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Bone 34 (2004) S46–S99
Abstracts from the Workshop: What is New in Bisphosphonates?
Preclinical
1
Histological Comparison of Two Calcium Phosphate
Cements
Naveed Akhtar; V.V. Khatsko, O.A. Dmytryukova, Ahsan
Ahmad, A.E. Kuzmenko, Naushad Alam. Donetsk Medical
State University.
A reasonable method of treating bone defects may be the
injection of a reasonable cement into the osseous defect.
Norian and Biobon are two injectable and resorbable calci-
um phosphate cements which have shown good osteointe-
gration properties. The purpose of the present study was to
compare histologically the osteoconductivity and biocom-
patibility of these two cements.
Each cement was implanted in distal femoral epiphyseal
defects in eight rabbits and evaluated at eight and sixteen
weeks postoperatively. Histological and histomorphometri-
cal examinations showed that both cements were osteocon-
ductive with Biobon-bone interface consisted of 84–2%
contact with mineralized bone by 8 weeks and increased to
92.8% by 16 weeks, while Norian showed 98.9% contact
with mineralized bone by 8 weeks and was completely
surrounded by mineralized bone at 16 weeks.Comparing to
Biobon, Norian has better osteoconductive properties due to
significantly more mineralized bone around the implant,
after 8 and 16 weeks respectively. The opposition of the new
bone on both cements was direct, with no associated
inflammatory tissue response and no interposition of fibrous
tissue, eventually reflecting biocompatibility of both
cements. The result of this study suggest that Norian and
Biobon have comparatively good biocompatibility, while
Norion has better initial osteoconductive properties in this
rabbit model. Both cements represent feasible materials for
repair of cancellous bone defects and merit further study.
2
A Single Systemic Dose of Pamidronate Increases Bone
Mineral Content and Strength Over Local Pamidronate
Administration or Saline in a Rat Fracture Model
N : Amanat,* R. Brown,y L. Bilston,z D. Little*,y
*University of Sydney, NSW AustraliayThe Children’s Hospital at Westmead, Sydney, NSW
AustraliazPrince of Wales Medical Research Institute, Randwick,
NSW Australia
8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2004.01.014
Purpose: An open rat femoral fracture model was used to
assess the effect of systemically and locally administered
pamidronate. Previous studies have shown that nitrogen-
containing bisphosphonates can increase callus bone min-
eral content (BMC) and strength in both fracture repair
and distraction osteogenesis. In this study we aimed to
confirm that a single parenteral dose of a nitrogen
containing bisphosphonate could increase BMC and
strength in fracture repair, and to compare this with the
bisphosphonate administered locally on the internal fixa-
tion device.
Method: 40 Wistar rats underwent open osteotomy of
the right femur. At the time of surgery, animals were
randomly allocated to receive either systemic saline,
systemic pamidronate 3mg/kg, or local pamidronate
0.1mg (low dose) or 1.0mg (high dose) in a poly(L-
lactide) (PLLA) coating present on the intramedullary
wire. Saline and systemic groups were fixated with a
PLLA coated wire which did not contain pamidronate.
At six weeks post op animals were culled and both
femora harvested, radiographed and analysed by QCT.
Specimens were then analysed by mechanical testing to
failure in torsion.
Results: There was no difference in rates of union between
experimental groups. BMC was increased by 34% in sys-
temic and 27% and 53% in low-dose and high-dose local
over saline respectively (p < 0.05). Bone mineral density
(BMD) was increased by 38% in systemic and 31% and
46% in low-dose and high-dose local over saline respec-
tively (p < 0.05). Total bone volume was not different
between the groups.
Torsional testing revealed that only the systemic group had
increased strength on the operated side with a 60%
increase in peak load (p = 0.05) and 129% increase in
stiffness over saline (p < 0.05). While saline operated
femurs were only 69% as strong as the non-operated side
at six weeks (p < 0.05), systemic dosed operated femurs
reached 111% of the strength of the non-operated controls.
There were non-significant trends toward increases in
strength and stiffness in the local dose groups over saline,
which were of a much smaller magnitude. No changes in
energy to failure were noted.
Conclusion: A single systemic dose of pamidronate resulted
in increased BMC and strength in an open rat femoral
fracture model. The increases in strength and stiffness were
not associated with decreases in energy to failure, i.e. the
R
L
1.0
0.8
0.6
0.4
0.2
0.0
Load
(N
m)
Saline Low High Systemic
Fig. 1.
ABSTRACTS / Bone 34 (2004) S46–S99 S47
bones were not brittle. Local administration via the fixation
device increased BMC, but no significant increase in
strength or stiffness could be demonstrated.
Significance: This study confirms that a single perioperative
dose of a nitrogen-containing bisphosphonate leads to a
significant increase in callus bone mineral content and
strength. No therapeutic advantage to local administration
could be determined. Perioperative dosing with nitrogen-
containing bisphosphonates remains a simple, viable adjunc-
tive therapy for increasing strength of union at early time
points in endochondral bone repair (Fig. 1).
3
Systemic Zolendronate Treatment Prevents Resorption
of Necrotic Bone During Revascularization
J orgen Astrand,*,y,z Magnus Tagil,*,y,z Per Aspenberg*,y,§
*MDyPhDzConsultant
Introduction: This abstract adresses the question whether
the bone collapse and joint destruction associated with
osteonecrosis can be prevented with bisphophonates. Bone
resorption is mediated by osteoclasts recruited from their
hematopoetic origin, and occurs during or following the
revascularization of the necrotic area. If the bone resorption
associated with osteonecrosis can be inhibited or delayed
with bisphosphonates until sufficient new bone has formed,
it would appear that structural failure and its consequences
could be avoided.
Material and methods: We used a model with a cancellous
graft in a bone conduction chamber. The chamber consists
of a threaded titanium cylinder, formed out of two half
cylinders held together by a hexagonal screw cap. The
interior of the chamber is 7 mm long and has a diameter
of 2 mm. One end of the implant is screwed into the
proximal tibia of a rat. At this end there are two ingrowth
openings where tissue can grow in from the subcortical
bone.
Grafts: Eight donor rats (female ca 200g) were killed and
bone grafts harvested from the proximal tibias. A cylindrical
cancellous bone rod was taken out from each tibia in the
axial direction, using a hole cutter, and frozen at �70jC. Atsurgery, the grafts were thawed and placed in the chambers,
which then were inserted in the recipient animals.
Surgical procedure: Sixteen male Sprague-Dawley rats
(382–425 grams) received one chamber each, containing
a bone graft. Under aseptic conditions, a longitudinal
incision was made over the antero-medial aspect of the
proximal tibial metaphysis. Holes were made with a drill
and the chambers were screwed into position with the
ingrowth holes situated subcortically.
Injections: Postoperatively 0.5 ml subcutaneous injections
with 1.05 ug zolendronate and saline for the controls was
given weekly until harvest.
Evaluation: The chambers were harvested after 6 weeks.
Three sections from each specimen were used for histologic
and histomorphometric analyses.
The bone density in the remodelled area was evaluated using
point counting and a Merz grid. The distinction between
living and dead bone was based on matrix staining, presence
of osteocytes and trabecular shape. The study was approved
by the local ethical committee.
Results: No infection occurred. In all specimens soft tissue
had invaded the whole grafts. In the bone density mea-
surement the total amount of bone within the remodeled
area was 22% in the zolendronate treated grafts and 8% in
the controls (p = 0.0003). The total amount of newly
formed bone was 14% in the treated and 6% in the controls
(p = 0.001).
Conclusion: Systemic bisphosphonate treatment can reduce
the resorption of necrotic bone. This could prove useful in
clinical situations, for example as a treatment to reduce the
risk of structural failure and joint surface collapse after
osteonecrosis of the knee and hip. Using zolendronate in a
weekly dose seems sufficient.
4
Evaluation of the Growth Inhibitory Effects of the
Bisphonate Zoledronic Acid on Myeloma Cells, and
Comparison with Other Inhibitors of the Mevalonate
Pathway
Cindy Baulch-Brown; Andrew Spencer. Myeloma Research
Group, The Alfred Hospital, Melbourne, Australia.
ABSTRACTS / Bone 34 (2004) S46–S99S48
Clinical studies have suggested that in addition to prevent-
ing osteoclast-mediated osteolytic bone disease,
bisphosphonates (BPs) may induce a reduction of the
tumour burden and prolong the survival of MM patients.
Evidence from in vitro studies by a number of groups
indicates that nitrogen-containing BPs such as zoledronic
acid (ZA) interfere with osteoclast recruitment, differenti-
ation and action, and induce apoptotic cell death of these
cells by disrupting the mevalonate pathway. We have
previously reported that ZA induces varying degrees of
apoptosis in human MM cell lines NCI-H929, RPMI-
8226, LP-1, OPM-2 and U266. Further work within the
laboratory has demonstrated that inhibitors of the meval-
onate pathway vary in their ability to inhibit cellular
proliferation and/or induce cell death in myeloma cell
lines. Using tetrazolium reduction assays to assess the
cytotoxic potential of mevalonate pathway inhibitors we
found that ZA inhibits proliferation of a number of
myeloma cell lines of different clinical origin more effec-
tively than the farnesyl transferase inhibitor SCH66336,
but less effectively than the HMGCoA reductase inhibitor
fluvastatin. LP-1, NCI-H929 and U266 cell lines were
selected for further analysis in order to evaluate the effects
of mevalonate pathway inhibitors on myeloma cells.
Following treatment with 100microM ZA, 10microM
SCH66336 or 25microM fluvastatin for 24 or 72h, each
of the cell lines was examined using Annexin V/propi-
dium iodide and cell cycle flow cytometry techniques.
Whole cell extracts were also prepared to assess changes
in proteins of interest. Using Annexin V/PI flow cytom-
etry we observed lower levels of cell death than those
observed using the tetrazolium reduction assays. Given
that the tetrazolium reduction assay is based on metabolic
activity, it was hypothesized that treatment with mevalo-
nate pathway inhibitors induced cell cycle arrest. Cell
cycle analysis demonstrated accumulation of cells in the
G0/G1 phase of the cell cycle indicating that the cell cycle
was inhibited. The degree of inhibition varied for each of
the three cell lines depending on the mevalonate pathway
inhibitor tested, reflecting the sensitivities originally ob-
served in the tetrazolium reduction assays. Immunoblot
analysis of retinoblastoma (Rb) family proteins supported
the observation of cell cycle inhibition, with the greatest
decrease in hyperphosphorylated Rb protein observed
following 72h treatment with fluvastatin. Changes in
prenylation status were detected by immunblot using
antibodies to lamin A/C and Rap1A in order to detect
changes in farnesylation and geranylgeranylation respec-
tively. Treatment of myeloma cell lines with ZA resulted
in an accumulation of unprenylated Rap1A in all three cell
lines while treatment with farnesyl transferase inhibitor
SCH66336 resulted in accumulation of prelamin A indi-
cating inhibition of farneyslation. Treatment with fluvas-
tatin inhibited farnesylation and geranylgeranylation. The
importance of farnesylated and gernaylgeranylated proteins
for myeloma cell survival was then assessed using the
tetrazolium assay. Cells were treated with each of the
inhibitors in the presence of 100microM mevalonic acid
lactone (MVA), 10microM farnesol (FOH) or 10microM
geranygeraniol (GGOH). Overall, the results indicated that
inhibition of geranylgeranylated proteins rather than farne-
sylated proteins results in suppression of apoptosis in
myeloma cell lines.
5
Zoledronate Up-Regulates Bone Sialoprotein Expression
in Saos-2 Cells Through RhoA Inhibition
Akeila Bellahcene,* Michael Chaplet,* Cedric Detry,*
Christophe Deroanne,y Larry W. Fisher,§
Vincent Castronovo*
*Metastasis Research Laboratory, University of Liege,
BelgiumyExperimental Cancer Research Center, University of Liege,
BelgiumzCraniofacial and Skeletal Diseases Branch, N.I.D.C.R.,
National Institutes of Health, H.H.S., Bethesda, Maryland,
USA
Zoledronate (ZOL), a new generation of nitrogen-contain-
ing bisphosphonate, is widely used for the therapeutic
management of osteoporosis and skeletal complications of
malignancy. Recent studies demonstrated that ZOL enhan-
ces bone formation, however a direct effect of this
compound on the expression of proteins playing a pivotal
role in the mineralization process has not been investigat-
ed yet. Several in vivo and in vitro studies indicate that
bone sialoprotein (BSP) is a bone matrix protein that plays
a major role during de novo bone formation. Because of
its bone-inducing activity, the osteosarcoma Saos-2 cell
line is considered as a model of osteoblastic function and
as a source of bone-related molecules including BSP. The
purpose of this study was to search for potential effects of
ZOL on the expression of BSP in Saos-2 cells. ZOL at a
concentration of 20 AM stimulated the synthesis of BSP
mRNA in Saos-2 cells treated for 48 hours. Previous
studies have shown that ZOL is able to interfere with
the mevalonate pathway and inhibits the synthesis of
mevalonate metabolites including farnesyldiphosphate
(FPP) and geranygeranyldiphosphate (GGPP) and thereby
impairs the prenylation of small GTPases such as Ras and
Rho. Therefore, we treated Saos-2 cells with ZOL in the
presence of geranylgeraniol (20 AM) or farnesylfarnesol
(20 AM) to test if these substances could overcome the
inducing effect of ZOL on BSP expression. The effect of
ZOL was reversed by geranylgeraniol but not by farne-
sylfarnesol. Because Rho GTPases undergo geranylgeranyl
modification, we investigated the contribution of Rho to
BSP mRNA up-regulation. Treatment with cytotoxic nec-
rotizing factor-1 (200 ng/ml), an activator of Rho
GTPases, decreased BSP mRNA expression. Furthermore,
inhibition of RhoA synthesis by short interfering (si)RNA
induced an increase of BSP mRNA expression. These
ABSTRACTS / Bone 34 (2004) S46–S99 S49
results are the first demonstration that ZOL up-regulates
BSP expression in Saos-2 cells suggesting a potential new
mechanism for its bone protective effects. This study also
emphasizes the role of the Rho GTPases in ZOL-mediated
BSP up-regulation.
6
Alendronate (ALN) Effects on Pre- and Post- Yield
Behavior of Cortical Bone in Ovariectomized (OX) Rats
may be Independent of Bone Mineralization
M :A: Chiappe,* G. Cointry,y R. Capozza,y G. Iorio,y
E. Alvarez,y J. Zanchetta,z J.L. Ferrettiy
*Faculty of Veterinary, National University of Buenos
Aires, ArgentinayFaculty of Medicine, National University of Rosario,
ArgentinazMetabolic Research Institute (IDIM), Buenos Aires,
Argentina
To describe ALN effects on cortical bone, fourty 3-month
old rats were OX and treated immediately with 0 (OX-C, n =
13), 5 (OX-5, n = 13) or 25 ug/kg sc (OX-25, n = 14) 2/wk
for 6 months, keeping further 15 as sham controls. Their
femurs were scanned by pQCT and tested in bending.
Despite not affecting bone mineralization (volumetric cor-
tical BMD) and cross-sectional geometry (diameters, bone
area, moment of inertia -MI-), OX impaired the stiffness of
both cortical tissue (elastic modulus, E) and diaphyses (load/
deformation ratio), and reduced the loads supported at yield
(Wy) and at fracture (Wf). Strikingly, the post-yield fraction
of Wf (Wp = Wf-Wy, indicative of bone toughness) was
significantly enhanced by OX, perhaps because of the
naturally inverse relationship between the tissue’s ability
to prevent crack generation (impaired) and progress (im-
proved). The highest ALN dose prevented all negative
effects of OX and improved Wf over sham values. No
changes in Wy were observed in treated rats (no effect on
crack generation). However, Wp (bone toughness) was
enhanced in a similar proportion than it was in OX rats.
The naturally negative correlations between MI (y, an
indicator of the efficiency of diaphyseal design) and E (x,
an indicator of bone material’s ‘‘quality’’) showed ‘‘anti-
anabolic’’ shifts (to lower-left region of the graph) for OX-C
and ‘‘anti-catabolic’’ displacements (to upper-right regions)
in OX-5 or -25 rats with respect to sham controls. This
would reflect negative or positive interactions of OX and
ALN, respectively, with the feed-back control of bone
architecture (spatial orientation of bone modeling) as a
function of bone stiffness and mechanical usage of the
skeleton (bone mechanostat theory).
In agreement with previous observations in intact rats
treated with Olpadronate, lack of effects on bone miner-
alization and geometry in this study suggests that both OX
and ALN treatment would have improved Wp (and
additionally ALN would have improved Wf) by affecting
some microstructural determinant(s) of bone material’s
stiffness and toughness (creeping factors) independently
of bone mineralization. These novel effects of bisphosph-
onates may explain the striking dissociation observed
between induced improvements in BMD and fracture
incidence in large studies with post-menopausal osteopo-
rotic women.
7
Effects of High Doses of Olpadronate (OPD) on the
Gustavo Cointry,* Nelida Mondelo,y Ricardo Capozza,*
Jose Luis Ferretti*
*Center for P-Ca Metabolism Studies, National University
of Rosario, ArgentinayGador SA, Buenos Aires, Argentina
High doses of OPD (Gador SA, Buenos Aires; 45–90
mg/kg/d � 3 months, from a carcinogenicity dose-range
finding study) were orally given to 20 male and 24 female
rats 4–5 weeks old (7 and 9 controls). The cortical vBMD,
cross-sectional perimeters (PM), area (CSA) and moment of
inertia (MI) of femur diaphyses and their structural stiffness
(load/deformation ratio) and strength during the successive
‘‘elastic’’, reversible (pre-yield,no microcracks)and ‘‘plas-
tic’’, irreversible (post-yield, microcrack accumulation) de-
formation periods were determined by pQCT and bending
tests. The pre-yield stiffness of cortical tissue (elastic mod-
ulus, E) and a Bone Strength Index, BSI = vBMD*MI
(which can predict ultimate strength but does not capture
any microstructural indicator of cortical tissue) were calcu-
lated from those data.
No effects on growth were observed. Treatment improved
significantly CSA and MI as a function of increases in
both endosteal and periosteal PMs, more evidently in male
than female rats, with no effects on cortical vBMD and E.
As a result, mild increases in structural diaphyseal stiffness
and strength at yield (only significant in males) were
observed. Diaphyseal ultimate strength was substantially
enhanced (males, +38%, p < 0.001; females, 17%, p <
0.01) chiefly because of a large increase in the post-yield
fraction of the ultimate load (males, +300%, p < 0.001,
+80%, p < 0.05). The BSI failed to predict ultimate load in
treated animals.
The possitive effects of the assayed OPD doses on the pre-
yield behavior of bones would reflect an anabolic improve-
ment in diaphyseal geometry induced independently of
bone material’s mineralization and elastic stiffness (i.e.
beyond the homeostatic control of bone structure as pre-
dicted by the mechanostat theory). The large effects on
post-yield behavior and ultimate strength of the bones
should be assigned to changes in other factors (not deter-
mined in the study) affecting the progress of cracks within
cortical tissue (‘‘plastic’’ deformation period) previously to
fracture. Failure of BSI to predict ultimate strength suggests
that the observed bone strengthening would have been
determined chiefly by mineralization-unrelated, microstruc-
tural factors in this study. These results point out some
ABSTRACTS / Bone 34 (2004) S46–S99S50
novel bisphosphonate effects on bone strength and mech-
anism of fracture with no apparent involvement of bone
mineralization.
8
Effects of Zoledronic Acid on the Adhesion and Invasion
of Prostate Cancer Cells and the Role of the
SDF-1/CXCR4 Chemokine Pathway on Cellular
Migration
Jonathan Coxon; Lisa Pickering, Claire Macdonald, Roger
Kirby, Kay Colston.
Introduction and objective: Prostate cancer (CaP) prefer-
entially metastasises to bone, where despite the formation of
sclerotic metastases it is believed to induce resorption, in
conjunction with new bone formation. Bisphosphonates
(BPs), which avidly localise to bone and are known to
inhibit osteoclasts, have also been shown to have direct
effects on cancer cell function. Potent nitrogen-containing
bisphosphonates such as zoledronic acid (ZOL) have been
shown to inhibit the mevalonate pathway, which normally
allows prenylation of G-proteins, required for their mem-
brane localisation and hence normal function. Other BPs
have previously been shown to inhibit adhesion and inva-
sion of CaP cells. In this study, we examined the effects of
ZOL on adhesion to mineralised matrix and invasion
through extracellular matrix material, of DU145 and PC3
CaP cells. We investigated how these effects were altered by
adding analogues of mevalonate pathway intermediates:
farnesol (FOH), or geranylgeraniol (GGOH), which allow
direct prenylation of proteins. We compared effects of ZOL
with those induced by manumycin and GGTI-298, inhib-
itors of farnesyl- and geranylgeranyltransferase (enzymes
responsible for prenylation), and C3-exoenzyme (C3X), an
inhibitor of Rho function. We also investigated the role of
the SDF-1/CXCR4 chemokine pathway in the migration of
prostate cancer cells. SDF-1 is found in high concentrations
in bone tissue, and is thought to influence the localising of
prostate cancer cells to bone.
Materials and methods: Cells were exposed for 24 hours
to various reagents before the assays were carried out. To
test adhesion, treated cells were seeded onto dentine slices
and left for 24 hours. For invasion, cells were added to
Matrigel Invasion Chambers, and left for 48 hours. Ad-
hering or invading cells were then fixed, stained and
counted.
Results: ZOL inhibited adhesion and invasion in a con-
centration-dependent fashion. The lowest dose used, 1AM,
inhibited adhesion by up to 38.3%, and invasion by up to
27.5%. 100AM ZOL inhibited adhesion by up to 85.4%,
invasion by as much as 89.9%. Effects of ZOL were
rescued to a greater degree by GGOH than FOH. GGTI-
298 had a greater inhibitory effect on adhesion and
invasion than manumycin. C3X also significantly inhibited
both properties. Invasion of both cell lines was increased
by adding SDF-1 to the lower part of the invasion
chambers. This effect was negated by adding either SDF-
1 or anti-CXCR4 antibody to the upper chamber, and
reduced by ZOL.
Discussion: ZOL inhibits adhesion and invasion of CaP
cells in vitro. The underlying mechanism appears to
involve inhibiting geranylgeranylation of proteins, such
as Rho—a G protein known to play a crucial role in
several cell functions involving re-organisation of the
cytoskeleton. The SDF-1/CXCR pathway has a clear
influence on the migration of CaP cells across extracellular
matrix material.
9
Structure-Activity Relationships for Inhibition of Rab
Prenylation and Inhibition of Bone Resorption by
Phosphonocarboxylate Drugs
Fraser Coxon,* Javier Rojas Navea,* F. Hal Ebetino,y
Michael Rogers*
*Bone Research Group, Institute of Medical Sciences,
University of Aberdeen, UKyProcter & Gamble Pharmaceuticals, Cincinnati, OH, USA
Nitrogen-containing bisphosphonates such as risedronate act
by inhibiting farnesyl diphosphate (FPP) synthase, thereby
preventing the synthesis of isoprenoid lipids required for
prenylation of Ras, Rho and Rab family proteins in osteo-
clasts. We recently found that a weakly anti-resorptive
phosphonocarboxylate analogue of risedronate, NE10790,
selectively prevents prenylation of Rabs in cells in vitro by
inhibiting Rab geranylgeranyl transferase (Rab GGTase).
This compound does not inhibit FPP synthase or protein:-
prenyl transferases other than Rab GGTase, and so has no
effect on prenylation of other small GTPases. We have now
examined the effect of two additional phosphonocarboxylate
analogues of NE10790: PGE1560036, which lacks the
geminal hydroxyl group of NE10790, and PGE-5752082,
in which the position of the nitrogen in the pyridyl ring of
the side-chain is altered (2-pyridyl vs 3-pyridyl in PGE-
1560036). PGE-1560036, like NE10790, specifically
inhibited the prenylation of Rab proteins in J774 macro-
phages and rabbit osteoclasts, demonstrated by metabolical-
ly labelling cells with [14C]mevalonate and by separating
prenylated Rabs from unprenylated Rabs by triton X-114
fractionation. Moreover, PGE-1560036 was of similar po-
tency to NE10790, effectively inhibiting Rab prenylation at
concentrations 0.5mM and above. By contrast, an analogue
of risedronate that lacks the geminal hydroxyl group
(NE58043) was approximately four-fold less potent at
inhibiting protein prenylation than risedronate. Like
NE10790, PGE-1560036 disrupted the golgi-localisation
of Rab6 in J774 cells and rabbit osteoclasts cultured on
plastic, visualised by immunofluorescence staining. How-
ever, PGE-1560036 was a weaker inhibitor of bone resorp-
tion in vitro than NE10790. This could be due to lack of
affinity of PGE-1560036 for bone mineral, since this
compound lacks a geminal hydoxyl group. In support of
ABSTRACTS / Bone 34 (2004) S46–S99 S51
this, we found that pre-treatment of dentine discs with
NE10790 was able to inhibit resorption by osteoclasts
cultured on these discs, whereas pre-treatment of discs with
PGE-1560036 had little effect. PGE-5752082, at concen-
trations up to 1.5mM, had no effect on prenylation of Rabs
or other proteins in J774 cells and did not affect bone
resorption by osteoclasts in vitro. In summary, these data
demonstrate that a geminal hydroxyl group is not required
for inhibition of Rab GGTase by phosphonocarboxylates,
but does contribute to the ability of bisphosphonates to
inhibit FPP synthase. As with bisphosphonates, the hydrox-
yl group also appears to be important for the anti-resorptive
action of phosphonocarboxylates in vitro by enabling them
to bind to bone mineral prior to uptake by osteoclasts. In
addition, as with bisphosphonates, the position of the
nitrogen in the side chain of phosphonocarboxylates appears
to be crucial for the ability of phosphonocarboxylates to
inhibit Rab GGTase and hence to inhibit bone resorption.
10
Visualisation of the Uptake of a Novel Fluorescent
Bisphosphonate by Resorbing Osteoclasts and J774 Cells
Fraser Coxon; Jonathan Langton, Michael Rogers. Bone
Research Group, Institute of Medical Sciences, University of
Aberdeen, UK.
Anti-resorptive Bisphosphonates (BPs) act by inhibiting the
intracellular enzyme farnesyl diphosphate (FPP) synthase in
osteoclasts, resulting in the inhibition of prenylation of
small GTP-binding proteins essential for osteoclast function.
BPs do not readily cross the cell membrane, and the exact
mechanism by which they are internalised by osteoclasts
remains unclear. To investigate this, we synthesised a
fluorescently-labelled BP (alendronate; ALN) by conjuga-
tion with alexa-fluor 488 succinimidyl ester. Labelled ALN
was separated from unreacted fluorochrome by calcium
precipitation. The resulting compound (ALN-AF488)
retained high affinity for bone mineral.
Confocal microscopy was used to examine the uptake of
ALN-AF488 by rabbit osteoclasts cultured for 24 hours on
dentine slices precoated with ALN-AF488. The ALN-
AF488 enabled visualisation of the dentine surface, which
together with TRITC-phalloidin staining allowed the iden-
tification of active osteoclasts. Resorption pits were also
intensely labelled when dentine was precoated with ALN-
AF488, but not when the protein on the dentine surface had
been labelled with carboxytetramethylrhodamine succini-
midyl ester. This indicates that ALN-AF488 binds back to
the bone surface following release from the surface by
resorbing osteoclasts, supporting the ‘recycling’ theory.
ALN-AF488 was internalised by resorbing osteoclasts into
distinct intracellular vesicles that became dispersed through-
out the cell. The presence of labelled vesicles at the baso-
lateral surface suggests that BPs may enter a transcytotic
pathway. Surprisingly, however, when the dentine was dual-
labelled the ALN-AF488 and surface protein labels were
largely confined to separate vesicles in osteoclasts, showing
little colocalisation. Non-resorbing osteoclasts showed no
evidence of uptake of ALN-AF488 or surface protein.
Although J774 macrophages could not internalise bone-
bound ALN-AF488, when cultured on glass coverslips these
cells internalised ALN-AF488 into discrete vesicles
throughout the cytoplasm in as little as five minutes. When
cultured in the presence of TRITC-dextran, a marker of
fluid-phase endocytosis, much of the ALN-AF488 and
dextran co-localised to the same vesicles and were taken
up at similar rates. By contrast, Alexa-fluor633-transferrin, a
marker of receptor-mediated endocytosis, localised to
vesicles that were completely distinct from those containing
dextran and/or ALN-AF488, indicating that ALN-AF488 is
internalised by fluid phase endocytosis. However, the up-
take of ALN-AF488 (but not that of TRITC-dextran) in
J774 cells was prevented by the presence of a ten fold
excess of clodronate. This suggests the involvement of an
additional stage of recognition of BP at the cell surface,
prior to internalisation by endocytosis.
These studies provide novel insights into the mechanism by
which BPs are internalised by cells, and indicate that ALN-
AF488 is a useful tool for studying the ability of different
cell types to take up BPs.
11
Pilot Studies of the Effect of Zoledronic Acid on
Tumour-derived Cells Ex Vivo in the ATP-Based Tumour
Chemosensitivity Assay (ATP-TCA)
Ian Cree; Louise Knight, Mark Conroy, Augusta Fernando.
Translational Oncology Research Centre, Department of
Histopathology, Queen Alexandra Hospital, Portsmouth
PO6 3LY.
Background: There is currently considerable debate regard-
ing the direct effect of bisphosphonates against visceral
metastases from solid tumours, despite their proven efficacy
against the skeletal complications of metastasis. Data from
cell lines suggests that such effects may exist, and may be
linked to inhibition of the mevalonate pathway.
Aim: The aim of this pilot study was to determine whether
zoledronic acid showed any direct activity against tumour-
derived cells in the ex vivo ATP-based tumour chemo-
sensitivity assay.
Methods: Cells were obtained from ascites or solid tumour
material by enzymatic digestion and tested with zoledronic
acid (Novartis), alendronate (Calbiochem) and clodronate
(Calbiochem) in the ATP-TCA. The results were expressed
as a percentage inhibition at each of six concentrations
tested to span the concentrations thought to be achievable
clinically. Zoledronic acid was tested in the ATP-TCA (6
day exposure) at 2.15–68.9 microM, alendronate at 1.93–
61.6 microM, and clodronate at 1.73–55.4 microM. A
total of five ovarian cancer cell lines (1847, 1847ad,
OVCAR, OVCA3, SKOV) and 17 tumours have been
tested to date, including 12 ovarian carcinomas, 3 cutane-
ABSTRACTS / Bone 34 (2004) S46–S99S52
ous melanomas, 1 uveal melanoma, and 1 carcinoma of
unknown primary site.
