Infant cynomolgus monkeys exposed to denosumab in utero exhibit an osteoclast-poor osteopetrotic-like skeletal phenotype at birth and in the early postnatal period

Abstract

RANKL is a key regulator of bone resorption and osteoclastogenesis. Denosumab is a fully human IgG2 monoclonal antibody that inhibits bone resorption by binding and inhibiting the activity of RANKL. To determine the effects of denosumab on pre- and postnatal skeletal growth and development, subcutaneous injections of 0 (control) or 50 mg/kg/month denosumab were given to pregnant cynomolgus monkeys from approximately gestation day (GD) 20 until parturition (up to 6 doses). For up to 6 months postpartum (birth day [BD] 180/181), evaluation of the infants included skeletal radiographs, bone biomarkers, and oral examinations for assessment of tooth eruption. Infant bones were collected at necropsy for densitometry, biomechanical testing, and histopathologic evaluation from control and denosumab-exposed infants on BD1 (or within 2 weeks of birth) and BD181, and from infants that died or were euthanized moribund from BD5 to BD69. In all denosumab-exposed infants, biomarkers of bone resorption and formation were markedly decreased at BD1 and BD14 and slightly greater at BD91 vs. control, then similar to control values by BD181. Spontaneous long bone fractures were detected clinically or radiographically in 4 denosumab-exposed infants at BD28 and BD60, with evidence of radiographic healing at ≥BD60. In BD1 infants exposed to denosumab in utero, radiographic evaluations of the skeleton revealed decreased long bone length; a generalized increased radio-opacity of the axial and appendicular skeleton and bones at the base of the skull with decreased or absent marrow cavities, widened growth plates, flared/club-shaped metaphysis, altered jaw/skull shape, and reduced jaw length; and delayed development of secondary ossification centers. Densitometric evaluations in these infants demonstrated a marked increase in bone mineral density at trabecular sites, but cortical bone mineral density was decreased. Histologically, long bone cortices were attenuated and there was an absence of osteoclasts. Bones with active endochondral ossification consisted largely of a dense network of retained primary spongiosa with reduced marrow space consistent with an osteopetrotic phenotype. A minimal increase in growth plate thickness largely due to the expansion of the hypertrophic zone was present. Retained woven bone was observed in bones formed by intramembranous ossification, consistent with absence of bone remodeling. These changes in bone tissue composition and geometry were reflected in reduced biomechanical strength and material properties of bones from denosumab-exposed infants. Material property changes were characterized by increased tissue brittleness reflected in reductions in calculated material toughness at the femur diaphysis and lack of correlation between energy and bone mass at the vertebra; these changes were likely the basis for the increased skeletal fragility (fractures). Although tooth eruption was not impaired in denosumab-exposed infants, the reduced growth and increased bone density of the mandible resulted in dental abnormalities consisting of tooth malalignment and dental dysplasia. Radiographic changes at BD1 persisted at BD28, with evidence of resumption of bone resorption and remodeling observed in most infants at BD60 and/or BD90. In 2 infants euthanized on BD60 and BD69, there was histologic and radiographic evidence of subphyseal/metaphyseal bone resorption accompanied by multiple foci of ossification in growth plates that were markedly increased in thickness. In infants necropsied at BD181, where systemic exposure to denosumab had been below limits of quantitation for approximately 3months, there was largely full recovery from all bone-related changes observed earlier postpartum, including tissue brittleness. Persistent changes included dental dysplasia, decreased bone length, reduced cortical thickness, and decreased peak load and ultimate strength at the femur diaphysis. In conclusion, the skeletal and secondary dental effects observed in infant monkeys exposed in utero to denosumab are consistent with the anticipated pharmacological activity of denosumab as a monoclonal antibody against RANKL and inhibitor of osteoclastogenesis. The resulting inhibition of resorption impaired both bone modeling and remodeling during skeletal development and growth. The skeletal phenotype of these infant monkeys resembles human infants with osteoclast-poor osteopetrosis due to inactivating mutations of RANK or RANKL.

Keywords: Brittle bone; Denosumab; Osteopetrosis; Pre-postnatal toxicity.