Diana Brickute

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.

Further evaluation of the carbon11-labeled D(2/3) agonist PET radiotracer PHNO: reproducibility in tracer characteristics and characterization of extrastriatal binding.

[11C]‐(+)‐PHNO is a new dopamine D2/3 receptor agonist radiotracer which has been successfully used to measure D2/3 receptor availability in experimental animals and man. Here we report in vivo evaluation in the rat of the biodistribution, metabolism, specificity, selectivity, and dopamine sensitivity of carbon11‐labeled PHNO ([11C]‐3‐PHNO) produced by an alternative radiochemical synthesis method. [11C]‐3‐PHNO showed rapid metabolism and clearance from most peripheral organs and tissues. [11C]‐3‐PHNO, but not its polar metabolite, readily crossed the blood‐brain barrier and showed high levels of uptake in the D2/3‐rich striatum. Pretreatment with unlabeled PHNO and the D2/3 receptor antagonist raclopride indicated that binding in the striatum was specific and selective to D2/3 receptors. PET studies in anesthetized rats revealed significant reductions in [11C]‐3‐PHNO binding in the striatum following amphetamine administration, indicating sensitivity to increases in endogenous dopamine concentrations. D2/3 antagonist pretreatment additionally indicated moderate levels of [11C]‐3‐PHNO specific binding in several extrastriatal brain areas—most notably the olfactory bulbs and tubercles, thalamus, and hypothalamus. Of particular interest, approximately 30% of [11C]‐3‐PHNO signal in the cerebellum—a region often used as a “low‐binding” reference region for PET quantification—was attributable to specific signal. These data demonstrate that [11C]‐3‐PHNO shows similar tracer characteristics to [11C]‐(+)‐PHNO, but additionally indicate that radiolabeled PHNO may be used to estimate D2/3 receptor availability in select extrastriatal brain regions with PET. Synapse 64:301–312, 2010.

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.

Radiosynthesis of the D2/3 Agonist [3-11C]-(+)- PHNO using [11C]Iodomethane

We report here a radiosynthesis for the D(2/3) agonist (+)-4-([3-(11)C]propyl)-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol (3-[(11)C]-(+)-PHNO) labelled at the terminal carbon of the N-propyl chain. The protocol is based on (11)C-methylation of an N-acetyl precursor. This initial step is followed by a reduction with LiAlH(4) to give ([3-(11)C]-(+)-PHNO). We first applied the method for the synthesis of a model compound, N-3-([(11)C]propyl)-1,2,3,4-tetrahydroisoquinoline, which we obtained in 77-97% analytical radiochemical yield (n=6) in 20 min. Similarly, we prepared ([3-(11)C]-(+)-PHNO) in 55-60% analytical radiochemical yield (n=5) using a one-pot procedure. We have also been able to implement the complete process on a semi-automated module. This platform delivered purified and formulated [3-(11)C]PHNO with an average radiochemical yield of 9% (n=13, range 2-30%, non-decay corrected), a radiochemical purity >95%, and a specific radioactivity of 26.8-81.1 GBq/μmol in a total time of 63-65 min.

Depicting changes in tumor biology in response to cetuximab mono- or combination therapy by apoptosis and proliferation imaging using 18F-ICMT-11 and 3′-Deoxy-3′-[18F]Fluorothymidine (18F-FLT) PET

