Lipoprotein targeting protease inhibitors and uses

Abstract

Described herein is the design and construction of a class of lipoprotein targeting protease inhibitors. Small peptides with protease inhibitor activity are conjugated to hydrophobic, lipoprotein targeting molecules using, for instance, amine reactive chemistry. Methods of use of the resultant lipoprotein targeting protease inhibitor (antiprotease) molecules are also described. Also described is the production and use of protease inhibitor enriched HDL particles, as well as A1AT-peptide-enriched HDL particles, and their use in various therapeutic contexts.

1 . A lipoprotein targeting protease inhibitor peptide having the following generic structure: T-I
 in which T is a hydrophobic entity comprising a vitamin E (VitE), an acyl chain, or cholesterol; and   I is a peptide-based protease inhibitor;   
       where T is covalently attached directly to I, or indirectly by way of a hydrophilic linker L. 
     
     
         2 . The lipoprotein targeting protease inhibitor peptide of  claim 1 , wherein T is a vitamin E selected from the group consisting of: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, or delta-tocotrienol. 
     
     
         3 . The lipoprotein targeting protease inhibitor peptide of  claim 1 , wherein T is an acyl chain 4 to 24 carbons in length, with any degree of hydrogen saturation. 
     
     
         4 . The lipoprotein targeting protease inhibitor peptide of  claim 1 , wherein I is a peptide-based inhibitor of elastase, matrix metalloprotease (MMP), cathepsin, chymase, thrombin, coagulation factor IX, coagulation factor X, urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), or a proteolytic component of the complement cascade (C1r, C1s, MASPs 1-3, C2, Factor B, Factor D or Factor I). 
     
     
         5 . The lipoprotein targeting protease inhibitor peptide of  claim 4 , wherein the peptide-based inhibitor I:
 inhibits elastase, and comprises the sequence Ala-Ala-Pro-Val;   inhibits elastase, and comprises the general structure R 1 -W p -X n -AA 1 -AA 2 -AA 3 -AA 4 -Y m-R 2 , in which AA 1  is -Arg-, -Phg- and -Nle- or is a bond; AA 2  is -Ala-, -Phg-, -Cit- and -Nle-; AA 3  is -Trp-, -Val- and -Tyr-; and AA 4 : -Phg- and -Gly-;   inhibits elastase, and comprises a constrained or β-hairpin peptide as shown in U.S. Pat. No. 8,658,604;   inhibits elastase, and comprises Pep4 (KRCCPDTCGIKCL; positions 3-16 pf SEQ ID NO: 8) or Pep4M (KRMMPDTMGIKML; positions 3-16 of SEQ ID NO: 9);   inhibits matrix metalloprotease, and comprises the sequence of an inhibitory peptide in Ndinguri et al.,  Molecules  17:14230-14248, 2012;   inhibits cathepsin, and comprises the structure Z-Phe-Gly-NHO-Bz, in which Z=carboxybenzyl and Bz=benzyl;   inhibits cathepsin, and comprises the structure Z-Phe-Phe-DK or Z-Phe-Phe-CHN 2 ;   inhibits chymase, and comprises the structure Z-Arg-Glu-Thr-Phep(OPh) 2 ;   inhibits thrombin and/or coagulation factors IX and X, and is selected from hirudin (MTYTDCTES GQNLCLCEGSNVCGQGNKCILGSDGEKNQCVTGEGTPKPQSHNDGD FEEIPEEYLQ; SEQ ID NO: 11) or a derivative thereof, such as lepirudin or desirudin; or   inhibits plasminogen activator, and comprises aprotinin (SEQ ID NO: 12;   RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGG A) or the plasminogen activator inhibitor type 1 (PAI-1)-derived peptide EEIIMD (positions 3-8 of SEQ ID NO: 10).   
     
     
         6 . The lipoprotein targeting protease inhibitor peptide of  claim 4 , wherein the peptide-based inhibitor inhibits elastase and comprises Ala-Ala-Pro-Val-chloromethylketone (AAPV-CMK) (SEQ ID NO: 3). 
     
     
         7 . The lipoprotein targeting protease inhibitor peptide of  claim 1 , comprising the linker L which is a hydrophilic linker comprising polyethylene glycol (PEG) or succinimide 
     
     
         8 . The lipoprotein targeting protease inhibitor peptide of  claim 1 , comprising the structure: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 4) 
                 
                     
                   VitE-AAPV; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 5) 
                 
                     
                   VitE-PEG-AAPV; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 6) 
                 
                     
                   VitE-PEG-AAPV-CMK; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 7) 
                 
                     
                   VitE-AAPV-CMK; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 8) 
                 
                     
                   VitE-PEG-KRCCPDTCGIKCL; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 9) 
                 
                     
                   VitE-PEG-KRMMPDTMGIKML; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 10) 
                 
                     
                   VitE-PEG-EEIIMD; 
                 
                     
                     
                 
                     
                   VitE-PEG-hirudin; 
                 
                     
                     
                 
                     
                   VitE-PEG-lepirudin; 
                 
                     
                     
                 
                     
                   VitE-PEG-desirudin; 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 13) 
                 
                     
                   VitE-PEG-KGSGAAPV-CMK;  
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 14) 
                 
                     
                   VitE-PEG-K(F)GSGAAPV-CMK. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         9 . A pharmaceutical composition, comprising the lipoprotein targeting protease inhibitor peptide of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         10 . A method, comprising administering the pharmaceutical composition of  claim 9  to a subject. 
     
     
         11 . The method of  claim 10 , which is a method of treating a protease-mediated disease or defect in the subject. 
     
     
         12 . The method of  claim 11 , wherein the I component in the lipoprotein targeting protease inhibitor peptide is selected to complement/treat the protease-mediated disease or defect of the subject. 
     
     
         13 . A method of producing HDL with enriched protease inhibitor activity, comprising contacting HDL with the lipoprotein targeting protease inhibitor peptide of  claim 1 . 
     
     
         14 . The method of  claim 13 , wherein the HDL is reconstituted HDL (rHDL) and the method is carried out ex vivo. 
     
     
         15 . The method of  claim 13 , in which contacting HDL with the lipoprotein targeting protease inhibitor peptide occurs in the bloodstream of a subject. 
     
     
         16 . A protease inhibitor enriched HDL produced by the method of  claim 13 . 
     
     
         17 . A protease inhibitor enriched HDL, comprising HDL and a lipoprotein targeting protease inhibitor peptide of  claim 1 . 
     
     
         18 . The protease inhibitor enriched HDL of  claim 17 , wherein the HDL is reconstituted HDL (rHDL).