Bone-targeted rapamycin analogs inhibit osteolysis and tumor growth in a PC-3-GFP intratibial bone cancer model in nude mice

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mTOR is a PI3K-like kinase that links mitogenic stimuli and nutrient status to cell growth, division and metabolism, and is an important target for anti-tumor therapy. Rapamycin is a natural product mTOR inhibitor that functions by inducing an inhibitory complex between mTOR and FKBP. Rapamycin and its derivatives also have inhibitory effects on osteoclasts and osteoclast cell precursors in vitro and in animal models of bone disease. These observations have prompted us to design bone-targeted analogs of rapamycin with the aim of enhancing the anti-tumor and anti-osteoclast effects at bone, for potential use in treating primary bone cancers and bone metastases. Previously we reported on several novel bone-targeted mTOR inhibitors, including AP23841, which demonstrated potent and sustained inhibition of PTH-induced hypercalcemia in mice. In this study, we synthesized a series of optimized analogs of AP23841 and tested them in a mouse model of bone-localized disease. Compounds were synthesized from common, activated rapamycin intermediates and various functionalized bone-targeted groups in good yield and high purity (>98%). Bone-targeted compounds varied according to in vitro hydroxyapatite (HA) affinity and linker functionality (e.g., cleavable or non-cleavable). Compounds were also tested for FKBP binding affinity using a fluorescence polarization assay. For in vivo testing, PC-3-GFP prostate cancer cells were injected intratibially into nude mice, and anti-tumor and anti-osteolytic effects assessed by GFP imaging, radiology and histology. One compound in particular, AP24170, demonstrated potent inhibitory effects on tumor growth (38% T/C) following 2-3 wks dosing (14 mg/kg iv, QDx5 and weekly) that were similar to rapamycin dosed equivalently (45% T/C). AP24170 also potently inhibited osteolysis in the mice, as scored semi-quantitatively by whole animal radiography with a statistically significant greater reduction in the osteolysis score vs. rapamycin (p <0.001, Dunnett’s test). No significant drop in body weights of the mice during AP24170 treatment was observed (2-3 wks). In a separate pharmacokinetic study, blood levels of rapamycin in AP24170 treated mice (28 mg/kg, iv) were at or below quantitation limits (3-5 ng/mL, 0.25-24 hr timepoints), consistent with a non-prodrug mechanism of action that does not involve systemic release of rapamycin. Further pre-clinical studies of AP24170 and its analogs are in progress.