Frazer Twyman

Evaluation of deuterated 18F- and 11C-labeled choline analogs for cancer detection by positron emission tomography

Purpose: 11C-Choline–positron emission tomography (PET) has been exploited to detect the aberrant choline metabolism in tumors. Radiolabeled choline uptake within the imaging time is primarily a function of transport, phosphorylation, and oxidation. Rapid choline oxidation, however, complicates interpretation of PET data. In this study, we investigated the biologic basis of the oxidation of deuterated choline analogs and assessed their specificity in human tumor xenografts. Experimental Design: 11C-Choline, 11C-methyl-[1,2-2H4]-choline (11C-D4-choline), and 18F-D4-choline were synthesized to permit comparison. Biodistribution, metabolism, small-animal PET studies, and kinetic analysis of tracer uptake were carried out in human colon HCT116 xenograft–bearing mice. Results: Oxidation of choline analogs to betaine was highest with 11C-choline, with reduced oxidation observed with 11C-D4-choline and substantially reduced with 18F-D4-choline, suggesting that both fluorination and deuteration were important for tracer metabolism. Although all tracers were converted intracellularly to labeled phosphocholine (specific signal), the higher rate constants for intracellular retention (Ki and k3) of 11C-choline and 11C-D4-choline, compared with 18F-D4-choline, were explained by the rapid conversion of the nonfluorinated tracers to betaine within HCT116 tumors. Imaging studies showed that the uptake of 18F-D4-choline in three tumors with similar radiotracer delivery (K1) and choline kinase α expression—HCT116, A375, and PC3-M—were the same, suggesting that 18F-D4-choline has utility for cancer detection irrespective of histologic type. Conclusion: We have shown here that both deuteration and fluorination combine to provide protection against choline oxidation in vivo. 18F-D4-choline showed the highest selectivity for phosphorylation and warrants clinical evaluation. Clin Cancer Res; 18(4); 1063–72. ©2012 AACR.

Biodistribution and Radiation Dosimetry of Deuterium-Substituted 18F-Fluoromethyl-[1, 2-2H4]Choline in Healthy Volunteers

11C-choline and 18F-fluoromethylcholine (18F-FCH) have been used in patients to study tumor metabolic activity in vivo; however, both radiotracers are readily oxidized to respective betaine analogs, with metabolites detectable in plasma soon after injection of the radiotracer. A more metabolically stable FCH analog, 18F-fluoromethyl-[1,2-2H4]choline (18F-D4-FCH), based on the deuterium isotope effect, has been developed. We report the safety, biodistribution, and internal radiation dosimetry profiles of 18F-D4-FCH in 8 healthy human volunteers. Methods: 18F-D4-FCH was intravenously administered as a bolus injection (mean ± SD, 161 ± 2.17 MBq; range, 156–163 MBq) to 8 healthy volunteers (4 men, 4 women). Whole-body (vertex to mid thigh) PET/CT scans were acquired at 6 time points, up to 4 h after tracer injection. Serial whole-blood, plasma, and urine samples were collected for radioactivity measurement and plasma radiotracer metabolites. Tissue 18F radioactivities were determined from quantitative analysis of the images, and time–activity curves were generated. The total numbers of disintegrations in each organ normalized to injected activity (residence times) were calculated as the area under the curve of the time–activity curve normalized to injected activities and standard organ volumes. Dosimetry calculations were performed using OLINDA/EXM 1.1. Results: The injection of 18F-D4-FCH was well tolerated in all subjects, with no radiotracer-related serious adverse event reported. The mean effective dose averaged over both men and women (±SD) was estimated to be 0.025 ± 0.004 (men, 0.022 ± 0.002; women, 0.027 ± 0.002) mSv/MBq. The 5 organs receiving the highest absorbed dose (mGy/MBq) were the kidneys (0.106 ± 0.03), liver (0.094 ± 0.03), pancreas (0.066 ± 0.01), urinary bladder wall (0.047 ± 0.02), and adrenals (0.046 ± 0.01). Elimination was through the renal and hepatic systems. Conclusion: 18F-D4-FCH is a safe PET radiotracer with a dosimetry profile comparable to other common 18F PET tracers. These data support the further development of 18F-D4-FCH for clinical imaging of choline metabolism.

