Pharmacological mechanisms of therapeutics

Chapter 73

Pharmacological mechanisms of therapeutics: receptor activator of nuclear factorekappa B ligand inhibition Elena Tsourdi1, 2, Michael S. Ominsky3, Tilman D. Rachner1, 2, 4, Lorenz C. Hofbauer1, 2, 5 and Paul J. Kostenuik6, 7 1

Department of Medicine III, Technische Universität Dresden, Dresden, Germany; 2Center for Healthy Aging, Technische Universität Dresden,

Dresden, Germany; 3Radius Health Inc., Waltham, MA, United States; 4German Cancer Consortium (DKTK), Partner site Dresden and German Cancer Research Center (DKFZ), Heidelberg, Germany; 5Center for Regenerative Therapies Dresden, Dresden, Germany; 6Phylon Pharma Services, Newbury Park, CA, United States; 7School of Dentistry, University of Michigan, Ann Arbor, MI, United States

Chapter outline History of osteoprotegerin/receptor activator of nuclear factorekappa B ligand-based drug development 1689 Physiologic mechanisms and effects of receptor activator of nuclear factorekappa B ligand inhibitors in bone 1691 Clinical studies demonstrating the effects of denosumab 1693 Osteoporosis indications 1693 Postmenopausal osteoporosis 1693 Male osteoporosis 1695 Use of denosumab in combination/sequence with other osteoporosis agents 1695 Cancer indications 1696 Denosumab for cancer treatmenteinduced bone loss 1696 Treatment of hypercalcemia of malignancy refractory to bisphosphonate therapy 1696 Denosumab for the treatment of metastatic bone disease, multiple myeloma, and giant cell tumors 1698 Additional denosumab data 1698

Glucocorticoid-induced osteoporosis 1698 Rheumatoid arthritis 1699 Other potential applications 1699 Denosumab safety 1700 Hypocalcemia 1700 Osteonecrosis of the jaw 1700 Atypical femoral fractures 1700 Denosumab discontinuation: effects on bone turnover, bone mass, and fracture risk 1701 Hypersensitivity, serious infections, and musculoskeletal pain 1701 Use in women of reproductive age 1702 Theoretical impact of receptor activator of nuclear factorekappa B ligand inhibition on insulin resistance and vascular calcification 1702 Summary 1702 References 1702

History of osteoprotegerin/receptor activator of nuclear factorekappa B ligandbased drug development Before the mid-1990s, the molecular mechanisms underlying osteoclastogenesis were not well understood, and in vitro culturing of osteoclasts was technically challenging. As genetic mapping approaches progressed, parallel efforts from several commercial and academic groups began to unravel components of a key pathway that drives osteoclast formation, function, and survival. At Amgen, phenotypic screening of transgenic mice engineered to overexpress previously unknown secreted factors via the liver resulted in the discovery of osteoprotegerin (OPG) (Simonet et al., 1997). OPG, a secreted member of the TNF receptor superfamily, was identified based on increased radiographic density in OPG-overexpressing transgenic mice (Simonet et al., 1997). Histology demonstrated that OPG transgenic mice had few osteoclasts and

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exhibited features reminiscent of human osteopetrosis. Subsequent experiments demonstrated that recombinant OPG inhibited osteoclast formation in vitro (Simonet et al., 1997), while genetic ablation of OPG resulted in severe osteopenia including fragility fractures (Bucay et al., 1998). Soon thereafter, independent investigators at the Snow Brand Milk Products Company reported an identical secreted protein with similar pharmacologic properties that they called osteoclast inhibitory factor (Yasuda et al., 1998). The Amgen investigators then used OPG to find its binding partner, which OPG was presumably blocking to inhibit osteoclastogenesis. A longer-circulating recombinant form of OPG called OPG-Fc, which comprises the cysteine-rich TNF receptor-like domains of native OPG fused to the Fc portion of human IgG1, was found to bind to a molecule expressed on the surface of a myelomonocytic cell line (Lacey et al., 1998). The Amgen investigators named this molecule OPG ligand (OPGL), which they showed to be a potent promoter of osteoclast differentiation and activation (Lacey et al., 1998). Independent research by the investigators at Snow Brand Milk showed that an identical molecule, which they named osteoclast differentiation factor (ODF), stimulated osteoclastogenesis in vitro and caused hypercalcemia in vivo (Yasuda et al., 1998). Meanwhile, investigators at Immunex Corporation had identified the same “OPGL/ODF” molecule by direct sequencing of a myeloid dendritic cell cDNA library (Dougall et al., 1999; Hsu et al., 1999). They had initially identified a new member of the TNF receptor family they called “receptor activator of nuclear factor kappa B” (RANK) based on the key role of the transcription factor NFekB in mediating its biological effects. Using an extracellular fragment of this receptor fused to Fc (RANK-Fc), they identified its binding partner via expression cloning and named this molecule RANK ligand (RANKL) (Anderson et al., 1997). The sequence of RANKL was identical to OPGL, and a nomenclature committee comprising representatives from the American Society for Bone and Mineral Research, along with esteemed advisors from the immunology field, proposed to adopt RANKL as the formal name with RANK as its receptor and OPG as the decoy receptor that binds RANKL and prevents it from activating RANK (American Society for Bone and Mineral Research President’s Committee on Nomenclature, 2000; Fig. 73.1). Shortly after the Immunex group characterized roles for RANK and RANKL in T cells and dendritic cells (Anderson et al., 1997), investigators from Amgen, the Amgen Institute, and the University of Toronto demonstrated that RANKL was a critical mediator of osteoclastogenesis and that the genetic ablation of RANKL caused osteopetrosis in mice (Kong et al., 1999b). RANKL has since been demonstrated to exist in both membrane-bound and soluble forms (Ikeda et al., 2001), and soluble RANKL levels in peripheral blood and bone marrow appear to be regulatable (Abrahamsen et al., 2005; Li et al., 2009), which may allow RANKL to act in an endocrine, juxtacrine, or paracrine manner (Lacey et al., 2012). Osteoclast Stimulation RANK RANKL

Osteoclast Inhibition OPG Denosumab

Calcium

Calcium

Osteoblasts Osteoid Osteoclasts

Resorption (weeks)

Reversal

Formation + Mineralization (months)

FIGURE 73.1 Role of the RANKL pathway and its inhibition on bone remodeling. Bone remodeling occurs as a coupled process of initiation of osteoclastic bone resorption followed by osteoblastic bone formation in the basic multicellular unit as shown here. Osteoclast formation, function, and survival are dependent on the binding of receptor activator of nuclear factorekappa B ligand (RANKL) to its surface receptor (RANK). Prevention of this binding by the naturally occurring protein osteoprotegerin (OPG) and/or the engineered RANKL antibody denosumab results in the disruption of osteoclasts and their formation. This inhibition greatly reduces the rate of bone remodeling, resulting in increased bone mass. Reproduced with Elsevier permission from Dempster et al., 2012. Clin. Ther. 34, 521e536.

