Natriuretic compounds, conjugates, and uses thereof

Abstract

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, enteral, subcutaneous, pulmonary, 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.

1 . A natriuretic compound conjugate comprising:
 (a) a biologically active natriuretic compound wherein the natriuretic compound is a brain natriuretic peptide (BNP), atrial natriuretic peptide, C-type natriuretic peptide, dendroaspis natriuretic peptide or a biologically active segment thereof wherein the biologically active fragment exhibits NPR-A receptor binding activity, and comprises:
 (i) a disulfide loop for binding to the NPR-A receptor; and 
 (ii) at least one modifying moiety conjugation site; and 
   (b) at least one modifying moiety attached to said modifying moiety conjugation site, wherein the modifying moiety comprises a polyalkylene glycol moiety “PAGn” moiety;   
       wherein the modifying moiety is a formula: 
       
         
           
           
               
               
           
         
         each C is independently selected and is an alkyl moiety having m carbons and m is 1 to 20; and each PAG is independently selected and is a polyalkylene glycol moiety having n subunits and n is 2-25; 
         each X is independently selected and is a linking moiety. 
       
     
     
         2 . The natriuretic compound conjugate of  claim 1  wherein at least one m is 1 to 12. 
     
     
         3 . The natriuretic compound conjugate of  claim 1  wherein at least one n is 2 -13. 
     
     
         4 . The natriuretic compound conjugate of  claim 1  wherein the natriuretic compound is hBNP. 
     
     
         5 . The natriuretic compound conjugate of  claim 1  wherein the natriuretic peptide units each comprise hBNP (SEQ ID NO. 99) or a biologically active analog, segment or segment analog thereof. 
     
     
         6 . The natriuretic compound conjugate of  claim 1  wherein each X is independently selected from the group consisting of —C—, —O—, —C(O)—, —NH—, —NHC(O)—, and —C(O)NH—. 
     
     
         7 . The natriuretic compound conjugate of  claim 1  wherein the natriuretic compound comprises a sequence: 
       
         
           
                 
               
                   (SEQ ID NO. 132) 
                 
                 
                 
               
                     
                   A 1 PX 1 MVQGSGCFGRX 2 MDRISSSSGLGCX 3 VLR. 
                 
             
                
               
            
             
                
               
            
           
         
       
       wherein
 A 1  is an amino acid or series of amino acids native to a natriuretic peptide, 
 X 1 , X 2  and X 3  are independently selected from the group consisting of Lys, Arg and Gly, and at least one of X 1 , X 2  and X 3  is a Lys. 
 
     
     
         8 . The natriuretic compound conjugate of  claim 1  wherein the natriuretic compound comprises a brain natriuretic peptide of SEQ ID NO. 99. 
     
     
         9 . The natriuretic compound conjugate of  claim 1  wherein the natriuretic compound comprises:
 a. an amino acid sequence   
       
         
           
                 
                 
                 
               
                     
                   X 1 -C 1 FGRX 2 MDRISSSSGLGC 2 -X 3   
                   (SEQ ID NO. 133) 
                 
             
                
               
            
           
         
       
       wherein
 X 1  is optionally present and when present is an amino acid sequence having from 1-10 amino acids; 
 X 2  is Gly, Arg, or Lys; and 
 X 3  is optionally present and when present is an amino acid sequence having from 1-10 amino acids. 
 b. a disulfide bond between C 1  and C 2  to form a loop. 
 
     
     
         10 . The natriuretic compound conjugate of  claim 9  wherein X 1  is Arg or Gly. 
     
     
         11 . The natriuretic compound conjugate of  claim 9  wherein X 1  is selected from the group consisting of:
 a. Lys;   b. Gly;   c. Arg;   d. SG-, GSG-, QGSG- (SEQ ID NO. 23), VQGSG- (SEQ ID NO. 24), MVQGSG- (SEQ ID NO. 25), PKMVQGSG- (SEQ ID NO. 27), and SPKMVQGSG- (SEQ ID NO. 28);   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.   
     
