Cypate and Cypate-Glucosamine as Near-Infrared Fluorescent Probes for In Vivo Tumor Imaging

Near infrared (NIR) imaging is a rapidly developing technique with promising use as a non-invasive, specific and sensitive diagnostic tool. While the NIR fluorescently labeled glucose analog, glucosamine (cypate-glucosamine), has been used for imaging purposes, the transport pathways and fate of this probe in tissues remain unaddressed. Here, we synthesized and characterized cypate and cypate-glucosamine conjugate (cy-2-glu), and investigated the probable transport pathways of these probes in vitro and in vivo. We compared their uptake in presence and absence of excess D-glucose, 9saturated cypate9 and palmitic acid in two normal - cancer cell line pairs: lung cancer (A549) - normal (MRC9), and prostate cancer (DU145) - normal (BPH). Breast cancer (MDA-MB-231) and liver cancer (HepG2) cell lines were also examined. Results support potential utilization of the glucose transport pathway by cy-2-glu and fatty acid transport pathway by cypate. Mass spectrometry data on the in vitro extracts revealed deamidation of cy-2-glu in prostate and liver cells, indicating release of glucosamine. In vivo biodistribution studies in mice engrafted with breast tumors showed a distinct accumulation of cy-2-glu in liver and tumors, and to a lesser extent in kidneys and spleen. A negligible accumulation of cypate alone in tumors was observed. Analysis of urine extracts revealed renal excretion of the cy-2-glu probe in the form of free cypate, suggesting deamidation of cy-2-glu in tissues. Thus, the investigation of metabolic pathways taken by NIR probes, opens new avenues for the detection and monitoring of the progression and regression of tumors in preclinical animal studies.