Radiosynthesis and in vivo tumor uptake of 2-deoxy-2-[(18)F]fluoro-myo-inositol.

Inositols play an important role in membrane lipid metabolism and mitogenic signaling of most cancer cells. There is paucity of data on the distribution of radiolabelled inositols. Based on work previously carried out on 1-deoxy-1-[(18)F]fluoro-scyllo-inositol ([(18)F]2), we began a program of work to label myo-inositol (2-deoxy-2-[(18)F]fluoro-myo-inositol, [(18)F]1), the most abundant inositol in cells. Fluorination of a triflate precursor 4 afforded the desired [(18)F]1 following deprotection with a radiochemical yield of 8% n.d.c. [(18)F]1 showed higher uptake in vivo in a human breast cancer xenograft model, MDA-MB-231, compared to [(18)F]2. Thus, we have developed a new inositol radiotracer that could have utility for studying inositol uptake in tumors.

Positron emission tomography imaging of drug-induced tumor apoptosis with a caspase-3/7 specific [18F]-labeled isatin sulfonamide.

Of the molecular biochemical alterations that occur during apoptosis, activation of caspases, notably caspase-3, is probably the most attractive for developing specific in vivo molecular imaging probes. We recently designed a library of isatin-5 sulfonamides and selected [18F]ICMT-11 for further evaluation on the basis of subnanomolar affinity for activated capsase-3, high metabolic stability, and facile radiolabeling. In this present study, we have demonstrated that [18F]ICMT-11 binds to a range of drug-induced apoptotic cancer cells in vitro and to 38C13 murine lymphoma xenografts in vivo by up to 2-fold at 24 h posttreatment compared to vehicle treatment. We further demonstrated that the increased signal intensity in tumors after drug treatment, detected by whole body in vivo microPET imaging, was associated with increased apoptosis. In summary, we have characterized [18F]ICMT-11 as a caspase-3/7 specific PET imaging radiotracer for the assessment of tumor apoptosis that could find utility in anticancer drug development and the monitoring of early responses to therapy.

Adaptation to HIF-1 deficiency by upregulation of the AMP/ATP ratio and phosphofructokinase activation in hepatomas.

BACKGROUND: HIF-1 deficiency has marked effects on tumour glycolysis and growth. We therefore investigated the consequences of HIF-1 deficiency in mice, using the well established Hepa-1 wild-type (WT) and HIF-1ß-deficient (c4) model. These mechanisms could be clinically relevant, since HIF-1 is now a therapeutic target. METHODS: Hepa-1 WT and c4 tumours grown in vivo were analysed by 18FDG-PET and 19FDG Magnetic Resonance Spectroscopy for glucose uptake; by HPLC for adenine nucleotides; by immunohistochemistry for GLUTs; by immunoblotting and by DIGE followed by tandem mass spectrometry for protein expression; and by classical enzymatic methods for enzyme activity. RESULTS: HIF-1ß deficient Hepa-1 c4 tumours grew significantly more slowly than WT tumours, and (as expected) showed significantly lower expression of many glycolytic enzymes. However, HIF-1ß deficiency caused no significant change in the rate of glucose uptake in c4 tumours compared to WT when assessed in vivo by measuring fluoro-deoxyglucose (FDG) uptake. Immunohistochemistry demonstrated less GLUT-1 in c4 tumours, whereas GLUT-2 (liver type) was similar to WT. Factors that might upregulate glucose uptake independently of HIF-1 (phospho-Akt, c-Myc) were shown to have either lower or similar expression in c4 compared to WT tumours. However the AMP/ATP ratio was 4.5 fold higher (p < 0.01) in c4 tumours, and phosphofructokinase-1 (PFK-1) activity, measured at prevailing cellular ATP and AMP concentrations, was up to two-fold higher in homogenates of the deficient c4 cells and tumours compared to WT (p < 0.001), suggesting that allosteric PFK activation could explain their normal level of glycolysis. Phospho AMP-Kinase was also higher in the c4 tumours. CONCLUSIONS: Despite their defective HIF-1 and consequent down-regulation of glycolytic enzyme expression, Hepa-1 c4 tumours maintain glucose uptake and glycolysis because the resulting low [ATP] high [AMP] allosterically activate PFK-1. This mechanism of resistance would keep glycolysis functioning and also result in activation of AMP-Kinase and growth inhibition; it may have major implications for the therapeutic activity of HIF inhibitors in vivo. Interestingly, this control mechanism does not involve transcriptional control or proteomics, but rather the classical activation and inhibition mechanisms of glycolytic enzymes.

