Bone 48 (2011) 677–692
Contents lists available at ScienceDirect
Bone
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
Review
Denosumab and bisphosphonates: Different mechanisms of action and effects
Roland Baron a,b,1, Serge Ferrari c,1, R. Graham G. Russell d,e,⁎,1
a
Department of Medicine, Harvard Medical School, Endocrine Unit, Massachusetts General Hospital, Boston, MA 02115, USA
Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA 02115, USA
c
Service des Maladies Osseuses, Département de Réhabilitation et Gériatrie, Hôpitaux Universitaires et Faculté de Médecine de Genève, CH-1211 Genève 14, Switzerland
d
Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Science, Oxford University Institute of Musculoskeletal Sciences (The Botnar Research Centre),
Nuffield Orthopaedic Centre, Oxford, OX3 7LD, UK
e
The Mellanby Centre for Bone Research, Department of Human Metabolism, Sheffield University Medical School, Beech Hill Road, Sheffield, S10 2RX, UK
b
a r t i c l e
i n f o
Article history:
Received 29 June 2010
Revised 30 November 2010
Accepted 30 November 2010
Available online 9 December 2010
Edited by: T. Jack Martin
Keywords:
Bisphosphonates
Denosumab
Osteoclast
Osteoporosis
RANK ligand
a b s t r a c t
To treat systemic bone loss as in osteoporosis and/or focal osteolysis as in rheumatoid arthritis or periodontal
disease, most approaches target the osteoclasts, the cells that resorb bone. Bisphosphonates are currently the
most widely used antiresorptive therapies. They act by binding the mineral component of bone and interfere
with the action of osteoclasts. The nitrogen-containing bisphosphonates, such as alendronate, act as inhibitors
of farnesyl-pyrophosphate synthase, which leads to inhibition of the prenylation of many intracellular
signaling proteins. The discovery of RANKL and the essential role of RANK signaling in osteoclast
differentiation, activity and survival have led to the development of denosumab, a fully human monoclonal
antibody. Denosumab acts by binding to and inhibiting RANKL, leading to the loss of osteoclasts from bone
surfaces. In phase 3 clinical studies, denosumab was shown to significantly reduce vertebral, nonvertebral and
hip fractures compared with placebo and increase areal BMD compared with alendronate. In this review, we
suggest that the key pharmacological differences between denosumab and the bisphosphonates reside in the
distribution of the drugs within bone and their effects on precursors and mature osteoclasts. This may explain
differences in the degree and rapidity of reduction of bone resorption, their potential differential effects on
trabecular and cortical bone, and the reversibility of their actions.
© 2010 Elsevier Inc. All rights reserved.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . .
Osteoclast differentiation and function . . . . . . . .
Osteoclast differentiation . . . . . . . . . . . . .
Osteoclast activity . . . . . . . . . . . . . . . .
Osteoclast survival and life-span. . . . . . . . . .
Osteoblast–osteoclast coupling and communication.
Preclinical pharmacology . . . . . . . . . . . . . . .
Mechanism of action of bisphosphonates. . . . . .
Mechanism of action of denosumab . . . . . . . .
Effects on osteoclastogenesis . . . . . . . . . . .
Other cell targets . . . . . . . . . . . . . . . . . .
Bone cells . . . . . . . . . . . . . . . . . . . .
Potential effects on osteoblasts. . . . . . . . .
Potential effects on osteocytes . . . . . . . . .
Effects on the immune and vascular systems . . . .
Immunomodulatory effects of RANKL inhibition.
Bisphosphonates and the acute-phase reaction .
Effects on the vascular system . . . . . . . . .
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⁎ Corresponding author. Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Science, Oxford University Institute of Musculoskeletal Sciences (The Botnar
Research Centre), Nuffield Orthopaedic Centre, Oxford, OX3 7LD, UK.
E-mail addresses: [email protected] (R. Baron), [email protected] (S. Ferrari), [email protected] (R.G.G. Russell).
1
All authors contributed equally.
8756-3282/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2010.11.020
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R. Baron et al. / Bone 48 (2011) 677–692
Pharmacokinetics and skeletal distribution . . . . . . . . . . . . . . . . . .
Animal models of bone loss . . . . . . . . . . . . . . . . . . . . . . . . .
Osteoprotegerin and bisphosphonates in rodents . . . . . . . . . . . . .
Single-dose and short-term effects . . . . . . . . . . . . . . . . . .
Long-term suppression of bone remodeling . . . . . . . . . . . . . .
Denosumab compared with bisphosphonates in preclinical models of bone loss
Human RANKL knock-in mouse model . . . . . . . . . . . . . . . . . .
Primate model . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of denosumab and bisphosphonates on bone histomorphometry . .
Safety and specific adverse events: denosumab vs bisphosphonates . . . .
Clinical perspectives and conclusions . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction
The development and activation of osteoclasts is an essential
process during skeletal growth in bone and mineral homeostasis.
These processes involve the removal of mineralized bone by
osteoclasts through resorption [1], followed by the formation and
subsequent mineralization of new bone matrix through the action of
osteoblasts [2,3]. In various situations, including after the menopause
and in osteoporosis, as well as in pathological conditions such as
osteolytic tumor metastases, rheumatoid arthritis, and periodontal
disease, the amount of bone removed during resorption by osteoclasts
exceeds that replaced during bone formation by osteoblasts, leading
to a net loss of bone mass, and increased skeletal fragility and risk of
fracture [4]. Accelerated or high bone remodeling usually causes an
earlier loss of trabecular bone compared with cortical bone; in the
elderly, or after a large amount of trabecular bone has already been
lost, more bone loss may occur in the cortical compartment [5–7].
Increased expression and production of RANK ligand (RANKL), as
seen with estrogen deprivation, is a central mechanism of osteoclast
activation and increased bone resorption [8,9]. Nearly all current
pharmacological treatments of osteoporosis utilize drugs that inhibit
bone resorption and these include bisphosphonates, raloxifene and
calcitonin. A novel and recent approach is to target the RANK/RANKL
system in a specific manner. The indispensable role of the RANK/
RANKL pathway in bone remodeling is illustrated by human
monogenic diseases in which absence of functional RANK or RANKL
results in osteoclast-poor osteopetrosis [10,11]. In early clinical
studies, inhibition of RANKL was achieved using the RANKL decoy
receptor, osteoprotegerin (OPG), which functions as the natural
inhibitor of/antagonist to RANKL [12,13]. Most recent clinical studies
have used the new osteoporosis drug denosumab – a fully human
monoclonal antibody – to bind to and inhibit RANKL.
This article describes how bisphosphonates and denosumab
reduce osteoclast-mediated bone resorption in fundamentally different ways. We review the process of osteoclastogenesis and bone
resorption, compare and contrast the pharmacokinetic/pharmacodynamic (PK/PD) profiles and the mechanisms of action of denosumab
and bisphosphonates, and summarize the similarities and differences
between these drugs based on evidence from preclinical and clinical
studies. We also discuss the implications that these differences may
have for the treatment of osteoporosis.
Osteoclast differentiation and function
Osteoclasts are multinucleated cells derived from hematopoietic
precursors in the monocyte–macrophage lineage. They are responsible for the degradation of mineralized bone matrix during bone
development and growth, and for skeletal homeostasis, repair and
bone remodeling throughout life. The overall rate of osteoclastic bone
resorption is regulated at least at three levels: 1) the differentiation
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rate of osteoclasts from their monocyte/macrophage precursor pool;
2) the activity of the individual mature osteoclast, through regulation
of key functional proteins; and 3) the survival rate of differentiated
and mature osteoclasts. Thus, reducing osteoclast numbers via
decreasing differentiation from precursors, or survival of mature
cells and/or decreasing the bone resorbing activity of osteoclasts
constitute three possible, yet not mutually exclusive, therapeutic
approaches for the treatment of hyper-resorptive skeletal diseases.
Osteoclast differentiation
Two cytokines are essential and sufficient to induce osteoclast
differentiation: the macrophage colony-stimulating factor (M-CSF)
[14,15] and RANKL [16] (Fig. 1). M-CSF binds to c-Fms, a single
transmembrane domain receptor of the tyrosine kinase family, and
RANKL binds to RANK, a single transmembrane receptor of the tumor
necrosis factor (TNF) receptor family, forming trimers upon ligand
binding. M-CSF first activates the proliferation and survival of cells of
the monocyte–macrophage lineage and the expression of RANK,
allowing the action of RANKL which, together with M-CSF, constitutes
an absolute requirement for commitment to and progression of early
precursors along the osteoclast lineage [17–19]. The differentiation
step from osteoclast precursors to multinucleated osteoclasts is then
induced by RANKL, again through the M-CSF-dependent expression of
RANK at the cell surface of these early precursors. Both M-CSF and
RANKL are secreted by bone marrow stromal cells and osteoblasts,
whereas RANKL is also secreted by T cells and, at lower levels, by B
cells, establishing a link to inflammation-induced osteolysis [17,20].
