Characterization of Sigma-2 Receptor-Specific Binding Sites Using [3H]DTG and [125I]RHM-4.

The sigma-2 receptor/transmembrane protein 97 (σ2R/TMRM97) is a promising biomarker of tumor proliferation and a target for cancer therapy. [3H]DTG has been used to evaluate σ2R/TMEM97 binding affinity in compound development studies. However, [3H]DTG has equal and moderate binding affinities to both sigma 1 receptor (σ1R) and σ2R/TMEM97. Furthermore, co-administration with the σ1R masking compound (+)-pentazocine may cause bias in σ2R/TMEM97 binding affinity screening experiments. We have developed a radioiodinated ligand, [125I]RHM-4, which has high affinity and selectivity for σ2R/TMEM97 versus σ1R. In this study, a head-to-head comparison between [3H]DTG and [125I]RHM-4 on the binding affinity and their effectiveness in σ2R/TMEM97 compound screening studies was performed. The goal of these studies was to determine if this radioiodinated ligand is a suitable replacement for [3H]DTG for screening new σ2R/TMEM97 compounds. Furthermore, to delineate the binding properties of [125I]RHM-4 to the σ2R/TMEM97, the structure of RHM-4 was split into two fragments. This resulted in the identification of two binding regions in the σ2R, the "DTG" binding site, which is responsible for binding to the σ2R/TMEM97, and the secondary binding site, which is responsible for high affinity and selectivity for the σ2R/TMEM97 versus the σ1R. The results of this study indicate that [125I]RHM-4 is an improved radioligand for in vitro binding studies of the σ2R/TMEM97 versus [3H]DTG.

Alanine and glycine conjugates of (2S,4R)-4-[18F]fluoroglutamine for tumor imaging.

INTRODUCTION: Glutamine is an essential source of energy, metabolic substrates, and building block for supporting tumor proliferation. Previously, (2S,4R)-4-[18F]fluoroglutamine (4F-Gln) was reported as a glutamine-related metabolic imaging agent. To improve the in vivo kinetics of this radiotracer, two new dipeptides, [18F]Gly-(2S,4R)4-fluoroglutamine (Gly-4F-Gln) and [18F]Ala-(2S,4R)4-fluoroglutamine (Ala-4F-Gln) were investigated. METHODS: Radiolabeling was performed via 2-steps 18F-fluorination. Cell uptake studies of Gly-4F-Gln and Ala-4F-Gln were investigated in 9 L cell lines. In vitro and in vivo metabolism studies were carried out in Fisher 344 rats. Biodistribution and microPET imaging studies were performed in 9 L tumor-bearing rats. RESULTS: In vitro incubation of these [18F]dipeptides in rat and human blood showed a rapid conversion to (2S,4R)-4-[18F]fluoroglutamine (t1/2 = 2.3 and 0.2 min for [18F]Gly-4F-Gln and [18F]Ala-4F-Gln, respectively for human blood). Biodistribution and PET imaging in Fisher 344 rats bearing 9 L tumor xenografts showed that these dipeptides rapidly localized in the tumors, comparable to that of (2S,4R)-4-[18F]fluoroglutamine (4F-Gln). CONCLUSIONS: The results support that these dipeptides, [18F]Gly-4F-Gln and [18F]Ala-4F-Gln, are prodrugs, which hydrolyze in the blood after an iv injection. They appear to be selectively taken up and trapped by tumor tissue in vivo. The dipeptide, [18F]Ala-4F-Gln, may be suitable as a PET tracer for imaging glutaminolysis in tumors.

Bacterial infection imaging with [18F]fluoropropyl-trimethoprim.

There is often overlap in the diagnostic features of common pathologic processes such as infection, sterile inflammation, and cancer both clinically and using conventional imaging techniques. Here, we report the development of a positron emission tomography probe for live bacterial infection based on the small-molecule antibiotic trimethoprim (TMP). [18F]fluoropropyl-trimethoprim, or [18F]FPTMP, shows a greater than 100-fold increased uptake in vitro in live bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) relative to controls. In a rodent myositis model, [18F]FPTMP identified live bacterial infection without demonstrating confounding increased signal in the same animal from other etiologies including chemical inflammation (turpentine) and cancer (breast carcinoma). Additionally, the biodistribution of [18F]FPTMP in a nonhuman primate shows low background in many important tissues that may be sites of infection such as the lungs and soft tissues. These results suggest that [18F]FPTMP could be a broadly useful agent for the sensitive and specific imaging of bacterial infection with strong translational potential.

[18F](2S,4R)4-Fluoroglutamine PET Detects Glutamine Pool Size Changes in Triple-Negative Breast Cancer in Response to Glutaminase Inhibition.

Glutaminolysis is a metabolic pathway adapted by many aggressive cancers, including triple-negative breast cancers (TNBC), to utilize glutamine for survival and growth. In this study, we examined the utility of [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) PET to measure tumor cellular glutamine pool size, whose change might reveal the pharmacodynamic (PD) effect of drugs targeting this cancer-specific metabolic pathway. High glutaminase (GLS) activity in TNBC tumors resulted in low cellular glutamine pool size assayed via high-resolution 1H magnetic resonance spectroscopy (MRS). GLS inhibition significantly increased glutamine pool size in TNBC tumors. MCF-7 tumors, with inherently low GLS activity compared with TNBC, displayed a larger baseline glutamine pool size that did not change as much in response to GLS inhibition. The tumor-to-blood-activity ratios (T/B) obtained from [18F]4F-Gln PET images matched the distinct glutamine pool sizes of both tumor models at baseline. After a short course of GLS inhibitor treatment, the T/B values increased significantly in TNBC, but did not change in MCF-7 tumors. Across both tumor types and after GLS inhibitor or vehicle treatment, we observed a strong positive correlation between T/B values and tumor glutamine pool size measured using MRS (r2 = 0.71). In conclusion, [18F]4F-Gln PET tracked cellular glutamine pool size in breast cancers with differential GLS activity and detected increases in cellular glutamine pool size induced by GLS inhibitors. This study accomplished the first necessary step toward validating [18F]4F-Gln PET as a PD marker for GLS-targeting drugs. Cancer Res; 77(6); 1476-84. ©2017 AACR.

Iodinated benzimidazole PARP radiotracer for evaluating PARP1/2 expression in vitro and in vivo.

BACKGROUND: PARP inhibitors (PARPi) have the potential to impact cancer therapy in a selective patient population; however, despite current patient selection methods clinical trials have shown mixed response rates. It is therefore clinically useful to determine which patients will respond prior to receiving PARPi therapy. One essential biomarker is to measure the level of PARP enzyme expression in tumors. Small molecule radiotracers have been developed to accurately quantify PARP-1 expression in vitro and in vivo. [125I]KX-02-019 is the first report of a radioiodinated analogue of the benzimidazole class of PARPi. Herein, we studied the pharmacological properties of [125I]KX-02-019 as well as the in vivo biodistribution. METHODS: [125I]KX-02-019 was evaluated in both cancer and non-cancer cell lines. We evaluated the pharmacologic properties of [125I]KX-02-019 in live cells by measuring enzyme association and dissociation kinetics, saturation, and specificity. In addition, competitive inhibition experiments were carried out with commercially available PARPi. Protein expression was analyzed by Western blot to compare PARP-1 and PARP-2 expression across cell lines studied. The biodistribution was studied in a mouse EMT6 tumor model at time points of 0.5, 1, 2, 4 and 6h. RESULTS: [125I]KX-02-019 showed subtle differences in pharmacological properties in the absence of PARP-2. In addition, [125I]KX-02-019 was competitively displaced by clinical PARPi. In vivo biodistribution studies showed an increasing tumor to muscle ratio over 6h as well as fast clearance from healthy tissues. CONCLUSION: [125I]KX-02-019 has binding sites in both PARP1 KO cells as well as PARP2 KO cells showing higher affinity for PARP-2. This observation is supported by a decrease in binding affinity in PARP2 KO cells compared to PARP1 KO cells. The pharmacologic and biological properties of [125I]KX-02-019 studied in vitro and in vivo showed that this analogue may be useful in determining pharmacokinetic and pharmacodynamic properties of clinical PARPi.

Comparative evaluation of 4 and 6-carbon spacer conformationally flexible tetrahydroisoquinolinyl benzamide analogues for imaging the sigma-2 receptor status of solid tumors.

INTRODUCTION: Nine novel analogues were synthesized including a 6-carbon spacer analogue of ISO-1 (7). They have moderate binding affinity for sigma-2 (σ2) receptors and high selectivity for σ2 receptors relative to sigma-1 (σ1) receptors. METHODS: ([18F]7) was synthesized and evaluated as a candidate ligand for positron emission (PET) imaging of the σ2 receptor in tumors. Radioligand [18F]7 was radiolabeled with 18F via displacement of the corresponding mesylate precursor with [18F]fluoride. Cellular uptake study of [18F]7 was performed in EMT-6 tumor cell, and in vivo biodistribution study of [18F]7 and microPET imaging study of [18F]3 and [18F]7 carried out in female Balb/c mice bearing EMT-6 tumors. RESULTS: [18F]7 had a respectable tumor uptake (1.55%ID/g at 60min post-injection) and high tumor/muscle ratios at 60 and 120min post-injection. MicroPET imaging of [18F]7 in tumor-bearing mice as above showed significant tumor localization and a high tumor/muscle ratio as well. CONCLUSIONS: These results are similar to or better than [18F]ISO-1 ([18F]3), which indicates that [18F]7 has potential for imaging the σ2 receptor status of solid tumors.

A Radiotracer Strategy to Quantify PARP-1 Expression In Vivo Provides a Biomarker That Can Enable Patient Selection for PARP Inhibitor Therapy.

Despite the availability of PARP inhibitors for cancer therapy, a biomarker to clearly stratify patients for selection of this treatment remains lacking. Here we describe a radiotracer-based method that addresses this issue, using the novel compound [(125)I] KX1: as a PARP-1-selective radiotracer that can accurately measure PARP-1 expression in vitro and in vivo The pharmacologic properties of the PARP radiotracer [(125)I] KX1: was characterized in multiple cell lines where single-agent sensitivity was correlated with [(125)I] KX1: binding to PARP-1. In vivo evaluation of [(125)I] KX1: verified in vitro results, validating PARP radiotracers to define PARP-1 enzyme expression as an in vivo biomarker. Notably, PARP-1 expression as quantified by [(125)I] KX1: correlated positively with the cytotoxic sensitivity of cell lines evaluated with PARP inhibitors. Overall, our results defined a novel technology with the potential to serve as a companion diagnostic to identify patients most likely to respond therapeutically to a PARP inhibitor. Cancer Res; 76(15); 4516-24. ©2016 AACR.

[(18)F]FluorThanatrace uptake as a marker of PARP1 expression and activity in breast cancer.

The nuclear enzyme PARP1 plays a central role in sensing DNA damage and facilitating repair. Tumors with BRCA1/2 mutations are highly dependent on PARP1 as an alternative mechanism for DNA repair, and PARP inhibitors generate synthetic lethality in tumors with BRCA mutations, resulting in cell cycle arrest and apoptosis. Zhou et al. recently synthesized an (18)F-labeled PARP1 inhibitor ([(18)F]FluorThanatrace) for PET, and demonstrated high specific tracer uptake in a xenograft model of breast cancer [1]. In the current study, we characterize the level of baseline PARP expression and activity across multiple human breast cancer cell lines, including a BRCA1 mutant line. PARP expression and activity, as measured by levels of PAR and PARP1, is correlated with in vitro [(18)F]FluorThanatrace binding as well as tracer uptake on PET in a xenograft model of breast cancer. Radiotracer uptake in genetically-engineered mouse fibroblasts indicates [(18)F]FluorThanatrace is selective for PARP1 versus other PARP enzymes. This motivates further studies of [(18)F]FluorThanatrace as an in vivo measure of PARP1 expression and activity in patients who would benefit from PARP inhibitor therapy.

The pre-clinical characterization of an alpha-emitting sigma-2 receptor targeted radiotherapeutic.

RATIONALE: The sigma-2 receptor is a protein with a Heme binding region and is capable of receptor-mediated endocytosis. It is overexpressed in many cancers making it a potential vector for therapeutic drug delivery. Our objective was to introduce an alpha-emitting radionuclide, astatine-211, into a selective sigma-2 ligand moiety to provide cytotoxic capabilities without adversely altering the pharmacological characteristics. In this study we investigated the in vitro/in vivo tumor targeting and estimated dosimetry of alpha-emitting sigma-2 ligand, 5-(astato-(211)At)-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2,3-dimethoxybenzamide ((211)At-MM3), in a pre-clinical human breast cancer model. METHODS: Astatine-211 was produced in a cyclotron and isolated by dry distillation. Radiosynthesis of (211)At-MM3 was performed using a tin precursor through radioastatodestannylation. In vitro sigma-2 binding experiments using (211)At-MM3 were carried out in live EMT6 and MDA-MB-231 breast cancer cells and liver homogenate tissue. In vivo biodistribution experiments were performed using EMT6 mouse breast cancer cells in BALB/c female mice. Approximately 370 kBq of (211)At-MM3 was administered intravenously and at time points of 5 min, 1, 2, 4, 8, and 24 h organs/tissue were harvested. Estimated human dosimetry was extrapolated from biodistribution data using OLINDA/EXM (VU e-Innovations). RESULTS: Astatine-211 was successfully produced and isolated in quantities suitable for in vitro and small animal in vivo experiments. Radiosynthesis of (211)At-MM3 was reproducible with high radiochemical purity. Astatine-211-MM3 exhibited picomolar affinity to the sigma-2 receptor in contrast to the iodinated analog that had nanomolar affinity. Prolonged tumor targeting was measured through biodistribution studies with a maximal tumor to muscle ratio of 9.02 at 4h. Estimated human dosimetry revealed doses of up to 370 MBq in an adult female patient were below organ radiation limits with the potential to provide a high therapeutic dose to tumors. CONCLUSION: The sigma-2 receptor could serve as a suitable targeting platform for designing radiotherapeutics. (211)At-MM3 showed tumor targeting properties in vitro/in vivo and favorable estimated human dosimetry establishing the proof of concept for future development as a radiotherapeutic for the treatment of breast cancer.

The sigma-2 receptor as a therapeutic target for drug delivery in triple negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is associated with high relapse rates and increased mortality when compared with other breast cancer subtypes. In contrast to receptor positive breast cancers, there are no approved targeted therapies for TNBC. Identifying biomarkers for TNBC is of high importance for the advancement of patient care. The sigma-2 receptor has been shown to be overexpressed in triple negative breast cancer in vivo and has been characterized as a marker of proliferation. The aim of the present study was to define the sigma-2 receptor as a target for therapeutic drug delivery and biomarker in TNBC. METHODS: Three TNBC cell lines were evaluated: MDA-MB-231, HCC1937 and HCC1806. Sigma-2 compounds were tested for pharmacological properties specific to the sigma-2 receptor through competitive inhibition assays. Sigma-2 receptor expression was measured through radioligand receptor saturation studies. Drug sensitivity for taxol was compared to a sigma-2 targeting compound conjugated to a cytotoxic payload, SW IV-134. Cell viability was assessed after treatments for 2 or 48 h. Sigma-2 blockade was assessed to define sigma-2 mediated cytotoxicity of SW IV-134. Caspase 3/7 activation induced by SW IV-134 was measured at corresponding treatment time points. RESULTS: SW IV-134 was the most potent compound tested in two of the three cell lines and was similarly effective in all three. MDA-MB-231 displayed a statistically significant higher sigma-2 receptor expression and also was the most sensitive cell line evaluated to SW IV-134. CONCLUSION: Targeting the sigma-2 receptor with a cytotoxic payload was effective in all the three cell lines evaluated and provides the proof of concept for future development of a therapeutic platform for the treatment of TNBC.

[(18)F](2S,4S)-4-(3-Fluoropropyl)glutamine as a tumor imaging agent.

Although the growth and proliferation of most tumors is fueled by glucose, some tumors are more likely to metabolize glutamine. In particular, tumor cells with the upregulated c-Myc gene are generally reprogrammed to utilize glutamine. We have developed new 3-fluoropropyl analogs of glutamine, namely [(18)F](2S,4R)- and [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 3 and 4, to be used as probes for studying glutamine metabolism in these tumor cells. Optically pure isomers labeled with (18)F and (19)F (2S,4S) and (2S,4R)-4-(3-fluoropropyl)glutamine were synthesized via different routes and isolated in high radiochemical purity (≥95%). Cell uptake studies of both isomers showed that they were taken up efficiently by 9L tumor cells with a steady increase over a time frame of 120 min. At 120 min, their uptake was approximately two times higher than that of l-[(3)H]glutamine ([(3)H]Gln). These in vitro cell uptake studies suggested that the new probes are potential tumor imaging agents. Yet, the lower chemical yield of the precursor for 3, as well as the low radiochemical yield for 3, limits the availability of [(18)F](2S,4R)-4-(3-fluoropropyl)glutamine, 3. We, therefore, focused on [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4. The in vitro cell uptake studies suggested that the new probe, [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, is most sensitive to the LAT transport system, followed by System N and ASC transporters. A dual-isotope experiment using l-[(3)H]glutamine and the new probe showed that the uptake of [(3)H]Gln into 9L cells was highly associated with macromolecules (>90%), whereas the [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, was not (<10%). This suggests a different mechanism of retention. In vivo PET imaging studies demonstrated tumor-specific uptake in rats bearing 9L xenographs with an excellent tumor to muscle ratio (maximum of ∼8 at 40 min). [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, may be useful for testing tumors that may metabolize glutamine related amino acids.

Synthesis and evaluation of ¹8F labeled FET prodrugs for tumor imaging.