Noninvasive imaging of cell proliferation following mitogenic extracellular kinase inhibition by PD0325901.

The mitogenic extracellular kinase 1/2 (MEK1/2) inhibitor, PD0325901, has potent activity in a number of cancer cell types in vitro. In SKMEL-28 human melanoma cells (BRAF mutant), the drug rapidly decreased phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1, and thymidine kinase 1 protein levels. We investigated if 3'-deoxy-3'-[18F]fluorothymidine-positron emission tomography ([18F]FLT-PET) could be used to image changes in cell proliferation following MEK1/2 inhibition in vivo. Mice bearing SKMEL-28 and human colon cancer HCT116 (K-RAS mutant) xenografts were treated daily with PD0325901 at 25 mg/kg and imaged by dynamic [18F]FLT-PET after 1 and 10 days of initiating treatment. The drug decreased tumor [18F]FLT uptake after 1 and 10 days of treatment compared with control animals. The normalized (maximal) [18F]FLT uptake in SKMEL-28 xenografts (at 60 minutes; NUVmax) after 1 day of vehicle or PD0325901 therapy was 1.81 +/- 0.18 versus 1.23 +/- 0.10, respectively (P = 0.03). In this model, NUVmax after 10 days was 2.07 +/- 0.40 versus 1.08 +/- 0.14, respectively (P = 0.03). The corresponding values for HCT116 tumors were 2.30 +/- 0.84 versus 1.88 +/- 0.36 (P = 0.045) after 1 day, and 1.97 +/- 0.13 versus 1.00 +/- 0.03 (P = 0.03) after 10 days. Similar changes were found for other [18F]FLT retention variables. The drug decreased phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1, and thymidine kinase 1 protein. Tumor [18F]FLT-PET variables correlated with proliferation as measured by Ki67 labeling index (r > or = 0.6; P > or = 0.003). In summary, [18F]FLT-PET is a sensitive imaging biomarker for detecting the antiproliferative effect of MEK1/2 inhibition by PD0325901.

In vivo biological activity of the histone deacetylase inhibitor LAQ824 is detectable with 3'-deoxy-3'-[18F]fluorothymidine positron emission tomography.

Histone deacetylase inhibitors (HDACI) are emerging as growth inhibitory compounds that modulate gene expression and inhibit tumor cell proliferation. We assessed whether 3'-deoxy-3'-[(18)F]fluorothymidine-positron emission tomography ([18F]FLT-PET) could be used to noninvasively measure the biological activity of a novel HDACI LAQ824 in vivo. We initially showed that thymidine kinase 1 (TK1; EC2.7.1.21), the enzyme responsible for [18F]FLT retention in cells, was regulated by LAQ824 in a drug concentration-dependent manner in vitro. In HCT116 colon carcinoma xenograft-bearing mice, LAQ824 significantly decreased tumor [18F]FLT uptake in a dose-dependent manner. At day 4 of treatment, [18F]FLT tumor-to-heart ratios at 60 minutes (NUV60) were 2.16 +/- 0.15, 1.86 +/- 0.13, and 1.45 +/- 0.20 in vehicle, and 5 and 25 mg/kg LAQ824 treatment groups, respectively (P < or = 0.05). LAQ825 at 5 mg/kg also significantly reduced both TK1 levels and [18F]FLT uptake at day 10 but not at day 2 (P < or = 0.05). [18F]FLT NUV60 correlated significantly with cellular proliferation (r = 0.68; P = 0.0019) and was associated with drug-induced histone H4 hyperacetylation. Of interest to [18F]FLT-PET imaging, both TK1 mRNA copy numbers and protein levels decreased in the order vehicle >5 mg/kg LAQ824 > 25 mg/kg LAQ824, providing a rationale for the use of [18F]FLT-PET in this setting. We also observed increases in Rb hypophosphorylation and p21 levels, factors that could have contributed to the alteration in TK1 transcription in vivo. In conclusion, we have shown the utility of [18F]FLT-PET for monitoring the biological activity of the HDACI, LAQ824. Drug-induced changes in tumor [18F]FLT uptake were due, at least in part, to reductions in TK1 transcription and translation.

