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 HA-GdDTPA-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 degrees C with peak activity at 25 degrees 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.

Figure 1

Temperature-dependent dynamics of hyaluronidase-mediated activation of HA-GdDTPA-beads. Bovine testes hyaluronidase was injected into NMR tubes that contained HA-GdDTPA-beads. Temporal changes in R₂ were determined from consecutive multi-echo images. Hyaluronidase activity was manifested by elevation in R₂. Activation of the contrast material was measured at four temperatures (7, 14, 24 and 36 °C).

Figure 2

Quantitative MRI-based dynamic analysis of enzymatic conversion of HA-GdDTPA-beads. Bovine testes hyaluronidase was injected into NMR tubes that contained HAGdDTPA-beads at 36 °C (as shown in Fig. 1). The concentrations of GdDTPA in intact contrast material, [Gd_B,t], and in HA-GdDTPA fragments, [Gd_D,t], were calculated using the difference in specific relaxivity of the two forms assuming complete degradation of the contrast material at steady state.

Figure 3

MRI analysis of the temperature dependence of hyaluronidase activity. Hyaluronidase was injected via a catheter into NMR tubes that contained HA-GdDTPA-beads. At each temperature the rate of enzymatic activity [k (min ^–1)] was calculated from the kinetics of degradation of HA-GdDTPA-beads (as shown in Fig. 2). The bell-shaped temperature dependence showed maximum enzymatic activity at 25 °C.

Figure 4

In vivo quantitative assessment of hyaluronidase activity in ovarian carcinoma tumors. HA-GdDTPA-beads were administered via a catheter to the hind limb in the vicinity of the ES-2 tumor and R₂ measurements were taken at different time points. (A) MRI images of the tumor area in different times: left, before contrast material injection; middle, time of interstitial administration of the HA-GdDTPA-beads; right, 2.4 min after injection. All the images were acquired under identical experimental conditions. (B) Corresponding R₂ maps. (C) Calculated differences in R₂ overlaid on the grays-scale images: left, difference between the relaxation rate at the time of interstitial administration of the HA-GdDTPA-beads and the relaxation rate before CM administration [ΔR = R (t = 2.4 min) – R (t = 0)]. The tumor can easily be seen as a bright spot in the center of the contrast enhancement area. (D)Left, contrast material concentration map overlaid on the gray-scale image; right, the percentage of activated contrast material. Most of the activation of the contrast material occurred at the rim of the tumor.