[99mTc]Tc-DTPA-Bis(cholineethylamine) as an Oncologic Tracer for the Detection of Choline Transporter (ChT) and Choline Kinase (ChK) Expression in Cancer.

OBJECTIVE: The elevated choline transporters (ChT), choline kinase (ChK), choline uptake, and phosphorylation in certain tumor cells have influenced the development of radiolabeled choline derivatives as diagnostic probes for imaging cell membrane proliferation. We, therefore, aimed to develop a choline-based moiety for imaging choline kinase-overexpressed tumors by single-photon emission tomography (SPECT). A novel choline-based diagnostic probe was synthesized and evaluated preclinically in various ChT- and ChK-overexpressed tumor models for SPECT imaging applications. METHODS: The synthesis of diethylenetriaminepentaacetic acid-bis-choline ethylamine [DTPA-bis(ChoEA)] featured the conjugation of dimethylaminoethanol to a bifunctional chelator DTPA anhydride. [99mTc]Tc-DTPA-bis(ChoEA) was prepared, and its in vivo characteristics were evaluated in BALB/c mice and tumor-xenografted PC3, A549, and HCT116 athymic mouse models. The in vitro parameters, including cell binding and cytotoxicity, were assessed in PC3, A549, and HCT116 cell lines. To evaluate the specificity of the radioprobe, competitive binding studies were performed. Small-animal SPECT/CT diagnostic imaging was performed for in vivo evaluation. The mouse biodistribution data was further investigated to estimate the radiation dose in humans. RESULTS: In silico studies suggested high binding with enhanced specificity. A standard radiolabeling procedure using stannous chloride as a reducing agent showed a labeling yield of 99.5 ± 0.5%. The in silico studies suggested high binding with enhanced specificity. [99mTc]Tc-DTPA-bis(ChoEA) showed high in vitro stability and specificity. The pharmacokinetic studies of [99mTc]Tc-DTPA-bis(ChoEA) in mice showed an increased tumor-to-background ratio after few minutes of intravenous administration. The first-in-human trial was also conducted. The effective dose was estimated to be 0.00467 mSv/MBq (4.67 mSv/GBq), resulting in a radiation dose of up to 1.73 mSv for the 370 MBq injection of [99mTc]Tc-DTPA-bis(ChoEA). CONCLUSIONS: The synthesized radioprobe [99mTc]Tc-DTPA-bis(ChoEA) accumulates specifically in choline kinase-overexpressed tumors with a high signal-to-noise ratio. The preclinical and first-in-man data suggested that [99mTc]Tc-DTPA-bis(ChoEA) could potentially be used as a diagnostic SPECT tracer in the monitoring and staging of cancer.

Radiolabeling and Preclinical Evaluation of a New S-Alkylated Cysteine Derivative Conjugated to C-Substituted Macrocycle for Positron Emission Tomography.

A new S-alkylated cysteine-derivatized tumor targeting agent, 2,2'-(12-(2-((2-acetamido-2-carboxyethyl)thio)acetamido)-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid was developed for positron emission tomography (PET) imaging. N-Acetyl cysteine (NAC) was conjugated to ATRIDAT as a specific targeting agent toward L-type and ASC amino acid transporter systems in the oncogenic cells. NAC was attached via S-alkylation to prevent its incorporation at undesired recognition sites affecting the signal-to-noise ratio. NAC-ATRIDAT was subjected to gallium-68 complexation with >75% radiolabeling yield. The radiocomplex was purified through the tc18 cartridge to obtain 99.89% radiochemical yield. IC-50 of the NAC-ATRIDAT conjugate was 0.8 mM in A549 cells as evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide assay. Binding affinity experiments on A549 cells showed noteworthy binding with KD in the nanomolar range. A time course study showed a Km value of 0.19 µM and Vmax value of 0.49 pmol/µg protein/min showing reasonable tumor kinetics. Efflux studies showed that the synthesized radioligand is transported majorly by LAT followed by the ASC system. Clearance was found to be renal with 7.67 ± 1.48% ID/g uptake at 30 min which substantially declined to 0.52 ± 0.% ID/g at 4 h. A significant uptake of 10.06 ± 1.056% ID/g was observed at the tumor site in mice at 1 h. µPET images revealed a high contrast with a tumor-to-kidney ratio of 4.8 and a tumor-to-liver ratio of 35.85 at 1 h after injection. These preclinical in vitro and in vivo evaluation supports its potential on the way of becoming a successful 68Ga-radiolabeled amino acid-based PET imaging agent.

[68Ga]/[188Re] Complexed [CDTMP] Trans-1,2-Cyclohexyldinitrilotetraphosphonic Acid As a Theranostic Agent for Skeletal Metastases.