Importantly, most of the cells producing RANKL also produce a decoyRANK receptor, OPG, which acts as an antagonist to RANK signaling
and osteoclastogenesis by scavenging RANKL in the extracellular
environment [21]. Ultimately, it is the ratio between RANKL and OPG
which determines the level of activation of RANK and, thereby, the
degree to which osteoclastogenesis is activated. All these molecules
are thought to act in a paracrine manner and are therefore regulating
bone resorption locally. The link between these local factors and the
endocrine regulation of skeletal homeostasis through bone resorption
and remodeling is, however, established by the fact that both RANKL
and OPG expression and secretion are regulated by several calciumregulating hormones, such as sex hormones, parathyroid hormone
(PTH), and vitamin D3 [22,23].
Deletion and overexpression of RANK/RANKL genes in rodents has
clearly established the essential role of this pathway on osteoclastogenesis and regulation of bone mass and strength [24].
Osteoclast activity
The functional features defining the activity of an osteoclast
include 1) the attachment of the cells to the bone surface 2) the
migration of osteoclasts along bone surfaces, 3) the synthesis and
R. Baron et al. / Bone 48 (2011) 677–692
679
Fig. 1. Osteoclast intracellular signaling and development.
directional secretion of hydrolytic enzymes, 4) the acidification of the
subosteoclastic bone-resorbing compartment by vacuolar proton
pumps (V-ATPase) and chloride channels and 5) efficient internalization of extracellular bone matrix degradation products. The
importance of these functional activities is demonstrated by the fact
that genetic alterations of these processes lead to defective bone
resorption and osteopetrosis [3].
Whereas M-CSF has been shown to favor the migratory function of
osteoclasts while decreasing their resorbing activity [25], RANKL
seems overall to contribute to the activation of osteoclast function, i.e.
bone resorption [26]. RANKL can activate mature osteoclasts in a
dose-dependent manner in vitro, and can rapidly increase the
resorption of bone in vivo by activating pre-existing osteoclasts.
Osteoclast survival and life-span
In vitro, the combination of RANKL and M-CSF is required for optimal
osteoclast survival [17]. Indeed, osteoclast numbers decline dramatically in the absence of M-CSF or RANKL, both from an impairment of
recruitment of precursors and a rapid apoptosis of existing osteoclasts.
In mice, a single dose of OPG led to a N90% loss of osteoclasts
within 48 h of exposure due to apoptosis and without affecting
osteoclast precursor cells. Therefore, RANKL is essential, but not
sufficient, for osteoclast survival and in mice endogenous M-CSF levels
are not sufficient to maintain osteoclast viability in the absence of
RANKL [27]. And while no factors appear capable of maintaining
osteoclasts in the absence of RANK/RANKL signaling, the important
role of M-CSF can be replaced by vascular endothelial growth factor
(VEGF) in mice that lack functional M-CSF [28,29].
Osteoblast–osteoclast coupling and communication
Bone homeostasis and bone mass are ensured by the proper
balance between bone resorption and bone formation during the bone
remodeling process. This balance between catabolic and anabolic
functions within bone is ensured by the coupling process — an array of
cross-talk networks between the osteoclasts and osteoblasts [30,31].
Any agent that affects one arm of the remodeling process usually also
alters the other arm and antiresorptives tend to also decrease bone
formation. This results primarily from inhibition of the activation
frequency of basic multicellular units (BMUs) (i.e. the decrease in
bone remodeling surfaces upon which secondary bone formation can
take place), whereas the intrinsic bone-forming ability of osteoblasts
may not be affected [32]. It is, however, well accepted that osteoclasts
produce and/or release several osteoblast-stimulating cytokines and
growth factors from the bone matrix. For example, insulin-like growth
factors (IGF) I and II, transforming growth factor beta (TGF-β) and
bone morphogenetic proteins (BMPs) are important regulators of
osteoblastic differentiation that are released from the matrix during
bone resorption. In addition, osteoclast-derived factors regulate
coupling with osteoblasts. Recent findings support the existence of
direct coupling between bone cells in the BMU. Both Wnt 10a and
sphingosine-1-phosphate (SIP) have been identified as osteoclastderived cytokines capable of activating bone formation [33]. In
addition, bidirectional signaling takes place in osteoclasts via the
transmembrane ephrinB2 ligand and in osteoblasts by EphB4, the
tyrosine kinase receptor of ephrinB2 [34,35].
In this physiological context, RANKL and OPG are both expressed
by stromal cells and osteoblasts, and their expression is regulated by
major calcium-regulating hormones, as well as by local inflammatory
cytokines. They constitute a major regulatory component of the crosstalk between osteoblasts and osteoclasts. Indeed, OPG expression is
often regulated in an opposite manner by factors that alter RANKL
expression: upregulation of RANKL is associated with downregulation
of OPG, or vice versa, altering the ratio of RANKL to OPG [24]. Thus,
inhibiting RANKL interrupts the osteoblast-dependent regulation of
osteoclastogenesis and osteoclast activity, thereby reducing bone
resorption. Due to the coupling process it is therefore expected that in
turn the reduction in the number and activity of osteoclasts will affect
both osteoclast-derived clastokines and the liberation of matrixderived growth factors during resorption, thereby decreasing bone
formation [36]. This phenomenon probably occurs with most
antiresorptive drugs, such as bisphosphonates. It is unclear at this
point whether the suppression of earlier osteoclast precursors will
also contribute to the diminished osteoblast activity [37,38].
Preclinical pharmacology
Mechanism of action of bisphosphonates
The clinical efficacy of bisphosphonates primarily stems from two
key properties: their ability to bind strongly to bone mineral and their
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R. Baron et al. / Bone 48 (2011) 677–692
inhibitory effects on mature osteoclasts [39]. The strong attachment of
bisphosphonates to bone gives bisphosphonates the unique property
of selective uptake by their intended target organ. As bisphosphonates
selectively bind to bone mineral, they enter and inhibit mature
osteoclasts at the sites of bone resorption. There is some evidence that
bisphosphonates may also have inhibitory effects on osteoclast
precursors [40–42], but this process does not appear to be as
complete as seen when RANKL is inhibited, because osteoclasts are
not ablated after exposure to bisphosphonates in either animals or
humans. Early studies with bisphosphonates in the 1960s showed
that these agents have multiple effects on hydroxyapatite (HAP): they
inhibit the de novo precipitation of calcium phosphate from solution,
delay the transformation of amorphous to crystalline HAP, and inhibit
the aggregation and dissolution of HAP crystals [43–45]. Subsequent
studies showed that bisphosphonates also inhibited bone resorption
in a range of experimental models [46], and led to the clinical use of
bisphosphonates to treat various hyper-resorptive bone disorders.
The effects of bisphosphonates on bone mineral have also been
modeled using carbonated apatite, which mimics natural bone more
closely than does HAP. Henneman et al. [47] found that bisphosphonates show concentration-dependent inhibition of carbonated
apatite dissolution in vitro, but concluded that the contribution of this
inhibitory action to the in vivo effects of bisphosphonates remains to
be determined. This inhibitory action on HAP and carbonated apatite
dissolution is a potential difference between bisphosphonates and
denosumab, as the latter is not expected to intrinsically affect crystal
dissolution. The bisphosphonates differ in their mineral binding
affinity [48], and this may determine their potency and duration of
action [39].
The adsorption of bisphosphonates to HAP bone mineral surfaces
brings them into close contact with osteoclasts and other cells in the
bone. During bone resorption, the acidic environment created by the
osteoclast in the resorption lacuna is thought to allow the dissociation
of bisphosphonates from HAP, as the ability of bisphosphonates to
bind to HAP decreases as pH is lowered [49]. The bisphosphonate
molecule – perhaps complexed with calcium and bone organic matrix
proteins – is then taken up by the osteoclast. Studies using
fluorescently labeled and radiolabeled bisphosphonates have shown
that the most likely mechanism of uptake is fluid-phase endocytosis
and that internalized bisphosphonates are initially localized within
intracellular endocytic vesicles [50,51]. Acidification of these endocytic vesicles seems to be required to allow the transfer of bisphosphonates from the vesicles into the cytosol [51,52], where they exert
their biochemical effect. Once in the cytosol, it is then likely that
bisphosphonates are imported into organelles such as the peroxisome
[51]. Studies to date suggest that the mechanisms by which bisphosphonates are internalized by osteoclasts are similar for different
bisphosphonates.
Bisphosphonates have a chemical structure similar to that of the
inorganic molecule pyrophosphate. Bisphosphonates, however, differ
from pyrophosphate in containing a phosphorus–carbon–phosphorus
(P–C–P) bond instead of the phosphorus–oxygen–phosphorus bond
that is present in pyrophosphate. Importantly, this P–C–P bond is
highly resistant to hydrolysis, thus making bisphosphonates resistant
to biological degradation. As well as being bonded to two phosphonate groups, the carbon atom in the P–C–P bond is also bonded to two
side chains, known as the R1 and R2 groups. Different bisphosphonates contain different R1 and R2 groups and, as a consequence,
vary in their binding affinity and biochemical potency [39,44,49,53].
Nitrogen-containing bisphosphonates, such as alendronate, ibandronate, minodronate, pamidronate, risedronate, neridronate and zoledronate, have a side chain that contains a nitrogen atom, whereas the
non-nitrogen-containing bisphosphonates, such as clodronate, tiludronate and etidronate, do not.
Once taken up by the osteoclast, all bisphosphonates inhibit bone
resorption through intracellular effects. Non-nitrogen-containing
bisphosphonates are metabolized in the osteoclast cytosol to
adenosine triphosphate (ATP) analogs that block osteoclast function
and induce osteoclast apoptosis [54]. In contrast, nitrogen-containing
bisphosphonates act principally by inhibiting farnesyl pyrophosphate
(FPP) synthase – an enzyme in the mevalonate pathway – thereby
preventing the post-translational modification (prenylation) of small
guanosine triphosphate (GTP)-binding proteins that are essential for
osteoclast function and survival (Fig. 2) [39].
Mechanism of action of denosumab
The identification of the RANK/RANKL pathway in the 1990s
opened up the possibility of developing novel agents that would
reduce osteoclastic bone resorption by inhibiting RANKL. The effect of
RANKL inhibition was first evaluated in preclinical and clinical studies
using Fc fusion proteins [13,55–59] (Fig. 3). With Fc-OPG and OPG-Fc,
Fig. 2. Osteoclast biology, RANK signaling pathway, and mevalonate pathway showing
the mechanism of action of nitrogen-containing bisphosphonates (NBPs). FPP: farnesyl
pyrophosphate.
R. Baron et al. / Bone 48 (2011) 677–692
681
Effects on osteoclastogenesis
Fig. 3. History of the development of RANKL antagonists.
OPG was fused to the Fc portion of human immunoglobulin G1 (IgG1)
[13,56,57]. A third molecule, RANK-Fc was formed by fusing the
extracellular domain of RANK (amino acids 22–201) to the Fc portion
of IgG1 [59]. Later studies investigated RANKL inhibition using
denosumab, a fully human monoclonal antibody that targets RANKL
[60]. Denosumab is an immunoglobulin of the IgG2 subclass, and this
isotype is relatively inactive, eliciting effector functions [61]. In vitro
binding assays have shown high-affinity binding of denosumab and
OPG to both soluble and membrane-bound forms of human RANKL
[62]. However, denosumab has several advantages over OPG-Fc or FcOPG constructs. First, in terms of its selectivity, denosumab does not
bind to TRAIL (TNF-related apoptosis-inducing ligand) or to other TNF
family members including TNF-α, TNF-β, and CD40 ligand, whereas
TRAIL binding has been observed with OPG [62,63]. Second, due to its
molecular mass, its half life is prolonged compared to the Fc
constructs. Finally, neutralizing antibodies against OPG-Fc could
have neutralizing effects on both the drug and OPG, which would
not be expected with denosumab. At present, no neutralizing
antibodies have been identified with denosumab [64].
Denosumab prevents RANKL from binding to its receptor, RANK,
thereby inhibiting the development, activation, and survival of
osteoclasts. This is different from the mechanism of action of
bisphosphonates, which bind to bone mineral and probably inhibit
osteoclast function mainly by being taken up by osteoclasts at sites of
bone resorption (Fig. 4).
Fig. 4. Osteoclast inhibition with denosumab vs bisphosphonates (BPs).
A major difference between denosumab and bisphosphonates on
osteoclasts is that bisphosphonates have to be internalized to act upon
cells, whereas denosumab acts in the extracellular milieu.
RANKL inhibition prevents the fusion of monocytes–macrophages
to become multinucleated osteoclasts, whereas long-term bisphosphonate treatment has been associated with an increase in the
number of osteoclasts, including giant, hypernucleated, detached
osteoclasts that undergo protracted apoptosis [65]. Inhibition of
osteoclast activity by bisphosphonates could trigger a feedback loop
that results in increased RANKL in the bone environment, which in
turn could help stimulate osteoclast precursors to fuse into multinucleated osteoclasts. This hypothesis is supported by the evidence of
increased osteoclast numbers on bone surfaces in ovariectomized
huRANKL mice (see below) treated with alendronate, whereas
denosumab caused a nearly complete disappearance of osteoclasts
in this model [32]. Preliminary results of a study that used a rat
arthritis model to compare the effects of RANKL inhibition and
bisphosphonate treatment on serum and bone proteins further
indicate that RANKL inhibition using OPG treatment significantly
reduced RANKL in bone, whereas zoledronic acid treatment caused a
two-fold increase in RANKL [66].
Other cell targets
Bone cells
Potential effects on osteoblasts
While data exist that OPG binds to membrane-bound RANKL on
osteoblasts, this does not apparently trigger intracellular signaling,
but may trigger internalization of the OPG/RANKL complex by a
clathrin-mediated mechanism followed by proteasomal degradation
[67]. Similarly, OPG exerts no direct effects on osteoblast survival, but
prevents TRAIL-induced apoptosis when added together with TRAIL to
osteoblast-like cell cultures [68]. Cultured murine bone marrow
stromal cells exhibited enhanced osteoblastic differentiation when
treated with OPG [69], but the mechanisms of these effects have not
been elucidated. It is therefore unlikely that the moderate decrease of
osteoblast numbers per bone surface observed for instance in
huRANKL mice treated with denosumab [62] and/or the profound
decrease in bone forming indices observed with denosumab in
rodents and humans (see below) [32] might be explained by the
effects of RANKL antagonists directly on osteoblasts, but rather by
their marked inhibition of bone remodeling.
Very low concentrations of bisphosphonates (as low as 10− 11 mol/L)
have been shown to stimulate osteoblasts in vitro to release factors that
inhibit bone resorption by osteoclasts [70]. In contrast, others have
suggested that continuous exposure of osteoblasts to high-dose
bisphosphonates could inhibit osteoblast function and/or survival [71].
However, despite many studies, direct effects of bisphosphonates on
osteoblasts, either positive or negative, have been difficult to demonstrate in vivo using clinically relevant doses [39]. Similar to RANKL
inhibitors, most of the clinical effects of bisphosphonates on osteoblast
function, such as suppression of osteoblast-derived biochemical
markers, can be attributed to indirect effects on the remodeling cycle
caused by the reduction in bone resorption and consequent reduction in
bone formation (see section ‘Osteoblast–osteoclast coupling and
communication’ above).
Potential effects on osteocytes
Osteocytes, which make up over 90–95% of all bone cells in the adult
skeleton, are important cells for bone health. [72]. Due to their
distribution throughout the bone matrix and extensive interconnectivity, osteocytes are thought to play a major role in sensing mechanical
strain and orchestrating signals of resorption and formation [73].
682
R. Baron et al. / Bone 48 (2011) 677–692
Osteocytes are a major regulator of OPG production locally, since
sclerostin – a main product of these osteocytes – inhibits the production
of OPG by differentiated osteoblasts [74]. A recent study has shown that
suppression of beta-catenin signaling in osteocytes (like in osteoblasts)
results in low bone mass mainly due to increased bone resorption,
because of diminished OPG levels [75]. Conversely, preliminary data
suggest that transgenic rats overexpressing OPG showed a slight but
significant increase in the percentage of trabecular lacunae occupied by
osteocytes in lumbar vertebrae compared with wild-type controls [76].
A modest but significant increase in the percentage of trabecular lacunae
occupied by osteocytes was also observed in the distal femur of
orchiectomized rats treated with OPG-Fc, compared with orchiectomized controls [77]. Whether these increases in the occupancy of
lacunae by osteocytes resulted from the direct effect of OPG on
osteoblast and/or osteocyte survival, or more likely from an overall
decrease of bone remodeling in these animals, is unknown. Whether
this phenomenon also happens with denosumab treatment is also
currently unknown. Eventually, it would be interesting to know
whether RANKL inhibition will affect osteocyte-mediated bone adaptation to mechanical strain.
