that is hydrolysable in vivo. 54. The natriuretic compound conjugate of claim 1 wherein the modifying moiety is coupled to the natriuretic compound by a bond that is hydrolysable in the bloodstream. 55. The natriuretic compound conjugate of claim 1 wherein the modifying moiety is coupled to the natriuretic compound by a bond that is not hydrolysable in vivo. 56. The natriuretic compound conjugate of claim 1 wherein the modifying moiety is coupled to the natriuretic compound by a bond that is not hydrolysable in the bloodstream. 57. The natriuretic compound conjugate of claim 1 wherein the modifying moiety is coupled to the natriuretic compound by a bond selected from the group consisting of ester, carbonate, carbamate, amide, ether, and amine. 58. The natriuretic compound conjugate of claim 1 wherein the modifying moiety is hydrolysable in vivo to yield a pegylated natriuretic compound. 59. The natriuretic compound conjugate of claim 58 wherein the modifying moiety is hydrolysable in vivo to yield a pegylated natriuretic compound comprising one or more PEG moieties having from 2 to 6 PEG units. 60. A pharmaceutical formulation comprising the natriuretic compound conjugate of claim 1. 61. The pharmaceutical formulation of claim 60 formulated for a route of delivery selected from the group consisting of enteral, perenteral, oral, subcutaneous, sublingual, buccal, nasal, intravenous and intramuscular. 62. A method of treating a condition characterized by an excessive level of extracellular fluid, the method comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a natriuretic compound conjugate of claim 1. 63. The method of claim 62 wherein the condition comprises congestive heart failure. 64. The method of claim 62 wherein the condition comprises chronic congestive heart failure. 65. The method of claim 62 wherein the condition comprises acute congestive heart failure. 66. The method of claim 62 wherein the natriuretic compound conjugate is self-administered. 67. The method of claim 62 wherein the natriuretic compound conjugate is orally administered. 68. The method of claim 62 wherein the natriuretic compound conjugate is administered via a route of administration selected from the group consisting of enteral, perenteral, oral, subcutaneous, sublingual, buccal, nasal, intravenous and intramuscular. 69. The method of claim 62 wherein the condition is hypertension. 70. A method of making the natriuretic compound conjugate of claim 1, the method comprising: (a) conjugating a natriuretic peptide multipeptide comprising two or more natriuretic compound units wherein the natriuretic peptide is selected from the group consisting of a brain natriuretic peptide, atrial natriuretic peptide, C-type natriuretic peptide, dendroaspis natriuretic peptide and a biologically active segment thereof; (b) cleaving the natriuretic peptide multipeptide to yield natriuretic compound conjugate; (c) oxidizing the cleaved natriuretic compound conjugate to form one or more disulfide bonds in the natriuretic compound conjugate. 71. The method of claim 70 wherein the natriuretic compound comprises Cys10 to Cys26 of hBNP (SEQ ID NO. 128) step 122(c) yields disulfide bond between the Cys10 and Cys26. 72. A method of making the natriuretic compound conjugate of claim 1, the method comprising: (a) making a multi-peptide natriuretic compound comprising two or more natriuretic compound units; (b) cleaving the natriuretic peptide multipeptide to yield natriuretic peptide compound; (c) conjugating the natriuretic compound to yield natriuretic compound conjugate; (d) oxidizing the cleaved natriuretic compound conjugate to form one or more disulfide bonds in the natriuretic compound conjugate. 73. The method of claim 72 wherein the natriuretic compound comprises Cys10 to Cys26 of hBNP (SEQ ID NO. 128) and step 124(c) yields a disulfide bond between the Cys10 and Cys26. 74. A method of making the natriuretic compound conjugate of claim 1, the method comprising: a. making a multi-peptide natriuretic compound comprising two or more natriuretic compound units selected from the group consisting of brain natriuretic peptide, atrial natriuretic peptide, C-type natriuretic peptide, dendroaspis natriuretic peptide and a biologically active segment thereof; b. cleaving the natriuretic peptide multipeptide to yield natriuretic compound; c. oxidizing the cleaved natriuretic compound to form one or more disulfide bonds in the natriuretic compound; and d. conjugating the natriuretic compound to the modifying moiety of claim 1. 