OBJECTIVE: Metastasis of the osseous tissue is one of the frequent and severe aggravations as a result of several neoplastic conditions, such as metabolic disorders, infections, and cancer. The objective of this study was to evaluate the pertinence of [68Ga]-trans-1,2-cyclohexyldinitrilo tetramethylene phosphonic acid (CDTMP) as a potential bone imaging agent for positron emission tomography (PET) applications as well as to assess [188Re]-CDTMP for bone pain palliation in metastatic skeletal disorders. METHODS: 68Ga complex of CDTMP was prepared at 80°C at pH 3.5, and 188Re complex of CDTMP was prepared at room temperature. [68Ga]-CDTMP complex was investigated as PET tracer while the therapeutic efficacy was assessed for [188Re]-CDTMP. Labeling efficiency, biodistribution, myelotoxicity, and imaging studies were carried out for the complexes synthesized. Both PET and MicroPET imaging studies were performed for [68Ga]-CDTMP whereas SPECT acquisitions were acquired for [188Re]-CDTMP. Data were analyzed semiquantitatively for all the scintigraphic scans obtained. RESULTS: The radiolabeling efficiency was observed to be >70% for [68Ga]-CDTMP. High bone uptake of [68Ga]-CDTMP as compared to contralateral tissue was found in PET imaging in Balb/C mice and New Zealand rabbit; the similar result for bone uptake was correlated in the biodistribution study of the compound in BALB/c mice at different time intervals. Biodistribution experiments carried out in mice showed maximum uptake of 6.12 ± 1.22%ID/g at 45 min postinjection. For [188Re]-CDTMP, total skeletal uptake was 8.12 ± 1.11%ID/g observed at 1 h postinjection from biodistribution data. High renal uptake confirms renal route of excretion. A good hydroxyapatite binding too was seen for both the complexes. No evidence of destruction or adverse functioning of vital organs was observed for the 188Re complex. CONCLUSION: [68Ga]-CDTMP complex can be used as a promising PET bone imaging agent and [188Re]-CDTMP as a surrogate moiety for therapeutic application. Owing to the short half-life of 68Ga (68 min), cyclotron-independent radiopharmacy, fast clearance, and rapid renal excretion as evidenced in preclinical animal models. Very low myelotoxicity and highly selective bone uptake prove the potential of [188Re]-CDTMP for therapeutic application.

Biotinidase Resistant 68Gallium-Radioligand Based on Biotin/Avidin Interaction for Pretargeting: Synthesis and Preclinical Evaluation.

A new macrocyclic system 2,2'-(12-amino-11,13-dioxo-1,4,7,10-tetraazacyclotridecane-4,7-diyl)diacetic acid (ATRIDAT) was designed for coordinating metals in +2 and +3 oxidation states particularly 68Ga(III), for PET imaging. ATRIDAT was conjugated to d-biotin for pretargeting via biotin-avidin interaction. This model provides high tumor targeting efficiency and stability to biotinidase activity leading to modest signal amplification at the tumor site. Cyclization of triethylenetetramine with protected diethylamino malonate resulted in the formation of 13 membered diamide ring. d-Biotin was then anchored on the pendant amine rendering α-methyne carbon to the biotinamide bond which blocks the biotinidase enzyme activity. Biotinidase stability assay showed remarkable stability toward the action of biotinidase with ∼95% remaining intact after treatment following 4 h. Binding affinity experiments such as HABA assay, competitive displacement studies with d-biotin and CD showed high binding affinity of the molecule with avidin in nanomolar range. Biotin conjugate was successfully radiolabeled with 68Ga(III) with radiolabeling efficiency of ∼70% and then purified to get 99.9% radiochemical yield. IC50 of the compound was found to be 2.36 mM in HEK cell line and 0.82 mM in A549 as assessed in MTT assay. In biodistribution studies, the major route of excretion was found to be renal. Significant uptake of 4.15 ± 0.35% was observed in tumor in the avidin pretreated mouse at 1 h. µPET images also showed a high tumor to muscle ratio of 26.8 and tumor to kidney ratio of 1.74 at 1 h post-injection after avidin treatment.

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

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.

LAT1 targeted delivery of methionine based imaging probe derived from M(III) metal ions for early diagnosis of proliferating tumours using molecular imaging modalities.

We investigated the potential of DTPA-bis(Methionine), a target specific amino acid based probe for detection of L-type amino acid transporters (LAT1) known to over express in proliferating tumours using multimodality imaging. The ligand, DTPA-bis(Met) was readily converted to lanthanide complexes and was found capable of targeting cancer cells using multimodality imaging. DTPA-bis(Met) complexes were synthesized and characterized by mass spectroscopy. MR longitudinal relaxivity, r1 = 4.067 ± 0.31 mM⁻¹s⁻¹ and transverse relaxivity, r2 = 8.61 ± 0.07 mM⁻¹s⁻¹ of Gd(III)-DTPA-bis(Met) were observed at pH 7.4 at 7 T. Bright, localized fluorescence of Eu(III)-DTPA-bis(Met) was observed with standard microscopy and displacement studies indicated ligand functionality. K(D) value determined for Eu(III)-DTPA-bis(Met) on U-87 MG cells was found to be 17.3 pM and showed appreciable fluorescence within the cells. Radio HPLC showed a radiochemical purity more than 95% (specific activity = 400-500 MBq/µmol, labelling efficiency 78 %) for 68Ga(III)-DTPA-bis(Met). Pre-treatment of xenografted U-87 MG athymic mice with 68Ga(III)-DTPA-bis(Met) following unlabelled L-methionine administration reduced tumour uptake by 10-folds in Micro PET. These data support the specific binding of 68Ga(III)-DTPA-bis(Met) to the LAT1 transporter. To summarize, this agent possesses high stability in biological environment and exhibits effective interaction with its LAT1 transporters giving high accumulation in tumour area, excellent tumour/non-tumour ratio and low non-specific retention in vivo.