Preclinical Evaluation of a Potential GSH Ester Based PET/SPECT Imaging Probe DT(GSHMe)2 to Detect Gamma Glutamyl Transferase Over Expressing Tumors

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.

γ-Glutamyltranspeptidase-Triggered Intracellular Gadolinium Nanoparticle Formation Enhances the T2-Weighted MR Contrast of Tumor

“…Conventional analytical methods such as colorimetric assay, high-performance liquid chromatography (HPLC), and electrochemistry have been used to detect GGT activity in vitro . Optical and nuclear techniques have been employed to image GGT activity in superficial xenografted tumor of living subjects. − However, optical imaging with shallow tissue penetration depth is not suit for the diagnosis of above-mentioned internal deep GGT-related cancers. , Although nuclear imaging offers high sensitivity, its spatial resolution is poor.…”

In vivo detection of γ-glutamyl-transferase up-regulation in glioma using hyperpolarized γ-glutamyl-[1-13C]glycine

“…After GGT removes the glutamyl group from GSH, cysteinylglycine is cleaved by an extracellular dipeptidase to the constituent amino acids cysteine and glycine, and the released amino acids are transported into the cell via dedicated transporters. We did not observe any consistent asymmetry in the lineshape of glycine peak, however, considering our line width (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and the relatively long T 1 of glycine (45 s at 9.4T 31 ) we cannot rule out that some of the HP γ-glutamyl-[1-13 C]glycine is intracellular. Importantly however, the precise compartmental localization of glycine would not be expected to affect our conclusions regarding its association with tumor.…”

“…The high level of expression of GGT in glioblastoma and its presence on the outer cell membrane make it an attractive molecular target for assessing redox and imaging GBMs. Non-invasive methods of monitoring GGT using radiolabeled or fluorescence probes have been previously described 18,[20][21][22] . However radiolabeled agents do not provide information regarding enzyme activity as they accumulate at the tumor site by diffusion, and the utility of fluorescent probes is limited by the depth of fluorescent light penetration through the skull.…”

Analysis of gamma-glutamyltransferase in acute promyelocytic leukemia patients undergoing arsenic trioxide treatment

“…In addition, a complementary increase in GGT level was observed in the gamma-glutamyl cycle for APL patients administered hepatoprotective agents. GSH and cysteine, major hepatoprotective agents, are also important substrates of GGT [23]. Therefore, metabolic interactions and mutual transformation occurs among most hepatoprotective agents.…”