Denosumab - a new option in the treatment of osteoporosis

Denosumab is the international name of a human, monoclonal antibody approved for the treatment of osteoporosis. This antibody is associated with RANK ligand (RANKL), inactivating it. In consequence, the formation and survival of osteoclasts are suppressed, leading to their apoptosis. All this results in lower bone resorption, while bone mineral density (BMD) increases. Denosumab also reduces the risk of vertebral and non-vertebral fractures. This agent is similarly effective in various stages of renal function impairment; it does not impair fracture healing processes nor contribute to atherosclerosis progression in patients with high cardiovascular risks. Following an analysis of adverse effects, performed in the FREEDOM study (in which it was demonstrated that the incidence of the majority of adverse effects observed in the course of denosumab use was similar to that in the placebo group), its safety for patients can definitely be confirmed.     

Denosumab--a new option in the treatment of osteoporosis

Denosumab is the international name of a human, monoclonal antibody approved for the treatment of osteoporosis. This antibody is associated with RANK ligand (RANKL), inactivating it. In consequence, the formation and survival of osteoclasts are suppressed, leading to their apoptosis. All this results in lower bone resorption, while bone mineral density (BMD) increases. Denosumab also reduces the risk of vertebral and non-vertebral fractures. This agent is similarly effective in various stages of renal function impairment; it does not impair fracture healing processes nor contribute to atherosclerosis progression in patients with high cardiovascular risks. Following an analysis of adverse effects, performed in the FREEDOM study (in which it was demonstrated that the incidence of the majority of adverse effects observed in the course of denosumab use was similar to that in the placebo group), its safety for patients can definitely be confirmed.     

Clinical development of anti-RANKL therapy

The receptor activator of nuclear factor-kappaB ligand (RANKL), its cognate receptor RANK, and its natural decoy receptor osteoprotegerin have been identified as the final effector molecules of osteoclastic bone resorption. This has provided an ideal target for therapeutic interventions in metabolic bone disease. As described in previous reviews in this supplement, RANKL signaling is required for osteoclast differentiation, activation, and survival. Furthermore, in vivo inhibition of RANKL leads to immediate osteoclast apoptosis, and there are no in vivo models of bone resorption that are refractory to RANKL inhibition. Thus, the only step remaining in the development of a clinical intervention is the generation of a safe, effective, and specific drug that can inhibit RANKL in humans. Here we review the clinical development of denosumab (formerly known as AMG 162), which is a fully human mAb directed against RANKL. This discussion includes the breadth of 21 human studies that have led to the current phase 3 clinical trials seeking approval for use of this agent to treat postmenopausal women with low bone mineral density (osteoporosis) and patients with metastatic lytic bone lesions (multiple myeloma, and prostate and breast cancer).     

First use of the RANKL antibody denosumab in Osteogenesis Imperfecta Type VI

Osteogenesis imperfecta (OI) is a genetically heterogeneous disease leading to bone fragility. OI-VI is an autosomal-recessive form caused by mutations in SERPINF1. There is experimental evidence suggesting that loss of functional SERPINF1 leads to an activation of osteoclasts via the RANK/RANKL pathway. Patients with OI-VI show a poor response to bisphosphonates. We report on four children with OI-VI who had shown continuously elevated urinary bone resorption markers during a previous treatment with bisphosphonates. We treated these children with the RANKL antibody denosumab to reduce bone resorption. Intervention and results: Denosumab (1 mg/kg body weight) was injected s.c. every 3 months. There were no severe side effects. Markers of bone resorption decreased to the normal range after each injection. N-terminal Propeptide of collagen 1 was measured in the serum during the first treatment cycle and decreased also. Urinary deoxypyridinoline/creatinine was monitored in a total of seven treatment cycles and indicated that bone resorption reached the pre-treatment level after 6-8 weeks. Conclusion: This was the first use of denosumab in children with OI-VI. Denosumab was well tolerated, and laboratory parameters provided evidence that the treatment reversibly reduced bone resorption. Therefore, denosumab may be a new therapeutic option for patients with OI-VI.     

