Development of a fluorine-18 radiolabelled fluorescent chalcone: evaluated for detecting glycogen"

Background: Glycogen is a multibranched polysaccharide of glucose produced by cells to store energy and plays a key role in cancer. A previously reported fluorescent probe (CDg4) was shown to selectively bind glycogen in mouse embryonic stem cells, however the molecule was not evaluated in cancer cells. We report the synthesis and biological evaluation of a dual-modality imaging probe based on CDg4, for positron emission tomography (PET) and fluorescence microscopy. Results: A fluorine-18 radiolabelled derivative of CDg4, ([18F]5) for in vivo quantification of total glycogen levels in cancer cells was developed and synthesised in 170 min with a non-decay corrected radiochemical yield (RCY n.d.c) of 5.1 ± 0.9 % (n = 4) in >98% radiochemical purity. Compound 5 and [18F]5 were evaluated in vitro for their potential to bind glycogen, but only 5 showed accumulation by fluorescence microscopy. The accumulation of 5 was determined to be specific as fluorescent signal diminished upon the digestion of carbohydrate polymers with α-amylase. PET imaging in non-tumour bearing mice highlighted rapid hepato-biliary-intestinal elimination of [18F]5 and almost complete metabolic degradation after 60 min in the liver, plasma and urine, confirmed by radioactive metabolite analysis. Conclusions: Fluorescent compound 5 selectively accumulated in glycogen containing cancer cells, identified by fluorescence microscopy; however, rapid in vivo metabolic degradation precludes further investigation of [18F]5 as a PET radiopharmaceutical

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

An improved automated radiosynthesis of [F-18]FET-beta AG-TOCA

The fluorine-18 radiolabelled octreotide derivative [18F]FET-βAG-TOCA targeting somatostatin receptor type 2, has been evaluated clinically for positron emission tomography (PET) imaging of neuroendocrine tumours (NETs). We report an improved automated radiosynthesis of [18F]FET-βAG-TOCA with several advantages over the current automated GMP synthesis: 1) cartridge-based purification of 2-[18F]fluoroethylazide ([18F]FEA); 2) simple set-up for the radiolabelling on a single cassette; 3) HPLC purification using a biocompatible mobile phase. [18F]FET-βAG-TOCA was produced with a radiochemical yield of 16.7 ± 0.6% (non-decay corrected) and radiochemical purity ≥98%. The automated synthesis produced multi-patient doses (900 MBq) that were radiochemically stable (≥98%) over 4 hours. In addition, the automated procedure described can be used, with minimal adaptation, to radiolabel any alkyne-containing peptide with [18F]FEA using the GE FASTlab™ platform

Solid-supported cyanoborohydride cartridges for automation of reductive amination radiochemistry

A solid-supported cyanoborohydride cartridge was designed to facilitate the automated production of positron emission tomography (PET) radiotracers synthesised via reductive amination chemistry. Two compounds, N-(4-fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)ethan-1-amine ([18F]2) and 1,3,4,6-tetra-O-acetyl-2-(4-fluorobenzylamine)-2-deoxy-β-D-glucopyranose ([18F]4) were radiosynthesised efficiently using a GE FASTlab™ platform, obtained in >98% RCP in a total synthesis time of 75 min (from the start of synthesis) with RCY (non-decay corrected) of 7.5 ± 2.5% and 6.0 ± 1.1%, respectively. The cartridge method provides a convenient alternative to conventional powdered reducing agents typically used in reductive amination radiochemistry

Synthesis and evaluation of 3’-[18F]fluorothymidine-5’-squaryl as a bioisostere of 3’-[18F]fluorothymidine-5’-monophosphate

The squaryl moiety has emerged as an important phosphate bioisostere with reportedly greater cell permeability. It has been used in the synthesis of several therapeutic drug molecules including nucleoside and nucleotide analogues but is yet to be evaluated in the context of positron emission tomography (PET) imaging. We have designed, synthesised and evaluated 3′-[18F]fluorothymidine-5′-squaryl ([18F]SqFLT) as a bioisostere to 3′-[18F]fluorothymidine-5′-monophosphate ([18F]FLTMP) for imaging thymidylate kinase (TMPK) activity. The overall radiochemical yield (RCY) was 6.7 ± 2.5% and radiochemical purity (RCP) was >90%. Biological evaluation in vitro showed low tracer uptake (<0.3% ID mg−1) but significantly discriminated between wildtype HCT116 and CRISPR/Cas9 generated TMPK knockdown HCT116shTMPK−. Evaluation of [18F]SqFLT in HCT116 and HCT116shTMPK− xenograft mouse models showed statistically significant differences in tumour uptake, but lacked an effective tissue retention mechanism, making the radiotracer in its current form unsuitable for PET imaging of proliferation

