Imaging of Alkaline Phosphatase Activity in Bone Tissue

Abstract

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with 19Flourine magnetic resonance spectroscopic imaging (19FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using 19Fluorine magnetic resonance spectroscopy (19FMRS) and 19FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. 19FMRS and 19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. 19FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized 19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of 19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, 19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.

Cathepsin-sensitive nanoscale drug delivery systems for cancer therapy and other diseases

“…have been exploited for cancer diagnosis. Furthermore, imaging probes utilizing these proteases have rapidly evolved [164][165][166][167]. It has been shown that the monitoring of protease activity was closely related to cancer progression especially in case of Cathepsin B [168].…”

Molecular Imaging: An Innovative Force in Musculoskeletal Radiology

“…The combination of PET/MRI scanning would present one possible method for simultaneously obtaining anatomic and physiologic information with regard to bone turnover. It may also be possible to simultaneously obtain anatomic and physiologic information about bone turnover with the use of emerging MRI techniques alone, as outlined in a recent article by Gade et al [28], who imaged the activity of alkaline phosphatase, a commonly used clinical marker of bone turnover, using 19 F-MR spectroscopic imaging. Alkaline phosphatase-mediated hydrolysis of a small imaging molecule (6,8-difluoro-4-methylumbelliferyl phosphate) was tested on osteoblastic cells and bone tissue.…”

Combined Fluorescence-Based in Vitro Assay for the Simultaneous Detection of Cell Viability and Alkaline Phosphatase Activity during Osteogenic Differentiation of Osteoblast Precursor Cells

“…As a substrate for ALP, we used 4-methylumbelliferone-phosphate (4-MUP). ALP converts non-fluorescent 4-MUP into blue-fluorescent 4-methylumbelliferone (4-MU) [ 18 ]. A substrate with blue fluorescence was selected to avoid interference with the green fluorescent fluorescein that is generated during the analysis of cell viability using FDA.…”