Mechanism of action of the Aurora kinase inhibitor CCT129202 and in vivo quantification of biological activity.

The Aurora family of serine/threonine kinases is important for the regulation of centrosome maturation, chromosome segregation, and cytokinesis during mitosis. Overexpression of Aurora kinases in mammalian cells leads to genetic instability and transformation. Increased levels of Aurora kinases have also been linked to a broad range of human tumors. Here, we describe the properties of CCT129202, a representative of a structurally novel series of imidazopyridine small-molecule inhibitors of Aurora kinase activity. This compound showed high selectivity for the Aurora kinases over a panel of other kinases tested and inhibits proliferation in multiple cultured human tumor cell lines. CCT129202 causes the accumulation of human tumor cells with >or=4N DNA content, leading to apoptosis. CCT120202-treated human tumor cells showed a delay in mitosis, abrogation of nocodazole-induced mitotic arrest, and spindle defects. Growth of HCT116 xenografts in nude mice was inhibited after i.p. administration of CCT129202. We show that p21, the cyclin-dependent kinase inhibitor, is induced by CCT129202. Up-regulation of p21 by CCT129202 in HCT116 cells led to Rb hypophosphorylation and E2F inhibition, contributing to a decrease in thymidine kinase 1 transcription. This has facilitated the use of 3'-deoxy-3'[(18)F]fluorothymidine-positron emission tomography to measure noninvasively the biological activity of the Aurora kinase inhibitor CCT129202 in vivo.

Early detection of tumor response to chemotherapy by 3'-deoxy-3'-[18F]fluorothymidine positron emission tomography: the effect of cisplatin on a fibrosarcoma tumor model in vivo.

We have assessed the potential of [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to measure early cytostasis and cytotoxicity induced by cisplatin treatment of radiation-induced fibrosarcoma 1 (RIF-1) tumor-bearing mice. Cisplatin-mediated arrest of tumor cell growth and induction of tumor shrinkage at 24 and 48 hours, respectively, were detectable by [18F]FLT-PET. At 24 and 48 hours, the normalized uptake at 60 minutes (tumor/liver radioactivity ratio at 60 minutes after radiotracer injection; NUV60) for [18F]FLT was 0.76 +/- 0.08 (P = 0.03) and 0.51 +/- 0.08 (P = 0.03), respectively, compared with controls (1.02 +/- 0.12). The decrease in [18F]FLT uptake at 24 hours was associated with a decrease in cell proliferation assessed immunohistochemically (a decrease in proliferating cell nuclear antigen labeling index, LI(PCNA), from 14.0 +/- 2.0% to 6.2 +/- 1.0%; P = 0.001), despite the lack of a change in tumor size. There were G1-S and G2-M phase arrests after cisplatin treatment, as determined by cell cycle analysis. For the quantitative measurement of tumor cell proliferation, [18F]FLT-PET was found to be superior to [18F]fluorodeoxyglucose-PET (NUV60 versus LIPCNA: r = 0.89, P = 0.001 and r = 0.55, P = 0.06, respectively). At the biochemical level, we found that the changes in [18F]FLT and [18F]fluorodeoxyglucose uptake were due to changes in levels of thymidine kinase 1 protein, hexokinase, and ATP. This work supports the further development of [18F]FLT-PET as a generic pharmacodynamic readout for early quantitative imaging of drug-induced changes in cell proliferation in vivo.

