MMPSense on the ASK


Overview


Matrix metalloproteinases (MMPs) play a central role in tumor growth, vascularization and metastasis. MMPs are a family of zinc containing endopeptidases known to degrade extracellular matrix proteins such as collagens, gelatins, fibronectin, and laminin. MMPs can be used as markers of cancer progression. MMP activity is involved in many disease-related processes including cancer progression, invasion and metastasis, rheumatoid arthritis, pulmonary diseases and areas of cardiovascular disease.

MMPSense agents are matrix metalloproteinase (MMP) activatable probes that are optically silent upon injection and produce fluorescent signals after cleavage by disease related MMPs. They are members of a family of activatable fluorescent imaging agents comprising a novel architecture termed F.A.S.T. (Fluorescent Activatable Sensor Technology) which confer an improved pharmacokinetic profile with earlier imaging time points. They may be used to monitor the progression of disease or to evaluate the potential therapeutic efficacy of drugs targeting the underlying mechanisms involved in a particular disease. Activation can occur by a broad range of MMPs including MMP 2, 3, 7, 9, 12, and 13.

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Figure 1: Schematic of MMPSense agents. Two quenched fluorophores are separated by a cleavable linker in the native state. MMPs recognize the cleavable linker and once the agent is cleaved, the fluorophores produce signal. The agents also contain a pharmacokinetic modifier (PKM) selected to provide optimal attributes for in vivo imaging.

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Figure 2: MMPSense 750 FAST (0.5 μM final concentration) was cleaved in the presence of 0.05-0.1 μM activated MMPs, Cathepsins B, D or urokinase-type plasminogen activator (uPA). These reactions were carried out in optimal buffers, pH and temperature. The fluorescence was read using a fluorescence microplate reader at 5 and 24 hours. The fold activation was measured by dividing the fluorescence released after cleavage with the enzyme by the fluorescence of the probe only. The control (VM281) is not significantly cleaved by any enzyme.


Products and catalog numbers


ProductCatalog NumberEx/Em wavelength (nm)Molecular weight (g/mol)Validated ExperimentsApplicationsStorage and Stability
MMPSense 645 FASTNEV10100649/66643,000In vivo/Ex vivoOncology
Pulmonary
Cardiovascular
Technical Data Sheet
MMPSense 680NEV10126680/700450,000In vivo/Ex vivoOncology
Pulmonary
Cardiovascular
Technical Data Sheet
MMPSense 750 FASTNEV10168749/77543,000In vivo/Ex vivoOncology
Pulmonary
Cardiovascular
Technical Data Sheet


Using MMPSense in vivo/ex vivo 


  • MMPSense 645 is administration via intravenous injection and imaging 6-72 hours post injection. MMPSense 645 will clear from tissues after approximately 96 hours. Repeat injection and imaging may be performed every four days for longitudinal studies. It is recommended that a pre-injection baseline image be taken prior to reinjection and imaging. MMPSense 645 enables imaging of tumors and inflammation in a broad range of oncology, pulmonary and cardiovascular applications.
  • MMPSense 680 is administration via tail vein injection and imaging 24 hours post tailvein injection. MMPSense 680 can be used as a marker for disease progression in animal tumor models.
  • MMPSense 750 FAST is administration via intravenous injection and imaging 6 hours post injection. MMPSense 750 FAST will clear from tissues after approximately 96 hours. Repeat injection and imaging may be performed every four days for longitudinal studies. It is recommended that a pre-injection baseline image be taken prior to reinjection and imaging. MMPSense 750 FAST enables imaging of tumors and inflammation in a broad range of oncology, pulmonary and cardiovascular applications
MMPSenseRoute of InjectionMouse Dose (25 g)Rat Dose (250 g)Blood t 1/2Tissue t 1/2Optimal imaging timeOptimal Re-injection Time (complete clearance)Route of Metabolism/ background tissueFMT and IVIS settings
MMPSense 645 FASTIV4 nmol12-40 nmol5 h72 h24 h (6-24)6-7 dLiver > kidneysFMT 635/655
IVIS 640/660 with spectral unmixing
MMPSense 680IV2 nmol6-20 nmol5 h72 h24 h (24-36)6-7 dLiverFMT 680/700
IVIS 675/720
MMPSense 750 FASTIV2 nmol6-20 nmol5 h72 h24 h (12-24)6-7 dLiver > kidneysFMT 750/770
IVIS 745/800

