Alanine and glycine conjugates of (2S,4R)-4-[18F]fluoroglutamine for tumor imaging☆
Introduction
Abnormal cellular energy metabolism is the defining characteristic of nearly all cancers. Under an abundantly oxygenated environment, tumor cells have the ability to use glucose to produce energy without the Krebs (TCA) cycle within the mitochondria, leading to an overproduction of lactic acid – the Warburg effect [[1], [2], [3]]. The increase in glucose utilization in tumors is linked to the stimulation of the PI3K/Akt/mTOR signal transduction pathways. This modification in cellular metabolism leads to the up-regulation of the glucose transporters and hexokinase II enzymes, which is the biochemical basis of using FDG-PET as a diagnostic tool for detection of tumors [[4], [5], [6]]. In addition, FDG-PET is useful for staging and assessing the extent of the disease, targeting and the delineation of radiation therapy planning, and predicting and evaluating the response to therapy [[7], [8], [9], [10]]. Despite the success of using FDG-PET in the staging and monitoring of tumors, there is a growing appreciation that some active tumors do not demonstrate high levels of FDG uptake [[6], [7], [8]]. Certain types of active tumors are not detected by FDG-PET, suggesting that the tumor cells may adapt a different metabolism profile using various metabolic substrates other than glucose. Glutamine is the second most abundant nutrient in human body. It provides a ready source of carbon and nitrogen for energy-generating and macromolecular biosynthesis to drive cancer cell growth. This could be exploited for diagnostic and therapeutic purposes. Developing 18F labeled glutamine as new PET agents for imaging FDG-negative tumors may improve cancer diagnosis and therapeutic development.
Recently, we published an efficient synthesis of the four stereo isomers of [18F]4-fluoroglutamine (4F-Gln, 1) [11]. We found that two of these isomers, (2S,4R) and (2S,4S) are excellent analogs for measuring changes in tumor metabolism. In 9 L and SF-188 cells, these new tracers showed significant cell uptake (5–15% dose/100 μg of protein), valued higher than that of FDG under identical in vitro incubation conditions. In vivo PET imaging studies of [18F]4F-Gln (1) in 9 L tumor xenografts in Fisher344 rats displayed high tumor uptake and retention [12]. This tracer is trapped inside the tumor tissue with a high percentage of radioactivity associated with intracellular macromolecules. It is likely that this glutamine analog, [18F]4F-Gln (1), is taken up by tumor cells through glutamine transporters. Recent reports showed that [18F]4F-Gln detected increases in cellular glutamine pool size induced by GLS inhibitors in breast cancers, which suggested the utility of [18F]4F-Gln in human breast cancers and other tumor types [13]. The clinical studies showed glutamine-based PET agent [18F]4F-Gln displayed a high tumor-to-brain ratio in glioma patients. It may serve as a valuable tool in the clinical management of gliomas [14,15].
In spite of these attributes of [18F]4F-Gln (1) there are several drawbacks to monitoring changes within tumor tissue. Mainly, in vitro instability, in vivo defluorination, and other metabolic processes may limit its potential for PET imaging. First, (2S,4R)-4-[18F]fluoroglutamine (4F-Gln, 1) appears to be unstable in vivo, releasing 18F fluoride. The fluoride ion might target bone surfaces that are not related to tumor metabolism. A new [18F]4F-Gln analog [18F](2S,4S)-4-(3-Fluoropropyl)glutamine (4F-PGln) has been developed in response to the drawbacks associated with defluorination and showed a slower defluorination rate in vivo [16]. However, [18F]4F-PGln displayed some specificity for LAT1 while [18F]4F-Gln appears to be transported by ASCT2. Secondly, it can hydrolyze to 4-fluoro-glutamic acid, which is taken up by tumor tissue via different mechanisms compared to that of glutamine [17] (Fig. 1). By blocking the 2-amino group of (2S,4R)-4-[18F]fluoroglutamine (1) (by formation of dipeptide, see discussion below), it is a posiblity that the rate of in vivo metabolic reactions can be changed, thus improving specificity to targeting the tumor. [18F]4F-Gln (1) may also cyclize to a pyroglutamic acid derivative or deaminated via glutaminase, after which it is no longer an analog of glutamine [18].
