Elsevier

Bone

Volume 49, Issue 2, August 2011, Pages 151-161
Bone

Decreased bone remodeling and porosity are associated with improved bone strength in ovariectomized cynomolgus monkeys treated with denosumab, a fully human RANKL antibody

https://doi.org/10.1016/j.bone.2011.03.769Get rights and content

Abstract

This study examined the effects of denosumab, an anti-RANKL antibody that inhibits bone resorption, on bone histomorphometry in adult ovariectomized cynomolgus monkeys (OVX cynos). A month after surgery, OVX cynos were treated with subcutaneous vehicle (OVX-Veh) or denosumab (25 or 50 mg/kg/month) for 16 months (n = 14–20/group). Sham controls were treated with vehicle (Sham-Veh; n = 17). Areal and volumetric BMD, urine NTx, and serum osteocalcin were measured at baseline and months 3, 6, 12, and 16. Double fluorochrome labels were injected prior to iliac and rib biopsies at month 6 and month 12, and prior to sacrifice at month 16. Histomorphometry was performed on these biopsies, the tibial diaphysis, the L2 vertebra, and the proximal femur. Strength of humeral cortical beams, femur diaphysis, femur neck, and trabecular cores of L5–L6 vertebrae was determined by destructive biomechanical testing. There was no evidence of woven bone, osteomalacia, or other bone histopathologic changes with OVX or with denosumab. OVX-Veh animals exhibited significantly greater bone remodeling at all skeletal sites relative to Sham-Veh controls. Both doses of denosumab markedly inhibited bone remodeling at all sites, including significant reductions in trabecular eroded surfaces (48–86% lower than OVX-Veh controls), cortical porosity (28–72% lower), and dynamic parameters of bone formation (81–100% lower). Decreased fluorochrome labeling with denosumab was related to reductions in cortical porosity and trabecular eroded surfaces, and regression analyses suggested that these reductions contributed to denosumab-related increments in BMD and bone strength. Denosumab-treated animals with the lowest levels of fluorescent labeling exhibited the greatest structural bone strength values at each site. Intracortical remodeling had no relationship with material properties including ultimate strength, elastic modulus or toughness (r2 = 0.00–0.01). These data suggest that remodeling inhibition with denosumab improved structural strength without altering material properties under these experimental conditions. Greater structural strength in the denosumab-treated animals can be primarily explained by the combined effects of increased trabecular and cortical bone mass, and reductions in trabecular eroded surfaces and cortical porosity.

Research highlights

► Denosumab markedly reduced cortical and trabecular bone remodeling in OVX cynos. ► Denosumab reduced trabecular eroded surfaces and cortical porosity. ► Lowest levels of remodeling at each site were associated with greatest structural strength. ► Cortical remodeling parameters had little or no relationship with cortical material properties. ► Denosumab effects on remodeling were fully reversible upon clearance of drug.

Introduction

Bone remodeling is a continuous lifelong process by which packets of bone matrix are resorbed by osteoclasts and then replaced by matrix-secreting osteoblasts [1]. Bone remodeling rate increases after the menopause in humans, and with ovariectomy in non-human primates, leading to reductions in bone mass that can reduce bone strength and increase fracture risk [2], [3]. Bone remodeling is reduced by antiresorptive agents, and clinical trials have shown that 3–5 years of reduced bone remodeling with antiresorptives decreases fracture risk in subjects with low bone mass [4], [5], [6]. Greater decreases in biochemical markers of bone turnover with various antiresorptive agents have been associated with greater fracture risk reduction [7], [8], [9], [10].

Most data examining the effects of remodeling inhibition on bone strength and fracture risk are from bisphosphonate studies. The recent introduction of a new class of antiresorptive agents, the RANKL inhibitors, provides new tools to re-examine relationships between bone remodeling and bone strength. RANKL inhibitors, including the fully human monoclonal antibody denosumab (Prolia®), can reduce bone remodeling to a greater extent than other antiresorptive agents, including bisphosphonates [11], [12], [13], [14], [15]. Denosumab was recently shown to reduce hip, spine and non-vertebral fractures in postmenopausal women [16]. Iliac crest biopsies from a subgroup of this trial population exhibited markedly reduced bone resorption and formation parameters after 2–3 years of denosumab treatment, but this sub-study was not powered to examine relationships between histomorphometric remodeling parameters and fracture risk [17]. And while the iliac crest is a common site for histomorphometry assessment, it is subjected to a different loading environment compared to sites where fragility fractures commonly occur. Non-clinical studies allow bone histomorphometry analyses at multiple sites including diaphyseal cortices, and relationships between tissue-level remodeling and bone strength can be examined to further explore potential safety signals related to remodeling inhibition.

This paper describes histology and histomorphometry analyses of cortical and trabecular bone from a number of skeletal sites after 6 to 16 months of denosumab administration to mature ovariectomized (OVX) cynomolgus monkeys. This animal model is widely used for bone quality assessments of osteoporosis agents due to their skeletal maturity, increased trabecular and intracortical remodeling, and systemic osteopenia that is typical of postmenopausal osteoporosis [18]. The denosumab dosing regimens (25 or 50 mg/kg/month, subcutaneous) resulted in systemic exposure levels that greatly exceeded those achieved with clinical dosing for osteoporosis, providing robust safety margins. The primary goal of the study was to determine whether denosumab's anti-remodeling effects were associated with any adverse effects on bone histology or on extrinsic (structural) or intrinsic (material) properties of bone strength.

