Cypate-2-Deoxy-d-glucose

Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets in small animals (1-3). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–650 nm) are used. Near-infrared (NIR) fluorescence (650–900 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a noninvasive complement to radionuclide imaging in small animals or with probes in close proximity to the target in humans (4). Among the various optical imaging agents, only indocyanine green (ICG), with NIR fluorescence absorption at 780 nm and emission at 820 nm, is approved by the United States Food and Drug Administration for clinical applications in angiography, blood flow evaluation, and liver function assessment (5-8). It is also under evaluation in several clinical trials for other applications, such as optical imaging and mapping of both the lymphatic vessels and lymph nodes in cancer patients for surgical dissection of tumor cells and endoscopic imaging of the pancreas and colon. The phosphorylation of glucose, an initial and important step in cellular metabolism, is catalyzed by hexokinases (HKs) (9). There are four HKs in mammalian tissues (HKI–HKIV). HKI, HKII, and HKIII have molecular weights of ~100,000 each; HKI is found mainly in the brain, and HKII is insulin-sensitive and is found in adipose and muscle cells. HKIV, also known as glucokinase, has a molecular weight of ~50,000 and is specific to the liver and pancreas. Most brain HK is bound to mitochondria, enabling coordination between glucose consumption and oxidation. Tumor cells are known to be highly glycolytic because of increased expression of glycolytic enzymes and HK activity (10), which was detected in tumors from patients with lung, gastrointestinal, and breast cancers. The HKs, by converting glucose to glucose-6-phosphate, help maintain the downhill gradient that results in the transport of glucose into cells through the facilitative glucose transporters (GLUT1–13) (11). GLUT1 is considered to be the main transporter of glucose uptake. GLUT4 and HKII are the major transporter and HK isoform in skeletal muscle, heart, and adipose tissue, wherein insulin promotes glucose utilization. HKIV is associated with GLUT2 in liver and pancreatic β cells. 2-Deoxy-d-glucose (2DG) was first developed to inhibit glucose utilization by cancer cells (12). HKs phosphorylate 2DG to 2DG-6-phosphate, which inhibits phosphorylation of glucose. 2-[F]Fluoro-2-deoxy-d-glucose ([F]FDG) was later developed for molecular imaging studies (13). FDG is moved into cells by glucose transporters, where it is phosphorylated by HK to FDG-6-phosphate. FDG-6-phosphate cannot be metabolized further in the glycolytic pathway and remains in the cells. Tumor cells do not contain a sufficient amount of glucose-6-phosphatase to reverse the phosphorylation. The elevated rates of glycolysis and glucose transport in many types of tumor cells and activated cells enhance the uptake of FDG in these cells relative to other normal cells. Positron emission tomography (PET) with [F]FDG has been used to assess alterations in glucose metabolism in brain, cancer, cardiovascular diseases, Alzheimer’s disease and other central nervous system disorders, and infectious, autoimmune, and inflammatory diseases (14-19). Various NIR dyes (such as cypate, Cy5.5, and IRDye800CW) were conjugated to 2DG (20-22) as optical imaging agents for imaging of glucose utilization in tumors in mice. Cypate is a reactive carbocyanine dye, which is derived from indocyanine green (ICG) (23). Cypate exhibits an absorbance maximum at 778 nm and an emission maximum at 805 nm, with a high extinction coefficient of 224,000 M−cm−. Cypate is a lipophilic dye and contains two carboxyl functional groups for covalent conjugation to the amino group of biomolecules. Guo et al. (24) evaluated cypate-2DG for NIR optical imaging in tumor-bearing mice.