- Email: [email protected]
Denosumab and bisphosphonates: Rivals or potential “partners”? A “hybrid” molecule hypothesis
Medical Hypotheses 77 (2011) 109–111
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Denosumab and bisphosphonates: Rivals or potential ‘‘partners’’? A ‘‘hybrid’’ molecule hypothesis Athanasios D. Anastasilakis a,⇑, Stergios A. Polyzos b, Chrysostomos D. Anastasilakis c, Konstantinos A. Toulis a, Polyzois Makras d a
Department of Endocrinology, 424 General Military Hospital, Thessaloniki, Greece Second Medical Clinic, Medical School, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece Department of Pharmacology, 424 General Military Hospital, Thessaloniki, Greece d Department of Endocrinology and Diabetes, 251 Hellenic Air Force & VA General Hospital, Athens, Greece b c
a r t i c l e
i n f o
Article history: Received 14 January 2011 Accepted 17 March 2011
a b s t r a c t Bisphosphonates are well established as the treatment of choice for disorders of excessive bone resorption, including osteoporosis. They bind bone mineral with high affinity and through internalization by the resorbing osteoclasts, affect their function and survival. Receptor activator of nuclear factor-jB ligand (RANKL) is a cytokine essential for osteoclast differentiation, activation, and survival. Denosumab, a human monoclonal antibody that neutralizes RANKL, constitutes a promising antiresorptive agent for osteoporosis treatment. However, its presumable interaction with the immune system could adversely affect immune response resulting in increased risk of infections. We hypothesize that bisphosphonates could serve as a vehicle for the delivery of denosumab selectively to the skeleton. Thus, the effect on the immune system could be minimized, along with a potential increase in the antiresorptive efficacy, as a result of the combined action of denosumab and the bisphosphonate on the earlier and later stages of osteoclast life, respectively. Ó 2011 Elsevier Ltd. All rights reserved.
Background Osteoporosis is the most common bone disease, caused by a relatively increased rate of bone resorption by the osteoclasts that exceeds the rate of bone formation by the osteoblasts, resulting in net loss of bone mass. To-date, antiresorptive agents, which inhibit osteoclast activity and induce their apoptosis, are considered as the cornerstone of osteoporosis prevention and treatment. Bisphosphonates (BPs) currently represent the first line antiresorptive agents for the management of disorders of excessive bone resorption, including osteoporosis, Paget’s disease of bone, myeloma and bone metastatic disease. They are stable analogs of the inorganic pyrophosphate (PPi) consisted by two phosphonate groups that share a common carbon atom (P–C–P), which are selectively uptaken and absorbed to mineral surfaces of the bone, where they bind to the hydroxyapatite (HAP) crystals. The two side-chains (R1 and R2) attached to the carbon atom, along with the two phosphonate groups, are essential for the BPs binding affinity to the bone and their biological activity. More specifically, the two phosphonate groups, together with a hydroxyl group at the R1 position, impart high affinity for bone mineral and act as a ⇑ Corresponding author. Address: Soulini 4, 566 25 Sykies, Greece. Tel.: +30 2310 639 027; fax: +30 2310 839 900. E-mail address: [email protected] (A.D. Anastasilakis). 0306-9877/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2011.03.039
‘‘bone hook’’, allowing rapid and efficient binding of bisphosphonates to bone mineral surfaces. Once ‘‘hooked’’ to the bone, the structure and three-dimensional conformation of the R2 side chain (as well as the phosphonate groups in the molecule) determine the biological activity of the molecule [1]. BPs are selectively internalized by the osteoclasts. During the process of bone resorption, the subcellular space beneath the osteoclast is acidified by the action of proton pumps in the ruffled border of the osteoclast membrane. The acidic pH of this microenvironment causes dissolution of the hydroxyapatite bone mineral, and should markedly increase the dissociation of BPs from HAP. This is followed by the uptake of the BP most likely by fluid-phase endocytosis. Inside the osteoclast, non-nitrogen containing BPs (e.g., clodronate, etidronate) are