Preclinical Evaluation of 3-18F-Fluoro-2,2-Dimethylpropionic Acid as an Imaging Agent for Tumor Detection

Deregulated cellular metabolism is a hallmark of many cancers. In addition to increased glycolytic flux, exploited for cancer imaging with 18F-FDG, tumor cells display aberrant lipid metabolism. Pivalic acid is a short-chain, branched carboxylic acid used to increase oral bioavailability of prodrugs. After prodrug hydrolysis, pivalic acid undergoes intracellular metabolism via the fatty acid oxidation pathway. We have designed a new probe, 3-18F-fluoro-2,2-dimethylpropionic acid, also called 18F-fluoro-pivalic acid (18F-FPIA), for the imaging of aberrant lipid metabolism and cancer detection. Methods: Cell intrinsic uptake of 18F-FPIA was measured in murine EMT6 breast adenocarcinoma cells. In vivo dynamic imaging, time course biodistribution, and radiotracer stability testing were performed. 18F-FPIA tumor retention was further compared in vivo to 18F-FDG uptake in several xenograft models and inflammatory tissue. Results: 18F-FPIA rapidly accumulated in EMT6 breast cancer cells, with retention of intracellular radioactivity predicted to occur via a putative 18F-FPIA carnitine-ester. The radiotracer was metabolically stable to degradation in mice. In vivo imaging of implanted EMT6 murine and BT474 human breast adenocarcinoma cells by 18F-FPIA PET showed rapid and extensive tumor localization, reaching 9.1% ± 0.5% and 7.6% ± 1.2% injected dose/g, respectively, at 60 min after injection. Substantial uptake in the cortex of the kidney was seen, with clearance primarily via urinary excretion. Regarding diagnostic utility, uptake of 18F-FPIA was comparable to that of 18F-FDG in EMT6 tumors but superior in the DU145 human prostate cancer model (54% higher uptake; P = 0.002). Furthermore, compared with 18F-FDG, 18F-FPIA had lower normal-brain uptake resulting in a superior tumor-to-brain ratio (2.5 vs. 1.3 in subcutaneously implanted U87 human glioma tumors; P = 0.001), predicting higher contrast for brain cancer imaging. Both radiotracers showed increased localization in inflammatory tissue. Conclusion: 18F-FPIA shows promise as an imaging agent for cancer detection and warrants further investigation.

The novel choline kinase inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth

The glycerophospholipid phosphatidylcholine is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. Inhibition of choline kinase alpha (CHKA), the first committed step to phosphatidylcholine synthesis, by the selective small-molecule ICL-CCIC-0019, potently suppressed growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM and inhibited tumor xenograft growth in mice. ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by [18F]-fluoromethyl-[1,2–2H4]-choline positron emission tomography. Herein, we reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased mitochondria function with associated reduction of citrate synthase expression and AMPK activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids.

Radiolabeled RGD Tracer Kinetics Annotates Differential αvβ3 Integrin Expression Linked to Cell Intrinsic and Vessel Expression

PurposeThe purpose of this paper is to study the association between RGD binding kinetics and αvβ3 integrin receptor density in the complex tumor milieu.ProceduresWe assessed αvβ3in vitro and by 68Ga-DOTA-[c(RGDfK)]2 positron emission tomography (PET) in tumors with varying αvβ3.ResultsIntrinsic αvβ3 expression decreased in the order of M21 >>> MDA-MB-231 > M21L in cells. Tumor volume of distribution by PET, VT, was significantly higher in M21 compared to isogenic M21L tumors (0.40 ± 0.01 versus 0.25 ± 0.02; p < 0.01) despite similar microvessel density (MVD) likely due to higher αvβ3. VT for MDA-MB-231 (0.40 ± 0.04) was comparable to M21 despite lower αvβ3 but in keeping with the higher MVD, suggesting superior tracer distribution.ConclusionsThis study demonstrates that radioligand binding kinetics of PET data can be used to discriminate tumors with different αvβ3 integrin expression—a key component of the angiogenesis phenotype—in vivo.

