Imaging agents for imaging protease activity and uses thereof
a protease activity and imaging technology, applied in the field of imaging agents for protease activity, can solve the problems of inability to fully investigate the drug candidate generated during in vitro screening, limited in vitro applications, inherent instability, etc., and achieve the effect of fast action and enhanced target-to-background ratio
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example 1
[0141]This example demonstrates the synthesis of a series of PEGylated imaging agent embodiments.
[0142]Synthesis of the agents began by preparing MMP activatable imaging agents without PEG, termed MMP-P0. The MMP-P0 consisted of Cy5.5 and BHQ-3 as a near-infrared dye / dark quencher pair, Cy5.5-GPLGVRGK(BHQ-3)GG (SEQ ID NO: 10). Next, PEGs of different sizes were conjugated to the C-termini of the MMP-P0 (MMP-Pn, where “n” represents the number of repeating ethylene glycol units (—CH2CH2O—) (Table 1 and FIGS. 1A and 1B). MMP-13 was used as a model MMP for in vitro screening since the imaging agent MMP-P0 showed highest specificity against MMP-13 (FIG. 2A). The activity of each MMP-Pn (n=0, 4, 12, 24, and 67) imaging agent was tested by incubating 15 nM of each agent in a 96-well microplate containing the reaction buffer and 40 nM of activated MMP-13, with and without a broad-spectrum hydroxamate-type MMP inhibitor (MMP-I). Fluorescence spectrometry clearly demonstrated that the imagin...
example 2
[0143]This example demonstrates that MMP-Pn imaging agents of the present invention can improve the visualization of overexpressed MMPs in vivo.
[0144]The MMP-Pns and MMPSense 680™ were administered intravenously into separate MMP-positive SCC-7 tumor-bearing mice. In vivo imaging was performed for 24 hours using a small-animal imaging system.
[0145]The inventors surprisingly found that modification of MMP-Pn imaging agents with small MW PEG showed significantly reduced activation time (ultrafast-acting and extended-use) in vivo. FIG. 3 shows representative serial images of mice at selected time points. In contrast to the in vitro activity, MMP-P4 and MMP-P12 clearly showed early onset of activation in vivo and provided high NIR fluorescence signals in the MMP-positive tumor region for a longer time compared to other MMP-Pns and MMPSense 680™. The activation of fluorescence signals in the tumor region was enhanced when the MW of the PEG moiety was increased up to 500 Da, and gradually...
example 3
[0147]This example demonstrates the ability to create video imaging of MMP activity in vivo using the imaging agents of the present invention.
[0148]The MMP-Pns imaging agents used in Example 2, were then used to create a video image of the activity of MMPs in vivo. To inhibit the activity of MMPs in SCC-7 tumor-bearing mice, MMP-I was intratumorally injected 30 minutes before the probe injection. MMP-P12 was injected into mice either directly or following MMP-I treatment, using a tail vein catheter during continuous imaging procedures. The animals were imaged every 10 to 20 seconds for 1 hour and video images were generated by using imaging software (DyCE, CRI, Woburn, Mass.). FIG. 5A shows a typical series of video images. The MMP-P12 generated strong NIR fluorescence signals as early as 20 to 30 minutes after the probe injection and enabled clear visualization of MMPs in the tumor region thereafter. In contrast, the overall NIR fluorescent signals were significantly decreased when...
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Abstract
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