Claudia Keller

5-(2-18F-Fluoroethoxy)-l-Tryptophan as a Substrate of System L Transport for Tumor Imaging by PET

Large neutral l-amino acids are substrates of system L amino acid transporters. The level of one of these, LAT1, is increased in many tumors. Aromatic l-amino acids may also be substrates of aromatic l-amino acid decarboxylase (AADC), the level of which is enhanced in endocrine tumors. Increased amino acid uptake and subsequent decarboxylation result in the intracellular accumulation of the amino acid and its decarboxylation product. 18F- and 11C-labeled neutral aromatic amino acids, such as l-3,4-dihydroxy-6-18F-fluorophenylalanine (18F-FDOPA) and 5-hydroxy-l-[β-11C]tryptophan, are thus successfully used in PET to image endocrine tumors. However, 5-hydroxy-l-[β-11C]tryptophan has a relatively short physical half-life (20 min). In this work, we evaluated the in vitro and in vivo characteristics of the 18F-labeled tryptophan analog 5-(2-18F-fluoroethoxy)-l-tryptophan (18F-l-FEHTP) as a PET probe for tumor imaging. Methods: 18F-l-FEHTP was synthesized by no-carrier-added 18F fluorination of 5-hydroxy-l-tryptophan. In vitro cell uptake and efflux of 18F-l-FEHTP and 18F-FDOPA were studied with NCI-H69 endocrine small cell lung cancer cells, PC-3 pseudoendocrine prostate cancer cells, and MDA-MB-231 exocrine breast cancer cells. Small-animal PET was performed with the respective xenograft-bearing mice. Tissues were analyzed for potential metabolites. Results: 18F-l-FEHTP specific activity and radiochemical purity were 50–150 GBq/μmol and greater than 95%, respectively. In vitro cell uptake of 18F-l-FEHTP was between 48% and 113% of added radioactivity per milligram of protein within 60 min at 37°C and was blocked by greater than 95% in all tested cell lines by the LAT1/2 inhibitor 2-amino-2-norboranecarboxylic acid. 18F-FDOPA uptake ranged from 26% to 53%/mg. PET studies revealed similar xenograft-to-reference tissue ratios for 18F-l-FEHTP and 18F-FDOPA at 30–45 min after injection. In contrast to the 18F-FDOPA PET results, pretreatment with the AADC inhibitor S-carbidopa did not affect the 18F-l-FEHTP PET results. No decarboxylation products of 18F-l-FEHTP were detected in the xenograft homogenates. Conclusion: 18F-l-FEHTP accumulates in endocrine and nonendocrine tumor models via LAT1 transport but is not decarboxylated by AADC. 18F-l-FEHTP may thus serve as a PET probe for tumor imaging and quantification of tumor LAT1 activity. These findings are of interest in view of the ongoing evaluation of LAT1 substrates and inhibitors for cancer therapy.

Imaging Tumour ATB0,+ Transport Activity by PET with the Cationic Amino Acid O-2((2-[18F]fluoroethyl)methyl-amino)ethyltyrosine

PurposeThe concentrative amino acid transporter ATB0,+ (SLC6A14) is under evaluation as a target for anticancer therapy. An ATB0,+-selective positron emission tomography (PET) probe could advance preclinical drug development. We characterised the cationic tyrosine analogue O-2((2-[18F]fluoroethyl)methyl-amino)ethyltyrosine ([18F]FEMAET) as a PET probe for ATB0,+ activity.ProceduresCell uptake was studied in vitro. ATB0,+ expression was quantified by real-time PCR. [18F]FEMAET accumulation in xenografts was investigated by small animal PET with mice.Results[18F]FEMAET accumulated in PC-3 and NCI-H69 cancer cells in vitro. As expected for ATB0,+ transport, uptake was inhibited by LAT/ATB0,+ inhibitors and dibasic amino acids, and [18F]FEMAET efflux was only moderately stimulated by extracellular amino acids. ATB0,+ was expressed in PC-3 and NCI-H69 but not MDA-MB-231 xenografts. PET revealed accumulation in PC-3 and NCI-H69 xenografts and significant reduction by ATB0,+ inhibition. Uptake was negligible in MDA-MB-231 xenografts.ConclusionATB0,+ activity can be imaged in vivo by PET with [18F]FEMAET.