metabolized to ATP analogs that induce osteoclast apoptosis, while the more potent amino-BPs (N-BPs) inhibit the enzyme farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway, thereby preventing the prenylation of small GTPase proteins essential for the function and survival of osteoclasts [2]. The BPs affinity for bone mineral has also been applied in bone scintigraphy, where medronic acid (MDA), the simplest BP, marked with the radionuclide technetium-99 m (99mTc) is used to reveal areas of increased bone turnover (bone lesions or tumors). In this case, bone imaging is based on the initial flow through the bone and absorption of covalently bonded 99mTc phosphate adduct to
110
A.D. Anastasilakis et al. / Medical Hypotheses 77 (2011) 109–111
the surface of hydroxyapatite crystal in bone. 99mTc is concentrated in the cement line located at the junction of osteoid and mineralized bone [3]. The receptor activator of nuclear factor-jB ligand (RANKL) is a member of the tumor necrosis factor receptor superfamily essential for osteoclastogenesis. It binds to its receptor RANK on the surface of osteoclast precursors and enhances their differentiation, survival and fusion, while it activates mature osteoclasts and inhibits their apoptosis. Denosumab (AMG-162), a fully human monoclonal IgG2 antibody against human RANKL, specifically binds and neutralizes RANKL in order to decrease bone resorption and subsequent bone loss. Subcutaneous administration of denosumab every six months has led to rapid and remarkable decrease in bone turnover markers, thereby resulting in a significant increase in bone mineral density (BMD) and reduction in fracture risk [4]. However, since RANK activation by RANKL is also essential for T-cell growth and dendritic-cell function [5,6], inhibition of its action could simultaneously affect the immune system, leading to susceptibility in infections or malignancies [7,8]. Additionally, denosumab binding to the TNF-related apoptosis-inducing ligand (TRAIL), which is a survival factor for tumor cells, may interfere with a natural defence mechanism against tumorigenesis [8,9]. Furthermore, findings from meta-analyses of randomized, controlled trials imply that the risk of serious infections may be increased [10,11].
Hypothesis The systemic effects of denosumab on the immune system and therefore their putative increased infection risk might be minimized if a way to specifically deliver the agent directly to the bone could be found. Considering the successful prior use of BPs in bone disease imaging and treatment, we propose binding of the molecule of denosumab on a bisphosphonate, similarly to the 99mTc binding on MDA in bone scintigraphy. The ‘‘hybrid’’ molecule would be incorporated into the bone mineral, and entombed in the bone, from where it could be gradually released and dissociated into its primary constituents during local bone resorption, ensuring a prolonged duration of action. Furthermore, locally released denosumab might demonstrate an increased efficacy in inhibiting osteoclast precursor differentiation and mature osteoclast activation on the bone surface, since it would be coupled with the BP inhibition on the mature osteoclast function and survival. Thus, this dual, concurrent action might affect osteoclast at all levels of differentiation and activation.
Discussion The natural compensatory mechanism against RANKL is osteoprotegerin (OPG), a decoy receptor, locally produced in bone microenvironment by the mature osteoblasts [12]. During the normal remodeling cycle, the resorption phase and the lacuna formation on bone surface, is followed by the reversal phase [13], where the osteoblasts produce OPG to neutralize the osteoclasts, so that they could fulfill the resorption lacuna undisturbed. Our model could mimic local OPG release from the osteoblasts: during the resorption the acidic pH in the resorption lacuna would cause the dissociation of the BP-denosumab complex from the hydroxyapatite, together with the dissociation of denosumab from the BP surface. Thus, denosumab could neutralize locally acting RANKL; however, denosumab is more potent and acts longer than natural OPG resulting in a more prolonged suppression of the osteoclasts [14]. Furthermore, the BP molecule could also act by being internalized by the mature osteoclasts.
Denosumab’s stereotactic configuration resembles that of the natural IgG2 immunoglobulin. Theoretically, denosumab could bind to a BP through several groups (hydroxyl-, amino-, carboxylgroup or combinations of them) of the phosphonate groups or the R1 and R2 side-chains. However, since the hydroxyl groups of the phosphonate groups and the R1 chain are essential for binding to the HAP crystals, the R2 chain is the preferable site for denosumab binding. Ideally, a specific site (hydroxyl-, amino-, carboxyl- group or combination of them) in the R2 chain could be selected. This could be achieved pharmacotechnically by covering all the other similar groups in the BP molecule by specific chemical reactions (esterification, chlorosis, halogenation, acetylation, etc.). Thus, only the selected group would be available for denosumab binding. Thereafter, the other groups in the molecule could be uncovered again, so that binding to the HAP crystals would remain unaffected. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action [2]. Therefore, the bisphosphonate used to bind denosumab should be carefully selected. Denosumab is composed of amino acids and carbohydrates as the natural immunoglobulin; consequently, it is subjected to proteolysis and, if binded in an orally administered BP, degradation in the stomach is very likely. Therefore, intravenously administered BPs (e.g., zoledronate, ibandronate) seem more suitable for its delivery to the skeleton. Our model, if feasible, might increase efficacy while minimizing the risk of immune adverse events (infections, malignancies). On the other hand, the risk of bone turnover oversuppresion might be maximized due to the synergistic actions of the BP and the denosumab. Therefore, adverse events observed with both agents and potentially attributed to suppressed bone metabolism, such as atypical fractures or osteonecrosis of the jaw, might occur sooner and/or more often. A lower dose of the BP could be used in order to avoid bone turnover oversuppresion. Although our hypothesis is appealing, this hybrid molecule might be ineffective if conformational changes in the denosumab molecule prevent it from binding to RANKL. Another problem could be the risk of further proteolysis of denosumab in smaller fragments in the acidic environment of the resorption cavity. Conflict of interest statement None of the authors has any conflict of interest to declare. References [1] Russell RG, Xia Z, Dunford JE, et al. Bisphosphonates: an update on mechanisms of action and how these relate to clinical efficacy. Ann N Y Acad Sci 2007;1117:209–57. [2] Russell RG, Watts NB, Ebetino FH, Rogers MJ. Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporosis Int 2008;19:733–59. [3] McDougall IR. Skeletal scintigraphy. West J Med 1979;130:503–14. [4] Cummings SR, San Martin J, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med 2009;361:756–65. [5] Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397:315–23. [6] Wong B, Josien R, Lee SY, et al. TRANCE (tumor necrosis factor [TNF]-related activation induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J Exp Med 1997;186:2075–80. [7] Schwartzman J, Yazici Y. Denosumab in postmenopausal women with low bone mineral density. N Engl J Med 2006;354:2390–1. [8] Wiley SR, Schooley K, Smolak PJ, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673–82. [9] Emery JG, McDonnell P, Burke MB, et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 1998;273:14363–7.
A.D. Anastasilakis et al. / Medical Hypotheses 77 (2011) 109–111 [10] Anastasilakis AD, Toulis KA, Goulis DG, et al. Efficacy and safety of denosumab in postmenopausal women with osteopenia or osteoporosis: a systematic review and a meta-analysis. Horm Metab Res 2009;41:721–9. [11] Toulis KA, Anastasilakis AD. Increased risk of serious infections in women with osteopenia or osteoporosis treated with denosumab. Osteoporosis Int 2010;21:1963–4. [12] Anastasilakis AD, Toulis KA, Polyzos SA, Terpos E. RANKL inhibition for the management of patients with benign metabolic bone disorders. Expert Opin Invest Drugs 2009;18:1085–102.
111
[13] Eriksen EF. Normal and pathological remodeling of human trabecular bone: three dimensional reconstruction of the remodeling sequence in normals and in metabolic bone disease. Endocr Rev 1986;7:379–408. [14] Bekker P, Holloway D, Rasmussen A, et al. A single-dose placebo-controlled study of AMG 162, a fully human monoclonal antibody to RANKL, in postmenopausal women. J Bone Miner Res 2004;19:1059–66.