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Figure 3: A) The pharmacokinetics is shown for MMPSense 750 FAST. BALB/c mice were injected intravenously with 2 nmoles of MMPSense 750 FAST and blood was drawn at various times. The plasma was obtained by centrifugation and the concentration of the agent was assessed using a competitive ELISA. As shown, MMPSense 750 FAST exhibits a plasma half-life of approximately 5 hours. B) The biodistribution of MMPSense 750 FAST is shown. Nu/Nu mice were injected subcutaneously in the mammary fat pads with human colorectal adenocarcinoma HT-29 tumor cells. Once tumors reached the desired volume, mice were injected intravenously with3 nmoles of MMPSense 750 FAST and sacrificed at 6 and 24 hours later. The organs were excised and imaged on the FMT 2500 system using the reflectance mode. Regions of interest (ROI) were drawn around each organ using the FMT software and the mean fluorescence (Counts/Energy) determined. This graph shows the mean fluorescence + SEM. The insert shows a representative image of the fluorescence detected in different organs at 6 hours after probe injection. The star (*) shows the tumor. As shown, the MMPSense 750 FAST preferentially localizes to tumors.

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Figure 4: Monitoring tumor growth. Nu/Nu mice were injected subcutaneously bilaterally in the mammary fat pads with mouse breast carcinoma 4T-1 cells. One week later, mice were injected intravenously with 2 nmoles of MMPSense 750 FAST and imaged 4-168 hours later by FMT quantitative tomography. Control mice were injected with 2 nmoles of VM281 (the control). In vivo imaging was performed using FMT 2500 under gas anesthesia. A) Representative volume rendering projections taken at the same color gating from mice injected with MMPSense 750 FAST and imaged at different time points thereafter, or a mouse injected with VM281 (the control) and imaged 6 hours later. B) The total amount of fluorescence (pmol) was quantified in specific ROIs for each tumor. Maximum signal was detected between 6-24 hours post agent injection.

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Figure 5: Mice were injected subcutaneously bilaterally on the chest with mouse breast carcinoma 4T-1 cells. One week later, mice were injected intravenously with 2 nmoles of MMPSense 750 FAST and imaged 4 -168 hours later by the FMT 2500. Control mice were injected with 2 nmoles of VM281 (as an inactivatable negative control using D-amino acids for the peptide sequence). In vivo imaging was performed using the FMT 2500 under gas anesthesia. A) Representative volume rendering projections taken at the same color gating. B) The total amount of fluorescence (pmol) was quantified in specific ROIs for each tumor. Maximum signal was detected between 6 - 24 hours post agent injection.

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Figure 6: Ex Vivo Imaging. Immediately following the FMT quantitative tomography imaging session (Fig 5), mice were sacrificed and tumors excised and snap-frozen in OCT for fluorescence microscopy. The distribution of NIR fluorescence was determined using a fluorescence microscope. Digital images were captured using appropriate filters for DAPI, and the near-infrared agent. The distribution of MMPSense 750 FASTis shown in red and the nuclei are counterstained with DAPI (shown in blue). Note that MMPSense 750 FAST localizes predominantly in the tumor margins.

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Figure 7: Anti-Matrix-Metalloproteinase Treatment: MMPSense 750 FAST can be used as an in vivo mechanistic biomarker of therapeutic efficacy in oncology. HT-29 tumor-bearing mice were randomized into 3 groups: 1) Doxycyline (Dox), 2) N-Acetyl-Cysteine (NAC) + pan-MMP Inhibitor (pan-MMPi) or 3) Vehicle. Mice in the Dox group received 1 mg Doxycyline/mouse subcutaneously, mice in NAC + pan-MMPi group received 3.2 g NAC/240 ml drinking water + 5 mg pan-MMPi/mouse intraperitoneally, and mice in the Vehicle group received only PBS. The following day, mice received the same treatment and were injected intravenously with 2 nmoles of MMPSense 750 FAST. The mice were imaged using FMT quantitative tomography 6 hours later. (Upper left panel) Schematic representation of the experimental protocol. (Upper right panel) Quantification of the tumor fluorescence in pmoles using FMT showing a significant decrease in MMPSense 750 FAST signal in the NAC+pan-MMPi and Dox groups (88% and 70%, respectively). (Lower panel) Representative volume rendering projections of a mouse having received Vehicle, a mouse treated with NAC + pan-MMPi and a mouse with Dox treatment.


Citations


Please visit our Citations Library for references using MMPSense on the IVIS or on the FMT.