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

Design, synthesis, and evaluation of a novel PET imaging agent targeting lipofuscin in senescent cells

Promoting a senescent phenotype to supress tumour progression may present an alternative strategy for treating cancer and encourages the development of positron emission tomography (PET) imaging biomarkers for assessing response to treatment. The accumulation of lipofuscin deposits in senescent cells are visualised using the pathology stain Sudan Black B (SBB) and is an emerging biomarker of senescence. We describe the design, synthesis and evaluation of [18F]fluoroethyltriazole-SBB ([18F]FET-SBB), a fluorine-18 radiolabelled derivative of SBB. The in vitro uptake of [18F]FET-SBB in a senescent cell line corelated with lipofuscin deposits; in vivo PET imaging and metabolite analysis confirms a favourable pharmacokinetic and metabolic profile for futher studies of in vivo models of senescence

Development and evaluation of an 18F-radiolabeled monocyclam derivative for imaging CXCR4 expression

In humans, C–X–C chemokine receptor type 4 (CXCR4) is a protein that is encoded by the CXCR4 gene and binds the ligand CXCL12 (also known as SDF-1). The CXCR4–CXCL12 interaction in cancer elicits biological activities that result in tumor progression and has accordingly been the subject of significant investigation for detection and treatment of the disease. Peptidic antagonists have been labeled with a variety of radioisotopes for the detection of CXCR4, but the methodology utilizing small molecules has predominantly used radiometals. We report here the development of a 18F-radiolabeled cyclam-based small molecule radioprobe, [18F]MCFB, for imaging CXCR4 expression. The IC50 value of [19F]MCFB for CXCR4 was similar to that of AMD3465 (111.3 and 89.8 nM, respectively). In vitro binding assays show that the tracer depicted a differential CXCR4 expression, which was blocked in the presence of AMD3465, demonstrating the specificity of [18F]MCFB. Positron emission tomography (PET) imaging studies showed a distinct uptake of the radioprobe in lymphoma and breast cancer xenografts. High liver and kidney uptakes were seen with [18F]MCFB, leading us to further examine the basis of its pharmacokinetics in relation to the tracer’s cationic nature and thus the role of organic cation transporters (OCTs). Substrate competition following the intravenous injection of metformin led to a marked decrease in the urinary excretion of [18F]MCFB, with moderate changes observed in other organs, including the liver. Our results suggest involvement of OCTs in the renal elimination of the tracer. In conclusion, the 18F-radiolabeled monocyclam, [18F]MCFB, has potential to detect tumor CXCR4 in nonhepatic tissues

Consideration of metabolite efflux in radiolabelled choline kinetics

Hypoxia is a complex microenvironmental condition known to regulate choline kinase α (CHKA) activity and choline transport through transcription factor hypoxia-inducible factor-1α (HIF-1α) and, therefore may confound uptake of choline radiotracer [ 18F]fluoromethyl-[1,2- 2H4]-choline ([ 18 F]-D4-FCH). The aim of this study was to investigate how hypoxia affects choline radiotracer dynamics. Three underlying mechanisms by which hypoxia could potentially alter the uptake of the choline radiotracer, [ 18 F]-D4-FCH, were investigated: 18F-D4-FCH import, CHKA phosphorylation activity, and efflux of [18 F]-D4-FCH and its phosphorylated product [ 18F]-D4-FCHP. Effects of hypoxia on [18 F]-D4-FCH uptake were studied in CHKA-overexpressing cell lines of prostate cancer, PC-3, and breast cancer, MDA-MB-231 cells. Mechanisms of radiotracer efflux were assessed by cell uptake and immunofluorescence in vitro, and examined in vivo (N=24). Mathematical modelling methodology was further developed to verify efflux hypothesis using [18 F]-D4-FCH dynamic PET scans from non-small cell lung cancer (NSCLC) patients (N=17). We report a novel finding involving export of phosphorylated [18F]-D4-FCH, [18 F]-D4-FCHP, via HIF-1α-responsive efflux transporters including ABCB4 when HIF-1α level is augmented. This is supported by graphical analysis of human data with a compartmental model (M2T6k+k5) that accounts for efflux. Hypoxia/HIF-1α increases the efflux of phosphorylated radiolabelled choline species, thus supporting consideration of efflux in modelling of radiotracer dynamics

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

Abstract 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.</jats:p