Osteocytes may be important target cells for bisphosphonates. In
cell lines and experimental animals, osteocyte apoptosis is induced by
many stimuli, including glucocorticoid exposure, mechanical loading,
microdamage, and weightlessness [78,79]. In contrast to the ability of
bisphosphonates to induce osteoclast apoptosis, very low concentrations of several bisphosphonates seem to protect osteocytes (and
osteoblasts) from glucocorticoid-induced apoptosis in vitro [80]. The
prevention of osteocyte apoptosis by bisphosphonates is mediated by
connexin 43 hemichannel opening and subsequent activation of ERKs
[81,82]. Bisphosphonates, such as amino-olpadronate, that have little
antiresorptive effect are able to inhibit osteocyte apoptosis, supporting the idea that the effects of bisphosphonates on osteocytes are
independent of their effects on osteoclasts [81,83,84]. In rats, low
doses of risedronate and alendronate have been shown to suppress
osteocyte apoptosis following cyclic mechanical loading [85]. The
canalicular compartment is bathed in an extracellular fluid that is
likely to be able to deliver drug to osteocytes. Studies using a
fluorescein-tagged risedronate analog have provided direct evidence
of distribution into this compartment [39,86]. The relevance of these
observations with regard to the effects of bisphosphonates on
targeted bone remodeling initiated by osteocytes remains to be
elucidated. Interestingly, alendronate together with treadmill exercise exerted synergistic effects on the mechanical properties of the
midshaft femur in rats, suggesting some common biological pathway
[87], whereas zoledronate did not exert synergistic/additive effects
with exercise or bone axial compression [88,89]. Whether these
observations pertain to differential effects of various bisphosphonates
on osteocyte function remains to be investigated.
OPG) inhibits dendritic-cell-dependent T-cell activation [91,92].
However, when RANKL is produced by activated T cells during
inflammatory processes such as colitis and rheumatoid arthritis (RA)
in the absence of other immune molecule deficits, antagonizing
RANK/RANKL signaling has no effect on proliferation or activation of
inflammatory cells [93–97]. Stolina et al. [98] have recently published
the first direct comparison of the effects of RANKL inhibition and of
TNF and IL-1 inhibition on inflammation and cytokine levels. Rats
with established adjuvant-induced arthritis or collagen-induced
arthritis were treated with vehicle, TNF-α inhibitor (pegsunercept),
IL-1 inhibitor (anakinra), or RANKL inhibitor (OPG-Fc). Anti-TNF-α or
anti-IL-1 therapy inhibited parameters of local inflammation (paw
swelling) and of systemic inflammation (serum levels of proinflammatory cytokines), and partially reduced local but not systemic bone
loss. In contrast, RANKL inhibition by OPG-Fc prevented local and
systemic bone loss in both arthritis models without inhibiting any
measured local or systemic parameter of inflammation. Furthermore,
a recent study showed that administration of RANK-Fc in mice did
not affect T-cell cytokine production or proliferation, nor did it modify
T-cell infiltration of inflammatory tissues in a model of Staphylococcus
aureus-induced arthritis [99]. For a more detailed review of RANKL
inhibitor effects on inflammation and immunity, see Ferrari-Lacraz
and Ferrari [100].
Bisphosphonates and the acute-phase reaction
The acute-phase reaction is a well-known adverse effect of
nitrogen-containing bisphosphonates and encompasses influenzalike symptoms such as fatigue, fever, chills, myalgia, and arthralgia.
These symptoms can occur with both oral and intravenous bisphosphonates, but are more common with the latter [101–103]. The
symptoms are transient and self-limiting, and usually do not recur
after subsequent drug administration [104]. Inhibition of FPP synthase
by nitrogen-containing bisphosphonates causes accumulation of
isopentenyl pyrophosphate (IPP), the metabolite immediately upstream of FPP synthase in the mevalonate pathway by cells (most
likely monocytes) in peripheral blood. Since IPP is a ligand for a
receptor on the most common subset of γ,δ-T cells in humans, Vγ9Vδ2
T cells, the accumulation of IPP leads to activation of these γ,δ-T cells,
which in turn causes the release of TNF-α, thus initiating the proinflammatory acute phase response [105,106]. Statins, which inhibit
an enzyme upstream of FPP synthase in the mevalonate pathway,
prevent the accumulation of IPP [105,107]. Co-administration of a
statin has therefore been suggested as a way to prevent the acutephase reaction in patients [105]. However, this strategy was not found
to be successful when it was tried in a small, randomized study in 12
children who were receiving intravenous bisphosphonates; in this
study, concurrent administration of atorvastatin did not significantly
reduce pain or analgesic/antipyretic usage [108].
Effects on the immune and vascular systems
Besides their effects on bone cells as reviewed above, both RANKL
inhibitors and bisphosphonates exert some non-skeletal effects,
particularly on immune and vascular cells, which are potentially of
clinical relevance.
Immunomodulatory effects of RANKL inhibition
Considering the role of RANK/RANKL in the ontogenesis of the
immune system [21,90] and the widespread expression of these
molecules during immune and inflammatory reactions, where they
appear to play a role as co-stimulatory factors for the activation of T
cells and dendritic cells [9], one would expect a RANKL antagonist to
influence immune and inflammatory processes. Thus, in severe
immune deficiency models, such as CD40 knockout mice, and
autoimmune disease models, such as IL-2 knockout mice that develop
spontaneous colitis, blockade of RANKL signaling (by RANK-Fc or Fc-
Effects on the vascular system
Epidemiological studies have found an association between
osteoporosis and vascular calcification in postmenopausal women
[109] and men [110]. The RANKL signaling pathway may have a role in
this association, since OPG knockout mice develop both osteoporosis
and calcification of the aorta and renal arteries [111]. Inactivation of
the OPG gene in ApoE knockout mice, which are prone to
atherosclerosis and vascular calcification, increased each of these
vascular phenotypes [112]. Therefore, the effects of RANKL inhibition
using denosumab on vascular calcium deposition following glucocorticoid exposure were studied in huRANKL mice [113]. Denosumab
treatment reduced aortic calcium deposition in prednisolone-treated
mice by up to 50%, based on calcium measurement. Aortic calcium
deposition was correlated negatively with bone mineral density
(BMD) at the lumbar spine and positively with urinary excretion of
deoxypyridinoline, a marker of bone resorption.
R. Baron et al. / Bone 48 (2011) 677–692
Bisphosphonates may also exert favorable effects on vascular
calcification. The earliest studies with bisphosphonates showed that
they can inhibit aortic and kidney calcification in rats, an effect which
was attributed to their direct inhibitory effects on crystal growth
[43,114]. More recently, in rat models, bisphosphonates inhibited
warfarin- and vitamin-D-induced artery calcification and calciphylaxis
at doses comparable to those that inhibit bone resorption [115,116].
Case reports have been published where bisphosphonates have been
used successfully to treat calciphylaxis in the clinical setting [117,118].
Some in vitro studies, particularly those studying the potential
antitumor effects of nitrogen-containing bisphosphonates, have found
that these agents, especially zoledronate and pamidronate, have
antiangiogenic properties [119,120]. Whether the concentrations necessary to exert these effects can be achieved in vivo remains unknown.
Pharmacokinetics and skeletal distribution
One way in which denosumab differs from bisphosphonates is in
its likely distribution in bone. As denosumab is an antibody, one
would expect this agent to be capable of distributing throughout the
extravascular space. In particular, there is no evidence of sustained
binding of denosumab to bone surfaces. The skeletal disposition of
denosumab has been visually assessed in huRANKL mice using
immunohistochemistry. In the proximal tibia of a denosumab-treated
huRANKL mouse, strong staining was observed within a major blood
vessel in the marrow diaphysis, and within a vessel penetrating the
cortex (Fig. 5) [62]. There was no apparent staining of bone matrix or
bone surfaces, suggesting that denosumab is primarily a circulating
soluble protein.
A single-dose, dose–response study of denosumab in healthy
postmenopausal women demonstrated a pharmacokinetic response
that was non-linear and dose-dependent [121] with the drug being
detectable in the circulation for up to several weeks; overall, the halflife of denosumab is approximately 26 days [121]. As seen with other
antibodies, clearance of denosumab occurs through the reticuloendothelial system, and is thus independent of renal clearance. This
study also assessed denosumab pharmacodynamics. At the clinically
relevant dose of 1 mg/kg, a single subcutaneous dose of denosumab
resulted in a rapid and profound decrease in urinary N-telopeptide/
creatinine that was 80% lower at 1 week and 59% lower at 6 months
after administration compared with baseline [121]. Furthermore, the
mean serum intact PTH level doubled from its baseline value within
4 days after administration of this dose of denosumab; it returned to
baseline levels during follow-up. This transient increase in PTH may
683
occur to mitigate the decrease in serum calcium. Whether it also
contributes to promote modeling-based bone mass gain remains
unknown at this time.