Section snippets

Materials and methods

All animal activities were approved by the Charles River Montreal Animal Care Committee and performed in an AAALAC-accredited facility. The study was performed under Good Laboratory Practice (GLP) conditions according to the protocol and Standard Operating Procedures established at Charles River Laboratories, Quebec, Canada, and Amgen Inc., Thousand Oaks, CA, USA.

Bone histology and histopathology assessments

Qualitative microscopic examination across all skeletal sites revealed no detrimental denosumab-related histological findings at either dose; the regular lamellar collagen arrangement was maintained and no osteoid accumulation was found. In many denosumab-treated monkeys, fluorochrome double labels were rare (Table 2) and had to be sought outside of the measured regions in order to confirm that the absence of labels in individual bones reflected very low bone formation rate and not an

Discussion

This paper provides the first description of cortical and trabecular bone histomorphometry data for adult OVX cynos treated with denosumab, a fully human anti-RANKL antibody that inhibits bone resorption and thereby reduces bone remodeling. The study's primary purpose was to determine whether the low-turnover state induced by high doses of denosumab led to detrimental effects on bone strength, in accordance with regulatory guidelines for new osteoporosis therapies [22], [23]. Other data from

Conflict of Interest

P.J.K., J.S., I.P., and M.S.O. are employees and own stock in Amgen Inc.

S.Y. Smith and J. Jolette are employees and own stock in Charles River Laboratories.

Charles River Laboratories received funding from Amgen Inc. for the study.

Acknowledgments

This study was supported by Amgen Inc. Rogely Boyce, D.V.M., Ph.D., provided helpful comments, and Michelle N. Bradley, Ph.D., provided editorial support on behalf of Amgen Inc. We are also grateful to the excellent technical expertise of the histomorphometry teams at Charles River Laboratories.

References (38)

  • D.M. Black et al.

    Fracture risk reduction with alendronate in women with osteoporosis: the fracture intervention trial

    J. Clin. Endocrinol. Metab.

    (2000)
  • M.R. McClung et al.

    Effect of risedronate on the risk of hip fracture in elderly women

    N. Engl. J. Med.

    (2001)
  • S. Sarkar et al.

    Relationship between changes in biochemical markers of bone turnover and BMD to predict vertebral fracture risk

    J. Bone Miner. Res.

    (2004)
  • R. Eastell et al.

    Relationship of early changes in bone resorption to the reduction in fracture risk with risedronate

    J. Bone Miner. Res.

    (2003)
  • D.C. Bauer et al.

    Change in bone turnover and hip, non-spine, and vertebral fracture in alendronate-treated women: the fracture intervention trial

    J. Bone Miner. Res.

    (2004)
  • P.D. Delmas et al.

    Effects of yearly zoledronic acid 5 mg on bone turnover markers and relation of PINP with fracture reduction in postmenopausal women with osteoporosis

    J. Bone Miner. Res.

    (2009)
  • R. Samadfam et al.

    Co-treatment of PTH with osteoprotegerin or alendronate increases its anabolic effect on the skeleton of oophorectomized mice

    J. Bone Miner. Res.

    (2007)
  • S. Morony et al.

    The RANKL inhibitor osteoprotegerin (OPG) causes greater suppression of bone resorption and hypercalcemia compared to bisphosphonates in two models of humoral hypercalcemia of malignancy

    Endocrinology

    (2005)
  • J.P. Brown et al.

    Comparison of the effect of denosumab and alendronate on bone mineral density and biochemical markers of bone turnover in postmenopausal women with low bone mass: a randomized, blinded, phase 3 trial

    J. Bone Miner. Res.

    (2008)
  • Cited by (95)

    • Osteoinductive potential of recombinant BMP-9 in bone defects of mice treated with antiresorptive agents

      2022, International Journal of Oral and Maxillofacial Surgery
      Citation Excerpt :

      In the present study, a mouse model of treatment with an anti-mouse RANKL neutralizing monoclonal antibody (mAb) was selected as an AMART model. Because denosumab does not recognize rodent RANKL and has no apparent effects in rodents, the evaluation of denosumab in vivo could be performed only with non-human primates19,20 or human RANKL-knock-in mice21,22. RANKL-knock-in mice exclusively express a chimeric (human/murine) form of RANKL that can maintain bone resorption in mice in a manner that can be fully inhibited by denosumab21.

    • On the evolution and contemporary roles of bone remodeling

      2020, Marcus and Feldman’s Osteoporosis
    • Denosumab for the treatment of osteoporosis

      2020, Marcus and Feldman’s Osteoporosis
    View all citing articles on Scopus

    Parts of the manuscript were presented at the 28th Annual Meeting of the American Society for Bone and Mineral Research in Philadelphia, PA in 2006, and at the 29th Annual Meeting of the American Society for Bone and Mineral Research in Honolulu, Hawaii in 2007.

    View full text