Imaging biomarkers must demonstrate their value in monitoring treatment. Two PET tracers, the caspase-3/7–specific isatin-5-sulfonamide 18F-ICMT-11 (18F-(S)-1-((1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2(2,4-difluoro-phenoxymethyl)-pyrrolidine-1-sulfonyl)isatin) and 18F-FLT (3′-deoxy-3′-18F-fluorothymidine), were used to detect early treatment-induced changes in tumor biology and determine whether any of these changes indicate a response to cetuximab, administered as monotherapy or combination therapy with gemcitabine. Methods: In mice bearing cetuximab-sensitive H1975 tumors (non–small lung cancer), the effects of single or repeated doses of the antiepidermal growth factor receptor antibody cetuximab (10 mg/kg on day 1 only or on days 1 and 2) or a single dose of gemcitabine (125 mg/kg on day 2) were investigated by 18F-ICMT-11 or 18F-FLT on day 3. Imaging was also performed after 2 doses of cetuximab (days 1 and 2) in mice bearing cetuximab-insensitive HCT116 tumors (colorectal cancer). For imaging–histology comparison, tumors were evaluated for proliferation (Ki-67 and thymidine kinase 1 [TK1]), cell death (cleaved caspase-3 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling [TUNEL]), and target engagement (epidermal growth factor receptor expression) by immunohistochemistry, immunofluorescence, and immunoblotting, respectively. Tumor and plasma were analyzed for thymidine and gemcitabine metabolites by liquid chromatography–mass spectrometry. Results: Retention of both tracers was sensitive to cetuximab in H1975 tumors. 18F-ICMT-11 uptake and ex vivo cleaved caspase-3 staining notably increased in tumors treated with repeated doses of cetuximab (75%) and combination treatment (46%). Although a single dose of cetuximab was insufficient to induce apoptosis, it did affect proliferation. Significant reductions in tumor 18F-FLT uptake (44%–50%; P < 0.001) induced by cetuximab monotherapy and combination therapy were paralleled by a clear decrease in proliferation (Ki-67 decrease, 72%–95%; P < 0.0001), followed by a marked tumor growth delay. TK1 expression and tumor thymidine concentrations were profoundly reduced. Neither imaging tracer depicted the gemcitabine-induced tumor changes. However, cleaved caspase-3 and Ki-67 staining did not significantly differ after gemcitabine treatment whereas TK1 expression and thymidine concentrations increased. No cetuximab-induced modulation of the imaging tracers or other response markers was detected in the insensitive model of HCT116. Conclusion: 18F-ICMT-11 and 18F-FLT are valuable tools to assess cetuximab sensitivity depicting distinct and time-variant aspects of treatment response.

Abstract 4235: Choline metabolism is an early predictor of EGFR-mediated survival in NSCLC

Oncogenic signalling and metabolic reprograming are hallmarks of tumour progression, yet little is known about the regulatory elements that coordinate their interface. Aberrant choline and phospholipid metabolism are strongly correlated to malignant progression in NSCLC and provide the essential components required by both hallmarks and yet mechanistic links to either remain scarce. Choline kinase alpha (ChoKα) regulates the conversion of choline to phosphocholine and although its regulatory cascade has not been described, it is thought to act in conjuction with EGFR. We used an integrated systems approach and queried whether pharmacoproteomic pathway mapping could identify regulators of the cholinic phenotype. Proteomic and phosphoproteomic Stable isotope labelling by amino acids in cell culture (SILAC) analysis was used to describe the interactome following ChoKα or EGFR inhibition. Bioinformatic analysis was used to identify the significant (Significance-B test) subset of targets for each condition. These subsets were clustered according to GeneOntology, Reactome and KEGG databases and the resulting maps identifed the potential regulators of choline metabolism. Choline uptake, phosphorylation and efflux were further evaluated in vitro in response to erlotinib, cisplatin, pemetrexed and paclitaxel using radio-labelled Choline analogues. Derived metabolites were characterised using radio-HPLC. Uptake was further characterised under hypoxic and nutrient deficient conditions. In vivo, [18F]-D4-Choline PET dynamic imaging was performed following treatment. Pharmacoproteomic analysis revealed a 40% overlap between ChoKα and EGFR inhibition providing direct evidence of the pathways and targets involved in, mostly, biosynthesis. Rapid modulation of the cholinic phenotype was directly dependent on ChoKα activity. Intracellular uptake was induced by nutrient deprivation, hypoxia and reversed through second messenger signalling or growth factor stimulation. Choline uptake within 3 hours of treatment correlated to survival at 72 hours. In vivo, [18F]-D4-Choline tracer kinetics were diagnostic of choline kinase expression and sensitive to treatment. Significant correspondence between ChoKα and EGFR inhibition provided mechanistic evidence that ChoKα and lipid metabolism are effectors of the EGFR signalling cascade in NSCLC. Choline can act as a sensor by synchronizing the survival response via metabolic and signalling reprograming and is thus an early predictor of therapeutic efficiency in vitro and in vivo. Citation Format: Rosy Favicchio, Nicos Angelopoulos, Diana Brickute, Robin Fortt, Frazer Twyman, Georgios Giamas, Juan Carlos Lacal, Eric O. Aboagye. Choline metabolism is an early predictor of EGFR-mediated survival in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4235.

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.