Results: Zoledronic acid showed concentration-dependent
inhibition with a median IC50 for all tumours tested of 31
microM. For alendronate, the IC50 was 54.8 and for
clodronate it was 80.3 microM. Cell lines were more
sensitive: the IC50 obtained for zoledronic acid was <8
microM in four of the five lines tested, while clodronate was
inactive in four lines, including three of those sensitive to
zeledronic acid.
Discussion: The ATP-TCA has been used to assess several
new drugs and has a strong track record in designing new
combinations. Effects observed tend to translate well to
clinical benefit in patients, and the assay can be used as a
predictive test to guide choice of chemotherapy. These
results of this pilot study therefore suggest a direct effect
of zoledronic acid and other bisphosphonates against neo-
plastic cells.
12
Evidence for A Specific Mechanism of Cellular Uptake
of Bisphosphonates
Julie C: Crockett; Keith Thompson, Fraser P. Coxon,
Michael J. Rogers. Bone Research Group, Department of
Medicine and Therapeutics, Institute of Medical Sciences,
University of Aberdeen, Foresterhill, Aberdeen, UK.
The exact mechanism by which bisphosphonates (BPs) are
internalised by osteoclasts remains unclear. We have previ-
ously shown that the non-nitrogen-containing BP (non-N-
BP) clodronate (CLO) antagonises the effects of nitrogen-
containing BPs (N-BPs) in J774 macrophages in vitro. We
demonstrated that, as well as preventing N-BP-induced
inhibition of protein prenylation, CLO partially prevented
the uptake of 14C ibandronate. We postulated that BP uptake
takes place via a membrane-bound transport protein. To
investigate this further, we synthesised fluorescently-la-
belled alendronate (ALN) by conjugation with Alexa-Flu-
or-488 succinimidyl ester (AF488).
The internalisation of ALN-AF488 was examined using
FACS analysis and confocal microscopy. Uptake of ALN-
AF488 could be seen in intracellular vesicles within 5
minutes of treatment of J774 cells. By FACS analysis,
J774 cells were found to markedly accumulate ALN-
AF488 within 4 hours and particularly after 8 hours. The
non-N-BP CLO dramatically inhibited the uptake of
100AM ALN-AF488. 0.25mM, 0.5mM and 1mM CLO
reduced ALN-AF488 uptake by 39%, 50%, and 70%
respectively at 4 hours, and reduced uptake by 45%, 68%
and 77% at 8 hours. The effectiveness of CLO for inhibit-
ing the uptake of ALN-AF488 was not due to inhibition of
fluid-phase pinocytosis, since CLO did not affect the
uptake of FITC-dextran.
Using confocal microscopy we examined the ability of BPs
and related compounds to affect uptake of ALN-AF488
after a 3-hour incubation. A ten-fold excess of the active N-
BP risedronate (RIS) completely inhibited uptake of ALN-
AF488 by J774 cells. The inactive N-BP NE10571 had
identical effects. By contrast, an active (NE10790) and an
inactive (PGE-5752082) phosphonocarboxylate only par-
tially inhibited ALN-AF488 uptake, while a monophosph-
onate analogue of risedronate (PGE-102784) had no effect.
None of these compounds affected uptake of TRITC-dex-
tran by J774 cells. 1mM EGTA had no effect on ALN-
AF488 uptake, indicating that the inhibitory effect of excess
BPs was not simply due to chelation of Ca2+. Hence, the
uptake of ALN-AF488 can be inhibited by the presence of
molar excess of other BPs, and to a lesser extent by
phosphonocarboxylates but not by monophosphonates.
1mM phosphonoformate (an inhibitor of the sodium/phos-
phate co-transporter) or 1mM probenecid (an inhibitor of
organic anion transporters) both failed to antagonise N-BP-
induced accumulation of unprenylated Rap1A (a marker of
N-BP uptake). The presence of excess pyrophosphate
(5mM) or phosphate (5mM) also failed to prevent N-BP-
induced inhibition of Rap1A prenylation, suggesting that
the mechanism of cellular uptake does not involve the PPi
transporter Ank. Taken together, these observations strongly
suggest that BPs are internalised by cells via a specific
transport mechanism that remains to be identified.
12a
Effects of pamidronate on experimental ectopic bone
formation in the rat
Debinski Andrzej*, Sawicki Andrzejy, Nowicka Grazyna*
*National Food and Nutrition InstituteyMedical Centre
Bisphosphonates are the group of drugs that inhibit bone
resorption but, N-containing bisphosphonates may modulate
osteoblasts activity. The pamidronate, N-containing
bisphosphonate, is well recognised as successful anti-re-
sorptive agent for therapy of bone lesions characterised by
excessive bone resorption (osteoporosis, Paget’s disease,
tumoral osteolysis with skeletal metastases), however data
on its influence on osteoblast activity are limited.
The aim of the study was to assess the influence of sodium
pamidronate on bone formation and resorption in the
process of experimental ectopic bone formation. Ectopic
bone formation was induced 10 days after the start of
treatment. A total of 30 rats divided into two groups. In
15 rats (treated group) receiving 1 mg/kg b.m/week sodium
pamidronate and in 15 control animals devitalised bone
matrix grafts was implanted intramuscularly into two thorax
regions. Six weeks later the ossicles were removed and
prepared, without decalcification, for histomorphometric
analysis. Bone area (B.Ar) and osteoblast perimeter
(Ob.Pm) were higher in experimental group as compared
to control group (35.6F 2,8 vs. 18,7F 3,4; p < 0.01:
22.5F 4.1 vs. 12.2F 2.9; p < 0.01, respectively). Osteoclast
number (Oc.N) was decreased in treated group (1.27F 0.30
vs 2.54F 0.31; p < 0.05).
ABSTRACTS / Bone 34 (2004) S46–S99 S53
It can be concluded, that pamidronate not only decreases
osteoclasts activity but also stimulates bone formation in
bone matrix grafts.
13
Effect of Bisphosphonates and Dexamethasone on Nitric
Oxide Production of Cartilage
E:J : Dombrecht; E.G. Neven, J.F. Van Offel,
A.J. Schuerwegh, C.H. Bridts, W.J. Stevens, L.S. De Clerck.
Immunology-Allergology-Rheumatology, University of
Antwerp, Belgium.
Introduction: Rheumatoid arthritis (RA) is characterized by
chronic inflammation in the joints accompanied by destruc-
tion of cartilage and bone. During inflammation, chondro-
cytes are exposed to pro-inflammatory cytokines such as IL-
1 and TNF-a that induce the synthesis of inflammatory
mediators such as reactive oxygen species, inflammatory
cytokines and nitric oxide (NO). It is known that NO plays
an important role in cartilage degeneration.
It has been suggested that bisphosphonates possess anti-
inflammatory properties and can be effective in the treat-
ment of RA.
The aim of this study was to investigate the influence of
bisphosphonates and dexamethasone on NO production of
bovine cartilage explants.
Methods: Full-thickness bovine cartilage explants were
obtained from the tarsometatarsal joints of 2–4 year-old
animals using a 4 mm biopsy punch. The cartilage discs
were preincubated during 48 h in Dulbecco’s modified
Eagle’s medium supplemented with 10% Fetal Bovine
Serum (FBS) alone as a control or in medium with varying
concentrations of the bisphosphonates clodronate (10–4,
10–6, 10–8 mol/L), pamidronate (10–6, 10–8 mol/L) or
risedronate (10–6, 10–8 mol/L), or with dexamethasone
(10–7 mol/L). After preincubation, the medium was
replaced by culture medium without FBS and with or
without bisphosphonates or dexamethasone. The cartilage
discs were stimulated with IL-1a and TNF-a each in a
concentration of 10 ng/mL for 48 h. NO was measured
using a spectrophotometric Griess assay.
Tissue wet weight was determined by individually weighing
the tissue from each well. The results were expressed in
Amol NO2-/L/mg cartilage.
Results: Clodronate and dexamethasone significantly
inhibited the NO production (p < 0.05). The amino-
bisphosphonates pamidronate and risedronate didn’t have
a significant effect on the NO production (p = 0.10 and 0.14
respectively).
Conclusion: The non-nitrogen containing bisphosphonate,
clodronate, and dexamethasone inhibit the NO production
of bovine cartilage. This suggests that these compounds
may have anti-inflammatory properties by inhibiting the
NO production resulting in reduced inflammation and
joint damage. The nitrogen-containing bisphosphonates
pamidronate and risedronate showed no anti-inflammatory
nor pro-inflammatory effect on the NO production by
cartilage.
14
In Vitro Antioxidant Profile of Bisphosphonates
E:J : Dombrecht,1 P. Cos,2 J.F. Van Offel,1 A.J. Schuerwegh,1
C.H. Bridts,1 D. Vanden Berghe,2 W.J. Stevens,1
L.S. De Clerck1
1Immunology-Allergology-Rheumatology2Pharmaceutical Microbiology, University of Antwerp,
Belgium
Introduction: Reactive oxygen species are believed to play
a major role in the inflammatory process in rheumatoid
arthritis (RA) and contribute to the destruction of cartilage
and bone. It has been suggested that bisphosphonates
possess anti-inflammatory properties and can be effective
in the treatment of RA.
The aim of this study was to investigate the in vitro
antioxidant profile of different bisphosphonates. Different
mechanisms of antioxidant activity have been described
such as inhibition of enzymes, free radical scavenging,
and chelation of transition metals.
Methods: Clodronate, pamidronate, risedronate, ibandro-
nate, zolendronate and pyrophosphate were tested for their
xanthine oxidase inhibiting capacity. The activity of xan-
thine oxidase was spectrophotometrically determined by
measuring the production of uric acid from xanthine at
290 nm. Microsomal lipid peroxidation inhibiting capacitiy
was spectrophotometrically measured using the thiobarbitu-
ric acid (TBA) test. Furthermore, the effect of these different
compounds on a stable and an unstable radical was inves-
tigated (radical scavenging activity). 1,1-Diphenyl-2-picryl-
hydrazyl free radical (DPPH) is a stable radical and the
decrease in DPPH absorption was measured at 517 nm.
Clodronate, risedronate and pyrophosphate were further
tested for their hydroxyl radical scavenging activity. The
unstable hydroxyl radicals were generated in a Fenton type
reaction and were visualized by 5,5-dimethyl-1-pyrroline-N-
oxide (DMPO) in an EPR instrument.
Results: None of the tested compounds showed xanthine
oxidase inhibiting activity nor DPPH scavenging activity at
concentrations up to 100 Amol/L. All the tested bisphosph-
onates and pyrophosphate exhibited inhibiting capacities
on the microsomal lipid peroxidation: the IC50 values
varied in a range from 0.7 Amol/L (pamidronate) and 9.4
Amol/L (pyrophosphate). Risedronate showed the highest
hydroxyl radical scavenging activity (80% inhibition),
while clodronate and pyrophosphate inhibited for 55%
and 38% respectively.
Conclusion: Bisphosphonates possess an inhibiting activity
on the microsomal lipid peroxidation and the Fenton
reaction. In these reactions iron plays an important role
suggesting that the selective in vitro antioxidant properties
of the bisphosphonates are due to their iron chelating
characteristics.
Table 1
Group Mean
OIS (%)
SD
Control (NACL 0.9%) 29.4 15.1
Continuous (25 MG/KG/D IB) 46.1 21.4
Single Shot (25 MG/KG � 27 IB) 45.3 24.1
ABSTRACTS / Bone 34 (2004) S46–S99S54
15
Bisphosphonates Inhibit the Mitogenic Effects of
Insulin-Like Growth Factor-1 on Prostate Cancer Cells
Jean-Claude Dumon,* Naıma Kheddoumi,*
Laurence Lagneaux,y Fabrice Journe,* Jean-Jacques Body*
*Lab. of Endocrinology and Bone Diseases, Inst. J. Bordet,
Free University of Brussels, BelgiumyLab. of Hematology, Inst. J. Bordet, Free University of
Brussels, Belgium
The high concentrations of growth factors, especially Insu-
lin-like Growth Factor-1 (IGF-1), released from bone matrix
during tumor-induced osteolysis provide a fertile soil for
metastatic prostate cancer cells growth. Besides their well-
known inhibitory effects on osteoclast-mediated bone re-
sorption, we and others recently showed that bisphospho-
nates (BPs), especially Zoledronic Acid, can also induce
prostate cancer cell death in vitro (Dumon et al., Eur Urol, in
press). In this study, we have investigated the effects of the
co-addition of BPs (Zoledronic acid, Zole; or Clodronate,
Clod) and IGF-1 on human PC-3 prostate cancer cells. PC-3
cells were cultured in Ham’s-F12/RPMI supplemented with
2mM L-Gln and 10% inactivated FCS that was also deplet-
ed of growth factors. Cell growth was evaluated at days 1, 2,
4 and 6 by crystal violet staining. We selected optimal
stimulatory concentrations of IGF-1 (100 ng/ml) and three
concentrations of BPs (10�6, 10�5 and 10�4 M). IGF-1
increased PC-3 cells growth by 28 F 5% (mean F SEM) at
day 2 and by 23 F 7% at day 6 (P < 0.001). At 10�6 M,
Zole did not affect cell growth but it completely abolished
the mitogenic effects of IGF-1. At 10�5 M, Zole inhibited
cell proliferation whether IGF-1 was present or not. At 10�4
M, Zole induced a marked decrease in cell number (at day 6,
cell population fell to 15% in comparison with the day 1
value). On the contrary, 10�6 M Clod did not affect cell
growth neither the stimulatory actions of IGF-1. At 10�5 M,
Clod was again without effect on growth but it abolished the
mitogenic actions of IGF-1. At 10�4 M, Clod weakly
inhibited cell proliferation and also suppressed the stimula-
tory actions of IGF-1. We previously reported that
bisphosphonates reduce prostate cancer survival by exerting
cytostatic and apoptotic effects. We assessed apoptotic cell
death by using annexin V/propidium iodide double staining
method based on apoptosis-related cell membrane modifi-
cations. Apoptosis was assessed when cells were co-treated
with 10�4 M bisphosphonates and 100 ng/ml IGF-1. Zole
induced apoptosis in PC-3 cells and this effect was en-
hanced if IGF-1 was simultaneously added. Clod did not
induce apoptosis in PC-3 cells, whether IGF-1 was present
or not. In conclusion, bisphosphonates can inhibit the
mitogenic effects of IGF-1 on prostate cancer cells, at
10�6 M for Zole and 10�5 M for Clod, while alone they
had no effect on cell proliferation at these concentrations.
Moreover, at higher concentrations, bisphosphonates actions
were not affected by IGF-1. Importantly, Zole induced
apoptosis even more in the presence of IGF-1. This could
represent a new mechanism of action of bisphosphonates in
their protective effects against prostate cancer-induced bone
disease and contribute to the beneficial effects of Zole for
the treatment of prostate cancer-induced bone disease.
16
Single Shot Ibandronat is as Effective as Continuous
Application in Implant Osseointegration
Christian Eberhardt,* Jochen Brankamp,*
Frieder Bauss,y Andreas A. Kurthz
*Dep. Orthopaedic Surgery, University Hospital Frankfurt/
MainyRoche Diagnostics GMBH, Pharma Research, PenzbergzDep. Orthopaedic Surgery, Orthopaedic Oncology Service,
University Hospital Frankfurt/Main
Uncemented total joint replacement is has an increasing
number of indications (e.g. younger patients). After implan-
tation initial mechanical stability is achieved by press-fit
implantation of the components. Secondary stability
depends on osseointegration of the implant. It is known
from experiments that by using a bisphosphonate after the
implantation of a cementless implant the amount of bone
attached to it can be increased. A recent study showed
ibandronate to be effective in reducing the time of osseoin-
tegration of HA-coated implants in an animal model. The
purpose of the present study was to evaluate whether a dose
equivalent single shot application of ibandronate is as
effective as a continuous daily application with regard to
osseointegration.
M&M: 60 S-D rats were randomly assigned to two treatment
groups and a control group. For a treatment period of 27 days
the first treatment group received daily s.c application of 25
Ag/kg ibandronate starting at day of surgery. The second
treatment group received a dose equivalent single shot
application of 25 Ag/kg � 27. The control group was treated
with NaCl 0.9%. Titanium and HA-coated titanium implants
were surgically inserted in press-fit technique into the med-
ullary canal of each femur through the intercondylar notch of
the knee. After harvesting the specimens radiographs were
taken and they were prepared for quantitative histomorph-
ometry. To quantify the extend of osseointegration the
osseointegrated implant surface (OIS) was determined. This
was defined as the ratio (%) of the implant surface in di-
rect contact with surrounding bone to the total implant
surface. Data were expressed as mean F standard deviation
(SD). Statistic was performed using the statistics package
SigmaStatk (SPSS Inc.). AWilk-Shapiro test for normality
was conducted on all parameters. To answer the research
Table 2
Difference (%) P < 0.05
Control vs. cont. +56.8 SIG.
Control vs. single shot +45.1 SIG.
Single shot vs. cont. +1.8 N.S.
ABSTRACTS / Bone 34 (2004) S46–S99 S55
questions, a one-way analysis of variance (ANOVA) was
carried out on all groups. A Tukey post-hoc analysis was
performed to determine with groups differed significantly
(p < 0.05).
Results: For HA-coated implants the OIS of both treatment
groups was significantly higher compared to the control
group. The comparison of both treatment groups showed no
statistical differences. For uncoated titanium-only implants
no significant effect on osseointegration was found.
Discussion: The present study demonstrates that a bis-
phosphonate treatment with the 25 Ag/kg ibandronate either
with daily application or with a dose equivalent single shot
application is effective to improve osseointegration of
hydoxyapatite-coated metal implants. The results of this
Fig. 1.
study indicate that administration of a bisphosphonate after
implantation of cementless endoprosthesis can improve early
secondary stability and by that probably the long time
survival rate of the implant. A clinically convenient dose
equivalent single shot application is shown to be as effective
as a continuous application (Tables 1 and 2).
17
Studies to Elucidate the Mechanism of Action of
Bisphosphonates in Osteoarthritis
F:H : Ebetino;M. Janusz, J.M. Meyer, W.F. Aronstein, M.D.
Francis. Procter & Gamble Pharmaceuticals, Mason, OH,
45040, USA.
We have shown that some pyridinyl bisphosphonates (BPs)
such as risedronate, are effective at inhibiting cartilage
lesion size, severity, and osteophytosis in the spontaneous
guinea pig model of primary osteoarthritis (Meyer JM et al
Arthritis Rheumatism, 2001) Efficacy in this model of
arthritis was observed even with compounds that exhibit
little antiresorptive activity. Therefore we have continued
ABSTRACTS / Bone 34 (2004) S46–S99S56
studies to investigate other potential mechanisms by which
BPs may attenuate arthritis.
Among these potential mechanisms, inflammatory process-
es were considered. Adjuvant induced arthritis in the rat is
characterized by severe intra- and peri-articular inflamma-
tion, which also appears coupled to bone lysis. A series of
BPs of varying antiresorptive potency was therefore inves-
tigated in this model. Modified Freunds adjuvant (MFA, 4.4
mg/kg) was injected into the tail of Lewis rats. Daily
subcutaneous (sc) injections of a variety of BP’s were
investigated starting on the day of MFA injection. Treatment
continued until Day 24. Risedronate and several analogs
were very effective at reducing paw volume swelling and
bone resorption in this model. At certain doses this effect
continues even after treatment is stopped. As found in the
guinea pig model, antiresorptive potency did not track the
effect of bisphosphonates in this model although all of the
potent antiresorptive analogs also demonstrated marked
reductions in paw volume swelling.
Bone affinity and antiresorptive properties are likely im-
portant components of BP effects, but these properties do
not fully predict efficacy in two models of arthritis. Inter-
estingly, from our preliminary study, potencies in the rat
adjuvant model and the guinea pig model do parallel,
suggesting a possible common mechanism beyond bone
resorption between two models of different types of arthri-
tis. Inflammation in the adjuvant RA model is well estab-
lished, but the role of inflammation in OA and the guinea
pig model is not clear. Thus, an inflammatory component in
OA, and the possible role of BPs in affecting this target
needs further study. Although their ability to inhibit bone
resorption may provide a useful therapeutic adjunct for
patients with arthritis, another activity, possibly anti-inflam-
matory, but yet to be elucidated, may also provide further
therapeutic benefit in both osteoarthritis and rheumatoid
arthritis (Fig. 1).
18
Zoledronic Acid and TRAIL Induce Synergistic Increase
in Apoptotic Tumor Cell Death
Alyson Evans; Helen Neville-Webbe, Robert E. Coleman,
Ingunn Holen. Clinical Oncology, Genomic Medicine,
School of Medicine and Biomedical Sciences, University
of Sheffield, UK.
Background: Cancer patients frequently receive a com-
bination of different therapies, but our understanding of
the potential benefits of such treatment combinations is
limited. We have investigated the effects on tumour cell
death using the potent bisphosphonate zoledronic acid
(ZOL) combined with TNF-related apoptosis inducing
ligand (TRAIL). Zoledronic acid is increasingly used to
treat metastatic bone disease in breast and prostate
cancer, and is currently in clinical trials in the adjuvant
setting. TRAIL is produced in tumours by invading
macrophages as part of the defence against cancer, and
acts through receptors with an intracellular death domain
(DR4/DR5). Based on its ability to selectively kill tumour
cells, TRAIL has been suggested to be utilised as a cyto-
toxic agent in the treatment of various tumour types.
In the current study, breast and prostate cancer cells were
exposed to a number of combinations of zoledronic acid and
TRAIL, and the levels of apoptotic cell death determined at
various time points.
Methods: The breast cancer lines MDA-MB-436 and
MCF7 and the prostate cancer cell line PC3 were treated
with ZOL (25mM) and TRAIL (10ng/ml) for varying
incubation periods and sequences as follows:
Group 1: TRAIL given first for 24 hrs followed by ZOL for
48 hrs.
Group 2: ZOL given first for 48 hrs followed by TRAIL for
24 hrs.
Group 3: ZOL and TRAIL given simultaneously for 24 hrs.
Group 4: TRAIL given first for 24 hrs then ZOL for 4 hrs
followed by drug-free medium for 48 hrs.
Group 5: ZOL given first for 4 hrs and cells maintained in
drug-free medium for 48 hrs followed by TRAIL for 24 hrs.
Results: The combination of ZOL and TRAIL had poten-
tially synergistic effects in inducing apoptotic death of
breast cancer cells, but the order in which the drugs are
given is important. Giving TRAIL before the ZOL (group 1)
increased apoptosis from 1.75% (TRAIL only) and 0.5%
(ZOL only) to 2.4% in the combined group. Likewise,
treating with ZOL and TRAIL together for 24 hrs (group
3) increased the level of apoptosis from 1.75% (TRAIL
only) and 0.7% (ZOL only) to 2.5% in the combined group.
When cells were treated with ZOL for 48 hrs followed by
TRAIL (group 2) results were indicative of synergy between
these two drugs. In the combined group there was 14.65%
apoptosis which was significantly greater than ZOL only,
(0.7% p < 0.001) and TRAIL only (2.7%, p < 0.001).
Similar results were obtained when a shorter incubation
period with ZOL (4hrs) was used, with apparent synergy
obtained only when ZOL was given before TRAIL. Prostate
cancer cells were also sensitive to the combination of ZOL
and TRAIL when these cells were treated as outlined for
group 5.
Conclusion: The combination of ZOL and TRAIL has
potential synergistic ability in inducing apoptosis of tumour
cells in vitro, but for maximum effect ZOL has to be given
prior to TRAIL treatment. The molecular mechanisms
underlying the synergistic effect of combining ZOL and
TRAIL are currently under investigation.
19
NE-10790, A Phosphonocarboxylate Analogue of the
Bisphosphonate Risedronate, Exhibits Direct Antitumor
Activity In Vivo
P:G: Fournier,* M.W. Lundy,y F.H. Ebetino,y P. Clezardin*
*INSERM U403, Laennec School of Medicine, Lyon,
FranceyProcter & Gamble Pharmaceuticals, Mason, OH, USA
ABSTRACTS / Bone 34 (2004) S46–S99 S57
In addition to being powerful inhibitors of bone resorp-
tion in vivo, bisphosphonates (BPs) also exhibit potent
antitumor activity. In vitro, BPs inhibit tumor cell adhe-
sion, invasion and proliferation, and they induce apopto-
sis of tumor cells. In vivo, BPs reduce skeletal tumor
growth. However, because of their high affinity for bone
mineral and rapid uptake in bone, tumor cells in the
bone marrow may be exposed to BPs for too short a
period to observe cytotoxicity. It is most likely that the
antitumor activity of BPs in bone is mediated through
inhibition of bone resorption which, in turn, deprives
tumor cells of bone-derived growth factors. Conversely, a
BP having a low bone affinity could act directly on
tumor cells in the bone marrow because of its rapid
release from bone mineral. To address this question, we
compared the antitumor potency of the nitrogen-contain-
ing BP risedronate with that of the phosphonocarboxylate
analogue of risedronate (NE-10790) in which one of the
phosphonate groups is substituted by a carboxyl group.
NE-10790 had a 15-fold lower affinity for bone mineral
compared to that observed with risedronate. In vitro, NE-
10790 and risedronate inhibited proliferation of GFP-
expressing B02 breast cancer cells (IC50s: 3.4 F 0.9
and 0.4 F 0.2 mM, respectively). Continuous treatment
of mice with risedronate (0.15 mg/kg/day) almost com-
pletely inhibited bone destruction caused by GFP-express-
ing B02 breast cancer cells (as judged by radiography)
and substantially reduced skeletal tumor burden (as
judged by external fluorescence imaging and histomorph-
ometry). NE-10790 (0.15 mg/kg/day), under similar ex-
perimental conditions, did not inhibit bone destruction
whereas it did drastically inhibit skeletal tumor burden
(70% reduction). This lack of inhibitory effect of NE-
10790 on bone destruction was consistent with the
observation that NE-10790 was 8,000-fold less potent
than risedronate (on a mg/kg basis) in inhibiting bone
resorption in ovariectomized rats. Moreover, a continuous
treatment of mice with NE-10790 (or risedronate), at a
daily dose (0.15 mg/kg) that inhibited skeletal B02 tumor
burden, did not inhibit the subcutaneous growth of GFP-
expressing B02 cells. Overall, these findings strongly
suggest that NE-10790 (because of its low bone affinity)
transiently accumulates in bone and subsequently act on
tumor cells to inhibit their growth.
20
Zoledronic Acid Blocks the Osteolytic Response Induced
by Intra-Tibial Inoculation of 4T1luc 2000 Mouse
Mammary Carcinoma Cells in Athymic Nude Mice
Konstantina Grosios; Fritz Wenger, Walter Tinetto, LaetitiaMartinuzzi-Duboc, Robert Cozens, Jonathan Green, Peter
Ingold, Juerg Gasser. NIBR.
Bone is a favoured location for several cancer metastases,
especially breast, prostate, kidney and myeloma. Bony
pathology in cancer patients represents a significant
source of morbidity and mortality. Studying bone metas-
tases in vivo is problematic, mainly due to the lack of
spontaneous tumour models that specifically and repro-
ducibly metastasise to the bone. This problem is partially
overcome by the use of experimental bone metastases
models which can provide valuable insight into the direct
effects of the tumour mass on bone tissue. In this study,
we examined the ability of the 4T1luc 2000 mouse
mammary carcinoma to colonise the tibia of athymic nude
mice and the effect of zoledronic acid (ZA) treatment
(0.1mg/kg, s.c., 2�/week). Tumour cells (1 � 105) were
injected into the tibia of mice, their growth was followed
in vivo by means of IVIS Xenogen imaging. The osteolytic
response was monitored non-invasively by quantitative
computed tomography (pQCT) and visualised by micro
computed tomography (VivaCT40). Dual-energy X-ray
absorptiometry (DEXA) measurements were performed ex
vivo on excised bones.
While no effect of ZA on 4T1luc2000 tumour growth in the
proximal tibia was observed, pQCT measurements carried
out at a distance of 3mm from the proximal end of the tibia
indicated that the bisphosphonate completely blocked the
decrease in bone mineral density of 5.5% observed in
vehicle-treated animals (p < 0.01). Ex vivo measurements
of BMD in the proximal tibia by DEXA indicated a 27.7%
(p < 0.05) decrease due to tumour-induced osteolysis, which
was completely prevented in the ZA-treated animals, in
which BMD actually increased to 7% above the level of the
control group. MicroCT images clearly showed that in
vehicle-treated mice osteolysis occurred in the cancellous
and cortical compartments; indeed in many cases the tumour
broke through the cortical shell to invade the tibia-fibular
space. Bi-weekly s.c. administration of ZA not only pro-
tected animals from cancellous and cortical bone erosions
but it also prevented the tumour from breaking through the
cortical shell in all 6 mice.
These data indicate that ZA, despite having no detectable
effect on tumour cell growth per se in this model, is a
highly effective treatment to prevent the tumour-induced
osteolysis occurring after intra-tibial inoculation of 4T1luc
2000 mouse mammary carcinoma cells in athymic nude
mice.