Quantitative positron emission tomography of mGluR5 in rat brain with [18F]PSS232 at minimal invasiveness and reduced model complexity

Imaging the density of metabotropic glutamate receptor 5 (mGluR5) in brain by positron emission tomography (PET) is of interest in relation to several brain disorders. We have recently introduced [18F]PSS232, an F‐18‐labeled analog of the mGluR5‐targeting [11C]ABP688. Quantitative PET requires kinetic modeling with an input function (IF) or an appropriate reference tissue model. We aimed at minimizing invasiveness of IF recording in rat and employing this protocol for mGluR5 quantitative PET with [18F]PSS232. We further aimed at defining models of low complexity for quantitative PET with [18F]PSS232. The IF was recorded in an arterio‐venous shunt applied by minimally invasive cannulation. PET data were analyzed with a modified two‐tissue compartment model including a single variable for radiometabolite correction in brain. We further evaluated a simple reference tissue model. Receptor‐dependent accumulation was similar to [11C]ABP688 at lower unspecific accumulation of unchanged [18F]PSS232, in agreement with its higher plasma protein binding and lower lipophilicity. The minimally invasive protocol revealed similar results as the invasive shunt method and parameters calculated with the modified two‐tissue compartment model were similar to those calculated with the standard model. The simple area under the curve ratios agreed with the Logan reference method. [18F]PSS232 is a promising radioligand for mGluR5 quantification.

Synthesis and biological evaluation of (18)F-labeled Fluoroethoxy tryptophan analogues as potential PET tumor imaging agents.

As a continuation of our research efforts toward the development of tryptophan-based radiotracers for tumor imaging with positron emission tomography (PET), three new fluoroethoxy tryptophan analogues were synthesized and evaluated in vivo. These new tracers (namely, 4-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]4-FEHTP), 6-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]6-FEHTP), and 7-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]7-FEHTP) carry the fluoroethoxy side chain either at positions 4-, 6-, or 7- of the indole core. Reference compounds and precursors were synthesized by multistep approaches. Radiosynthesis was accomplished by no-carrier-added nucleophilic (18)F-fluorination following either an indirect approach (O-alkylation of the corresponding hydroxytryptophan with [(18)F]fluoroethyltosylate) or a direct approach (nucleophilic [(18)F] fluorination using a protected mesyl precursor). Radiochemical yields (decay corrected) for both methods were in the range of 10-18%. Small animal PET imaging with xenograft-bearing mice revealed the highest tumor/background ratio for [(18)F]6-FEHTP which, in a direct comparison, outperformed the other two tryptophan tracers and also the well-established tyrosine analogue O-(2-[(18)F]fluoroethyl)-l-tyrosine ([(18)F]l-FET). Investigation of the transport mechanism of [(18)F]6-FEHTP in small cell lung cancer cells (NCI-H69) revealed that it is most probably taken up exclusively via the large neutral amino acid transporter(s) (LAT).

Synthesis and biological evaluation of 18F-labeled fluoropropyl tryptophan analogs as potential PET probes for tumor imaging

In the search for an efficient, fluorine-18 labeled amino acid based radiotracer for tumor imaging with positron emission tomography (PET), two new tryptophan analogs were synthesized and characterized in vitro and in vivo. Both are tryptophan alkyl-derivatives, namely 2-(3-[(18)F]fluoropropyl)-DL-tryptophan ([(18)F]2-FPTRP) and 5-(3-[(18)F]fluoro-propyl)-DL-tryptophan ([(18)F]5-FPTRP). Standard reference compounds and precursors were prepared by multi step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [(18)F]fluorination in 29-34% decay corrected yields with radiochemical purity over 99%. In vitro cell uptake assays showed that both compounds are substrates for amino acid transport and enter small cell lung cancer cells (NCI-H69) most probably almost exclusively via large neutral amino acids transporter(s) (LAT). Small animal PET imaging with xenograft bearing mice revealed high tumor/background ratios for [(18)F]2-FPTRP comparable to the well established tyrosine analog O-(2-[(18)F]fluroethyl)-L-tyrosine ([(18)F]FET). Radiometabolite studies showed no evidence of involvement of a biotransformation step in tumor accumulation.

