Abstract
Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers. The redox processes ensuing from GGT-mediated metabolism of extracellular GSH are implicated in critical aspects of tumor cell biology. Reportedly, Glutathione monoethyl ester (GSHMe) is a substrate of GGT, which has been used for its rapid transport over glutathione. Exploring GGT to be an important target, a homobivalent peptide system, DT(GSHMe)2 was designed to target GGT-over expressing tumors for diagnostic purposes. DT(GSHMe)2 was synthesized, characterized and preclinically evaluated in vitro using toxicity, cell binding assays and time dependent experiments. Stable and defined radiochemistry with 99mTc and 68Ga was optimized for high radiochemical yield. In vivo biodistribution studies were conducted for different time points along with scintigraphic studies of radiolabeled DT(GSHMe)2 on xenografted tumor models. For further validation, in silico docking studies were performed on GGT (hGGT1, P19440). Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration. Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min. Quantitative estimation of GGT expression from western blot experiments showed substantial expression with 41.6 ± 7.07 % IDV for tumor. Small animal micro PET (Positron Emission Tomography)/CT(Computed Tomography) coregistered images depicted significantly high uptake of DT(GSHMe)2 at the BMG-1 tumor site. ROI analysis showed high tumor to contra lateral muscle ratio of 9.33 in PET imaging studies. Avid accumulation of radiotracer was observed at tumor versus inflammation site at 2 h post i.v. injection in an Ehrlich Ascites tumor (EAT) mice model, showing evident specificity for tumor. We propose DT(GSHMe)2 to be an excellent candidate for prognostication and tumor imaging using PET/SPECT.
Highlights
Radiolabeled receptor targeting peptides have emerged as a class of radiopharmaceuticals for cancer diagnosis and therapy
The concept of in vivo imaging is based on ligand-receptor interaction which lies in choosing an appropriate target which is found to be invariably expressing in the diseased condition
There is evidence that Gamma Glutamyl Transferase (GGT) is up regulated in malignant cells and by producing reactive oxygen species causes tumor progression towards more aggressive phenotypes associated with a worse prognosis
Summary
Radiolabeled receptor targeting peptides have emerged as a class of radiopharmaceuticals for cancer diagnosis and therapy. The design of these radiopharmaceuticals require meticulous ligand design and metal coordination chemistry [1,2]. A large number of peptides have been designed and synthesized that are specific for certain receptors or target enzymes localized on the cell surface. Their expression has been found to be invariably high in diseased condition. Radiolabeled peptide chelator systems are used commonly to non-invasively visualize tissue over expressing the receptor or enzyme using molecular imaging modalities with high target to non target ratio. Diagnostic radiopharmaceutical provides a non-invasive method for assessing and estimating the diseased condition by studying its distribution pattern and kinetics inside the body [4,5,6,7]
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