21
Bisphosphonate Bone Affinity, Differences Predicted by
In Vitro Binding to Carbonated Apatite
Z:J : Henneman,* R. Tang,* S. Gulde,* G.H. Nancollas,*
R.J. Phipps,y R.G.G. Russell,z F.H. Ebetinoy
*University at Buffalo, Buffalo, NY, USAyProcter & Gamble Phamaceuticals, Mason, OH, USAzUniversity of Oxford, Oxford, UK
Bisphosphonates (BPs) are effective inhibitors of bone
resorption. An important component in their ability to
inhibit bone resorption is their affinity for calcium
phosphate surfaces that allows them to target bone
ABSTRACTS / Bone 34 (2004) S46–S99S58
mineral. This can be predicted from in vitro studies of
their effects on the dissolution and growth of calcium
phosphates. The order of affinity predicted from recent in
vitro growth inhibition studies on hydroxyapatite (HAP)
at pH = 7.4 was Zol > Aln > IbanfRis > Etid >
Clo. All binding affinities (KL’s) were significantly
different (P < 0.002) with the exception of Risedronate
(Ris) vs Ibandronate (Iban) We have now also studied
the effects of BP’s on carbonated apatite (CAP), which is
more relevant to bone composition in vivo. These effects
have been studied by a constant composition method at
both physiological ionic strength and temperature, 0.15M
and 37jC. Adsorption affinity constants were calculated
from the kinetics data. For HAP dissolution at pH =
5.50, the rank order of inhibition (high to low) was
zoledronate > alendronate > risedronate, but was less
marked than at pH 7.4. For CAP dissolution at pH =
5.50, the rank order of inhibition was as observed with
HAP, but with a greater discrimination between zoledro-
nate, alendronate, and risedronate. The degree of inhibi-
tion was dependent on the relative undersaturation, s,
with respect to CAP. At a physiologically relevant under-
saturation, sCAP = �.56, KL’s for risedronate (3.90 �105 M-1) and alendronate (5.40 � 105 M-1) were only
34% and 48% of that for zoledronate (1.12 � 106 M-1),
respectively. The inhibition of CAP dissolution by the
BPs was also related to the carbonate content of the
crystallites. Greater dissolution rates were obtained with
crystallites containing more carbonate. Thus, at sCAP =
�0.56 and pH = 5.50, KLs for zoledronate, at a concentra-
tion of 1.0� 10�6 M, were 1.11� 106 and 3.56� 105 M-1
for CAP containing 3.1 F 0.1% and 8.0 F 0.1% carbonate,
respectively. These results confirm the importance of car-
bonate in distinguishing differences in the ability of BPs to
target and affect bone mineral under physiological condi-
tions. They further substantiate the significantly lower bone
affinity of risedronate compared to alendronate and zoledr-
onate. These differences in bone affinity may contribute to
the observed differences in pharmacokinetics among these
three BPs in the clinic. Partial support by NIH/NIDCR grant
#DE03223.
22
Bone Regeneration with Resorbable Polylactide
Membrane and Sponge in an Unstable Fracture Model
in Rabbit Radius
Wing Yuk Ip*;y Gogolewski Sylwester z,§
*Department of Orthopaedic SurgeryyThe University of Hong KongzPolymer Research§AO Research Institute
Background: Healing of segmental diaphyseal bone
defects in animals can be enhanced by covering the defects
with resorbable polylactide membranes. Based on the
results of bone healing in defects 10mm long in the rabbit
radii, it was suggested that the membranes prevents muscle
and soft tissue from invading the defect and maintains
osteogenic cells and osteogenic substances within the
space covered with membrane, thus promoting new bone
formation.
Objectives:
1. To investigate and compare bone regeneration with
resorbable polylactide membrane and polylactide sponge
in a 20 mm bone defect in rabbit radii.
2. To determine and compare the biomechanical strength of
the bone fixation construct with reinforcement by membrane
and sponge of such bone defect which were rendered
unstable by ulnar osteotomy.
Material and method: The material used was poly (l/DL-
lactide) 80/20% in the form of membranes and sponges. The
membrane was o.1mm thick and was porous in the defect
side. The sponge has an average pore size of 300–500 Ym
with pore to volume ratio 95%. 20mm long diaphyseal
segmental defects were made in the left radii of adult New
Zealand rabbits.Transverse ulnar osteotmies were made at
midshaft to make the forearm unstable. The rabbits were
divided into 5 groups. In Group one, no fixation of the bone
were performed and the limbs were immobilized in a plaster
for 8 weeks. In Group two, the bone defects were fixed with
1.5 AO miniplate, with 2 screws on each side of the defect.
In Group 3, the bone defects were fixed similarly and
polylactide membranes were used to cover up the bony
defect. In Group 4, the bone defects were fixed similarly to
Group 2 and the defects were bridged by sponge of 20mm
long, 3 mm in diameter, In Group 5, the bones were fixed
similarly and the defects were bridged by a sponge of same
dimensional and wrapped by polylactide membrane same as
theses in Group 2 from rabbit of same species. Histological
study and biomechanical study were performed on the
explanted forearm at 8 weeks.
Results: In Group 1, there was bone healing bridging the
bone ends at 8 weeks. However, there was marked short-
ening of the limbs and all the limbs were deformed.
In Group 2, there was bone formation at the ends of both
proximal bone stumps and distal bone stumps at 8 weeks.
There was no bone bridging the defect.
In Group 3,4,5, there was bone formation dispersed across
the defect. There was more abundant bone regeneration in
Group 4 and 5. Evaluation at 1 year revealed good bone
formation in the groups with sponges.
Conclusion and discussion: Polylactide membrane and
sponge promote bone regeneration in 20 mm bone defect
in the rabbit radii model.
It was postulated that membrane serves as a barrier to
prevent fibrous tissue to grow into the bone defect and
preserve the osteogenic factors and cells. It was highly
likely that membrane surface served as an anchorage site
for osteoblasts so they can proliferate and express their
phenotype sponge provided more surface area for osteo-
blasts to proliferate and differentiate so more bone regener-
ation occurred. The size, 20 mm, well exceeded the critical
ABSTRACTS / Bone 34 (2004) S46–S99 S59
size defect of the rabbit so resorbable polylactide sponge
can be used to regenerate long segmental defect.
23
Ibandronate Inhibits the Proliferation of Estrogen
Receptor-Positive Breast Cancer Cells: Evidence for
Additivity with Antiestrogens
Fabrice Journe,* Carole Chaboteaux,* Guy Laurent,y
Jean-Claude Dumon,* Jean-Jacques Body*
*Laboratory of Endocrinology and Bone Diseases, Institut
Bordet, Universite Libre de Bruxelles, Brussels, BelgiumyLaboratory of Histology, Faculty of Medicine and
Pharmacy, Universite de Mons-Hainaut, Mons, Belgium
Bone is the site most commonly colonized by breast
cancers, notably those expressing estrogen receptor alpha
(ER). Metastatic cells stimulate osteoclast-mediated bone
resorption leading to osteolysis. Bisphosphonates are
potent inhibitors of osteoclast-mediated osteolysis and
have emerged as a rational approach for the management
of bone metastases. In addition to their direct effects on
osteoclasts, they also induce growth inhibition and apo-
ptosis of other cell types including breast cancer cells.
Bisphosphonates are often combined with conventional
endocrine agents in the treatment of metastatic bone
diseases, but it is unclear which kinds of interaction
could occur as a result of combination therapy. In the
present study, we assessed the anti-proliferative properties
of ibandronate (Iba) on ER-positive MCF-7 breast cancer
cells, cultured in steroid-free medium (SFM) to allow for
the measurement of ER expression and activity, and we
tested Iba with or without antiestrogens. In SFM, Iba
inhibited cancer cell growth in a dose-dependent manner,
as assessed by crystal violet staining (approx. IC50: 10�4
M). Electronic cell count after trypsination gave closely
similar results. As revealed by time-course experiments,
Iba drastically affected cell growth kinetics (population
doubling time increased to 50 hours versus 20 hours for
untreated cells), suggesting cytostatic effects of the
bisphosphonate in MCF-7 cells. Pulse exposure studies
examining drug effect on cell growth at 72 hours
indicated that 2 hours of exposure to 10�4 M Iba did
not affect cell growth while 4 and 8 hours induced
partial and complete effects, respectively. At 10�4 M, Iba
completely abolished the mitogenic effect induced by
10�9 M 17beta-estradiol (E2), but affected neither ER
level nor estrogen-inducible progesterone receptor expres-
sion. Moreover, Iba enhanced the growth inhibitory
action of partial (tamoxifen) and pure (fulvestrant, Fas-
lodex) antiestrogens at 10�7 M. Isobologram analysis
identified additive interactions between Iba and ER
antagonists. In conclusion, 10�4 M Iba clearly decreases
MCF-7 cell growth in SFM and this inhibition does not
require continuous drug exposure. Iba prevents E2 stim-
ulatory effects without affecting ER level or activity. It
also exerts additive effects with antiestrogens in vitro.
23a
New cellular effects of clodronate: stimulation of estro-
gen receptor-positive breast cancer cell proliferation in
steroid-free medium
F: Journe*, C. Chaboteaux*, G. Laurenty, J.C. Dumon*,J.J. Body*
*Laboratory of Endocrinology and Bone Diseases, Institut
J. Bordet, Centre des Tumeurs de l’Universite Libre de
Bruxelles, Brussels, and yLaboratory of Histology, Faculty
of Medicine and Pharmacy, Universite de Mons-Hainaut,
Mons, Belgium
Aromatase inhibitors (AIs) induce complete estrogen
deprivation and could replace tamoxifen as the first line
endocrine therapy for breast cancer in the metastatic and
adjuvant setting. Nevertheless, AIs lead to clinically
significant bone loss. Several trials are ongoing to com-
bine AIs with bisphosphonates, even more that adjuvant
clodronate (Clod) has been shown to reduce the incidence
of bone metastases in the adjuvant setting. We and others
have shown that bisphosphonates can inhibit breast cancer
cell growth in vitro, but they have never been tested in
steroid-free medium (SFM), an in vitro condition that
mimics the effects of AIs. Quite surprisingly, in SFM,
Clod stimulated MCF-7 cell growth by up to two-fold
(crystal violet staining assay), whereas it had no detect-
able mitogenic activity in complete medium. 17� -estradiolalso stimulated MCF-7 cell proliferation in SFM. Partial
(4OH-tamoxifen) and pure antiestrogens (fulvestrant), si-
multaneously added with Clod, completely suppressed the
mitogenic effects of the bisphosphonate, suggesting that it
is mediated by an activation of estrogen receptor (ER). In
accordance with this view, Clod induced ER downregu-
lation, weakly increased progesterone receptor (PgR)
expression, and stimulated the transcription of an estro-
gen-responsive reporter gene. The estrogenic stimulation
induced by Clod was also observed with IBEP-2 cells, a
new ER-positive breast cancer cell line established in our
laboratory and recently characterized as an estrogen
responsive cell line closely resembling MCF-7 cell line
with regard to ER expression, cell mitogenic response to
estrogenic stimulation, and sensitivity to antiestrogens.
Thus, in SFM, Clod also stimulated IBEP-2 cell prolifer-
ation, downregulated ER, and induced PgR expression.
Lastly, we investigated the mitogen-activated protein
kinase pathway as a new potential target of this
bisphosphonate in MCF-7 cells. Clod increased extracel-
lular signal-regulated kinase (ERK1/2) phosphorylation
and its mitogenic effect was prevented by ERK kinase
inhibition. In conclusion, we report a previously unknown
stimulating effect of Clod on ER-positive breast cancer
cells growth in SFM, a condition that is potentially,
relevant to the use of AIs for breast cancer. Our data
suggest that ER specifically mediates these effects of
Clod on cell growth and that ERK1/2 activation is pivotal
for cell proliferative response.
ABSTRACTS / Bone 34 (2004) S46–S99S60
24
Effect of Zoledronat on Human Osteosarcoma Cells
Bernd Kubista; Klemens Trieb*
Introduction: In recent years, the long-term survival of
patients with osteosarcoma has improved due to the use of
neoadjuvant chemotherapy. However, there is still a certain
number of non-responders who do not benefit from these
improvements. The use of bisphosphonates could be bene-
ficial for this group of patients.
The aim of this study was to examine the effects of
Zoledronateon viability, proliferation and expression of
alkaline phosphatase of a human osteosarcoma cell line.
The cell cultures were treated with different concentrations
of Zoledronate and experiments were performed after 24, 48
and 72 hours. Furthermore we investigated whether Zoledr-
onate induces apoptosis in human osteosarcoma cells.
Material & methods:
Cell culture: For the experiments the osteosarcoma cell line
MG-63 was used. The cells were treated for 24, 48, and 72
hours with various concentrations of Zoledronate.
Cell viability: The amount of LDH was used to measure
death of the osteosarcoma cell-line in culture after treatment
with Zoledronate. To measure LDH, a Promega Cyto Tox96
kit was used.
Assessment of cell proliferation: The medium containing
various concentrations of Zoledronate and the cultures were
pulsed with H3-Thymidine, followed by a 24, 48 and 72
hours incubation period. The results are expressed as counts/
minute using a microplate liquid-scintillation counter.
Modulation of Alkaline Phosphatase (ALP) Activity: After
24, 48 and 72 hours incubation with Zoledronate the 96
multiwell-microplates were used for determination of ALP-
activity of the osteosarcoma cells. The cells were incubated
for 15 minutes with 150ml 10mmol/L p-nitrophenylphos-
phate (P-nPP). The absorption was measured in a microplate
reader.
Induction of Apoptosis: Programmed cell death was moni-
tored microscopically following Hoechst and Trypan Blue
staining. The percentage of apoptotic cells was determined in
a total of 500 cells after 48 hours of incubation with various
concentrations of Zoledronat. Induction of apoptosis was
also investigated by caspase substrate (PARP, lamin, caspase
3, 8, 9) cleavage using western blot analysis.
Results:
Effect of Zoledronate on cell viability: A dose and time
dependent effect of Zoledronate on cell viability of osteo-
sarcoma cells was observed. Treatment of osteosarcoma
cells with 100AM Zoledronate for 72h, significanrly in-
creased mean cytotoxicity.
Effect of Zoledronate on cell proliferation: Incubation of the
cells at concentrations of 100AM Zoledronate for 72 hour
significantly decreased cell proliferation compared to un-
treated control.
Effect of Zoledronate on alkaline-phosphatase activity:
Alkaline phophatse activity could be detected in all exper-
imental settings. Treatment with 100AM Zoledronate de-
creased alkaline phophatase activity.
Induction of Apoptosis: After treatment of osteosarcoma
cells with 50mM Zoledronate for 48hours, the proportion of
apoptotic cells significantly increased from 2% to 15%.
Induction of apoptosis could also be detected by caspase
substrate cleavage using western blot analysis.
Discussion: In our study, we demonstrate that the new
Bispophonate Zoledronate can reduce cell growth and
alkaline phosphatase production of human osteosarcoma
cells in vitro.
We also found that Zoledronate induces apoptosis in human
Osteosarcoma cells.
The results of our study could lead to new therapeutic
strategies in the treatment of osteosarcoma in the future.
25
How toMaximise Pamidronate Delivery to BoneWithout
Renal Burden?
Divesh Kumar*,y,z,§,b
*V. KumaryR. Howman-GileszD.G. Little*§T. KitsosbDepartment of Nuclear Medicine, *Orthopaedic Research
Unit, Westmead, and The Children’s Hospital at Westmead,
Sydney, Australia
Aim: Bisphosphonates (BPs) are stable analogs of pyro-
phosphate (PUOUP), a physiological regulator of calcifica-
tion and bone resorption. They play a major therapeutic role
in bone diseases and in orthopaedic surgery. The major
disadvantage of the clinically utilized BPs, especially
Pamidronate (APD) is associated with nephrocalcinosis in
patients, when administered intravenously for therapeutic
purposes. We administered 99mTc-APD by three different
methods in vivo to study renal uptake and biodistribution.
Method: Rats were injected with 99mTc-APD (1) intrave-
nously (2) subcutaneously and (3) direct application on
surgically exposed femur. The rats were sacrificed at
different time points and various organs harvested &
counted for radioactivity to establish the biodistribution.
Results:
Method 1: When 99mTc-APD was injected intravenously,
there was impressive skeletal uptake with no significant soft
tissue uptake, especially by the liver or the kidneys. 99mTc-
APD was cleared by the kidneys within 2h post-injection.
The femur uptake was 2.5% injected dose per gram of tissue
(%ID/g).
Method 2: When 99mTc-APD was injected subcutaneously,
majority of the compound was retained at the site of injection.
There was significantly less impressive skeletal uptake com-
pared to Method 1. The femur uptake was 0.5%ID/g.
Method 3: When 99mTc-APD was applied directly on the
right femur, it retained >8%ID/g at the site of application
with very little uptake in the kidneys (0.05% ID/g).
Table 4
Group comparison SIG Diff %
Sham/saline vs. ovx/saline �56.5
Ovx/1 AG IB vs. ovx/saline +113.5
Ovx/25 AG IB vs. ovx/saline +185.0
ABSTRACTS / Bone 34 (2004) S46–S99 S61
Conclusion: The results clearly indicate that by Method 3,
maximum dose of 99mTc-APD can be delivered to the bone
for therapeutic purposes with very little renal uptake.
Method 3 would be advantageous to orthopaedic surgeons
in treating local conditions.
26
Ibandronate Improves Implant Integration in
Experimental Osteoporosis
Andreas A: Kurth,* Christian Eberhardt,*
Markus Schwarz,y Frieder Baussz
*Dep. of Orthopaedic Surgery, Orthopaedic Oncology
Service, University Hospital Frankfurt/MainyDep. of Orthopaedic Surgery, University Hospital
MannheimzRoche Diagnostics GMBH, Pharma Research, Penzberg,
Germany
Uncemented total joint replacement has a wide range of
indications. As the mean life expectancy increases an
increasing number of uncemented total joint replacements
are being performed on patients with osteopenia/osteoporo-
sis. It is known that ovariectomy (ovx) in rats not only
causes osteopenia but also reduces the ossointegration of
implants in bone. Bisphosphonates are a standard of treat-
ment and prophylaxis for osteoporosis. Preclinical experi-
ments have confirmed that bisphosphonates administered
after the implantation of a cementless implant can increase
the amount of bone attached to it. The purpose of the current
study was to investigate the effects of experimental osteo-
porosis on the osseointegration of metal implants and if
different doses of the bisphosphonate ibandronate can
overcome the effects of osteopenic bone and improve
osseointegration of the implants.
M&M: 84 S-D rats were randomly assigned to different
groups. One group underwent a sham-ovx, three an ovx.
Two groups received daily saline s.c. and served as control
groups (sham control and ovx control). Two groups received
daily s.c. ibandronate (1.0, 25.0 Ag/kg) starting at the day of
surgery. The bone mineral density (mg/cm2) of lumbar
vertebrae was assessed before surgery (12 weeks after
ovx) and before sacrifice. Titanium and HA-coated titanium
implants were surgically inserted in press-fit technique into
the medullary canal of each femur. After 27 days of
treatment all animals were killed in a CO-chamber. The
specimens were prepared for quantitative histomorphome-
try. To quantify the extent of osseointegration, osseointe-
grated implant surface (OIS) was determined. This was
Table 3
Group Mean
% OIS
STD
DEV
Sham/saline 54.5 25.5
Ovx/saline 23.7 18.6
Ovx/1 AG IB 50.6 24.4
Ovx/25 AG IB 67.6 26.8
defined as the ratio (%) of the implant surface in direct
contact with surrounding bone to the total implant surface.
Results: The BMD values of the lumbar vertebrae increased
significantly in all groups over time. After ovx BMD signif-
icantly decreased after 12 weeks. In the groups treated with
Ibandronate for 27 days, regardless the dose, BMD was
significantly higher than in the ovx-control group. For tita-
nium-only implants, no significant effect on osseointegration
was found. For HA-coated implants the OIS for the ovx group
decreased significantly. Both ibandronate treatment groups
were significantly higher compared to the ovx-control group.
Discussion: Experimental osteoporosis has a negative effect
not only on bone mass and bone architecture, but also on
osseointegration of metal implants. Administration of dif-
ferent doses of ibandronate improves the osteopenia in ovx
animals. Furthermore an osteoporosis equivalent dose of
ibandronate equals out the negative effect of ovx on
osseointegration. A considerable higher dose of ibandronate
improves the osseointegration even more but not statistically
significant. In conclusion, this is the first study showing the
possitive effect of a bisphosphonate treatment on osseointe-
gration of implants in experimental osteoporosis. This
suggests that recipients of total joint replacements with high
risk of osteoporosis have a benefit from a bisphosphonate
treatment with respect to bone mass and early stabilization
of the implants (Tables 3 and 4).
27
Treatment of Aggressive Osteolytic Benign Bone Lesions
with Topical Antiresorptive Agents: Part I. A Scientific
Rationale
Francis Young-In Lee; John Yu, Seong Sil Chang, Sanjeev
Suratwala, Peter Abdelmessieh, Koko Murakami. Columbia
University.
Introduction: Benign osteolytic bone tumors are character-
ized by overly activated osteoclastogenesis in a well-demar-
cated region and frequent recurrence. Tumor-induced
osteclastogenesis is triggered by mesenchymal stromal cells
and excessive osteolysis may lead to pathologic fracture and
pain. Topical adjuvant agents, such as liquid nitrogen,
phenol, alcohol or hydrogen peroxides have been used to
prevent recurrence. Bisphosphonates are known to induce
apoptosis in osteoclasts and certain neoplastic cells, sug-
gesting bisphosphonates as one of possible specific thera-
peutic agents for benign osteolytic diseases. However, the
potential therapeutic effects of bisphosphonates for localized
bone diseases are unknown. We determined the therapeutic
effect of bisphosphonates on the benign aggressive osteo-
lytic diseases prior to possible clinical applications.
ABSTRACTS / Bone 34 (2004) S46–S99S62
Materials and methods: (1) Cell Culture: We established
cultures from 4 cases of giant cell tumor (GCT), 5 cases of
unicameral bone cyst (UBC) and 1 case of nonossifying
fibroma (NOF) that showed extensive bone destruction on
radiographs. Conditioned media from the culture was added
to human peripheral mococytes to confirm osteoclasto-
genesis. 30 nM and 100 nM of zolendronate and pamidr-
onate were added to the GCT and UBC cultures. Cells were
prepared for flowcytometry, RT-PCR and western blotting.
Normal bone marrow stromal cells and fibroblasts were
used as controls. (2) RT-PCR, Western Blotting and Flow-
cytometry: RNAs were extracted from the tumor tissues
and cells and real-time RT-PCR was performed using
primers for RANKL, TNF alpha, cbfa-1, osterix, osteocal-
cin and SDF-1. The cells were stained with annexin V and
propidium iodide. Flowcytometry was performed using
samples from day 1 and 3 cultures. Western blotting was
performed to confirm the activation of caspase 3 and
degradation of poly (ADP-ribose) polymerase (PARP). In
addition, GGOH and FFP were added to determine the
therapeutic targets.
Results: Conditioned media from GCT, UBC and NOF
cultures induced osteoclastogenesis in the human monocyte
culture in the presence or absence of high concentration of
osteoprotegerin. RANKL, TNF, cbfa-1 and SDF-1 were
consistently expressed by stromal cells while Osterix gene
expression was barely detectable or absent. SDF-1, a chemo-
kine released by stromal cells, was present in a physiologi-
cally active concentration. Bisphosphonates induced death of
neoplastic stromal cells in a dose and time dependent manner.
Bisphosphonates induced activation of caspase 3 and degra-
dation PARP suggesting activation of apoptosis. GGOH
blocked bisphosphonate-induced apoptosis in UBC cultures
not in GCTs. Bisphosphonates did not affect expression of
genes for RANKL, SDF-1 and other adhesion molecules.
Discussion: These findings indicate that stromal cells of
aggressive osteolytic lesions are of early osteoblastic lineage
with maturational arrest and are capable of inducing osteo-
clastogenesis and actively recruiting ostoclast precursors.
Bisphosphonates effectively and selectively induced apo-
ptosis in the stromal cells of the human osteolytic diseases.
The specific pahramacologic action may be different among
different types of tumors. Our data provide a scientific
rationale for the use of bisphosphonates for benign osteo-
lytic diseases in addition to known inhibitory effects on
osteoclasts and osteoclastogenesis. Further study on the
drug delivery and clinical trial will be necessary to deter-
mine the safety and therapeutic efficacy.
27a
Relative Binding Affinities of Bisphosphonates for
Human Bone
C.-T. Leu, E. Luegmayr, Leonard P. Freedman,
A.A. Reszka*
Department of Molecular Endocrinology, Merck Research
Laboratories, West Point, PA 19038, USA
Potent bisphosphonates (BPs) inhibit bone resorption by
binding bone at sites of active resorption. There they are
taken up by osteoclasts and act intracellularly to inhibit
function or to induce apoptosis. Rank ordering is generally
consistent between in vivo and in vitro cellular BP effects,
although clodronate (CL2), which is comparable in poten-
cy to alendronate (ALN) in vitro, is roughly two orders of
magnitude less potent and about half as efficacious in
increasing bone mineral density in clinical tests. We
hypothesized that the relative binding affinites of these
and other BPs for human bone could play a role in clinical
efficacy and potency. Scatchard analyses indicated that
[14C]-ALN bound to human bone with an apparent Kd
of 140 � M. As expected, binding was competitively
inhibited by pyrophosphate (IC50: 287 � M) or by ALN
(157-165 � M). Most hydroxyl-bearing BPs (OH-group at
the R2 position) showed relative binding that was com-
parable to ALN, including etidronate (200 � M), ibandr-
onate (243 � M), pamidronate (178 � M), risedronate
(RIS, 174 � M) and zoledronate (176 � M). Slightly
weaker, tiludronate (H at R2) was most comparable to
pyrophosphate with an IC50 of 331 � M. Interestingly,
CL2 (Cl at both R1 and R2) displaced [14C]-ALN
binding with a ten-fold higher IC50 of 1719 � M. When
unlabeled BP was preincubated with human bone, relative
binding affinites were increased approximately two-fold
(ALN, 73 � M; clodronate, 668 � M; etidronate, 116 � M;
ibandronate, 98 � M; pamidronate, 97 � M, RIS, 94, � M;
Tiludronate, 188 � M; and zoledronate, 109 � M). We also
tested the efficacy of ALN, RIS and CL2 in an vitro
resorption assay using precoated bovine bone slices that
were either washed or left unwashed prior to addition of
mouse osteoclast cultures. In comparison to the unwashed
bone slices, resorption inhibition was completely lost after
washing of the CL2-treated bone. However, with ALN and
RIS there was only a slight loss of efficacy by washing.
Together these findings suggest that BPs can differ in their
binding to human bone. The hydroxyl bearing bisphospho-
nates, such as ALN and RIS, bind with similar and some-
what enhanced affinities vs. that seen with pyrophosphate or
tiludronate, while CL2 binds most weakly. Reduced bone
affinity of CL2 vs. these hydroxyl-bearing bisphosphonates,
may account for its lower potency in vivo.
28
Long-Term Efficacy and Safety of Zoledronic Acid in
Patients With Bone Metastases From Renal Cell
Carcinoma
Allan Lipton,* John Seaman,y Ming Zhengy
*Milton S. Hershey Medical Center, Hershey, PA, USAyNovartis Pharmaceuticals Corporation, East Hanover, NJ,
USA
Background: We recently reported a retrospective subset
analysis of the efficacy and safety of 4 mg zoledronic acid
for preventing skeletal complications from bone metastases
Fig. 1. Zolendronic acid administration resulted in significant improvement
in EQ. The number of flat femoral heads was significantly reduced.
ABSTRACTS / Bone 34 (2004) S46–S99 S63
in patients with renal cell carcinoma (RCC) in the 9-month
core analysis of a multicenter, randomized, placebo-con-
trolled, phase III trial (Lipton et al. Cancer. 2003;98:962–
969). Herein we report the analysis of the 21-month core +
extension data for the patients with RCC in this trial.
Patients and methods: Patients with bone metastases
secondary to lung cancer or other solid tumors (not includ-
ing breast cancer or prostate cancer) (N = 773) were
randomized to treatment with either zoledronic acid (4
mg) or placebo. Zoledronic acid was administered as a
15-minute infusion every 4 weeks for 9 months (core
phase), and patients could elect to continue treatment for
up to 21 months. Patients were monitored for skeletal
complications (pathologic fractures, palliative radiotherapy
to bone, orthopedic surgery to prevent or treat a pathologic
fracture, or spinal cord compression) and safety parameters.
Results for the subset of patients with RCC (n = 46) treated
with 4 mg zoledronic acid (n = 27) or placebo (n = 19) are
reported. Of these, 13 patients completed the core phase,
and 9 entered the extension phase.
Results: Long-term treatment with zoledronic acid (4 mg)
significantly reduced the risk of skeletal complications in
patients with RCC by 58% compared with placebo, as
assessed by multiple event analysis (risk ratio = 0.418;
P = .010). The proportion of patients with RCC who
experienced 1 or more skeletal complications was signif-
icantly lower in the zoledronic acid group compared with
the placebo group (41% versus 79%, respectively; P =
.011), and the mean annual incidence of skeletal compli-
cations was significantly reduced (2.58 versus 3.13
events/year, respectively; P = .009). Treatment with 4
mg zoledronic acid also significantly extended median
time to first skeletal complication by almost 1 year (424
versus 72 days for placebo; P = .007). The median time
to progression of bone lesions was also significantly
longer in the zoledronic acid group (256 versus 89 days
for placebo; P = .014), suggesting potential antitumor
effects. A nonsignificant trend toward improved survival
was also observed for patients treated with zoledronic acid
(median, 347 versus 216 days for placebo; P = .104).
Zoledronic acid (4 mg via 15-minute infusion) was well
tolerated. The only adverse events occurring at notably
higher incidence compared with placebo were fatigue,
pyrexia, abdominal pain, hypocalcemia, and dyspepsia.
Bone pain and constipation were more common in the
placebo group. There were no significant between-group
differences in the proportions of patients who experienced
renal adverse events.
Conclusions: The final 21-month analysis of this trial
demonstrated that among patients with RCC, 4 mg zole-
dronic acid significantly reduced the risk of skeletal com-
plications, prolonged the time to first skeletal complication,
and delayed bone lesion progression compared with place-
bo. Therefore, zoledronic acid is effective and safe for long-
term therapy in patients with bone metastases secondary to
RCC.
29
Zoledronic Acid Improves Femoral Head Sphericity in a
Rat Model of Perthes Disease
David G: Little; Michelle M. McDonald, Ian Sharpe,
Rachel A. Peat, Paul R. Williams, Anthony McEvoy. The
Childrens Hospital Westmead.
Purpose: Perthes disease is a common idiopathic form of
childhood osteonecrosis. It is suggested to result from
ischaemia following a vascular insult with subsequent
osteonecrosis leading to deformity and collapse of the
growing femoral head epiphysis. As the optimal treatment
for Perthes disease remains unclear, a pharmaceutical ther-
apy was investigated. We hypothesized that the bisphosph-
onate zoledronic acid (ZA) would protect the epiphysis
against deformation following ischaemia, maintaining fem-
oral head architecture during development. In this study, the
effect of ZA was tested in an established model of Perthes
disease, the spontaneously hypertensive rat (SHR).
Method: 120, 4-week old SHR rats were divided into three
groups of 40: Saline monthly (Saline), 0.05 mg/kg ZA
monthly or 0.015 mg/kg ZA weekly. At 15 weeks, femora
were harvested. Radiographs were taken and a modified
epiphyseal quotient (EQ), central epiphyseal height over
width, was measured (EQ = 0.5, spherical). DXA analysis
was performed on 16 specimens/group prior to processing
for un-decalcified histomorphology. Remaining specimens
were embedded decalcified for histology. Results are
expressed as mean [SE].
Results: ZA treated femoral head BMD was increased by
18% and 21% over controls in weekly and monthly treat-
ment groups, respectively (p < 0.01). As only non-signifi-
cant variations were observed between both ZA dosing
regimes, results were combined (ZA). Radiographs revealed
increased mineralization and improved sphericity of femoral
heads in ZA samples. Overall EQ was increased in ZA
compared to saline with the mean EQ rising from 0.43
[0.008] to 0.47 [0.004] (p < 0.01) (Fig. 1). The proportion of
ABSTRACTS / Bone 34 (2004) S46–S99S64
‘‘flat’’ heads (EQ < 0.40) was significantly reduced from
33% in saline to 10% in ZA (p < 0.01).
Histological analysis revealed that affected femoral heads
showed osteonecrosis, ossification delay, or both. There was
a similar prevalence of osteonecrosis in Saline (53%) and
ZA (58%) animals. Where both osteonecrosis and ossifica-
tion delay were present, ZA treatment protected against
femoral head deformity. Saline animals showing reduced
sphericity EQ 0.40 [0.01] as compared to ZA treatment EQ
0.49 [0.02], almost completely preserving femoral head
sphericity (p < 0.01). Interestingly, the prevalence of ossi-
fication delay in the epiphysis was also significantly re-
duced in ZA (16%) compared to Saline (60%) (p < 0.01).
Static histomorphometric variables were also affected by
ZA treatment. There was an 11% increase in BV/TV in ZA
(p < 0.02) based on a 42% increase in trabecular number
(p < 0.01).
Conclusion: Zoledronic acid favourably preserved femoral
head sphericity in this spontaneous model of osteonecrosis
in growing rats. The data suggest this was achieved
through a reduction in the prevalence of ossification delay
and thus an increase in the content and amount of
trabecular bone. Such a preservation of normal ossifica-
tion is consistent with improved vascularity resulting from
ZA-induced preservation of epiphyseal structures during
development.
Significance: Application of these results to Perthes dis-
ease could mean that Zoledronic acid treatment may
modify the deformity of the femoral head in many chil-
dren, therefore reducing the need for surgical intervention
in these patients.
30
Zoledronic Acid Increases Total Bone Volume in OP-1
Mediated Bone Formation in a Segmental Rat Femoral
Defect Model
David G: Little; Rick Bransford, Rachael Bugler, Julie
Briody. Childrens Hospital Westmead.
Purpose: It is estimated that up to 10% of fractures have
difficulty healing. Many new therapies, including recombi-
nant bone morphogenetic proteins (BMPs), are becoming
Fig. 1. Addition of ZA at either surgery or 2 week
available for the prevention or treatment of non-union. Use
of BMPs has yet to be optimised. BMPs enhance osteo-
blastogenesis, however it is known that they also up-
regulate osteoclasts, directly through BMP receptors and
indirectly via osteoblasts through RANK/RANKL. In some
clinical situations this may produce high bone turnover,
which could limit the volume of callus produced. Premature
catabolic resorption of the calcified matrix scaffold may also
be undesirable. We hypothesised that, if the catabolic
osteoclastic upregulation is modulated by zoledronic acid
(ZA, Novartis), the anabolic response to BMPs would be
optimised. Thus the combination of BMP and ZA should
produce increased callus over BMP alone.
Method: A rat 6mm critical size defect model was utilized.
Control animals and those with carrier alone do not heal this
defect. The BMP used was the OP-1 device, 50 micrograms
of rhBMP-7 in 15mg bovine collagen per rat (Stryker
Biotech). Groups consisted of collagen carrier alone, carrier
plus ZA, OP-1 alone and OP-1 with ZA either given
systemically at surgery or systemically 2 weeks post sur-
gery. ZAwas given at 0.1 mg/kg as a single parenteral dose.
Plain radiographic and QCT analysis of the gap was used to
define outcome.
Results: Carrier alone and carrier ZA groups had not united
by 8 weeks on plain radiographs. Radiological union
occurred in all OP-1 groups, but was clearly more robust
in the combined OP-1/ZA groups.
QCT analysis revealed that addition of ZA increased BMC in
the 6mm defect by 45% in the ZA at surgery group and 93%
in the ZA at 2 weeks group over OP-1 alone (p < 0.01). Callus
volume increased by 47% in the ZA at surgery group and 82%
in the ZA at 2 weeks group over OP-1 alone (p < 0.01).
Conclusion: In line with our hypothesis, zoledronic acid
significantly increased the amount of OP-1 induced net
callus formation in a femoral critical size defect in rats.
Timing of ZA administration to reduce catabolism may be
important in maximising the anabolic effect of OP-1.
Significance: Modulation of the high bone turnover state
induced by pharmacotherapeutic doses of BMPs may opti-
mise the amount and density of callus produced, which
could be of clinical benefit in obtaining initial bone repair in
difficult situations (Fig. 1).
s later significantly increases bone volume.
ABSTRACTS / Bone 34 (2004) S46–S99 S65
31
Zoledronic Acid Results in Increased Total Bone Volume
in a Rabbit Spine Fusion Model
David G: Little; Rick Bransford, Elisabeth Goergens,
Rachael Bugler, Julie Briody, Andrew Cree. Childrens
Hospital Westmead.
Purpose: A NZW rabbit model of L6/L7 intertransverse
process arthrodesis was used to evaluate the effect of single
dose zoledronic acid on autograft fusion mass. Previous
studies have shown zoledronic acid administration can
increase mineral content and strength in distraction osteo-
genesis. Few studies have examined the use of bisphosph-
onates in spinal arthrodesis, and none have looked at the
effect of a single perioperative dose.
Method: 48 New Zealand white rabbits had an L6–L7
intertransverse process fusion, which has a much lower
fusion rate than the usual rabbit model at L5–6, due to
smaller transverse processes and proximity to the sacrum.
Animals were randomly allocated to one of three groups,
one received iliac bone graft alone, one group received
iliac crest bone graft with locally administered ZA, 20 Ag,and one group received iliac crest bone graft with a single
dose of systemically administered ZA at 100 Ag/kg at the
time of surgery. 24 rabbits were culled at 6 weeks and 24
rabbits were culled at 12 weeks. Specimens were manu-
ally tested to determine whether they were fused, were
evaluated radiographically, and underwent quantitative CT
analysis.
Results: In the 6 week group, only 5 out of the 24 spines
fused with no noticeable trend in respect to treatment.
Radiographs at 12 weeks revealed that remodelling was
significantly delayed (P < 0.05) in the Local ZA group,
but there was no significant difference between Saline and
Systemic ZA in respect to remodelling scores. In the 12 week
group there was a trend toward increased fusion in the
Systemic ZA group (63%) versus the other two groups
(25%) but this was not statistically significant. On quantita-
tive CT analysis, bone mineral content (BMC) in the 12-
week systemic ZA group was increased by 87% over saline
controls (p < 0.01). There was also a 33% increase in
volumetric bone mineral density (vBMD), and a 41% in
total fusion mass volume in the Systemic ZA group at 12
weeks (p < 0.01). The 12-week local ZA group also showed
a significant increase in BMC, BMD, and total area as
well with increases of 69%, 31%, and 29% respectively
(p < 0.05).
Conclusion: A single systemic dose of zoledronic acid
significantly increased fusion mass BMC, vBMD and
bone volume, and led to a fusion rate of 63% by 12
weeks in an L6/L7 rabbit model of spinal arthrodesis
using autograft. A larger study is needed to evaluate the
increase in fusion rates definitively, and also look at the
optimal timing and dosing of zoledronic acid in this and
other spinal fusion models. Locally applied zoledronic
acid significantly delayed remodelling at the dose given
in this study.
Significance: Zoledronic acid may enlarge fusion mass
volume and fusion rates in posterolateral spinal arthrodesis.
32
Zoledronic Acid Inhibits Neoangiogenesis by Interacting
with Bone Marrow-Derived Cells
Amanda Littlewood-Evans; Lorenza Wyder, Christian
Schnell, Adrian Zumsteg, Beatrice Probst, Tim De Waal,
Jonathan Green, Jeanette Wood. Novartis, NIBR, Basel,
Switzerland.
Neovascularisation is a hallmark of cancer and many
inflammatory diseases. Elucidation of the precise mecha-
nism of this process is therefore important in determining
rational therapeutic strategies for these diseases. Until
recently it was thought that blood vessel formation in
postnatal life was mediated mainly by a process termed
angiogenesis, ie the formation of new blood vessels by
sprouting from the preexisting vasculature. However,
recent studies have shown that endothelial stem cells
persist into adult life and that endothelial cell precursors
are present in peripheral blood and bone marrow. These
precursors home from the bone marrow to the site of
neovascularisation and contribute to the formation of new
vessels.
We have previously shown that the bisphosphonate,
Zoledronic acid has antiangiogenic effects in a growth
factor containing chamber model implanted subcutane-
ously in mice. Since this compound is rapidly targeted
to hydroxyapatite containing organs, we surmised that
Zoledronic acid could be affecting cells within the
bone marrow that contribute to the angiogenic process.
We can now show that in our VEGF or bFGF driven
neovascularization model, blood vessel formation is
impaired even if the drug is administered 3 days
before implantation of the growth factor containing
chamber.
In order to clarify the role of bone marrow derived cells
in neovascularsation we set up a model to distinguish
between bone marrow derived cells and cells originating
from the tissue surrounding the tumor. For this purpose
receiver mice were treated with Busulfan to deplete their
stem cells and thereafter transplanted with bone marrow
from a mouse ubiquitously expressing GFP under the
beta-actin promoter. Thus, in the resulting chimeric mice,
only bone marrow derived cells will be GFP positive.
Using double immunofluorescence we could determine
the effects of Zoledronic acid on the contribution of
different bone marrow derived cells in our chamber
model.
Elucidating the effect of Zoledronic acid on blood vessel
formation can lead to novel indications for bisphospho-
nate therapy as well as providing insights towards the
contribution of bone marrow derived cells in various
diseases where neovascualrization plays an important
role.
ABSTRACTS / Bone 34 (2004) S46–S99S66
33
Anti-Tumour Potential of Bisphosphonates on Human
Lung Carcinoma Cell Lines
Claire Macdonald; Lisa Pickering, Janine Mansi, Kay
Colston. St. George’s Hospital Medical School, London, UK.
Lung carcinoma is one of the most commonly diagnosed
cancers world-wide. The survival rate of this disease is
however one of the lowest: only 20% of patients survive 5
years after diagnosis. One of the major complications
associated with lung carcinoma is systemic spread of the
primary tumour: 80% of lung carcinomas metastasise to
bone where they may cause intractable pain, fracture, cord
compression and hypercalcaemia. This results in increased
morbidity and mortality; the median survival after diagnosis
of bone metastasis is six months. Lung carcinomas are
classified according to histology and fall into two main
groups: non small cell and small cell carcinomas. Small cell
carcinomas are highly metastatic and are associated with a
particularly poor prognosis.
Bisphosphonates (BPs) have been shown to have a wide
range of direct effects on a number of cancer cell types in
vitro, including on breast, prostate and myeloma tumour
cells. They can directly induce apoptosis and alter adhesive
and invasive properties as well as modulate growth factor
secretion, matrix metalloprotein production and inhibit
angiogenesis. Furthermore, in clinical studies, BPs have
been shown to reduce the formation and progression of
bone metastasis in many carcinomas including lung cancer
and to reduce the symptoms associated with osteolytic
lesions. However, the molecular mechanisms by which
BPs mediate these effects are incompletely understood and
there is little data about the in vitro, effects of bisphosph-
onates in lung cancer cells. This study therefore aimed to
determine whether bisphosphonates have direct inhibitory
effects on lung cancer cell lines in vitro.
In this study we investigated the effect of zoledronic acid
(ZOL), pamidronate (APD) and clodronate (CLOD) on a
variety of human lung carcinoma cell lines including both
non small and small cell lines. We found that BPs reduced
cell viability and number in a concentration and time
dependant manner. The order of potency of the BPs was
cell line dependent. The nitrogen containing BPs ZOL and
APD were equipotent, with LD50s between 10 and >100AMacross the different cell lines, while CLOD, an antimetab-
olite, was less potent with LD50s ranging from 500 to
>2000AM. There was no distinguishable difference in sus-
ceptibility between non small cell and small cell carcinoma
cell lines. All three BPs were found to induce apoptosis by
either day two or three of treatment at the doses studied by
both caspase dependent and independent mechanisms.
In conclusion, BPs can directly induce apoptosis in human
lung carcinoma cell lines. The mechanisms involved in this
process may be cell line dependent. This study provides a
scientific basis for the in vivo effects of BP therapy. We
intend to further investigate the mechanisms involved by
elucidating the pathways affected by these BPs, principally
concentrating on cell signalling molecules and G proteins
involved in adhesion and invasion.
34
A New Mechanism of Action for Bisphosphonates:
ApppI Mediated Cytotoxity of N-BPs
H : Monkkonen,* P.P. Lehenkari,y M. Kellinsalmi,y
I.E. Hassinen,z S. Auriola,§ J. Vepsalainen,b J. Monkkonen*
*Department of Pharmaceutics, University of Kuopio,
FinlandyDepartment of Surgery, University of Oulu, FinlandzDepartment of Medical Biochemistry, University of
Oulu, Finland§Department of Pharmaceutical Chemistry, University of
Kuopio, FinlandbDepartment of Chemistry, University of Kuopio, Finland
Bisphosphonates (BPs) are a class of drugs developed for
the treatment of metabolic bone diseases with high bone
turnover, such as Paget’s disease, tumor associated osteol-
ysis and osteoporosis. Despite their common clinical out-
come, inhibition of bone resorption, bisphosphonates can be
divided into two classes with distinct molecular mechanisms
of action. Nitrogen-containing BPs (N-BPs), such as alen-
dronate, risedronate, and zoledronic acid inhibit at least
one enzyme of the intracellular mevalonate pathway,
thereby preventing the modification of important signalling
proteins with isoprenoid lipids. Loss of prenylated proteins
causes a loss of osteoclast function and consequently, apop-
totic cell death. The less potent non-nitrogen-containing BPs
(non-N-BPs), such as clodronate, do not inhibit isoprenyla-
tion, but are metabolised by osteoclasts to analogues of
adenosine triphosphate (ATP), which accumulate in the cell
cytoplasm and cause apoptosis (1,2).
Recently, we found that N-BPs induce production of an
intracellular ATP-analogue (ApppI) via inhibition of meval-
onate pathway and accumulation of isopentenyl diphosphate
(IPP) in vitro by mammalian cells, including osteoclasts.
These results strongly suggest that the ApppI production is
due to inhibition of mevalonate pathway. However, inhib-
itors of HMG-CoA, farnesyl transferase, and geranylgeranyl
transferase I, (lovastatin, FTI-277, and GGTI-298), did not
cause ApppI production in J774 macrophage cells, indicat-
ing that ApppI production is an unique effect of N-BPs.
We reported earlier that the ATP-analogue of clodronate,
AppCCl2p, inhibits mitochondrial ATP/ADP translocase,
causes a loss of mitochondrial membrane potential and,
thus, direct apoptosis of the cells (1). Since we were able
to synthetize ApppI, we tested its effect on mitochondrial
ADP/ATP translocase. The results show that ApppI inhib-
its ADP/ATP translocase in similar manner and efficacy
than the clodronate metabolite, AppCCl2p. N-BPs them-
selves did not have any effect on ADP/ATP translocase.
Taken together, in addition to the prevention of post-
translational modification of GTP-binding proteins with
ABSTRACTS / Bone 34 (2004) S46–S99 S67
isoprenoid lipids, potent N-BPs cause intracellular accumu-
lation of ApppI via inhibition of mevalonate pathway.
ApppI inhibits ADP/ATP translocase in mitochondria, and
could induce direct apoptosis, similarly to AppCCl2p. The
caspase activation and apoptosis studies are in progress. It
seems thus, that potent N-BPs could present a ‘‘third’’ class
of bisphosphonates in terms of the molecular mechanism of
action, by combining the indirect and direct induction of
apoptosis.
References
[1] Lehenkari P, et al. Mol Pharmacol 2002;61(5):1255.
[2] Monkkonen H, et al. Pharm Res 2001;18(11):1550.
35
Nitrogen-Containing Bisphosphonates Cause
Intracellular Accumulation of Isopentenyl Diphosphate
(IPP) and Biosynthesis of ApppI
H : Monkkonen,* P.P. Lehenkari,y M. Kellinsalmi,y
I.E. Hassinen,z S. Auriola,§ J. Vepsalainen,b
J. Monkkonen*
*Department of Pharmaceutics, University of Kuopio,
FinlandyDepartment of Surgery, University of Oulu, FinlandzDepartment of Medical Biochemistry, University of Oulu,
Finland§Department of Pharmaceutical Chemistry, University of
Kuopio, FinlandbDepartment of Chemistry, University of Kuopio, Finland
Bisphosphonates (BPs) are a class of drugs developed for
the treatment of metabolic bone diseases with high bone
turnover, such as Paget’s disease, tumor associated osteol-
ysis and osteoporosis. Despite their common clinical out-
come, inhibition of bone resorption, bisphosphonates can be
divided into two classes with distinct molecular mechanisms
of action. Nitrogen-containing BPs (N-BPs), such as alendr-
onate, risedronate, and zoledronic acid inhibit at least one
enzyme of the intracellular mevalonate pathway, thereby
preventing the modification of important signalling proteins
with isoprenoid lipids. Loss of prenylated proteins causes a
loss of osteoclast function and apoptotic cell death. The less
potent non-nitrogen-containing BPs (non-N-BPs), such as
clodronate, do not inhibit isoprenylation but are metabolised
to a cytotoxic analogue of adenosine triphosphate (ATP),
which accumulates in the cell cytoplasm and cause apopto-
sis at high doses, but has specific anti-inflammatory action
at lower doses.
It has been previously shown that N-BP:s are not metabolised
to any ATP-analogue. We report here, however, that N-BPs
induce formation of a novel ATP-analogue (ApppI) produc-
tion via inhibition of mevalonate pathway in vitro by mam-
malian cells, such as macrophages, gliomas, and osteoclasts.
The formation and structure of ApppI (triphosphoric acid 1-
adenosin-5V-yl ester 3-(3-methylbut-3-enyl) ester) was deter-
mined by using mass spectrometry (MS) and nuclear mag-
netic resonance (NMR). Farnesol or geranylgeraniol,
intermediate products of mevalonate pathway, decreased
the ApppI production after co-treatment of J774 macro-
phages with N-BP and these intermediates. Pure farnesol or
geranylgeraniol did not have any effect on ApppI production.
We also measured the IPP concentrations in the cells by MS,
and only N-BPs induced elevation of IPP levels caused by
inhibition of FPP synthase. ApppI production correlates well
with the capacity of N-BPs to inhibit FPP synthase and,
consequently, with the capacity to increase IPP concentration
in the cells. Non-N-BPs do not induce ApppI production.
It has been reported that the inhibition of protein prenylation
in osteoclasts and J774 cells caused by N-BPs is prevented
by cotreatment with clodronate. Furthermore, induction of
apoptosis of J774 cells by N-BPs was significantly inhibited
by clodronate (1). In present study, clodronate decreased the
ApppI production and reciprocally risedronate decreased the
metabolism of clodronate to AppCCl2p after co-treatment of
the J774 macrophages. This strongly suggests that amino-
acyl-tRNA-synthetases, which catalyze the formation of the
AppCp-type metabolite of clodronate, are also involved in
ApppI production induced by N-BPs.
The novel ApppI molecule has not been reported earlier,
and the concept that drug treatment induce a formation of
new chemical entity in the cells not containing the drug
itself is a new and interesting mechanism of action. ApppI
could also mediate the ‘‘third ’’ mechanism of action of
bisphosphonates.
Reference
[1] Frith JC, Rogers MJ. J Bone Miner Res 2003;18(2):204.
36
Paclitaxel and Zoledronic Acid Induce Synergistic
Increase in Apoptotic Tumour Cell Death at Clinically
Relevant Concentrations
Helen Neville-Webbe; Robert E. Coleman, Ingunn Holen.
Clinical Oncology, School of Medicine and Biomedical
Sciences, University of Sheffield, UK.
Introduction: We have investigated the ability of combi-
nations of the commonly used chemotherapy agent Pacli-
taxel (PAC) and the potent anti-resorptive agent Zoledronic
acid (ZOL) to induce apoptosis of breast cells in vitro.
Whereas previously published studies have used high doses
of ZOL for 72h, we applied clinically relevant doses and
exposure times. We also determined the importance of drug
sequencing, and found that the order in which the drugs
were given significantly affects the maximum level of
apoptosis achieved.
Method: MCF7 cells were treated with ZOL and PAC
according to the sequences and concentrations shown be-
low, and apoptosis determined by evaluation of nuclear
morphology.
ABSTRACTS / Bone 34 (2004) S46–S99S68
Experiment 1: Group 1: 25mM of ZOL (1 hour on day 1)
followed by 2nM PAC (4 hours day 2).
Group 2: 2nM PAC (4 hours on day 1) followed by 25mM
of ZOL (1 hour on day 2).
Following the last drug exposure the cells were incubated in
drug free medium to 48hrs (both groups).
Experiment 2: 2nM of PAC (4 hours on day 1) followed by
1mM ZOL (1 hour on day 2). Following the last drug
exposure cells were incubated in drug free medium to 48hrs.
Experiment 3: 2nM of PAC, (4 hours on day 1) followed by
25mM or 1mM ZOL, with or without geranylgeraniol
(GGOH) 50mM (added and removed simultaneously with
ZOL), followed by incubation in drug free medium to
48hrs.
Results:
Experiment 1 results: Apoptosis was induced in a synergis-
tic fashion in MCF7 cells but order of drug exposure was
important for maximal apoptosis. Giving ZOL (25mM)
before PAC (group 1) induced 2.4% apoptosis, whereas
maximal induction of apoptosis was seen in cells treated
with PAC on day 1 followed by ZOL on day 2 (6.1%, p <
0.001 compared to ZOL or PAC alone).
Experiment 2 results: Clinically relevant doses of ZOL are
effective at inducing apoptosis.
Following infusion of ZOL in vivo peak plasma concentra-
tion is 1-2mM for a few hours.
As was found using higher doses, MCF7 cells treated with
PAC (day 1) followed by 1mM ZOL (day 2) also induced
apoptosis in a synergistic fashion, with 4.1% in combined
group, compared to PAC alone (1.25%, p = 0.004) and ZOL
alone (0.25%, p = 0.004).
Experiment 3 results: ZOL induces apoptosis of MCF7 cells
via inhibition of the MVA pathway.
Geranylgeraniol is an intermediary of the mevalonate path-
way, which reverses the effects of ZOL. Treating MCF7
cells with PAC followed by ZOL combined with geranyl-
geraniol (GGOH, 50mM) prevented the synergistic increase
in apoptotic cell death by 70–80%.
Discussion: Jagdev et al previously reported that high doses
of ZOL and PAC given simultaneously for 72 hours had
synergistic activity. We found that synergy is also achiev-
able with shorter time points and clinically relevant con-
centrations of ZOL. Additionally, for maximal induction of
apoptosis cells must be exposed to PAC first followed by
ZOL, preferably on separate days. Induction of apoptosis is
mainly via inhibition of the MVA pathway. Our results
suggest combining PAC and clinically relevant doses of
ZOL does induce apoptosis of tumour cells, and that the
drug sequence is important for obtaining the maximum
effect of combined treatment.
37
Bisphosphonates Inhibit Glycerophosphate-Induced
Calcification of Human Urogenital Smooth Muscle Cells,
In Vitro
Vasilis Paspaliaris; Gregory Anderson, Carl Wood
Bisphosphonates (BP) have been shown to have a number
of pharmacological effects on vascular smooth muscle, from
L-type calcium channel inhibitor properties to inhibition of
calcification induced by glycerophosphate (GP). While the
pharmacological effects of BPs have led to their investiga-
tion into the treatment of pathogenic calcification such as
atherosclerosis; less looked at are the effects on non-vascu-
lar smooth muscle. Since pathogenic calcification of the
urogenital tract can be a consequence of many diseases, we
have investigated whether BPs can inhibit GP-induced
calcification of human myometrial and bladder smooth
muscle cells, in vitro. Etidronate, clodronate and pamidro-
nate inhibited calcification induced by GP in cultured
myometrial and bladder smooth muscle cells in a concen-
tration-dependent manner, at effective concentrations well
below an expected calcium chelating property. In terms of
potency, pamidronate > clodronate > etidronate in human
myometrial cells; while in bladder smooth muscle clodro-
nate > pamidronate > etidronate. Further investigation into
this pharmacological effect of BPs may lead to the plausi-
bility of BPs being therapeutically used for pathogenic
urogenital calcification.
38
Zoledronic Acid Inhibits Adhesion of Breast Cancer Cells
to Protein Matrices in a Caspase Dependent Manner
Lisa M : Pickering; Claire D. Macdonald,
Jonathan P. Coxon, Janine L. Mansi, Kay W. Colston. St.
George’s Hospital Medical School, London, UK.
Breast cancers preferentially metastasise to bone. The
mechanisms associated with breast cancer bone metastasis
development are incompletely understood but the molecular
events that facilitate adhesion of breast cancer cells to bone
matrix are thought to be critical. Nitrogen-containing
bisphosphonates (NBPs), such as zoledronic acid (ZOL),
have been shown to impair breast cancer cell adhesion and
invasion in vitro and are known to limit skeletal morbidity
in patients with osteolytic breast cancer metastases. NBPs
also induce caspase-dependent apoptosis in osteoclasts and
malignant cells by mevalonate pathway inhibition. The
mevalonate pathway is responsible for the generation of
the isoprenoid lipids farnesyl and geranylgeranyl pyrophos-
phate (FPP and GGPP) which are required for post-transla-
tional prenylation and membrane localisation of G proteins.
NBPs inhibit enzymes within this pathway resulting in
reduced G protein prenylation and activation. This study
aimed to investigate characteristics of ZOL-induced inhibi-
tion of breast cancer cell adhesion to protein matrices.
Adhesion of MCF-7 and MDA-MB-231 breast cancer cells
was assessed. Cells were exposed to investigational agent(s)
for 24 hours. Equal concentrations of viable cells were then
seeded onto mineralised dentine and unmineralised fibro-
nectin, vitronectin, laminin and collagen protein matrices for
1–24 hours. Adherent cells were washed, fixed, stained and
the dye eluted or cells counted.
ABSTRACTS / Bone 34 (2004) S46–S99 S69
MCF-7 and MDA-MB-231 breast cancer cell lines adhered
to all protein matrices under control conditions. Exposure to
ZOL for 24 hours at doses from 100nM–100AM was
associated with 40–80% impaired adhesion to all matrices.
This abrogation of adhesion could be largely overcome by
co-treatment of each cell line for 24 hours with ZOL and the
broad spectrum caspase inhibitor Z-vad-FMK. This was
observed with all protein matrices tested. Adhesion was also
almost entirely restored by co-incubation of breast cancer
cells with ZOL and either farnesol or geranylgeraniol. These
cell-permeable isoprenoid intermediates are converted intra-
cellularly into FPP or GGPP respectively and thus overcome
ZOL-induced mevalonate pathway inhibition.
In conclusion, these findings confirm that breast cancer cell
adhesion to a variety of extracellular matrix proteins is
inhibited by ZOL and demonstrate that this occurs at
concentrations that are likely to be biologically relevant.
Furthermore, these results suggest that ZOL-induced inhi-
bition of breast cancer cell adhesion occurs by a caspase-
dependent mechanism and support the hypothesis that
mevalonate pathway inhibition is a critical mechanism for
the ZOL-induced inhibition of breast cancer cell adhesion in
vitro. Future studies will assess the time course of caspase
activation by ZOL in these breast cancer cells and will
further investigate the molecular basis of ZOL-induced
inhibition of breast cancer cell adhesion.
39
Modifications to the Phosphonate Groups of
Bisphosphonates Affects their Potency and Target
Enzyme Specificity
Javier Rojas Navea,* Michael Rogers,* F. Hal Ebetino,y
Fraser Coxon*
*Bone Research Group, Institute of Medical Sciences,
University of Aberdeen, UKyProcter & Gamble Pharmaceuticals, Cincinnati, OH, USA
We and others have demonstrated that nitrogen-containing
BPs inhibit bone resorption by inhibiting farnesyl diphos-
phate (FPP) synthase, thereby preventing the synthesis of
isoprenoid lipids required for prenylation of small GTPase
proteins such as Rap1A and Rab6 in bone-resorbing osteo-
clasts. More recently, we showed that a weak anti-resorptive
phosphonocarboxylate analogue of risedronate, NE10790,
inhibits Rab geranylgeranyl transferase (Rab GGTase),
thereby selectively preventing prenylation of Rab proteins
in cells in vitro. We have now examined the effects of other
modifications to the phosphonate groups of BPs ie removal
of one of the phosphonate groups and the geminal hydroxyl
group in risedronate (RIS), alendronate (ALN) and pamidr-
onate (PAM), and replacement of one of the hydroxyl
groups on one or both of the phosphonate moieties.
