Gadolinium-tetraazacyclododecane tetraacetic acid coupled with folate bis(aminoethyl)ethylene glycol linker

Gadolinium-tetraazacyclododecane tetraacetic acid (Gd.DOTA) coupled with folate the bis(aminoethyl)ethylene glycol linker, abbreviated as Gd.DOTA. Folate, was synthesized by Kalber et al. for folate receptor (FR)-targeted magnetic resonance imaging (MRI) of FR-positive tumors (1). Folate (folic acid) is an essential vitamin for cell synthesis of nucleotide bases. Cellular uptake of the folate is mediated either by membrane transport proteins or by FR. The human FR has three isoforms: FR-α-isoform, FR-β-isoform, and FR-γ-isoform. The FR-α isoform is expressed on the apical (luminal) surface of epithelial cells in limited tissues, and because of no vascular supply, it is inaccessible to exogenous folate conjugates with the exception of the FR-α isoforms on the proximal tubules of kidneys (1-3). The FR-β isoform is observed mainly on activated macrophages, whereas the FR-γ isoform is rarely detected in human tissues (1, 4). Several features of FR make it a valuable target for developing FR-targeted imaging and therapeutic agents (2, 3, 5). First, FR has a high affinity for the exogenous folate conjugates ( = ~100 pM), and the conjugates do not bind to most normal cells. Second, the FR-α isoform is overexpressed in ~40% of human cancer tissues, where it is completely accessible to folate conjugates. Third, folate conjugates are taken up by cancer cells FR-mediated endocytosis, and FR recycles actively to the cell surface with a frequency of 5.7–20 h, depending on cell types. Fourth, conjugation of imaging labels the γ-carboxyl group of folate has no apparent effects on the ligand-binding affinity to FR. Furthermore, folate is a small molecule (MW = ~441) and exhibits rapid and complete penetration of solid tumors and rapid clearance from FR-negative tissues ( = <10 min). However, the MRI contrast can be limited by the relatively low density of FR on the tumor cell surface (1–3 million FR/cell) and by the rapid saturation of binding (2, 3). Nevertheless, most studies demonstrate that cellular uptake of the Gd- or iron oxide-folate conjugates in FR-positive tumors is much higher than in FR-negative tumors. Normal organs except for the kidneys have minimal uptake (1, 2, 6). Kalber et al. synthesized the novel compound Gd.DOTA.Folate by coupling folate to Gd.DOTA bis(aminoethyl)ethylene glycol, a very short linker (1). MRI with Gd.DOTA.Folate in tumor-bearing mice showed an increase in with clear enhancement on the MRI images in a human ovarian carcinoma (IGROV-1) xenograft overexpressing the FR-α isoform, but no change was observed in a FR-α–negative human ovarian carcinoma (OVCAR-3) xenograft. However, contrast enhancement with Gd.DOTA.Folate was not observed in either IGROV-1 cells or OVCAR-3 cells (i.e., the MRI data did not support the findings (1)). A similar discrepancy has been reported in other studies when comparing and results with FR-targeted agents (7, 8). Studies with Gd-folate dendrimer and P866 low molecular weight dimeric Gd.DOTA indicate that the discrepancy may be caused by the potential sensitivity limitation , perhaps due to the marked cellular variability in FR expression (5, 8). Miotti et al. have shown that the degree of internalized folate is not completely proportional to the degree of expression for the FR-α isoform because cell lines with low expression of the FR-α isoform are still capable of low levels of uptake (7). This appears to be supported by the studies by Kalber et al. in that, although the FR-α–positive IGROV-1 cells had a much higher degree of Gd.DOTA.Folate internalization than did the FR-α–negative OVCAR-3 cells, there was still a degree of uptake within the OVCAR-3 cells as measured with inductively coupled mass spectrometry (ICP-MS) (1).