In contrast, some of the bisphosphonate that is originally adsorbed
on to bone mineral surfaces becomes buried within bone by being
covered by bone that is synthesized de novo at the original site of bone
resorption [122]. Whether this accumulation leads to a continuous
and progressive decrease in bone remodeling has been studied in
animal models and clinical studies. Using a rat model, Reitsma et al.
showed that inhibition of bone resorption by pamidronate was dosedependent, appeared to reach a steady-state level within days, and
did not become progressively lower, even with repeated dosing [123].
In long-term clinical studies of alendronate, ibandronate, risedronate
and zoledronate, a new steady state of bone turnover was achieved
within 1–6 months of starting treatment, and this reduced level of
bone resorption remained constant for as long as treatment continued
[101,124–128]. The findings of these studies suggest that bisphosphonate buried in bone does not affect resorption for at least as long
as it remains buried there. The pharmacokinetic half-lives for the
elimination of bisphosphonates that are retained in bone are thus not
equivalent to the half-lives of bisphosphonates' biochemical effects.
The elimination of bisphosphonate from bone depends greatly on
remodeling and resorption [122,129]. After release from the skeleton
mainly as a result of resorptive action, bisphosphonates are excreted
via the kidneys. Indeed, urinary excretion of small amounts of
bisphosphonate that has been released from the skeleton can be
measured over many weeks, months, or even years after stopping
bisphosphonate treatment [130,131]. This means that bisphosphonate must be present in the circulation and available for re-uptake
into bone for prolonged periods. This recycling of bisphosphonates
back onto bone mineral surfaces has been proposed as the reason for
the long duration of action of zoledronate in particular, and also of
alendronate [39]. This helps to explain why the effects of bisphosphonates are less rapidly reversible after stopping treatment than
those of denosumab (see section ‘Clinical trials’).
Animal models of bone loss
Osteoprotegerin and bisphosphonates in rodents
Single-dose and short-term effects
In rodents, blocking RANKL was first achieved by using OPG, because
denosumab – which is highly specific for human RANKL – does not
recognize mouse or rat RANKL and requires genetically modified mice
Fig. 5. Immunohistochemical assessment of denosumab disposition in the proximal tibia of a huRANKL mouse. Left panel: Negligible specific staining was seen in bones from
huRANKL mice treated with phosphate-buffered saline (PBS). Right panel: In a denosumab (DMab)-treated huRANKL mouse, strong staining was observed within a major blood
vessel in the marrow diaphysis, and within a vessel penetrating the cortex (circled).
Reproduced with permission from [62].
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R. Baron et al. / Bone 48 (2011) 677–692
expressing huRANKL. In intact female rats, a single injection of
recombinant human OPG caused a transient decrease in the serum
osteoclastic marker TRACP between 2 and 20 days after injection. The
decrease in TRACP returned to a level similar to the control by day 30.
Similarly, osteoclast-associated bone surfaces decreased by approximately 80% by day 2, and by up to 90% between days 5 and 10, after
which a recovery was noted with more than 50% of the original
osteoclastic surfaces being present again 30 days after the injection.
Although no detectable change in serum calcium levels was noted
throughout the duration of the experiment, there was a transient
increase in serum PTH levels in the first 24 h after injection. The amount
of bone mineral at the proximal tibia metaphysis increased markedly
over 30 days after a single injection of recombinant OPG, which was
paralleled by a near doubling of the trabecular bone volume at this site
[132]. Histomorphometrical bone turnover indices were not reported in
this experiment. In another experiment in ovariectomized rats, 6 weeks'
administration of recombinant OPG markedly decreased osteoclast and
osteoblast surfaces at the distal femoral metaphyses. This was associated
with significantly greater bone mass and trabecular microarchitecture,
as well as with increased bone strength at other skeletal sites (vertebral
and femoral neck) [133].
A similar experimental design has been used to assess the effects of a
single dose of a bisphosphonate. Female rats received a single
intravenous injection of zoledronic acid 4 days before bilateral
ovariectomy [71]. Bone mass and density and serum bone markers
were then measured every 4 weeks for 32 weeks. Zoledronate given at
100 μg/kg (equivalent to the human 5-mg dose) completely prevented
the decreases in total BMD and cortical thickness that occurred after
ovariectomy in untreated (vehicle) animals, and this persisted for the
32 weeks of the study. This dose also increased cancellous bone mass at
32 weeks vs vehicle. Zoledronate given at a 100 μg/kg dose significantly
suppressed osteocalcin (a serum marker of bone formation) by about
30% for the entire study duration, and reduced TRACP5b activity by up to
90% from week 4 to week 8, when TRACP5b levels began to rise
gradually; however, TRACP5b activity was still significantly suppressed
for the entire duration of the study (weeks 4 to 32). This prolonged
inhibition of bone turnover markers by a single injection of zoledronate
was reflected by a significant reduction in mineralizing surface, mineral
apposition rate, and formation rate of cancellous bone at 32 weeks. Of
note, this study provided no information about the effects of zoledronate
on osteoclast or osteoblast numbers on bone surfaces.
Comparing the results of these two single-dose experiments
indicates that robust inhibition of bone remodeling by a single dose
of either a RANKL inhibitor or a bisphosphonate increases bone mass
and preserves bone structure in rodents.
Although rodent models do not spontaneously develop cortical
porosity nor cortical thinning, transgenic mice which express a
constitutively active PTH/PTH-related protein (PTH/PTHrP) receptor
in osteoblasts exhibit not only increased osteoblast and osteoclast
numbers on bone surfaces, with a resulting higher cancellous bone
volume, but also prominent cortical porosity as well as fibroblastoid
transformation of the bone marrow, which are consistent with the
phenotype of hyperparathyroidism [134]. OPG, zoledronate, or
alendronate all similarly increased trabecular bone volume in these
mice compared with vehicle-treated transgenic mice, suggesting that
trabecular bone resorption was comparably suppressed by these
agents [135]. In contrast, only OPG greatly reduced osteoclast
numbers and abrogated the increased cortical porosity and bone
marrow fibrosis in this particular model, indicating a key role for
osteoclastic cells (rather than bone resorption per se) in these two
pathological processes. Whether the observation in this particular
model has clinical relevance remains uncertain.
Long-term suppression of bone remodeling
To study the effects of continuous long-term inhibition of RANKL
on bone physical properties, transgenic rats were engineered to
overexpress OPG [76]. In these rats, 1 year of continuous overexpression
of OPG was found to markedly suppress bone resorption, and also
resulted in significant increases in bone mass, cortical area and
thickness, and trabecular bone volume, density, and breaking strength
in lumbar vertebrae, compared with wild-type rats. There was an overall
reduction in femur bending strength, which – as in other rodent
osteopetrosis models [136] – appeared to be caused by abnormal long
bone geometry and not by any changes in bone material properties [76].
However, considering the lack of haversian bone remodeling in rodents
under normal conditions, there is a question of whether this model is
fully appropriate to evaluate the consequences of suppressed bone
remodeling on cortical bone strength.
Long-term administration of alendronate in rodents has proven
safe and effective at increasing bone mass and strength. Dogs, rather
than rodents, have been used to assess the relationship between bone
microdamage and the suppression of remodeling by bisphosphonates.
Studies using a beagle dog model have shown that long-term
suppression of bone turnover by bisphosphonates is associated with
microdamage accumulation in the ribs and vertebrae [137–140].
However, data from the dog model suggest that the level of
microdamage accumulation in animals treated with anti-remodeling
agents (bisphosphonates or raloxifene) does not correlate with bone
toughness nor does it interfere with breaking strength [141].
Intriguingly, a 3-year course of high-dose alendronate therapy in
these young gonad-intact dogs did not increase vertebral BMD or
vertebral strength compared to vehicle controls [137], which differs
from the typical response of mouse, rat, or primate vertebrae to longterm antiresorptive therapy. The applicability of these animal studies
to humans remains unclear, as they used dogs that were not estrogen
deficient and did not have low BMD [142]. In addition, reduced
material properties with bisphosphonates have not been corroborated in any of the numerous long-term studies conducted in adult
ovariectomized primates. Whether microdamage accumulation
occurs in postmenopausal women treated long term with bisphosphonates remains controversial [143,144].
In summary, long-term inhibition of bone turnover by antiresorptives in preclinical experiments (rodents and dogs) seems to increase
cancellous bone mass and strength, whereas its effects on cortical
bone remain uncertain.