NE10788, the monophosphonate analogue of RIS, inhibited
the prenylation of Rab6 and other Rabs in J774 macro-
phages and RAW 264 osteoclast-like cells but had no effect
on the prenylation of Rap1A, demonstrated by separating
prenylated from unprenylated Rab6/Rap1A by triton X-114
fractionation and by metabolically labelling cells with
[14C]mevalonate. This compound, like NE10790, therefore
inhibits Rab GGTase rather than FPP synthase, although it
was around four-fold less potent than NE10790 (effective at
4mM compared to 1mM for NE10790). However, 4mM
NE10788 had little effect on bone resorption by rabbit
osteoclasts in vitro. This might be explained by the reduced
bone affinity of this compound (due to the lack of both a
phosphonate group and the geminal hydroxyl group). By
contrast with NE10788, neither of the monophosphonate
analogues of PAM or ALN inhibited the prenylation of
either Rab6 or Rap1A in J774 cells at concentrations up to
4mM. These monophosphonates therefore do not appear to
inhibit Rab GGTase or FPP synthase.
We also studied the ability of a bisphosphonate (NE97220)
and its phosphonoalkylphosphinate (NE58029) and
bisphosphinate (NE58052) analogue to inhibit prenylation
in J774 macrophages and rabbit osteoclasts. NE97220
inhibited prenylation of Rap1A and Rab6 with similar
potency to RIS (complete inhibition with 100mM).
NE58029 also inhibited prenylation of Rap1A and Rab6,
but was approximately 4-fold less potent than NE97220. By
contrast, NE58052 had no effect on prenylation at concen-
trations up to 1mM.
In summary, the ability of bisphosphonates to inhibit FPP
synthase and hence to inhibit bone resorption requires the two
phosphonate moieties in the P-C-P structure and is dramat-
ically affected by modifications such as 1) the replacement of
a hydroxyl group with an alkyl group on one or both of the
phosphonate moieties, 2) the conversion of one phosphonate
group to a carboxylate, 3) removal of one phosphonate group.
Modification of RIS by replacement of a phosphonate group
with a carboxylate, or removal of a phosphonate group,
generates compounds that specifically inhibit Rab GGTase.
40
Action of the Novel Bisphosphonate Lidadronate in
Animal Models and Osteoblast-Like Cells
G: Santillan,* S. Morelli,* S. Katz,* N. Mondelo,y
R. Boland,* R. Puche,z E. Roldany
*Universidad Nacional del Sur, San Juan 670, (8000)-Bahıa
Blanca, ArgentinayGador S.A., Darwin 429, (1414)-Buenos Aires, ArgentinazFacultad de Ciencias Medicas, Universidad Nacional de
Rosario (2000)-Rosario, Argentina
The exact molecular mechanism by which bisphosphonates
(BPs) inhibit bone resorption is not yet completely under-
stood. From in vitro studies with the R1-amino substituted
analogue of Olpadronate (OPD), Lidadronate (LID), Van
Beek et al. (1996) concluded that LID lacks any antiresorp-
tive action. Studies with ovariectomized rats treated with
oral BPs do not show significant effects on various bone
parameters of LID in comparison with Alendronate (ALE)
and OPD (Mondelo et al., unpublished). However, these
ABSTRACTS / Bone 34 (2004) S46–S99S70
BPs, including lidadronate, have been recently shown to
suppress glucocorticoid-induced osteocyte apoptosis hold-
ing a normal number of functional osteoblast/osteocyte cells
(Plotkin et al., 1999), in part through a mitogenic action on
the osteoblast dependent on Ca2+ influx into the cell.
Interestingly, like olpadronate, lidadronate reduces bone
periodontal resorption in rats as revealed by measurements
of conventional resorptive parameters.
Moreover, an evaluation of the effects of OPD and LID on
intracellular Ca2+ in an osteoblastic cell line show that both
bisphosphonates modulate cytosolic Ca2+ levels through a
mechanism dependent of purinergic activation and which
involves the participation of phospholipase C and Ca2+
influx through VDCC channels from the L-type. In addition,
both agents significantly stimulated osteocalcin release, a
marker of osteoblast differentiation.
It is possible that lidadronate exerts an anabolic effect on
certain types of bone tissue. Future studies should address
the exact role of the calcium messenger system in LID
signaling in bone cells.
41
Alendronate Prevents Subchondral Collapse in
Mechanically Loaded Osteochondral Grafts
Magnus Tagil,* Jorgen Astrand,* Per Aspenbergy
*Department of Orthopedics, Lund University Hospital, 221
85, Lund, SwedenyDepartment of Orthopedics, Linkoping University
Hospital, Linkoping, Sweden
Introduction: Remodeling of avascular bone like an osteo-
chondral graft can lead to temporary weakening and, in a
load bearing joint, subchondral collapse with joint surface
Fig. 1. Alendronate treated specimen to the left with new bone and graft remaining
untreated control to the right the bone has been resorbed and a marrow cavity ha
incongruity as consequence. Bisphosphonates inhibit bone
resorption by adhering to the bone mineral inactivating the
osteoclasts once they start to resorb. Since the bisphospho-
nates have a high affinity to bone, grafts can be treated
locally with bisphosphonates simply by soaking the graft in
a bisphosphonate solution (Aspenberg and Astrand 2002).
Our hypothesis was that local bisphosphonate treatment of
an osteochondral graft, in a high load environment, would
protect the subchondral bone from collapse and maintain the
graft volume during remodeling.
Material and methods: We used a loaded bone chamber
(Fig. 1 in Tagil and Aspenberg 1999), which is 7 mm long
and has a diameter of 2 mm. One end of the implant is
screwed into the proximal tibia of a rat and tissue can grow
in through two ingrowth openings from the subcortical bone
into the graft. The tissue within the chamber can be exposed
to a load through the moving piston inside the chamber.
Osteochondral cylinders, 3–4 mm long, were taken from
the patellar groove of female Sprague Dawley rats perpen-
dicular to the joint surface using a hole-cutter and frozen at
�70jC. After thawing, the grafts were placed in alendronatesolution (1 mg/mL) for ten minutes and placed in the
chambers with the cartilage surface facing the loading
piston. The chamber was mounted, a single full push with
the loading device was done to seat the graft and the graft
length was measured with a caliper before inserted into male
Sprague-Dawley rats (361–395 grams).
Loading: The chambers were left unloaded for two weeks
allowing revascularization of the graft and then loaded for
four weeks. Pressure was applied by hand using a specially
designed dynamometer. This was held to the top of the
chamber, outside the intact skin, during 3 seconds followed
by an unloaded interval of another 3 seconds. This cycle
in the remodeled area close to the ingrowth openings in the bottom. In the
s formed.
Fig. 2.
ABSTRACTS / Bone 34 (2004) S46–S99 S71
was repeated 10 times, once a day. The pressure beneath the
piston was calculated to be about 2 MPa.
Evaluation: The rats were killed, the chambers harvested
and the graft length again measured with a caliper. After
fixation and decalcification, the specimens were embedded,
cut and stained.
Results: One rat was killed because the chamber loosened
and one rat died of unrelated causes; both were in the
control group. No wound infection occurred. At harvest, 5
of the 6 remaining control grafts had collapsed, i.e. the graft
length had decreased, whereas this occurred in only 2 of
8 alendronate treated grafts (Fischer exact test = 0.05).
In the alendronate treated specimens the graft prevailed in
the remodeling area and the graft trabeculas were lined with
new bone.
Conclusion: Local treatment of an osteochondral graft with
a bisphosphonate diminishes the risk for joint line collapse
during revascularization. This might be important to avoid
the subchondral collapse in osteochondral grafts but also in
different forms of avascular necrosis (Fig. 1).
42
Zoledronate Precoating of a Bone Graft Reduces Bone
Resorption During Remodeling
Magnus Tagil,* Jorgen Astrand,* Per Aspenbergy
*Department of Orthopedics, Lund University Hospital,
Lund, SwedenyDepartment of Orthopedics, Linkoping University
Hospital, Linkoping, Sweden
Remodeling of an avascular bone graft may lead to
temporary mechanical weakening as the revascularization
Fig. 1.
front advances into the necrotic bone. Especially when
the graft is bearing a high mechanical load, the necrotic
bone might collapse and loose height, shape or form. It
would be advantageous to have the whole graft or the
bone bed pretreated with an anti-resorptive agent, such as
a bisphosphonate, during the whole revascularization
period.
Bisphosphonates bind to the bone mineral and can
practically be considered to be permanent until the bone
is resorbed. However, only vascularized bone is reached
by the circulating bisphosphonates. With large grafts, the
patient would have to be treated continuously as long as
the revascularization front is advancing into the graft. We
tried an to treat the living vascularized bone while still at
the donor site, so that it would be protected from
resorption during the whole remodeling period after
transplantation.
Material and methods: We used a model with a cancellous
graft in the bone conduction chamber (Wang and Aspenberg
1996). The chamber is 7 mm long and has a diameter of 2
mm. One end of the implant is screwed into the proximal
tibia of a rat. At this end there are two ingrowth openings
where tissue can grow in from the subcortical bone.
Donor rats were given either a single dosis of 0.7 ug
zolendronate subcutaneously or a saline injection. After 24
hours, the rats were killed and bone grafts harvested from
the proximal tibias and frozen.
ABSTRACTS / Bone 34 (2004) S46–S99S72
Sixteen male Sprague-Dawley rats (382–425 grams) re-
ceived one chamber each, containing either a treated graft or
a control graft.
The chambers were harvested after 6 weeks. The specimens
were fixed, decalcified, embedded, cut and stained with
hematoxylin and eosin. The bone density in the remodelled
area was evaluated using point counting and a Merz grid.
Results: No infection occurred. Histologically, the whole
graft was invaded by fibrous tissue and new bone had
invaded the graft with a clear ossification front in all grafts.
In the remodeled area of the controls, the graft was almost
totally resorbed and replaced by a fatty bone marrow. In the
alendronate treated specimens the graft prevailed in the
remodeling area and the graft trabeculas were lined with
new bone. The increased bone density was obvious to the
naked eye.
In the bone density measurement the total amount of bone
within the remodeled area was 16% in the zolendronate
treated grafts and 5% in the controls (p = 0.001). The total
amount of living newly formed bone was 10% in the treated
and 4% in the controls (p = 0.002).
Conclusion: We show that by treating bone graft donor rats
with a potent bisphosphonate, resorption of the graft after
transferal to the recipient can be effectively hindered. This
would be useful in procedures where the graft has to bear
load and must not collapse during remodeling, as in toe to
finger joint transfers, in mosaicplasty and in primary and
revision arthroplasty (Figs. 1, 2).
43
Cellular Resistance to Bisphosphonates is Associated
with Decreased Drug Uptake
Keith Thompson;Michael J. Rogers. Bone Research Group,
Department of Medicine & Therapeutics, Institute of Medical
Sciences, University of Aberdeen, Foresterhill, Aberdeen,
UK.
Clinically, bisphosphonates have been reported to have
varying efficacy between patients. Furthermore, different
cell types exhibit different sensitivities to bisphosphonates
(BPs) in vitro. To identify possible routes by which cells
could become resistant to BPs, we generated a BP-resistant
strain of J774 macrophages. J774 cells were cultured in
increasing concentrations of ibandronate (IBA), from 0.1AMto 5AM, which caused extensive (>95%) apoptosis. The
surviving cells were further cultured in IBA (5AM) for 3
weeks, until recovery occurred.
The resulting cells (J774-RES) were resistant to the cyto-
toxic effects of BPs in an MTT assay (IC50 >100AM for
IBA, vs 59AM in wild-type cells) but grew at a comparable
rate to the parental cells, although the protein content of the
J774-RES cells was significantly lower than in the parental
strain. J774-RES cells were also found to be cross-resistant
to the cytotoxic effects of another nitrogen-containing-BP
(N-BP) zoledronic acid (ZOL) (IC50 69AM in J774-RES vs
18AM in parental cells) but were equally susceptible to the
non-N-BP clodronate, the HMG-CoA reductase inhibitor
mevastatin and the topoisomerase II inhibitors etoposide and
doxorubicin.
Western blotting for unprenylated Rap1A and metabolic
labelling with [14C]mevalonolactone were used to determine
the effect of IBA and ZOL on protein prenylation in the
J774-RES cells. IBA and ZOL were markedly less effective
at inhibiting protein prenylation in J774-RES cells com-
pared to the parental cells. FPP synthase activity in lysates
of the J774-RES cells was not significantly different to the
activity in parental cells and there was no difference in the
degree of inhibition of FPP synthase activity in lysates by
IBA or ZOL. Furthermore, the level of HMG-CoA reduc-
tase did not differ between the strains.
However, J774-RES cells were found to accumulate a
fluorescently-labelled BP (ALN-AF488) substantially less
than parental cells, indicating that resistance could be due to
decreased cellular uptake of BP. However, the J774-RES
cells did not differ in their ability to internalise FITC-
dextran, a marker of fluid phase pinocytosis, suggesting
that the J774-RES cells may express lower levels of a
putative BP transporter and/or have greater capacity to
export intracellular BP. Preliminary DNA microarray anal-
ysis also revealed that the J774-RES cells exhibit a differ-
ence in the level of expression of a small number of genes
compared to the parental cells. Proteomic analysis also
revealed several down-regulated proteins in J774-RES cells,
which are currently being characterised further.
This study supports the notion that cells can become
resistant to BPs via a mechanism that may involve decreased
uptake of BP and/or increased efflux, although the exact
molecular basis for this resistance remains to be determined.
44
Statins Prevent Bisphosphonate-Induced V;9VD2-T Cell
Activation and Proliferation In Vitro
Keith Thompson;Michael J. Rogers. Bone Research Group,
Department of Medicine & Therapeutics, Institute of
Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen, UK.
An acute phase response is the major adverse effect of
intravenously-administered nitrogen-containing bisphosph-
onates (N-BPs). This febrile reaction is characterised by
an increase in circulating levels of IL-6, TNFa and IFNg
and has been attributed to a direct agonistic action of N-
BPs on the g,y-T cell receptor (g,y-TCR), thereby acti-
vating and causing the proliferation of the major subset
of g,y-T cells in humans (Vg9Vy2+-T cells). However,
we and others recently found that these effects on g,y-Tcells are more likely due to inhibition of FPP synthase,
an enzyme in the mevalonate pathway. Inhibition of this
enzyme in PBMCs causes the accumulation and release
of upstream isoprenoid lipids such as IPP (known anti-
gens for g,y-T cells) that then directly activate g,y-Tcells. By this mechanism, N-BPs stimulate cytokine
ABSTRACTS / Bone 34 (2004) S46–S99 S73
release and the proliferation of g,y-T cells in human
PBMC cultures in a manner consistent with the potency
for inhibition of FPP synthase; zoledronic acid (ZOL) >
alendronatefibandronate > pamidronate.
Since the mevalonate pathway is also inhibited by choles-
terol-lowering statins, which inhibit HMG-CoA reductase
(the most proximal enzyme of the pathway), statins could
prevent the accumulation of IPP that occurs in PBMCs
following N-BP treatment. To test this hypothesis, human
PBMC cultures were treated with a pharmacologically
relevant dose of 1AM N-BP in the absence or presence of
mevastatin. The stimulatory effect of N-BPs on prolifera-
tion of CD3+ g,y-T cells was dose-dependently inhibited by
mevastatin at concentrations from as low as 10nM and was
abrogated by 1AM mevastatin. This abrogative effect of
mevastatin was not due to cytotoxicity, since 1AM mevas-
tatin did not affect the proliferation of g,y-T cells in
response to a synthetic agonist of Vg9Vy2-T cells, bromo-
hydrin pyrophosphate (BrHPP) or to anti-CD3 antibody.
Furthermore, 1AM mevastatin did not affect the stimulation
of TNFa and IFNg release in response to BrHPP treatment,
but completely inhibited TNFa and IFNg release following
treatment of PBMCs with ZOL for 48 hours. As well as
inhibiting HMG-CoA reductase, statins can interfere with
lymphocyte adhesion and co-stimulation via binding to a
novel allosteric site within LFA-1 and disrupting ICAM-1
binding. However, 1AM desoxolovastatin (an analogue of
lovastatin that binds LFA-1 but does not inhibit HMG-CoA
reductase) had no effect on ZOL-induced g,y-T cell prolif-
eration, whereas 1AM lovastatin significantly decreased
g,y-T cell proliferation following ZOL treatment. The
ability of fluvastatin (FLU) to prevent ZOL-induced g,y-Tcell proliferation was investigated in more detail. FLU
concentration-dependently inhibited ZOL-induced g,y-Tcell proliferation and TNFa release at concentrations
>10nM, with 1AM FLU consistently abrogating ZOL-
induced g,y-T cell proliferation and TNFa release in
PBMC cultures.
These observations demonstrate that statins can prevent
N-BP-induced g,y-T cell proliferation and release of pro-
inflammatory cytokines in human PBMC cultures,
through inhibition of HMG-CoA reductase and a subse-
quent decrease in the accumulation of isoprenoid lipids
upstream of FPP synthase in the mevalonate pathway. Co-
administration of a statin such as FLU with intravenous
N-BP may therefore provide a novel and effective means
for preventing the acute-phase response to bisphosphonate
therapy in vivo.
45
Differentiating the Mechanisms of Antiresorptive Action
of Nitrogen Containing Bisphosphonates
E: van Beek,* L.H. Cohen,y I.M. Leroy,y F.H. Ebetino,z
C.W. Lowik,* S.E. Papapoulos*
*Dept. of Endo., Leiden Univ. Med. Cent., Leiden, The
Netherlands
y2Gaubius Lab., TNO Prev. and Health Leiden, The
NetherlandszProct & Gamb Pharmac., Health Care Res. Center,
Mason, USA
Bisphosphonates (BPS) are distinguished into two classes
according to their chemical structure and mechanism of
action. First, non nitrogen containing BPS such as etidronate
and clodronate that are of low potency and inhibit osteoclast
function via metabolism into toxic ATP-metabolites. Sec-
ond, nitrogen-containing BPS (NBPS), such as alendronate
and risedronate that inhibit the enzyme of the mevalonate
biosynthetic pathway farnesyl pyrophosphate synthase
(FPPS), resulting in inhibition of the prenylation of small
GTP-binding proteins in osteoclasts and disruption of their
cytoskeleton. Previous, studies in various cell types sug-
gested, however, that pamidronate functions by mecha-
nism(s) additional or independent of the mevalonate
pathway. To examine if such mechanism(s) are also in-
volved in the action of NBPS on osteoclastic bone resorp-
tion, we examined the action of alkyl- and heterocyclic
NBPS with close structural homology on FPPS/isopentenyl
pyrophosphate isomerase (IPPI) activity, on osteoclastic
resorption and on reversibility of this effect with GGOH.
As expected, both pamidronate and alendronate suppressed
bone resorption and FPPS/IPPI activity, the latter with
greater potency than the first (EC50 values for suppression
of 45Ca-release; 0.6 and 4 uM and for FPPS/IPPI activity;
1.2 and 27 uM, respectively). Surprisingly, however, unlike
alendronate, the antiresorptive effect of pamidronate was
only partially reversible with GGOH (EC50 values for
inhibition of 45Ca-release by pamidronate and alendronate
in the presence of GGOH; 15 and 100 uM, respectively),
indicating the involvement of mechanism(s) of action addi-
tional to that of suppression of FPPS. Comparable results
were obtained with the heterocyclic NBP NE-21650, a
structural analog of risedronate. Thus, despite an effect on
FPPS, the actions on bone resorption of some NBPS may
involve mechanisms additional to suppression of FPPS.
These findings may lead to identification of additional
pathways that are important for bone resorption and may
help to differentiate among members of the NBP class,
which are currently distinguished only according to their
potency to inhibit bone resorption.
45a
Geranylgeranyl pyrophospate and Geranylgeraniol
Stimulate Osteoclastic Bone Resorption mediated by
the Retinoic Acid Receptor
Ermond van Beek, Clemens Lowik, Socrates Papapoulos
Department of Endocrinology and Metabolic Diseases,
Leiden University Medical Center, Albinusdreef 2, 2333
ZA Leiden the Netherlands
Intermediates of the mevalonate biosynthetic pathway are
essential for osteoclast function, and inhibition of their
ABSTRACTS / Bone 34 (2004) S46–S99S74
synthesis by statins or nitrogen-containing bisphosphonates
(NBPS) inhibit osteoclastic resorption. In particular, gera-
nylgeranyl pyrophosphate (GGPP) and all-trans geranylger-
aniol (GGOH) are utilized for the prenylation of proteins
that are essential for the integrity of the cytoskeleton and
intracellular signalling. Previously we have indicated that
GGOH may also stimulate bone resorption, but the mech-
anism is not known. It has previously been shown that all-
trans GGOH is metabolised to all-trans geranygeranoic acid
(GGA) and that this metabolite can stimulate retinoic acid
receptor (RAR) expression and pRARCAT activity. There-
fore, we hypothesized that GGOH stimulates bone resorp-
tion via GGA that subsequently activates RAR. GGOH,
GGPP, GGA all stimulated osteoclastic bone resorption in a
bone explants in vitro, assessed biochemically and histolog-
ically. This action could be totally blocked by co-treatment
with the RAR antagonist AGN-193109 (obtained from
Allergan Inc). This antagonist had no effect on PTHrP or
calcitriol stimulated bone resorption, but reduced the effec-
tive concentration of retinoic acid (RA) required to stimulate
resorption by 10-fold.
We further examined the reversibility of NBP (ibandronate)
inhibited bone resorption by GGOH, GGPP and GGA.
Whereas, as expected, GGOH and GGPP rescued osteo-
clasts and reversed the antiresorptive effect of ibandronate,
GGA had no effect similar to RA. This suggests that GGA is
not involved in protein prenylation.
Thus, intermediates of the mevalonate pathway such as
GGPP and GGOH can modulate bone resorption not only
by the prenylation of proteins, but also by another mecha-
nism involving RAR.
46
Inhibition of Growth of Breast Cancer Cells by Low
Dose Risedronate
Lois Witters,* Stephen Morris,y F. Hal Ebetino,z
Allan Lipton*
*The Milton S.Hershey Medical Center, Hershey, PA, USAyAventis Pharmaceuticals, Bridgewater, NJ, USAzP&G Pharmaceuticals, Mason, OH, USA
Risedronate (RIS) is a third generation nitrogen containing
bisphosphonate (N-BP) used to prevent and treat postmen-
opausal and steroid-induced osteoporosis. Treatment of
human breast cancer cell lines with RIS (1–20 uM) resulted
in dose-dependent growth inhibition (MCF-7: IC50 = 6 uM;
HER-2/neu transfected MCF-7: IC50 = 11 uM) (Morris, et
al., Int Conf. on Cancer-induced Bone Diseases, 2003).
These levels of RIS are not normally achievable in the
plasma of patients receiving RIS. Bisphosphonates bind
avidly to hydroxyapatite (HA), an experimental model of
bone. The purpose of this study was to assess the effects of
RIS bound to hydroxyapatite on the growth of breast cancer
cells.
Methods: MCF-7 cells were grown on HAwafers pretreated
with 0.5 uM or 20 uM of RIS. Cells were also grown on HA
wafers not pretreated with RIS but where RIS was added
after attachment. Growth effects under these conditions were
compared to the effects of RIS in culture media in 24 well
plates. Cell number was determined after a 3 day exposure to
RIS by the colorimetric MTT tetrazolium dye assay.
Results:MCF-7 cells grown in culture media were inhibited
by 20 uM RIS (78%) or conditioned media from HAwafers
pretreated with 20 uM RIS (71%). HA wafers pretreated
with 20 uM RIS also inhibited cell growth but to a lesser
extent (41%). MCF-7 cells were not inhibited by 0.5 uM
RIS in culture media (7%) nor conditioned media from HA
wafers pretreated with 0.5 uM RIS (4%). HA wafers pre-
treated with 0.5 uM RIS, however, inhibited MCF-7 cell
growth by 29%.
Conclusions: High doses (20 uM) of the N-BP, risedro-
nate, inhibited growth of MCF-7 breast cancer cells in
culture media, in conditioned media from HA wafers
pretreated with 20 uM RIS, and to a lesser extent on the
HA wafers pretreated with 20 uM RIS. Low doses (0.5
uM) of RIS caused negligible growth inhibition when
added to culture media or in the conditioned media from
HA wafers pretreated with 0.5 uM RIS. The pretreated HA
wafers, however, had the ability to inhibit MCF-7 cells
suggesting that interactions of N-BPs with tumor cells
attached to bone may magnify the effectiveness beyond
what may be expected from BP serum concentrations. This
study raises the possibility that early intervention with
risedronate, prior to metastatic disease, may well protect
against this progression.
47
Different Effects of Olpadronate in Osteopenic Rats with
a Normal or Deficient State of Vitamin D. Preliminary
Study
Susana Zeni,*,y Silvina Mastaglia,* Patricia Mandalunis,z
Marıa del C Degrandi,* Somoza Julia,* Silvia Friedmany
*Seccion Osteopatıas Medicas-Hospital de Clınicas-Fac. de
Medicina-UBAyCatedra de Bioquımica General y Bucal. Facultad de
Odontologıa. UBAzCatedra de Fisiologıa. Facultad de Odontologia. UBA
Background: Ovariectomized (OVX) rats are often used as
an experimental model to study estrogen deficiency as well
as to test the efficacy of different therapies in preventing
bone loss. Bisphosphonates (BPs) are useful antiresortive
agents to inhibit resorption in several bone diseases. Vitamin
D insufficiency or deficiency has negative effects on skel-
etal metabolism. There is controversy related to the effect of
BPs in vitamin D depletion state.
Objective: The aim of the present preliminary study was to
investigate if olpadronate (OPD) has different effects in
recovery bone mass in OVX rats with established osteope-
nia and different vitamin D state.
Material and Methods: A total of 24 female Wistar rats
(250–300 g) were OVX and divided in three groups: 1) +
Table 1
BMDT BMDLS BMDPT BALP SCTX 25OHD(TF)
+VITD+OPD 12F 4A 7.4F 3.0A 20.0F 8.1A 1.2F 3.1A � 94.0F 12.5A 59.0F 3.5A
�VITD+OPD 5F 3A 1.2F 2.0B 6.8F 3.1B � 0.3F 3.3A � 57.0F 9.6B 9.2F 0.8B
+/�VITD+OPD 7F 1A 5.5F 3.1A 16.2F 4.3A 2.1F 2.8A � 106F 10.8A 58.0F 3.2A
ABSTRACTS / Bone 34 (2004) S46–S99 S75
VitD: animals fed with a synthetic diet that covered all
adequate requirements, 2) �VitD (laking of dietary vitamin
D and skin production): Animals were housed under red
light and fed with the same synthetic diet without Vitamin
D, 3)+/�VitD: these rats were laking of skin production and
fed with dietary Vitamin D. During 60 days rats lost
approximately 15% of bone mass, after that (To) all animals
received during a 45-day period (Tf) 16 ug OPD/100 g rat
weekly. Bone mineral density (BMD), 25OHvitamin D (ng/
ml), bone alkaline phosphate (bALP) (mUI/ml) and serum
CTX (ng/ml) were assessed at To and Tf and tibia histo-
morphometry was made at Tf.
Results: D:Changes (Tf-To) in total skeleton (te), lumbar
spine (ls), proximal tibia (pt) BMD (mg/cm2, bALP and
sCTX were calculated (Table 1).
Different letters indicated a p < 0.05.
Histological sections of tibia in �VitD group evidenced
greater thickness of metaphyseal cartilage and sealed trabec-
ulae immediately below it. The presence of bone trabeculae
defined osteoid surfaces. The +VitD group had greater bone
volume and a decrease of the sealed trabeculae and osteoid
surface compared to the �VitD group.
Conclusion: Bone resorption inhibition, bone volume and
positive changes in BMD were higher in the +VitD group
suggesting that OPD effects is less beneficial in vitamin D
deficient state (Table 5).
Clinical (general)
48
Patient Compliance and Preference of Alendronate’s
Once Weekly Administration in Comparison with
Alendronate’s Daily Administration in Osteoporosis in
Postmenopausal Women
D: Babiolakis; A. Stamatiadou, C. Andronis, I. Piskontaki,F. Xristogiannis, E. Kousta, D. Spanoudis, I. Athanasiou,
P. Basilakis, A.E. Georgiadis. Osteoporosis Center. Lito
Gynecological Hospital.
It is widely accepted that the end therapeutic result appears
to be closely connected to the good or bad compliance of the
patient during treatment of chronic diseases. However com-
pliance studies in Osteoporosis (OP) are very rare in the
international literature although OP is a chronic disease.
The presence lately in the market of two biocompatible
dosages of the same drug (Alendronate) e.g. Once Weekly
and daily, give us a good opportunity to measure the
compliance of the OP patient in two different treatment
schedules without having problems from drug therapeutic
action. For this reason an open label, randomized, crossover
12 months study has been performed at 107 Rheuma or
Endocrino centers all over Greece. 723 patients (mean age
61,97 years) were treated for 6 ms with Alendronate 10mg/d
and then replaced the above drug with Alendronate 70 Once
Weekly (OW) for another six months.
After experiencing both regimens, a questionnaire was ad-
ministered which addressed compliance, preference, conve-
nience and willingness to take the regimens long term.
Results were evaluated using �2 and Gart’s test (Odds ratio
test).
After the analysis of the results, the overall compliance
during daily treatment was 79, 4% and during OW treatment
96, 5%. In addition 99, 4% of the patients preferred the OW
regimen while the 99, 9% of them found that OW regimen
was more convenient and the 99, 5% chose the OW regimen
as the one that they would be more willing to use in long
term. All the results were statistically significant ( p < 0,001
for each of them).
In conclusion this study demonstrates that the patient’s
compliance is much better with the Alendronate OW dosing
regimen than with the daily regimen and that the patients
preferred this regimen from the daily one.
49
Ibandronate does not Affect Time to Renal Function
Deterioration over 2 Years of Treatment: Phase III Trial
Results
Richard Bell,* Jean-Jacques Body,y Debu Tripathy,z
B Bergstrom§
*The Andrew Love Cancer Centre, Geelong, Victoria,
AustraliayInst. Jules Bordet, Universite Libre de Bruxelles,
Brussels, BelgiumzUniversity of Texas Southwestern Medical Center, Dallas,
Texas, USA§Hoffmann-La Roche Inc., Nutley, New Jersey, USA
Background: As patients with metastatic bone disease
typically receive long-term treatment with bisphosphonates,
drug-related safety is of considerable importance. There are
increasing concerns in relation to renal adverse events
associated with intravenous aminobisphosphonates that
are used to manage this condition.1–2 Clinical trial data
for intravenous ibandronate, a new aminobisphosphonate
for the treatment of bone metastases, suggests that its
nephrotoxic potential is comparable to placebo.3 To inves-
tigate this further, we conducted a Kaplan-Meier analysis
of time to renal function deterioration with intravenous
ibandronate.
