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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

In vivo biodistribution and scintigraphy DT(GSHMe)2 on experimental models.In vivo biodistribution and scintigraphy of DT(GSHMe)2 on experimental models. A) Bar graph representing biodistribution of 68Ga-DT(GSHMe)2 on BALB/c mice bearing EA tumors. B) Coregistered microPET/CT whole body scan was performed on tumor models after 2 h of i.v. injection with and without co-administration of blocking dose of GSH. C) Biodistribution of 99mTc-DT(GSHMe)2 in BALB/c mice bearing EA tumors. D (a & b) Whole body SPECT image of BALB/c mice bearing EA tumor in right hind limb and a site of inflammation in the left forelimb acquired at 30 min and 2 h post i. v. injection of 99mTc-DT(GSHMe)2.
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pone.0134281.g005: In vivo biodistribution and scintigraphy DT(GSHMe)2 on experimental models.In vivo biodistribution and scintigraphy of DT(GSHMe)2 on experimental models. A) Bar graph representing biodistribution of 68Ga-DT(GSHMe)2 on BALB/c mice bearing EA tumors. B) Coregistered microPET/CT whole body scan was performed on tumor models after 2 h of i.v. injection with and without co-administration of blocking dose of GSH. C) Biodistribution of 99mTc-DT(GSHMe)2 in BALB/c mice bearing EA tumors. D (a & b) Whole body SPECT image of BALB/c mice bearing EA tumor in right hind limb and a site of inflammation in the left forelimb acquired at 30 min and 2 h post i. v. injection of 99mTc-DT(GSHMe)2.

Mentions: Biodistribution studies were performed for 68Ga-DT(GSHMe)2 in BALB/c mice bearing tumors. Distribution data revealed a similar trend to that compared with 99mTc-DT(GSHMe)2 but in early time points cardiac activity was visible with 68Ga-DT(GSHMe)2 as the tracer. At 1 and 2 h, there was a decline in activity found in blood. (Fig 5A) Major accumulation of radioconjugate was observed in kidneys at 30 min with 11.077 ± 0.64% ID/g. Rapid washout of tracer was seen 1 h post injection. Liver shows uptake of the radiotracer 2.95 ± 0.236% ID/g and 0.868 ± 0.098% ID/g, as observed at 30 min and 1 h respectively. Uptake was insignificant which was well correlated with SPECT and PET images.In vivo biodistribution was also done for 99mTc-DT(GSHMe)2 in tumor bearing BALB/c mice 30 min, 1, 2 and 4 h post i.v. injection of the radiotracer. Major accumulation was seen in kidneys with 16.78 ± 1.148% ID/g within 1 h. Furthermore, rapid washout of the radioactivity from kidneys was observed at 4 h with 6.43 ± 0.51% ID/g. At 1 and 2 h, minimal radioactivity 1.09 ± 0.059% and 0.96 ± 0.046% ID/g was found associated with liver tissue, which almost cleared off within 4 h. (Fig 5C) The comparison of the biodistribution with different isotopes was done using two tailed t- test and P <0.05 for all the organs except for the heart where significant difference was found in the accumulation of radioactivity where P>0.05.


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)

In vivo biodistribution and scintigraphy DT(GSHMe)2 on experimental models.In vivo biodistribution and scintigraphy of DT(GSHMe)2 on experimental models. A) Bar graph representing biodistribution of 68Ga-DT(GSHMe)2 on BALB/c mice bearing EA tumors. B) Coregistered microPET/CT whole body scan was performed on tumor models after 2 h of i.v. injection with and without co-administration of blocking dose of GSH. C) Biodistribution of 99mTc-DT(GSHMe)2 in BALB/c mice bearing EA tumors. D (a & b) Whole body SPECT image of BALB/c mice bearing EA tumor in right hind limb and a site of inflammation in the left forelimb acquired at 30 min and 2 h post i. v. injection of 99mTc-DT(GSHMe)2.
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Related In: Results  -  Collection

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

pone.0134281.g005: In vivo biodistribution and scintigraphy DT(GSHMe)2 on experimental models.In vivo biodistribution and scintigraphy of DT(GSHMe)2 on experimental models. A) Bar graph representing biodistribution of 68Ga-DT(GSHMe)2 on BALB/c mice bearing EA tumors. B) Coregistered microPET/CT whole body scan was performed on tumor models after 2 h of i.v. injection with and without co-administration of blocking dose of GSH. C) Biodistribution of 99mTc-DT(GSHMe)2 in BALB/c mice bearing EA tumors. D (a & b) Whole body SPECT image of BALB/c mice bearing EA tumor in right hind limb and a site of inflammation in the left forelimb acquired at 30 min and 2 h post i. v. injection of 99mTc-DT(GSHMe)2.
Mentions: Biodistribution studies were performed for 68Ga-DT(GSHMe)2 in BALB/c mice bearing tumors. Distribution data revealed a similar trend to that compared with 99mTc-DT(GSHMe)2 but in early time points cardiac activity was visible with 68Ga-DT(GSHMe)2 as the tracer. At 1 and 2 h, there was a decline in activity found in blood. (Fig 5A) Major accumulation of radioconjugate was observed in kidneys at 30 min with 11.077 ± 0.64% ID/g. Rapid washout of tracer was seen 1 h post injection. Liver shows uptake of the radiotracer 2.95 ± 0.236% ID/g and 0.868 ± 0.098% ID/g, as observed at 30 min and 1 h respectively. Uptake was insignificant which was well correlated with SPECT and PET images.In vivo biodistribution was also done for 99mTc-DT(GSHMe)2 in tumor bearing BALB/c mice 30 min, 1, 2 and 4 h post i.v. injection of the radiotracer. Major accumulation was seen in kidneys with 16.78 ± 1.148% ID/g within 1 h. Furthermore, rapid washout of the radioactivity from kidneys was observed at 4 h with 6.43 ± 0.51% ID/g. At 1 and 2 h, minimal radioactivity 1.09 ± 0.059% and 0.96 ± 0.046% ID/g was found associated with liver tissue, which almost cleared off within 4 h. (Fig 5C) The comparison of the biodistribution with different isotopes was done using two tailed t- test and P <0.05 for all the organs except for the heart where significant difference was found in the accumulation of radioactivity where P>0.05.

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