Molecular transporter compositions comprising dendrimeric oligoguanidine with a tri-functional core that facilitates delivery into cells in vivo

Abstract

Novel molecular transporter compositions and their use for transporting bioactive substances into cells in living animals are disclosed. To afford in vivo delivery, the composition is covalently linked to the bioactive substance and the resultant composite structure is introduced into the subject. The transporter composition includes multiple guanidine moieties on a dendrimeric scaffold having a tri-functional core. The tri-functional core is a phosphorodiamidate or phosphoramide moiety.

1 . A method for preparing a precursor transporter composition containing a tri-functional core scaffold for dendrimeric assembly, comprising:
 (a) using two reactive sites of phosphorus oxychloride to prepare an oligomer having 4, 6, 8, 12, or 16 side chains with terminal primary amino groups in a chemically protected form; and,   (b) using a third reactive site of phosphorus oxychloride to install a linker for covalent coupling with a bioactive substance.   
   
   
       2 . The method of  claim 1 , wherein the protecting groups on each of the chemically protected amines on the side chains are trifluoroacetyl groups. 
   
   
       3 . The method of  claim 1 , wherein two sites of the tri-functional core are used for multiplying the number of side chains. 
   
   
       4 . The method of  claim 1 , wherein one site of the tri-functional core is used for a linking group to conjugation with a bioactive substance. 
   
   
       5 . A method for preparing a conjugate containing a tri-functional core with an oligoguanidine transporter composition and a bioactive substance, comprising:
 (a) contacting a precursor transporter composition having a reactive linker with the bioactive substance still on its synthesis resin under conditions effective for covalent coupling;   (b) contacting the conjugate containing the precursor transporter composition and the bioactive substance with a protecting group removal agent to remove said protecting groups to generate free amino groups on the side chains; and,   (c) contacting the conjugate containing the oligomer having multiple free primary amines on the side chains with a guanidinylation reagent to convert each of said free primary amines to a guanidinyl group to produce an oligoguanidine compound.   
   
   
       6 . The method of  claim 5 , wherein the precursor transporter composition has a tri-functional core scaffold. 
   
   
       7 . The method of  claim 5 , wherein both removal of protecting groups and guanidinylation are conducted in an ammonia medium and the guanidinylation is carried out subsequent to the removal of protecting groups without intermediate purification. 
   
   
       8 . The method of  claim 5 , wherein the guanidinylation reagent is a salt of □-methylisourea. 
   
   
       9 . The method of  claim 5 , wherein the linkage between the transporter composition and a bioactive substance is a permanent connection under biological conditions. 
   
   
       10 . The method of  claim 5 , wherein the linkage between the transporter composition and the bioactive substance is a cleavable connection under biological conditions. 
   
   
       11 . The method of  claim 5 , wherein the terminal side chains are derived from a triamine. 
   
   
       12 . The method of  claim 11 , wherein the triamine has two primary amines and one secondary amine. 
   
   
       13 . The method of  claim 12 , wherein the nitrogen atoms of the triamine can be symmetric. 
   
   
       14 . The method of  claim 12 , wherein the nitrogen atoms of the triamine can be non-symmetric. 
   
   
       15 . The method of  claim 11 , wherein the triamine is selectively protected for its two primary amines and the unprotected secondary amine is used for contacting. 
   
   
       16 . The method of  claim 15 , wherein the protecting group for primary amine is trifluoroacetyl. 
   
   
       17 . The method of  claim 5 , wherein the transporter composition has at least four guanidine residues. 
   
   
       18 . The method of  claim 17 , wherein the transporter composition has at least eight guanidine residues. 
   
   
       19 . The method of  claim 5 , wherein the conjugate comprises the transporter composition chemically connected to the bioactive substance. 
   
   
       20 . The composition of  claim 19 , wherein the transporter composition contains 4, 6, 8, 12, 16 guanidine head groups assembled from the tri-functional core as a dendrimeric scaffold. 
   
   
       21 . The composition of  claim 20 , wherein the transporter composition has the following structure: 
     
       
         
         
             
             
         
       
     
   
   
       22 . The composition of  claim 20 , wherein the transporter composition is chemically linked with a bioactive substance. 
   
   
       23 . The composition of  claim 22 , wherein the bioactive substance is a Morpholino antisense oligo. 
   
   
       24 . The composition of  claim 23 , wherein the morpholino antisense agent is effective to modify splicing of a selected pre-mRNA. 
   
   
       25 . The composition of  claim 23 , wherein the morpholino antisense agent is effective to block translation of a selected mRNA. 
   
   
       26 . The composition of  claim 22 , wherein the bioactive substance is a therapeutic agent for use in animals. 
   
   
       27 . The composition of  claim 26 , wherein the animal is a human. 
   
   
       28 . The use of  claim 26 , wherein a therapeutic agent is administered intravenously. 
   
   
       29 . The use of  claim 26 , wherein a therapeutic agent is administered intraperitoneally. 
   
   
       30 . The composition of  claim 1 , wherein the tri-functional core is phosphoramide. 
   
   
       31 . The composition of  claim 1 , wherein the tri-functional core is phosphorodiamidate.