Abstract
Multiple myeloma (MM) leads to high risk for bone pain and fractures. MM-induced bone disease is due to acute degradation of bone matrix by osteoclasts, and absence of repair by bone forming osteoblasts. It is currently treated with bisphosphonates, highly effective bone resorption inhibitors, which do not promote but rather inhibit bone formation and may cause renal damage and osteonecrosis of the jaw. Thus, it is important to reconsider the management of MM bone disease in long-term treatment. Recent preclinical studies reported that the proteasome inhibitor Bortezomib (V) used for the treatment of MM patients can stimulate bone formation, and that in MM patients treated with V, serum levels of bone formation markers are increased. The present study aims at investigating if V may inhibit osteoclast activity.
Methods: Osteoclasts were differentiated from pure populations of blood derived CD14-positive monocytes cultured with M-CSF and RANKL for 6–7 days, and treated continuously with V at various concentrations. As prolonged inhibition of proteasome activity has been reported to be toxic for any cell type, and in vivo pharmacodynamic studies have shown V to be eliminated from the vascular compartment as soon as 30min after intravenous injection, displaying maximal inhibitory activity of the proteasome within 24 hours subsiding rapidly thereafter, V was also given intermittently, to mimick the in vivo situation. Osteoclast differentiation and activity were assessed by measuring Tartrate-Resistant Acid Phosphatase (TRACP) activity in the medium. Cell viability was determined with Celltiter Blue measuring metabolic activity. To extend our observations to the clinical situation, serum levels of CTX-I, a bone resorption marker, were measured during the 3 days following therapeutic V administration in a single patient.
Results: A continuous treatment of cultures with V at 4 nM and higher concentrations proved to be highly toxic for differentiating osteoclasts but also monocytes. A 3-hour-pulse treatment with V followed by a 3-day culture in the absence of V, was not toxic neither to monocytes nor to osteoclasts, even at a concentration as high as 100 nM. This 3-hour pulse was however highly toxic for myeloma cells. Interestingly, a 3-hour pulse with 25 nM V induced a 50% inhibition of the resorptive activity of osteoclasts, as assessed by culturing them for 3 days on bone slices and measuring the formation of resorption pits. The release of TRACP in the medium was inhibited to a similar extent within the first 24 hours post-pulse, but tended to return to the control level during the next 2 days. This 3-hour pulse with 25 nM V inhibited strongly RANKL-induced translocation of NF-KB in the osteoclast nuclei, an event dependent on proteasome function and critical for osteoclastic activity. Serum CTX-I levels decreased during the first 48 hours after each V injection (n = 3), and tended to increase again after 72 hours suggesting a partial recovery of osteoclast activity between each administration.
Conclusions: Our results suggest that Bortezomib temporarily inhibits osteoclast activity in vitro and in vivo. This effect is linked to RANKL-induced translocation of NF-KB in the osteoclast nuclei and proteasome function. Since recent reports suggested that formation of new bone requires at least a transient activity of osteoclasts transient inhibition of osteoclasts could be an advantage compared to the more persistent inhibition of osteoclast activity by bisphosphonate.
Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation
