Methods and compositions 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 pertains to 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 a preferred 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 conjugate comprised of a biologically active agent covalently attached to a polymeric carrier having a non-peptide backbone composed of from about 6 to about 25 subunits, at least 50% of which are substituted with a guanidino or amidino sidechain moiety, wherein adjacent subunits are linked through a covalent linkage selected from amido, N-substituted amido, ester, methylenecarbonyl, methyleneimino, thioamido, phosphinate, phosphonamidate, phosphonamidate ester, retropeptidyl, trans-alkenyl, fluoroalkenyl, ethylene, thioether, hydroxyethylene, methyleneoxy, tetrazolyl, retrothiamido, retromethyleneimino, sulfonamido, methylenesulfonamido, retrosulfonamido, alkylene, sulfonyl, azo, and imino linkages, and combinations thereof, with the proviso that when the subunits are amino acids, the covalent linkages are other than amido linkages.  
     
     
         2 . The conjugate of  claim 1 , wherein the subunits are selected from N-substituted amino acids, hydroxy amino acids, and amino aldehydes.  
     
     
         3 . The conjugate of  claim 2 , wherein the polymeric carrier is composed of 7 to 15 subunits.  
     
     
         4 . The conjugate of  claim 3 , wherein the subunits are N-substituted amino acids.  
     
     
         5 . The conjugate of  claim 4 , wherein the subunits are N-substituted glycine residues.  
     
     
         6 . The conjugate of  claim 5 , wherein adjacent subunits are linked through N-substituted amide linkages, such that the polymeric carrier is a peptoid.  
     
     
         7 . The conjugate of  claim 6 , wherein the polymeric carrier is composed of 7 to 15 N-substituted glycine residues.  
     
     
         8 . The conjugate of  claim 4 , wherein the N-substituted amino acids are N-substituted arginine residues.  
     
     
         9 . The conjugate of  claim 8 , wherein at least one of the N-substituted arginine residues has a D-configuration.  
     
     
         10 . The conjugate of  claim 9 , wherein all of the N-substituted arginine residues have a D-configuration.  
     
     
         11 . The conjugate of  claim 8 , wherein the polymeric carrier is composed of 7 to 15 N-substituted arginine residues.  
     
     
         12 . The conjugate of  claim 1 , wherein the carrier is effective to increase the rate at which the conjugated biologically active agent is transported through a biological membrane relative to the rate at which the biologically active agent can be transported through the biological membrane in unconjugated form.  
     
     
         13 . The conjugate of  claim 1 , wherein the carrier is effective to increase the amount of conjugated biologically active agent that is transported through a biological membrane relative to the amount of biologically active agent that can be transported through the biological membrane in unconjugated form.  
     
     
         14 . The conjugate of  claim 1 , wherein the carrier is effective to increase the rate at which the conjugate is transported through a biological membrane relative to the rate at which the biologically active agent conjugated to a basic HIV tat peptide consisting of residues 49-57 can be transported through the biological membrane.  
     
     
         15 . The conjugate of  claim 1 , wherein at least 70% of the subunits contain a guanidino or amidino sidechain moiety.  
     
     
         16 . The conjugate of  claim 15 , wherein at least 90% of the subunits contain a guanidino or amidino sidechain moiety.  
     
     
         17 . The conjugate of  claim 16 , wherein all of the subunits contain a guanidino or amidino sidechain7moiety.  
     
     
         18 . The conjugate of  claim 1 , wherein the polymeric carrier includes at least 6 contiguous subunits substituted with guanidino sidechain moieties.  
     
     
         19 . The conjugate of  claim 1 , wherein the guanidino sidechain moieties have the structure  
       
         
           
           
               
               
           
         
       
       and the amidino sidechain moieties have the structure  
       
         
           
           
               
               
           
         
       
       wherein n is 2, 3, 4 or 5.  
     
     
         20 . The conjugate of  claim 19 , wherein n is 3.  
     
     
         21 . The conjugate of  claim 1 , wherein the biologically active agent is covalently attached to the polymeric carrier through a linker.  
     
     
         22 . The conjugate of  claim 21 , wherein the linker contains a linkage that is chemically or enzymatically cleaved in vivo.  
     
     
         23 . The conjugate of  claim 22 , wherein the linkage is a carbamate, ester, thioether, disulfide or hydrazone linkage.  
     