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Recognizing the key and essential role of RANKL for bone resorption and bone loss, efforts were initiated to develop inhibitors of the RANKeRANKL pathway using a variety of therapeutic strategies. Investigators at Immunex developed RANK-Fc, which was an effective inhibitor of bone resorption, but preclinical studies indicated that immune responses arising against RANK-Fc had the potential to cause hypercalcemia via the activation of endogenous RANK, leading to the discontinuation of its development (Lacey et al., 2012). At Amgen, Fc-OPG was tested in Phase 1 clinical trials in postmenopausal women. Fc-OPG proved to be an effective inhibitor of bone resorption, with reductions in biochemical markers of bone resorption lasting for several weeks depending on dose (Bekker et al., 2001). In the pursuit of longeracting versions that could be dosed less frequently for patient convenience, Amgen pivoted toward the development of OPG-Fc (AMGN-0007). OPG-Fc had a longer circulating half-life than that of Fc-OPG and also achieved more sustained reductions in bone resorption markers in subjects with breast cancer or multiple myeloma, with inhibitory effects lasting beyond the trial’s 2-month endpoint (Body et al., 2003). However, concerns regarding potential anti-OPG-Fc immune responses that could also adversely affect the bioavailability of endogenous OPG led to the discontinuation of OPG-Fc development in favor of other approaches (Lacey et al., 2012). OPG-Fc remains a commonly used RANKL inhibitor in animal studies due to its ability to recognize and inhibit RANKL from various species including mice (Bonnet et al., 2016), rats (Ominsky et al., 2008), rabbits (Rodeo et al., 2007), cynomolgus monkeys (cynos) (Ominsky et al., 2007), and pigs (Sipos et al., 2011). Pharmacologic strategies were then initiated at Amgen to develop antibodies to inhibit RANKL (Lacey et al., 2012). The XenoMouse (Green, 1999) allowed the rapid generation of high-affinity, fully human monoclonal antiRANKL antibodies including denosumab (Lacey et al., 2012). As outlined below, this RANKL-Ab strategy became the preferred therapeutic approach due to improved pharmacokinetics requiring less frequent administration compared with FcOPG or OPG-Fc, and the lack of potential for adverse immune-mediated perturbations of endogenous OPG or RANK activity. To date, there have been few if any cases of patients developing neutralizing immune responses against the fully human denosumab molecule, and the hypothetical impact of such a response might only involve loss of denosumab efficacy. In addition, unlike OPG, which can bind to TNF family member TRAIL, denosumab does not bind TRAIL or other TNF family members (Kostenuik et al., 2009). Beyond RANK, OPG, and antibody-based approaches to antagonize RANKL, there have been efforts to develop small-molecule (Idris et al., 2010) and peptide-based (Ta et al., 2010) approaches to inhibit RANKL. Although these modalities inhibit bone resorption in vitro and in animals, they have yet to demonstrate clinical efficacy. The section below summarizes some of the preclinical and clinical data generated during the development of the RANKL antibody denosumab.

Physiologic mechanisms and effects of receptor activator of nuclear factorekappa B ligand inhibitors in bone Numerous studies have examined the pharmacologic effects of RANKL inhibitors in healthy animals and in animal models of bone disease. The first such publications demonstrated that Fc-OPG and OPG-Fc inhibited RANKL-induced osteoclast formation in vitro and in vivo (Simonet et al., 1997; Lacey et al., 1998). Subsequent studies in bone disease models including rodent and nonhuman primate models of postmenopausal osteoporosis further demonstrated the impact of RANKL inhibitors on bone turnover, bone mass, bone microarchitecture, bone strength, and bone quality. Rodent studies have generally relied on recombinant RANK-Fc or OPG-based RANKL inhibitors because denosumab does not recognize mouse or rat RANKL (Kostenuik et al., 2009), though a number of pharmacology studies with denosumab were performed in nonhuman primates (Ominsky et al., 2011; Kostenuik et al., 2011, 2015) and in mice genetically engineered to express human or partially humanized RANKL (huRANKL) mice (Kostenuik et al., 2009; Rinotas et al., 2014). The osteoclast-inhibiting effects of RANKL inhibitors are reflected in rapid decreases in biochemical markers of bone resorption followed by a delayed reduction in bone formation markers that reflects the coupled process of bone remodeling. In postmenopausal women with low bone mass, denosumab decreased bone resorption markers compared with placebo within 24 h after a single dose, and bone formation markers showed significant decreases several weeks later (Bekker et al., 2004). In growing male rats, a single injection of recombinant human OPG-Fc resulted in a significant decrease in the resorption marker serum TRACP within 48 h and a decline in the formation marker serum osteocalcin by day 10 (Capparelli et al., 2003). These inhibitory effects on bone turnover were associated with decreases in histological indices of osteoclasts and osteoblasts, effects that reversed over time as OPG-Fc concentrations diminished. Similar rapid declines in bone resorption markers were observed with OPG-Fc in OVX rats (Ominsky et al., 2008) and with denosumab in huRANKL mice (Li et al., 2009). Histomorphometry demonstrated that these changes corresponded to >95% reductions in the extent of trabecular osteoclast surfaces. Similar robust reductions in bone resorption and formation markers were reported in cynos treated with OPG-Fc (Ominsky et al., 2007) and denosumab (Ominsky et al., 2011) in association with marked

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reductions in histomorphometric indices of resorption and formation at trabecular and cortical sites (Kostenuik et al., 2011). Bone resorption and formation in adult animals occur predominantly in a coupled process known as remodeling, with resorption preceding formation as bone matrix is continually removed and replaced. Thus, the delayed reduction in bone formation indices after denosumab-mediated inhibition of bone resorption was expected, reflecting more remodeling spaces having completed their refilling with new bone matrix. In contrast, bone modeling represents temporally and spatially distinct mechanisms by which bone is formed or removed via uncoupled processes. Although bone modeling occurs predominantly during growth, there is evidence for continued bone modeling in adulthood. As denosumab was not thought to affect osteoblasts directly, its effects on bone modeling were investigated in OVX cynos (Ominsky et al., 2015). These results indicated that in the near absence of bone resorption and remodeling-based bone formation, modeling-based bone formation was present in the femur neck and ribs of denosumab-treated monkeys to a similar extent as that found in vehicle-treated controls. The extent of this modeling-based bone formation was small and location-dependent, but its presence and persistence over the 16-month treatment duration supported the hypothesis that bone modeling contributes to the uniquely progressive hip and femoral neck bone mineral density (BMD) gains observed in postmenopausal women receiving denosumab over a 10-year treatment period (Bone et al., 2017; Ominsky et al., 2015). In contrast, bisphosphonates do not induce incremental BMD gains at the hip or femoral neck beyond the first 3e4.5 years of therapy (Reid, 2015). The reductions in bone turnover observed with RANKL inhibitors result in significant gains in bone mass and bone strength in various animal models. In OVX rats, 6 weeks of OPG-Fc resulted in increased in vivo DXA BMD and ex vivo bone volume by micro-CT at the lumbar vertebra and femur neck relative to OVX controls (Ominsky et al., 2008). Trabecular microarchitecture was also improved as reflected in increases in trabecular number and thickness. These improvements resulted in significant elevations in strength endpoint peak load in lumbar vertebrae and the femoral neck. Similar observations were made after 16 months of denosumab administration to OVX cynos, with denosumab-mediated gains in trabecular and cortical bone mass resulting in significantly greater peak load at the femoral neck (up to 34%), L3eL4 vertebral bodies (up to 55%), and L5eL6 cancellous cores (up to 82%) compared with OVX-Veh (Ominsky et al., 2011). In this study, microarchitectural improvements included increased cortical thickness and reduced cortical porosity as well as increased trabecular number. Bone remodeling has long been considered by many to be essential for maintaining bone health via the removal and replacement of older bone matrix; as such, the longer-term effect of antiresorptives on bone quality has been a longstanding concern. Therefore, the demonstration that the near absence of bone turnover with up to 16 months of highdose denosumab therapy led to gains in whole bone strength was reassuring. Linear regressions were performed to assess relationships between bone mass and bone strength variables, as such analyses were previously shown capable of identifying drug-related impairments in bone quality (Lafage et al., 1995). Regression analyses demonstrated that bone quality was maintained at the lumbar vertebra, femur neck, and femur diaphysis after 16 months of denosumab (Ominsky et al., 2011). In addition, calculated material properties from these tests, and destructive testing results for size-matched cortical samples machined from the humeral cortex, confirmed the maintenance of bone quality. A subsequent OVX cyno study verified these findings after 12 months of denosumab while also showing a lack of impaired bone quality measures with the bisphosphonate alendronate (Kostenuik et al., 2015). In addition, finite element strength estimates generated from vertebral CT images from the 16-month study strongly predicted measured bone strength, further supporting the interpretation that bone quality is maintained with denosumab (Lee et al., 2016). Reduced bone remodeling, including that which results from RANKL inhibition, is expected to alter matrix mineralization properties primarily by affording remodeling sites more time to achieve a fuller degree of matrix mineralization. This effect often leads to a reduction in the heterogeneity of matrix mineralization distribution within the bone that some observational studies have associated with bone fragility (Lloyd et al., 2017). Aged OVX rats treated with OPG exhibited a higher average degree of matrix mineralization in femoral cancellous bone along with reduced heterogeneity of mineralization (Valenta et al., 2005), and the same femurs used for those analyses showed improved bone biomechanical properties with OPG treatment (Kostenuik et al., 2001). Vertebrae and femurs from huRANKL mice treated with denosumab or alendronate for 6 months also showed reduced heterogeneity of matrix mineralization, which was associated with improved biomechanical properties compared with vehicle-treated controls (Misof et al., 2011; Ominsky et al., 2008b). The degree of matrix mineralization in the vertebrae of OPG-treated OVX rats bore no relationship with vertebral bone strength, which was significantly increased in close association with increments in bone volume (Ominsky et al., 2008a). Increased matrix mineralization and reduced heterogeneity of mineralization is also observed in postmenopausal women treated with denosumab for up to 10 years (Dempster et al., 2018) in association with persistently reduced fracture rates (Bone et al., 2017). Together, these preclinical and clinical findings lend little support for the theory that potent inhibition

