Characteristic Evaluation of a 11C-Labeled Leucine Analog, l-α-[5-11C]methylleucine, as a Tracer for Brain Tumor Imaging by Positron Emission Tomography.

Amino acid transporters are upregulated in many cancer cells, and system L amino acid transporters (LAT1-4), in particular, LAT1, which preferentially transports large, neutral, and branched side-chain amino acids, are considered a primary target for cancer positron emission tomography (PET) tracer development. Recently, we developed a 11C-labeled leucine analog, l-α-[5-11C]methylleucine ([5-11C]MeLeu), via a continuous two-step reaction of Pd0-mediated 11C-methylation and microfluidic hydrogenation. In this study, we evaluated the characteristics of [5-11C]MeLeu and also compared the sensitivity to brain tumors and inflammation with l-[11C]methionine ([11C]Met) to determine its potential for brain tumor imaging. Competitive inhibition experiments, protein incorporation, and cytotoxicity experiments of [5-11C]MeLeu were performed in vitro. Further, metabolic analyses of [5-11C]MeLeu were performed using a thin-layer chromatogram. The accumulation of [5-11C]MeLeu in tumor and inflamed regions of the brain was compared with [11C]Met and 11C-labeled (S)-ketoprofen methyl ester by PET imaging, respectively. Transporter assay with various inhibitors revealed that [5-11C]MeLeu is mainly transported via system L amino acid transporters, especially LAT1, into A431 cells. The protein incorporation assay and metabolic assay in vivo demonstrated that [5-11C]MeLeu was neither used for protein synthesis nor metabolized. These results indicate that MeLeu is very stable in vivo. Furthermore, the treatment of A431 cells with various concentrations of MeLeu did not change their viability, even at high concentrations (∼10 mM). In brain tumors, the tumor-to-normal ratio of [5-11C]MeLeu was more elevated than that of [11C]Met. However, the accumulation levels of [5-11C]MeLeu were lower than those of [11C]Met (the standardized uptake value (SUV) of [5-11C]MeLeu and [11C]Met was 0.48 ± 0.08 and 0.63 ± 0.06, respectively). In brain inflammation, no significant accumulation of [5-11C]MeLeu was observed at the inflamed brain area. These data suggested that [5-11C]MeLeu was identified as a stable and safe agent for PET tracers and could help detect brain tumors, which overexpress the LAT1 transporter.

Synthesis and Preclinical Evaluation of 18F-Labeled Ketoprofen Methyl Esters for Cyclooxygenase-1 Imaging in Neuroinflammation.

Cyclooxygenase (COX) is a rate-limiting enzyme in the synthesis of proinflammatory prostanoids from arachidonic acid. In vivo imaging of COX by PET is a potentially powerful tool for assessing the inflammatory response to injury, infection, and disease. We previously reported on a promising PET probe for COX imaging, 11C-labeled ketoprofen methyl ester, which can detect COX-1 activation in models of neuroinflammation and neurodegenerative disorders. In the current study, we aimed to design a fluorine-substituted benzoyl group of ketoprofen (FKTP) and to evaluate its racemate and enantiomers (18F-labeled ketoprofen methyl ester, [18F]FKTP-Me) as PET proradiotracers, potential radiopharmaceuticals for in vivo PET study of COX-1. Methods: We performed nucleophilic aromatic 18F-fluorination to obtain the desired racemic radiolabeled probe, (RS)-[18F]FKTP-Me, at a radiochemical yield of 11%-13%. Subsequent high-performance liquid chromatography separation with a chiral column yielded the desired enantiomerically pure (R)- and (S)-[18F]FKTP-Me. We examined the in vivo properties of (RS)-, (R)-, and (S)-[18F]FKTP-Me in PET studies using rats in which hemispheric inflammation was induced by intrastriatally injecting a lipopolysaccharide. Results: Racemic (RS)-[18F]FKTP-Me and enantiomeric (R)- or (S)-[18F]FKTP-Me were synthesized with radiochemical and chemical purities of more than 99%. The metabolite analysis revealed that the racemic (RS)-[18F]FKTP-Me crossed the blood-brain barrier and entered the brain, where it was subsequently hydrolyzed to its pharmacologically active acid form. PET images revealed a high accumulation of (R)-, (S)-, and (RS)-[18F]FKTP in the inflamed regions in rat brain. Moreover, the accumulated radioactivity of (S)-[18F]FKTP-Me was higher than that of (RS)-[18F]FKTP-Me and (R)-[18F]FKTP-Me, which was correlated with the stereospecific inhibitory activity of FKTP against COX-1. Conclusion: From the results of this study, we conclude that racemic (RS)-[18F]FKTP-Me and its enantiomers could act as proradiotracers of neuroinflammation in rat brain by the association of their hydrolyzed acid forms with COX-1 in inflamed regions. In particular, (S)-[18F]FKTP-Me demonstrated suitable properties as a COX-1-specific probe in PET imaging of neuroinflammation.

Synthesis of L-[5-11 C]Leucine and L-α-[5-11 C]Methylleucine via Pd0 -mediated 11 C-Methylation and Microfluidic Hydrogenation: Potentiality of Leucine PET Probes for Tumor Imaging.

The efficient synthesis of L-[5-11 C]leucine and L-α-[5-11 C]methylleucine has been investigated using a continuous two-step sequence of rapid reactions consisting of Pd0 -mediated 11 C-methylation and microfluidic hydrogenation. The synthesis of L-[5-11 C]leucine and L-α-[5-11 C]methylleucine was accomplished within 40 min with a decay-corrected radiochemical yield of 15-38 % based on [11 C]CH3 I, radiochemical purity of 95-99 %, and chemical purity of 95-99 %. The Pd impurities in the injectable solution measured using inductively coupled plasma mass spectrometry met the international criteria for human use. Positron emission tomography scanning after an intravenous injection of L-[5-11 C]leucine or L-α-[5-11 C]methyl leucine in A431 tumor-bearing mice was performed. As a result, L-α-[5-11 C]methylleucine was found to be a potentially useful probe for visualizing the tumor. Tissue distribution analysis showed that the accumulation value of L-α-[5-11 C]methylleucine in tumor tissue was high [12±3% injected dose/g tissue (%ID/g)].