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Denosumab and bisphosphonates: Rivals or potential ‘‘partners’’? A ‘‘hybrid’’ molecule hypothesis Athanasios D. Anastasilakis a,⇑, Stergios A. Polyzos b, Chrysostomos D. Anastasilakis c, Konstantinos A. Toulis a, Polyzois Makras d a
Department of Endocrinology, 424 General Military Hospital, Thessaloniki, Greece Second Medical Clinic, Medical School, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece Department of Pharmacology, 424 General Military Hospital, Thessaloniki, Greece d Department of Endocrinology and Diabetes, 251 Hellenic Air Force & VA General Hospital, Athens, Greece b c
a r t i c l e
i n f o
Article history: Received 14 January 2011 Accepted 17 March 2011
a b s t r a c t Bisphosphonates are well established as the treatment of choice for disorders of excessive bone resorption, including osteoporosis. They bind bone mineral with high affinity and through internalization by the resorbing osteoclasts, affect their function and survival. Receptor activator of nuclear factor-jB ligand (RANKL) is a cytokine essential for osteoclast differentiation, activation, and survival. Denosumab, a human monoclonal antibody that neutralizes RANKL, constitutes a promising antiresorptive agent for osteoporosis treatment. However, its presumable interaction with the immune system could adversely affect immune response resulting in increased risk of infections. We hypothesize that bisphosphonates could serve as a vehicle for the delivery of denosumab selectively to the skeleton. Thus, the effect on the immune system could be minimized, along with a potential increase in the antiresorptive efficacy, as a result of the combined action of denosumab and the bisphosphonate on the earlier and later stages of osteoclast life, respectively. Ó 2011 Elsevier Ltd. All rights reserved.
Background Osteoporosis is the most common bone disease, caused by a relatively increased rate of bone resorption by the osteoclasts that exceeds the rate of bone formation by the osteoblasts, resulting in net loss of bone mass. To-date, antiresorptive agents, which inhibit osteoclast activity and induce their apoptosis, are considered as the cornerstone of osteoporosis prevention and treatment. Bisphosphonates (BPs) currently represent the first line antiresorptive agents for the management of disorders of excessive bone resorption, including osteoporosis, Paget’s disease of bone, myeloma and bone metastatic disease. They are stable analogs of the inorganic pyrophosphate (PPi) consisted by two phosphonate groups that share a common carbon atom (P–C–P), which are selectively uptaken and absorbed to mineral surfaces of the bone, where they bind to the hydroxyapatite (HAP) crystals. The two side-chains (R1 and R2) attached to the carbon atom, along with the two phosphonate groups, are essential for the BPs binding affinity to the bone and their biological activity. More specifically, the two phosphonate groups, together with a hydroxyl group at the R1 position, impart high affinity for bone mineral and act as a ⇑ Corresponding author. Address: Soulini 4, 566 25 Sykies, Greece. Tel.: +30 2310 639 027; fax: +30 2310 839 900. E-mail address: [email protected] (A.D. Anastasilakis). 0306-9877/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2011.03.039
‘‘bone hook’’, allowing rapid and efficient binding of bisphosphonates to bone mineral surfaces. Once ‘‘hooked’’ to the bone, the structure and three-dimensional conformation of the R2 side chain (as well as the phosphonate groups in the molecule) determine the biological activity of the molecule [1]. BPs are selectively internalized by the osteoclasts. During the process of bone resorption, the subcellular space beneath the osteoclast is acidified by the action of proton pumps in the ruffled border of the osteoclast membrane. The acidic pH of this microenvironment causes dissolution of the hydroxyapatite bone mineral, and should markedly increase the dissociation of BPs from HAP. This is followed by the uptake of the BP most likely by fluid-phase endocytosis. Inside the osteoclast, non-nitrogen containing BPs (e.g., clodronate, etidronate) are metabolized to ATP analogs that induce osteoclast apoptosis, while the more potent amino-BPs (N-BPs) inhibit the enzyme farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway, thereby preventing the prenylation of small GTPase proteins essential for the function and survival of osteoclasts [2]. The BPs affinity for bone mineral has also been applied in bone scintigraphy, where medronic acid (MDA), the simplest BP, marked with the radionuclide technetium-99 m (99mTc) is used to reveal areas of increased bone turnover (bone lesions or tumors). In this case, bone imaging is based on the initial flow through the bone and absorption of covalently bonded 99mTc phosphate adduct to