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of bone turnover can impair bone biomechanics via changes in matrix mineralization parameters. Ultimately, even 5 years of denosumab therapy in postmenopausal women caused relatively modest absolute changes in matrix mineralization parameters, with no further changes between 5 and 10 years of therapy, despite extremely and persistently low remodeling rates (Dempster et al., 2018). Such findings may suggest the existence of self-regulatory mechanisms within bone that resist large excursions from biomechanically optimal matrix mineralization characteristics.

Clinical studies demonstrating the effects of denosumab (Table 73.1) Osteoporosis indications Postmenopausal osteoporosis The efficacy and safety of denosumab administered as a 60 mg subcutaneous injection every 6 months was evaluated in several large clinical trials. Based on these data, described below, this regimen was approved by the FDA and EMA (trade name Prolia) for the treatment of postmenopausal women with osteoporosis at high risk for fracture. The pivotal randomized, double-blind, placebo-controlled “FREEDOM” trial underscored the efficacy of denosumab treatment over 3 years to reduce the risk of new vertebral fractures by 68%, nonvertebral fractures by 20%, and hip fractures by 40% compared with placebo in postmenopausal women with osteoporosis (Cummings et al., 2009). Interestingly, the fracture reduction effect was independent of patient characteristics for vertebral fractures but was influenced by baseline body mass index and femoral neck T-score for nonvertebral fractures as depicted by a subgroup analysis (McClung et al., 2012). A post hoc analysis evaluating fracture incidence in women with known risk factors for fractures revealed that denosumab significantly reduced the risk of new vertebral and hip fractures in women at high fracture risk, whereas only new vertebral fractures were significantly reduced in women at low fracture risk (Boonen et al., 2011). Another post hoc analysis showed that the antifracture efficacy of denosumab was not dependent on renal function (Jamal et al., 2011). Long-term data support the antifracture efficacy of denosumab for up to 10 years according to data from the open-label extension of FREEDOM (Bone et al., 2017). Women originally randomized to denosumab (years 1e3) who remained on the drug during the extension (years 4e10) continued to exhibit low yearly rates of new vertebral, nonvertebral, and hip fracture, which were similar to the rates observed in denosumab-treated subjects during the first 3 years of the FREEDOM trial. The effects of denosumab in FREEDOM were largely reproduced in the DIRECT study, which was a randomized, double-blind, placebo- and active comparator-controlled phase 3 trial in Japanese patients with osteoporosis (95% of whom were postmenopausal women) (Nakamura et al., 2014). Over 2 years, a 74% reduction in the risk of new vertebral fracture with denosumab versus placebo was noted, but the study was underpowered to assess differences in nonvertebral fracture risk. The antifracture benefit of denosumab was sustained in Japanese female and male patients with osteoporosis who continued to receive the drug in a 1-year open-label extension of DIRECT, while patients who switched from placebo to denosumab at the start of the extension had fracture rates similar to those of denosumab recipients in the original DIRECT trial (Sugimoto et al., 2015). BMD, as evaluated by DXA, was significantly improved at various skeletal sites (total hip, lumbar spine, femoral neck) with denosumab treatment compared with placebo in the original FREEDOM trial (Bolognese et al., 2013). A substudy of FREEDOM analyzing changes in volumetric BMD as measured by quantitative CT yielded similar results (McClung et al., 2013). Furthermore, in the FREEDOM extension, BMD at the spine, hip, and femoral neck continued to progressively increase over a period of 10 years without reaching a plateau (Bone et al., 2017). Data from the 2-year DIRECT trial (Nakamura et al., 2014) conducted in Japan, and its 1-year extension (Sugimoto et al., 2015), were generally concordant with results of the FREEDOM trial while providing new evidence of a greater reduction in vertebral fracture risk with denosumab compared with open-label alendronate therapy. Several studies have investigated the effect of denosumab compared with bisphosphonates in improving BMD in postmenopausal women with osteopenia or osteoporosis. Denosumab significantly increased BMD at all sites when compared with once-weekly oral alendronate (Brown et al., 2009), once-monthly oral risedronate (Roux et al., 2014), and once-monthly oral ibandronate (Recknor et al., 2013). In a head-tohead study with once-yearly intravenous zoledronic acid in postmenopausal women with osteoporosis who were previously treated with oral bisphosphonates, denosumab significantly increased BMD at all skeletal sites compared with zoledronic acid (Miller et al., 2016). Consistent with improved BMD, various subgroup analyses of the FREEDOM trial documented increased bone strength with denosumab treatment. In a subgroup of 99 patients in whom estimated bone strength was evaluated via QCTbased finite element analysis, denosumab treatment significantly increased bone strength in both trabecular and cortical

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TABLE 73.1 Seminal denosumab clinical studies. Results Phase

n

BTM

BMD

Fx/SRE

References

Treatment of postmenopausal osteoporosis (FREEDOM)

3

7868

Y

[

Y

Cummings et al. (2009)

Treatment of postmenopausal osteoporosisdextension study (FREEDOM extension)

4

2626

Y

[

Y

Bone et al. (2017)

Comparison with alendronate in postmenopausal women with low BMD (DECIDE)

3

1189

Y

[

NA

Brown et al. (2009)

Comparison with zoledronic acid in postmenopausal women with osteoporosis

3

643

Y

[

NA

Miller et al. (2016)

Transition to denosumab after teriparatide treatment in women with postmenopausal osteoporosis (DATA)

4

94

Y

[

NA

Leder et al. (2014, 2015)