Evaluation of [11C]Me-NB1 as a potential PET radioligand for measuring GluN2B-containing NMDA receptors, drug occupancy and receptor crosstalk

Clinical and preclinical research with modulators at the N-methyl-d-aspartate (NMDA) receptor GluN2B N-terminal domain (NTD) aims for the treatment of various neurologic diseases. The interpretation of the results is hampered by the lack of a suitable NMDA PET tracer for assessing the receptor occupancy of potential drugs. We have developed 11C-Me-NB1 as a PET tracer for imaging GluN1/GluN2B-containing NMDA receptors and used it to investigate in rats the dose-dependent receptor occupancy of eliprodil, a GluN2B NTD modulator. Methods: 11C-Me-NB1 was synthesized and characterized by in vitro displacement binding experiments with rat brain membranes, in vitro autoradiography, and blocking and displacement experiments by PET and PET kinetic modeling. Receptor occupancy by eliprodil was studied by PET with 11C-Me-NB1. Results: 11C-Me-NB1 was synthesized at 290 ± 90 GBq/μmol molar activity, 7.4 ± 1.9 GBq total activity at the end of synthesis (n = 17), and more than 99% radiochemical purity. 11C-Me-NB1 binding in rat brain was blocked in vitro and in vivo by the NTD modulators Ro-25-6981 and eliprodil. Half-maximal receptor occupancy by eliprodil occurred at 1.5 μg/kg. At 1 mg/kg of eliprodil, a dose with reported neuroprotective effects, more than 99.5% of binding sites were occupied. In vitro, 11C-Me-NB1 binding was independent of the σ-1 receptor (Sigma1R), and the Sigma1R agonist (+)-pentazocine did not compete for high-affinity binding. In vivo, a 2.5 mg/kg dose of (+)-pentazocine abolished 11C-Me-NB1–specific binding, indicating an indirect effect of Sigma1R on 11C-Me-NB1 binding. Conclusion: 11C-Me-NB1 is suitable for the in vivo imaging of NMDA GluN1/GluN2B receptors and the assessment of receptor occupancy by NTD modulators. GluN1/GluN2B NMDA receptors are fully occupied at neuroprotective doses of eliprodil. Furthermore, 11C-Me-NB1 enables imaging of GluN1/GluN2B NMDA receptor cross talk.

Synthesis, Radiolabeling, and Biological Evaluation of 5-Hydroxy-2-[18F]fluoroalkyl-tryptophan Analogues as Potential PET Radiotracers for Tumor Imaging

Aiming at developing mechanism-based amino acid (18)F-PET tracers for tumor imaging, we synthesized two (18)F-labeled analogues of 5-hydroxy-l-[β-(11)C]tryptophan ([(11)C]5HTP) whose excellent in vivo performance in neuroendocrine tumors is mainly attributed to its decarboxylation by aromatic amino acid decarboxylase (AADC), an enzyme overexpressed in these malignancies. Reference compounds and precursors were synthesized following multistep synthetic approaches. Radiosynthesis of tracers was accomplished in good radiochemical yields (15-39%), high specific activities (45-95 GBq/μmol), and excellent radiochemical purities. In vitro cell uptake was sodium-independent and was inhibited ≥95% by 2-amino-2-norbornanecarboxylic acid (BCH) and ∼30% by arginine. PET imaging in mice revealed distinctly high tumor/background ratios for both tracers, outperforming the well-established O-(2-[(18)F]fluoroethyl)tyrosine ([(18)F]FET) tracer in a head-to-head comparison. Biological evaluation revealed that the in vivo performance is most probably independent of any interaction with AADC. Nevertheless, the excellent tumor visualization qualifies the new tracers as interesting probes for tumor imaging worthy for further investigation.

CD80 Is Upregulated in a Mouse Model with Shear Stress-Induced Atherosclerosis and Allows for Evaluating CD80-Targeting PET Tracers

PurposeA shear stress-induced atherosclerosis mouse model was characterized for its expression of inflammation markers with focus on CD80. With this model, we evaluated two positron emission tomography (PET) radiotracers targeting CD80 as well as 2-deoxy-2-[18F]fluoro-d-mannose ([18F]FDM) in comparison with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG).ProcedureA flow constrictive cuff implanted around the common carotid artery in apolipoprotein E knockout mice resulted in plaque formation. CD80 expression levels and plaque histopathology were evaluated. Serial PET/X-ray computed tomography scans were performed to follow inflammation.ResultsPlaque formation with increased levels of CD80 was observed. Histologically, plaques presented macrophage-rich and large necrotic areas covered by a thin fibrous cap. Of the CD80-specific tracers, one displayed an increased uptake in plaques by PET. Both [18F]FDG and [18F]FDM accumulated in atherosclerotic plaques.ConclusionThis mouse model presented, similar to humans, an increased expression of CD80 which renders it suitable for non-invasively targeting CD80-positive immune cells and evaluating CD80-specific radiotracers.

