Preparation and Evaluation of Molecular Imaging Probes Targeting the Urokinase Plasminogen Activator System

Abstract

The aim of this thesis was to develop a molecular imaging probe for the urokinase plasminogen activator (uPA) system, which has been shown to play a critical role in cancer metastasis, tumour aggressiveness and likelihood of progression. Two classes of small molecule inhibitors carrying isotopes of iodine were synthesized and evaluated using in vitro assays and in vivo studies. Lead compounds showed high affinity for the target with Ki values in the low nanomolar range (1b = 1.4 nM, 1e = 6.1 nM, 1g = 2.6 nM and 2a = 2.1 nM). Biodistribution studies of the reversible compounds (1b, 1e, 1g) showed rapid clearance, accumulation in the gall bladder and intestines and little to no tumour uptake (<1 %ID/g). The irreversible inhibitor (2a) showed specificity for the target through SDS-PAGE and biodistribution studies. Analysis of the biodistribution pattern showed retention in the tumour over time reaching a maximum at 24 h post-injection of 1.95 %ID/g with tumour-to-blood ratio being 0.65 at 24 h, 1.13 at 48 h and 1.09 at 96 h post-injection. A parallel strategy reported involved targeting the uPA receptor (uPAR) through the use of antibodies and bioorthogonal chemisty based on radiolabeled tetrazines and transcyclooctene (TCO) functionalized biomolecules. A new tetrazine synthon was developed that can be readily labeled with both 99mTc and 18F where the products were produced in 75 and 31 % radiochemical yields. Stability studies showed the compounds are suitable for use in vivo. Biodistribution studies were carried out in CD1 mice and results showed that both probes had sufficient distribution patterns to warrant use in pre-targeting strategies. Their reactivity with TCO, including functionalized derivatives such as TCO-anti-uPAR, was also demonstrated creating the means to develop PET and SPECT probes for imaging the urokinase system using a single prosthetic group.