Clinical Translation of a ‘Click’ Labeled 18F-Octreotate Radioligand for Imaging Neuroendocrine Tumors

We conducted the first-in-human study of 18F-fluoroethyl triazole [Tyr3] octreotate (18F-FET-βAG-TOCA) in patients with neuroendocrine tumors (NETs) to evaluate biodistribution, dosimetry, and safety. Despite advances in clinical imaging, detection and quantification of NET activity remains a challenge, with no universally accepted imaging standard. Methods: Nine patients were enrolled. Eight patients had sporadic NETs, and 1 had multiple endocrine neoplasia type 1 syndrome. Patients received 137–163 MBq (mean ± SD, 155.7 ± 8 MBq) of 18F-FET-βAG-TOCA. Safety data were obtained during and 24 h after radioligand administration. Patients underwent detailed whole-body PET/CT multibed scanning over 4 h with sampling of venous bloods for radioactivity and radioactive metabolite quantification. Regions of interest were defined to derive individual and mean organ residence times; effective dose was calculated with OLINDA 1.1. Results: All patients tolerated 18F-FET-βAG-TOCA with no adverse events. Over 60% parent radioligand was present in plasma at 60 min. High tumor (primary and metastases)-to-background contrast images were observed. Physiologic distribution was seen in the pituitary, salivary glands, thyroid, and spleen, with low background distribution in the liver, an organ in which metastases commonly occur. The organs receiving highest absorbed dose were the gallbladder, spleen, stomach, liver, kidneys, and bladder. The calculated effective dose over all subjects (mean ± SD) was 0.029 ± 0.004 mSv/MBq. Conclusion: The favorable safety, imaging, and dosimetric profile makes 18F-FET-βAG-TOCA a promising candidate radioligand for staging and management of NETs. Clinical studies in an expanded cohort are ongoing to clinically qualify this agent.

Correction: Microwave gallium-68 radiochemistry for kinetically stable bis(thiosemicarbazone) complexes: Structural investigations and cellular uptake under hypoxia (Dalton Transactions (2016) 45 (144-155))

We report the microwave synthesis of several bis(thiosemicarbazones) and the rapid gallium-68 incorporation to give the corresponding metal complexes. These proved kinetically stable under ‘cold’ and ‘hot’ biological assays and were investigated using laser scanning confocal microscopy, flow cytometry and radioactive cell retention studies under normoxia and hypoxia. Ga complex retention was found to be 34% higher in hypoxic cells than in normoxic cells over 30 min, further increasing to 53% at 120 min. Our data suggests that this class of gallium complexes show hypoxia selectivity suitable for imaging in living cells and in vivo tests by microPET in nude athymic mice showed that they are excreted within 1 h of their administration.

Abstract C118: Choline kinase inhibition with the novel pharmacological inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth

The glycerophospholipid phosphatidylcholine (PC) is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. PC is synthesized via the CDP-choline pathway, whereby choline kinase alpha (CHKA) denotes the first committed step in this sequence of enzymatic reactions. CHKA phosphorylates choline to form phosphocholine and its overexpression in many solid tumors is linked to progression of normal cells to malignancy. We developed the highly selective, choline-competitive small-molecule ICL-CCIC-0019 (IC50 of 0.27±0.06 μM). Across a panel of 131 human kinases, the inhibitor showed minimal off-target effects (only 5 kinases were inhibited more than 20% at a concentration of 10 μM). ICL-CCIC-0019 potently inhibited growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM. Importantly, proliferation of normal cells was only minimally affected (MCF-10A and ST-T1b: GI50 30-120 μM). ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by 18F-fluoromethyl-[1,2-2H4]-choline positron emission tomography. Pharmacokinetic modeling revealed that the macro parameter Ki denoting the net irreversible uptake rate was significantly decreased in tumor after 48 hours (Ki (1/min): control, 0.0054±0.00060; ICL-CCIC-0019, 0.0032±0.00064), confirming in vivo target inhibition. This resulted in potent antitumor activity in HCT116 xenografts. We further reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased TCA cycle activity, oxygen consumption rate and elevated extracellular acidification rate. This was associated with a reduction of citrate synthase expression and AMP kinase activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition is a valid therapeutic strategy and critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids. This work was supported in part by Cancer Research UK-Engineering and Physical Sciences Research Council Grant C2536/A10337. Citation Format: Sebastian Trousil, Maciej Kalisczcak, Zachary Schug, Quang-De Nguyen, Giampaolo Tomasi, Rosy Favicchio, Diana Brickute, Robin Fortt, Frazer J. Twyman, Laurence Carroll, Andrew Kalusa, Naveenan Navaratnam, Thomas Adejumo, David Carling, Eyal Gottlieb, Eric O. Aboagye. Choline kinase inhibition with the novel pharmacological inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C118.