Treatment of bone loss in men on androgen-deprivation treatment for nonmetastatic prostate cancer (HALT)

3

1468

NA

[

Y

Smith et al. (2009)

Treatment of bone loss in women on aromatase inhibitors for nonmetastatic breast cancer

3

252

NA

[

NA

Ellis et al. (2008)

Treatment of bone loss in women on aromatase inhibitors for early hormone receptorepositive breast cancer (ABCSG18)

3

3425

NA

[

Y

Gnant et al. (2015)

Denosumab versus zoledronic acid for the treatment of bone metastases in advanced breast cancer

3

2046

Y

NA

Y

Stopeck et al. (2010)

Denosumab versus zoledronic acid for the treatment of bone metastases in castration-resistant prostate cancer

3

1904

Y

NA

Y

Fizazi et al. (2011)

Denosumab versus zoledronic acid for the treatment of bone disease in patients with multiple myeloma

3

1718

NA

NA

Y

Raje et al. (2018)

3

242

Y

[

NA

Orwoll et al. (2012)

3

795

Y

[

NA

Saag et al. (2018)

Name Postmenopausal osteoporosis

Malignant bone disease

Male osteoporosis Treatment of men with low BMD Glucocorticoid-induced osteoporosis Comparison with risedronate in patients with glucocorticoid-induced osteoporosis

BMD, bone mineral density; BTM, bone turnover marker; Fx, fracture; NA, not available; SRE, skeletal-related event. Arrows indicate a significant effect of denosumab versus the comparator group.

departments compared with the effects of placebo (Keaveny et al., 2014). Similar results were noted for estimated strength of the radius (Simon et al., 2013). Furthermore, denosumab treatment over 3 years led to a significant increase in cortical thickness and mass and reduced cortical porosity at the proximal femur, which also correlated with increased bone strength (Zebaze et al., 2016). Nevertheless, these QCT-based findings were not reproduced in histological and micro-CT-based analyses of 112 iliac crest biopsies from FREEDOM, where significant differences between denosumab and placebo with regard to cortical thickness and porosity were not consistently observed (Chapurlat, 2017; Reid et al., 2010). Consistent with the mechanism of action of denosumab in inhibiting osteoclast formation, function, and survival, clinical studies revealed a rapid decrease in serum markers of bone resorption after denosumab administration that was followed by later reductions in serum markers of bone formation (Bekker et al., 2004). In the pivotal FREEDOM trial, denosumab was associated with median reductions of 86%, 72%, and 72% in concentrations of the bone resorption marker C-telopeptide of type 1 collagen at months 1, 6, and 36, respectively. Concentrations of the bone formation marker

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procollagen type 1 N-terminal propeptide decreased by 18%, 50%, and 76% at months 1, 6, and 36, respectively (Cummings et al., 2009). Bone turnover marker reductions were sustained for up to 10 years in patients who continued to receive denosumab treatment in the FREEDOM extension trial (Bone et al., 2017). In 1-year active comparator-controlled trials conducted in postmenopausal women with low bone mass or osteoporosis, denosumab was generally more effective than oral or intravenous bisphosphonates at reducing bone turnover markers (Brown et al., 2009; Roux et al., 2014; Recknor et al., 2013; Miller et al., 2016). These bone turnover findings were mirrored in data obtained from iliac crest biopsies from 92 participants of the pivotal FREEDOM trial, where denosumab reduced histomorphometric measures of bone resorption (e.g., eroded surface, erosion depth, and osteoclast number) and measures of bone formation (osteoid surface, width, and volume) without impairing bone mineralization or microarchitecture (Chapurlat, 2017; Reid et al., 2010). Normal bone microarchitecture and sustained reduced bone turnover were also documented in 22 biopsies of patients who continued denosumab treatment over 10 years in the FREEDOM extension (Bone et al., 2017; Dempster, 2018, p. 39).

Male osteoporosis Although women are more commonly affected by osteoporosis than men, up to 30%e40% of all osteoporotic fractures worldwide occur in men (Johnell and Kanis, 2006), underlining the necessity of screening and treating male patients with osteoporosis. Despite the fact that BMD measurements are less well standardized in men than in women, men with low BMD are at risk for fragility fractures, as shown in the large prospective analysis of the MrOS cohort (Cummings et al., 2006). Most pharmacological treatments for male osteoporosis have been initially approved for postmenopausal osteoporosis and then replicated in smaller randomized controlled trials in men, with change in BMD as a primary endpoint (Gennari and Bilezikian, 2018). The study that led to the approval of denosumab (Prolia) by the FDA and EMA for the treatment of male osteoporosis was a phase 3 randomized-controlled trial in men with low BMD that showed significant increases in BMD at the lumbar spine and total hip region (5.7% and 2.4% increases, respectively, at 12 months) versus placebo (Orwoll et al., 2012). Although antiresorptive drugs are generally currently recommended as first line pharmacotherapy in male osteoporosis (Watts et al., 2012), considering that primary male osteoporosis is predominantly characterized by impaired bone formation, a case for the early application of osteoanabolic substances can be made (Gennari and Bilezikian, 2013).

Use of denosumab in combination/sequence with other osteoporosis agents The sequence in which osteoporosis therapies are utilized in patients can be critical to their impact on BMD (Cosman et al., 2017) and transitions to and from denosumab have been tested in numerous studies. The transition from alendronate to denosumab has been examined in both OVX cynos and humans. In OVX cynos that initially received 6 months of ALN, transition to denosumab further decreased bone resorption and cortical porosity, and increased BMD and bone strength, without deleterious effects on serum calcium or bone quality (Kostenuik et al., 2015). In postmenopausal women receiving alendronate for at least 6 months, transition to denosumab also resulted in further decreases in bone turnover by serum biomarkers and histomorphometry, while BMD increased (as measured by DXA) and cortical porosity decreased (as measured by HRpQCT) (Reid et al., 2010; Kendler et al., 2010; Zebaze et al., 2014). A separate clinical study demonstrated that transitioning from alendronate to denosumab yielded larger BMD changes than transitioning from alendronate to zoledronic acid (Miller et al., 2016). Some of these differences have been attributed to greater inhibition of intracortical remodeling with denosumab; this greater inhibition may reflect denosumab’s effects on osteoclastogenesis and may also relate to the limited uptake of bisphosphonates in dense cortical bone, which could contribute to the more pronounced effects of denosumab in the cortex (Zebaze et al., 2014). Transition from the anabolic agent teriparatide to denosumab was also examined, as antiresorptive therapy is generally thought to be necessary to maintain bone mass gains after teriparatide therapy is discontinued. In the DATA study, the transition to 24 months of denosumab after 24 months of teriparatide resulted in positive gains in BMD at all sites examined (Leder et al., 2015) in association with improvements in finite elementeestimated bone strength at the distal tibia and radius as evidenced by HRpQCT (Tsai et al., 2017). In the same study, the transition from denosumab to teriparatide was detrimental to estimated bone strength at these sites, further demonstrating the importance of sequence in the use of osteoporosis therapies. The addition of denosumab to teriparatide as a combination therapy was also examined in the DATA study. Previous studies suggested that the addition of antiresorptive agents (oral bisphosphonates) did not consistently improve BMD when combined with parathyroid hormonedPTH(1e84) and/or teriparatide, PTH(1e34)din postmenopausal women

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(Black et al., 2003) or men (Finkelstein et al., 2003). In contrast, the DATA study demonstrated that the combination of denosumab and teriparatide for 24 months increased BMD to a significantly greater extent than either monotherapy alone (Leder et al., 2014). Similar findings were observed in rodent models when RANKL inhibitors were combined with PTH (Samadfam et al., 2007; Kostenuik et al., 2001; Pierroz et al., 2010). Transition from denosumab to other antiresorptives has also been examined because most BMD gains are lost within the first 12 months after denosumab discontinuation if no follow-on osteoporosis therapy is taken (Bone et al., 2011; Miller et al., 2008; Popp et al., 2018), as discussed below. In general, follow-on bisphosphonate therapy after denosumab discontinuation appears to prevent or partially mitigate bone loss (Freemantle et al., 2012; Lehmann and Aeberli, 2017; Reid et al., 2017), though the effects of these and other followon therapies on fracture risk remain unknown.