Denosumab compared with bisphosphonates in preclinical
models of bone loss
Human RANKL knock-in mouse model
As denosumab does not recognize murine RANKL, a mouse model
expressing a chimeric mouse/human RANKL gene (huRANKL mouse)
was developed. These mice have a slightly lower osteoclast number
per bone surface, and an approximately 50% greater trabecular bone
volume fraction (BV/TV) compared with normal mice. This may be
because the affinity of human RANKL is likely to be less for mouse
RANK. In intact adult huRANKL mice, short-term administration of
denosumab reduced bone resorption and markedly increased cortical
and cancellous bone mass and trabecular BV/TV (N2× increase) [62].
In ovariectomized huRANKL mice, denosumab administered at the
time of ovariectomy was more effective than alendronate in
increasing BMD and preserving trabecular architecture and cortical
thickness in vertebrae and distal femur after 4 and 8 weeks [32]. In
this experiment, BMD and trabecular bone volume fraction (BV/TV)
was higher in estrogen-deficient mice after 8 weeks of denosumab
treatment than in intact mice without treatment, indicating that
RANKL inhibition had not only prevented the ovariectomy-induced
increase in bone turnover, as observed with alendronate, but lowered
bone turnover below baseline levels, with a further beneficial effect on
bone mass and microstructure. Histomorphometrical analysis showed
an increase in osteoclast number per bone surface in alendronate-
R. Baron et al. / Bone 48 (2011) 677–692
treated mice, but a marked decrease with denosumab. There was a
significant decrease of bone remodeling indices with alendronate and
the absence of detectable mineralizing surfaces with denosumab.
Bone turnover, as evaluated both systemically (circulating levels of
TRACP5b) and at the tissue level (bone formation rate [BFR]), was
inversely correlated with BMD, trabecular BV/TV and vertebral
cortical thickness (R values ≥ 0.7). Of note, administration of intermittent daily PTH concomitantly with alendronate or denosumab
resulted in similar mineral apposition rates in both groups, indicating
the intact response of osteoblastic bone formation. However, the PTHstimulated BFR remained significantly lower in the denosumab group
than in the alendronate group, probably reflecting the smaller
remodeling space with denosumab. In addition, there are a number
of experiments in rodents combining alendronate (and/or zoledronate) with PTH, which confirm that in general those drugs exert
additive effects on the skeleton [69,145,146]. This is in contrast to
clinical studies with alendronate and PTH, which show limited
additive effects with this combination [147]. The reasons why the
response to PTH may be smaller in patients who have received or are
receiving alendronate is unknown. The interaction between denosumab and PTH in clinical studies has not been studied to date.
Male huRANKL mice were used to study the effects of denosumab
on glucocorticoid-induced bone loss. In adult male huRANKL mice
treated with prednisolone, bone loss was associated with suppressed
vertebral bone formation and increased bone resorption [148].
Denosumab prevented this prednisolone-induced bone loss through
a pronounced antiresorptive effect. Biomechanical compression tests
of lumbar vertebrae revealed a detrimental effect of prednisolone on
bone strength that was prevented by denosumab.
Male huRANKL mice were also used to compare the effects of
denosumab and alendronate on fracture healing [149]. Unilateral
transverse femur fractures were generated and the mice were then
treated with twice weekly doses of alendronate (0.1 mg/kg),
denosumab (10 mg/kg), or phosphate buffered saline (control
group). Within each treatment group, the mice were further divided
into subgroups receiving treatment for either 21 or 42 days.
Denosumab treatment almost completely suppressed serum TRACP5b
levels, whereas alendronate reduced the levels of this osteoclastic
marker by only about 25%. Qualitative histological analysis showed
that the 21- and 42-day alendronate and denosumab groups had
greater amounts of unresorbed cartilage or mineralized cartilage
matrix compared with the controls, and unresorbed cartilage could
still be seen in the denosumab group at 42 days. Although alendronate
and denosumab delayed the removal of cartilage and the remodeling
of the fracture callus, this did not diminish the mechanical integrity of
the healing fractures. In contrast, strength and stiffness were
enhanced in alendronate- and denosumab-treated animals at day 42
compared with the control group. This study shows that neither the
suppression of osteoclasts by denosumab nor inhibition of osteoclastic
activity by alendronate impairs the fracture consolidation by the
callus process. Whether anti-resorptives delay osteonal healing by
interfering with remodeling processes at the fractured bone ends,
however, remains controversial.
Primate model
In a long-term (16-month) toxicology safety study of denosumab
in ovariectomized adult cynomolgus monkeys (available in abstract
format only), once-monthly subcutaneous injection of denosumab
significantly increased bone mineral content in vertebrae and the
proximal femur, as well as in the more cortical femur diaphysis [150].
Preliminary evidence indicates that peak load was significantly
increased in both vertebrae and the femur neck upon compression
testing, whereas the intrinsic properties of the bone materials
remained unaltered, suggesting that denosumab improved bone
strength at these sites primarily by increasing bone mass. Denosumab
685
markedly inhibited histomorphometrical indices of bone remodeling
after 6 months and until study completion, as observed on both rib
biopsies and tibia metaphyses. One of the mechanisms by which
denosumab could have increased cortical volumetric BMD in these
animals is by inhibiting haversian remodeling with a significant
reduction of intracortical porosity compared with ovariectomized
controls, which was inversely correlated with femur bone strength
[151]. In a subsequent study, ovariectomized cynomolgus monkeys
were switched to once-monthly denosumab after 6 months of
continuous alendronate treatment [152]. Switching to denosumab
further decreased serum CTx (C-terminal telopeptide of type I
collagen) to virtually undetectable levels, and also further decreased
bone-specific alkaline phosphatase (BSAP) and osteocalcin. This
decrease in biochemical markers of bone turnover with denosumab
reflected a complete disappearance of osteoclasts from cancellous
bone surfaces, i.e. in contrast to the continuous alendronate group in
which osteoclast surfaces remained virtually unchanged. In this
experiment, both denosumab and alendronate markedly inhibited
bone remodeling indices in cancellous bone, although switching to
denosumab led to a significant improvement of the compressive
strength of vertebral trabecular cores. Both drugs also reduced cortical
porosity at the rib and tibial diaphysis by about 50%, although cortical
porosity was non-significantly lower in monkeys switched from
alendronate to denosumab. Cortical bone turnover was also more
greatly suppressed in monkeys switched from alendronate to
denosumab than in monkeys treated with alendronate alone. Bone
mass and strength remained well correlated with 12 months of
denosumab or 12 months of alendronate therapy in ovariectomized
cynomolgus monkeys. This observation suggests that bone quality
was not adversely affected by either agent, and that increments in
bone mass with either agent should translate to proportional
improvements in bone strength. Whether denosumab exerts structural effects on cortical bone that are different from bisphosphonates,
however, remains unclear.
Clinical trials
In randomized controlled trials in postmenopausal women with
osteoporosis, bisphosphonates have significantly reduced the risk of
vertebral fractures by 39–70% compared with placebo [101,153–159].
Analyses of randomized controlled trials with alendronate, risedronate, and zoledronic acid have also shown significant antifracture
efficacy at nonvertebral sites and the hip, with up to 25% and 40%
relative risk reduction, respectively [101,154,157–159]. Similar
results were found in the FREEDOM trial, after 3 years of treatment,
where denosumab (60 mg subcutaneously every 6 months) significantly reduced the risk of vertebral fractures by 68%, nonvertebral
fractures by 20%, and hip fractures by 40%, compared with placebo
[160]. Comparative fracture trials of denosumab vs bisphosphonates
have not been performed.
In two double-blind, randomized clinical studies, the efficacy and
safety of denosumab vs alendronate were evaluated in treatmentnaïve patients or those previously treated with alendronate. Both
studies showed significantly greater BMD increases at all measured
sites (including the one-third distal radius, which is considered a
predominantly cortical site) in denosumab-treated patients compared
to those treated with alendronate (approximately + 1% BMD with
denosumab vs alendronate) [161,162]. Decreases in concentrations of
bone turnover markers (serum CTX and P1NP) were also significantly
greater with denosumab than with alendronate treatment. Whether
these differences will translate into clinically relevant benefits is
unclear.
Furthermore, the effects of discontinuing denosumab or alendronate treatment in postmenopausal women with low BMD were
studied in a phase 2 trial [163]. Decreases in BMD at the lumbar spine
and total hip were faster in patients who discontinued denosumab
686
R. Baron et al. / Bone 48 (2011) 677–692
than in patients who discontinued alendronate, suggesting that
denosumab has a faster offset of action than oral weekly alendronate
at these sites. Whether the rapid reversal of bone turnover that
occurred with denosumab withdrawal could negatively affect bone
structure and strength remains to be investigated.
