Peptides which enhance transport across tissues and methods of identifying and using the same

Abstract

A method of identifying a peptide which permits or facilitates the transport of an active agent through a human or animal tissue. A predetermined amount of phage from a random phage library or preselected phage library is plated unto or brought into contact with a first side, preferably the apical side, of a tissue sample or polarized tissue cell culture. At a predetermined time, the phage which is transported to a second side of the tissue opposite the first side, preferably the basolateral side, is harvested to select transported phage. This modified phage is amplified in a host. This cycle of events is repeated (using the transported phage produced in the most recent cycle) a predetermined number of times to obtain a selected phage library containing phage which can be transported from the first side to the second side. Lastly, the identity of at least one peptide coded by phage in the selected phage library is determined to identify a peptide which permits or facilitates the transport of an active agent through a human or animal tissue.

1 . A method of identifying a peptide which permits or facilitates the transport of an active agent through a human or animal tissue, comprising the steps of: 
 (a) contacting a predetermined amount of phage from a random phage library or a preselected phage library to a first side of a tissue sample or polarized tissue cell culture;    (b) at a predetermined time harvesting phage which is transported to a second side of the tissue sample or culture to select transported phage, which second side is opposite the first side;    (c) amplifying the transported phage in a host;    (d) repeating in order step (a) using the transported phage obtained in step (b) and amplified in step (c) and steps (b) and (c) a predetermined number of times to obtain a selected phage library containing phage which can be transported from the first side of the tissue sample or culture to the second side; and    (e) determining the identity of at least one peptide coded by phage in the selected phage library to identify a peptide which permits or facilitates the transport of an active agent through a human or animal tissue.    
     
     
         2 . The method of  claim 1 , wherein step (d) is repeated from 0 to 30 times.  
     
     
         3 . The method of  claim 1 , wherein the tissue sample is selected from the group consisting of duodenum, jejunum, ilium, ascending colon, transverse colon, desending colon, pelvic colon, vascular endothelium which lines the vascular system, vascular endothelium of the blood brain barrier, vascular smooth muscle, alveolar, liver, kidney, bone marrow, heart, spleen, pancreas, thymus, brain, neuronal or retinal eye tissue.  
     
     
         4 . The method of  claim 1 , wherein the tissue sample comprises epithelial cells lining the lumenal side of the gastro-intestinal track.  
     
     
         5 . The method of  claim 4 , wherein the tissue sample is derived from the colon.  
     
     
         6 . The method of  claim 1 , wherein the polarized tissue cell culture is cultured from gastro-intestinal track epithelial cells, alveolar cells, endothelial cells of the blood-brain barrier, vascular smooth muscle cells, Caco-2 cells, or T-84 cells.  
     
     
         7 . The method of  claim 1 , wherein the first side of the tissue sample or culture is the apical side and the second side is the basolateral side.  
     
     
         8 . The method of  claim 1 , wherein the active agent is a drug or antigen.  
     
     
         9 . The method of  claim 1 , wherein the active agent is a nano- or microparticle.  
     
     
         10 . The method of  claim 9 , wherein the peptide is coated onto or adsorbed onto or covalently bonded to the surface of the nano- or microparticle.  
     
     
         11 . The method of  claim 9 , wherein the nano- or microparticle is formed from the peptide.  
     
     
         12 . The method  claim 9 , wherein the nano- or microparticle is a drug-loaded or drug-encapsulated nano- or microparticle.  
     
     
         13 . A method of identifying a peptide which permits or facilitates the transport of an active agent through a human or animal tissue, comprising the steps of: 
 (a) contacting a predetermined amount of phage from a random phage library or a preselected phage library to the apical side of a tissue sample or polarized tissue cell culture;    (b) at a predetermined time harvesting phage which is transported to the basolateral side of the tissue sample or culture to select transported phage;    (c) amplifying the transported phage in a host;    (d) repeating in order step (a) using the transported phage obtained in step (b) and amplified in step (c) and steps (b) and (c) a predetermined number of times to obtain a selected phage library containing phage which can be transported from the apical side of the tissue sample or culture to the basolateral side; and    (e) determining the identity of at least one peptide coded by phage in the selected phage library to identify a peptide which permits or facilitates the transport of an active agent through a human or animal tissue.    
     
