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P&F Grant Awards

Year 9


Grant # 19

Subclinical Doses of Nitrogen-containing Bisphosphonates Produce Clinical Response on Bone.

PI: Timothy Peterson, PhD

 

Specific Aims

New therapies for bone are hard to come by. Bisphosphonates, especially the nitrogen-containing species (NBPs), are the standard of care for many diseases involving bone1,2. They are inexpensive and work well. However, because they cause rare yet devastating side-effects such as atypical fractures and osteonecrosis of the jaw many patients avoid taking them1,3,4. This fear over side effects is so great and is growing that it has caused the number of prescriptions to plummet 50% in the last four years5. Moreover, several drug companies and funding agencies have stalled their support for bone research and development6. Thus, there is a pressing need for the bone community to work with what we’ve got. Our preliminary data suggests that NBPs used at concentrations 1,000-fold less than those currently clinically used increase bone density and strength and could be protective against fracture. The impetus for this work revolves around poorly appreciated aspects of NBP drug actions. The widely accepted mechanism for the NBPs is that they inhibit the function of the bone resorptive cell, osteoclasts7. In osteoclasts the targets for NBPs are the cholesterol biosynthetic enzymes, farnesyl pyrophosphate synthase and geranyl-geranyl phosphate synthase, FPPS and GGPS, respectively8. These enzymes are inhibited by μM doses of NBPs in cell culture contexts. Interestingly, in vitro and ex vivo experiments with the bone depositing cell type, osteoblasts, suggest that very low doses of NBPs (in the nM range, i.e., 1,000-fold less than those used to inhibit cultured osteoclasts) can have osteoblast differentiation-potentiating effects9-11. These same very low doses don’t impair osteoclasts in vitro12,13. Lastly, it has also been shown that NBPs can maintain osteocyte viability depending on the dose14. The mechanisms for these effects are unclear. Considering the number of patients at stake (hundreds of millions), it is surprising that few in vivo studies exist to clarify the effects of the various NBPs at varying doses on the major bone cell types: osteoclasts, osteoblasts, and osteocytes. To add clarity to the NBPs mechanism of action, we’ve recently identified two poorly characterized genes15,16 we named TBONE1 and TBONE2 (Target of BisphOsphonate NitrogEnous 1 and 2), that are critical to the bone protective effects of NBPs at clinical doses. We hypothesize that NBPs given at very low doses – up to 10,000-fold lower than those typically given – increase bone density and strength without increasing fragility by potentiating osteoblast function in a TBONE1/2-dependent manner.

Aim 1.  Determine the doses of NBPs that increase bone density and strength without increasing fragility.
Though NBPs improve bone density and reduce fracture risk in most people, they can cause atypical fractures which is highly undesirable. In mice, the doses that are normally used in people increase bone fragility. Making use of this predictable response in mice, in this Aim we will seek to define doses of NBPs that don’t increase fragility yet still provide robust therapeutic benefit. The implication is that using these doses in people would reduce the occurrence of side effects to a negligible amount. In preliminary work, we have shown that 1,000-fold less Alendronate than that is normally given to mice (0.1μg/kg/wk vs. 100μg/kg/wk) increases bone density and strength while not increasing fragility as higher doses do. In this aim, we will systematically approach NBP dosing by performing titrations over a 10,000-fold concentration range with two commonly prescribed NBPs, Zolendronate and Alendronate17. We will measure bone structure and strength using μCT and three-point bending tests, respectively. Successful completion of this aim will define the NBPs doses that improve bone structure and strength.

Aim 2. Identify the bone cell type(s) that respond to very low dose bisphosphonates.
Here we propose that subclinical doses improve bone strength by improving bone formation in the absence of inhibiting bone resorption. At micromolar (μM) doses that are typical used in cell-based assays, NBPs negatively regulate osteoclasts, i.e., inhibit protein prenylation, yet also inhibit osteoblast viability and differentiation18-20. However, at nanomolar (nM) doses, NBPs promote osteoblast function. In preliminary work, we have shown that 10,000- fold less Alendronate than what is normally used to inhibit protein prenylation, potentiates osteoblast differentiation. Successful completion of this aim will define the doses that regulate the major bone cell types: osteoclasts, osteoblasts and osteocytes.