Progesterone-targeted magnetic resonance imaging probes

Abstract

Determination of progesterone receptor (PR) status in hormone-dependent diseases is essential in ascertaining disease prognosis and monitoring treatment response. The development of a noninvasive means of monitoring these processes would have significant impact on early detection, cost, repeated measurements, and personalized treatment options. Magnetic resonance imaging (MRI) is widely recognized as a technique that can produce longitudinal studies, and PR-targeted MR probes may address a clinical problem by providing contrast enhancement that reports on PR status without biopsy. Commercially available MR contrast agents are typically delivered via intravenous injection, whereas steroids are administered subcutaneously. Whether the route of delivery is important for tissue accumulation of steroid-modified MRI contrast agents to PR-rich tissues is not known. To address this question, modification of the chemistry linking progesterone with the gadolinium chelate led to MR probes with increased water solubility and lower cellular toxicity and enabled administration through the blood. This attribute came at a cost through lower affinity for PR and decreased ability to cross the cell membrane, and ultimately it did not improve delivery of the PR-targeted MR probe to PR-rich tissues or tumors in vivo. Overall, these studies are important, as they demonstrate that targeted contrast agents require optimization of delivery and receptor binding of the steroid and the gadolinium chelate for optimal translation in vivo.

Figure 1

Structures of PR-targeted contrast probes, 1–4. The alkane linker between the PR-targeting moiety and Gd(III) chelate was modified to elucidate the effect of linker length on toxicity, receptor binding, and tissue distribution in vivo.

Figure 2

Relative binding affinity of complexes 1–4, compared with an unmodified progesterone control (P4), to progesterone receptor. As the linker length increases, the binding affinity of the probe is improved. Error bars indicate ±SEM.

Figure 3

Cytotoxicity of complexes 1–4 as obtained through MTS assay on the PR(+) T47D cell line. Complex 1 is the least toxic, followed by 2, 3, and 4. Error bars indicate ±SEM.

Figure 4

Complexes accumulate preferentially in cells that express PR. Time-dependent uptake experiments were performed in PR(−) MDA-MB-231 cells (A) and PR(+) T47D cells (B). Error bars indicate ±SEM. Statistical difference determined using two-way ANOVA test. * p < 0.05.

Figure 5

Complexes retain the ability to activate PR after chemical modification: the addition of the Gd(III) chelate to the PR-targeting moiety. Incubation with complexes resulted in transcriptional activation of the luciferase reporter gene (A). Transcription of an endogenous PR-inducible gene was monitored (B). Complex 4 was the most potent in both assays. Error bars indicate ±SEM. Statistical differences as indicated by asterisks measured by one-way ANOVA test. ** p < 0.05, * p < 0.05 (when compared to samples treated with only DMSO).

Figure 6

Complex 1 accumulates in tissues that have high concentrations of PR such as the uterus and ovary. 1 was injected either i.v. or i.p. into CD-1 female mice, and organs were harvested after 6 (A) or 24 (B) h. Data is presented normalized to saline ICP values. Error bars indicate ±SEM. Statistical differences determined through two-way ANOVA test. *** p < 0.0001, ** p < 0.001, * p < 0.05.

Figure 7

In vivo imaging at 9.4 T after injection of complex 1 i.p. (A) MR image before injection, on the left, and 6 h after injection, on the right. PR(+) and PR(−) tumors are indicated by arrows. Positive enhancement is observed in the tumors. (B) Gd(III) concentration in the tissues harvested as quantified by ICP-MS. Gd(III) concentration was not significantly different between the tumor types.