Physiologically Based Pharmacokinetic Modelling with Dynamic PET Data to Study the In Vivo Effects of Transporter Inhibition on Hepatobiliary Clearance in Mice

Physiologically based pharmacokinetic modelling (PBPK) is a powerful tool to predict in vivo pharmacokinetics based on physiological parameters and data from in vivo studies and in vitro assays. In vivo PBPK modelling in laboratory animals by noninvasive imaging could help to improve the in vivo-in vivo translation towards human pharmacokinetics modelling. We evaluated the feasibility of PBPK modelling with PET data from mice. We used data from two of our PET tracers under development, [11C]AM7 and [11C]MT107. PET images suggested hepatobiliary excretion which was reduced after cyclosporine administration. We fitted the time-activity curves of blood, liver, gallbladder/intestine, kidney, and peripheral tissue to a compartment model and compared the resulting pharmacokinetic parameters under control conditions ([11C]AM7 n = 2; [11C]MT107, n = 4) and after administration of cyclosporine ([11C]MT107, n = 4). The modelling revealed a significant reduction in [11C]MT107 hepatobiliary clearance from 35.2 ± 10.9 to 17.1 ± 5.6 μl/min after cyclosporine administration. The excretion profile of [11C]MT107 was shifted from predominantly hepatobiliary (CLH/CLR = 3.8 ± 3.0) to equal hepatobiliary and renal clearance (CLH/CLR = 0.9 ± 0.2). Our results show the potential of PBPK modelling for characterizing the in vivo effects of transporter inhibition on whole-body and organ-specific pharmacokinetics.

Identification and Preclinical Evaluation of a Radiofluorinated Benzazepine Derivative for Imaging the GluN2B Subunit of the Ionotropic NMDA Receptor

The previously reported 11C-labeled GluN2B PET radioligand 11C-Me-NB1 served as a starting point for derivatization and led to the successful development of a radiofluorinated analog designated (R)-18F-OF-Me-NB1. Given the short physical half-life of 20.3 min for 11C, (R)-18F-OF-Me-NB1 with a physical half-life of 109.8 min would allow satellite distribution to nuclear medicine facilities without an on-site cyclotron. Methods: Two fluorinated Me-NB1 derivatives, OF-Me-NB1 and PF-Me-NB1, were synthesized. On chiral resolution, the respective enantiomers were radiolabeled with 11C and assessed in a proof-of-concept study by applying in vitro autoradiography on rodent brain sections. On the basis of the autoradiograms, (R)-OF-Me-NB1 was selected for radiofluorination and preclinical evaluation by ex vivo autoradiography, PET imaging, biodistribution, and metabolite studies on Wistar rats. To rule out off-target binding to the σ1 receptor (σ1R), brain uptake of (R)-18F-OF-Me-NB1 in wild-type mice was compared with that in σ1R knock-out mice. Results: Autoradiographic assessment revealed that both enantiomers of 11C-PF-Me-NB1 distributed homogeneously across all brain regions on rodent brain sections. In contrast, the 2 enantiomers of 11C-OF-Me-NB1 exhibited an entirely different behavior. Although (S)-11C-OF-Me-NB1 bound to virtually all brain regions, with considerable σ1R binding, (R)-11C-OF-Me-NB1 exhibited high selectivity and specificity for the GluN2B-rich rat forebrain. These findings were confirmed for the radiofluorinated analog (R)-18F-OF-Me-NB1, which was obtained via copper-mediated radiofluorination in radiochemical yields of 13%–25% and molar activities ranging from 61 to 168 GBq/μmol. PET imaging and biodistribution studies on Wistar rats indicated an appropriate pharmacokinetic profile and high in vivo specific binding of (R)-18F-OF-Me-NB1 as revealed by blocking studies with the GluN2B antagonist CP101,606. Off-target binding to the σ1R was excluded by PET imaging of σ1R knock-out mice. Half-maximal receptor occupancy by CP101,606 occurred at 8.3 μmol/kg (intravenous). Conclusion: (R)-18F-OF-Me-NB1 is a promising radiofluorinated probe that exhibits specificity and selectivity for the GluN2B-containing N-methyl-d-aspartate complex and enables in vivo target occupancy studies on rodents.