In vivo positron emission tomography imaging of protease activity by generation of a hydrophobic product from a noninhibitory protease substrate.
OBJECTIVE
To develop an imaging technology for protease activities in patients that could help in prognosis prediction and in design of personalized, protease-based inhibitors and prodrugs for targeted therapy.


METHODS
Polyethylene glycol (PEG) was covalently attached to the N-terminus of a hydrophilic peptide substrate (GPLGVR) for matrix metalloproteinase (MMP) to increase hydrophilicity. PEG-peptide was then linked to a hydrophobic tetramethylrhodamine (TMR) domain and labeled with (18)F to form a PEG-peptide-(18)F-TMR probe. Specific cleavage of the probe by MMP2 was tested in vitro by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF). In vivo imaging of MMP2-expressing tumors was evaluated by micro-PET.


RESULTS
The hydrophobic TMR fragment (948 Da) was specifically generated by MMP2 enzymes and MMP-expressing HT1080 cells but not control MCF-7 cells. MMP-expressing HT1080 cells and tumors selectively accumulated the hydrolyzed, hydrophobic TMR fragment at sites of protease activity. Importantly, we found that (18)F-labeled probe ((18)F-TMR) preferentially localized in HT1080 tumors but not control MCF-7 tumors as shown by micro-PET. Uptake of the probe in HT1080 tumors was 18.4 ± 1.9-fold greater than in the MCF-7 tumors 30 minutes after injection. These results suggest that the PEG-peptide-(18)F-TMR probe displays high selectivity for imaging MMP activity.


CONCLUSIONS
This strategy successfully images MMP expression in vivo and may be extended to other proteases to predict patient prognosis and to design personalized, protease-based inhibitors and prodrug-targeted therapies.
Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan.
Chih-Hung
CH
Chuang
Kuo-Hsiang
KH
Chuang
Hsin-Ell
HE
Wang
Steve R
SR
Roffler
Jen-taie
JT
Shiea
Shey-Cherng
SC
Tzou
Ta-Chun
TC
Cheng
Chien-Han
CH
Kao
Shih-Yen
SY
Wu
Wei-Lung
WL
Tseng
Chiu-Min
CM
Cheng
Ming-Feng
MF
Hou
Ju-Ming
JM
Wang
Tian-Lu
TL
Cheng
D000230:Adenocarcinoma / Q000000981:diagnostic imaging* / Q000378:metabolism / Q000473:pathology; D000818:Animals; D001943:Breast Neoplasms / Q000000981:diagnostic imaging* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D003952:Diagnostic Imaging; D005260:Female; D005354:Fibrosarcoma / Q000000981:diagnostic imaging* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010446:Peptide Fragments / Q000378:metabolism* / Q000493:pharmacokinetics; D011092:Polyethylene Glycols / Q000737:chemistry; D049268:Positron-Emission Tomography; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014018:Tissue Distribution
D061965:Matrix Metalloproteinase Inhibitors; D010446:Peptide Fragments; D011092:Polyethylene Glycols