Evaluation of 2-deoxy-2-[18F]fluoro-D-glucose- and 3'-deoxy-3'-[18F]fluorothymidine-positron emission tomography as biomarkers of therapy response in platinum-resistant ovarian cancer.

PURPOSE: We evaluated whether 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) positron emission tomography (PET) could be used as imaging biomarkers of platinum resensitization in ovarian cancer. PROCEDURES: Paired platinum-sensitive and platinum-resistant ovarian cancer cells from the same patient, PEO1 and PEO4, grown as tumor xenografts in nude mice, were assessed by PET. RESULTS: The AKT inhibitor, API-2, resensitized platinum-resistant PEO4 tumors to cisplatin, leading to a markedly lower Ki67 labeling index (p ≤ 0.006, n = 6 per group). [(18)F]FDG-PET and [(18)F]FLT-PET imaging variables were lower after combination treatment compared with vehicle treatment (p ≤ 0.006, n = 6 per group). No changes were seen with either drug alone. PRAS40 phosphorylation status was a sensitive biochemical marker of pathway inhibition, whereas reductions thymidine kinase 1 expression defined the [(18)F]FLT response. CONCLUSIONS: Therapeutic inhibition of AKT activation in acquired platinum-resistant disease can be imaged noninvasively by [(18)F]FDG-PET and [(18)F]FLT-PET warranting further assessment.

Glucose metabolism measured by [¹8F]fluorodeoxyglucose positron emission tomography is independent of PTEN/AKT status in human colon carcinoma cells.

The phosphoinositide 3-kinase (PI3K) signaling pathway is one of the most altered in cancer, leading to a range of cellular responses including enhanced proliferation, survival, and metabolism, and is thus an attractive target for anticancer drug development. Stimulation of the PI3K pathway can be initiated by alterations at different levels of the signaling cascade including growth factor receptor activation, as well as mutations in PIK3CA, PTEN, and AKT genes frequently found in a broad range of cancers. Given its role in glucose metabolism, we investigated the utility of [(18)F]fluorodeoxyglucose positron emission tomography ([(18)F]FDG PET) as a pharmacodynamic biomarker of PI3K pathway-induced glucose metabolism. PTEN deletion in human colon carcinoma cells led to constitutive AKT activation but did not confer a phenotype of increased cell proliferation or glucose metabolism advantage in vivo relative to isogenic tumors derived from cells with a wild-type allele. This was not due to the activation context, that is, phosphatase activity, per se because PIK3CA activation in xenografts derived from the same lineage failed to increase glucose metabolism. Acute inhibition of PI3K activity by LY294002, and hence decreased activated AKT expression, led to a significant reduction in tumor [(18)F]FDG uptake that could be explained at least in part by decreased membrane glucose transporter 1 expression. The pharmacodynamic effect was again independent of PTEN status. In conclusion, [(18)F]FDG PET is a promising pharmacodynamic biomarker of PI3K pathway inhibition; however, its utility to detect glucose metabolism is not directly linked to the magnitude of activated AKT protein expression.

Radiosynthesis and pre-clinical evaluation of [(18)F]fluoro-[1,2-(2)H(4)]choline.

