1--(4-(2-[F]Fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)--β-d-glucopyronuronate

The β-glucuronidase (β-GUS; EC 3.2.1.31) is a lysosomal enzyme that is known to activate prodrugs (PDs) for the treatment of cancer. β-GUS has been used to track the path of gene delivery vehicles, and there is evidence that it can be used as a tumor marker (1). The primary function of the enzyme is to catalyze the hydrolysis of β-glucuronic acid residues from the cell-surface glycosaminoglycans for normal restructuring of the extracellular matrix (ECM) (2), and the enzyme is believed to participate in the processes of angiogenesis, cancer metastasis, and inflammation (3). Normal tissues have low levels of β-GUS in the ECM, but tissues under pathological stress, such as bacterial infection, fibrosis, and malignancy, show elevated levels of the enzyme (4). Intracellular β-GUS is released from necrotic cells, and its activity in these lesions has been used to activate anti-cancer PDs to treat cancers (2). Chemotherapeutic anti-cancer drugs are generally nonselective and toxic to healthy cells; thus, they are of limited efficacy to the patient due to their side effects. The conversion of a toxic drug into a non-toxic PD that can be activated only under specific conditions (e.g., enzyme catalysis or chemical hydrolysis) would facilitate drug activation only in tissues that provide the specialized microenvironment and improve its concentration and efficacy at the desired location in the body (5, 6). For example, glucuronide PDs (drugs that are linked to a glucuronic acid moiety with or without a linker) have been shown to have superior anti-tumor activity compared with the parent drugs because the activated drugs are released from the PDs by the β-GUS activity in a site-specific manner (7, 8). β-GUS activity varies among individuals, and its expression or accumulation in tumor tissues may change depending on the location in the body or the type of neoplasm (2, 9). Fluorescent or bioluminescent substrates were developed to determine the expression of β-GUS with optical imaging in various tissues of mice (1). However, this imaging modality is suitable for the detection of fluorescence or bioluminescence signals generated only in the superficial tissues of small animals such as rodents; the low depth of light penetration in tissues is a limitation for its application in large animals and humans (2, 4, 10). Imaging modalities such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), which use radionuclides to generate tracer signals, can be used to detect and determine the activity of enzymes such as the β-GUS because signals generated by radiolabeled probes can be detected even in deep tissues of the body (11). In general, PET imaging has a higher sensitivity than SPECT and has been used to investigate drug kinetics in preclinical and clinical settings (11). A I-labeled phenolphthalein glucuronide PD probe ([I]-PTH-G) was developed and evaluated with microPET for the detection of xenograft tumors that express β-GUS in mice (9). Although [I]-PTH-G was suitable for the detection of tumors in the rodents, biodistribution studies of the tracer in these animals revealed that, even at 20 h postinjection (p.i.), higher levels of the label could be detected in the liver, gallbladder, stomach, and intestines than in the tumors. Therefore, the investigators concluded that [I]-PTH-G is probably unsuitable for the imaging of tumors that express β-GUS. Antunes et al. synthesized 1--(4-(2-[F]fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)--β-d-glucopyronuronate ([F]-FEAnGA) as a PD in an effort to develop a probe that could be used with PET to detect and visualize β-GUS activity in tissues (4). The mechanism of or activation of [F]-FEAnGA is described elsewhere (4). Briefly, the hydrolysis of [F]-FEAnGA by β-GUS results in the production of glucuronic acid, 4-hydroxy-3-nitrobenzyl alcohol (HNBA; this is the spacer in the intact FEAnGA molecule, and the concentration of HNBA in the reaction mixture can be measured with ultraviolet (UV) spectroscopy at 402 nm after the FEAnGA has been hydrolysed), and 2-[F]fluoroethylamine ([F]-FEA). [F]-FEA subsequently accumulates in the cells (attributed to passive diffusion into the cells) and is detected with PET imaging. [F]-FEAnGA has been evaluated for the detection of tumors that expressed β-GUS (2, 4) and inflammation (2, 10) in mice.