Radiopharmacological evaluation of 6-deoxy-6-[18F]fluoro-d-fructose as a radiotracer for PET imaging of GLUT5 in breast cancer

https://doi.org/10.1016/j.nucmedbio.2010.11.004Get rights and content

Abstract

Introduction

Several clinical studies have shown low or no expression of GLUT1 in breast cancer patients, which may account for the low clinical specificity and sensitivity of 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) used in positron emission tomography (PET). Therefore, it has been proposed that other tumor characteristics such as the high expression of GLUT2 and GLUT5 in many breast tumors could be used to develop alternative strategies to detect breast cancer. Here we have studied the in vitro and in vivo radiopharmacological profile of 6-deoxy-6-[18F]fluoro-d-fructose (6-[18F]FDF) as a potential PET radiotracer to image GLUT5 expression in breast cancers.

Methods

Uptake of 6-[18F]FDF was studied in murine EMT-6 and human MCF-7 breast cancer cells over 60 min and compared to [18F]FDG. Biodistribution of 6-[18F]FDF was determined in BALB/c mice. Tumor uptake was studied with dynamic small animal PET in EMT-6 tumor-bearing BALB/c mice and human xenograft MCF-7 tumor-bearing NIH-III mice in comparison to [18F]FDG. 6-[18F]FDF metabolism was investigated in mouse blood and urine.

Results

6-[18F]FDF is taken up by EMT-6 and MCF-7 breast tumor cells independent of extracellular glucose levels but dependent on the extracellular concentration of fructose. After 60 min, 30±4% (n=9) and 12±1% (n=7) ID/mg protein 6-[18F]FDF was found in EMT-6 and MCF-7 cells, respectively. 6-deoxy-6-fluoro-d-fructose had a 10-fold higher potency than fructose to inhibit 6-[18F]FDF uptake into EMT-6 cells. Biodistribution in normal mice revealed radioactivity uptake in bone and brain. Radioactivity was accumulated in EMT-6 tumors reaching 3.65±0.30% ID/g (n=3) at 5 min post injection and decreasing to 1.75±0.03% ID/g (n=3) at 120 min post injection. Dynamic small animal PET showed significantly lower radioactivity uptake after 15 min post injection in MCF-7 tumors [standard uptake value (SUV)=0.76±0.05; n=3] compared to EMT-6 tumors (SUV=1.23±0.09; n=3). Interestingly, [18F]FDG uptake was significantly different in MCF-7 tumors (SUV15 min 0.74±0.12 to SUV120 min 0.80±0.15; n=3) versus EMT-6 tumors (SUV15 min 1.01±0.33 to SUV120 min 1.80±0.25; n=3). 6-[18F]FDF was shown to be a substrate for recombinant human ketohexokinase, and it was metabolized rapidly in vivo.

Conclusion

Based on the GLUT5 specific transport and phosphorylation by ketohexokinase, 6-[18F]FDF may represent a novel radiotracer for PET imaging of GLUT5 and ketohexokinase-expressing tumors.

Introduction

Breast cancer represents the second leading cause of cancer related deaths in women. Advances in early diagnosis and treatment have led to a decline of mortality, despite an increase in breast cancer incidence. Breast cancer remains a major health care problem in women, and early detection in order to improve prognosis remains the cornerstone of breast cancer research and clinical applications. Most primary cancers are detected by physical examination or mammography, although mammography is limited by only moderate sensitivity and specificity. Therefore, other imaging methodologies like ultrasound and magnetic resonance imaging have been investigated to complement and increase the diagnostic accuracy for breast cancer [1].