INTRODUCTION: Choline radiotracers are widely used for clinical PET diagnosis in oncology. [(11)C]Choline finds particular utility in the imaging of brain and prostate tumor metabolic status, where 2-[(18)F]fluoro-2-deoxy-D-glucose ('FDG') shows high background uptake. More recently we have extended the clinical utility of [(11)C]choline to breast cancer where radiotracer uptake correlates with tumor aggressiveness (grade). In the present study, a new choline analog, [(18)F]fluoro-[1,2-(2)H(4)]choline, was synthesized and evaluated as a potential PET imaging probe. METHODS: [(18)F]Fluorocholine, [(18)F]fluoro-[1-(2)H(2)]choline and [(18)F]fluoro-[1,2-(2)H(4)]choline were synthesized by alkylation of the relevant precursor with [(18)F]fluorobromomethane or [(18)F]fluoromethyl tosylate. Radiosynthesis of [(18)F]fluoromethyl tosylate required extensive modification of the existing method. [(18)F]Fluorocholine and [(18)F]fluoro-[1,2-(2)H(4)]choline were then subjected to in vitro oxidative stability analysis in a chemical oxidation model using potassium permanganate and an enzymatic model using choline oxidase. The two radiotracers, together with the corresponding di-deuterated compound, [(18)F]fluoro-[1-(2)H(2)]choline, were then evaluated in vivo in a time-course biodistribution study in HCT-116 tumor-bearing mice. RESULTS: Alkylation with [(18)F]fluoromethyl tosylate proved to be the most reliable radiosynthetic route. Stability models indicate that [(18)F]fluoro-[1,2-(2)H(4)]choline possesses increased chemical and enzymatic (choline oxidase) oxidative stability relative to [(18)F]fluorocholine. The distribution of the three radiotracers, [(18)F]fluorocholine, [(18)F]fluoro-[1-(2)H(2)]choline and [(18)F]fluoro-[1,2-(2)H(4)]choline, showed a similar uptake profile in most organs. Crucially, tumor uptake of [(18)F]fluoro-[1,2-(2)H(4)]choline was significantly increased at late time points compared to [(18)F]fluorocholine and [(18)F]fluoro-[1-(2)H(2)]choline. CONCLUSIONS: Stability analysis and biodistribution suggest that [(18)F]fluoro-[1,2-(2)H(4)]choline warrants further in vivo investigation as a PET probe of choline metabolism.

Targeting somatostatin receptors: preclinical evaluation of novel 18F-fluoroethyltriazole-Tyr3-octreotate analogs for PET.

UNLABELLED: The incidence and prevalence of gastroenteropancreatic neuroendocrine tumors has been increasing over the past 3 decades. Because of high densities of somatostatin receptors (sstr)--mainly sstr-2--on the cell surface of these tumors, (111)In-diethylenetriaminepentaacetic acid-octreotide scintigraphy has become an important part of clinical management. (18)F-radiolabeled analogs with suitable pharmacokinetics would permit PET with more rapid clinical protocols. METHODS: We compared the affinity in vitro and tissue pharmacokinetics by PET of 5 structurally related (19)F/(18)F-fluoroethyltriazole-Tyr(3)-octreotate (FET-TOCA) analogs: FET-G-polyethylene glycol (PEG)-TOCA, FETE-PEG-TOCA, FET-G-TOCA, FETE-TOCA, and FET-ßAG-TOCA to the recently described (18)F-aluminum fluoride NOTA-octreotide ((18)F-AIF-NOTA-OC) and the clinical radiotracer (68)Ga-DOTATATE. RESULTS: All (19)F-fluoroethyltriazole-Tyr(3)-octreotate compounds retained high agonist binding affinity to sstr-2 in vitro (half-maximal effective concentration, 4-19 nM vs. somatostatin at 5.6 nM). Dynamic PET showed that incorporation of PEG linkers, exemplified by (18)F-FET-G-PEG-TOCA and (18)F-FETE-PEG-TOCA, reduced uptake in high sstr-2-expressing AR42J pancreatic cancer xenografts. (18)F-FET-ßAG-TOCA showed the lowest nonspecific uptake in the liver. Tumor uptake increased in the order (68)Ga-DOTATATE < (18)F-AIF-NOTA ≤ (18)F-FET-ßAG-TOCA < (18)F-FET-G-TOCA. The uptake of (18)F-FET-ßAG-TOCA was specific: a radiolabeled scrambled peptide, (18)F-FET-ßAG-[W-c-(CTFTYC)K], did not show tumor uptake; there was lower uptake of (18)F-FET-ßAG-TOCA in AR42J xenografts when mice were pretreated with 10 mg of unlabeled octreotide per kilogram; and there was low uptake of (18)F-FET-ßAG-TOCA in low sstr-2-expressing HCT116 xenografts. CONCLUSION: We have developed novel fluoroethyltriazole-Tyr(3)-octreotate radioligands that combine high specific binding with rapid target localization and rapid pharmacokinetics for high-contrast PET. (18)F-FET-ßAG-TOCA and (18)F-FET-G-TOCA are candidates for future clinical evaluation.