     
         12 . The natriuretic compound conjugate of  claim 8  wherein X 3  is selected from the group consisting of:
 a. Lys;   b. Gly;   c. Arg;   d. hBNP segments KV, KVL, KVLR (SEQ ID NO. 30), KVLRR (SEQ ID NO.31), and KVLRRH (SEQ ID NO. 32); 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.   
     
     
         13 . The natriuretic compound conjugate of  claim 8  wherein the natriuretic compound comprises a sequence selected from the group consisting of:
 a. SPKMVQGSGCCFGRKMDRISSSSGLGCKVL (SEQ ID NO. 134);   b. SPKMVQGSGCFGRKMDRISSSSGLGC (SEQ ID NO. 135); and   C. segments a or b comprising a substitution selected from the group consisting of Lys-to-Gly and Lys-to-Arg .   
     
     
         14 . The natriuretic compound conjugate of  claim 1  wherein the compound comprises a Cys 10  to Lys 27  segment of hBNP, wherein said natriuretic compound is a monoconjugate including a single modifying moiety coupled thereto at Lys 27  of the segment. 
     
     
         15 . The natriuretic compound conjugate of  claim 1  wherein the natriuretic compound comprises a Cys 10  to His 32  segment of hBNP and a disulfide bond coupling the Cys 10  to Cys 26  of the segment, wherein said natriuretic compound is a monoconjugate including a single modifying moiety coupled thereto at Lys 27  of the segment.natriuretic 
     
     
         16 . 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:
 i. a modifying moiety coupled to the natriuretic peptide at Lys 3  of the hBNP amino acid sequence; and 
 ii. a modifying moiety coupled to the natriuretic peptide at Lys 14  of the hBNP amino acid sequence. 
   
     
     
         17 . The natriuretic compound conjugate of  claim 1  wherein:
 a. the natriuretic compound is hBNP; and   b. the natriuretic compound conjugate is a diconjugate comprising:
 i. a modifying moiety coupled to the natriuretic peptide at Lys 3  of the hBNP amino acid sequence; and 
 ii. a modifying moiety coupled to the natriuretic peptide at Lys 27  of the hBNP amino acid sequence. 
   
     
     
         18 . The natriuretic compound conjugate of  claim 1  wherein the natriuretic compound sequence comprises an N-terminal tail and the modifying moiety is coupled to an amino acid which is positioned in the N-terminal tail. 
     
     
         19 . The natriuretic compound conjugate of  claim 18  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. 
     
     
         20 . 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. 
     
     
         21 . 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. 
     
     
         22 . 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. 
     
     
         23 . 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. 
     
     
         24 . 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. 
     
     
         25 . A pharmaceutical formulation comprising the natriuretic compound conjugate of  claim 1 . 
     
     
         26 . The pharmaceutical formulation of  claim 25  formulated for a route of delivery selected from the group consisting of enteral, perenteral, oral, subcutaneous, sublingual, buccal, nasal, pulmonary, intravenous and intramuscular. 
     
     
         27 . 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 the BNP natriuretic compound conjugate of  claim 1 . 
     
     
         28 . The method of  claim 27  wherein the condition comprises congestive heart failure. 
     
     
         29 . The method of  claim 27  wherein the condition comprises chronic congestive heart failure. 
     
     
         30 . The method of  claim 27  wherein the condition comprises acute congestive heart failure. 
     
     
         31 . The method of  claim 27  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, pulmonary, intravenous and intramuscular. 
     
     
         32 . A method of making the natriuretic compound conjugate of  claim 1 , the method comprising:
 making a multi-peptide natriuretic compound comprising two or more natriuretic compound units;   cleaving the natriuretic peptide multipeptide to yield natriuretic compound;   oxidizing the cleaved natriuretic compound to form one or more disulfide bonds in the natriuretic compound;   activating the modifying moiety using an activating agent selected from the group consisting of disuccinimidyl carbonate, paranitrochloroformate, phosgene and N-hydroxysuccinimide; and   conjugating the natriuretic compound to the following activated structure.   
     
     
         33 . The method of  claim 32  wherein the activated modifying moiety has the following structure: