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.

Monoamine oxidase binding not expected to significantly affect [18F]flortaucipir PET interpretation.

PURPOSE: [18F]-labeled positron emission tomography (PET) radioligands permit in vivo assessment of Alzheimer's disease biomarkers, including aggregated neurofibrillary tau (NFT) with [18F]flortaucipir. Due to structural similarities of flortaucipir with some monoamine oxidase A (MAO-A) inhibitors, this study aimed to evaluate flortaucipir binding to MAO-A and MAO-B and any potential impact on PET interpretation. METHODS: [18F]Flortaucipir autoradiography was performed on frozen human brain tissue slices, and PET imaging was conducted in rats. Dissociation constants were determined by saturation binding, association and dissociation rates were measured by kinetic binding experiments, and IC50 values were determined by competition binding. RESULTS: Under stringent wash conditions, specific [18F]flortaucipir binding was observed on tau NFT-rich Alzheimer's disease tissue and not control tissue. In vivo PET experiments in rats revealed no evidence of [18F]flortaucipir binding to MAO-A; pre-treatment with MAO inhibitor pargyline did not impact uptake or wash-out of [18F]flortaucipir. [18F]Flortaucipir bound with low nanomolar affinity to human MAO-A in a microsomal preparation in vitro but with a fast dissociation rate relative to MAO-A ligand fluoroethyl-harmol, consistent with no observed in vivo binding in rats of [18F]flortaucipir to MAO-A. Direct binding of flortaucipir to human MAO-B was not detected in a microsomal preparation. A high concentration of flortaucipir (IC50 of 1.3 µM) was found to block binding of the MAO-B ligand safinamide to MAO-B on microsomes suggesting that, at micromolar concentrations, flortaucipir weakly binds to MAO-B in vitro. CONCLUSION: These data suggest neither MAO-A nor MAO-B binding will contribute significantly to the PET signal in cortical target areas relevant to the interpretation of [18F]flortaucipir.

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.

Quantitative PET Reporter Gene Imaging with [11C]Trimethoprim.

There is a need for improved methods to image genetically engineered cells, including immune cells used for cell-based therapy. Given the genetic manipulation inherent to gene therapy, the use of a reporter protein is a logical solution and positron emission tomography (PET) can provide the desired sensitivity and spatial localization. We developed a broadly applicable PET imaging strategy based on the small bacterial protein E. coli dihydrofolate reductase (Ec dhfr) and its highly specific small molecule inhibitor, trimethoprim (TMP). The difference in TMP affinity for bacterial compared to mammalian DHFR suggests that a TMP radioligand would have a low background in unmodified mammalian tissues and high retention in Ec dhfr engineered cells, providing high contrast imaging. Here, we describe the in vitro properties of [11C]TMP and show over 10-fold increased signal in transgenic Ec dhfr cells compared to control. In a mouse xenograft model, [11C]TMP rapidly accumulated in Ec dhfr carrying cells within minutes of intravenous administration. Moreover, [11C]TMP can identify less than a million xenografted cells in a small volume in tissues other than the abdominal compartment. This limit of detection is a clinically relevant number and bodes well for clinical translation especially given that [11C]TMP is an isotopologue of clinically approved antibiotic.

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.

A systematic exploration of the effects of flexibility and basicity on sigma (σ) receptor binding in a series of substituted diamines.

The sigma-1 receptor (S1R) has attracted a great deal of attention as a prospective drug target due to its involvement in numerous neurological disorders and, more recently, for its therapeutic potential in neuropathic pain. As there was no crystal structure of this membrane-bound protein reported until 2016, ligand generation was driven by pharmacophore refinements to the general model suggested by Glennon and co-workers. The generalised S1R pharmacophore comprises a central region where a basic amino group is preferred, flanked by two hydrophobic groups. Guided by this pharmacophore, S1R ligands containing piperazines, piperazinones, and ethylenediamines have been developed. In the current work, we systematically deconstructed the piperazine core of a prototypic piperazine S1R ligand (vide infra) developed in our laboratories. Although we did not improve the affinity at the S1R compared to the lead, we identified several features important for affinity and selectivity. These included at least one basic nitrogen atom, conformational flexibility and, for S1R, a secondary or tertiary amine group proximal to the anisole. Furthermore, S2R selectivity can be tailored with functional group modifications of the N-atom proximal to the anisole.

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.

Characterization of FlipIDAM as a SERT-selective SPECT imaging agent.

INTRODUCTION: Biological evaluation of ([(125)I]4), a new single-photon emission computed tomography (SPECT) radioligand for imaging the serotonin transporter (SERT) which displayed improved in vivo kinetics for mapping SERT binding sites in the brain. METHODS: In vitro binding studies of [(125)I]4 were performed with membrane homogenates of LLC-PK1 cells stably transfected and overexpressing one of the monoamine transporter (SERT, DAT or NET) and rat cortical homogenates. Biodistribution and ex vivo autoradiography studies were carried out in rats. In vivo competition experiments were evaluated to determine the SERT selectivity of [(125)I]4 vs. ([(125)I]1). RESULTS: In vitro binding studies of 4 showed excellent binding affinity (Ki,SERT=0.90 ± 0.05 nM) and excellent selectivity over the other monoamine transporters (100 fold and >4000 fold for NET and DAT respectively). Scatchard analysis of saturation binding of [(125)I]4 to rat cortical homogenates gave a Kd value of 0.5 ± 0.09 nM and a Bmax value of 801.4 ± 58.08 fmol/mg protein. The biodistribution study showed rapid high brain uptake (3.09 ± 0.11% dose/organ at 2 min) and a good target to non-target ratio (hypothalamus to cerebellum) at 30 min (2.62) compared to [(125)I]1 (2.19). Ex vivo autoradiography showed that FlipIDAM localizes in accordance with SERT distribution patterns in the brain. In vivo and ex vivo competition experiments with specific and non-specific SERT compounds also showed that [(125)I]4 binds specifically to SERT rich regions. CONCLUSIONS: The biological evaluation of [(125)I]4 demonstrates that [(123)I]4 would be a good candidate for SPECT imaging of SERT.

A new single-photon emission computed tomography (SPECT) imaging agent for serotonin transporters: [(125)I]Flip-IDAM, (2-((2-((dimethylamino)methyl)-4-iodophenyl)thio)phenyl)methanol.

New ligands for in vivo brain imaging of serotonin transporter (SERT) with single photon emission tomography (SPECT) were prepared and evaluated. An efficient synthesis and radiolabeling of a biphenylthiol, FLIP-IDAM, 4, was accomplished. The affinity of FLIP-IDAM was evaluated by an in vitro inhibitory binding assay using [(125)I]-IDAM as radioligand in rat brain tissue homogenates (K(i) = 0.03 nM). New [(125)I]Flip-IDAM exhibited excellent binding affinity to SERT binding sites with a high hypothalamus to cerebellum ratio of 4 at 30 min post iv injection. The faster in vivo kinetics for brain uptake and a rapid washout from non-specific regions provide excellent signal to noise ratio. This new agent, when labeled with (123)I, may be a useful imaging agent for mapping SERT binding sites in the human brain.

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.

Imaging of VMAT2 binding sites in the brain by (18)F-AV-133: the effect of a pseudo-carrier.

OBJECTIVES: Recently, 9-[(18)F]fluoropropyl-(+)-dihydrotetrabenazine ((18)F-AV-133) was reported as a new vesicular monoamine transporter (VMAT2) imaging agent for diagnosis of Parkinson's disease (PD). To shorten the preparation of (18)F-AV-133 and to make it more widely available, we evaluated a simple, rapid purification with a solid-phase extraction method (SPE) using an Oasis HLB cartridge instead of high pressure liquid chromatography (HPLC). The SPE method produced doses containing a pseudo-carrier, 9-hydroxypropyl-(+)-dihydrotetrabenazine (AV-149). METHODS: To test the possible side effects of this pseudo-carrier, comparative dynamic PET scans of the brains of normal monkeys (2 each) and uni-laterally 6-OH-dopamine-lesioned PD monkeys (2 each) were performed using (18)F-AV-133 doses prepared by either SPE (containing pseudo-carrier) or HPLC (containing no pseudo-carrier). Autoradiographs of post mortem monkey brain sections were evaluated to confirm the relative (18)F-AV-133 uptake in the PD monkey brains and the effects of the pseudo-carrier on VMAT2 binding. RESULTS: The radiochemical purity of the (18)F-AV-133, whether prepared by SPE or by HPLC, was excellent (>99%). PET scans of normal and PD monkey brains showed an expected reduction of VMAT2 in the lesioned areas of the striatum. It was not affected by the presence of the pseudo-carrier, AV-149 (maximally 250 µg/dose). The reduced uptake in the striatum of the lesioned monkey brains was confirmed by autoradiography. Ex vivo inhibition studies of (18)F-AV-133 binding in rat brains, conducted with increasing amounts of AV-149, suggested that at the highest concentration (3.5mg/kg) the VMAT2 binding in the striatum was only moderately blocked (20% reduction). CONCLUSIONS: The pseudo-carrier, AV-149, did not affect the (18)F-AV-133/PET imaging of VMAT2 binding sites in normal or uni-laterally lesioned monkey brains. The new streamlined SPE purification method will enable (18)F-AV-133 to be widely available for routine clinical application in determining changes in monoamine neurons for patient with movement disorders or other psychiatric illnesses.

Synthesis and evaluation of novel tropane derivatives as potential PET imaging agents for the dopamine transporter.

A novel series of tropane derivatives containing a fluorinated tertiary amino or amide at the 2ß position was synthesized, labeled with the positron-emitter fluorine-18 (t(1/2)=109.8 min), and tested as potential in vivo dopamine transporter (DAT) imaging agents. The corresponding chlorinated analogs were prepared and employed as precursors for radiolabeling leading to the fluorine-18-labeled derivatives via a one-step nucleophilic aliphatic substitution reaction. In vitro binding results showed that the 2ß-amino compounds 6b, 6d and 7b displayed moderately high affinities to DAT (K(i)<10nM). Biodistribution studies of [(18)F]6b and [(18)F]6d showed that the brain uptakes in rats were low. This is likely due to their low lipophilicities. Further structural modifications of these tropane derivatives will be needed to improve their in vivo properties as DAT imaging agents.