110
A.D. Anastasilakis et al. / Medical Hypotheses 77 (2011) 109–111
the surface of hydroxyapatite crystal in bone. 99mTc is concentrated in the cement line located at the junction of osteoid and mineralized bone [3]. The receptor activator of nuclear factor-jB ligand (RANKL) is a member of the tumor necrosis factor receptor superfamily essential for osteoclastogenesis. It binds to its receptor RANK on the surface of osteoclast precursors and enhances their differentiation, survival and fusion, while it activates mature osteoclasts and inhibits their apoptosis. Denosumab (AMG-162), a fully human monoclonal IgG2 antibody against human RANKL, specifically binds and neutralizes RANKL in order to decrease bone resorption and subsequent bone loss. Subcutaneous administration of denosumab every six months has led to rapid and remarkable decrease in bone turnover markers, thereby resulting in a significant increase in bone mineral density (BMD) and reduction in fracture risk [4]. However, since RANK activation by RANKL is also essential for T-cell growth and dendritic-cell function [5,6], inhibition of its action could simultaneously affect the immune system, leading to susceptibility in infections or malignancies [7,8]. Additionally, denosumab binding to the TNF-related apoptosis-inducing ligand (TRAIL), which is a survival factor for tumor cells, may interfere with a natural defence mechanism against tumorigenesis [8,9]. Furthermore, findings from meta-analyses of randomized, controlled trials imply that the risk of serious infections may be increased [10,11].
Hypothesis The systemic effects of denosumab on the immune system and therefore their putative increased infection risk might be minimized if a way to specifically deliver the agent directly to the bone could be found. Considering the successful prior use of BPs in bone disease imaging and treatment, we propose binding of the molecule of denosumab on a bisphosphonate, similarly to the 99mTc binding on MDA in bone scintigraphy. The ‘‘hybrid’’ molecule would be incorporated into the bone mineral, and entombed in the bone, from where it could be gradually released and dissociated into its primary constituents during local bone resorption, ensuring a prolonged duration of action. Furthermore, locally released denosumab might demonstrate an increased efficacy in inhibiting osteoclast precursor differentiation and mature osteoclast activation on the bone surface, since it would be coupled with the BP inhibition on the mature osteoclast function and survival. Thus, this dual, concurrent action might affect osteoclast at all levels of differentiation and activation.
Discussion The natural compensatory mechanism against RANKL is osteoprotegerin (OPG), a decoy receptor, locally produced in bone microenvironment by the mature osteoblasts [12]. During the normal remodeling cycle, the resorption phase and the lacuna formation on bone surface, is followed by the reversal phase [13], where the osteoblasts produce OPG to neutralize the osteoclasts, so that they could fulfill the resorption lacuna undisturbed. Our model could mimic local OPG release from the osteoblasts: during the resorption the acidic pH in the resorption lacuna would cause the dissociation of the BP-denosumab complex from the hydroxyapatite, together with the dissociation of denosumab from the BP surface. Thus, denosumab could neutralize locally acting RANKL; however, denosumab is more potent and acts longer than natural OPG resulting in a more prolonged suppression of the osteoclasts [14]. Furthermore, the BP molecule could also act by being internalized by the mature osteoclasts.