INTRODUCTION: O-(2-[(18)F]fluoroethyl)-L-tyrosine (FET, [(18)F]1) is a useful amino-acid-based imaging agent for brain tumors. This paper reports the synthesis and evaluation of three FET prodrugs, O-(2-[(18)F]fluoroethyl)-L-tyrosyl-L-glycine (FET-Gly, [(18)F]2), O-(2-[(18)F]fluoroethyl)-L-tyrosyl-L-alanine (FET-Ala, [(18)F]3) and N-acetyl O-(2-[(18)F]fluoroethyl)-L-tyrosine (AcFET, [(18)F]4), which could be readily hydrolyzed to FET in vivo for tumor imaging. We investigated their metabolism in the blood and imaging properties in comparison to FET ([(18)F]1). METHODS: Three new [(18)F]FET derivatives, 2-4, were prepared from their corresponding tosylate-precursors through nucleophilic fluorination and subsequent deprotection reactions. In vitro uptake studies were carried out in 9L glioma cancer cell lines. In vitro and in vivo hydrolysis studies were conducted to evaluate the hydrolysis of FET prodrugs in blood and in Fisher 344 rats. Biodistribution and PET imaging studies were then performed in rats bearing 9L tumors. RESULTS: New FET prodrugs were prepared with 3-28% decay corrected radiochemical yields, good enantiomeric purity (>95%) and high radiochemical purity (>95%). FET-Gly ([(18)F]2), FET-Ala ([(18)F]3), and AcFET ([(18)F]4) exhibited negligible uptake in comparison to the high uptake of FET ([(18)F]1) in 9L cells. Metabolism studies of FET-Gly ([(18)F]2), FET-Ala ([(18)F]3), and AcFET ([(18)F]4) in rat and human blood showed that FET-Ala ([(18)F]3) was hydrolyzed to FET ([(18)F]1) faster than FET-Gly ([(18)F]2) or AcFET ([(18)F]4). Most of the FET-Ala (79%) was converted to FET ([(18)F]1) within 5min in blood in vivo. Biodistribution studies demonstrated that FET-Ala ([(18)F]3) displayed the highest tumor uptake. The tumor-to-background ratios of FET-Ala ([(18)F]3) and FET ([(18)F]1) were comparable and appeared to be better than those of FET-Gly ([(18)F]2) and AcFET ([(18)F]4). PET imaging studies showed that both FET ([(18)F]1) and FET-Ala ([(18)F]3) could visualize tumors effectively, and that they share similar imaging characteristics. CONCLUSIONS: FET-Ala ([(18)F]3) demonstrated promising properties as a prodrug of FET ([(18)F]1), which could be used in PET imaging of tumor amino acid metabolism.

Comparative evaluation of 18F-labeled glutamic acid and glutamine as tumor metabolic imaging agents.

UNLABELLED: (18)F-labeled (2S,4R)-4-fluoro-l-glutamine (4F-GLN) has demonstrated high uptake in tumor cells that undergo high growth and proliferation. Similar tumor targeting properties have also been observed for (18)F-labeled (2S,4R)-4-fluoro-l-glutamate (4F-GLU), suggesting that both are useful imaging agents. A new labeling procedure facilitates the preparation of (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU with confirmed radiochemical and enantiomeric purity. Here, we report the preparation and comparative evaluation of (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU as tumor metabolic imaging agents. METHODS: Uptake of enantiomerically pure (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU was determined in 3 tumor cell lines (9L, SF188, and PC-3) at selected time points. The in vitro cell uptake mechanism was evaluated by inhibition studies in 9L cells. In vivo biodistribution and PET studies were performed on male F344 rats bearing 9L tumor xenografts. RESULTS: In vitro cell uptake studies showed that (18)F-(2S,4R)4F-GLN displayed higher uptake than (18)F-(2S,4R)4F-GLU. Amino acid transport system ASC (alanine-serine-cysteine-preferring; in particular, its subtype ASCT2 [SLC1A5 gene]) and system X(c)(-) (SLC7A11 gene) played an important role in transporting (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU, respectively, across the membrane. After being transported into cells, a large percentage of (18)F-(2S,4R)4F-GLN was incorporated into protein, whereas (18)F-(2S,4R)4F-GLU mainly remained as the free amino acid in its original form. In vivo studies of (18)F-(2S,4R)4F-GLN in the 9L tumor model showed a higher tumor uptake than (18)F-(2S,4R)4F-GLU, whereas (18)F-(2S,4R)4F-GLU had a slightly higher tumor-to-background ratio than (18)F-(2S,4R)4F-GLN. Imaging studies showed that both tracers had fast accumulation in 9L tumors. Compared with (18)F-(2S,4R)4F-GLU, (18)F-(2S,4R)4F-GLN exhibited prolonged tumor retention reflecting its incorporation into intracellular macromolecules. CONCLUSION: Differences in uptake and metabolism in tumor cells were found between (18)F-(2S,4R)4F-GLN and (18)F-(2S,4R)4F-GLU. Both agents are potentially useful as metabolic tracers for tumor imaging.

Synthesis and evaluation of 18F labeled alanine derivatives as potential tumor imaging agents.

INTRODUCTION: This paper reports the synthesis and labeling of (18)F alanine derivatives. We also investigate their biological characteristics as potential tumor imaging agents mediated by alanine-serine-cysteine preferring (ASC) transporter system. METHODS: Three new (18)F alanine derivatives were prepared from corresponding tosylate-precursors through a two-step labeling reaction. In vitro uptake studies to evaluate and to compare these three analogs were carried out in 9L glioma and PC-3 prostate cancer cell lines. Potential transport mechanisms, protein incorporation and stability of 3-(1-[(18)F]fluoromethyl)-L-alanine (L-[(18)F]FMA) were investigated in 9L glioma cells. Its biodistribution was determined in a rat-bearing 9L tumor model. PET imaging studies were performed on rat bearing 9L glioma tumors and transgenic mouse carrying spontaneous generated M/tomND tumor (mammary gland adenocarcinoma). RESULTS: New (18)F alanine derivatives were prepared with 7%-34% uncorrected radiochemical yields, excellent enantiomeric purity (>99%) and good radiochemical purity (>99%). In vitro uptake of the L-[(18)F]FMA in 9L glioma and PC-3 prostate cancer cells was higher than that observed for the other two alanine derivatives and [(18)F]FDG in the first 1h. Inhibition of cell uptake studies suggested that L-[(18)F]FMA uptake in 9L glioma was predominantly via transport system ASC. After entering into cells, L-[(18)F]FMA remained stable and was not incorporated into protein within 2h. In vivo biodistribution studies demonstrated that L-[(18)F]FMA had relatively high uptake in liver and kidney. Tumor uptake was fast, reaching a maximum within 30 min. The tumor-to-muscle, tumor-to-blood and tumor-to-brain ratios at 60 min post injection were 2.2, 1.9 and 3.0, respectively. In PET imaging studies, tumors were visualized with L-[(18)F]FMA in both 9L rat and transgenic mouse. CONCLUSION: L-[(18)F]FMA showed promising properties as a PET imaging agent for up-regulated ASC transporter associated with tumor proliferation.

Preparation and characterization of L-[5-11C]-glutamine for metabolic imaging of tumors.

UNLABELLED: Recently, there has been a renewed interest in the study of tumor metabolism above and beyond the Warburg effect. Studies on cancer cell metabolism have provided evidence that tumor-specific activation of signaling pathways, such as the upregulation of the oncogene myc, can regulate glutamine uptake and its metabolism through glutaminolysis to provide the cancer cell with a replacement of energy source. METHODS: We report a convenient procedure to prepare l-[5-(11)C]-glutamine. The tracer was evaluated in 9L and SF188 tumor cells (glioma and astrocytoma cell lines). The biodistribution of l-[5-(11)C]-glutamine in rodent tumor models was investigated by dissection and PET. RESULTS: By reacting (11)C-cyanide ion with protected 4-iodo-2-amino-butanoic ester, the key intermediate was obtained in good yield. After hydrolysis with trifluoroacetic and sulfonic acids, the desired optically pure l-[5-(11)C]-glutamine was obtained (radiochemical yield, 5% at the end of synthesis; radiochemical purity, >95%). Tumor cell uptake studies showed maximum uptake of l-[5-(11)C]-glutamine reached 17.9% and 22.5% per 100 µg of protein, respectively, at 60 min in 9L and SF188 tumor cells. At 30 min after incubation, more than 30% of the activity appeared to be incorporated into cellular protein. Biodistribution in normal mice showed that l-[5-(11)C]-glutamine had significant pancreas uptake (7.37 percentage injected dose per gram at 15 min), most likely due to the exocrine function and high protein turnover within the pancreas. Heart uptake was rapid, and there was 3.34 percentage injected dose per gram remaining at 60 min after injection. Dynamic small-animal PET studies in rats bearing xenografted 9L tumors and in transgenic mice bearing spontaneous mammary gland tumors showed a prominent tumor uptake and retention. CONCLUSION: The data demonstrated that this tracer was favorably taken up in the tumor models. The results suggest that l-[5-(11)C]-glutamine might be useful for probing in vivo tumor metabolism in glutaminolytic tumors.

PET imaging of glutaminolysis in tumors by 18F-(2S,4R)4-fluoroglutamine.

UNLABELLED: Changes in gene expression, metabolism, and energy requirements are hallmarks of cancer growth and self-sufficiency. Upregulation of the PI3K/Akt/mTor pathway in tumor cells has been shown to stimulate aerobic glycolysis, which has enabled (18)F-FDG PET tumor imaging. However, of the millions of (18)F-FDG PET scans conducted per year, a significant number of malignant tumors are (18)F-FDG PET-negative. Recent studies suggest that several tumors may use glutamine as the key nutrient for survival. As an alternative metabolic tracer for tumors, (18)F-(2S,4R)4-fluoroglutamine was developed as a PET tracer for mapping glutaminolytic tumors. METHODS: A series of in vitro cell uptake and in vivo animal studies were performed to demonstrate tumor cell addiction to glutamine. Cell uptake studies of this tracer were performed in SF188 and 9L glioblastoma tumor cells. Dynamic small-animal PET studies of (18)F-(2S,4R)4-fluoroglutamine were conducted in 2 animal models: xenografts produced in F344 rats by subcutaneous injection of 9L tumor cells and transgenic mice with M/tomND spontaneous mammary gland tumors. RESULTS: In vitro studies showed that both transformed 9L and SF188 tumor cells displayed a high rate of glutamine uptake (maximum uptake, ≈ 16% dose/100 µg of protein). The cell uptake of (18)F-(2S,4R)4-fluoroglutamine by SF188 cells is comparable to that of (3)H-L-glutamine but higher than that of (18)F-FDG. The tumor cell uptake can be selectively blocked. Biodistribution and PET studies showed that (18)F-(2S,4R)4-fluoroglutamine localized in tumors with a higher uptake than in surrounding muscle and liver tissues. Data suggest that certain tumor cells may use glutamine for energy production. CONCLUSION: The results support that (18)F-(2S,4R)4-fluoroglutamine is selectively taken up and trapped by tumor cells. It may be useful as a novel metabolic tracer for tumor imaging.

Multidentate (18)F-polypegylated styrylpyridines as imaging agents for Aß plaques in cerebral amyloid angiopathy (CAA).

ß-Amyloid plaques (Aß plaques) in the brain are associated with cerebral amyloid angiopathy (CAA). Imaging agents that could target the Aß plaques in the living human brain would be potentially valuable as biomarkers in patients with CAA. A new series of (18)F styrylpyridine derivatives with high molecular weights for selectively targeting Aß plaques in the blood vessels of the brain but excluded from the brain parenchyma is reported. The styrylpyridine derivatives, 8a-c, display high binding affinities and specificity to Aß plaques (K(i) = 2.87, 3.24, and 7.71 nM, respectively). In vitro autoradiography of [(18)F]8a shows labeling of ß-amyloid plaques associated with blood vessel walls in human brain sections of subjects with CAA and also in the tissue of AD brain sections. The results suggest that [(18)F]8a may be a useful PET imaging agent for selectively detecting Aß plaques associated with cerebral vessels in the living human brain.

Facile synthesis [5-(13)C-4-(2)H(2)]-L-glutamine for hyperpolarized MRS imaging of cancer cell metabolism.

RATIONALE AND OBJECTIVES: Recent reports suggest that cancer cells may use glutamine, instead of glucose, as an alternative source of metabolic energy. This suggests that hyperpolarized (13)C glutamine may be useful as a magnetic resonance spectroscopy (MRS) imaging agent for detecting changes in glutamine metabolism in cancerous cells or tissues. MATERIALS AND METHODS: Synthesis of [5-(13)C-4-(2)H(2)]-L-glutamine was accomplished through a seven-step synthetic pathway with a 44% overall yield. The introduction of two stable isotopes was performed by a NaB(2)H(4)-mixed anhydride reduction and K(13)CN-nuclophilic substitution, respectively. The desired [5-(13)C-4-(2)H(2)]-L-glutamine was successfully obtained by a one-pot reaction of deprotection and controlled cyanide hydrolysis. Hyperpolarized [5-(13)C-4-(2)H(2)]-L-glutamine samples were tested in human glioma cells (myc upregulated glia cells, SF188-Bcl-x(L)). MRS signals were obtained with a 9.4 Tesla 89-mm bore nuclear magnetic resonance spectrometer and a direct-detection multi-nuclear probe. RESULTS: The initial degree of polarization for [5-(13)C-4-(2)H(2)]-L-glutamine was ~5% and the initial (13)C signal to noise ratio was ~100:1. Glutamate was detected within seconds after the injection of hyperpolarized glutamine into the cells. The ratio of glutamate to glutamine was very high, indicating rapid conversion to glutamate. Similar cell uptake studies using [(3)H]-L-glutamine also demonstrated cell uptakes higher than that of [(18)F]fluorodeoxyglucose. CONCLUSION: We are reporting the first example of using specifically deuterated [5-(13)C-4-(2)H(2)]-L-glutamine in conjunction with hyperpolarized MRS for studying "glutaminolysis" in proliferating tumor cells.