The effects of denosumab and alendronate in preventing microarchitectural deterioration of cortical and trabecular bone were
compared in a placebo-controlled, randomized, double-blind, phase
2 study in postmenopausal women with moderately low bone mass
[164]. Morphologic changes were assessed using high-resolution
peripheral quantitative computed tomography (HR-pQCT). In the
placebo group, both cortical and trabecular volumetric BMD
decreased over 1 year at the distal radius (by 1.5% and 2%,
respectively). These changes were prevented by alendronate and
denosumab. In cortical bone, there was actually a small net gain of
volumetric BMD with denosumab that was significantly greater than
with alendronate. More surprisingly, cortical thickness at the distal
radius, which did not significantly change from baseline in the placebo
group, increased by 2% with alendronate and 3% with denosumab.
Cortical thickness at the distal tibia increased by approximately 1% in
the placebo group, and by 4% with alendronate and 5% with
denosumab. Consequently, the calculated polar moment of inertia,
an index of bone strength at the distal radius, significantly improved
with both agents, more significantly so with denosumab.
In a recent study by Genant and colleagues of 332 postmenopausal
osteopenic women, using QCT, the decrease in cortical thickness at the
distal radius observed in the placebo group was prevented in the
group treated with denosumab [165] whereas volumetric BMD and
the polar moment of inertia tended to be greater after 24 months of
denosumab than at baseline, particularly at the ultradistal site. These
results are consistent with dual-energy X-ray absorptiometry results
previously reported from the same study, in which areal BMD
increased by 1.4% at the one-third radius in women receiving
denosumab and decreased by 2.1% in those receiving placebo. A
possible explanation for the increase in bone mass and structure seen
with denosumab at the radius could be due to a reduction in cortical
porosity as shown by the monkey studies (above). Although QCT has
limited spatial resolution and precision in the evaluation of cortical
and trabecular bone structure [166], these results could indicate
potential differences between denosumab and bisphosphonates,
which have not been shown to produce such large changes at the
radius. Whether this will lead to detectable differences in nonvertebral fracture risk reduction is currently uncertain. Effects on cortical
porosity have been observed in iliac crest bone biopsies from women
treated with risedronate [167].
In summary, whereas there is accumulating evidence that
denosumab could improve bone mass and estimated strength at
cortical bone sites (particularly the distal radius) potentially more
than alendronate, the exact structural mechanisms by which
denosumab exerts these effects, for instance, through a greater
suppression of cortical porosity and/or endocortical resorption remain
unclear.
Effects of denosumab and bisphosphonates on bone histomorphometry
Bone histology is used in the evaluation of drugs used in
osteoporosis to assess both efficacy and safety. It is well known
from studies of different bisphosphonates that there is a reduction in
activation frequency and bone turnover as assessed by the BFR and
mineralizing surfaces, using quantitative histomorphometric techniques. However, the mineral apposition rate has been shown to
decrease, not change or even increase with bisphosphonates,
depending on the method used to evaluate (or discard) biopsies
where double tetracycline labels were undetectable. Results from iliac
crest biopsies from patients treated with denosumab have recently
been published by Reid and colleagues, and as expected showed that
denosumab produces a marked and sustained inhibition of osteoclasts, activation frequency and bone turnover [38]. The bone
microarchitecture remains normal and there was no evidence of
adverse effects on mineralization or the formation of lamellar bone.
The inhibition of bone turnover with denosumab was greater than
with alendronate, which is consistent with the reduction in
biochemical markers of bone resorption. Hence, in a comparative
study with alendronate and denosumab, histomorphometry analysis
revealed that all biopsies from alendronate-treated subjects showed
double label in trabecular and cortical bone whereas this was reduced
in denosumab-treated subjects (trabecular bone: 90% alendronate vs
20% denosumab and cortical bone: 100% vs 53%, respectively) [38].
Table 1 provides a summary of the results obtained with different
bisphosphonates compared with denosumab.
Safety and specific adverse events: denosumab vs bisphosphonates
The safety profile of denosumab has been well-documented in the
phase 3 FREEDOM trial with no increase in the risk of cancer,
cardiovascular disease, fracture healing, hypocalcemia or infection
observed in women treated with denosumab vs those receiving
placebo. However, compared with placebo, a higher number of
serious adverse events of cellulitis were reported in denosumabtreated women (0.3% [n = 12] vs b0.1% [n = 1]) [160]. In phase 3
clinical trials and clinical practice, bisphosphonates have been shown
to be generally well tolerated [168]. Some of the most commonly
reported side effects include those on the gastrointestinal tract and
kidney function as well as transient acute phase reactions (extensively reviewed by Pazianas et al. [168]).
There is currently concern regarding the decrease in bone
resorption associated with bone loss therapy and conditions such as
osteonecrosis of the jaw (ONJ) and atypical subtrochanteric fractures.
Bisphosphonate-related ONJ was originally reported in patients
receiving treatment to prevent hypercalcemia or other skeletal
complications of cancer [169,170]. For patients with metastatic bone
disease receiving denosumab, the incidence of ONJ observed, to date,
is similar to the incidence observed with bisphosphonates [171,172].
In postmenopausal osteoporosis, rare cases of ONJ have been reported
in both women receiving bisphosphonates as well as denosumab
[64,170,173], but no causal association has been established. In
patients receiving long-term bisphosphonate (particularly alendronate) therapy for 5–10 years, there have been reports of atypical
subtrochanteric femoral fractures, which have been linked with
impaired bone remodeling [174]. However, these fractures are rare,
even after 10 years of treatment [175], and a causal association with
bisphosphonate treatment remains unproven. To date, there have
been no reports of subtrochanteric fractures with denosumab [160].
Both these topics have been extensively reviewed elsewhere [168].
Another area of interest and possible distinction between
denosumab and bisphosphonates is in their effects on the kidney. As
an antibody, denosumab is not known to have effects on the kidneys,
nor have adverse events been identified. In contrast, bisphosphonates
are known to be excreted unaltered through the kidneys via filtration
and possibly by proximal tubular secretion. Intravenous administration produces exposure to high initial concentrations of bisphosphonates in the kidney and may be associated with acute renal injury
[176] and nephrotoxicity [177]. Although this has not been shown for
oral bisphosphonates when used as labeled for the treatment of
osteoporosis, the administration of bisphosphonates is contraindicated in patients with severe renal impairment (creatinine clearance
b30 mL/min) due to “lack of sufficient clinical experience”, meaning
that patients with at least stage 4 renal insufficiency were excluded
from the registration studies with these drugs [178–182]. Recent
publications indicate that oral bisphosphonates, including alendronate [183] and risedronate [184,185], may be safe and effective in
reducing fractures in patients with glomerular filtration rates less
R. Baron et al. / Bone 48 (2011) 677–692
687
Table 1
Effects of antiresorptives on the main histomorphometrical dynamic and structural bone parameters.a
Trabecular MAR (μm/day)
Bone forming rate (μm3/μm2/day)
MS/BS (%)
Activation frequency (/year)
BV/TV (%)
2D
3D
Trabecular number (/mm)
2D
3D
Cortical thickness (mm)
2D
3D
Alendronateb
PBO
Zoledronic acid
PBO
Risedronate
PBO
Ibandronatec
PBO
Denosumabd
PBO
0.63–0.70
0.003–0.019
0.25–2.30
0.035–0.223
0.59
0.039
6.37
0.451
0.60
0.05
0.45
0.1
0.53
0.15
4.79
0.27
0.55
BFR/BV 0.085
1.1
0.15
0.58
(/year) 0.18
5.5
0.34
0.48
0.01
2.0
0.1
0.40
0.02
3.1
0.2
0.30
0.004
0.12
0.002
0.75
0.14
3.08
0.2
17.1
19.4
13.4
16.2
16.9
16.2
14.2
12.9
19.3
20.4–22.7e
19.0
23.0–20.0e
15.3
NA?
12.6
NA?
13.5
25.2
12.5
16.2
1.19
1.46
1.07
1.31
NA
1.36
NA
1.22
1.4
1.32–1.31e
1.3
1.50–1.31e
1.2
NA
1.1
NA
NA
1.46
NA
1.31
NA
NA
NA
NA
0.624
0.72 (0.79)f
0.511
0.63
1.1
0.91–0.83e
1.0
0.86–0.74e
NA
NA
NA
NA
0.658
0.565
0.765
0.710
Table adapted from Ferrari [188] (with kind permission from Springer Science + Business Media B.V.), with data from [189].
