SA3 - Development of a Cause-based Treatment for NBP-induced MRONJ

Abstract: The problem: MRONJ is a devastating side effect of nitrogen-containing bisphosphonate (NBPs) use in patients with osteoporosis or cancer, resulting in painful lesions of exposed necrotic bone. The incidence of MRONJ in cancer patients is over 12%.1 Current treatment protocols call for debridement of the lesion and a drug “holiday” to promote healing. This cessation of treatment can often result in fractured mandibles. There is no current cause-based treatment to speed healing and reduce morbidity. Most MRONJ is caused by IV-administered nitrogen-containing bisphosphonates (NBPs). NBPs are known to inhibit the enzyme farnesyl diphosphate synthase (FDPS) in the mevalonate-cholesterol synthesis pathway.2 FDPS is necessary for the prenylation of small GTPases such as Rac, Rho, and Cdc42, which are required for osteoclast function.
Hypothesis: Geranyl-geraniol (GGOH), a metabolite directly downstream of FDPS in the mevalonate pathway, will rescue osteoclasts and speed the healing of MRONJ lesions in a rat model. GGOH can be incorporated into and released from an osteoconductive, tunable, and resorbable carrier in concentrations that can rescue osteoclasts from the toxic effects of NBPs.
Materials and methods: In-vitro expts: viability: 1.5-2.0 x 104 Raw 264.7 osteoclasts were grown on 24 well plates for 24 hours after which media containing 20ng/ml of RANKL with or without GGOH in pellets and pamidronate were incubated for 72 hours. Cell viability was quantitated, using Cell Titer 96 kit (Promega) by measuring optical density at 490nm after subtracting background absorption at 650nm. Each time point had between 3 and 5 replicates, which were averaged.
Function: Corning Osteo Assay 24 well plate (3987) was used to measure osteoclast function along with reagents from the Cosmo Bio Bone resorption Assay kit (CSR-BRA-24). The coated calcium phosphate is first bound to fluoresceinamine-labeled chondroitin sulfate (FACS), which is released from the calcium phosphate layer into conditioned medium by osteoclastic resorption activity. Bone resorption activity is evaluated by simply measuring the fluorescence intensity of the conditioned medium.
In vivo expts: 3 Sprague Dawley 10 week old rats were ovariectomized and given 100ug/kg zoledronic acid 1x/week for 7 weeks. At time 0, animals were anesthetized with a full thickness flap created in the diastema between mandibular molars and incisors. A 1mm-diameter, 1mm-deep bony defect was created on the left and right sides. On the right side, bone cement alone was inserted. On the left side, bone cement+ 4mM GGOH was placed. After 9 days, animals were sacrificed, mandibles resected and uCT analysis performed.

Results:
1) Exposure of osteoclasts to 400 and 800uM pamidronate over 72 hours showed a 49% and 69% loss of viability, respectively (P < .0001 for each concentration); 2mM GGOH incorporated into bone cement increased survival by 247% (P = .0001) for the cells treated with 800uM pamidronate and 10mM GGOH by 450% (P < .0001). Pamidronate at both concentrations reduced osteoclast function and 4mM GGOH completely restored resorptive function.
2) After 9 days of treatment, bone density in the region surrounding the bony defect increased selectively on the side of mandibles treated with GGOH containing cement by 15% (P = .0031) compared to control side treated with cement alone.

Conclusion: GGOH contained in a bone cement carrier can rescue osteoclast function from some of the toxic effects of NBPs in vitro and in vivo. This carrier may be the first step in local delivery of a metabolite in the oral cavity that might reverse the effects of MRONJ.

References: 1. Hoff AO, Toth B, Hu M, et al: Ann N Y Acad Sci 1218:47-54, 2011
2. Dunford JE, Thompson K, Coxon FP, Luckman SP, Hahn FM, Poulter CD, et al: J Pharmacol Exp Ther 296:235-42, 2001