In the clinic, increased glucose uptake and metabolism in cancer cells is used to identify tumors in patients and to assess tumor metabolism in response to therapy by using 18F-labeled 2-deoxy-2-fluoro-d-glucose ([18F]FDG) with positron emission tomography (PET) [2], [3], [4]. [18F]FDG is the most commonly used PET radiotracer for diagnosis and management of a variety of cancers [5], [6], [7], [8]. The uptake of this radiolabeled hexose analogue into malignant cells is facilitated by the increased expression of several members of the facilitative hexose transporter (GLUT) family. It is evident that malignant transformation results in the altered expression of genes encoding members responsible not only for hexose transport but also metabolism [9]. Fourteen genes encoding facilitative glucose transporter proteins have been identified (GLUT1–13, and HMIT) [10], [11]. It has been suggested that overexpression of the GLUT1 and GLUT3 proteins is responsible for the increased uptake of glucose and [18F]FDG in malignancies [12]. Like glucose, [18F]FDG uptake in cells is followed by phosphorylation to [18F]FDG 6-phosphate through hexokinase, the first enzymatic step in glycolysis. [18F]FDG 6-phosphate is not further metabolized, which leads to its metabolic trapping and accumulation within malignant cells, as it is unable to be transported back out of the cell. [13], [14], [15], [16].

[18F]FDG displays some important limitations for tumor detection which has led to the clinical use of alternative PET tracers [17], [18]. Macrophages and other immune cells readily transport high levels of glucose and [18F]FDG, and uptake into these cells has been implicated in the generation of false positive diagnoses [19], [20], [21]. An additional limitation of [18F]FDG in tumor diagnosis is increased uptake in inflammatory lesions, restricting the distinction between inflammation and tumor tissue and frequently leading to an overestimation in tumor size and to complications for assessment of cancer treatment efficacy.

A recent review assessing the clinical value of [18F]FDG-PET in breast cancer diagnosis indicated 76–89% sensitivity and 73–80% specificity for the diagnosis of primary breast cancer [22]. Low and very variable sensitivity (20–50%) was observed for the detection of auxillary lymph node metastases. Several clinical studies have investigated GLUT1 expression in breast cancers, revealing that 28–47% of selected patient samples were GLUT1 negative [23], [24], [25], [26]. The low or absent tumor expression of GLUT1 in these patients seems to account for the low sensitivity of [18F]FDG-PET in detecting these breast cancers.

About 15 years ago, Zamora-Leon et al. postulated that the relatively high-affinity fructose transporter GLUT5 expressed in human breast cancer cells could provide an interesting alternative targeting strategy for earlier diagnosis and treatment of breast cancer [27]. Recently, it has been shown that the fructose transporting Class I facilitative hexose transporter GLUT2 and the Class II facilitative hexose transporter GLUT5 are overexpressed in breast as well as other cancers [28]. The authors found that 31% of the breast tumor tissue samples studied expressed GLUT2 and 37% expressed GLUT5. It has been suggested that increased fructose metabolism may play an important role in cancer progression [29]. It has also been postulated that tumor cells can switch or supplement their nutrient pool through an increase of GLUT2 and GLUT5 expression, thus allowing a larger array of substrates to enter their metabolic pathways. However, GLUT5 overexpression in these tumors does not contribute to the utility of [18F]FDG, as it is not a substrate for GLUT5. This makes GLUT5 a promising molecular target for the PET imaging of breast cancer and other cancers by means of radiolabeled fructose derivatives.

Rational design of 18F-labeled fructose derivatives is essential because incorporation of fluorine is critical for both proper binding and trafficking across the membrane via GLUT2 and GLUT5, as well as for its intracellular metabolism. Intracellular phosphorylation of fructose occurs via two distinct enzymes: either hexokinase II at the 6-position, or ketohexokinase (fructokinase) at the 1-position. Haradahira et al. described the labeling of fructose with 18F at the 1-position to yield 1-deoxy-1-[18F]fluoro-d-fructose (1-[18F]FDF) which would be susceptible to phosphorylation by hexokinase II. 1-[18F]FDF was evaluated in fibrosarcoma tumor-bearing mice; however, no trapping of 1-[18F]FDF in the tumor was observed [30]. More recently, Levi et al. labeled fructose with small fluorophores at the 1-position and apparently showed uptake in GLUT5-expressing human breast cancer cells versus no uptake in cells lacking GLUT5 [31].

Alternatively, labeling of fructose with 18F could be performed at the 6-position. Previous work by Yang and coworkers indicated that a compound labeled at the 6-position would still be handled properly by the transporter (GLUT5) and may actually increase the compound's affinity for binding [32]. In addition to the reported labeling of position 1 of fructose with fluorine and fluorophores, we have synthesized the fructose analogue 6-deoxy-6-fluoro-d-fructose (6-FDF; [33]). Initial experiments using 6-FDF have shown its transport into two human breast cancer cell lines and dose-dependent competitive inhibition of d-fructose transport, as well as transport of 14C-labeled 6-FDF via GLUT5 in a cell culture transport model [33].

Herein we describe the synthesis and radiopharmacological evaluation of 6-[18F]fluoro-6-deoxy-d-fructose (6-[18F]FDF) as a novel radiotracer for PET imaging of GLUT5 expression. We have analyzed the in vitro transport of 6-[18F]FDF and [18F]FDG in two different breast cancer cell lines known to express GLUT5. Biodistribution and metabolism of 6-[18F]FDF was studied in wild-type BALB/c mice. Furthermore, we have studied solid tumor uptake of 6-[18F]FDF and of [18F]FDG in a murine (EMT-6) and human (MCF-7) breast tumor-bearing mouse model using dynamic small animal PET.

Section snippets

Radiotracer synthesis

6-[18F]FDF was synthesized in an automated Eckert & Ziegler Modular-Lab synthesis unit (Berlin, Germany). The synthesis of reference compound 6-FDF was accomplished in eight steps in 15% overall yield starting with readily available d-fructose [32]. This route provides the triflate labeling precursor (methyl 1,3,4-tri-O-acetyl-6-O-(trifluoromethanesulfonyl)-α/β-d-fructofuranoside) for radiofluorinations in milligram-scale quantities. Since the triflate is not stable to prolonged storage, it had

Radiotracer synthesis

The radiosynthesis of 6-[18F]FDF was performed in a remotely-controlled synthesis unit via a two-step procedure through treatment of a triflate precursor 1 with no-carrier-added potassium [18F]fluoride and kryptofix K222 in acetonitrile at 85°C. Intermediate 2 was subsequently deprotected by treatment with 2 N HCl at 110°C for 8 min (Fig. 1). [18F]Fluoride incorporation by nucleophilic displacement of the triflate leaving group in the labeling precursor 1 and subsequent acidic hydrolysis of

Discussion

The goal of the present study was to investigate the radiopharmacological profile of the fructose derivative 6-[18F]FDF in vitro and in vivo. We found that (i) uptake of 6-[18F]FDF in mouse EMT-6 as well as human MCF-7 breast tumor cells is mediated via GLUT5, (ii) 6-FDF possesses a 10-fold higher potency than fructose to inhibit 6-[18F]FDF uptake in EMT-6 cells via GLUT5, (iii) 6-[18F]FDF is rapidly cleared from the body and radioactivity is accumulated in the bladder, (iv) 6-[18F]FDF

Conclusion

6-[18F]FDF represents a novel PET radiotracer for imaging of GLUT5 expression in vivo. It is a substrate for human ketohexokinase and it is rapidly metabolized in mice. Radiopharmacological evaluation in vitro and in vivo has demonstrated radioactivity uptake in murine and human breast tumor models, indicating its potential application for molecular imaging of cells expressing GLUT5. However, after 2 h, 6-[18F]FDF showed no advantages over [18F]FDG for imaging in the two mouse models and even

Acknowledgments

The authors would like to thank John Wilson, David Clendening and Jayden Sader from the Edmonton PET Center for providing 18F made on a biomedical cyclotron and Monica Wang for assisting in the cell experiments. In addition, Dan McGinn and Gail Hipperson from the Vivarium of the Cross Cancer Institute provided expertise in animal handling. This work was supported by grants from the Canadian Breast Cancer Foundation, Canadian Institutes of Health Research and the Natural Sciences and Engineering

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