ABSTRACTS / Bone 34 (2004) S46–S99S76
Methods: Women aged z18 years of age with histologi-
cally confirmed breast cancer and bone metastases were
included in this 96-week, randomized, double-blind, place-
bo-controlled, parallel-group, multicenter, phase III study.
Patients were received intravenous ibandronate 6mg infused
over 1–2 hours every 3–4 weeks. A post-hoc Kaplan-Meier
analysis assessed time to deterioration in renal function over
2 years of treatment. A renal event was considered to occur
if there was an increase in serum creatinine of 0.5mg/dL
from baseline, if baseline serum creatinine was <1.4mg/dL;
1.0mg/dL from baseline, if baseline serum creatinine was
z1.4mg/dL; or if serum creatinine increased to twice the
baseline value.
Results: A total of 152 patients were randomized to
treatment with intravenous ibandronate 6mg and included
in this safety analysis. The Kaplan-Meier analysis showed
that the proportion of patients with a pre-defined increase in
serum creatinine was 4% after 1 year of ibandronate
treatment and 6% after 2 years, versus 6% for placebo at
both 1 year and 2 years.
Conclusions: Few patients treated with i.v. ibandronate 6mg
over 2 years of treatment experienced renal function dete-
rioration. No patients were withdrawn from the study
because of renal adverse events. There was no evidence
for drug-related renal toxicity over time with ibandronate.
This is consistent with pre-clinical ibandronate data.4
Kaplan-Meier analysis using identical renal event endpoints
in a trial of i.v. zoledronic acid 4mg in metastatic bone
disease due to breast cancer and multiple myeloma reported
a 9% incidence of renal function deterioration over 1 year of
treatment, increasing to 11% at 2 years.1,5 Direct compar-
isons of the renal safety of ibandronate and other intrave-
nous bisphosphonates are warranted.
References
[1] Rosen LS, et al. Cancer J 2001;7:377–87.
[2] Chang JT, et al. N Engl J Med 2003;349:1676–9.
[3] Body JJ, et al. Ann Oncol 2003;14:1399–405.
[4] Pfister T, et al. Toxicology 2003;191:159–67.
[5] Rosen LS, et al. Cancer 2003;96:1735–44.
50
Alendronate is Safe and Effective During Long-Term
Therapy in Patients with Osteoporosis and Impaired
Fasting Glucose
Miro Cokolic,* Rok Hreny
*Department of Endocrinology and Diabetology, Internal
Clinic, Teaching Hospital, Maribor, SloveniayInstitute of Mathematics, Physics, and Mechanics,
University of Ljubljana, Ljubljana, Slovenia
While osteoporosis can nowadays be effectively treated, it is
important that osteoporosis treatment regimen does not
worsen patients’ concomitant diseases. The aim of this
study was to monitor in patients with impaired fasting
glucose (IFG; serum levels of fasting glucose > 6.1 and <6.9
mmol/l) that are treated for postmenopausal osteoporosis
both bone mineral density (BMD) and levels of blood
glucose and HbA1c. Eleven women with postmenopausal
osteoporosis (T-score below �2.5 SD) and IFG were
enrolled in a five-year prospective study. Patients were
59 to 74 years old (mean: 65 years) and 8 to 25 years
(mean: 16 years) after the menopause. They were treated
with alendronate (10 mg/d during the first four years, 70
mg/w during the fifth year) in combination with 500-mg/d
elemental calcium. During the five-year follow-up, the
BMD in the lumbar spine (L1–L4) and left hip was
measured in all patients using dual energy X-ray densi-
tometry (Hologic QDR 2000+). The serum levels of
glucose, HbA1c, Ca, alkaline phosphatase (ALP) and
creatinine were measured every 6 months. All patients
were treated for IFG only with diabetic diet. In 5 years,
BMD increased on average by 8.1% (range 0.2–9.6%) in
the lumbar spine (L1–L4) and by 4.8% (range 0.5–8.7%)
in the left hip. Levels of Ca, ALP and creatinine were
within normal limits during the treatment, and no clinical
side effects were observed during the study. Serum levels
of fasting glucose showed no statistically significant
changes during the alendronate treatment, with an average
value of 6.3 mmol/l (range 6.1–6.6 mmol/l) in the begin-
ning of the treatment and 6.5 mmol/l (range 6.4–6.8
mmol/l) after 5 years of the treatment. Similarly, the
average level of HbA1c was 6.5% (range 6.2–6.6%) at
the start of the treatment and 6.7% (range 6.2–6.8%) after
5 years of the treatment. Results of our study indicate that
osteoporosis can be effectively treated in patients with IFG
for up to five years, while maintaining (with little varia-
tion) serum levels of fasting glucose and HbA1c.
51
Renal Excretion of Alendronate in Relation to Bone
Turnover in Patients with Crohn’s Disease
Serge Cremers,* Denise Banffer,y Jan den Hartigh,*
Pieter Vermeij,* Ruud van Hogezand,z
Socrates Papapoulos,y Neveen Hamdyy
*Dept Clin Pharmacy and ToxicologyyDept Endocrinology and Metabolic DiseaseszDept Gastroenterology, Leiden University Medical center
Aims: Patients with Crohn’s disease may have osteoporosis
which can be treated with bisposphonates. Considering the
gastrointestinal pathology in Crohn’s disease it is conceiv-
able that bioavailability of bisphosphonates is altered, which
could have an effect on clinical outcome. The aim of the
present study was to investigate if the oral bisphosphonate
alendronate was absorbed by patients with Crohn’s disease,
and whether this had effect on bone turnover.
Methods: Urinary excretion of alendronate and biochemical
parameters of bone turnover (uNTx/Cr and bsAP) were
studied at 3 and 6 months after start of treatment (10 mg/
day) in 19 patients with Crohn’s disease.
ABSTRACTS / Bone 34 (2004) S46–S99 S77
Results: The average excretion per 24h of alendronate in
Crohn patients was 0.5–0.6% of the dose administered,
which is comparable to excretion in non-Crohn-patients.
The excretion was not influenced by gut resection or by
localisation site of the disease. Bone turnover in patients
with Crohn’s disease was decreased to the same extent as in
other types of osteoporosis.
Conclusions: Our data suggest that in patients with Crohn’s
disease orally administered alendronate is adequately
absorbed and taken up by the skeleton, its efficacy being
demonstrated by adequate suppression of parameters of
bone turnover. This sends a clear clinical message that in
patients with Crohn’s disease alendronate can be effectively
used in the management of osteoporosis.
52
Short-Term Whole Body Retention in Relation to Rate
of Bone Resorption and Cartilage Degradation After
Intravenous Bisphosphonate (Pamidronate) in
Rheumatoid Arthritis
Serge Cremers,* Mariette Lodder,y Jan den Hartigh,*
Pieter Vermeij,* Philomena van Pelt,y Willem Lems,y
Socrates Papapoulos,z Ben Dijkmansy
*Dept. of Clin Pharmacy and Toxicology, Leiden University
Medical CenteryDept. of Rheumatology, VU Medical CenterzDept. of Endocrinology and Metabolic Diseases, Leiden
University Medical Center
Aims: Bisphosphonates (BPs) inhibit osteoclast-mediated
bone resorption, and have been reported to decrease the rate
of cartilage degradation. The antiresorptive effect of BPs is
determined by the amount of BP retained by the skeleton. In
rheumatoid arthritis (RA) the uptake is not confined only to
the skeleton, but BP is also retained in the joints, which
could have implications for dose regimens. In the present
study we investigated the whole body retention (WBR) of
pamidronate and its relationship to bone resorption and
cartilage degradation in patients with active RA.
Methods: 26 patients received placebo, 45mg or 90 mg
intravenous pamidronate. Serum and urine samples were
collected before and during 12 days after drug administra-
tion. Rate of bone resorption was assessed by sCTx, uCTx/
Cr and uNTx/Cr, and rate of cartilage degradation by
uCTxII/Cr. WBR was derived from urinary excretion data
of pamidronate.
Results: Pamidronate induced a rapid and sustained
decrease in the level of biochemical markers of bone
resorption and cartilage degradation. The mean WBR of
pamidronate was 69% of the administered dose, and
showed a remarkably wide range (41–96%). The decrease
in rate of bone resorption, but also rate of cartilage
degradation appeared to be related to the WBR of
pamidronate.
Conclusions: This is the first study in which the effect of
BP treatment has been studied in relation to the amount of
BP retained by the body in patients with active RA. The
total amount of BP retained by the body shows a remarkably
wide range and is comparable with literature data on patients
with osteoporosis indicating no substantial involvement of
the joints in binding of the BP. The apparent relationships
between the amount of BP retained by the body and the
effect could have implications for therapeutic regimens in
patients with RA.
53
Low Nephrotoxicity of Ibandronate: Evidence from
Clinical Trials in Metastatic Bone Disease
Ingo Diel,* Jean-Jacques Body,y B Bergstromz
*CGG-Klinik GmbH, Mannheim, GermanyyInstitut Jules Bordet, Universite Libre de Bruxelles,
Brussels, BelgiumzHoffmann-La Roche Inc., Nutley, New Jersey, USA
Background: Many patients with metastatic bone disease
will receive bisphosphonates for several years to reduce
skeletal morbidity. In addition to efficacy against skeletal
complications, the long-term tolerability of bisphospho-
nates can affect clinical outcomes. The nephrotoxic po-
tential of certain intravenous (i.v.) bisphosphonates was
highlighted by published reports of renal adverse events
(AEs) in patients receiving i.v. zoledronic acid or pamidr-
onate.1–3 Some patients developed acute renal failure that
required immediate drug discontinuation.3 This abstract
reviews renal safety data for ibandronate, a new amino-
bisphosphonate for the prevention of metastatic bone
events.
Methods: Three 96-week, randomized, double-bind, place-
bo-controlled trials were conducted in women with meta-
static breast cancer. In a trial of i.v. ibandronate, a 6mg dose
(n = 154) was compared with placebo (n = 158) infused over
1–2 hours every 3–4 weeks. In a pooled analysis of data
from two further trials, oral ibandronate 50mg (n = 286) was
compared with placebo administered once daily (n = 287).
Two-year, non-controlled, follow-up studies have also
assessed renal safety in 62 patients receiving i.v. ibandronate
6mg and 115 patients receiving oral ibandronate 50mg (total
exposure to ibandronate = 4 years).
Results: Over 96 weeks, the incidence of renal AEs was
comparable between the intravenous ibandronate 6mg
(4.5%) and placebo (4.0%) groups. Few patients had
decreased creatinine clearance (ibandronate 6mg, 2.6%;
placebo, 1.3%). None of the renal AEs with i.v. ibandronate
were graded serious, or led to withdrawal from treatment.
In the pooled oral trials, a similar percentage of patients in
the ibandronate 50mg and the placebo groups had renal
AEs (5.2% versus 4.7%). The incidence of creatinine
increase was similar with oral ibandronate 50mg (0.7%)
and placebo (1.4%). Over 4 years of treatment (2-year
follow-up), there were no renal AEs or laboratory changes
indicating renal impairment in patients treated with i.v. or
oral ibandronate.
ABSTRACTS / Bone 34 (2004) S46–S99S78
Conclusion: In patients with bone metastases due to breast
cancer, i.v. and oral ibandronate have a similar renal safety
profile to placebo over 96 weeks. The absence of renal AEs
over 4 years of treatment suggests that long-term use of
ibandronate does not lead to drug-related accumulation of
renal toxicity. The relatively low incidence of renal AEs
with ibandronate compared with other i.v. bisphosphonates
indicates that the risk of renal toxicity differs between
agents. This is supported by pre-clinical data showing that,
unlike zoledronic acid, there is no accumulation of renal
tubular damage with ibandronate.4 Differences in nephro-
toxic potential are also reflected in the product labelling.
Unlike zoledronic acid, ibandronate may be used in patients
with severe renal function deterioration, and there are no
restrictions on the use of ibandronate with nephrotoxic
medications or renally-metabolized medications.5,6
References
[1] Rosen LS, et al. Cancer J 2001;7:377–87.
[2] Stein SH, et al. Proc ASCO 2003;22:745 [Abstract 2997].
[3] Chang JT, et al. N Engl J Med 2003;349:1676–9.
[4] Pfister T, et al. Toxicology 2003;191:159–67.
[5] Bondronat product label. Basel, Switzerland: F. Hoffmann-La Roche
Ltd; Oct 2003.
[6] Zometa product label. Basel, Switzerland: Novartis; Sept 2003.
54
Early Experience of Bisphosphonate Use in Adolescents
with Osteonecrosis
Elisabeth Goergens; Richard Brown, Chris Cowell,
David G. Little
Traumatic osteonecrosis of the femoral head in adoles-
cents has a poor prognosis due to collapse and degen-
erative change. We hypothesised that early bisphospho-
nate treatment to reduce osteoclast activity could allow
revascularisation and repair with maintenance of joint
congruity.
Nine patients with documented osteonecrosis were trea-
ted with intermittent intravenous pamidronate (Aredia,
Novartis) commencing within a mean 1 month of
diagnosis (range, 5 to 91 days). There were 7 boys
and 2 girls with mean age 13 years. The dosing
protocol has evolved over two years with the current
dose being 9 mg/kg/year for 18 months. Mean follow
up is 25.3 months (range, 18 to 38 months) with all
patients followed for more 18 months. There were 6
patients, who presented after unstable SCFE. Of these
the index procedure had failed in three, requiring
multiple early operations. The other three patients had
sustained an intertrochanteric fracture with a pelvic
fracture, a traumatic hip dislocation and a femoral neck
fracture respectively.
Eight of the patients are painfree. Only one occasionally
uses crutches. Three have a leg length discrepancy greater
than 2 cm. The mean Harris Hip score is 96.8 (91.7–100)
and Iowa Hip rating 96.8 (88–100). Seven of 9 patients do
not show significant resorption of the femoral heads at the
most recent follow up. Of the two patients with significant
resorption, one patient began to resorb on a lower dose of
bisphosphonate, so he received the higher dose (9 mg/kg/
year). The other patient had resorption of a section of the
femoral head, which had not revascularised by 18 months,
and this was elevated and bone grafted. These two hips are
pain free and functional in the short term, but their defor-
mity is expected to bring about early osteoarthritis in adult
life.
This early experience lays the foundation for prospective
clinical trials of bisphosphonate therapy in adolescents with
osteonecrosis. It appears that bisphosphonate treatment
protocols for adolescents will need to be prolonged. Our
current practice is for a duration of around 18 months with
normalisation of uptake on bone scan as the end point for
therapy.
55
Short-Term Safety Assessment in the Use of Intravenous
Zoledronic Acid in Children
Wolf gang Hogler,* Fabian Yap,* David Little,§
Geoffrey Ambler,* Mary McQuade,* Christopher Cowell*
*Institute of Endocrinology and Diabetes, The Childrens
Hospital at Westmead, Sydney, AustraliayDepartment of Paediatrics and Adolescent Medicine,
University of Innsbruck, AustriazDepartment of Paediatrics, KK Womens and Childrens
Hospital, Singapore§Department of Orthopaedics, The Childrens Hospital at
Westmead, Sydney, Australia
Background: Zoledronic acid, a nitrogen-containing bis-
phosphonate, is used in the treatment of malignancy-induced
hypercalcemia and metastatic bone disease in adults with
similar long-term efficiency and safety compared to pamidr-
onate. Both drugs are associated with renal deterioration. In
children, pamidronate has been successfully used off-label
for a variety of bone disorders, particularly osteogenesis
imperfecta. The clinical and metabolic side effects of
pamidronate in children are well described. So far, no
comparable information is available for the more potent drug
zoledronic acid. We report our experience with clinical side
effects of zoledronic acid in children, the frequency and
pattern of post-infusion hypocalcemia and hypophosphate-
mia and the short-term effects on renal function.
Subjects and Methods: Thirty-four children (aged 2–17
years, 22 males) received at least one infusion of zoledronic
acid (0.02 to 0.05mg/kg). The children were suffering from
osteoporotic conditions or severe localised bone disease.
Inclusion criteria involved normal Vitamin D, creatinine and
urea levels and a normal renal ultrasound. All patients
received calcium supplements prior to and after the infusion.
The clinical and metabolic effects of first-, second- and
ABSTRACTS / Bone 34 (2004) S46–S99 S79
third-dose infusions were reviewed. Serum total calcium
and inorganic phosphorus were measured at baseline, 48
and 72 hours after infusion. Longitudinal data was available
for second- and third dose zoledronic acid on a subset of 14
patients. Creatinine and urea levels were measured at
baseline, 48 hours and after the third infusion.
Results: The most frequent first-dose clinical side effects
were flu-like symptoms and myalgia (85%), fever (68%),
localised bone pain (44%) and nausea or vomiting (41%).
All side effects were managed on an outpatient basis with
standard doses of paracetamol. Only 15% of subjects had
no side effects. Calcium and phosphorus levels decreased
significantly between baseline and 48 hours post-infusion
(p < 0.001) and were still below baseline after 72 hours
(p < 0.001). Hypocalcaemia (<2.1mmol/L) was observed
in 74% of subjects (nadir 2.02, range 1.76–2.31) and
hypophosphataemia (<1mmol/L) in 82% (nadir 0.74, range
0.43–1.25). No clinical side effects were reported for sub-
sequent infusions. Pre-infusion calcium and phosphorus
concentrations were not different between the first, second
and third infusion (6 week intervals). The% fall in calcium
and phosphorus was significantly lower (p < 0.001) after the
third (0.05mg/kg) compared to the first infusion (0.025mg/
kg). Creatinine and urea levels were not different from
baseline both after the first and the third infusion and no
individual had levels outside the normal range.
Discussion: Our results show a high frequency of first-dose
clinical side effects resembling those reported for pamidro-
nate. The side effects were easily manageable and did not
recur with subsequent infusions. The frequency of post-
infusion hypocalcaemia and hypophosphataemia in our
cohort appears of greater magnitude compared to pamidro-
nate. The short-term administration of zoledronic acid was
safe and did not affect renal function. We conclude that
sufficient oral calcium intake and close clinical supervision
are required, particularly for the first infusion, when hypo-
calcaemia is most pronounced. The use of zoledronic acid in
children requires thorough consideration of treatment indi-
cation, dosing, benefits and potential side effects.
56
Tolerability and Renal Safety of Intravenous Zoledronic
Acid in Benign Metabolic Bone Disease
Dianne Hughes,* Julie Hetherington,* Klaus Sommer,*
Bronwyn Crawford,*,y Michael Hooper*,y
*Endocrinology and Metabolism, Central Sydney Area
Health ServiceyThe University of Sydney
Intravenous (IV) bisphosphonate therapy may be followed
by symptoms associated with an acute phase reaction and a
recent report has associated IV zoledronic acid (ZA:
Zometa, Novartis Pharmaceuticals) with renal impairment
in cancer patients. (1) We have treated 256 patients, 19–95
years of age, F:M 2:1, with benign bone disease in routine
clinical practice with IV ZA (4 mg) either as a single annual
infusion for osteoporosis (193) or 1.5–3 monthly for other
benign bone diseases (63).
In 171 patients clinical tolerability data was collected by
telephone interview 7–14 days post infusion on a stand-
ardised form. Symptoms, scored on a linear analogue scale
of 1–10, were grouped as mild (1–5) and severe (6–10).
52% had no symptoms. At least one severe symptom was
reported by 28% of patients. Generalised bone and muscle
aches and pain occurred in 38% patients (16% mild, 22%
severe), fever in 16% (7%, 9%), headache in 15% (9%, 6%),
chills/shakes in 12% (5%, 7%), nausea/vomiting in 9% (6%,
3%), breathlessness in 2% (1%, 1%) and chest pain in 2%
(0%, 2%). 67% had been treated previously with an oral
bisphosphonate (15%) or by IV pamidronate (53%). Severe
symptoms tended to occur more frequently in patients not
previously treated with bisphosphonates.
Serum creatinine concentration was routinely measured
before the first and each subsequent infusion. The laboratory
computer database was also interrogated for interval results.
Baseline and subsequent values were available in 128
patients. 119 patients had a normal serum creatinine prior
to the first infusion and 9 an elevated serum creatinine (115–
185 Amol/L, RR < 110 Ammol/l). In those with a normal
serum creatinine at baseline, 4 had a subsequent creatinine
value that was more than 30% higher at any subsequent time
point. In these 4 patients the serum creatinine remained
within the normal range. In the 9 patients who had an
elevated serum creatinine at baseline, 8 had no significant
change in subsequent values. In one patient, who received a
single infusion, the values were lower 1.5 and 3 months later
but became significantly elevated 6 months after the infusion
associated with an intercurrent illness.
One or more severe symptoms were reported by 28% of
patients interviewed. Most of these transient symptoms can
be attributed to an acute phase response and this frequency
and severity would appear to justify prophylactic adminis-
tration of a NSAID to minimise the likelihood of the acute
phase reaction occurring. The data supports the renal safety
of ZA infusions in benign bone disease but in view of the
reports of renal deterioration in patients with cancer, who
are likely to have other risk factors, adequate hydration and
monitoring of renal function is appropriate.
Reference
[1] Chang JT, et al. NEMJ 2003; 394:1676–1679.
57
Quantification of Metaphyseal Remodelling in Children
Treated with Bisphosphonates
David G: Little; Kate Ward. Childrens Hospital Westmead.
Purpose: There has been recent concern in the literature that
the treatment with bisphosphonates in children can have an
adverse effect on metaphyseal remodelling leading to
‘‘drug-induced osteopetrosis’’. We created a normal data-
ABSTRACTS / Bone 34 (2004) S46–S99S80
base called metaphyseal index in the distal femur so that we
could quantify the inwasting remodelling process in chil-
dren on bisphosphonates.
Method: Radiographs of the distal femur of 185 normal
children who had presented to our institution for orthopae-
dic trauma were examined. Medical records were checked
and 30 children were excluded as they were found to have
underlying bone disorders or had had surgery. A measure-
ment of the distal femoral growth plate width (GPW) was
recorded. The femoral width at an interval of 0.5GPW
proximal to the distal femoral growth plate was also
recorded (0.5W). The metaphyseal index was defined as a
ratio of 0.5W/GPW. A graph of the means, one and two
standard deviations from the mean, was constructed using
the data obtained from this cohort.
Results: We found this ratio to be constant with minimal
variability regardless of the age or sex of the child (Fig. 1).
We then charted the metaphyseal index for the patient in the
study by Whyte et al in the New England Journal of
Medicine—a 12 year old boy with drug induced osteopet-
rosis. His metaphyseal index was calculated using the distal
femoral radiographs taken both before and after the admin-
istration of pamidronate. His pre-treatment index at age 7 3/
4 was at the upper limit of normal, but post treatment was
7.6 standard deviations above the mean (Fig. 1). This patient
was given a total of 2800 mg for a diagnosis of ‘‘bone pain’’
over 4 years. We also plotted a patient of ours, MM, a 10
year old girl with tibial pseudarthrosis who was treated with
bisphosphonates at our Hospital from August 1999 to
February 2003. Over this time she underwent 13 doses of
IV pamidronate, totalling 350mg. Her clinical outcome was
successful. Her metaphyseal index is unchanged from pre-
treatment (Fig. 1).
Conclusion: Metaphyseal remodelling in the distal femur is
constant through age and sex, resulting in a similar shape of
the distal femur throughout childhood. Patients given very
large doses of bisphosphonates outside the clinical range
display disordered metaphyseal remodelling. Clinically rel-
evant doses given for appropriate indications do not neces-
Fig. 1.
sarily disturb this process, while the beneficial clinical effect
is maintained.
Significance: Distal femoral metaphyseal index can be used
to plot the remodelling of individual children over time. In
this way, monitoring of remodelling can assist clinicians in
titrating relevant doses. Further studies are planned as well
as the creation of a database for the wrist.
58
Gastrointestinal Adverse Events CanReduce Compliance
With Oral Bisphosphonate Therapy and Result in Early
Study Termination
Pierre Major . McMaster University, Hamilton, Ontario,
Canada.
Oral bisphosphonate therapy is limited by poor bioavail-
ability and gastrointestinal (GI) adverse events (AEs).
Because <5% of the oral dose is absorbed, bisphospho-
nates must be administered at high doses to achieve
clinical activity, and ingestion of food or beverages
further reduces or blocks absorption. Moreover, the
dosing regimen requires patients to remain upright for
at least 30 minutes and to drink 6 to 8 ounces of water
to prevent epigastric pain. Oral clodronate therapy is
often further complicated by large tablet size. The effect
of the treatment regimen and AEs on compliance with
oral bisphosphonate therapy has been evaluated. In a
clinical trial of oral clodronate in metastatic breast cancer
(N = 173), 26% of patients evaluable for compliance
were noncompliant (Paterson et al. J Clin Oncol. 1993;
11:59–65). In another study of oral clodronate in
patients with metastatic bone pain (N = 55), despite
overall compliance of approximately 90%, 11% of
patients taking clodronate and 25% of patients taking
placebo withdrew from the study because they had
difficulty swallowing the capsules (Robertson et al. J
Clin Oncol. 1995;13: 2427–2430). Noncompliance with
bisphosphonate therapy can also be expected to have
negative effects on clinical outcomes. The IMPACT
Table 6
Crystallinity and collagen cross-links ratio
Time Cryst:
placebo
Cryst:
risedronate
Collagen:
placebo
Collagen:
risedronate
0-Yr 0.92 (0.06) 1.00 (0.11) 1.40 (0.20) 1.61 (0.40)
3-Yr 1.22 (0.04)* 0.93 (0.06) 1.90 (0.04)* 1.61 (0.86)
5-Yr 0.93 (0.04) 1.64 (0.52)
Mean (SD).
*P < 0.05.
ABSTRACTS / Bone 34 (2004) S46–S99 S81
study evaluated the effects of compliance using markers
of bone resorption as surrogate markers of biologic
activity (Eastell et al. 30th European Symposium on
Calcified Tissues. 2003.). At week 22, >50% reductions
of serum C-telopeptide were achieved in 60% of com-
pliant patients versus only 20% of noncompliant patients.
In the Spanish PROCUOS osteoporosis trial in postmen-
opausal women, the most common reason for discontin-
uation of alendronate was AEs (45.1%) (Turbi et al. 30th
European Symposium on Calcified Tissues. 2003). In a
similar study in women with postmenopausal osteoporo-
sis (N = 247), 7% of patients, all of whom reported GI
AEs, abandoned alendronate after 6 months (Bandeira et
al. 30th European Symposium on Calcified Tissues.
2003). These reports suggest that GI toxicity from oral
bisphosphonates can cause early study discontinuation.
Overall, 11% to 47% of patients report upper-GI AEs
after oral bisphosphonate therapy. In a study of long-
term oral clodronate (1,600 mg/day) as adjuvant therapy
for breast cancer (N = 1,079), the reason for study
termination was AEs for 58 (10.7%) patients in the
clodronate group versus 43 (7.9%) patients in the pla-
cebo group, and GI disorders were significantly more
common with clodronate (66% versus 56.2% for place-
bo; P < .05) (Atula et al. Drug Safety. 2003;26:661–
671). In particular, diarrhea was significantly more com-
mon among patients treated with oral clodronate (15.1%
versus 6.8% for placebo) and abdominal pain was also
more frequent in the clodronate group (7.2%) compared
with placebo (5%) during the medication period. How-
ever, the incidence of upper-GI AEs was similar for both
groups (22.3% for clodronate, 19.3% for placebo)
(Powles et al. J Clin Oncol. 2002;20:3219–3224). In
summary, the complicated treatment regimen and GI AEs
have been shown to reduce patient compliance and cause
early study withdrawal in patients undergoing oral
bisphosphonate therapy. P.
59
5-Years Treatment with Risedronate Preserves Bone
Mineral Crystallinity and Collagen Cross-Links Ratio
Elef therios Paschalis,* Roger Phippsy
*Ludwig Boltzman Institute, Vienna, AustriayProcter & Gamble Pharmaceuticals, Mason, OH, USA
Treatment of postmenopausal osteoporotic subjects with
risedronate reduces vertebral and nonvertebral fractures
while concomitantly preserving bone microarchitecture
and increasing bone mineral density (BMD). However, in
addition to BMD and architecture, parameters of bone
quality are important determinants of bone strength and
fracture risk. In this study we analyzed the effects of 5-yr
treatment with risedronate on two parameters of bone
quality, mineral crystallinity (crystallite size) and collagen
cross-links ratio (pyridinoline/dehydro-dihydroxylysinonor-
leucine or pyr/deH-DHLNL) via Fourier transform infrared
microscopic imaging (FTIRI). These two parameters are
indicators of the maturity (tissue age) of the bone composite
(pyr and deH-DHLNL are abundant in mature and young
collagen, respectively).
Paired iliac crest biopsies were obtained from postmeno-
pausal osteoporotic subjects at baseline and after 3-yr
treatment with placebo (n = 8) or risedronate (5 mg/day
po; n = 11). Biopsies were also obtained after 5-yr treatment
with risedronate from 8 of these 11 subjects. Biopsies were
embedded in methylmethacrylate, and the trabecular bone
region was analyzed by FTIRI in f4 um thick sections.
Spectroscopic images were obtained via a step-scanning
FTIR spectrometer with an MCT array detector placed at an
image focal plane of an IR microscope. Analysis of the
FTIR spectra was focused on the apatite composite peaks
corresponding to mineral crystallinity (mineral crystallite
size in the crystallographic c-axis) and maturity (extent of
carbonate substitution and calcium/phosphate stoichiome-
try), and on perturbations in the Amide I carbonyl peak
corresponding to pyr and deH-DHLNL cross links. Analysis
was limited to trabeculae devoid of resorbing surfaces. Three
images per section were acquired (each image 400 � 400
um2 area or >2000 pixels with a spatial resolution of 7 um).
Mineral crystallinity and collagen cross-link ratio were
compared before and after treatment.
Subjects treated for 3-yr with placebo had significant
increases in both mineral crystallinity and collagen cross-
link ratio, a pattern consistent with untreated osteoporosis
and representing maturation of the bone composite matrix.
In contrast, 3-yr and 5-yr treatment with risedronate pre-
served mineral crystallinity and collagen cross-link ratio of
trabecular bone.
