Method for enhancing transport across biological membranes

Abstract

Methods and compositions for transporting drugs and macromolecules across biological membranes are disclosed. In one embodiment, the invention includes a method for enhancing transport of a selected compound across a biological membrane, wherein a biological membrane is contacted with a conjugate containing a biologically active agent that is covalently attached to a transport polymer. In one embodiment, the polymer consists of from 6 to 25 subunits, at least 50% of which contain a guanidino or amidino sidechain moiety. The polymer is effective to impart to the attached agent a rate of trans-membrane transport across a biological membrane that is greater than the rate of trans-membrane transport of the agent in non-conjugated form.

1 . A method for enhancing transport of a selected compound across a biological membrane, comprising 
 contacting a biological membrane with a conjugate containing a biologically active agent that is covalently attached to a transport polymer,    wherein said polymer consists of from 6 to 25 subunits, at least 50% of which contain a guanidino or amidino sidechain moiety, and the polymer contains at least 6 contiguous guanidino and/or amidino sidechain moieties, and    whereby said contacting is effective to promote transport of said conjugate across said biological membrane at a rate that is greater than the trans-membrane transport rate of the biological agent in non-conjugated form.    
     
     
         2 . The method of  claim 1 , wherein at least 70% of the subunits in the polymer contain a guanidino sidechain moiety.  
     
     
         3 . The method of  claim 1 , wherein no guanidino or amidino sidechain moiety is separated from another such moiety by more than one non-guanidino or non-amidino subunit.  
     
     
         4 . The method of  claim 1 , wherein said polymer consists of from 7 to 20 subunits.  
     
     
         5 . The method of  claim 1 , wherein each subunit contains a guanidino group.  
     
     
         6 . The method of  claim 1 , wherein said polymer comprises a peptide containing at least six contiguous arginine residues.  
     
     
         7 . The method of  claim 6 , wherein said polymer consists of from 7 to 20 subunits.  
     
     
         8 . The method of  claim 6 , wherein all of said subunits are arginine residues.  
     
     
         9 . The method of  claim 6 , wherein at least one of said arginine residues has a D-configuration.  
     
     
         10 . The method of  claim 6 , wherein all of said arginine residues have a D-configuration.  
     
     
         11 . The method of  claim 1 , wherein said conjugate contains at least two such transport polymers.  
     
     
         12 . The method of  claim 1 , wherein said biological membrane is a eukaryotic cell membrane.  
     
     
         13 . The method of  claim 1 , wherein said biological membrane is a prokaryotic cell membrane.  
     
     
         14 . The method of  claim 1 , wherein said agent is a nucleic acid or nucleic acid analog.  
     
     
         15 . The method of  claim 14 , wherein said agent is a peptide nucleic acid.  
     
     
         16 . The method of  claim 1 , wherein said agent is a polypeptide.  
     
     
         17 . The method of  claim 16 , wherein said polypeptide is a protein antigen and said biological membrane is a cell membrane of an antigen-presenting cell.  
     
     
         18 . The method of  claim 17 , wherein said agent is a tumor antigen.  
     
     
         19 . The method of  claim 1 , wherein said agent is a taxane compound.  
     
     
         20 . The method of  claim 1 , wherein said agent is a metal ion.  
     
     
         21 . The method of  claim 1 , wherein said agent is an antimicrobial compound.  
     
     
         22 . The method of  claim 1 , wherein said polymer is conjugated to said agent by a cleavable linker.  
     
     
         23 . The method of  claim 22 , wherein said cleavable linker contains an ester group.  
     
     
         24 . The method of  claim 22 , wherein said cleavable linker contains a disulfide group.  
     
     
         25 . The method of  claim 22 , wherein said cleavable linker contains a first cleavable group that is distal to the agent, and a second cleavable group that is proximal to the agent, such that cleavage of the first cleavable group yields a linker-agent conjugate containing a nucleophilic moiety capable of reacting intramolecularly to cleave the second cleavable group, thereby releasing the agent from said linker and polymer.  
     
     
         26 . The method of  claim 25 , wherein said first group is an amide group and said second group is an ester group, and cleavage of the amide group yields a free amino group that reacts intramolecularly to cleave the ester group.  
     
     
         27 . The method of  claim 25 , wherein said first group is a phosphate ester group and said second group is a carboxylate ester group, such that cleavage of the phosphate ester group yields a free hydroxyl group that reacts intramolecularly to cleave the carboxylate ester group.  
     
     
         28 . The method of  claim 1 , for use in screening a plurality of conjugates for a selected biological activity, wherein said conjugates are formed from a plurality of candidate agents, and said contacting includes contacting each conjugate with a cell that exhibits a detectable signal upon uptake of the conjugate into the cell, such that the magnitude of the signal is indicative of the efficacy of the conjugate with respect to the selected biological activity.  
     
     
         29 . The method of  claim 28 , wherein said candidate agents are selected from a combinatorial library.  
     
     
         30 . A pharmaceutical composition for delivering a biologically active agent across a biological membrane, comprising: 
 a conjugate in accordance with  claim 1 , and    a pharmaceutically acceptable excipient.