     
         24 . The conjugate of  claim 1 , wherein the polymeric carrier is attached on at least one terminus to a flanking moiety that does not significantly affect the delivery of the biologically active agent across biological membranes.  
     
     
         25 . The conjugate of  claim 22 , wherein the polymeric carrier is attached on at least one terminus to a flanking moiety that does not significantly affect the delivery of the biologically active agent across biological membranes.  
     
     
         26 . The conjugate of  claim 25 , wherein the polymeric carrier has a first terminus and a second terminus, with the first terminus attached to a first flanking moiety and the second terminus conjugated to the biologically active agent through the linker.  
     
     
         27 . The conjugate of  claim 26 , wherein a second flanking moiety is attached to the second terminus, such that the linker is attached to the second flanking moiety.  
     
     
         28 . The conjugate of  claim 26 , wherein the first flanking moiety comprises one or more subunits that do not contain said guanidino sidechain or said amidino sidechain.  
     
     
         29 . The conjugate of  claim 27 , wherein the first flanking moiety and the second flanking moiety comprise one or more subunits that do not contain said guanidino sidechain or said amidino sidechain.  
     
     
         30 . The conjugate of  claim 26 , wherein the first flanking moiety is a blocking group effective to prevent ubiquitination in vivo.  
     
     
         31 . The conjugate of  claim 30 , wherein the blocking group is an acetyl or benzyl group.  
     
     
         32 . The conjugate of  claim 1 , wherein the biologically active agent is a therapeutic compound whose efficacy in non-conjugated form is limited by its solubility in aqueous liquid or its inability to cross biological membranes to manifest biological activity.  
     
     
         33 . The conjugate of  claim 1 , wherein the biologically active agent is an antimicrobial agent.  
     
     
         34 . The conjugate of  claim 1 , wherein the biologically active agent is an anticancer agent.  
     
     
         35 . The conjugate of  claim 1 , wherein the biologically active agent is comprised of a metal.  
     
     
         36 . The conjugate of  claim 1 , wherein the biologically active agent is a macromolecule.  
     
     
         37 . The conjugate of  claim 36 , wherein the macromolecule is selected from the group consisting of nucleic acids, oligonucleotides, polynucleotides, peptides, proteins, peptide nucleic acids, and polysaccharides.  
     
     
         38 . The conjugate of  claim 37 , wherein the macromolecule is a protein.  
     
     
         39 . The conjugate of  claim 38 , wherein the protein is an enzyme, an antigen, an antibody, or an antibody fragment.  
     
     
         40 . A pharmaceutical composition comprising the conjugate of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         41 . A pharmaceutical composition for administration, to a human subject, of a biologically active agent whose efficacy in non-conjugated form is limited by its aqueous solubility, said composition comprising the conjugate of  claim 32  and a pharmaceutically acceptable carrier.  
     
     
         42 . A method for enhancing transport of a selected biologically active agent across a biological membrane, comprising contacting a biological membrane with a conjugate comprised of the biologically active agent and a polymeric carrier covalently attached thereto, wherein the polymeric carrier has a non-peptide backbone composed of from about 6 to about 25 subunits, at least 50% of which are substituted with a guanidino or amidino sidechain moiety, wherein adjacent subunits are linked through a covalent linkage selected from amido, N-substituted amido, ester, methylenecarbonyl, methyleneimino, thioamido, phosphinate, phosphonamidate, phosphonamidate ester, retropeptidyl, trans-alkenyl, fluoroalkenyl, ethylene, thioether, hydroxyethylene, methyleneoxy, tetrazolyl, retrothiamido, retromethyleneimino, sulfonamido, methylenesulfonamido, retrosulfonamido, alkylene, sulfonyl, azo, and imino linkages, and combinations thereof, with the proviso that when the subunits are amino acids, the covalent linkages are other than amido linkages.  
     
     
         43 . The method of  claim 42 , wherein the subunits are selected from N-substituted amino acids, hydroxy amino acids, and amino aldehydes.  
     
     
         44 . The method of  claim 43 , wherein the biological membrane is a eukaryotic cell membrane.  
     
     
         45 . The method of  claim 44 , wherein the biological membrane is a prokaryotic cell membrane.  
     
