Dampening of the bone formation response following repeat dosing with sclerostin antibody in mice is associated with up-regulation of Wnt antagonists

Abstract

Administration of antibodies to sclerostin (Scl-Ab) has been shown to increase bone mass, bone mineral density (BMD) and bone strength by increasing bone formation and decreasing bone resorption in both animal studies and human clinical trials. In these studies, the magnitude and rate of increase in bone formation markers is attenuated upon repeat dosing with Scl-Ab despite a continuous and progressive increase in BMD. Here, we investigated whether the attenuation in the bone formation response following repeated administration of Scl-Ab was associated with increased expression of secreted antagonists of Wnt signalling and determined how the circulating marker of bone formation, P1NP, responded to single, or multiple doses, of Scl-Ab four days post-dosing. Female Balb/c mice were treated with Scl-Ab and we demonstrated that the large increase in serum P1NP observed following the first dose was reduced following administration of multiple doses of Scl-Ab. This dampening of the P1NP response was not due to a change in the kinetics of the bone formation marker response, or differences in exposure to the drug. The abundance of transcripts encoding several secreted Wnt antagonists was determined in femurs collected from mice following one or six doses of Scl-Ab, or vehicle treatment. Compared with vehicle controls, expression of SOST, SOST-DC1, DKK1, DKK2, SFRP1, SFRP2, FRZB, SFRP4 and WIF1 transcripts was significantly increased (approximately 1.5-4.2 fold) following a single dose of Scl-Ab. With the exception of SFRP1, these changes were maintained or further increased following six doses of Scl-Ab and the abundance of SFRP5 was also increased. Up-regulation of these Wnt antagonists may exert a negative feedback to increased Wnt signalling induced by repeated administration of Scl-Ab and could contribute to self-regulation of the bone formation response over time. After an antibody-free period of four weeks or more, the P1NP response was comparable to the naïve response, and a second phase of treatment with Scl-Ab following an antibody-free period elicited additional gains in BMD. Together, these data demonstrate that the rapid dampening of the bone formation response in the immediate post-dose period which occurs after repeat dosing of Scl-Ab is associated with increased expression of Wnt antagonists, and a treatment-free period can restore the full bone formation response to Scl-Ab.

Gene Expression Profile and Acute Gene Expression Response to Sclerostin Inhibition in Osteogenesis Imperfecta Bone

“…(39,53,(58)(59)(60) It has been proposed that the dampening effects following long-term SclAb treatment may be caused by a large and acute upregulation in inhibitory regulators of bone formation (SOST, DKK1). (38) We observed a similar upregulation of SOST and DKK1 with SclAb treatment. This compensatory response has been documented in the acute phase of treatment with significant upregulation observed following a single dose of SclAb.…”

“…The panel represents a subset of markers in the bone remodeling cycle-many of which have been identified as key targets for SclAb therapy in prior animal studies. (38)(39)(40)44) Because of the rarity of the OI bone tissue and the size of the available harvested bone (which affected the amount of total nucleic acid we were able to extract), we chose to analyze only one housekeeping gene (HPRT1), which has been documented in the literature as a stable gene across experimental conditions in human bone studies. (45,46) Pooled, purified RNA samples underwent reverse transcription using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) using 1.5 μg of retro-transcribed RNA per reaction followed by thermocycling (C1000 Thermal Cycler; Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's recommendations.…”

“…Understanding the transcriptional response to treatment in the diseased target tissue is of great interest. Gene expression response following SclAb treatment has been reported in rat models of postmenopausal osteoporosis and in female Balb/c mice, (38)(39)(40)(41) highlighting the unique signaling events and compensatory response occurring in the osteoblast lineage as a result of SclAb. However, patterns of gene expression response caused by treatment in human OI bone tissue remain unknown and difficult to assess clinically.…”

Sclerostin neutralization unleashes the osteoanabolic effects of Dkk1 inhibition

“…The role of other secreted inhibitors in the compensatory expression mechanism are unknown. Canonical WNT signaling antagonists such as WISE (65), sFRP4 (66), and NOTUM (67), are associated with skeletal phenotypes in mice when mutated either globally or selectively in bone cells, and sclerostin inhibition enhances a number of these transcripts in mouse bone (68). Whether pharmacologic modulation of these or other members of the secreted WNT inhibitory milieu can improve or otherwise tailor the efficacy of sclerostin and/or Dkk1 neutralizing therapy is not known.…”

Scl-Ab reverts pro-osteoclastogenic signaling and resorption in estrogen deficient osteocytes

“…Scl-Ab administration down-regulated both WIF1 and SFRP2 expression in estrogen-treated cells, however it appeared to have no signi cant in uence on estrogen de cient cells. This may be a result of our experimental timelines, in which we analysed WNT antagonist expression after only 2 days of Scl-Ab treatment, whereas an in vivo study showed an upregulation in Wnt antagonist expression after 1 week of Scl-Ab administration [64].…”