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

Khurana H, Meena VK, Prakash S, Chuttani K, Chadha N, Jaswal A, Dhawan DK, Mishra AK, Hazari PP - PLoS ONE (2015)

Bottom Line: Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Delhi, India; Department of Biophysics, Biomedical Sciences Block, Panjab University, Chandigarh, India.

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.

No MeSH data available.


Related in: MedlinePlus

Cell binding assay and uptake kinetics of DT(GSHMe)2 A&B) Representative scatchard plot resulting from competitive binding assay of 99mTc-DT(GSHMe)2 on BMG-1 and EAT cells.Resulting Kd of 22.9 nM and 156 nM was observed in BMG-1 and EAT cells respectively C) Graphical representation of time course and kinetics of 99mTc- DT(GSHMe)2 on EAT cells.
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pone.0134281.g003: Cell binding assay and uptake kinetics of DT(GSHMe)2 A&B) Representative scatchard plot resulting from competitive binding assay of 99mTc-DT(GSHMe)2 on BMG-1 and EAT cells.Resulting Kd of 22.9 nM and 156 nM was observed in BMG-1 and EAT cells respectively C) Graphical representation of time course and kinetics of 99mTc- DT(GSHMe)2 on EAT cells.

Mentions: The ability of 99mTc-DT(GSHMe)2 conjugate to bind target enzyme GGT on the surface of tumor cell lines was examined by binding assay. Nonspecific binding was determined using 100-fold excess of unlabeled glutathione. Examination of binding curves showed significant external binding of the labeled 99mTc-DT(GSHMe)2 conjugate. Fig 3A and 3B shows the scatchard plot of DT(GSHMe)2 on BMG and EAT cells respectively. The Scatchard plot shows affinity of the labeled 99mTc-DT(GSHMe)2 on tumor cells. Kd was derived graphically and was found to be 156 nM in EAT tumor cells and 22.39 nM in BMG cells respectively. Bmax was found to be 17.2 nM and 91.02 nM for BMG-1 and EAT cells respectively.


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

Khurana H, Meena VK, Prakash S, Chuttani K, Chadha N, Jaswal A, Dhawan DK, Mishra AK, Hazari PP - PLoS ONE (2015)

Cell binding assay and uptake kinetics of DT(GSHMe)2 A&B) Representative scatchard plot resulting from competitive binding assay of 99mTc-DT(GSHMe)2 on BMG-1 and EAT cells.Resulting Kd of 22.9 nM and 156 nM was observed in BMG-1 and EAT cells respectively C) Graphical representation of time course and kinetics of 99mTc- DT(GSHMe)2 on EAT cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519333&req=5

pone.0134281.g003: Cell binding assay and uptake kinetics of DT(GSHMe)2 A&B) Representative scatchard plot resulting from competitive binding assay of 99mTc-DT(GSHMe)2 on BMG-1 and EAT cells.Resulting Kd of 22.9 nM and 156 nM was observed in BMG-1 and EAT cells respectively C) Graphical representation of time course and kinetics of 99mTc- DT(GSHMe)2 on EAT cells.
Mentions: The ability of 99mTc-DT(GSHMe)2 conjugate to bind target enzyme GGT on the surface of tumor cell lines was examined by binding assay. Nonspecific binding was determined using 100-fold excess of unlabeled glutathione. Examination of binding curves showed significant external binding of the labeled 99mTc-DT(GSHMe)2 conjugate. Fig 3A and 3B shows the scatchard plot of DT(GSHMe)2 on BMG and EAT cells respectively. The Scatchard plot shows affinity of the labeled 99mTc-DT(GSHMe)2 on tumor cells. Kd was derived graphically and was found to be 156 nM in EAT tumor cells and 22.39 nM in BMG cells respectively. Bmax was found to be 17.2 nM and 91.02 nM for BMG-1 and EAT cells respectively.

Bottom Line: Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Delhi, India; Department of Biophysics, Biomedical Sciences Block, Panjab University, Chandigarh, India.

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.

No MeSH data available.


Related in: MedlinePlus