BFR: bone forming rate; BV: bone volume; BV/TV: trabecular bone volume fraction; MAR: mineral apposition rate; MS/BS: mineralizing surface per bone surface; NA: not available;
PBO: placebo.
a
As evaluated on iliac crest bone biopsies after 3 years of treatment in postmenopausal women.
b
5 or 10 mg/day for 2 and 3 years.
c
Results are shown only for the 2.5 mg oral daily dose. Greater inhibition of bone turnover indices may be achieved with 3 mg IV every 3 months [190].
d
Dynamic indices could be evaluated on 7/37 biopsies pooled from 2 and 3 years; 3D structural indices at 3 years.
e
The two numbers for each treatment indicate values that were evaluated separately in a subgroup of women with low or high turnover, respectively (MS/BS b or N median).
f
The result in parentheses is from a subgroup with previous non-bisphosphonate antiresorptive treatment.
than 30 mL/min. Similarly, denosumab also reduces fractures in
patients with impaired renal function [186]. However, in clinical
practice, because of the possible presence of adynamic bone disease in
patients with severe renal impairment, in whom any further
reduction of bone turnover might be detrimental, antiresorptive
drugs should only be used with caution.
Clinical perspectives and conclusions
Treatment with denosumab appears to have at least three major
points of difference compared with bisphosphonate treatment. Firstly,
in treatment-naïve women and those transitioning to denosumab from
alendronate, who thus already have low levels of bone turnover
markers, biochemical bone turnover markers are reduced. This could
reflect either a more profound inhibition of bone remodeling (i.e., a
more profound inhibition of the activation frequency of new BMUs)
with denosumab at any skeletal site or a difference in their inhibitory
effects depending on their ability to gain access to different sites within
the skeleton. The strong affinity of bisphosphonates for HAP and bone
mineral may limit their even distribution throughout the skeleton,
particularly to sites deep within the bone. However, bisphosphonates
with lower affinity for bone mineral appear to penetrate deeper into the
network [187]. Secondly, compared with alendronate, denosumab
treatment results in greater gains in BMD after 1 year at all sites,
including cortical bone sites such as the one-third radius (Table 2). As
denosumab is a circulating antibody, it is expected to reach all sites
within bone, including intracortical sites such as the distal radius, which
is not typically seen with bisphosphonates, as summarized in Table 2.
Whether denosumab has significantly greater effects on cortical
porosity and/or thickness than bisphosphonates, however, remains
unclear. Lastly, the effects of denosumab on bone turnover are quickly
reversible with discontinuation, actually leading to a transient rebound
phenomenon, and can be restored with subsequent retreatment. Results
of the phase 2 trial in postmenopausal women with low BMD also
suggest that denosumab has a faster offset of action at the lumbar spine
and total hip than weekly oral alendronate. However, the speed of offset
of action of bisphosphonates varies depending on their chemical
structure, dose, and frequency of administration.
Other novel approaches to the treatment of osteoporosis include
cathepsin-K inhibitors, such as odanacatib, and new selective estrogen
receptor modulators, such as bazedoxifene and lasofoxifene. The place of
denosumab and other novel agents in the management of osteoporosis
Table 2
Comparison of RANKL inhibitors and bisphosphonates on cortical bone.
Study design
Results
OPG in rodent models
Transgenic mice expressing constitutively
active PTH/PTH-related
protein receptors on cells of the
osteoblast lineage [135]
Intracortical porosity:
OPG ↓↓↓
Alendronate ↓
Zoledronate ↓
Denosumab in rodent models
Knock-in mice expressing a chimeric
human/mouse RANKL gene
(huRANKL mice) [191]
Ovariectomized huRANKL mice [32]
Cortical volumetric bone density
and thickness:
Denosumab ↑
Alendronate ↑
Cortical thickness in vertebrae and
distal femur:
Denosumab ↑↑a
Alendronate ↑
Denosumab in cynomolgus monkey models
Cortical porosity at the rib and tibial
Ovariectomized cynomolgus monkeys
switched to denosumab after 6 months of diaphysis, and cortical bone
turnover:
continuous alendronate treatment [152]
Switched to denosumab ↓↓b
Alendronate alone ↓
Clinical studies of denosumab vs alendronate
DECIDE — 12-month, randomized, double- BMD at all sites including
predominantly cortical (hip and
blind, phase 3 trial in postmenopausal
distal radius):
women with low BMD [161]
Denosumab ↑↑c
Alendronate ↑
STAND — randomized, double-blind, phase Femoral neck and one-third radius
BMD:
3 trial in postmenopausal women with
Switched to denosumab ↑↑c
low BMD switching from alendronate
[162]
Continued alendronate ↑
HR-pQCT phase 2 study in postmenopausal Cortical volumetric BMD and
women with low BMD
thickness at distal radius and tibia:
Denosumab ↑↑d
Alendronate ↑
Key: ↑ = increased; ↓ = decreased; a greater number of arrows indicates a greater
effect. BMD: bone mineral density; HR-pQCT: high-resolution peripheral quantitative
computed tomography; OPG: osteoprotegerin; PTH: parathyroid hormone.
a
Significantly better than alendronate at vertebra.
b
Not significantly better than alendronate.
c
Significantly better than alendronate.
d
Significantly better than alendronate on cortical volumetric BMD.
688
R. Baron et al. / Bone 48 (2011) 677–692
Table 3
Summary of differences between bisphosphonates and denosumab.
Characteristics
Bisphosphonate
Denosumab
Chemistry
Targets
Chemical agent
Selective uptake by hydroxyapatite. Inhibition of FPP synthase
(for nitrogen bisphosphonates) [129]
Bone mineral surface [129,193]
Mature osteoclasts. Possible effects on osteoclast precursors
and osteocytes [41]
Inhibits osteoclast resorptive function and survival by disrupting
intracellular signaling pathways. Dysfunctional osteoclasts may
persist [39,195]
Oral or IV (depending on the bisphosphonate). Daily, weekly, or
monthly (oral), quarterly or yearly (IV)
Fast onset of action if given parenterally, slower orally
Monoclonal antibody
Selectively binds RANKL [192]
Distribution
Major bone target cells
Mechanism of action
Mode of administration
Onset of action (serum CTX)
Inhibition of bone resorption (serum CTX)
Circulating in blood and extracellular fluid [194]
Osteoclast precursors and mature osteoclasts [194]
Prevents the formation, function, and survival of osteoclasts.
Depletes osteoclasts [194]
Subcutaneous (60 mg 6 monthly)
Faster onset of action than oral alendronate [192]
Appears to reduce bone resorption to a greater extent than
alendronate, e.g. DECIDE study [161]
Effect on BMD
Denosumab produces significantly greater gains in BMD at all
measured skeletal sites when compared with alendronate [161]
Duration of action and reversibility of effect Depends on type of BP and length of treatment. Slow offset of action Fully reversible and relatively rapid offset of action [192]
after stopping treatment
Contraindications
Pregnancy
Pregnancy
Severe renal impairment
Hypocalcemia
Abnormalities of the esophagus which delay esophageal emptying Hypersensitivity to the active substance or to any of the excipients
(tablet formulations)
Inability to stand or sit upright for at least 30–60 min
(tablet formulations)
Patients at increased risk of aspiration (alendronate oral solution)
Hypocalcemia
Hypersensitivity to any component of the product
BP: bisphosphonate; CTX: C-terminal telopeptide; FPP: farnesyl pyrophosphate; IV: intravenous.
will be determined by these agents' long-term effectiveness and safety
compared with existing treatments.
In summary, by targeting the RANKL pathway, denosumab provides
a new approach for the treatment of postmenopausal osteoporosis
(Table 3). Denosumab has a fundamentally different mechanism of
action from that of bisphosphonates. This may explain differences in the
degree and rapidity of reduction of bone resorption, their potential
differential effects on trabecular and cortical bone, and the reversibility
of their actions. In phase 3 studies, denosumab has shown efficacy
against vertebral, nonvertebral, and hip fractures compared with
placebo. Targeting the RANKL pathway represents a significant example
of the rapid translation of advances in basic science into a novel clinical
therapy. Whether this novel approach will result in further benefit for
women with osteoporosis will be seen as clinicians gather experience
with this new drug.
Acknowledgments
The authors drafted and wrote this review article. Writing/editing
assistance was also provided by Reza Sayeed (Bioscript Stirling Ltd.)
funded by Amgen (Europe) GmbH and GlaxoSmithKline. Graphics
support was provided by Jennifer Keysor (Surfmedia). Thanks also to
Antonia Panayi, Robert Stad, and Nathalie Franchimont of Amgen
(Europe) GmbH and Paul Kostenuik and Cesar Libanati of Amgen Inc
for providing many helpful discussions and comments during the
preparation of this review.
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