Lack of increases in mineral crystallinity and collagen cross-
link ratio coupled with increased BMD and preservation of
microarchitecture suggests that risedronate suppresses oste-
oclastic activity relatively more than osteoblastic activity.
These results contrast to previously reported changes in
crystallinity and/or collagen cross-link ratio seen with other
antiresorptive therapies (Table 6).
60
Head-to-Head Comparison of Risedronate and
Alendronate Pharmacokinetics at Clinical Doses
Roger Phipps,* Robert Lindsay,z David Burgio,* Amy Sun,*
Darrell Russell,* Barbara Kuzmak,* Brad Keck,*
Claus Christianseny
*P&G Pharmaceuticals, USA
ABSTRACTS / Bone 34 (2004) S46–S99S82
yCenter for Clinical and Basic Research, Ballerup,
DenmarkzHelen Hayes Hospital, West Havershaw, NY, USA
Previous studies suggest there are differences in bisphos-
phonate pharmacokinetics. In clinical studies, urinary ex-
cretion during the first 24 hr post-dose was <50% of the
administered dose for zoledronic acid and alendronate, but
>60% for risedronate. However, these were separate
studies with different study designs and different dose
levels. The purpose of the current study was to compare
head-to-head the pharmacokinetics of risedronate and
alendronate following single intravenous doses (at clinical
dose levels).
This is a 52-week, open label, randomized study in post-
menopausal women with osteopenia or osteoporosis (age
range 43–73 yr). Subjects were administered single intra-
venous doses of 14C-risedronate sodium (230 ug; 2.9
uCi) or 14C-alendronic acid (450 ug; 3.2 uCi), each
approximately equivalent to an oral weekly dose, followed
by weekly oral doses of unlabeled Actonel (35 mg) or
Fosamax (70 mg), respectively. Concentrations in serum
and urine are being measured over the 52-week study
period. 14C-risedronate and 14C-alendronate concentra-
tions are measured in serum and urine by liquid scintil-
lation counting as long as the samples contain adequate
radioactivity, and thereafter by accelerator mass spectrom-
etry (AMS). Use of AMS allows measurement of these
compounds at concentrations of 10 pg/mL and lower. The
study in-life is on-going; presented here are urinary
excretion results from the first 27-days (the primary
end-point).
The cumulative urinary excretion of risedronate (66%) was
significantly higher than that of alendronate (55%) over the
first 27 days. This resulted in one-third more of drug
retained on bone for alendronate compared with risedronate
(34% risedronate; 45% alendronate).
The absolute amount of drug retained from the initial dose at
27 days was 78 ug risedronate sodium and 202 ug alen-
dronic acid. This difference (2.5� at 27 days) is estimated to
increase disproportionately with repeated dosing (to >2.8�at 6-mo). This suggests a smaller skeletal burden is associ-
ated with long-term risedronate use, and indicates the
potential for improved control of therapy for risedronate
compared to alendronate (Table 7).
Table 7
Percent of dose excreted in urine (median)
Time
interval
Risedronate
(N = 15)
Alendronate
(N = 17)
P value*
0–24 HR 49.0 44.5 0.0966
0–72 HR 54.6 48.2 0.0235
0–336 HR 63.5 52.6 0.0025
0–648 HR 66.2 55.1 0.0020
*Wilcoxon rank sum test.
Clinical (cancer)
61
Safety and Convenience of Bisphosphonate Infusions
James Berenson,* Allan Lipton,y Pierre Major,§ Lee Rosenz
*Cedars-Sinai Medical Center, Los Angeles, CA, USAyMilton S. Hershey Medical Center, Hershey, PA, USAzCancer Institute Medical Group, Los Angeles, CA, USA
The introduction of new-generation bisphosphonates has
expanded the use of bisphosphonates in oncology. First-
generation intravenous (IV) bisphosphonates were less po-
tent and required long infusion times to assure renal safety.
Among the newer IV bisphosphonates (ie, pamidronate,
ibandronate, and zoledronic acid) approved for the treatment
of hypercalcemia of malignancy or bone metastases in
patients with breast cancer, zoledronic acid has the shortest
infusion time (ie, 15 minutes compared with 1 to 2 hours
required for other agents). Moreover, zoledronic acid is the
only IV bisphosphonate to receive regulatory approval for
the treatment of bone metastases secondary to prostate
cancer, lung cancer, and a broad range of solid tumors.
Based on available data, the safety and convenience of these
new-generation bisphosphonates are described. The efficacy
and tolerability of pamidronate (90 mg via 2-hour infusion)
and zoledronic acid (4 mg via 15-minute infusion) every 3
to 4 weeks were compared directly in a 2-year, randomized,
phase III trial in patients with breast cancer and multiple
myeloma (Rosen et al. Cancer. 2003;98:1735–1744). The
efficacy of 4 mg zoledronic acid was at least comparable
with that of pamidronate in the overall patient population
and superior in patients with breast cancer. Adverse event
profiles were similar between treatment groups. The overall
incidence of National Cancer Institute Common Toxicity
Criteria (CTC) grade 3 (greater than or equal to 3.6 to 7.2
mg/dL) or grade 4 (>7.2 mg/dL) serum creatinine (using an
upper limit of normal of 1.2 mg/dL) was 0.4% for 4 mg
zoledronic acid and 1.9% for pamidronate. Among patients
with breast cancer, there were no CTC grade 3 or 4 serum
creatinine increases in the 4-mg zoledronic acid group; only
1 (0.5%) patient in the 90-mg pamidronate group developed
CTC grade 4 serum creatinine. Recently, Body et al (Ann
Oncol. 2003;14:1399–1405) reported findings from a trial
of ibandronate (6 mg via 1- to 2-hour infusion) every 3 to 4
weeks in patients with bone metastases from breast cancer.
The proportion of patients with serum creatinine >300
micromolar (ie, >3.4 mg/dL) was 2.6% for 6 mg ibandro-
nate versus 1.3% for placebo [NOTE: the criteria for
elevated serum creatinine in this study is presumed to be
>300 micromolar; however, the publication reported the
criteria to be >300 mM]. Zoledronic acid has a shorter
infusion time compared with pamidronate and ibandronate
and could, therefore, provide quality-of-life benefits to
patients. In a patient preference study reported by Joseph
et al (Blood. 2002;100:388b. Abstract 5114), patients were
treated with alternating monthly infusions of zoledronic acid
(4 mg over 15 minutes) and pamidronate (90 mg over 2
ABSTRACTS / Bone 34 (2004) S46–S99 S83
hours), and zoledronic acid was preferred by 86% of
patients. Therefore, zoledronic acid has a safety profile
comparable with other IV bisphosphonates and may be
preferred because of the shorter infusion time.
62
Relief from Metastatic Bone Pain with Standard and
Intensive Ibandronate Dosing
Jean-Jacques Body,* Debu Tripathy,y B Bergstromz
*Inst. Jules Bordet, Universite Libre de Bruxelles, Brussels,
BelgiumyUniversity of Texas Southwestern Medical Center, Dallas,
Texas, USAzHoffmann-La Roche Inc., Nutley, New Jersey, USA.
Background:Metastatic bone pain has severe consequences
for patient well-being, with impaired mobility, poor quality
of life, and greater dependence on others. Bisphosphonates
can provide incremental bone pain relief, and augment the
effect of palliative radiation. The effect on bone pain of
ibandronate, a new aminobisphosphonate for the prevention
of bone events in metastatic bone disease, has been evaluated
in phase III trials and independent studies.
Methods: In three 96-week, randomized, double-blind
trials, patients received intravenous (i.v.) ibandronate 6mg
(n = 154) or placebo (n = 158) infused over 1–2 hours
every 3–4 weeks, or oral ibandronate 50mg (n = 287) or
placebo (n = 277) daily. Bone pain was assessed on a 5-
point scale from 0 = none to 4 = intolerable. Alternatively,
in two independent studies using an intensive i.v. ‘loading
dose’ in patients with bone metastases, pain was assessed
on a visual analog scale (VAS) from 0 = no pain to 10 =
maximum pain. In a pilot study of 18 patients with opioid-
resistant pain from breast cancer and other tumor types, i.v.
ibandronate 4mg was infused over 2 hours for 4 consecu-
tive days (total loading dose = 16mg). Patients were
assessed for up to 6 weeks. The second trial was a pros-
pective study of 53 patients with painful osseous metastases
due to urologic cancer that investigated the effect on bone
pain of i.v. ibandronate 6mg infused over 1 hour for 3
consecutive days (total loading dose = 18mg). Treatment
was continued every 4 weeks for 20 weeks.
Results: In phase III trials, standard doses of ibandronate
reduced bone pain within 4 weeks (mean change from
baseline: i.v. ibandronate 6mg, �0.23; placebo, �0.06; oral
ibandronate 50mg, 0.15; placebo, �0.03), with significant
and sustained reductions over 2 years of treatment com-
pared with placebo (i.v. ibandronate 6mg, �0.28 versus
+0.21, p < 0.001; oral ibandronate 50mg, �0.10 versus
+0.20, p = 0.001). In patients with metastatic breast cancer
and other tumor types, loading-dose ibandronate signifi-
cantly reduced mean VAS pain score within 7 days (p <
0.001), an effect that was maintained throughout the 6-week
study period (p < 0.05). In the metastatic urologic cancer
study, 44/53 patients (83%) had pain relief from Day 2,
defined as both a 3-point reduction in VAS and a 50%
reduction in analgesic use. Twenty-five percent of the
patients were completely pain free. The mean VAS score
on Day 3 was significantly reduced from baseline (2.5 versus
6.8, p < 0.001), and remained below baseline throughout
the 20-week study period.
Conclusions: In clinical trials of patients with metastatic
breast cancer, ibandronate maintained bone pain levels
below baseline for over 2 years. This has not been reported
previously with other bisphosphonates. Ibandronate pro-
vided greater bone pain reductions than placebo within 4
weeks, with maximal results at 3 months. Intensive, loading-
dose i.v. ibandronate therapy effectively alleviated mode-
rate-to-severe pain (including opioid-resistant pain) from
osseous lesions. Maximal, statistically significant pain relief
occurred very rapidly, within 3–7 days. The benefits of in-
tensive ibandronate therapy for bone pain palliation should
be investigated in controlled clinical trials.
63
Data from an Italian, Multicenter Clinical Trial in 312
Breast Cancer Patients with Newly Diagnosed Bone
Metastases Confirms the Renal Safety of Zoledronic
Acid (ZometaRRRR)G: Carten; R. Bordonaro, G. Colucci, V. Lorusso, A.
Veronesi, M.C. Marre, Brunenghi, R. Rondena, V. Vinaccia
and D. Amadori on behalf of CZO446EIT01 working group
A.O.R.N. A. Cardarelli.
Key words: zoledronic acid, bone metastases, breast cancer,
bisphosphonate
ZometaR (zoledronic acid, Zol), a new, highly potent,
heterocyclic nitrogen-containing bisphosphonate, was re-
cently approved in Europe and the USA for the prevention
of skeletal related events in a wide variety of solid tumors
and multiple myeloma. Zol has demonstrated to significant-
ly reduce the risk of developing skeletal-related events
(SREs) compared with pamidronate in breast cancer patients
with bone metastases.
In 2001 we started a multicenter, open-label, prospective
clinical trial with the primary objective to describe the course
over 12months in SREs (radiation therapy to bone, surgery to
bone, pathologic bone fracture, spinal cord compression,
hypercalcemia of malignancy), in breast cancer patients with
newly diagnosed bone metastases treated with Zol 4 mg
every 3 or 4 weeks via 15-min infusion, in addition to anti-
neoplastic therapy. The secondary objectives included: bone
lesions and overall tumour response, bone pain and analgesic
use evaluation, quality of life, performance status and toler-
ability of Zol 4 mg. We report here the renal safety data.
We enrolled 312 patients over 60 centers with histologically
confirmed breast cancer and at least one bone metastasis
diagnosed no more than 6 weeks prior to the inclusion in the
study. Previous treatment with bisphosphonates was not
allowed.
The median number of Zol infusions within the study was
12 (mean + sd: 9.5 + 3.8); 59.3% of patients completed 12
ABSTRACTS / Bone 34 (2004) S46–S99S84
infusions and at investigator’s discretion had the possibility
to continue the therapy with Zol outside the protocol.
No NCIC-CTG grade 3–4 serum creatinine increases were
reported. In 15/312 patients (4.8%) a NCIC-CTG grade 1
serum creatinine increase was reported with a mean creat-
inine value of 1.5 mg/dl (range: 1.32–1.59). The median
number of Zol infusions within the study in this group of
patients was 12 (mean + sd: 10.5 + 2.3). In 1/312 patients
(0.3%) a NCIC-CTG grade 2 serum creatinine increase with
a creatinine level of 2.1 mg/dl was observed at the 11th
infusion of Zol.
According to our data, Zol is safe at renal level, when
administered according to the registered label.
64
Effect of Zoledronic Acid on Bone Loss Associated With
Androgen Blockade for Prostate Cancer
Richard Casey, MD,*,y William Love, MD,* Debra
Pearson, MSc,* Didier Reymond, MD,z Marc Zarenda,
MSc,§ and the CMX Research Investigator Group*
*CMX Research Inc.yThe Male Health CentreszNovartis Pharmaceuticals Canada Inc.§AstraZeneca Canada Inc.
Background: The primary treatment for locally extensive
or metastatic hormonal-dependant prostate cancer is andro-
gen suppression. Studies have demonstrated that goserelin
acetate (Zoladex) is a well tolerated, potent synthetic LHRH
analogue which, when administered in a 10.8 mg depot
formulation every three months, will reduce serum testos-
terone to levels similar to those found after orchiectomy. A
potential consequence of prolonged androgen deprivation
therapy with LHRH analogues is an increase in the risk for
bone loss. Zoledronic acid (Zometa) is a potent intravenous
bisphosphonate indicated for treatment of bone metastases
and it has been shown to increase bone mineral density
(BMD).
Study Objective: To determine if treatment with Zometa
can prevent bone loss in prostate cancer patients undergoing
androgen blockade with Zoladex.
Study Design: Open-label, controlled, multi-center study
conducted in 25 sites across Canada. Two hundred (200)
hormone naıve patients with locally advanced prostate
cancer (no bone metastases) will be randomized in a 1:1
ratio into two groups. The Control group will receive
Zoladex alone every three months and the Treatment group
will receive Zoladex plus Zometa every 3 months for one
year. Primary endpoint: percent change from baseline in
lumbar-spine BMD measurement at one year. Secondary
endpoints: percent change from baseline in lumbar-neck
BMD; change in height; development of bone metastases;
and safety.
Baseline Data: Ninety (90) men between the ages of 56 and
91 have been randomized into the study (data for 150
patients will be available at the time of poster presentation).
Of the BMD measures collected to date, preliminary base-
line measures show that the average lumbar spine (L2–L4)
is 1.281 g/cm2, the average femoral neck is 0.935 g/cm2
and the average total hip is 0.999g/cm2.
Interim Data: Six month BMD results will be available for
approximately 60 patients at the time of poster presentation.
Conclusion: This study will give physicians valuable
information for the treatment and prevention of cancer-
treatment induced bone loss (CTIBL) in patients with
prostate cancer undergoing androgen-blockade with LHRH
analogues.
65
Zoledronic Acid Has Broad Long-term Efficacy in
Reducing Skeletal Complications in Patients With Bone
Metastases From Breast Cancer, Prostate Cancer, and
Other Solid Tumors
Robert Coleman,* Lee Rosen,y Donald M. Gleason,z
Ming Zheng§
*Academic Unit of Oncology, Weston Park Hospital,
Sheffield, EnglandyCancer Institute Medical Group, Los Angeles, California,
USAzAdvanced Clinical Therapeutics, Tucson, Arizona, USA§Novartis Pharmaceuticals Corporation, East Hanover,
New Jersey USA.
Background: The efficacy and safety of 4 mg zoledronic
acid for the prevention of skeletal complications from bone
metastases in patients with breast cancer, prostate cancer, or
other solid tumors were demonstrated in 3 large, multicen-
ter, randomized, phase III trials. We report here the long-
term results of these 3 trials.
Patients and Methods: Patients received 4 mg zoledronic
acid via 15-minute infusion every 3 or 4 weeks for up to 2
years. Comparators were 90 mg pamidronate via 2-hour
infusion (patients with breast cancer and multiple myeloma)
or placebo (patients with prostate cancer or other solid
tumors). Data are presented from the stratified subset of
766 patients with breast cancer who were treated with 4 mg
zoledronic acid or pamidronate. All data from core and
extension phases are reported.
Results: After long-term treatment, zoledronic acid re-
duced the incidence of skeletal complications (primarily
pathologic fractures and the need for radiation or surgery
to bone) in patients with all types of solid tumors. A
preplanned multiple event analysis, which provides a
comprehensive assessment of skeletal morbidity, demon-
strated that 4 mg zoledronic acid significantly reduced
the risk of developing skeletal complications. Among
766 patients with breast cancer (454 completed core
and 279 entered extension), zoledronic acid reduced the
risk of skeletal complications by an additional 20% over
that achieved with pamidronate (hazard ratio [HR] =
0.799; P = .025). In this trial, 4 mg zoledronic acid
also reduced the risk of skeletal complications by 7%
ABSTRACTS / Bone 34 (2004) S46–S99 S85
(nonsignificant) compared with pamidronate in patients
with multiple myeloma. Among patients with advanced
prostate cancer (n = 422: 147 completed core and 133
entered extension) or other solid tumors (n = 507: 131
completed core and 69 entered extension), zoledronic
acid reduced the risk of skeletal complications compared
with placebo by 36% (HR = 0.64; P = .002) and 31%
(HR = 0.693; P = .003), respectively. Zoledronic acid
also significantly reduced the proportion of prostate
cancer patients with a skeletal complication (38% versus
49% for placebo; P = .028) and delayed time to first
event by >5 months (median, 488 versus 321 days for
placebo; P = .009). In poor-prognosis patients with lung
cancer or other solid tumors, zoledronic acid significantly
delayed skeletal complications by >2 months (median,
236 versus 155 days; P = .009). Zoledronic acid was
well tolerated, with a long-term safety profile similar to
that of pamidronate and other intravenous bisphospho-
nates across all tumor types.
Conclusions: These data indicate that zoledronic acid is
more effective than pamidronate for reducing the long-term
risk of skeletal complications in patients with breast cancer.
Zoledronic acid is the only bisphosphonate to show supe-
riority in a direct comparison with another active agent (ie,
pamidronate). Zoledronic acid also demonstrated favorable
long-term efficacy compared with placebo in patients with
bone metastases from prostate cancer or other solid tumors.
Consequently, zoledronic acid is the only bisphosphonate
approved for these indications.
Key Words: bone metastases; solid tumors; Zoledronic acid
66
Zoledronic Acid Is Being Investigated for the Prevention
of Bone Metastases in Patients With Early-Stage Breast
Cancer
Robert Coleman,* Julie Gralow,y Richard Bell,z Allan
Lipton,§ on behalf of the AZURE and SWOG (0307)/
Intergroup investigators
*Weston Park Hospital, Sheffield, England, UKyUniversity of Washington Medical Center, Seattle, Wash,
USAzAndrew Love Cancer Centre, Geelong, Victoria, Australia§Milton S. Hershey Medical Center, Hershey, Pa, USA
Based on evidence that daily oral clodronate may benefit
patients with early-stage breast cancer, studies are ongoing
to investigate the potential of intravenous (IV) zoledronic
acid to prevent metastasis to bone. Zoledronic acid is the
most potent bisphosphonate currently available and is
highly effective for the treatment of bone metastases in
patients with breast cancer. Preclinical studies with the
MDA-MB-231 breast cancer cell line have shown that
bisphosphonates have direct antitumor effects. Zoledronic
acid exhibited a 50% inhibitory concentration (IC50) of 15
micromoles, which was approximately 50-fold lower than
the IC50 of clodronate (700 micromoles). Zoledronic acid
also demonstrated synergistic antitumor effects when com-
bined with paclitaxel and doxorubicin. Moreover, in ani-
mal models of breast cancer metastasis to bone, zoledronic
acid significantly reduced skeletal tumor burden and pre-
vented the formation of new bone metastases at lower
concentrations than any other bisphosphonate tested. These
effects appear to result from inhibition of osteolysis and
osteoclastogenesis, thereby rendering the bone marrow a
less favorable site for tumor cell growth. These studies
further suggest that the achievable concentrations of zole-
dronic acid in bone tissue can inhibit tumor growth.
Therefore, studies are planned or ongoing to investigate
the clinical benefit of zoledronic acid and other bisphosph-
onates in the adjuvant setting in early-stage breast cancer.
The AZURE study is now recruiting patients and is
evaluating the effect of zoledronic acid on disease-free
survival in 3,400 patients with stage II/III node-positive
breast cancer receiving standard adjuvant chemotherapy
and/or hormonal therapy. Patients are being randomized to
placebo versus zoledronic acid (monthly for 6 months,
then every 3 months for 8 doses [approximately 2 years],
then every 6 months for 5 doses [approximately 2.5
years]). This study is exploring a suitable dose interval
and duration of IV zoledronic acid, which may be effective
with less frequent dosing, thus providing greater conve-
nience than daily oral bisphosphonate therapy. In addition,
the Southwest Oncology Group will soon commence a
large, randomized trial to compare the benefits of oral
clodronate (1,600 mg/day), oral risedronate (30 mg/day),
and IV zoledronic acid (4 mg every 4 weeks for 6 months
and every 3 months thereafter) for 3 years as an adjunct to
standard adjuvant therapy in approximately 6,000 women
with stage I, II, or IIIA breast cancer. These studies are
critical to better define the role of bisphosphonates, in-
cluding IV zoledronic acid, as an adjunct to standard
adjuvant therapy and to determine if bisphosphonates can
improve outcomes in patients with early-stage breast
cancer.
67
Pharmacokinetics and Pharmacodynamics of
Intravenous Pamidronate in Patients with Breast Cancer
and Bone Metastases
Serge Cremers,* Lia van Zuylen,y Hans Gelderblom,z
Carolien Seynaeve,y Jan den Hartigh,* Huub Pols,§
Pieter Vermeij,* C van der Rijt,y Socrates Papapoulosb
*Dept. Clin Pharmacy and Toxicology, Leiden University
Medical CenteryDept. Medical Oncology, Erasmus Medical CenterzDept. Clinical Oncology, Leiden University Medical Center§Dept. Internal Medicine, Erasmus Medical CenterbDept. Endocrinology and Metabolic Diseases, Leiden
University Medical Center
Introduction: Bisphosphonates (BPs) given i.v. every 3 to 4
weeks are effective in the management of metastatic bone
ABSTRACTS / Bone 34 (2004) S46–S99S86
disease from breast cancer but responses among patients
vary and it is not known whether current dose and dose
intervals are appropriate for an individual patient. An
influence of PK of BP on its antiresorptive action may
contribute to this variation. To test this hypothesis we
determined the skeletal retention of intravenous pamidro-
nate and its association to the rate of bone resorption in
patients with bone metastases from breast cancer.
Methods: In a cross-sectional study, 24 hr urinary excretion
of pamidronate and NTx were measured in 28 patients with
bone metastases from breast cancer at the beginning, after
3–6 months or after 1 year of treatment with iv pamidronate
90 mg every 3–4 weeks.
Results: Skeletal retention (dose–amount excreted into
urine) varied between 12–98% (mean 59%) of the admin-
istered dose but there were no differences in retention
between patients receiving pamidronate for the first time
or after one year of treatment. Retention of pamidronate was
related to the prevalent rate of bone resorption in previously
untreated patients whereas no such relationship was found
in previously treated patients. The rate of bone resorption
normalized in all patients, but mean NTx levels started to
increase again before the next dose. Retention of pamidro-
nate and the specific pattern of bone resorption were
described adequately by a physiology-based 3-compartment
PK/PD model, in which the rate of bone resorption depends
on the amount of BP attached to bone. This model enables
now the prediction of the rate of bone resorption induced by
different treatment regimens.
Conclusions: During 1 year of treatment with i.v. pamidr-
onate, skeletal retention of the BP does not change,
indicating no saturation of skeletal binding sites with
treatment. The variability in retention among patients can
be attributed to the number of potential binding sites. This
is related, however, to bone resorption only before start of
pamidronate treatment. The PK/PD model described can
help to optimize BP treatment of both a population and
individual patients with bone metastases from breast
cancer.
68
Effects of Skeletal Complications on Total Medical Care
Costs in Women with Bone Metastases of Breast Cancer
Thomas Delea,* McKiernan James,y Martin Liss,*
Jane Brandman,z Jennifer Sung,z Monika Raut,z
John Edelsberg,* Gerry Oster*
*Policy Analysis Inc. (PAI)yColumbia University College of Physicians and Surgeons,zNovartis Pharmaceuticals Corp
Background:Women with bone metastases of breast cancer
often experience skeletal-related complications including
pathological fracture, spinal cord compression, hypercalce-
mia, or pain requiring surgery, radiotherapy, or opioid
analgesics (collectively, skeletal-related events [SREs]).
Previous studies have estimated the costs of SREs by
tallying costs for services specifically attributable to these
events. This approach may underestimate costs if SREs have
effects on other services.
Methods: We used a large US health insurance claims
database (7/94–6/02) to quantify impact of SREs on total
medical care costs in women with bone metastases of breast
cancer. Subjects included in this analysis had >2 encounters
with a diagnosis of primary breast cancer and >2 encounters
with a diagnosis of metastases to bone. SREs were identi-
fied based on the occurrence on or after the date of first
diagnosis of bone metastases, of (1) >1 encounter with a
diagnosis of pathological fracture, spinal cord compression,
or hypercalcemia, or (2) >1 bone surgery or radiotherapy
procedure, or (3) initiation of opioid analgesic therapy.
Each subject with an SRE was matched to one without
an SRE based on a propensity score, which was estimated
using logistic regression with occurrence of an SRE as the
dependent variable and baseline clinical and demographic
characteristics as independent variables. For SRE patients,
the ‘‘index date’’ was defined as the date of first SRE. For
no-SRE patients, the index date was defined as the date of
first bone metastasis plus the number of days from first
bone metastases to the date of first SRE for the matched
SRE patient. Total medical care costs post index were
compared for SRE and no-SRE groups using Kaplan Meier
methods.
Results: We identified 617 breast cancer patients with bone
metastases, including 321 (52%) with >1 SRE. After match-
ing, there were 201 patients each in the SRE and no-SRE
groups. The two groups were well matched with respect to
baseline characteristics. Mean follow-up was 15 in SRE
patients and 12 months in no-SRE patients (p = .031).
Estimated median survival was 19 and 17 months in the
two groups respectively (p = .498). Estimated total costs per
patient were $52,099 greater in SRE patients vs no-SRE
patients ($119,798 vs $67,699). Costs of care directly
attributable to SREs were $14,580 per patient. Costs of
non-SRE-related care were $37,519 greater in the SRE
group vs no SRE group ($105,218 vs $67,699). Approxi-
mately 50% of SRE-related costs were due to radiotherapy.
Fifty six percent of SRE-related costs were incurred during
inpatient hospitalizations. Almost 50% of the difference
(SRE group vs no-SRE group) in non-SRE-related costs
were incurred during physicians’ office visits.
Conclusions: Costs of SREs in patients with breast cancer
and bone metastases are substantial and potentially greater
than previously estimated. Treatments which prevent SREs,
such as intravenous bisphosphonates, may reduce these
costs.
69
Circulating Level Modification of Angiogenic Factors,
Metalloproteinases, Proinflammatories Cytokines by
Zoledronic Acid in Advanced Breast Cancer
�������������Gianluigi Ferretti , Alessandra Fabi, Paola Cordiali Fei,
Paolo Carlini, Serena Di Cosimo, Nello Salesi,
ABSTRACTS / Bone 34 (2004) S46–S99 S87
Valentina Bordignon, Diana Giannarelli,
Paola Papaldo,*,y Andrea Alimonti,*,y Alain Gelibter,
Barbara Di Cocco, Mariangela Ciccarese,
Alessandra Felici, Chiara Nardoni,
Simona Gasparro, Francesco Cognetti,
Division of Medical Oncology ‘‘A’’, Regina Elena Cancer
Institute, Rome, Italy
Background: Zoledronic acid, a third generation bisphosph-
onate, may have an an antitumor activity through its anti-
angiogenic property and metalloproteinases inhibition. The
purpose of this study was to evaluate the modifications of
angiogenic cytokines and metalloproteinases after its first
single i.v. infusion.
Methods: Eighteen consecutive breast cancer patients with
bone metastases receiving endocrine therapy were monthly
given zoledronic acid at the same single center and were
prospectively evaluated for circulating levels of vascular
endothelial growth factor (VEGF), basic fibroblast growth
factor (bFGF), metalloproteinase 1 (MMP1), metalloprotei-
nase 2 (MMP2), interleukin-1beta, -6 and -8 (IL1a, IL6,
IL8), interferon gamma (IFNa), tumor necrosis factor alfa
(TNFa), and tumor growth factor beta (TGFa) just before
and at 2 and 7 days after zoledronic acid infusion.
Results: The MMP2 basal value showed a statistically
significant decrease 48 hours after the infusion of zole-
dronic acid (P = 0.008), being at 7 days after the
infusion higher than the value registered after 48 hours
(P = 0.03) and before infusion (P = 0.55). The VEGF
basal value showed a statistically significant decrease 48
hours after the single infusion of zoledronic acid (P =
0.03), being increased at 7 days after the infusion
compared with the basal value (P = 0.07). The bFGF
basal level almost significantly decreased 2 days after
zoledronate infusion (P = 0.06), being higher at 7 days
after infusion than the basal level (P = 0.09). The IL1a,
IL6, IL8 basal values did not show any statistically
significant modification at 2 and 7 days after zoledronic
acid infusion. The tumor growth factor beta (TGFa)
basal value showed a statistically significant decrease
48 hours after the single infusion of zoledronic acid (P
= 0.04). Comparing the 2-day values with basal ones,
the linear regression model showed a significant positive
correlation between IL8 and bFGF values (P = 0.02),
IL8 and TNFa (P < 0.0001), IL8 and IL1a (P = 0.02),
TNFa and bFGF (P = 0.01), TNFa and IL-1a (P =
0.04), TNFa a and MMP1 (P = 0.02), bFGF and IL1a
(P = 0.02). Comparing the 3-days values with initial
ones, the linear regression model showed a significant
positive correlation between IL8 and TNFa (P = 0.02),
MMP2 and TNFa (P = 0.01).