Cancer indications Denosumab for cancer treatmenteinduced bone loss Maintenance of bone health and strength is a continuous challenge in the adjuvant setting of cancer patients. In particular, patients with prostate or breast cancer receiving sex hormone ablation therapies are prone to excessive bone loss and osteoporotic fractures (Hadji et al., 2017; Adler, 2011), and several studies have addressed the ability of denosumab at 60 mg every 6 months to preserve or increase bone mass and reduce fracture risk in such patients. Based on the data below, Prolia was approved by the FDA and EMA as a treatment to increase bone mass in men at high risk for fracture who are receiving androgen deprivation therapy for nonmetastatic prostate cancer and in women at high risk for fracture who are receiving adjuvant aromatase inhibitor therapy for breast cancer. Androgen deprivation in men with prostate cancer In men with prostate cancer undergoing adjuvant hormone ablation therapy via gonadotropin-releasing hormone analogues, denosumab increased lumbar spine BMD by 5.6% compared with a loss of 1% in the placebo group after 24 months. In addition, vertebral fractures were significantly reduced in the denosumab group (1.5%) compared with placebo (3.9%) after 36 months (Smith et al., 2009). Aromatase inhibitors in women with breast cancer Aromatase inhibitors are commonly used as adjuvant hormone ablation therapies for the treatment of hormone receptorepositive breast cancer in postmenopausal women. While aromatase inhibitors significantly improve the prognosis of affected patients (Burstein et al., 2014), they exert distinct negative effects on bone mass and increase the risk of fractures (Hadji, 2015). Denosumab was the first osteoporosis drug to undergo clinical trials to specifically assess its efficacy in fracture risk reduction in breast cancer patients treated with aromatase inhibitors, which is a growing population. In an initial smaller trial with 252 patients, denosumab was shown to have positive effects on BMD compared with placebodBMD at the lumbar spine increased by 5.5% and 7.6% in the denosumab group compared with placebo after 12 and 24 months, respectively, and denosumab also increased BMD at all other measured sites (Ellis et al., 2008). The larger ABCSG-18 phase 3 trial (n ¼ 3425 subjects) was designed to assess the effects of adjuvant denosumab versus placebo on fracture risk reduction in postmenopausal women with early hormone-responsive breast cancer receiving aromatase inhibitor treatment. Denosumab reduced the number of fractures compared with placebo (92 vs. 172) and significantly delayed the time to first clinical fracture. Notably, fracture risk reduction was observed independent of baseline T-score, with significant reductions in the patient subgroup with a T-score of 1 or higher as well as in the subgroup with a T-score lower than 1 (Gnant et al., 2015).

Treatment of hypercalcemia of malignancy refractory to bisphosphonate therapy Hypercalcemia of malignancy is a complication of patients with advanced cancer that denotes a poor prognosis. It is caused by enhanced osteoclast activity through either a humoral mechanism or local cancer-induced osteolysis. Optimal therapy requires treatment of the underlying malignancy, but in case of extensive disease a palliative approach to alleviate patients’ symptoms is warranted. In addition to the initial correction of volume depletion through intravenous saline infusions and loop diuretics to promote renal calcium excretion, intravenous bisphosphonates have been the treatment of choice (Stewart, 2005). Alternatively, in cases of severe renal insufficiency or refractory hypercalcemia despite bisphosphonate use, denosumab was shown to be effective in lowering serum calcium in a single-arm international study (Hu et al., 2014). That study involved the subcutaneous administration of 120 mg denosumab on days 1, 8, 15, and 29 and then every 4 weeks to

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subjects with BP-refractory hypercalcemia of malignancy. Denosumab reduced serum calcium in 64% of patients within 10 days, with an estimated median response duration of 104 days. Based on those findings, denosumab (XGEVA) received regulatory approval from the FDA under an orphan drug designation for the treatment of BP-refractory hypercalcemia of malignancy. Breast cancer Bone metastases are a frequent and dire complication in breast cancer patients with advanced disease, leading to a variety of SREs that include pathological fractures, radiation and/or surgery to bone, and spinal cord compression (Irelli et al., 2016). A phase 3 noninferiority study in patients with breast cancer and bone metastases assessed the effects of monthly denosumab (120 mg, n ¼ 1026) or zoledronic acid (4 mg, n ¼ 1020), with the primary endpoint being the time to first onstudy SRE (Stopeck et al., 2010). Denosumab was shown to be noninferior and also superior to zoledronic acid in delaying the time to first on-study SRE. Denosumab was associated with fewer renal adverse events and fewer acute-phase reactions compared with zoledronic acid, whereas there were no significant differences in overall survival, breast cancer disease progression, or the occurrence of ONJ between the two treatment groups (Stopeck et al., 2010). Prostate cancer Bone metastases in men with prostate cancer are also associated with substantial morbidity and significant adverse impacts on health-related quality of life (Saad et al., 2017). A phase 3 trial was conducted in men with castration-resistant prostate cancer and bone metastases who were randomized to receive denosumab (n ¼ 950) or zoledronic acid (n ¼ 951), with the primary endpoint being the time to first on-study SRE (Fizazi et al., 2011). Denosumab significantly improved the time to first on-study SRE by 3.6 months compared with zoledronic acid. Hypocalcemia was more common in the denosumab group, while osteonecrosis of the jaw (ONJ) occurred at similarly infrequent rates in the denosumab (2%) and zoledronic acid (1%) arms (Fizazi et al., 2011). Preclinical data suggest that at least some human cancer cells express functional RANK and that RANKL can promote the migration and invasion of various cancer cell types including prostate cancer cells (Jones et al., 2006; Mori et al., 2007), suggesting a potential role for RANKL in the promotion of metastasis, and bone metastasis in particular. A phase 3 trial was conducted to assess the ability of denosumab to prevent bone metastases in men with nonmetastatic castration-resistant prostate cancer at high risk of developing bone metastases (Smith et al., 2012). Denosumab improved bone metastasesefree survival by 4.2 months compared with placebo (median 29.5 vs. 25.2 months). Overall survival remained unaffected by the treatment. ONJ developed in 5% of patients on denosumab compared with none on placebo (Smith et al., 2012). While these results suggest efficacy of denosumab in preventing bone metastases, there is currently no FDA- or EMA-approved indication for the adjuvant treatment of prostate cancer patients with denosumab. Other solid tumors A third phase 3 trial compared denosumab and zoledronic acid in patients with bone metastases secondary to solid tumors (excluding breast and prostate) and myeloma (Henry et al., 2011). The primary endpoint was time to first on-study SRE, and denosumab proved noninferior to zoledronic acid in delaying time to first SRE. There were no differences in overall survival between the groups. A subanalysis of the lung cancer cohort within this trial revealed a significant survival benefit for patients with any form of lung cancer receiving denosumab, with an overall survival of 8.9 months compared with 7.7 months for those receiving zoledronic acid. Survival benefits were more pronounced in the non-small-cell lung cancer group (9.5 vs. 8.0 months), with the most pronounced effect on overall survival seen in patients with squamous cell carcinoma (8.6 vs. 6.4 months) (Scagliotti et al., 2012). Multiple myeloma Osteolytic lesions are a hallmark of multiple myeloma, which can also lead to SREs. In contrast to solid tumors, multiple myeloma is a hematological disease, and as such the classic metastatic pathway is not required for bone manifestations to occur. For the assessment of denosumab, patients with multiple myeloma were first included in the above-mentioned trial in combination with patients who had bone metastases secondary to solid tumor malignancies other than prostate or breast (Henry et al., 2011). While the primary endpoint of this trial, reduction in first on-trial SRE, was successfully reached by showing noninferiority over zoledronic acid, an ad hoc analysis of the myeloma subgroup (n ¼ 180) revealed a lower overall survival in the patients who received denosumab compared with those who received zoledronic acid. Based on these findings, denosumab was approved by the FDA and EMA for the treatment of bone metastases from solid tumors but not multiple myeloma.