75. A natriuretic compound conjugate comprising: (a) a natriuretic compound comprising: i. a natriuretic molecule NPR-A binding site; and ii. at least one modifying moiety conjugation site wherein the natriuretic compound comprises a peptide or a biologically active peptide segment of brain natriuretic peptide, atrial natriuretic peptide, C-type natriuretic peptide, or dendroaspis natriuretic peptide; and (b) at least one modifying moiety attached to said modifying moiety conjugation site, wherein the modifying moiety has a formula: each C is independently selected and is an alkyl moiety having m carbons and m is from 1 to 20; and each PAG is independently selected and is a polyalkylene glycol moiety having n subunits and n is from 2 to 25; each X is independently selected and is a linking moiety selected from the group consisting of —C —, —O—, —C(O)—, —NH—, —NHC(O)—, and —C(O)NH—, and o is from 1 to 15, and wherein said natriuretic compound retains a therapeutically significant percentage of cGMP stimulating activity relative to a corresponding unconjugated natriuretic compound. 76. A natriuretic compound conjugate comprising: (a) a natriuretic compound comprising: i. a natriuretic molecule NPR-A binding site; and ii. at least one modifying moiety conjugation site wherein the natriuretic compound comprises a peptide or a biologically active peptide segment of brain natriuretic peptide, atrial natriuretic peptide, C-type natriuretic peptide, or dendroaspis natriuretic peptide; and (b) at least one modifying moiety attached to said modifying moiety conjugation site, wherein the modifying moiety has a formula: each C is independently selected and is an alkyl moiety having m carbons and m is from 1 to 20; and each PAG is independently selected and is a polyalkylene glycol moiety having n subunits and n is from 2 to 25; each X is independently selected and is a linking moiety selected from the group consisting of —C—, —O—, —C(O)—, —NH—, —NHC(O)—, and —C(O)NH—, and o is from it 1 to 15, and wherein said natriuretic compound conjugate retains at least 50% of the cGMP stimulating activity of a corresponding unconjugated natriuretic compound. 77. A natriuretic compound conjugate comprising: (a) a natriuretic compound comprising: i. a natriuretic molecule NPR-A binding site; and ii. at least one modifying moiety conjugation site wherein the natriuretic compound comprises a peptide or a biologically active peptide segment of brain natriuretic peptide, atrial natriuretic peptide, C-type natriuretic peptide, or dendroaspis natriuretic peptide; and (b) at least one modifying moiety attached to said modifying moiety conjugation site, wherein the modifying moiety has a formula: each C is independently selected and is an alkyl moiety having m carbons and m is from 1 to 20; and each PAG is independently selected and is a polyalkylene glycol moiety having n subunits and n is from 2 to 25; each X is independently selected and is a linking moiety selected from the group consisting of —C—, —O—, —C(O)—, —NH—, —NHC(O)—, and —C(O)NH—, and o is from 1 to 15, and wherein said natriuretic compound conjugate is more hydrophilic than a corresponding unconjugated natriuretic compound. 78. A natriuretic compound conjugate comprising: (a) a natriuretic compound comprising: i. a natriuretic molecule NPR-A binding site; and ii. at least one modifying moiety conjugation site wherein the natriuretic compound comprises a peptide or a biologically active peptide segment of brain natriuretic peptide, atrial natriuretic peptide, C-type natriuretic peptide, or dendroaspis natriuretic peptide; and (b) at least one modifying moiety attached to said modifying moiety conjugation site, wherein the modifying moiety has a formula: each C is independently selected and is an alkyl moiety having m carbons and m is from 1 to 20; and each PAG is independently selected and is a polyalkylene glycol moiety having n subunits and n is from 2 to 25; each X is independently selected and is a linking moiety selected from the group consisting of —C—, —O—, —C(O)—, —NH—, —NHC(O)—, and —C(O)NH—, and o is from 1 to 15, and wherein said natriuretic compound conjugate is more amphiphilic than a corresponding unconjugated natriuretic compound.