OPG/RANKL: role and therapeutic target in osteoporosis

Given the increasing risk of fractures with aging in western countries, there is a need for the development of safe and efficient anti-osteoporotic drugs for the prevention and treatment of osteoporosis. Recent studies have provided evidence for an essential role of RANKL (Receptor Activator of Nuclear Factor-kappa B Ligand) and its decoy receptor osteoprotegerin in the control of osteoclast differentiation and survival. Post-menopausal osteoporosis results from an imbalance between resorption and formation associated with decreased OPG/RANKL. Targeting the OPG/RANKL system may therefore have a beneficial impact in osteoporosis. Accordingly, the development of novel strategies targeting OPG/RANKL using anti-RANKL or therapeutic intervention proved to be efficient to reduce bone resorption and to prevent bone loss in postmenopausal osteoporosis. This opens the way for novel therapeutic strategies for correcting bone metabolism in various pathologic disorders characterized by increased bone remodelling and bone loss.     

The development of denosumab for the treatment of diseases of bone loss and cancer-induced bone destruction

Denosumab is a fully human monoclonal antibody against RANK ligand (RANKL), an essential cytokine for the formation, function, and survival of osteoclasts. The role of excessive RANKL as a contributor to conditions characterized by bone loss or bone destruction has been well studied. With its novel mechanism of action, denosumab offers a significant advance in the treatment of postmenopausal osteoporosis; bone loss associated with hormone ablation therapy in women with breast cancer and men with prostate cancer; and the prevention of skeletal-related events in patients with bone metastases from solid tumors by offering clinical benefit to these patients in need.     

Denosumab

Denosumab is an anti-receptor activator of nuclear factor (NF)-kappaB (RANK) ligand human monoclonal antibody studied as a treatment for postmenopausal osteoporosis (PMO) and bone destruction due to rheumatoid arthritis (RA) or metastatic cancers. As of February 2009, the candidate was undergoing US Food and Drug Administration review, and might be approved by October 2009. Late phase clinical trials demonstrated that denosumab possesses a similar safety profile to bisphosphonates and that it can be either equally or more effective than bisphosphonates at preventing bone loss due to PMO, RA or cancer treatment and metastases.Key Words: monoclonal antibody, RANKL, bone loss, osteoporosis, breast cancer, rheumatoid arthritis     

In vitro stoichiometry of complexes between the soluble RANK ligand and the monoclonal antibody denosumab

The in vitro binding stoichiometry of denosumab, an IgG2 fully human monoclonal therapeutic antibody, to RANK ligand was determined by multiple complementary size separation techniques with mass measuring detectors, including two solution-based techniques (size-exclusion chromatography with static light scattering detection and sedimentation velocity analytical ultracentrifugation) and a gas-phase analysis by electrospray ionization time-of-flight mass spectrometry from aqueous nondenaturing solutions. The stoichiometry was determined under defined conditions ranging from small excess RANK ligand to large excess denosumab (up to 40:1). High concentrations of denosumab relative to RANK ligand were studied because of their physiological relevance; a large excess of denosumab is anticipated in circulation for extended periods relative to much lower concentrations of free soluble RANKL. The studies revealed that an assembly including 3 denosumab antibody molecules bound to 2 RANKL trimers (3D2R) is the most stable complex in DPBS at 37 °C. This differs from the 1:1 binding stoichiometry reported for RANKL and osteoprotegerin (OPG), a soluble homodimeric decoy receptor which binds RANKL with high affinity. Denosumab and RANKL also formed smaller assemblies including 1 denosumab and 2 RANKL trimer molecules (1D2R) under conditions of excess RANKL, 3 denosumab molecules and 1 RANKL trimer (3D1R) under conditions of excess denosumab, and larger assemblies, but these intermediate species were only present at lower temperatures (4 °C), shortly after mixing denosumab and RANKL, and converted over time to the more stable 3D2R assembly.     

Anti-resorptive therapies for osteoporosis

The treatment of osteoporosis is largely based around the use of agents that inhibit bone resorption by osteoclasts. The main classes of anti-resorptives currently in use are calcium, bisphosphonates, estrogen, selective estrogen receptor modulators (SERMs) and calcitonin. Novel agents in development are: inhibitors of the osteoclast enzyme, cathepsin K; and a monoclonal antibody against receptor activator of NFkappaB-ligand (RANKL), a factor made by osteoblasts which stimulates osteoclast development. Potent anti-resorptive agents decrease numbers of vertebral fractures by about 50%, and non-vertebral fractures by only 25%. Whether the newer agents can improve on this remains to be seen, though it is possible that anabolic agents which increase bone mass more substantially will be needed to achieve greater reductions in all fracture numbers.     