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Further assessment of the myeloma subgroup revealed imbalances between the groups with regard to baseline risk characteristics such as ECOG score, renal function, and form of treatment, such as stem cell transplantation. A higher rate of early withdrawals was also noted in the zoledronic acid group (Raje et al., 2016). These results prompted the initiation of a larger (n ¼ 1718) trial specific to patients with multiple myeloma. Patients were randomized to receive denosumab (n ¼ 859) or zoledronic acid (n ¼ 859), and the primary endpoint of noninferiority of denosumab compared with zoledronic acid was met. Adverse events were comparable between groups, although fewer renal treatment-emergent adverse events were observed in the denosumab group. ONJ occurrence was 4% and 3% in the denosumab and zoledronic acid groups, respectively. Based on these results, denosumab was approved by the FDA and EMA in early 2018 for the treatment of skeletal complications arising from multiple myeloma. Giant cell tumors GCTBs are benign tumors associated with an osteolytic phenotype and substantial skeletal morbidity (Sobti et al., 2016). In a phase 2 trial, 120 mg of denosumab was given to 37 patients with unresectable or recurrent GCTBs at days 1, 8, 15, and 28 followed by additional doses every 4 weeks. A tumor response was observed in 30 of 35 patients (Thomas et al., 2010). In a larger subsequent trial, 163/169 (96%) of surgically unsalvageable GCBT patients had no disease progression after a median follow-up 13 months after denosumab administration. In a separate cohort of this study, 74% (74/100) of patients with salvageable GCTBs whose surgery was associated with severe morbidity did not require surgery a median of 9.2 months after denosumab initiation; 62% (16/26) of those that did require surgery underwent a less extensive procedure than initially planned prior to therapy. Osteonecrosis occurred in 1% of all patients (Chawla et al., 2013).

Denosumab for the treatment of metastatic bone disease, multiple myeloma, and giant cell tumors The efficacy and safety of denosumab has been extensively studied in patients with malignancy-associated bone lesions administered denosumab subcutaneously at a dose of 120 mg every 4 weeks. Based on the data described below, the FDA and EMA approved this regimen of denosumab (trade name XGEVA) for the prevention of skeletal-related events (SREs) in patients with multiple myeloma and those with bone metastases from solid tumors as well as for the treatment of adults and skeletally mature adolescents with giant cell tumor of bone (GCTB) that is unresectable or where surgical resection is likely to result in severe morbidity. For the latter indication, additional 120 mg doses are administered subcutaneously on Days 8 and 15 of the first month of therapy.

Additional denosumab data Glucocorticoid-induced osteoporosis Glucocorticoids exert manifold adverse effects on bone tissue, and while physiological concentrations of glucocorticoids are indispensable for bone accrual and growth, supraphysiological concentrations, either endogenously or more commonly iatrogenically, have detrimental effects on bone (Hofbauer and Rauner, 2009). Glucocorticoids dose-dependently inhibit the differentiation and proliferation of cultured osteoblasts, promote apoptosis of cultured osteoblasts and osteocytes, and slow the rate of bone matrix mineralization. In addition, glucocorticoids transiently enhance the activity of osteoclasts through upregulation of RANKL (Hofbauer and Rauner, 2009). In vitro data show that the treatment of osteoblast-like cells with glucocorticoids upregulates their expression of RANKL and downregulates OPG expression (Hofbauer et al., 1999). Denosumab was shown to prevent reductions in bone mass and bone strength in a mouse model of glucocorticoid-induced osteoporosis (GIOP) (Hofbauer et al., 2009). Antiresorptive and osteoanabolic drugs constitute the current therapeutic options for patients with GIOP. Clinical studies show that bisphosphonates are effective in reducing the occurrence of fractures in patients with GIOP to a degree comparable to that observed in trials of patients with postmenopausal osteoporosis (Kanis et al., 2007). A recently published double-blind, active-controlled study investigated the efficacy of denosumab compared with risedronate in patients with osteoporosis either initiating or continuing glucocorticoid treatment (Saag et al., 2018). At 12 months, treatment with denosumab (60 mg every 6 months) led to a significant BMD increase at the lumbar spine (4.4% vs. 2.3%) and total hip (2.1% vs. 0.6%) compared with risedronate. Based on these results, denosumab was approved by the FDA and EMA in 2018 for the treatment of bone loss associated with long-term systemic glucocorticoid therapy.

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Rheumatoid arthritis Inflammation in arthritic joints leads to joint destruction, in part through increased RANKL expression by activated T cells (Kong et al., 1999a). Therefore, the effects of RANKL inhibitors have been examined in multiple models of rheumatoid arthritis (RA) and in clinical trials of patients with RA. In adjuvant- and collagen-induced arthritis models in rats, RANKL inhibition via OPG treatment inhibited bone loss in arthritic joints without affecting local or systemic inflammation parameters (Kong et al., 1999a; Romas et al., 2002; Stolina et al., 2009). In clinical studies, denosumab was effective at inhibiting the progression of bone erosions (via Sharp erosion score) in RA patients receiving methotrexate, though denosumab did not affect joint space narrowing or RA disease activity (Cohen et al., 2008; Takeuchi et al., 2016, 2017). The combined use of RA biologics and denosumab did not lead to greater rates of serious infection in this patient population (Lau et al., 2018), which had been a potential concern based on results from the FREEDOM trial in postmenopausal women with osteoporosis, as described below. Based on these studies, in 2017 Daiichi Sankyo received regulatory approval from Japan’s PMDA for the use of denosumab to inhibit the progression of bone erosions associated with RA. Although the pathogenesis of osteoarthritis (OA) differs somewhat from that of RA, the OPG/RANKL axis has also been implicated in OA. Early changes in subchondral bone have also been hypothesized to contribute to OA joint pathology, and therefore the prevention of these changes via RANKL inhibition may affect OA progression. OPG was shown to ameliorate pain in the monosodium iodoacetate OA rat model but did not improve pathology in joints with established damage (Sagar et al., 2014). Clinical data of the effects of denosumab on OA are limited, though at least one clinical trial is ongoing to examine denosumab’s effects on erosive OA (NCT02771860).

