Binding Kinetics of a Fluorescently Labeled Bisphosphonate as a Tool for Dynamic Monitoring of Bone Mineral Deposition In Vivo

Abstract

Bone mineral deposition during the modeling of new bone and remodeling of old bone can be perturbed by several pathological conditions, including osteoporosis and skeletal metastases. A site-specific marker depicting the dynamics of bone mineral deposition would provide insight into skeletal disease location and severity, and prove useful in evaluating the efficacy of pharmacological interventions. Fluorescent labels may combine advantages of both radioisotope imaging and detailed microscopic analyses. The purpose of this study was to determine if the fluorescent bisphosphonate OsteoSense could detect localized changes in bone mineral deposition in established mouse models of accelerated bone loss (ovariectomy) (OVX) and anabolic bone gain resulting from parathyroid hormone (PTH) treatment. We hypothesized that the early rate of binding, as well as the total amount of bisphosphonate, which binds over long periods of time, could be useful in evaluating changes in bone metabolism. Evaluation of the kinetic uptake of bisphosphonates revealed a significant reduction in both the rate constant and plateau binding after OVX, whereas treatment with PTH resulted in a 36-fold increase in the bisphosphonate binding rate constant compared with untreated OVX controls. Localization of bisphosphonate binding revealed initial binding at sites of ossification adjacent to the growth plate and, to a lesser extent, along more distal trabecular and cortical elements. Micro-computed tomography (CT) was used to confirm that initial bisphosphonate binding is localized to sites of low tissue mineral density, associated with new bone mineral deposition. Our results suggest monitoring binding kinetics based on fluorescently labeled bisphosphonates represents a highly sensitive, site-specific method for monitoring changes in bone mineral deposition with the potential for translation into human applications in osteoporosis and bone metastatic processes and their treatment.

Utilizing time-lapse micro-CT-correlated bisphosphonate binding kinetics and soft tissue-derived input functions to differentiate site-specific changes in bone metabolism in vivo

“…Bone loss expected as a result of OVX was confirmed by BMD assessment which showed significantly reduced values in both the spine and knee after long-term OVX (data not shown) and serum marker analyses [30]. Baseline and follow-up scans were overlaid to ensure analyses of similar regions and to visualize regions of bone formation and resorption which occurred between imaging time points and pre-OVX baseline scan (Fig.…”

“…To examine region-specific differences in uptake, changes in fluorescent bisphosphonate kinetic parameters obtained for the knee (as previously published [30]) and spine for each group were compared. Overall, changes in kinetic parameters were similar for both the knee and spine in response to OVX.…”

“…For long-term anesthetization, additional administration of ketamine and xylazine at half dose was administered upon initial signs of mouse waking. Animals were separated into 3 groups (n = 9/group): i) non-operated, control animals, ii) ovariectomized (OVX) animals, imaged 3 days (shortterm) and 14 days (long-term) after OVX, and iii) PTH-treated mice, which were subjected to OVX, then received daily PTH injections for 3 days beginning 11 days post-surgery (total of 14 days OVX and 3 days PTH treatment) as previously described [30]. Animals were ovariectomized via their dorsal side.…”

“…Recent radionuclide studies in humans have also suggested that significant overall changes in skeletal activity, such as those seen in the case of treatment with parathyroid hormone (PTH), may alter the plasma time-activity curve [2]. This effect would alter both the rate and total binding values of a fluorescently-labeled bisphosphonate as discussed previously [30].…”

“…Conjugation of bisphosphonates with fluorescent dyes is currently under great scrutiny to help characterize bisphosphonate binding [11,27], as well as in the use as a bone-specific turnover marker [11,14]. While early works focused on bisphosphonate binding days after injection, new data suggests that monitoring the kinetics of early binding (as little as 5 min after injection) may provide additional information about the metabolic status of the bone [30]. One limitation in previous in vivo kinetic assessments has been the lack of indicators of blood pool bisphosphonates available for bone binding, which are used in more refined, quantitative, multi-compartment analyses commonly seen in the context of radiolabeled bisphosphonates [18].…”