Contribution of bone mineral density and bone turnover markers to the estimation of risk of osteoporotic fracture in postmenopausal women

In osteoporosis, the main cause for concern is the increase in the risk of fractures. The level of bone mineral density (BMD) measured by various techniques has been shown to be a strong predictor of fracture risk in postmenopausal women. However, half of patients with incident fractures have BMD value above the diagnostic threshold of osteoporosis defined as a T-score of -2.5 SD or more below the average value of young healthy women. Clearly there is a need for improvement in the identification of patients at risk for fracture. Several prospective studies have shown that an increased bone resorption evaluated by specific biochemical markers was associated with increased risk of the hip, spine and non-vertebral fractures independently of BMD. The use of bone markers in individual patients may be appropriate in some situations, especially in women who are not detected at risk by BMD measurements. For example, in the OFELY study including 668 postmenopausal women followed prospectively over 9 years, we found that among the 115 incident fractures, 54 (47%) actually occurred in non-osteoporotic women. Among these women, the combination of bone markers and history of previous fracture was highly predictive of fracture risk. Thus, bone markers may be used in the assessment of fracture risk in selected cases in which BMD and clinical risk factors are not enough to take a treatment decision. Advances in our knowledge of bone matrix biochemistry, most notably of post-translational modifications in type I collagen, may allow identification of biochemical markers that reflect changes in the material property of bone, which is an important determinant of bone strength. Preliminary in vitro studies indicate that the extent of post-translational modifications of collagen--which can be reflected in vivo by the measurement of the urinary ratio between native and isomerised type I collagen--play a role in determining the mechanical competence of cortical bone, independently of BMD. Further studies in osteoporosis should explore the changes in these biochemical parameters of bone matrix as they may represent a key component of bone quality.     

Strontium ranelate in post-menopausal osteoporosis

Strontium ranelate is one of the first-line agents with proven anti-fracture activity used in the therapy of post-menopausal osteoporosis. Its mechanism of action makes it, however, different from other drugs, since it simultaneously stimulates two reverse processes: bone formation and bone resorption. The action of the agent depends on various mechanisms, including the activation of calcium receptors, localised on osteoblasts and osteoclasts, and on the influence on the OPG/RANKL system. The drug effectively prevents spinal, hip and extravertebral fractures. The agent's anti-fracture efficacy within the spine does not depend on the patient's age, or on base BMD values, or on the concentration of bone metabolism markers. As to the anti-fracture efficacy in the hip, it concerns women with an increased bone fracture risk. Strontium ranelate increases bone mineral density within the lumbar spine and the hip, decreases the concentrations of bone resorption markers, and increases the concentrations of bone formation markers. The drug is administered in a daily 2.0 g oral dose. This paper presents indications to therapy with strontium ranelate, specifying also its side effects and contraindications. We compare the anti-fracture efficacy of strontium ranelate to the efficacy of other agents of proven anti-fracture activity, based on published clinical studies.     

Osteoimmunology: The correlation between osteoclasts and the Th17/Treg balance in osteoporosis

Osteoporosis is a bone disease that is caused by disorder of the skeletal microenvironment, and it characterized by a high disability rate and the occurrence of low energy fractures. Studies on osteoporosis and related treatment options have always been hot spots in the field of bone biology. In the past, the understanding of osteoporosis has been rather limited; research has only shown that osteoporosis involves the imbalance of bone resorption and bone formation, and recent studies have not provided cutting‐edge theories of the basic understanding of osteoporosis. Recent studies have shown crosstalk between bone and immune responses. RANKL, an essential factor for osteoclasts (OCs), is associated with the immune system. T helper (Th17)/regulatory T (Treg) cells are two different kinds of T cells that can self‐interact and regulate the differentiation and formation of OCs. Therefore, understanding the correlation between the skeletal and immune systems and further revealing the roles and the cooperation between RANKL and the Th17/Treg balance will help to provide new insights for the treatment of osteoporosis.     