Denosumab’s stereotactic configuration resembles that of the natural IgG2 immunoglobulin. Theoretically, denosumab could bind to a BP through several groups (hydroxyl-, amino-, carboxylgroup or combinations of them) of the phosphonate groups or the R1 and R2 side-chains. However, since the hydroxyl groups of the phosphonate groups and the R1 chain are essential for binding to the HAP crystals, the R2 chain is the preferable site for denosumab binding. Ideally, a specific site (hydroxyl-, amino-, carboxyl- group or combination of them) in the R2 chain could be selected. This could be achieved pharmacotechnically by covering all the other similar groups in the BP molecule by specific chemical reactions (esterification, chlorosis, halogenation, acetylation, etc.). Thus, only the selected group would be available for denosumab binding. Thereafter, the other groups in the molecule could be uncovered again, so that binding to the HAP crystals would remain unaffected. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action [2]. Therefore, the bisphosphonate used to bind denosumab should be carefully selected. Denosumab is composed of amino acids and carbohydrates as the natural immunoglobulin; consequently, it is subjected to proteolysis and, if binded in an orally administered BP, degradation in the stomach is very likely. Therefore, intravenously administered BPs (e.g., zoledronate, ibandronate) seem more suitable for its delivery to the skeleton. Our model, if feasible, might increase efficacy while minimizing the risk of immune adverse events (infections, malignancies). On the other hand, the risk of bone turnover oversuppresion might be maximized due to the synergistic actions of the BP and the denosumab. Therefore, adverse events observed with both agents and potentially attributed to suppressed bone metabolism, such as atypical fractures or osteonecrosis of the jaw, might occur sooner and/or more often. A lower dose of the BP could be used in order to avoid bone turnover oversuppresion. Although our hypothesis is appealing, this hybrid molecule might be ineffective if conformational changes in the denosumab molecule prevent it from binding to RANKL. Another problem could be the risk of further proteolysis of denosumab in smaller fragments in the acidic environment of the resorption cavity. Conflict of interest statement None of the authors has any conflict of interest to declare. References [1] Russell RG, Xia Z, Dunford JE, et al. Bisphosphonates: an update on mechanisms of action and how these relate to clinical efficacy. Ann N Y Acad Sci 2007;1117:209–57. [2] Russell RG, Watts NB, Ebetino FH, Rogers MJ. Mechanisms of action of bisphosphonates: similarities and differences and their potential influence on clinical efficacy. Osteoporosis Int 2008;19:733–59. [3] McDougall IR. Skeletal scintigraphy. West J Med 1979;130:503–14. [4] Cummings SR, San Martin J, McClung MR, et al. Denosumab for prevention of fractures in postmenopausal women with osteoporosis. N Engl J Med 2009;361:756–65. [5] Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999;397:315–23. [6] Wong B, Josien R, Lee SY, et al. TRANCE (tumor necrosis factor [TNF]-related activation induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. J Exp Med 1997;186:2075–80. [7] Schwartzman J, Yazici Y. Denosumab in postmenopausal women with low bone mineral density. N Engl J Med 2006;354:2390–1. [8] Wiley SR, Schooley K, Smolak PJ, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673–82. [9] Emery JG, McDonnell P, Burke MB, et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem 1998;273:14363–7.
A.D. Anastasilakis et al. / Medical Hypotheses 77 (2011) 109–111 [10] Anastasilakis AD, Toulis KA, Goulis DG, et al. Efficacy and safety of denosumab in postmenopausal women with osteopenia or osteoporosis: a systematic review and a meta-analysis. Horm Metab Res 2009;41:721–9. [11] Toulis KA, Anastasilakis AD. Increased risk of serious infections in women with osteopenia or osteoporosis treated with denosumab. Osteoporosis Int 2010;21:1963–4. [12] Anastasilakis AD, Toulis KA, Polyzos SA, Terpos E. RANKL inhibition for the management of patients with benign metabolic bone disorders. Expert Opin Invest Drugs 2009;18:1085–102.
111
[13] Eriksen EF. Normal and pathological remodeling of human trabecular bone: three dimensional reconstruction of the remodeling sequence in normals and in metabolic bone disease. Endocr Rev 1986;7:379–408. [14] Bekker P, Holloway D, Rasmussen A, et al. A single-dose placebo-controlled study of AMG 162, a fully human monoclonal antibody to RANKL, in postmenopausal women. J Bone Miner Res 2004;19:1059–66.