Imaging agents for imaging protease activity and uses thereof

Abstract

Disclosed are imaging agents having the following Formula I: (I); wherein F is a near infrared fluorophore, S is an enzymatically cleavable oligopeptide, Q is a fluorescence quencher molecule, and M is a moiety selected from the group consisting of PEG or derivative thereof and a targeting ligand, and wherein F, Q and M are linked to separate amino acids of the enzymatically cleavable oligopeptide. Compositions comprising such compounds, as well as methods of use, methods of identifying a cell or a population of cells in vivo expressing a protease of interest, and methods of treating a disease through imaging are also disclosed.

1 . An imaging agent of formula I: 
       
         
           
           
               
               
           
         
         wherein: 
         F is a near infrared fluorophore; 
         S is an enzymatically cleavable oligopeptide; 
         Q is a fluorescence quencher molecule; and 
         M is a moiety selected from the group consisting of polyethylene glycol (PEG) or a derivative thereof and a targeting ligand, wherein PEG or a derivative thereof is a polyethylene glycol polymer molecule or a derivative thereof, and wherein F, Q and M are linked to separate amino acids of the enzymatically cleavable oligopeptide. 
       
     
     
         2 . The imaging agent of  claim 1 , wherein M is PEG or a derivative thereof, wherein PEG or derivative thereof is a polyethylene glycol polymer molecule or a derivative thereof having a molecular weight of 1200 Daltons or less. 
     
     
         3 . (canceled) 
     
     
         4 . The imaging agent of  claim 1 , wherein M is a targeting ligand selected from the group consisting of cytokines, chemokines, growth factors, interferons, erythropoietin (EPO), TNFα, interleukins, growth hormone, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), gonadotropins, insulin, integrins, immunoglobulins, hormones, peptides, proteins that interact with a cell surface molecule or with a pattern recognition receptor, tumor receptor binding molecules, and molecules involved in vascular lesions. 
     
     
         5 . (canceled) 
     
     
         6 . The imaging agent of  claim 4 , wherein the ligand is cyclic Arg-Gly-Asp-Tyr-Lys peptide, c(RGDyK) (SEQ ID NO: 2). 
     
     
         7 . The imaging agent of  claim 1 , wherein F comprises a near infrared fluorescent dye selected from the group consisting of fluorescein isothiocyanate (FITC, green), cyanine dyes Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, Texas Red, IRDye800CW, and derivatives thereof. 
     
     
         8 . (canceled) 
     
     
         9 . The imaging agent of  claim 7 , wherein the near infrared fluorescent dye is selected from the group consisting of Cy5.5, Cy7, Cy7.5, IRDye800CW and derivatives thereof. 
     
     
         10 . (canceled) 
     
     
         11 . The imaging agent of  claim 1 , wherein S comprises between 4 and 100 amino acids. 
     
     
         12 . (canceled) 
     
     
         13 . The imaging agent of  claim 11 , wherein S comprises an amino acid sequence cleavable by a protease selected from the group consisting of: Arg-C proteinase, Asp-N endopeptidase, chymotrypsin-high specificity, chymotrypsin-low specificity, clostripain, enterokinase, Factor Xa, glutamyl endopeptidase, granzyme B, LysC, pepsin (pH 1.3), pepsin (pH>2), proline-endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin, matrix metalloproteinases (MMPs), MMP-1, MMP-2, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19, MMP-20, MMP-21, MMP-22, MMP-23, MMP-24, MMP-25, MMP-26, MMP-27, MMP-28, MMP-29, MMP-30, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, cathepsin-B, cathepsin-C, cathepsin-D, cathepsin-E, cathepsin-G, cathepsin-H, and cathepsin-L, and dipeptidyl peptidase. 
     
     
         14 . The imaging agent of  claim 11 , wherein S is an oligopeptide comprising an amino acid sequence motif selected from the group consisting of Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys (SEQ ID NO: 3), Val-Pro-Leu-Ser-Leu-Thr-Met (SEQ ID NO: 4), Pro-Tyr-Ala-Leu-Trp-Ala (SEQ ID NO: 5), Pro-Leu-Ala-Tyr-Trp-Ala-Arg (SEQ ID NO: 6), Leu-Pro-Lys-Gly-Leu (SEQ ID NO: 7), Arg-Pro-Lys-Pro-Val-Glu (SEQ ID NO: 8), Val-Pro-Arg (SEQ ID NO: 9). 
     
     
         15 . The imaging agent of  claim 11 , wherein S comprises an oligopeptide comprising the core amino acid motif X-Pro-Leu-Gly-Val-Arg-X-X-X-X, wherein X is any natural or synthetic amino acid. 
     
     
         16 . The imaging agent of  claim 15 , wherein S is Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys-Gly-Gly (SEQ ID NO: 10). 
     
     
         17 .- 18 . (canceled) 
     
     
         19 . The imaging agent of  claim 1 , wherein Q is a non-fluorescent fluorescence quencher selected from the group consisting of DABCYL, DABSYL, Eclipse® DarkQuencher, ElleQuencher™, Iowa Black® Dark Quenchers, Black Hole Quencher-1 (BHQ-1), Black Hole Quencher-2 (BHQ-2), Black Hole Quencher-3 (BHQ-3), Blackberry Quencher 650, QSY-7, QSY-9, and QSY-21. 
     
     
         20 .- 23 . (canceled) 
     
     
         24 . A composition comprising the imaging agent of  claim 1 , and a carrier. 
     
     
         25 . A method of identifying a cell or a population of cells in vivo expressing a protease of interest comprising:
 a) contacting the cell or a population of cells expressing a protease of interest with the imaging agent of  claim 1 , which is selectively cleavable by a protease of interest;   b) allowing the imaging agent to be selectively cleaved by the protease of interest in the cell or population of cells; and   c) detecting the fluorescence of the fluorophore in the imaging agent after being cleaved by the protease of interest in the cell or population of cells.   
     
     
         26 . The method of  claim 25 , wherein the cell or population of cells is a tumor cell. 
     
     
         27 . A method of diagnosing a disease overexpressing a protease in a subject comprising:
 a) administering to a subject suspected of having said disease, an imaging agent which is selectively cleavable by a protease of interest, the cleavage of which indicates the presence of the disease, wherein said imaging agent is an imaging agent of  claim 1 ;   b) allowing the imaging agent to be cleaved by the protease of interest;   c) detecting the fluorescence of the fluorophore in the imaging agent binding the protease of interest in the subject; and   d) determining whether the subject has a disease overexpressing a protease.   
     
     
         28 . The method of  claim 27 , wherein the protease of interest is associated with a disease of pulmonary fibrosis, liver fibrosis, or cancer. 
     
     
         29 . The method of  claim 27 , wherein the protease of interest is a matrix metalloproteinase. 
     
     
         30 . A method of treating a disease in a subject comprising:
 a) administering to a subject suspected of having said disease, one or more imaging agents of  claim 1 ;   b) allowing the imaging agent to be cleaved by the protease of interest;   c) detecting the presence of the fluorophore in the imaging agent which is being cleaved by the protease of interest in the patient; and   d) performing image-guided surgery, image-guided microsurgery, image-guided photo dynamic therapy, or image-guided surgery on the subject.