     
         46 . A conjugate comprised of a biologically active agent covalently attached to a polymeric carrier having a non-peptide, non-peptoid, backbone composed of from about 6 to about 25 subunits, at least 50% of which are substituted with a guanidino or amidino sidechain moiety, wherein adjacent subunits are linked through a covalent linkage selected from amido, N-substituted amido, ester, methylenecarbonyl, methyleneimino, thioamido, phosphinate, phosphonamidate, phosphonamidate ester, retropeptidyl, trans-alkenyl, fluoroalkenyl, ethylene, thioether, hydroxyethylene, methyleneoxy, tetrazolyl, retrothiamido, retromethyleneimino, sulfonamido, methylenesulfonamido, retrosulfonamido, alkylene, sulfonyl, azo, and imino linkages, and combinations thereof.  
     
     
         47 . The conjugate of  claim 46 , wherein the sidechain moieties are selected from amino acid sidechain moieties, N-substituted amino acid sidechain moieties, hydroxy amino acid sidechain moieties, and amino aldehyde sidechain moieties.  
     
     
         48 . The conjugate of  claim 47 , wherein the polymeric carrier is composed of 7 to 15 subunits.  
     
     
         49 . The conjugate of  claim 48 , wherein the sidechain moieties are N-substituted amino acid sidechain moieties.  
     
     
         50 . The conjugate of  claim 48 , wherein the sidechain moieties are amino acid sidechain moieties.  
     
     
         51 . The conjugate of  claim 50 , wherein the polymeric carrier is composed of 7 to 15 subunits having sidechains composed of amino acid sidechain moieties.  
     
     
         52 . The conjugate of  claim 50 , wherein the amino acid sidechain moieties are arginine sidechain moieties.  
     
     
         53 . The conjugate of  claim 52 , wherein at least one of the arginine sidechain moieties has a D-configuration.  
     
     
         54 . The conjugate of  claim 53 , wherein all of the arginine sidechain moieties have a D-configuration.  
     
     
         55 . The conjugate of  claim 52 , wherein the polymeric carrier is composed of 7 to 15 subunits having sidechains composed of arginine sidechain moieties.  
     
     
         56 . The conjugate of  claim 46 , wherein the carrier is effective to increase the rate at which the conjugated biologically active agent is transported through a biological membrane relative to the rate at which the biologically active agent can be transported through the biological membrane in unconjugated form.  
     
     
         57 . The conjugate of  claim 46 , wherein the carrier is effective to increase the amount of conjugated biologically active agent that is transported through a biological membrane relative to the amount of biologically active agent that can be transported through the biological membrane in unconjugated form.  
     
     
         58 . The conjugate of  claim 46 , wherein the carrier is effective to increase the rate at which the conjugate is transported through a biological membrane relative to the rate at which the biologically active agent conjugated to a basic HIV tat peptide consisting of residues 49-57 can be transported through the biological membrane.  
     
     
         59 . The conjugate of  claim 46 , wherein at least 70% of the subunits contain a guanidino or amidino sidechain moiety.  
     
     
         60 . The conjugate of  claim 59 , wherein at least 90% of the subunits contain a guanidino or amidino sidechain moiety.  
     
     
         61 . The conjugate of  claim 60 , wherein all of the subunits contain a guanidino or amidino sidechain moiety.  
     
     
         62 . The conjugate of  claim 46 , wherein the polymeric carrier includes at least 6 contiguous subunits having guanidino sidechain moieties.  
     
     
         63 . The conjugate of  claim 46 , wherein the guanidino sidechain moieties have the structure  
       
         
           
           
               
               
           
         
       
       and the amidino sidechain moieties have the structure  
       
         
           
           
               
               
           
         
       
       wherein n is 2, 3, 4 or 5.  
     
     
         64 . The conjugate of  claim 65 , wherein n is 3.  
     
     
         65 . The conjugate of  claim 46 , wherein the biologically active agent is covalently attached to the polymeric carrier through a linker.  
     
     
         66 . The conjugate of  claim 65 , wherein the linker contains a linkage that is chemically or enzymatically cleaved in vivo.  
     
     
         67 . The conjugate of  claim 66 , wherein the linkage is a carbamate, ester, thioether, disulfide, or hydrazone linkage.  
     
