Detection of in vivo enzyme activity with CatalyCEST MRI

Abstract

Purpose: CatalyCEST MRI compares the detection of an enzyme-responsive chemical exchange saturation transfer (CEST) agent with the detection of an unresponsive "control" CEST agent that accounts for other conditions that influence CEST. The purpose of this study was to investigate the feasibility of in vivo catalyCEST MRI.

Methods: CEST agents that were responsive and unresponsive to the activity of urokinase plasminogen activator were shown to have negligible interaction with each other. A CEST-fast imaging with steady state precession (FISP) MRI protocol was used to acquire MR CEST spectroscopic images with a Capan-2 pancreatic tumor model after intravenous injection of the CEST agents. A function of (super)-Lorentzian line shapes was fit to CEST spectra of a region-of-interest that represented the tumor.

Results: The CEST effects from each agent showed the same initial uptake into tumor tissues, indicating that both agents had the same pharmacokinetic transport rates. Starting 5 min after injection, CEST from the enzyme-responsive agent disappeared more quickly than CEST from the unresponsive agent, indicating that the enzyme responsive agent was being catalyzed by urokinase plasminogen activator, while both agents also experienced net pharmacokinetic washout from the tumor.

Conclusion: CatalyCEST MRI demonstrates that dynamic tracking of enzyme-responsive and unresponsive CEST agents during the same in vivo MRI study is feasible.

Figure 1

Chemical structures of imaging contrast agents. a) The ZGGR peptide ligand is cleaved from the 7-amino-4-methylcoumarin (AMC) by urokinase Plasminogen Activator (uPA). The ligand-free AMC has bright fluorescence, which can be temporally monitored to detect uPA enzyme activity. b) ZGGR-α-amino-(Tm-DOTA) has the same design as ZGGR-AMC for detecting uPA activity. A water molecule fills the ninth coordinate site of the Tm-DOTA chelate. However, the CEST effect at −54 ppm from this agent originates from the amide nearest the Tm(III) ion. c) Eu-DOTA-Gly₄ does not contain a ZGGR peptide and is therefore unresponsive to uPA activity. A water molecule fills the ninth coordinate site of the Eu-DOTA chelate. The CEST effect at +54 ppm from this agent originates from the bound water molecule.

Figure 2

Solution-state CEST MRI of two PARACEST agents. a) The horizontal reaction shows MR saturation of the amide proton of ZGGR-α-amino-(Tm-DOTA) (gray), followed by chemical exchange with water, which transfers the MR saturation to water and decreases the water signal. The vertical reaction shows cleavage of the ZGGR peptide from α-amino-(Tm-DOTA) by uPA, which converts an amide to an amine that does not generate CEST. b) ZGGR-α-amino-(Tm-DOTA) showed CEST with MR saturation at −54.1 ppm before adding uPA (black) but not after adding uPA (gray). A control agent, Eu-DOTA-Gly₄, showed no change in CEST at +53.9 ppm.

Figure 3

Quantitative evaluation of the concentration dependence of CEST for each agent. a) Serially-diluted chemical solutions, with each solution containing both contrast agent at the same concentration, were used to acquire 31 CEST-FISP MR images with selective saturation ranging from +200 to −200 ppm. Only 7 of the 31 images are shown. b) A function of three Lorentzian line shapes was fit to each CEST spectrum to measure each CEST effect of ZGGR-α-amino-(Tm-DOTA) (red) and Eu-DOTA-Gly₄ (blue). Fitting residuals are shown as triangles with a dashed line. The residuals had a standard deviation ranging from 1.1% to 2.5% water signal for each fitting, which attested to the excellent quality of the fittings. c) The CEST results and concentrations were fit with a Hanes-Woolf-like linear analysis method based on a two-pool model of chemical exchange between only one agent and water. The excellent fitting indicated that each CEST effect can be approximated as a two-pool model, so that the direct interaction of the two CEST agents can be considered to be negligible. d) Based on results from the Hanes-like analysis method, the CEST effects of each PARACEST agent showed a typical correlation with concentration.

Figure 4

Development of a pancreatic tumor model with high uPA activity. a) Total uPa activity was analyzed using the PAI Activity Assay Kit (ECM610 Kit, Millipore, Inc.) and a Synergy 2 microplate reader (Biotek Instruments Inc., Winooski, VA). Paired media samples without chromogenic substrate added were used for subtracting background absorbance. b) To ensure that enzyme concentration measured by this assay quantified enzyme activity, the initial rate of substrate cleavage was monitored as a function of substrate concentration and the results were analyzed using a Lineweaver-Burk plot. c) uPA activity was measured following two hours of incubation of the substrate with each cell type during in vitro studies conducted in normoxic and hypoxic conditions. The amount of uPa activity was normalized to total protein in each sample. Error bars represent the standard deviation of six trials for each cell type. d) The uPA activity was measured in blood plasma and homogenized tumor tissue. The amount of uPa activity in the samples was normalized to total protein in each sample. Error bars represent the standard deviation of results from four mice.

Figure 5

In vivo CEST MRI of two PARACEST agents. a) A CEST image and CEST maps obtained at 3.6 minutes post-injection showed selective detection of each agent in the tumor and bladder. b) CEST spectra of the tumor were fit with Lorentzian and super-Lorentzian line shapes. The two Lorentzian line shapes corresponding to the CEST effects of the two agents are shown. c) CEST from each agent was quantified from each Lorentzian line shape. Residuals of the line shape fitting are shown as a dashed line.

Figure 6

CatalyCEST MRI. a) A function of three Lorentzian line shapes and one super-Lorentzian line shape was fit to each CEST spectrum (circles). The Lorentzian line shapes corresponding to ZGGR-α-amino-(Tm-DOTA) and Eu-DOTA-Gly₄ are shown, the sum of the four (super)-Lorentzian line shapes are also shown, and the sum of the super-Lorentzian line shape and the Lorentzian line shape of the direct saturation of water are also shown. b) Each agent showed the same initial tumor uptake rates, c) but the rates of CEST disappearance from the tumor were different for each agent. The faster CEST disappearance from ZGGR-α-amino-(Tm-DOTA) was attributed to uPA activity. d) The agents had the same pharmacokinetics in the bladder and muscle.