Development of a Gd(III)-based receptor-induced magnetization enhancement (RIME) contrast agent for ß-glucuronidase activity profiling.

ß-Glucuronidase is a key lysosomal enzyme and is often overexpressed in necrotic tumor masses. We report here the synthesis of a pro receptor-induced magnetization enhancement (pro-RIME) magnetic resonance imaging (MRI) contrast agent ([Gd(DOTA-FPßGu)]) for molecular imaging of ß-glucuronidase activity in tumor tissues. The contrast agent consists of two parts, a gadolinium complex and a ß-glucuronidase substrate (ß-d-glucopyranuronic acid). The binding association constant (KA) of [Gd(DOTA-FPßGu)] is 7.42 × 10(2), which is significantly lower than that of a commercially available MS-325 (KA = 3.0 × 10(4)) RIME contrast agent. The low KA value of [Gd(DOTA-FPßGu)] is due to the pendant ß-d-glucopyranuronic acid moiety. Therefore, [Gd(DOTA-FPßGu)] can be used for detection of ß-glucuronidase through RIME modulation. The detail mechanism of enzymatic activation of [Gd(DOTA-FPßGu)] was elucidated by LC-MS. The kinetics of ß-glucuronidase catalyzed hydrolysis of [Eu(DOTA-FPßGu)] at pH 7.4 best fit the Miechalis-Menten kinetic mode with Km = 1.38 mM, kcat = 3.76 × 10(3), and kcat/Km = 2.72 × 10(3) M(-1) s(-1). The low Km value indicates high affinity of ß-glucuronidase for [Gd(DOTA-FPßGu)] at physiological pH. Relaxometric studies revealed that T1 relaxivity of [Gd(DOTA-FPßGu)] changes in response to the concentration of ß-glucuronidase. Consistent with the relaxometric studies, [Gd(DOTA-FPßGu)] showed significant change in MR image signal in the presence of ß-glucuronidase and HSA. In vitro and in vivo MR images demonstrated appreciable differences in signal enhancement in the cell lines and tumor xenografts in accordance to their expression levels of ß-glucuronidase.

Synthesis and characterization of a new bioactivated paramagnetic gadolinium(III) complex [Gd(DOTA-FPG)(H2O)] for tracing gene expression.

A smart contrast agent for magnetic resonance imaging (MRI) can be used to exploit an enzymatic activity specific to the tissue or disease state signified by converting an MRI-inactivated agent to an activated MRI agent. In this study, a beta-galactopyranose-containing gadolinium(III) complex [Gd(DOTA-FPG)(H 2O)] was designed, synthesized, and characterized as being potentially suitable for a bioactivated MRI contrast agent. The (17)O NMR experiments were conducted to estimate the water exchange rate k e x 298 and rotational correlation time tau R 298 . The k ex 298 value of [Gd(DOTA-FPG)(H 2O)] is similar to that of [Gd(DO3A-bz-NO 2)(H 2O)]. The rotational correlation time value of [Gd(DOTA-FPG)(H 2O)] is dramatically longer than that of [Gd(DOTA)(H 2O)] (-) Relaxometric studies show that the percentage change in the T 1 value of [Gd(DOTA-FPG)(H 2O)] decreases dramatically in the presence of beta-galactosidase and human serum albumin. The T(1) change percentage of [Gd(DOTA-FPG)(H 2O)] (60%) is significantly higher than those of Egad and gadolinium(III)-1-(4-(2-(1-(4,7,10-triscarboxymethyl-(1,4,7,10-tetraazacyclododecyl)))-ethylcarbamoyloxymethyl)-2-nitrophenyl)-beta- d-glucopyronuronate. The signal intensity of the MR image for [Gd(DOTA-FPG)(H 2O)] in the presence of human serum albumin and beta-galactosidase (2670 +/- 210) is significantly higher than that of [Gd(DOTA-FPG)(H 2O)] in the sodium phosphate buffer solution (1490 +/- 160). In addition, the MR images show a higher-intensity enhancement in CT26/beta-gal tumor with beta-galactosidase gene expression but not for the CT26 tumor without beta-galactosidase gene expression. We conclude that [Gd(DOTA-FPG)(H 2O)] is a suitable candidate for a bioactivated MRI contrast agent in tracing gene expression.