Synthesis and comparative biological evaluation of L- and D-isomers of 18F-labeled fluoroalkyl phenylalanine derivatives as tumor imaging agents.

INTRODUCTION: L-amino acid-based tracers have established their important role as tumor metabolic imaging agents. Recently, a number of studies demonstrated that D-amino acids may have improved imaging properties than their corresponding L-isomers. We synthesized and evaluated the D-isomer of a new phenylalanine derivative, p-(2-[(18)F]fluoroethyl)-phenylalanine ([(18)F]FEP), in comparison to its L-isomer and previously reported the L- and D-isomers of O-(2-[(18)F]fluoroethyl)-tyrosine ([(18)F]FET). METHODS: L- and D-Isomers of [(18)F]FET and [(18)F]FEP were successfully synthesized via a rapid and efficient two-step nucleophilic fluorination and deprotection reaction. In vitro studies were carried out in 9L glioma cells. In in vivo studies, Fisher 344 rats bearing the 9L tumor model were used. RESULTS: L- and D-Isomers of (18)F-fluoroalkyl tyrosine and phenylalanine derivatives were efficiently labeled with high enantiomeric purity (>95%), good yield (11-45%) and high specific activity (21-75 GBq/µmol). D-[(18)F]FEP showed a similar linear time-dependent uptake as D-[(18)F]FET, while their corresponding L-isomers had much faster and higher uptake (4.3- to 16.0-fold at maximum uptake). The maximum uptake of the new compounds, L- and D-[(18)F]FEP, was 1.4- and 5.2-fold of that reported for L- and D-[(18)F]FET, respectively. Transport characterization studies indicated that both L- and D-[(18)F]FEP were selective substrates for system L. While L-[(18)F]FEP exhibited preference towards one subtype of system L, LAT1, D-[(18)F]FEP did not exhibit the same preference. Small animal PET imaging studies showed that both L- and D-[(18)F]FEP had higher uptake in 9L tumor compared to surrounding tissues, but D-isomer had lower tumor-to-muscle ratio in comparison with its L-isomer. CONCLUSION: Both L- and D-[(18)F]FEP are substrates for system L amino acid transporter with different preference toward its subtypes. Small animal imaging studies of 9L tumor showed that D-[(18)F]FEP did not show better imaging properties than their corresponding L-isomer.

Synthesis, uptake mechanism characterization and biological evaluation of (18)F labeled fluoroalkyl phenylalanine analogs as potential PET imaging agents.

INTRODUCTION: Amino acids based tracers represent a promising class of tumor metabolic imaging agents with successful clinical applications. Two new phenylalanine derivatives, p-(2-[(18)F]fluoroethyl)-L-phenylalanine (FEP, [(18)F]2) and p-(3-[(18)F]fluoropropyl)-L-phenylalanine (FPP, [(18)F]3) were synthesized and evaluated in comparison to clinically utilized O-(2-[(18)F]fluoroethyl)-L-tyrosine (FET, [(18)F]1). METHODS: FEP ([(18)F]2) and FPP ([(18)F]3) were successfully synthesized by a rapid and efficient two-step nucleophilic fluorination of tosylate precursors and deprotection reaction. In vitro cell uptake studies were carried out in 9L glioma cells. In vivo studies, 9L tumor xenografts were implanted in Fisher 344 rats. RESULTS: FEP ([(18)F]2) and FPP ([(18)F]3) could be efficiently labeled within 90 min with good enantiomeric purity (>95%), good yield (11-37%) and high specific activity (21-69 GBq/µmol). Cell uptake studies showed FEP had higher uptake than FPP as well as reference ligand FET ([(18)F]1). Uptake mechanism studies suggested that FEP is a selective substrate for system L and prefers its subtype LAT1. In vivo biodistribution studies demonstrated FEP had specific accumulation in tumor cells and tumor to background ratio reached 1.45 at 60 min. Small animal positron emission tomography (PET) imaging studies showed FEP was comparable to FET for imaging rats bearing 9L tumor model. FEP had high uptake in 9L tumor compared to surrounding tissue and was quickly excreted through urinary tract. CONCLUSION: Biological evaluations indicate that FEP ([(18)F]2) is a potential useful tracer for tumor imaging with PET.