System a amino acid transport-targeted brain and systemic tumor PET imaging agents 2-amino-3-[18 F]fluoro-2-methylpropanoic acid and 3-[18 F]fluoro-2-methyl-2-(methylamino)propanoic acid
Introduction
Cancer is the second leading cause of death in the United States, exceeded only by heart disease [1], and imaging plays key roles the clinical diagnosis, staging, and/or treatment monitoring of most human malignancies. Alterations in cellular metabolism including amino acid transport (AAT) and metabolism are hallmarks of cancer cells and can be targeted for diagnostic imaging and for therapy [2], [3]. Metabolic positron emission tomography (PET) imaging of cancer targets the altered metabolism of cancer cells, and the glucose analogue 2-[18 F]fluoro-2-deoxy-d-glucose (FDG) has been successfully employed for the clinical imaging of a wide range of cancers based on the increased rates of glucose transport and glycolysis that occur in many human tumors. Despite its success, FDG has important limitations that potentially can be overcome with PET tracers targeting different aspects of tumor metabolism and biochemistry. Glycolysis reflects only one facet of tumor metabolism and may not be the most effective molecular imaging target for certain cancers and may not be adequate to predict and monitor response to certain metabolic and molecular therapies. Additionally, FDG has high uptake in inflammatory processes which limits its specificity [4], [5], [6], [7], [8], [9], [10]. Finally, certain types of cancer, including prostate cancer, have relatively low uptake of FDG which limits its sensitivity for detecting these neoplasms [11].
Radiolabeled α-amino acids (AAs) represent a diverse and useful class of PET tracers that target the increased rates of AAT exhibited by many cancer cells. AA-based radiotracers enter and accumulate in tumor calls through membrane-associated AAT systems. For molecular imaging, tumor uptake of radiolabeled AAs is primarily determined by AAT rather than protein synthesis [12], [13]. The most extensively evaluated AAs for human tumor imaging include L-[11C]methionine ([11C]MET) [14], [15], [16], [17], O-(2-[18 F]fluoroethyl)-L-tyrosine ([18 F]FET) [18], [19] and 3,4-dihydroxy-6-[18 F]-fluoro-L-phenylalanine ([18 F]FDOPA) [20], [21]. All these amino acids are primarily system L (leucine-preferring) substrates and these studies have implicated increased system L expression in tumor cells. System L transport is active at the normal blood–brain barrier (BBB) which makes these system L tracers particularly well-suited for brain tumor imaging, but their performance for detecting other tumor types outside of the brain has been relatively poor in part due to low tumor to background ratios limiting the sensitivity of these tracers [12].
System A (alanine-preferring) AAT is an important target of oncogene action and a crucial regulator of cell growth based on its response to environmental stimuli, growth factors, oncogenic transformation and the dependency of its synthesis on protein kinase C [22], [23], [24], [25], [26], [27], [28], [29], [30]. System A transporters have been shown to be upregulated in several human cancers including breast cancer, hepatocellular carcinoma and cholangiocarcinoma [29], [31], [32]. Unlike system L transporters, system A AAT can concentrate substrates in tumor cells through secondary active transport. This concentrative property of system A transport has the potential to provide higher tumor to normal tissue ratios and more persistent tumor uptake, leading to superior tumor visualization and detection. To date, there are relatively few preclinical/clinical studies involving system A substrates since the chemical structures of the system A substrates are restricted to α-AAs with short, neutral side chains. The reported PET tracer examples for system A are limited to carbon-11 labeled 2-amino isobutyric acid (AIB) and its N-methyl derivative 2-(methylamino)isobutryic acid (MeAIB) [9], [10], [33], [34]. The limited evaluation of [11C]AIB/MeAIB has shown promise in clinical oncology for PET tumor imaging in patients with brain and systemic tumors. However the short-lived carbon-11 label (T1/2 = 20 min) greatly limits their suitability for routine clinical use since an on-site cyclotron and radiosynthetic capabilities are needed to produce 11C, and batch production for multiple patients and remote distribution is challenging.
