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 AlAT-peptide-enriched HDL particles, and their use in various therapeutic contexts.

1 . A lipoprotein targeting protease inhibitor peptide comprising the structure:
 a vitamin E (VitE); and   a peptide-based protease inhibitor, wherein the peptide-based protease inhibitor:
 inhibits elastase and comprises Ala-Ala-Pro-Val-chloromethylketone (AAPV-CMK) (SEQ ID NO: 3); 
 inhibits elastase, and comprises the sequence Ala-Ala-Pro-Val (SEQ ID NO: 1); 
 inhibits elastase, and comprises Pep4 (KRCCPDTCGIKCL; SEQ ID NO: 8) or Pep4M (KRMMPDTMGIKML; SEQ ID NO: 9); 
 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-fluoromethylketone (FMK) or Z-Phe-Phe-CHN 2 , wherein Z is carboxybenzyl; 
 inhibits chymase, and comprises the structure Z-Arg-Glu-Thr-Phep(OPh) 2 ; 
 inhibits thrombin and/or coagulation factors IX and X, and is hirudin (MTYTDCTESGQNLCLCEGSNVCGQGNKCILGSDGEKNQCVT GEGTPKPQSHNDGDFEEIPEEYLQ; SEQ ID NO: 11), lepirudin, or desirudin; or 
 inhibits plasminogen activator, and comprises aprotinin (SEQ ID NO: 12; RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCM RTCGGA) or the plasminogen activator inhibitor type 1 (PAI-1)-derived peptide EEIIMD (SEQ ID NO: 10); 
   where the vitamin E is covalently attached directly to the peptide-based protease inhibitor, or indirectly by way of a hydrophilic linker.   
     
     
         2 . The lipoprotein targeting protease inhibitor peptide of  claim 1 , wherein the vitamin E is selected from the group consisting of alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol. 
     
     
         3 . The lipoprotein targeting protease inhibitor peptide of  claim 1 , comprising a hydrophilic linker, wherein the hydrophilic linker comprises polyethylene glycol (PEG) or succinimide. 
     
     
         4 . A pharmaceutical composition, comprising the lipoprotein targeting protease inhibitor peptide of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         5 . The pharmaceutical composition of  claim 4 , formulated for injection or infusion. 
     
     
         6 . A protease inhibitor enriched high density lipoprotein (HDL), comprising HDL and a lipoprotein targeting protease inhibitor peptide, comprising the structure:
 VitE-PEG-AAPV (SEQ ID NO: 5);   VitE-PEG-AAPV-CMK (SEQ ID NO: 6);   VitE-AAPV-CMK (SEQ ID NO: 7);   VitE-PEG-KRCCPDTCGIKCL (SEQ ID NO: 8);   VitE-PEG-KRMMPDTMGIKML (SEQ ID NO: 9);   VitE-PEG-EEIIMD (SEQ ID NO: 10);   VitE-PEG-hirudin (SEQ ID NO: 11);   VitE-PEG-lepirudin;   VitE-PEG-desirudin;   VitE-PEG-KGSGAAPV-CMK (SEQ ID NO: 13); or   VitE-PEG-K (F) GSGAAPV-CMK (SEQ ID NO: 14).   
     
     
         7 . The protease inhibitor enriched HDL of  claim 6 , wherein the HDL is reconstituted HDL (rHDL). 
     
     
         8 . The protease inhibitor enriched HDL of  claim 6 , wherein the vitamin E is selected from the group consisting of: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, and delta-tocotrienol. 
     
     
         9 . A pharmaceutical composition, comprising the protease inhibitor enriched HDL of  claim 6 , and a pharmaceutically acceptable carrier. 
     
     
         10 . The pharmaceutical composition of  claim 9 , formulated for injection or infusion.