Other potential applications The effects of RANKL inhibitors have been examined in other conditions affecting bone including immobilization-induced bone loss (disuse osteopenia), periprosthetic osteolysis, fracture repair, and osteogenesis imperfecta (OI). Mechanical loading affects RANKL expression in bone; increased loading-induced strain reduced RANKL expression in marrowderived stromal cell cultures (Rubin et al., 2000), while in vivo studies indicate that immobilization resulted in increased RANKL expression in murine bone (Aliprantis et al., 2012). Inhibition of RANKL by genetic modification (Xiong et al., 2011) or administration of OPG (Bateman et al., 2000) protected against disuse osteopenia in mice. Similarly, OPG ameliorated bone loss due to sciatic nerve damage (Bateman et al., 2001), transient muscle paralysis (Aliprantis et al., 2012), and space flight (Lloyd et al., 2015). Limited clinical data are available regarding the effects of denosumab on bone mass in immobilized patients, though clinical studies are ongoing (NCT01983475 and NCT03029442). One year of denosumab reduced bone turnover markers and increased BMD in a cohort of 14 patients with recent spinal cord injury (mean: 15 months postinjury) compared with their baseline values (Gifre et al., 2016). Aseptic loosening due to wear debriseinduced osteoclastic bone resorption is the most common cause of implant failure, and increased production of RANKL likely plays a critical role in this process (Haynes et al., 2001). Administration of RANKL inhibitors was able to prevent wear debris osteolysis in a mouse calvarial model (Childs et al., 2002; Tsutsumi et al., 2008) and improve screw fixation strength in a nonosteolysis model in rats (Bernhardsson et al., 2015). Initial results from a clinical trial of denosumab in postmenopausal women undergoing cementless total hip replacement indicated that although treatment ameliorated the loss of periprosthetic BMD, it did not prevent stem migration or improve functional recovery indices (Aro, 2017). These data remain preliminary and did not specifically examine the effect of denosumab in patients with aseptic loosening, which remains undetermined. A small clinical study in patients with established aseptic loosening is ongoing (NCT02299817). Bone resorption plays a key role in the fracture repair process, primarily through callus remodeling that restores the fractured bone’s original mass and shape. Because denosumab is primarily used in patients at increased risk of fracture, it is important to understand the effect of RANKL inhibitors on fracture healing. Studies in rodent models indicated that RANKL inhibitors do not impair fracture union or reduce callus structural strength (Gerstenfeld et al., 2009; Delos et al., 2008; Ulrich-Vinther and Andreassen, 2005). High-dose denosumab administration to human RANKL knock-in mice with a closed femoral fracture led to near-total ablation of callus osteoclasts that was predictably associated with delayed callus remodeling, a finding also seen in fractured rodents receiving other RANKL inhibitors (Gerstenfeld et al., 2009; Delos et al., 2008; Ulrich-Vinther and Andreassen, 2005). Delayed callus remodeling in denosumab-treated mice was associated with greater callus structural strength (Gerstenfeld et al., 2009). A subset of patients that experienced nonvertebral fractures during the FREEDOM study showed no indication of increased incidence of delayed healing or nonunion in the denosumab group (Adami et al., 2012).

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OI is a genetic disorder that results in increased bone fragility. The effects of RANKL inhibitors have been examined in animals and humans with OI. In young oim/oim mice treated from 2 to 14 weeks of age, RANK-Fc improved bone microarchitecture and decreased fracture incidence compared with controls (Bargman et al., 2012). To date, clinical reports of denosumab use in OI patients have been limited, though current data indicate that lumbar spine BMD is increased (Li et al., 2018). In children with OI type IV treated with 1 mg per kg body mass denosumab every 3 months, hypercalcemia and hypercalciuria ensued, indicating that the dosing strategy may need adjustment in rapidly growing children with this unique form of OI (Trejo et al., 2018). A larger Amgen-sponsored clinical study of denosumab in OI patients is ongoing (NCT02352753). Bone marrow edema syndrome is a painful and often difficult-to-treat condition characterized by an increase of interstitial fluid within bone. Since this “bone bruise” condition is accompanied by local bone resorption (Thiryayi et al., 2008), the use of an antiresorptive agent seems reasonable. Intravenous bisphosphonates were shown to be effective in reducing bone marrow edema in professional athletes (Simon et al., 2014). More recently, denosumab was used in a caseseries of 14 patients with bone marrow edema, with an overall treatment success rate of 93% based on MRI findings (Rolvien et al., 2017). Denosumab also has been recently used to treat alveolar bone destruction, which characterizes periodontitis. In a rodent model of periodontitis, systemic delivery of OPG-Fc preserved alveolar bone volume and decreased bone resorption markers (Jin et al., 2007). Similarly, rats treated with OPG-Fc displayed enhanced postorthodontic tooth stability (Hudson et al., 2012).

Denosumab safety As with any drug, the clinical benefits of RANKL inhibitors should be weighed against their potential risk. The warnings and precautions section of the US label for Prolia and XGEVA indicate potential risks including hypocalcemia, ONJ, atypical subtrochanteric femoral fractures (AFFs), multiple vertebral fractures following discontinuation, hypersensitivity, serious infections, dermatologic adverse reactions, and musculoskeletal pain (Prolia/XGEVA PI).

Hypocalcemia Decreases in serum calcium with denosumab secondary to decreased bone resorption may lead to or exacerbate hypocalcemia (Prolia/XGEVA PI). Higher baseline bone formation may contribute to hypocalcemia after denosumab therapy is initiated (Kinoshita et al., 2016; Kostenuik et al., 2015). Clinical monitoring of serum calcium is recommended within 14 days of denosumab administration for patients predisposed to hypocalcemia or altered mineral metabolism, including patients with renal impairment. Denosumab is contraindicated in patients with preexisting hypocalcemia. In contrast, discontinuation of denosumab in XGEVA-treated patients with growing skeletons should involve monitoring for hypercalcemia, which has been reported.

Osteonecrosis of the jaw ONJ has been reported in patients taking Prolia and XGEVA, with a higher incidence reported for XGEVA. ONJ, which can occur spontaneously, is generally associated with tooth extraction and/or local infection with delayed healing. Therefore, a dental examination is recommended prior to treatment in patients who may require such procedures or have other risk factors that may predispose them to ONJ (Prolia/XGEVA PI). During the long-term extension of the FREEDOM trial in postmenopausal women with osteoporosis, in which all subjects received open-label denosumab, 12 cases of ONJ occurred, all but 1 of which followed invasive oral dental procedures or events comprising scaling or root planning, tooth extraction, dental implant, natural tooth loss, or jaw surgery. A total of 11 subjects developed ONJ among the 1621 subjects who underwent or experienced such procedures or events, for a rate of 0.7%, compared with 1 ONJ case among the 1970 subjects who did not undergo or experience such procedures or events (0.05%) (Watts et al., 2017). The pathophysiology of the increased incidence of ONJ is not clear, though increased ONJ risk is also associated with other antiresorptive agents (i.e., bisphosphonates) (Khan et al., 2017). The recent development of nonclinical models demonstrating induction of ONJ in the presence of periapical disease may provide additional insights (Aghaloo et al., 2014).

Atypical femoral fractures AFFs have also been reported with denosumab (Prolia/XGEVA PI). First reported in patients receiving bisphosphonates, these transverse fractures occur in the femoral diaphysis below the lesser trochanter and are associated with minimal

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trauma or comminution. Prodromal pain and bilaterality are frequent or occasional features of AFFs, and therefore patients on denosumab who experience new thigh or groin pain should seek medical attention, and the contralateral limb should be assessed should an AFF occur. The pathophysiology of these fractures is not clear, though the evidence suggests they may occur as stress or insufficiency fractures (Shane et al., 2014). Large-scale studies have reported that most radiographically confirmed AFFs occur in patients who were never dispensed any potent antiresorptive therapies (Feldstein et al., 2012), and that long-term bisphosphonate use is not associated with an overall increased risk of fractures in the specific femoral region where AFFs occur (Abrahamsen et al., 2016). These observations have led to suggestions that potent antiresorptives may lead to an atypical radiographic fracture pattern without necessarily causing the fracture itself (Feldstein et al., 2012). In support of that possibility, tibiae from OPG-treated mice subjected to destructive biomechanical testing exhibited more transverse and distal failure patterns than vehicle-treated mice, and these shifts were associated with greater, not lesser, bone strength (Bonnet et al., 2016).