[18F]fluoromethyl-[1,2-2H4]-choline: a novel radiotracer for imaging choline metabolism in tumors by positron emission tomography.

Current radiotracers for positron emission tomography imaging of choline metabolism have poor systemic metabolic stability in vivo. We describe a novel radiotracer, [(18)F]fluoromethyl-[1,2-(2)H(4)]-choline (D4-FCH), that employs deuterium isotope effect to improve metabolic stability. D4-FCH proved more resistant to oxidation than its nondeuterated analogue, [(18)F]fluoromethylcholine, in plasma, kidneys, liver, and tumor, while retaining phosphorylation potential. Tumor radiotracer levels, a determinant of sensitivity in imaging studies, were improved by deuterium substitution; tumor uptake values expressed as percent injected dose per voxel at 60 min were 7.43 +/- 0.47 and 5.50 +/- 0.49 for D4-FCH and [(18)F]fluoromethylcholine, respectively (P = 0.04). D4-FCH was also found to be a useful response biomarker. Treatment with the mitogenic extracellular kinase inhibitor PD0325901 resulted in a reduction in tumor radiotracer uptake that occurred in parallel with reductions in choline kinase A expression. In conclusion, D4-FCH is a very promising metabolically stable radiotracer for imaging choline metabolism in tumors.

Design, synthesis, and biological characterization of a caspase 3/7 selective isatin labeled with 2-[18F]fluoroethylazide.

Imaging of programmed cell death (apoptosis) is important in the assessment of therapeutic response in oncology and for diagnosis in cardiac and neurodegenerative disorders. The executioner caspases 3 and 7 ultimately effect cellular death, thus providing selective molecular targets for in vivo quantification of apoptosis. To realize this potential, we aimed to develop 18F-labeled isatin sulfonamides with high metabolic stability and moderate lipophilicity while retaining selectivity and affinity for caspase 3/7. A small library of isatins modified with fluorinated aromatic groups and heterocycles was synthesized. A lead compound incorporating 2'-fluoroethyl-1,2,3-triazole was identified with subnanomolar affinity for caspase 3. "Click labeling" provided the 18F-labeled tracer in 65 +/- 6% decay-corrected radiochemical yield from 2-[18F]fluoroethylazide. The compound showed high stability in vivo with rapid uptake and elimination in healthy tissues and tumor. The novel 18F-labeled isatin is a candidate radiotracer for further preclinical evaluation for imaging of apoptosis.

Imaging pharmacodynamics of the alpha-folate receptor-targeted thymidylate synthase inhibitor BGC 945.

The assessment of tissue-specific pharmacodynamics is desirable in the development of tumor-targeted therapies. Plasma deoxyuridine (dUrd) levels, a measure of systemic thymidylate synthase (TS) inhibition, has limited application for studying the pharmacodynamics of novel TS inhibitors targeted to the high affinity alpha-folate receptor (FR). Here, we have evaluated the utility of [(18)F]fluorothymidine positron emission tomography ([(18)F]FLT-PET) for imaging the tissue pharmacodynamics of BGC 945, an FR-targeted antifolate TS inhibitor; the nontargeted antifolate BGC 9331 was used for comparison. TS inhibition by both drugs induced a concentration-dependent increase in [(3)H]thymidine uptake in FR-positive human epidermoid KB cells. Membrane-associated equilibrative nucleoside transporter type 1 levels increased from 55,720 +/- 6,101 to 118,700 +/- 5,193 and 130,800 +/- 10,800 per cell at 100 mug/mL of BGC 9331 and BGC 945, respectively, suggesting this as a potential mechanism of increased nucleoside uptake. In keeping with these in vitro findings, tumor [(18)F]FLT accumulation in KB xenografts increased by >/=2-fold after drug treatment with maximal levels at 1 to 4 hours and 4 to 24 hours after BGC 9331 and BGC 945 treatment, respectively. Of interest to FR targeting, BGC 9331, but not BGC 945, induced accumulation of [(18)F]FLT uptake in intestine, a proliferative and TS-responsive tissue. For both drugs, quantitative changes in tumor [(18)F]FLT uptake were associated with increased tumor dUrd levels. In conclusion, we have validated the utility of [(18)F]FLT-PET to image TS inhibition induced by antifolates and shown the tumor-specific activity of BGC 945. This imaging biomarker readout will be useful in the early clinical development of BGC 945.

Redistribution of nucleoside transporters to the cell membrane provides a novel approach for imaging thymidylate synthase inhibition by positron emission tomography.

Thymidylate synthase (EC 2.1.1.45) is a key enzyme for the de novo synthesis of DNA and as such a target for anticancer drug development. There is a need to develop noninvasive methods for assessing thymidylate synthase inhibition in tumors. The aim of this study was to assess the potential of 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) positron emission tomography (PET) for early measurement of thymidylate synthase inhibition and to elucidate the cellular mechanisms involved. Radiation-induced fibrosarcoma-1 tumor-bearing mice were injected with a single i.p. dose of the thymidylate synthase inhibitor 5-fluorouracil (5-FU; 165 mg/kg) and imaged by [(18)F]FLT-PET at 1 to 2 hours after treatment. Deoxyuridine, thymidine kinase 1 (cytoplasmic thymidine kinase; EC2.7.1.21), and ATP levels in excised tumors were measured. Cellular assays for membrane transport were also done. There was a 1.8-fold increase in the 60-minute [(18)F]FLT tumor/heart radioactivity ratio in drug-treated mice compared with vehicle controls (P = 0.0016). Plasma and tumor deoxyuridine levels increased significantly but thymidine kinase and ATP levels were unchanged. Whole-cell assays implicated a (low level) functional role for the type-1 equilibrative nucleoside transporter (ENT). There was an increase in type-1 ENT-binding sites per cell from 49,110 in untreated cells to 73,142 (P = 0.03) in cells treated with 10 microg/mL 5-FU for 2 hours, without a change in transporter affinity (P = 0.41). We conclude that [(18)F]FLT-PET can be used to measure thymidylate synthase inhibition as early as 1 to 2 hours after treatment with 5-FU by a mechanism involving redistribution of nucleoside transporters to the plasma membrane.

The uptake of 3'-deoxy-3'-[18F]fluorothymidine into L5178Y tumours in vivo is dependent on thymidine kinase 1 protein levels.

PURPOSE: The aim of this study was to investigate the role of thymidine kinase 1 (TK1) protein in 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography (PET) studies. METHODS: We investigated the in vivo kinetics of [18F]FLT in TK1+/- and TK1-/- L5178Y mouse lymphoma tumours that express different levels of TK1 protein. RESULTS: [18F]FLT-derived radioactivity, measured by a dedicated small animal PET scanner, increased within the tumours over 60 min. The area under the normalised tumour time-activity curve were significantly higher for the TK1+/- compared with the -/- variant (0.89+/-0.02 vs 0.79+/-0.03 MBq ml(-1) min, P=0.043; n=5 for each tumour type). Ex vivo gamma counting of tissues excised at 60 min p.i. (n=8) also revealed significantly higher tumour [18F]FLT uptake for the TK1+/- variant (6.2+/-0.6 vs 4.6+/-0.4%ID g(-1), P=0.018). The observed differences between the cell lines with respect to [18F]FLT uptake were in keeping with a 48% higher TK1 protein in the TK1+/- tumours versus the -/- variant (P=0.043). On average, there were no differences in ATP levels between the two tumour variants (P=1.00). A positive correlation between [18F]FLT accumulation and TK1 protein levels (r=0.68, P=0.046) was seen. Normalisation of the data for ATP content further improved the correlation (r=0.86, P=0.003). CONCLUSION: This study shows that in vivo [18F]FLT kinetics depend on TK1 protein expression. ATP may be important in realising this effect. Thus, [18F]FLT-PET has the potential to yield specific information on tumour proliferation in diagnostic imaging and therapy monitoring.