Through our efforts to develop PET tracers targeting system A AAT with greater potential clinical utility, we have prepared fluorine-18 (T1/2 = 110 min) labeled AIB analogs (R)- and (S)-2-amino-3-[18 F]fluoro-2-methylpropanoic acid ((R)- and (S)-[18 F]FAMP, 1) and fluorine-18 labeled MeAIB analogs (R)- and (S)-3-[18 F]fluoro-2-methyl-2-N-(methylamino)propanoic acid ((R)- and (S)-[18 F]FMeAMP, 2), which chemical structures are shown in Fig. 1. In our initial study with a rat brain tumor model, these fluorinated AIB/MeAIB analogs showed high and selective in vitro uptake by the system A AAT, and demonstrated excellent tumor to normal brain ratios of 20:1 to 115:1 in vivo in part due to the lack of system A transport at the normal BBB [35], [36]. To test the tumor imaging potential of these tracers for a wider range of human cancers including prostate cancer, breast cancer, and non-small cell lung cancer, we further evaluated (R)- and (S)- FAMP 1 and (R)- and (S)- MeFAMP 2 through in vitro AA uptake assays and in tumor-bearing mice implanted with human derived cancer cell lines.
Section snippets
Synthesis of tracers
Enantiomers of [18 F]FAMP 1 and [18 F]MeFAMP 2 used for in vitro and in vivo studies performed at Emory University were synthesized according to the method previously described [35], [36]. Briefly, the automated radio-syntheses of (R)-[18 F]1, (S)-[18 F]1, (R)-[18 F]2 and (S)-[18 F]2 were carried out with no-carrier-added (NCA) nucleophilic fluorination using the cyclic sulfamidate precursors (S)-3, (R)-3, (S)-4, and (R)-4, respectively (Scheme 1). The synthesis was completed within 90 min after the
In vitro uptake and inhibition
To measure the contribution of system A AAT to the uptake of (R)- and (S)-[18 F]1 and (R)- and (S)-[18 F]2 by human A549, DU145, U87, MDA MB468 and SKOV3 tumor cells, inhibition assays were performed using cultured tumor cells in the presence and absence of amino acid transporter inhibitors. MeAIB is a selective competitive inhibitor of system A while BCH is an inhibitor of system L AAT that also inhibits system B0-like AAT in the presence of sodium [39], [40], [41]. The transport assays were all
Conclusion
The current study demonstrates that radiofluorinated amino acids (R)- and (S)-[18 F]1 and (R)- and (S)-[18 F]2 have high uptake in tumor cells in vitro and in vivo. The inhibition experiments showed that (R)- and (S)-[18 F]1 entered these tumor cells in vitro via mixed system A AAT (inhibited by MeAIB) and non-system A AAT (inhibited by BCH), likely including system L transport. Future studies are planned to better define the non-system A transport systems mediating the uptake of the enantiomers
Acknowledgment
The authors would like to thank Vernon M. Camp, Zhaobin Zhang, Larry Williams, and Eugene J. Malveaux of Emory University, for the assistance in the cell and the animal experiments. This study was supported by the NIH grant (R21 CA098891-02) and Nihon Mediphyiscs Co., Ltd., Japan.
References (48)
- et al.
Differentiation between malignancy and inflammation in pulmonary ground-glass nodules: The feasibility of integrated (18)F-FDG PET/CT
Lung Cancer
(2009) - et al.
O-(2-[18 F]fluoroethyl)-L-tyrosine: uptake mechanisms and clinical applications
Nucl Med Biol
(2006) - et al.
The regulation of amino acid transport in animal cells
Biochim Biophys Acta
(1978) - et al.
Characteristics and hormonal regulation of amino acid transport system A in isolated rat hepatocytes
Curr Top Cell Regul
(1985) - et al.
Overexpression of ATA1/SLC38A1 predicts future recurrence and death in Chinese patients with hilar cholangiocarcinoma
J Surg Res
(2011) - et al.
Stereoselective synthesis and biological evaluation of syn-1-amino-3-[18 F]fluorocyclobutyl-1-carboxylic acid as a potential positron emission tomography brain tumor imaging agent
Bioorg Med Chem
(2009) - et al.
Synthesis and biological evaluation of anti-1-amino-2-[18 F]fluoro-cyclobutyl-1-carboxylic acid (anti-2-[18 F]FACBC) in rat 9 L gliosarcoma
Bioorg Med Chem Lett
(2010) - et al.
Synthesis and quality assurance of [11C]alpha-aminoisobutyric acid (AIB), a potential radiotracer for imaging and amino acid transport studies in normal and malignant tissues
Int J Nucl Med Biol
(1984) American Cancer Society
- et al.
Radiolabeled amino acids for oncologic imaging
J Nucl Med
(2013)