     
         68 . The conjugate of  claim 46 , wherein the polymeric carrier is attached on at least one terminus to a flanking moiety that does not significantly affect the delivery of the biologically active agent across biological membranes.  
     
     
         69 . The conjugate of  claim 66 , wherein the polymeric carrier is attached on at least one terminus to a flanking moiety that does not significantly affect the delivery of the biologically active agent across biological membranes.  
     
     
         70 . The conjugate of  claim 69 , wherein the polymeric carrier has a first terminus and a second terminus, with the first terminus attached to a first flanking moiety and the second terminus conjugated to the biologically active agent through the linker.  
     
     
         71 . The conjugate of  claim 70 , wherein a second flanking moiety is attached to the second terminus, such that the linker is attached to the second flanking moiety.  
     
     
         72 . The conjugate of  claim 70 , wherein the first flanking moiety comprises one or more subunits that do not contain said guanidino sidechain moiety or said amidino sidechain moiety.  
     
     
         73 . The conjugate of  claim 71 , wherein the first flanking moiety and the second flanking moiety comprise one or more subunits that do not contain said guanidino sidechain moiety or said amidino sidechain moiety.  
     
     
         74 . The conjugate of  claim 70 , wherein the first flanking moiety is a blocking group effective to prevent ubiquitination in vivo.  
     
     
         75 . The conjugate of  claim 74 , wherein the blocking group is an acetyl or benzyl group.  
     
     
         76 . The conjugate of  claim 46 , wherein the biologically active agent is a therapeutic compound whose efficacy in non-conjugated form is limited by its solubility in aqueous liquid or its inability to cross biological membranes to manifest biological activity.  
     
     
         77 . The conjugate of  claim 46 , wherein the biologically active agent is an antimicrobial agent.  
     
     
         78 . The conjugate of  claim 46 , wherein the biologically active agent is an anticancer agent.  
     
     
         79 . The conjugate of  claim 46 , wherein the biologically active agent is comprised of a metal.  
     
     
         80 . The conjugate of  claim 46 , wherein the biologically active agent is a macromolecule.  
     
     
         81 . The conjugate of  claim 80 , wherein the macromolecule is selected from the group consisting of nucleic acids, oligonucleotides, polynucleotides, peptides, proteins, peptide nucleic acids, and polysaccharides.  
     
     
         82 . The conjugate of  claim 81 , wherein the macromolecule is a protein.  
     
     
         83 . The conjugate of  claim 82 , wherein the protein is an enzyme, an antigen, an antibody, or an antibody fragment.  
     
     
         84 . A pharmaceutical composition comprising the conjugate of  claim 46  and a pharmaceutically acceptable carrier.  
     
     
         85 . A pharmaceutical composition for administration, to a human subject, of a biologically active agent whose efficacy in non-conjugated form is limited by its aqueous solubility, said composition comprising the conjugate of  claim 76  and a pharmaceutically acceptable carrier.  
     
     
         86 . A method for enhancing transport of a selected biologically active agent across a biological membrane, comprising contacting a biological membrane with a conjugate comprised of the biologically active agent and a polymeric carrier covalently attached thereto, wherein the polymeric carrier has a non-peptide, non-peptoid, backbone composed of from about 6 to about 25 subunits, at least 50% of which are substituted with a guanidino or amidino sidechain moiety, wherein adjacent subunits are linked through a covalent linkage selected from amido, N-substituted amido, ester, methylenecarbonyl, methyleneimino, thioamido, phosphinate, phosphonamidate, phosphonamidate ester, retropeptidyl, trans-alkenyl, fluoroalkenyl, ethylene, thioether, hydroxyethylene, methyleneoxy, tetrazolyl, retrothiamido, retromethyleneimino, sulfonamido, methylenesulfonamido, retrosulfonamido, alkylene, sulfonyl, azo, and imino linkages, and combinations thereof.  
     
     
         87 . The method of  claim 86 , wherein the sidechain moieties are selected from amino acid sidechain moieties, N-substituted amino acid sidechain moieties, hydroxy amino acid sidechain moieties, and amino aldehyde sidechain moieties.  
     
     
         88 . The method of  claim 87 , wherein the biological membrane is a eukaryotic cell membrane.  
     
     
         89 . The method of  claim 87 , wherein the biological membrane is a prokaryotic cell membrane.