Kinetic analysis of hyaluronidase activity using a bioactive MRI contrast agent

Abstract

One of the attractions of molecular imaging using 'smart' bioactive contrast agents is the ability to provide non-invasive data on the spatial and temporal changes in the distribution and expression patterns of specific enzymes. The tools developed for that aim could potentially also be developed for functional imaging of enzyme activity itself, through quantitative analysis of the rapid dynamics of enzymatic conversion of these contrast agents. High molecular weight hyaluronan, the natural substrate of hyaluronidase, is a major antiangiogenic constituent of the extracellular matrix. Degradation by hyaluronidase yields low molecular weight fragments, which are proangiogenic. A novel contrast material, HA-GdDTPA-beads, was designed to provide a substrate analog of hyaluronidase in which relaxivity changes are induced by enzymatic degradation. We show here a first-order kinetic analysis of the time-dependent increase in R 2 as a result of hyaluronidase activity. The changes in R 2 and the measured relaxivity of intact HAGdDTPA-beads (r 2B ) and HA-GdDTPA fragments (r 2D ) were utilized for derivation of the temporal drop in concentration of GdDTPA in HA-GdDTPA-beads as the consequence of the release of HA-GdDTPA fragments. The rate of dissociation of HA-GdDTPA from the beads showed typical bell-shaped temperature dependence between 7 and 36 °C with peak activity at 25°C. The tools developed here for quantitative dynamic analysis of hyaluronidase activity by MRI would allow the use of activation of HA-GdDTPA-beads for the determination of the role of hyaluronidase in altering the angiogenic microenvironment of tumor micro metastases.

Detecting Enzyme Activities with Exogenous MRI Contrast Agents

“…[50] Similarly, a highly aggregated bead with hyaluronan linkers becomes less aggregated when the linkers are cleaved by hyaluronidase, leading to decreased T2* relaxation time. [51] These last examples demonstrate that careful optimization of contrast agent formulations can be critical for generating an enzyme-dependent response, because other hydrogels or beads with less initial aggregation may follow the behavior of the other agents in this classification.…”

Emerging Roles for Hyaluronidase in Cancer Metastasis and Therapy

“…Hyal1 and Hyal2 activity in the ovarian tumor cells were both found to contribute in vitro to the degradation of the HA carrier, liberating the environmentally sensitive contrast agent (Shiftan et al, 2005). The HA-GdDTPA-bead targeting agent was further found to be sufficiently sensitive to report apparent kinetics of hyaluronidase activity in the tumors, and found the initial activity was localized primarily to the peripheral tumor, possibly concentrated in peritumoral lymphatics (Shiftan and Neeman, 2006). Since hyaluronidases are elevated in the stromal space and/or at the tumor cell surface, and may be involved in the cellular uptake of HA, imaging probes that give no signal until activated by hyaluronidase cleavage offer higher levels of sensitivity.…”

Imaging aspects of the tumor stroma with therapeutic implications

“…When conjugated, this complex showed low relaxivity, while after degradation by hyal the relaxivity increased resulting in enhanced contrast (Shiftan, et al, 2005; Shiftan & Neeman, 2006). HA was conjugated to Gd-DTPA on agarose beads.…”

“…In vivo, signal significantly elevated after Hyal digestion, in the tumors injected with HA-Gd-DTPA-beads, compared to HA-Gd-DTPA-beads with no tumor (Shiftan, et al, 2005). Analysis based on in vitro studies was used to quantify the activity of Hyal in the tumor (Shiftan & Neeman, 2006). …”