In vitro hydrolysis of glutamine to glutamic acid will have a strong negative effect on tumor imaging. A significantly lower tumor cell uptake of [18F](2S,4R)4-fluoroglutamic acid vs. (2S,4R)-4-[18F]fluoroglutamine (1) was found in rat glioma 9 L, human glioblastoma SF-188, and human prostate cancer cells (PC-3), using [3H]glutamine as internal reference ligand [17]. As represented in 9 L cells, [18F]4F-Gln (1) had a high and linearly increasing uptake over a 120 min time period, in contrast to [3H]glutamine. The cell uptake of the corresponding [18F](2S,4R)4-fluoro-pyroglutamic acid is not known because the pure form is not easily isolated. In addition, it was often observed that upon standing in saline, [18F]4F-Gln (1) showed varying degrees of decomposition. So through blocking the 2-amino group of (2S,4R)-4-[18F]fluoroglutamine (1) by glycine or alanine, the vitro stability may also be enhanced.
Glutamine and its dipeptides have long been recognized as a potential nutrient supplement for patients under critical care. Naturally occurring glutamine is known to have low water solubility and is unstable in water [[19], [20], [21]]. The instability of glutamine makes it unsuitable as a parenteral solution, especially when the solution requires a terminal sterilization by autoclaving. To circumvent the in vitro instability and relatively low solubility of using glutamine as parenteral solution directly, dipeptides (most commonly L-Ala-L-glutamine dipeptide, marketed as Dipeptiven by Hospira) are used as a prodrug of glutamine [[20], [21], [22], [23], [24]]. It was found that the dipeptide, L-Ala-L-glutamine (Dipeptiven), showed excellent in vitro stability and high water solubility (solubility 550 mg/cm3, a factor of 15 improvement over glutamine, 36 mg/cm3). In a group of ICU patients during an infusion period, a steady state in plasma concentration was reached for L-Ala-l-glutamine dipeptide after intravenous infusion, and the glutamine concentration in the blood increased comparable to that of the dipeptide concentration [25]. The half-life of this dipeptide, as a prodrug, in the human plasma was relatively short - 16 min [24].
It is important to note that [18F]4-fluoroalkylglutamic acid has been previously reported as a tumor imaging agent. The uptake mechanism of this radiotracer is predominantly based on the upregulation of the cysteine/glutamic acid transporter of tumor cells [26,27]. The uptake and retention mechanisms of labeled glutamic acids in tumor cells are not the same as the glutamine derivatives [[28], [29], [30]]. It is likely that the glutamic acid imaging agents are not mechanistically related to glutamine metabolism for fueling tumor proliferation [17].
We evaluated two new dipeptides, glycine- and alanine-(2S,4R)-4-[18F]fluoroglutamine, [18F]Gly-4F-Gln (2), and [18F]Ala-4F-Gln (3), as potential prodrugs useful for overcoming the drawbacks (in vivo instability and side reactions mentioned above) for delivering [18F]4F-Gln (1), in vivo (Fig. 2). This approach would avoid some of the unfavorable pharmacokinetics mentioned above. Reported herein is the synthesis and characterization of dipeptides containing (2S,4R)-4-[18F]fluoroglutamine, [18F]Gly-4F-Gln (2), and [18F]Ala-4F-Gln (3) as potential tumor imaging agents.
Section snippets
General
All chemicals were purchased from Aldrich Chemical (St. Louis, MO), and other commercially available materials were used without further purification unless otherwise indicated. Solvents were purified and dried through a molecular sieve system (Pure Solve Solvent Purification System; Innovative Technology, Inc.). 1H and 13C NMR spectra were recorded by a Bruker DPX spectrometer at 200 MHz and 50 MHz respectively and referenced to NMR solvents as indicated. Chemical shifts are reported in ppm
Chemistry
Starting materials 4 and 5 were synthesized according to the reported scheme [11]. The synthesis of new dipeptides is summarized in Scheme 1. Selective removal of the N-Boc protection group was achieved by using TFA/CH2Cl2/thioanisole at 0 °C. The free amino group was reacted with Boc-Gly-OSu or Boc-Ala-OSu to give the tosylate precursors, 8a and 8b (yields of this two-step reaction were 69–85%). The fluorinated intermediates 9a and 9b were prepared from 5, following the same procedure as
Acknowledgments
This work was supported in part by grants from Stand-Up 2 Cancer grant (SU2C), PA Health Department and National Institutes of Health (CA-164490).
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Financial support: This work was supported by grants awarded from the National Institutes of Health ROI-CA-164490; SU2C and PA-Health grant.