     
         14 . A peptide which permits or facilitates the transport of an active agent through a human or animal tissue, the peptide being identified by a method comprising the steps of: 
 (a) contacting a predetermined amount of phage from a random phage library or a preselected phage library to a first side of a tissue sample or polarized tissue cell culture;    (b) at a predetermined time harvesting phage which is transported to a second side of the tissue to select transported phage, which second side is opposite the first side;    (c) amplifying the transported phage in a host;    (d) repeating in order step (a) using the transported phage obtained in step (b) and amplified in step (c) and steps (b) and (c) a predetermined number of times to obtain a selected phage library containing phage which can be transported from the first side of the tissue sample or culture to the second side; and    (e) determining the identity of at least one peptide coded by phage in the selected phage library to identify a peptide which permits or facilitates the transport of an active agent through a human or animal tissue.    
     
     
         15 . The peptide of  claim 14 , wherein step (d) is repeated from 0 to 30 times.  
     
     
         16 . The peptide of  claim 14 , wherein the tissue sample is selected from the group consisting of duodenum, jejunum, ilium, ascending colon, transverse colon, desending colon, pelvic colon, vascular endothelium which lines the vascular system, vascular endothelium of the blood brain barrier, vascular smooth muscle, alveolar, liver, kidney, bone marrow, heart, spleen, pancreas, thymus, brain, neuronal or retinal eye tissue.  
     
     
         17 . The peptide of  claim 14 , wherein the tissue sample comprises epithelial cells lining the lumenal side of the gastro-intestinal track.  
     
     
         18 . The peptide of  claim 17 , wherein the tissue sample is derived from the colon.  
     
     
         19 . The peptide of  claim 14 , wherein the polarized tissue cell culture is cultured from gastro-intestinal track epithelial cells, alveolar cells, endothelial cells of the blood-brain barrier, vascular smooth muscle cells, Caco-2 cells, or T-84 cells.  
     
     
         20 . The peptide of  claim 14 , wherein the first side of the tissue sample or culture is the apical side and the second side is the basolateral side.  
     
     
         21 . The peptide of  claim 14 , wherein the active agent is a drug or antigen.  
     
     
         22 . The peptide of  claim 14 , wherein the active agent is a nano- or microparticle.  
     
     
         23 . The peptide of  claim 22 , wherein the peptide is coated onto or adsorbed onto or covalently bonded to the surface of the nano- or microparticle.  
     
     
         24 . The peptide of  claim 22 , wherein the nano- or microparticle is formed from the peptide.  
     
     
         25 . The peptide  claim 22 , wherein the nano- or microparticle is a drug-loaded or drug-encapsulated nano- or microparticle.  
     
     
         26 . A peptide comprising SEQ ID NO: 12 or a fragment thereof which contains at least 6 amino acid residues.  
     
     
         27 . A peptide comprising SEQ ID NO: 14 or a fragment thereof which contains at least 6 amino acid residues.  
     
     
         28 . A peptide comprising SEQ ID NO: 16 or a fragment thereof which contains at least 6 amino acid residues.  
     
     
         29 . A method of identifying a peptide motif which when present in a peptide permits or facilitates the transport of an active agent through a human or animal tissue, comprising the steps of: 
 (a) contacting a predetermined amount of phage from a random phage library to the apical side of a tissue sample or polarized tissue cell culture sample;    (b) at a predetermined time harvesting phage which is transported to the basolateral side of the tissue to select modified phage;    (c) amplifying the modified phage in a host;    (d) repeating in order step (a) using the modified phage obtained in step (b) and amplified in step (c) and steps (b) and (c) a predetermined number of times to obtain a selected phage library containing phage which can be transported from the first side to the second side; and    (e) determining the identity of a plurality of peptides coded by phage in the selected phage library to identify a peptide motif common to at least two of the peptides which permit or facilitate the transport of an active agent through a human or animal tissue.