Objectives: Bisphosphonates (BPs) are a class of compounds that selectively bind to bone and can act as potent inhibitors of osteoclast-mediated bone resorption. Current imaging probes for bone diseases suffer from lack of specificity or lack of resolution and well-defined chemical species. We have combined the bone-targeting of BPs with the sensitivity and resolution of F-18 positron emission tomography (PET) imaging. In this study, we describe an ¹⁸F-labeled bisphosphonate-based PET imaging probe designed to enable early detection and diagnosis of bone turnover sites.

Methods: We covalently introduced F-18 into the bisphosphonate scaffold to enable PET imaging targeted to bone using rapid reactions consistent with the 110 min half-life of F-18. We first created an optimized F-19 synthesis, starting with the fluorination of a diazomethylenebisphosphonate ester using Olah’s reagent, followed by refluxing with zinc in acetic acid to reduce the C-Cl bond and subsequent treatment with bromotrimethylsilane (BTMS) to yield monofluoromethylenebisphosphonic acid (FMBP). The entirely cartridge-based purification avoids the length and inconvenience of HPLC. We then adapted this method for the radiofluorination of the BP scaffold to synthesize [¹⁸F]FMBP. To evaluate the in vivo PET imaging potential of this novel probe, normal nude mice were intravenously injected with [¹⁸F]FMBP and imaged using animal PET.

Results: The rapid fluorination (< 1 min) of the diazomethylenebisphosphonate ester precursor produced a mixture of the desired fluorochloromethylene- and a side product, dichloromethylenebisphosphonate ester. Optimized solvent conditions minimized the formation of the side product, which was eliminated following the subsequent Zn/AcOH reduction step by facile silica cartridge purification which gave the labeled product in high purity, confirmed by NMR. Dealkylation was achieved quantitatively by reaction with BTMS, resulting in the final compound (FMBP) which was isolated by ion exchange chromatography. These conditions were successfully translated to the radiochemical preparation of [¹⁸F]FMBP with a radiochemical purity of >98% within less than two F-18 half-lives. The serum stability of [¹⁸F]FMBP at 37 °C at 2 h was over 95% with no detectable defluorination. Following administration to nude mice, vivid PET images were acquired demonstrating highly selective binding of [¹⁸F]FMBP to bone. The joints and skeleton were clearly visible with high contrast to the contralateral background at all imaging time points. Predominant uptake of radioactivity was observed in the bladder, indicating that [¹⁸F]FMBP is excreted via the renal system.

Conclusions: A novel bone-specific ¹⁸F-labeled PET probe has been successfully synthesized using rapid and robust fluorination chemistry. The procedure was optimized for radiosynthesis and simplified to avoid HPLC, facilitating future clinical translation.

Image/Figure:
Click to View

Image/Figure Caption:

Figure: (A) Synthetic scheme of FMBP intermediates. (B) Synthetic scheme of [¹⁸F]FMBP. (C) Radio-HPLC chromatograms of a crude reaction mixture of [¹⁸F]2, pure [¹⁸F]2 after C18 cartridge purification, pure [¹⁸F]FMBP after rapid dealkylation with BTMS, and co-injection of [¹⁸F]FMBP with [¹⁸F]Fluoride. (D) Maximum intensity projection (MIP) PET images of a nude mouse at 1 h post-injection of [¹⁸F]FMBP.

Author