Strontium ranelate in post-menopausal osteoporosis

Strontium ranelate is one of the first-line agents with proven anti-fracture activity used in the therapy of post-menopausal osteoporosis. Its mechanism of action makes it, however, different from other drugs, since it simultaneously stimulates two reverse processes: bone formation and bone resorption. The action of the agent depends on various mechanisms, including the activation of calcium receptors, localised on osteoblasts and osteoclasts, and on the influence on the OPG/RANKL system. The drug effectively prevents spinal, hip and extravertebral fractures. The agent's anti-fracture efficacy within the spine does not depend on the patient's age, or on base BMD values, or on the concentration of bone metabolism markers. As to the anti-fracture efficacy in the hip, it concerns women with an increased bone fracture risk. Strontium ranelate increases bone mineral density within the lumbar spine and the hip, decreases the concentrations of bone resorption markers, and increases the concentrations of bone formation markers. The drug is administered in a daily 2.0 g oral dose. This paper presents indications to therapy with strontium ranelate, specifying also its side effects and contraindications. We compare the anti-fracture efficacy of strontium ranelate to the efficacy of other agents of proven anti-fracture activity, based on published clinical studies.     

Recommendations on the management of fragility fracture risk in women younger than 70 years

The risk for fragility fracture represents a problem of enormous magnitude. It is estimated that only a small fraction of women with this risk take the benefit of preventive measures. The relationship between estrogen and bone mass is well known as they are the other factors related to the risk for fracture. There are precise diagnostic methods, including a tool to diagnose the risk for fracture. Yet there continues to be an under-diagnosis, with the unrecoverable delay in instituting preventive measures. Women under the age of 70 years, being much more numerous than those older, and having risk factors, are a group in which it is essential to avoid that first fragility fracture. Today it is usual not to differentiate between the treatment and the prevention of osteoporosis since the common aim is to prevent fragility fractures. Included in this are women with osteoporosis or with low bone mass and increased risk for fracture, for whom risk factors play a primary role. There is clearly controversy over the type of treatment and its duration, especially given the possible adverse effects of long-term use. This justifies the concept of sequential treatment, even more so in women under the age of 70, since they presumably will need treatment for many years. Bone metabolism is age-dependent. In postmenopausal women under 70 years of age, the increase in bone resorption is clearly predominant, related to a sharp drop in estrogens. Thus a logical treatment is the prevention of fragility fractures by hormone replacement therapy (HRT) and, in asymptomatic women, selective estradiol receptor modulators (SERMs). Afterwards, there is a period of greater resorption, albeit less intense but continuous, when one could utilise anti-resorptive treatments such as bisphosphonates or denosumab or a dual agent like strontium ranelate. Bone formation treatment, such as parathyroid hormone (PTH), in women under 70 years will be uncommon. That is because it should be used in cases where the formation is greatly diminished and there is a high risk for fracture, something found in much older women.     

Mangiferin attenuates osteoclastogenesis,bone resorption,and RANKL‐induced activation of NF‐κB and ERK

Osteolytic bone diseases such as osteoporosis have a common pathological feature in which osteoclastic bone resorption outstrips bone synthesis. Osteoclast formation and activation are regulated by receptor activator of nuclear factor κB ligand (RANKL). The induction of RANKL‐signaling pathways occurs following the interaction of RANKL to its cognate receptor, RANK. This specific binding drives the activation of downstream signaling pathways; which ultimately induce the formation and activation of osteoclasts. In this study, we showed that a natural immunomodulator, mangiferin, inhibits osteoclast formation and bone resorption by attenuating RANKL‐induced signaling. Mangiferin diminished the expression of osteoclast marker genes, including cathepsin K, calcitonin receptor, DC‐STAMP, and V‐ATPase d2. Mechanistic studies revealed that mangiferin inhibits RANKL‐induced activation of NF‐κB, concomitant with the inhibition of IκB‐α degradation, and p65 nuclear translocation. In addition, mangiferin also exhibited an inhibitory effect on RANKL‐induced ERK phosphorylation. Collectively, our data demonstrates that mangiferin exhibits anti‐resorptive properties, suggesting the potential application of mangiferin for the treatment and prevention of bone diseases involving excessive osteoclastic bone resorption. J. Cell. Biochem. 112: 89–97, 2011. © 2010 Wiley‐Liss, Inc.     

