Targeting vector-phospholipid conjugates

Abstract

Peptide vectors having high KDR binding affinity and processes for making such vectors are provided. The peptide vectors may be conjugated to phospholipids and included in ultrasound contrast agent compositions. Such ultrasound contrast agents are particularly useful in therapeutic and diagnostic methods, such as in imaging KDR-containing tissue and in the evaluation and treatment of angiogenic processes associated with neoplastic conditions. The present invention also provides processes for the large scale production of highly pure dimeric and monomeric peptide phospholipid conjugates as well as precursor materials used to form the conjugates. The present invention further provides processes for the large scale production of highly pure peptide phospholipid conjugates which contain very low levels of TFA.

1 .- 37 . (canceled) 
     
     
         38 . A method for preparing a gas-filled microvesicle comprising a phospholipid which comprises the steps of:
 a. preparing an aqueous-organic emulsion comprising i) an aqueous medium including water, ii) an organic solvent substantially immiscible with water, iii) a phospholipid, iv) a peptide-phospholipid conjugate selected from the group consisting of:   
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         and v) a lyoprotecting agent; 
         b. lyophilizing said emulsion, to obtain a lyophilized matrix comprising said phospholipid; 
         c. contacting said lyophilized matrix with a biocompatible gas; and 
         d. reconstituting said lyophilized matrix by dissolving it into a physiologically acceptable aqueous carrier liquid, to obtain a suspension of said gas-filled microvesicles. 
       
     
     
         39 . The method of  claim 38  wherein step a comprises the steps of:
 a1. preparing an aqueous suspension comprising a pegylated phospholipid and said peptide-phospholipid conjugate; 
 a2. preparing an aqueous-organic emulsion comprising the aqueous medium, the organic solvent, the phospholipid, and the lyoprotecting agent; and 
 a3. admixing said aqueous suspension of step a1 with the emulsion of step a2. 
 
     
     
         40 . A method of making a peptide phospholipid conjugate comprising, conjugating a peptide selected from the group consisting of SEQ ID NO. 2, Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH 2 cyclic (6-13) disulfide, Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH 2 cyclic (2-12) disulfide, SEQ ID NO. 5, and Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH 2  cyclic (2-12) disulfide]-NH 2  cyclic (6-13) disulfide with a phospholipid. 
     
     
         41 . The method of  claim 40 , wherein the phospholipid is pegylated. 
     
     
         42 . The method of  claim 41 , wherein the pegylated phospholipid is DSPE-PEG2000-NH 2 . 
     
     
         43 . The method of  claim 42 , wherein the conjugation comprises reacting a mono NHS ester of the peptide with a free amino group of the phospholipid salt. 
     
     
         44 .- 49 . (canceled) 
     
     
         50 . A method of making a peptide-phospholipid conjugate having low levels of TFA comprising eluting a peptide phospholipid conjugate and TFA ions in a size exclusion column. 
     
     
         51 . The method of  claim 50 , wherein said eluting is in the presence of ammonium bicarbonate. 
     
     
         52 . The method of  claim 50 , wherein the peptide phospholipid conjugate comprises a pegylated phospholipid. 
     
     
         53 . The method of  claim 52 , wherein the pegylated phospholipid is DSPE-PEG2000-NH 2 . 
     
     
         54 . The method of  claim 50 , wherein the peptide-phospholipid conjugate comprises a peptide dimer. 
     
     
         55 . The method of  claim 54 , wherein the peptide dimer comprises Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH 2  cyclic (2-12) disulfide]-NH 2  cyclic (6-13) disulfide. 
     
     
         56 . The method of  claim 50 , wherein the peptide-phospholipid conjugate comprises a peptide monomer. 
     
     
         57 . The method of  claim 56 , wherein the monomer is selected from the group consisting of: SEQ ID NO. 2, Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH 2  cyclic (6-13) disulfide, Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH 2  cyclic (2-12) disulfide, and SEQ ID NO. 5.