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Cat K FAST on ASK


Overview


Cathepsin K (Cat K) is a lysosomal cysteine protease expressed in osteoclasts, chondrocytes, and synovial fibroblasts and is involved in bone resorption and collagen degradation. Women with osteoporosis, breast cancer and bone metastases have high levels of Cat K and therefore, Cat K expression is a good marker for these diseases. In addition, researchers have been looking for inhibitors of Cat K for potential therapeutics. Cat K is also expressed by a broad range of other cell types including ovarian cells, colonic tissue, alveolar and bronchial epithelial cells, synovial fibroblasts, and macrophages.

Cat K 680 FAST was developed as an imaging tool for monitoring and quantifying Cat K activity. Cat K 680 FAST is an activatable agent that is optically silent upon injection and only produces fluorescent signal after cleavage by Cathepsin K. Cat K 680 FAST contains a human Cat K-cleavable sequence with a pair of self-quenching NIRF fluorochomes. In addition, a pharmacokinetic modifier was added to confer increased blood half-life. Disease status and progression can easily be monitored in vivo with this non-invasive agent. Cat K 680 FAST offers higher target specific signal with reduced background while also reducing the optimal imaging time after injection.

catkfastfig1.jpg
Figure 1: Cat K 680 FAST is comprised of a Cat K-specific peptide sequence flanked by two near-infrared (NIR) fluorochromes. It includes a pharmacokinetic modifier (PKM) selected to confer increased blood half-life. The agent is quenched (no fluorescence) until cleavage by Cat K when it becomes highly fluorescent.

catkfastfig2.jpg
Figure 2: Cat K 680 FAST is selectively cleaved by Cat K over a variety of other enzymes. Cat K 680 FAST (0.5 µM) was incubated with a variety of enzymes and the fluorescence released was measured after 5 hours.


Products and catalog numbers


ProductCatalog NumberEx/Em wavelength (nm)Molecular weight (g/mol)Validated ExperimentsApplicationsStorage and Stability
Cat K 680 FASTNEV11000674/6928500In vivo/Ex vivo
Flow cytometry
In vitro microscopy
Oncology
Atherosclerosis
Arthritis
Osteoporosis
Lung remodeling
Obesity
Technical Data Sheet


Using Cat K 680 FAST in vivo/ex vivo


The recommended procedure for in vivo imaging with Cat K 680 FAST is administration via intravenous injection and imaging 6 hours post injection. Optimal imaging time is 6-24 hours after injection for Cat K 680 FAST depending on the model.

Route 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
IV2 nmol6-20 nmol30 min36 h6-24 h3 dKidney > liverFMT 680/700
IVIS 675/720

In Vivo/Ex vivo Imaging

catkfastfig3.jpg
Figure 3: ApoE-/- mice were placed on a high-cholesterol diet for 30 weeks (n =5). These mice and the control mice (n = 3) were injected with 8 nmols per mouse Cat K 680 FAST and imaged by FMT 2500. A) The representative FMT 2500 images from control and apoE-/- mice. Images were taken 6 hours following injection and tissues were collected from perfused animals immediately. B) The quantification of FMT aortic arch region fluorescence from non-invasive imaging datasets (used optimal threshold to minimize background fluorescence). C) The corresponding ex vivo FRI images of aortic tissue (from the same mice). D) The corresponding ex vivo FRI analysis of aortic arch tissue, focusing the quantification on the aortic arch region only.


Using Cat K 680 FAST in vitro


Cat K 680 FAST can be used for detecting and monitoring Cat K activity in synovial fibroblasts from rheumatoid arthritis (RA) and bone marrow-derived macrophages.

Flow cytometry and in vitro microscopy

We have validated Cat K 680 FAST for use with fluorescence microscopes and flow cytometers. Here is a brief protocol with a recommended concentration of agent to use:

  1. Culture cells in standard TC plate or chamber slide.
  2. Incubate cells with 0.5 uM Cat K 680 FAST for 6 hours at 37 °C. 
  3. Wash 1x with PBS. For flow cytometry, detach and resuspend cells in PBS. 
  4. Flow cytometry filter settings: 712/21
    Fluorescence micrsocopy filter: Cy5.5

In Vitro Imaging

catkfastfig4.jpg
Figure 4: A) Cells (human synovial fibroblasts (HSF) from a patient with RA (upper) and rabbit synoviocytes (HIG-62) (lower)) were cultured in the presence of 1 µM Cat K 680 FAST for 6 hours. Cat K 680 FAST activation is shown in red and DAPI nuclear stain is shown in blue and the final magnification is 40x. B) As a control, HSF cells were pre-incubated in the absence (top) or presence (lower panel) of a specific Cathepsin K inhibitor (L7, 200 nM) for 1 hour before addition of the Cat K 680 FAST. The cells were cultured for an additional 6 hours and as shown in these panels, Cat K 680 FASTactivation is inhibited. The final magnification for these panels is 20x.

catkfastfig5.jpg
Figure 5: Human osteoclasts were cultured on bovine cortical bone for seven days. Cat K 680 FAST (1 µM) was added for 24 hours and then the cells were visualized by confocal microscopy. Using activated Cat K 680 FAST (blue), Cathepsin K activity was easily detected in the resorption lacunae and intracellularly in both mononuclear multinucleated cells. Acidic vesicles in lysozomes were stained using Lysotracker (shown in red) and osteoclast actin rings were visualized using FITC-phalloidin (shown in green).


Application notes and posters


  • Poster: Imaging of Cathepsin K activity in rodent models of bone turnover and soft tissue calcification 
  • Poster: Development of a Fast Activating New Near Infrared-Labeled Agent for Detecting Cathepsin K Activity


FAQs


Q. Can I use Cat K 680 FAST in humans?

A. No, Cat K 680 FAST is intended for animal research only and not for use in humans.


Q. What is the difference between Cat K 680 FAST and the OsteoSense agents?

A. Cat K is useful in pathological models, such as Osteoporosis, where bone resorption and soft tissue calcification occurs. OsteoSense, on the other hand, specifically targets area of bone growth and loss. It does this by binding to hydroxyapatite, a major mineral product of osteoblasts and, thus, marker of osteoblast activity.


Citations


Please visit our Citations Library for references using Cat K 680 FAST on the IVIS or on the FMT.