Biomarkers for osteoporosis management: utility in diagnosis, fracture risk prediction and therapy monitoring

Osteoporosis is a systemic disease characterized by low bone mass and microarchitectural deterioration of bone tissue, resulting in an increased risk of fracture. While the level of bone mass can be estimated by measuring bone mineral density (BMD) using dual X-ray absorptiometry (DXA), its measurement does not capture all the risk factors for fracture. Quantitative changes in skeletal turnover can be assessed easily and non-invasively by the measurement of serum and urinary biochemical markers; the most sensitive markers include serum osteocalcin, bone specific alkaline phosphatase, the N-terminal propeptide of type I collagen for bone formation, and the crosslinked C- (CTX) and N- (NTX) telopeptides of type I collagen for bone resorption. Advances in our knowledge of bone matrix biochemistry, most notably of post-translational modifications in type I collagen, are likely to lead to the development of new biochemical markers that reflect changes in the material property of bone, an important determinant of bone strength. Among those, the measurement of the urinary ratio of native (alpha) to isomerized (beta) CTX - an index of bone matrix maturation - has been shown to be predictive of fracture risk independently of BMD and bone turnover.In postmenopausal osteoporosis, levels of bone resorption markers above the upper limit of the premenopausal range are associated with an increased risk of hip, vertebral, and nonvertebral fracture, independent of BMD. Therefore, the combined use of BMD measurement and biochemical markers is helpful in risk assessment, especially in those women who are not identified as at risk by BMD measurement alone. Levels of bone markers decrease rapidly with antiresorptive therapies, and the levels reached after 3-6 months of therapy have been shown to be more strongly associated with fracture outcome than changes in BMD. Preliminary studies indicate that monitoring changes of bone formation markers could also be useful to monitor anabolic therapies, including intermittent parathyroid hormone administration and, possibly, to improve adherence to treatment. Thus, repeated measurements of bone markers during therapy may help improve the management of osteoporosis in patients.     

The Role of PINP in Diagnosis and Management of Metabolic Bone Disease

Serum procollagen type I N-propeptide (PINP) is designated the reference marker of bone formation in osteoporosis; the reference marker for resorption is C-terminal telopeptide of type I collagen (CTX). PINP has very low circadian and biological variation, is not affected by food intake, and is very stable in serum after venepuncture. The two automated commercial assays for PINP provide similar results in subjects with normal renal function, allowing reference intervals to be used interchangeably. Bone turnover markers (BTM) are currently not recommended for fracture risk assessment and therefore not included in fracture risk calculators. In the management of osteoporosis, the main utility of BTM including PINP is for monitoring therapy, both antiresorptive as well as anabolic agents; monitoring is thought to help improve adherence. PINP as well as CTX may also be used in assessing offset of drug action following a pause in bisphosphonate therapy, to help decide when to re-instate therapy, or following cessation of denosumab therapy to assess efficacy of follow-on bisphosphonate therapy. PINP may also be used in the diagnosis of Paget’s disease of bone as well as in monitoring response to therapy and for recurrence. Although BTM other than bone alkaline phosphatase are currently not recommended for use in metabolic bone disease of chronic kidney disease, PINP measured by assays specific to the intact molecule has potential in this condition. Further studies are needed to examine this area, as well as in malignant bone disease.     

Parathyroid hormone and its analogues - molecular mechanisms of action and efficacy of osteoporosis therapy

Most medical agents currently applied in osteoporosis therapy act by inhibiting bone resorption and reducing bone remodelling, i.e. they inhibit the process of bone mass loss by suppressing bone resorption processes. These drugs provide an ideal therapeutic option to prevent osteoporosis progression. They however have a rather limited usefulness when the disease has already reached its advanced stages with distinctive bone architecture lesions. The fracture risk reduction rate, achieved in the course of anti-resorptive therapy, is insufficient for patients with severe osteoporosis to stop the downward spiral of their quality of life (QoL) with a simultaneously increasing threat of premature death. The activity of the N-terminal fragment of 1-34 human parathormone (teriparatide - 1-34 rhPTH), a parathyroid hormone (PTH) analogue obtained via genetic engineering , is expressed by increased bone metabolism, while promoting new bone tissue formation by stimulating the activity of osteoblasts more than that of osteoclasts. The anabolic activity of PTH includes both its direct effect on the osteoblast cell line, and its indirect actions exerted via its regulatory effects on selected growth factors, e.g. IGF-1 or sclerostin. However, the molecular mechanisms responsible for the actual anabolic effects of PTH remain mostly still unclear. Clinical studies have demonstrated that therapeutic protocols with the application of PTH analogues provide an effective protection against all osteoporotic fracture types in post-menopausal women and in elderly men with advanced osteoporosis. Particular hopes are pinned on the possibility of applying PTH in the therapy of post-steroid osteoporosis, mainly to suppress bone formation, the most important pathological process in this regard. The relatively short therapy period with a PTH analogue (24 months) should then be replaced and continued by anti-resorptive treatment.     

Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha

The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.