Denosumab discontinuation: effects on bone turnover, bone mass, and fracture risk Cessation of therapy with denosumab is associated with rapid reversal of BMD gains and an increase in bone turnover markers to above pretreatment baseline levels from around month 8 to around month 24 after the last denosumab dose, followed by a return of turnover markers to baseline levels (Bone et al., 2011; Miller et al., 2008). BMD levels also tend to return to pretreatment baseline levels after discontinuing denosumab but generally remain above the levels of patients who never received denosumab (Bone et al., 2011; Miller et al., 2008). Recent post hoc analyses of patients from FREEDOM and its open-label extension indicate that for subjects who discontinue denosumab without any follow-on osteoporosis therapy, overall fracture risk resumes at levels similar to those of subjects who never received denosumab. One analysis showed that for subjects in FREEDOM who discontinued placebo or denosumab after having received at least two doses of study drug and continued to participate in the study for at least 7 months after the last dose received, the exposure-adjusted rates of new vertebral fractures were 9.3 vs. 5.6 per 100 subject-years, respectively (Brown et al., 2013). A subsequent analysis wherein subjects from the FREEDOM extension study were also included reported similar exposure-adjusted rates of new or worsening vertebral fractures after discontinuing denosumab or placebo (Cummings et al., 2018). However, the latter study also reported that the rate of multiple new or worsening vertebral fractures was somewhat higher for subjects discontinuing denosumab than for those who discontinued placebo (4.2 vs. 3.2 per 100 subject-years, respectively). And among subjects from FREEDOM and its extension who experience at least one new off-treatment vertebral fracture, 60.7% of those who discontinued denosumab experienced multiple vertebral fractures, compared with 34.5% of such subjects who discontinued placebo (Cummings et al., 2018). Several published case series have also described multiple vertebral fractures in patients discontinuing denosumab (Popp et al., 2016; Aubry-Rozier et al., 2016; Anastasilakis et al., 2017). Accordingly, professional committees and national guidelines have issued statements advising against cessation of denosumab without an alternative treatment, especially in patients at high fracture risk (Tsourdi et al., 2017; Meier et al., 2017). Mechanisms underlying the risk of multiple vertebral fractures after denosumab discontinuation are unclear, but a cogent pathophysiological hypothesis should reconcile with observations that overall vertebral and nonvertebral fracture risks were similar after discontinuing denosumab or placebo. One potential hypothesis relates to the potential for perturbed spinal alignment after an index off-treatment (or prevalent) vertebral fracture. Altered spinal alignment in subjects with vertebral fractures has been associated with increased mechanical loading on the other vertebrae (Briggs et al., 2006), and for subjects experiencing high turnover after discontinuing denosumab therapy, those increased mechanical loads may act on vertebrae that bear microstructural hallmarks of high bone turnover, such as more extensive resorption cavities and trabecular disconnections. Microstructural features of high turnover can be particularly deleterious to bone strength when placed in regions of higher strain or lower bone volume (Hernandez et al., 2006; Slyfield et al., 2012). This hypothesis suggests that the risk of multiple vertebral fractures after denosumab discontinuation may abate to some degree as transiently higher turnover subsides and biomechanically detrimental resorption cavities are refilled. On the other hand, index or prevalent vertebral fractures may continue to adversely influence spine alignment and vertebral biomechanics independent of bone turnover rates, which could represent an ongoing risk for additional vertebral fractures.

Hypersensitivity, serious infections, and musculoskeletal pain Hypersensitivity has been reported with denosumab use, and treatment should be discontinued if clinically significant allergic reactions occur. Increased risk of serious infection that required hospitalization including endocarditis and infections of the skin, abdomen, urinary tract, and ear were reported more frequently in the Prolia group in the FREEDOM

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trial (Prolia PI). Higher rates of skin reactions such as dermatitis, eczema, and rashes also occurred with Prolia in FREEDOM. Severe bone, joint, and/or muscle pain has also been reported in patients taking Prolia.

Use in women of reproductive age Denosumab is contraindicated in patients who are pregnant based on data from animal studies suggesting that denosumab may cause fetal harm (Prolia PI/XGEVA). Administration of high-dose denosumab to cynos throughout pregnancy resulted in increased fetal loss, stillbirths, and postnatal mortality with absent peripheral lymph nodes, abnormal bone growth, and decreased neonatal growth. Some of these abnormalities improved following a recovery period from birth through 6 months of age (Boyce et al., 2014). Nonetheless, for women of reproductive age receiving denosumab, contraception is recommended for at least 5 months after the last dose.

Theoretical impact of receptor activator of nuclear factorekappa B ligand inhibition on insulin resistance and vascular calcification Additional animal studies have suggested the potential for RANKL inhibitors to affect other pathological processes including insulin resistance and vascular calcification. In a series of experiments in mouse models of type 2 diabetes, genetic inhibition of RANKL signaling or OPG administration improved insulin sensitivity and glucose metabolism (Kiechl et al., 2013). These data were supported by the authors’ observation that high serum RANKL concentration was as an independent risk predictor of T2DM in humans. A separate set of studies demonstrated that RANKL inhibitors could induce b-cell proliferation in vitro and in vivo (Kondegowda et al., 2015). A post hoc analysis of the FREEDOM study did not demonstrate a clear effect of denosumab on fasting serum glucose in postmenopausal osteoporotic women with prediabetes or diabetes; however, some improvement was observed in a subset of diabetic patients not receiving antidiabetic medication (Napoli et al., 2018). Early genetic studies suggested a link between RANKL signaling and vascular calcification based on the presence of calcified arteries in osteoporotic OPG-deficient mice (Bucay et al., 1998). A subsequent study demonstrated that denosumab reduced aortic calcium deposition in mice receiving prednisolone (Helas et al., 2009). A post hoc analysis of the FREEDOM study did not show an effect of treatment on the progression of aortic calcification (by lateral spine X-ray) or the incidence of cardiovascular adverse events, compared with placebo (Samelson et al., 2014).

Summary Denosumab is an effective therapeutic agent for increasing BMD and reducing vertebral and nonvertebral fracture risk in patients with postmenopausal osteoporosis, glucocorticoid osteoporosis, and male osteoporosis. Denosumab also delays SREs, reduces the onset of bone metastases, and prevents fractures in patients with bone metastases from breast and prostate cancer. Denosumab’s subcutaneous mode of administration renders it a practical and well-tolerated drug because it circumvents the gastrointestinal tract, and no infusion equipment or facility is required for denosumab administration. A further advantage lies in the possible utilization of denosumab in patients with renal insufficiency, a common condition in the elderly and/or patients with cancer. Denosumab displays a favorable safety profile, and the risks of hypocalcemia and ONJ can be reduced by preventive measures. Since denosumab is not deposited in the skeleton, there is no sustained effect on bone metabolism after its discontinuation. Therefore, regular denosumab administration during therapy is warranted, and a long-term therapeutic strategy is recommended if denosumab is discontinued, particularly in patients at increased risk for fracture.

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1710 PART | III Pharmacological mechanisms of therapeutics

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