Conclusion: Our study shows that zoledronic acid may
exert a transient antiangiogenic activity and inhibition of
tumor cell bone invasiveness by a significant reduction of
VEGF, bFGF and MMP2 serum levels 48 hours after its
infusion.
70
Treatment of Aggressive Osteolytic Benign Bone Lesions
with Topical Antiresorptive Agents: Part II. Preliminary
Clinical Results
Francis Young-InLee; Sanjeev Suratwala, John Yu. Co-
lumbia University.
Introduction: Giant cell tumor (GCT), unicameral bone
cyst (UBC), aneurysmal bone cyst (ABC) of bone are
locally aggressive benign bone lesions that are characterized
by extensive bone destruction and a high recurrence rate.
These tumors or lesions consist of mesenchymal stromal
cells that can induce active osteoclastogenesis. Many non-
specific local adjuvant therapies such as phenol, hydrogen
peroxide or liquid nitrogen have been used but specific
adjuvant therapies to target osteoclastogenesis have not been
described. Bisphosphonates are ideal candidate agents that
can target osteoclastogenesis and neoplastic stromal cells, as
previously described in other types of tumors. Topical
application of bisphosphoante is a very logical therapeutic
approach for localized osteolytic bone diseases. The purpose
of our retrospective clinical study is to determine the safety
and therapeutic efficacy of topical bisphosphonates for
aggressive osteolytic lesions.
Materials and Methods: Ten benign osteolytic lesions were
treated with conventional surgical curettage in conjunction
with topical pamidronate or zolendronate. The lesions were 4
GCTs with extensive bone destruction, 4 UBCs in the femur,
1 aneurysmal bone cyst (ABC) in the proximal tibia, and 1
extensive fibrous dysplasia in the femur. Two of 4 UBCs
recurred three times at presentation. There were four patho-
logic fractures. After surgical curettage, the cavitary lesions
were soaked with 30 mg of pamidronate or 1 mg of zometa
for 1 minute and then packed with allograft bone chips that
were soaked with the same amount of bisphosphonate
solution. Metal implants were used as indicated to stabilize
the fracture. The cases were followed with serial radiographs
for one year. Recurrence of osteolytic lesions, bone graft
incorporation, resorption of bone grafts and complications
were assessed.
Results: Clinical data showed no recurrences of GCT or
UBC after the use of topical bisphosphonates one year
postoperatively. There was one case of ABC that showed
focal recurrence at the periphery of the lesion. Overall, bone
graft incorporation and fracture healing were not affected by
bisphosphonates and the integrity of the allografts appeared
to be better maintained. Seven patient developed postoper-
ative fever that resolved within one to three days after the
surgery. There were 2 patients with partial wound dehis-
cence of which significance is not certain in relation to the
use of bisphosphonates.
Discussion: To our knowledge, this is the first clinical
series on the safety and therapeutic efficacy on the topical
use of bisphosphonates for the locally aggressive osteolytic
disorders. Topical application is a logical therapeutic ap-
proach for the localized aggressive osteolytic disorders
ABSTRACTS / Bone 34 (2004) S46–S99S88
without affecting other uninvolved skeletons. Transient
fever was present in our series. Although anesthesia and
surgery can cause fever, fever has been described as one of
possibe side effects following the intravenous use of
bisphosphoantes for metastatic bone cancers. The prelimi-
nary results were very encouraging based on the preclinical
scientific data as well as clinical results. Patients with
recurrent, aggressive osteolytic lesions in the weight-bear-
ing bones would benefit from the specific pahramcologic
agents that can target an autocrine loop of osteoclasto-
genesis among neoplastic stromal cells and hematopoietic
osteoclast precursors.
71
Survival-Adjusted Cumulative Event Analysis of
Skeletal-Related Events in Patients With Cancer
Metastatic to Bone in Trials of Zoledronic Acid
�������������Pierre P: Major,* Richard J. Cook,y Bee-Lian Chen,z
Ming Zhengz
*McMaster University, Hamilton, Ontario, CanadayUniversity of Waterloo, Waterloo, Ontario, CanadazNovartis Pharmaceuticals, East Hanover, New Jersey
Background: Bone metastases are common in patients with
breast or prostate cancer and can cause significant pain and
skeletal morbidity. After diagnosis of bone metastasis, many
patients survive for months or years and are at risk for
developing multiple skeletal complications. Therefore, mul-
tiple event analyses are appropriate to determine the effect
of treatment on the cumulative incidence of skeletal mor-
bidity over the course of disease. Recently new methods
became available (Cook et al. Stat Med. 1997;16;911–924;
Ghosh et al. Biometrics. 2000;56:554–562) that provide a
tool to look at the data from a different perspective and
display the results graphically. These robust nonparametric
methods take into account patient survival and provide a
visual representation of cumulative burden of SREs over the
entire course of follow-up. Therefore, we used these new
methods to evaluate the effect of zoledronic acid (4 mg) on
the cumulative incidence of SREs in patients with breast,
prostate, or lung cancer and other solid tumors enrolled in 3
large, randomized clinical trials.
Patients and Methods: These trials were GCP compliant,
and patients gave informed written consent. Patients re-
ceived 4 mg zoledronic acid via 15-minute infusion every 3
or 4 weeks for up to 2 years. Comparators were 90 mg
pamidronate via 2-hour infusion (breast cancer) or placebo
(prostate cancer and other solid tumors). Skeletal-related
events were defined as pathologic fractures, spinal cord
compression, radiation or surgery to bone, and hypercalce-
mia. Change in antineoplastic therapy for bone pain was
considered an SRE in men with prostate cancer. A 21-day
window (bootstrap sampling method) was used to exclude
linked events. Results are expressed as survival-adjusted
cumulative incidence of SREs (Cook et al. Stat Med.
1997;16;911–924), and between-group differences were
analyzed by the method of Ghosh and Lin (Ghosh et al.
Biometrics. 2000;56:554–562).
Results: Our analyses show that in patients with breast
cancer (n = 766), 4 mg zoledronic acid was superior to 90
mg pamidronate and significantly reduced the cumulative
incidence of SREs (P = .046). In patients with prostate cancer
or with lung cancer and other solid tumors, 4 mg zoledronic
acid significantly reduced the cumulative incidence of SREs
compared with placebo (P = .004 and P = .010, respectively).
These results concur with those obtained in the protocol-
specified Andersen-Gill multiple event analysis (Andersen et
al. Ann Stat. 1982;10:1100–1120) which provides the rela-
tive risk of developing skeletal complications.
Conclusions: These new and robust methods provide
another way to look at the data and provide simple
graphical summaries of cumulative disease burden and
absolute reduction of skeletal complications achieved with
bisphosphonate therapy. When used in combination with
survival information, useful clinical insight can be gained
about the effects of investigational agents for the treatment
and prevention of skeletal complications arising from bone
metastases.
72
Effect of Bisphosphonate Treatment on Serum Levels of
Osteoprotegerin and Soluble RANK-Ligand in Patients
with Tumor-Associated Hypercalcemia
Gudrun Pohl,*,y Karin Brenner,*,y,z,§,b Gerhard Hawa,z,§
Martin Pecherstorfer*,y
*1.Department of Medicine and Medical OncologyyWilhelminenspital, Vienna, AustriazBiomedica Medizinprodukte§Vienna, Austria
Background: Recent research points to a crucial role of
RANK, RANK-ligand (RANKL) and osteoprotegerin
(OPG) system in the physiopathology of neoplastic bone
involvement: Tumor cells stimulate osteoclast-mediated
bone resorption by directly expressing RANKL or increas-
ing the expression of RANKL on osteoblasts or bone
marrow stromal cells.
Aim of the study: We evaluated whether treatment with
the nitrogen-containing bisphosphonate ibandronate
affects the serum concentration of OPG or soluble
RANKL.
Patients and methods: 16 hypercalcemic cancer patients
(10 females, 6 males; median age 68.5 years) received one
infusion of 6 mg ibandronate on day 0. Serum levels of
soluble RANKL, OPG, creatinine and calcium were deter-
mined before ibandronate treatment and daily until day 5.
Both soluble RANKL and OPG were measured by com-
mercially available enzyme immunoassays (Biomedica
Medizinprodukte GmbH & Co KG, Vienna, Austria). These
assays measure the total (free and bound) amount of OPG
ciruclating in blood and the concentration of free, soluble
RANKL.
ABSTRACTS / Bone 34 (2004) S46–S99 S89
Results: Ibandronate treatment led to a decrease in the
median serum calcium concentration (day 0: 3.06 mmol/l,
day 5: 2.41; p < 0.0001) whereas the serum concentrations
of OPG (day 0: 8.45 pmol/L, day 5: 8.59 pmol/L; p =
0.05224), of soluble RANKL (day 0: 0.2950 pmol/L, day 5:
0.2475 pmol/L; p = 0.2017) and of creatinine (day 0: 0.5
mg/dL, day 5: 0.3 mg/dL; p = 0.0776), as well as the soluble
RANKL/ OPG ratio (day 0: 0.028, day 5: 0.023; p = 0.1692)
did not change.Conclusion: Bisphosphonate treatment of
neoplastic bone disease does not appear to affect the RANK,
RANKL, OPG system.
73
Long-term Zoledronic Acid Therapy Is Effective and
Safe for Reducing the Risk of Skeletal Complications in
Patients With Non-Small Cell Lung Cancer (NSCLC)
and Bone Metastases
Lee Rosen,* David Gordon,y Simon Tchekmedyian,z
Vera Hirsh,§ Ronald Yanagihara,b Robert ColemanO
*Cancer Institute Medical Group, Santa Monica, Calif, USAyUS Oncology, San Antonio, Tex, USAzPacific Shores Medical Group, Long Beach, Calif, USA§McGill University, Montreal, Quebec, CanadabSt. Louise Hospital, Gilroy, Calif, USAOYorkshire Cancer Research, Weston Park Hospital,
Sheffield, England, UK
Background: Lung cancer is the most prevalent cancer
worldwide, and patients with advanced lung cancer frequent-
ly develop painful and debilitating bone metastases. Median
survival for patients with lung cancer after the development
of bone metastases is only 6 to 10 months; however, the
morbidity resulting from bone metastases can have a signif-
icant negative effect on health-related quality of life. We
have previously reported the efficacy and safety of 4 mg
zoledronic acid administered every 3 weeks for 9 months
(core phase) in preventing skeletal complications in patients
with lung cancer and other solid tumors (Rosen et al. J Clin
Oncol. 2003;21:3150–3157). Herein, we report the results
from the 21-month core + extension phase of this study in
patients with NSCLC.
Methods: A subgroup of 264 patients with NSCLC and at
least 1 bone metastasis (a stratified subgroup) was random-
ized to 4 mg zoledronic acid via 15-minute infusion (n =
134; 18 entered extension phase) or to placebo (n = 130; 17
entered extension phase). The primary efficacy endpoint
was the percentage of patients who experienced at least 1
skeletal-related event (SRE), defined as pathologic fracture,
spinal cord compression, radiation therapy to bone, or
surgery to bone. Secondary endpoints included median
time to first SRE, mean annual incidence of SREs, and
Andersen-Gill multiple event analysis. Hypercalcemia of
malignancy was included as an SRE in all secondary
analyses.
Results: Although this study was not designed to detect
statistically significant between-group differences in the
NSCLC stratum, Andersen-Gill multiple event analysis
demonstrated a significant 32% reduction in the risk of
developing SREs for NSCLC patients treated with zole-
dronic acid versus placebo (risk ratio = 0.675 [95%
confidence interval = 0.490, 0.929]; P = .016). This
pre-planned analysis accounts for all SREs and the timing
of SREs, thus providing a comprehensive assessment of
the risk of skeletal complications between treatment
groups throughout the course of the study. Conversely,
traditional conservative efficacy analyses (ie, percentage
of patients with at least 1 SRE and time to first SRE) did
not detect significant between-group differences in the
NSCLC stratum. The adverse-event profiles were similar
for patients treated with zoledronic acid or placebo.
Moreover, there were no significant differences between
zoledronic acid (4 mg via 15-minute infusion) and place-
bo groups in time to first serum creatinine increase (log-
rank P = .920), indicating that 4 mg zoledronic acid does
not significantly affect renal function when infused over
15 minutes.
Conclusions: These 21-month follow-up data demonstrate
that long-term therapy with 4 mg zoledronic acid is effective
for preventing skeletal complications in patients with bone
metastases from NSCLC. Finally, long-term zoledronic acid
therapy is safe and well tolerated in this patient population
and has demonstrated overall and renal safety profiles
similar to placebo.
74
The Combination of Autologous Stem Cell
Transplantation with Zoledronic Acid Normalises
Abnormal Bone Remodelling in Multiple Myeloma
Evangelos Terpos; Marianna Politou, Richard Szydlo,
Elizabeth Nadal, Sharon Avery, Eduardo Olavarria,
Edward Kanfer, John M Goldman, Jane F Apperley,
Amin Rahemtulla. Department of Haematology, Faculty of
Medicine Imperial College London, Hammersmith Hospital,
London, United Kingdom.
Bone disease in multiple myeloma (MM) is mainly due to
increased osteoclastic activity, which is not accompanied
by a comparable increase in bone formation. The osteo-
protegerin(OPG)/receptor activator of NF-kappa B ligand
(RANKL) system has a major role in osteoclastogenesis.
Our group has recently shown that the ratio of sRANKL/
OPG correlates with the extent of bone disease and
survival in MM. High dose chemotherapy (HDT) and
autologous stem cell transplantation (ASCT) is the treat-
ment of choice for eligible patients with MM, but their
effect on bone turnover in MM has not been completely
evaluated. Bisphosphonates are potent inhibitors of osteo-
clastic activity. Zoledronic acid, a third generation bisphos-
phonate, seems to have not only antiresorptive activity but
an antimyeloma effect as well. The aim of this study was
to evaluate the effect of ASCT in combination with zole-
dronic acid on bone remodelling in MM. We have studied
ABSTRACTS / Bone 34 (2004) S46–S99S90
51 patients with MM undergoing ASCT conditioned with
high dose melphalan. Markers of bone resorption [tart-
rate-resistant acid phosphatase isoform-5b (TRACP-5b)
and N-terminal cross-linking telopeptide of type-I colla-
gen (NTX)], of bone formation [bone alkaline phospha-
tase (bALP), osteocalcin (OC)], OPG and sRANKL were
measured pre- and every month post-ASCT. The median
age of patients (35M/16F) was 58 years and the median
follow-up was 12 months. All patients received zoledronic
acid, 4mg, IV, every month, both pre- and post-ASCT.
Thirty-nine patients (76%) had multiple lytic lesions or
fractures at the time of transplant. Eight patients were
transplanted in CR or very good PR, 38 were transplanted
in 1st or 2nd PR and 5 patients had progressive or resistant
disease. At baseline the majority of patients had increased
sRANKL/OPG ratio, TRACP-5b and NTX levels, while
markers of bone formation were strongly suppressed,
compared with controls. HDT and ASCT produced a
significant reduction of sRANKL/OPG ratio, with a con-
comitant decrease of NTX, and TRACP-5b levels, starting
the 2nd month post ASCT. The reduction of RANKL/OPG
strongly correlated with the reduction of TRACP-5b and
NTX. Bone formation markers, OC and bALP, started to
Fig. 1.
increase after the 9th and 11th month post ASCT, respec-
tively, while the increase of OPG preceded this. These
results are depicted in the table and provide biochemical
evidence that the combination of ASCT and zoledronic
acid normalises the abnormal bone resorption in MM
patients through the decrease of RANKL/OPG ratio, while
bone formation requires a longer period to return to normal
(Fig. 1).
75
The Evolving Role of Bisphosphonates for the Prevention
of Cancer Treatment-Induced Bone Loss in Patients
With Breast Cancer
Richard Theriault ,* Raimund Jakesz,y Michael Gnant,y
Julie Gralowz
*The University of Texas MD Anderson Cancer Center,
Houston, Tex, USAy2University of Vienna, Vienna, AustriazUniversity of Washington Medical Center, Seattle, Wash,
USA
Cancer treatment-induced bone loss (CTIBL) is an emerg-
ing concern in the oncology setting. Patients with breast
ABSTRACTS / Bone 34 (2004) S46–S99 S91
cancer often receive long-term adjuvant therapy with
aromatase inhibitors or ovarian-ablative chemotherapy,
resulting in a high incidence of CTIBL. Bone loss places
patients at increased risk for skeletal complications, which
may substantially reduce quality of life. Studies have
shown that bisphosphonates can preserve bone mineral
density (BMD) in patients receiving estrogen-ablative
therapies, indicating a role for bisphosphonates in main-
taining bone health in patients with breast cancer (Eastell.
Med Pediatr Oncol. 2003;41:222–227). Zoledronic acid,
the most potent bisphosphonate available, may preserve
or potentially increase BMD in patients receiving adju-
vant therapy for breast cancer. In the non-oncology
setting, zoledronic acid has been shown to significantly
preserve BMD in postmenopausal women compared with
placebo when administered as infrequently as once a year
(Reid et al. N Engl J Med. 2002;346:653–661). The
infrequent intravenous dosing schedule of zoledronic acid
may improve both convenience and compliance with
therapy.
Recent evidence from clinical trials of patients receiving
hormonal therapy for breast or prostate cancer suggests
that zoledronic acid is effective at limiting CTIBL. Haus-
maninger et al (8th International Conference: Primary
Therapy of Early Breast Cancer. 2003) reported the re-
sults of a randomized, placebo-controlled trial of zole-
dronic acid (4 mg every 6 months) in premenopausal
patients receiving standard hormonal therapy with either
tamoxifen or anastrozole (both in combination with goser-
elin). After 6 months of therapy, patients treated with
zoledronic acid had significantly higher lumbar-spine
BMD compared with the placebo group (P < .0001) for
both therapeutic regimens. Patients receiving anastrozole
appeared to have more significant BMD loss than those
receiving tamoxifen (P = .0125). Zoledronic acid (4 mg
every 3 months for 1 year) has also been shown to sig-
nificantly increase BMD compared with placebo in patients
with nonmetastatic prostate cancer receiving long-term an-
drogen-deprivation therapy (Smith et al. J Urol. 2003;169:
2008–2012).
Based on these promising results, we have initiated the Z-
FAST and ZO-FAST trials. In Z-FAST (United States and
Canada), postmenopausal women with hormone receptor-
positive breast cancer receiving initial adjuvant therapy
with the aromatase inhibitor letrozole (2.5 mg/day) for 5
years are being randomized to receive 4 mg zoledronic
acid every 6 months, either starting with the initiation of
letrozole or at the time of a significant decline in BMD
(lumbar-spine BMD T-score >2.0 standard deviations
below normal). Accrual to the Z-FAST trial in the United
States is complete. The ZO-FAST trial has a parallel
study design and will be conducted in approximately 30
countries outside the United States. ZO-FAST will also
include patients in whom menopause has recently been
induced by medical intervention. These studies will
investigate the safety and efficacy of intravenous zole-
dronic acid for the prevention of CTIBL during adjuvant
aromatase inhibitor therapy and may provide a clinical
strategy for maintaining bone health in patients with
breast cancer.
76
Assessing the Efficacy of Ibandronate for the Prevention
of Skeletal-Related Events (SREs) in Metastatic Bone
Disease: A Methodological Comparison
Debu Tripathy,* M. Buddey
*University of Texas Southwestern Medical Center, Dallas,
Texas, USAyF. Hoffmann-La Roche Ltd, Basel, Switzerland
Background: A significant proportion of patients with
advanced malignancy will develop bone metastases.
SREs such as pathological fractures, radiotherapy or
surgery to bone, spinal cord compression, and hypercal-
cemia, are a significant cause of morbidity. In clinical
trials of bisphosphonates in metastatic bone disease,
different statistical methods have been used to evaluate
the occurrence of SREs, making it difficult to compare
efficacy between agents. This abstract reports the results
of two alternative methods used to investigate the impact
of intravenous (i.v.) and oral ibandronate on SREs in
phase III trials of women with breast cancer and bone
metastases.
Methods: Three multicenter, randomized, double-blind,
placebo-controlled trials were conducted. In one study,
i.v. ibandronate 6mg (n = 154) was compared with
placebo (n = 158) infused over 1–2 hours every 3–4
weeks. In two trials of oral ibandronate (data pooling
prespecified), a 50mg daily dose (n = 287) was compared
with placebo (n = 277). The occurrence of SREs was
analysed prospectively using multivariate Poisson regres-
sion modelling of the number of new bone events. This
model determines the effect of treatment on SREs, while
accounting for differences in the baseline characteristics of
patients as covariate factors, and adjusting for time on
study. Model input variables included age, time on study,
time since diagnosis of breast cancer or bone metastases,
prior chemotherapy or hormone therapy, bone lesion type/
size, severity of bone pain and performance status. A post-
hoc analysis using the Andersen-Gill method (time to
multiple SREs) was also performed. This method factors
in the number and timing of SREs, but does not fully
account for patients’ time on study.1 Andersen-Gill was
used previously as a specified post-hoc analysis1 to assess
new bone events in a 2-year trial of i.v. zoledronic acid in
metastatic bone disease.2
Results: By multivariate Poisson regression analysis, i.v.
ibandronate 6mg significantly reduced the risk of SREs
compared with placebo (40% reduction, risk reduction [RR]
0.60, 95% CI = 0.43, 0.85; p = 0.0033). The effect of oral
ibandronate 50mg on the risk of SREs was similar (38%
reduction versus placebo, RR 0.62, 95% CI = 0.48, 0.79;
ABSTRACTS / Bone 34 (2004) S46–S99S92
p < 0.0001). Compared with placebo, the post-hoc Ander-
sen-Gill analysis showed a 29% reduction in SREs for i.v.
ibandronate 6mg (RR 0.71, p = 0.0183) and a 38% reduction
for oral ibandronate 50mg (RR 0.62, p V 0.0001).
Conclusions: In patients with metastatic breast cancer, i.v.
ibandronate 6mg and oral ibandronate 50mg led to similar,
statistically significant reductions in the occurrence of SREs.
The post-hoc Andersen-Gill analysis confirms the multivar-
iate Poisson regression results. As the Poisson regression
model is the only method to fully account for between-
group differences of time on study and possible deviations
from general assumptions, it may be the most robust model
for assessing the occurrence of SREs. Direct comparative
trials are warranted to compare the reductions in the risk of
new bone events with i.v. and oral ibandronate and other
bisphosphonates, such as i.v. zoledronic acid.
References
1. Major PP, et al. Am J Clin Oncol 2002;25(6 Suppl. 1):S10–18.
2. Rosen LS, et al. Cancer 2003;98;1735–44.
Clinical (fracture and osteoporosis)
77
Alendronate Reduces Fracture Risk in Lowest Bone
Turnover Group
������������Douglas Bauer,* Arthur Santora,y Marc Hochberg,z
Mary Melton,y Philip Rossy
*University of California, San Francisco, CAyMerck Research Laboratories, Rahway, NJzUniversity of Maryland, Baltimore, MD§
Alendronate reduces the rate of bone turnover to within the
premenopausal range, increases BMD, and reduces the risk
of both vertebral and nonvertebral fractures. We compared
the incidence of fractures among alendronate (ALN) treated
women with the lowest bone turnover to other women who
received alendronate in the Fracture Intervention Trial.
Women with femoral neck T-scores <�1.6 were randomized
to alendronate or placebo for 3 years (women with prior
vertebral fracture) or 4–4.5 years (no prior vertebral frac-
ture). We used a per-protocol analysis of the turnover
marker, bone-specific alkaline phosphatase (BSAP), at 12
months, because the full effect of ALN on turnover markers
occurs within a few months and is subsequently main-
tained. The number and % incidence (95% CI) of women
with new nonspine fractures after month 12 (and new
Table 8
Nonspine fracture Nonspine fracture
Group Lowest 10% Upper 90%
PBO N= 2711 N= 31 11.4% (8.1, 15.9) N = 273 11.2% (10.0
ALN N= 2769 N= 16 5.8% (3.5, 9.3) N = 230 9.2% (8.2, 1
vertebral fractures at any time) was calculated separately
for women in the lowest 10% of BSAP values in the
alendronate group (BSAP < 5.5 ng/mL) and in the placebo
group (BSAP < 7.9), and compared to all other women in
each treatment arm (Table). The sample size in the low
turnover group is small; hence, statistical tests were not
performed. The fracture incidence in the 10% of ALN-
women with the lowest BSAP was similar or lower than
that in other women. Thus, there is no evidence of an
increase in risk among women with the lowest BSAP
levels, suggesting that bone quality is not impaired during
ALN treatment, including women with the lowest bone
turnover Table 8.
78
Using in VIH-Infected with Bone Mass Loss of Calcitonin
and Alendronate
Antonio Bazarra-Fernandez,* Elena Casary
*University of La CorunayHospital de Monforte
Objective: Determining if the combined use of calcitonin
and alendronate influences on bone mass loss.
Material and method: We studied for 6 months 21 women
who were 44 to 64 years old at base line, were within 2 and
11 years of menopause, and had a bone mineral density at
the lumbar spine between 145 mg/cc and 50 mg/cc mea-
sured by the QBMAP system with a spiral CT Picker PQ-S
densitometer at L2, L3, L4 and L5. Of all the women, 10
were assigned to 10 mg of alendronate, 800 IU of vitamin
D3 and 1 g of calcium carbonate supplementation. 11 were
treated with 10 mg of alendronate, 200 UI of intranasal
calcitonin, 800 IU of vitamin D3 and 1 g of calcium
carbonate supplementation. The SPSS programme was used
for statiscal analysis.
Results: The characteristics of the women recruited for both
groups were similar. Mean mineral bone density at the
lumbar spine was between 1 and 3 DS below the mean
value for 30 years old normal premenopausal women. After
a treatment of 12 months no statistically significant differ-
ence was found among both groups as for the bone mineral
density at the lumbar spine.
Conclusions: It is necessary to carry out a wider and
longer study, among VIH-patient, but it seems that
alendronate contribute advantages to decrease bone mass
loss, at least, at lumbar spine, without calcitonin. This
results can be interesting for VIH-infected, who have a lot
of medication.
Vertebral fracture Vertebral fracture
Lowest 10% Upper 90%
, 12.5) N = 20 7.4% (4.8, 11.2) N = 173 7.1% (6.1, 8.2)
0.4) N = 10 3.6% (1.9, 6.7) N = 89 3.6% (2.9, 4.4)
ABSTRACTS / Bone 34 (2004) S46–S99 S93
79
Bone Loss in Long Term Cancer Survivors. A Pilot
Study to Evaluate the Effect of Annual Intravenous
Zoledronic Acid on Bone Mineral Density (BMD)
and Bone Turnover Markers
Janet Brown , James Lester, Susan Ellis, Sandra Gutcher,
Leslie Turner, Omprakash Purohit, Barry Hancock,
Robert Coleman. Academic Unit of Clinical Oncology,
Weston Park Hospital, Sheffield, UK.
Cure rates and survival times for certain malignancies have
increased markedly over recent decades and there are
increasing numbers of patients who will be long-term
survivors after receiving curative therapy. Such therapies
may accelerate the normal process of bone loss resulting in
an increased risk of osteoporosis. The bisphosphonates, as
potent inhibitors of bone resorption, have emerged as an
important class of agents in managing postmenopausal
osteoporosis, but there is a need for more studies addressing
the effectiveness of bisphosphonates in the management of
cancer treatment-induced bone loss. We are currently carry-
ing out a two year pilot study in long term cancer survivors
to determine the effect of a single annual infusion of
Zoledronic acid on BMD assessed at baseline and then 6
monthly by DEXA scan of the lumbar spine and hip. The
main objectives are to determine the effect of an annual
infusion of Zoledronic acid on bone mass in osteoporotic
patients and the magnitude and duration of action of a single
infusion in osteopenic patients.
Patients who are osteopenic on DEXA scanning at baseline
receive a single 4 mg infusion of Zoledronic acid with
follow-up for 2 years; those who are osteoporotic receive
two such annual infusions. The bone turnover markers
urinary N-telopeptide (Ntx) and serum bone specific alka-
line phosphatase (BAP) are measured at baseline, 6 weeks
and then 3-monthly.
71 patients have now been enrolled (46 breast cancer,13
lymphoma and 12 testicular cancer) and 34 patients have
now been on study for 12 months or longer. In this group the
mean percentage increases in BMD at the lumbar spine and
hip were 2.8 (range 12.9, �4.5) and 3.7 (13.7, �5.9)
respectively. For 17 patients with measurements out to 18
months, the corresponding values were 2.6 (range 11.5,
�2.9) and 5.5 (range 15.2, �1.4) respectively. For the
osteopenic subgroup (14 patients) mean BMD gains 18
months after Zoledronic acid infusion were 2.0% and
4.9% at the spine and hip respectively.
Analyses of Ntx data for the 34 patients who had been on
the study for at least 12 months showed a marked initial
suppression of the baseline level of 60% at 6 weeks. At one
year the mean Ntx increased but remained about 40% below
baseline. Interestingly, for the 12 osteopenic patients with
data at 18 months, the mean Ntx level remained suppressed
at 33% below baseline.
Extended results will be presented, but the initial data
suggest that an annual intravenous infusion of Zoledronic
acid may not only arrest the decline, but may also induce
an increase in BMD in these patients. Furthermore both
BMD and Ntx data suggest that a single infusion of
Zoledronic acid may have beneficial effects beyond one
year.
80
Efficacy of Alendronate in a Clinical Setting: An
Observational Study of 500 Unselected Patients
A: Joseph Foldes; Marina Weisman, Amir Bar-Shai.
Osteoporosis Center, Hadassah University Hospital,
Jerusalem, Israel.
Background: Alendronate sodium (ALN) is the most
widely used anti-osteoporosis medication. Several clinical
trials have established its anti-fracture efficacy, as well as
its favourable effect on bone mineral density (BMD). One
of the outstanding features emerging from clinical trials
with ALN is the high response rate and the persistent
magnitude of response in terms of BMD. Indeed, across
many controlled trials, mean BMD increments of about 5%
in the lumbar spine and 3% in the femoral neck (FN) were
observed after one year of ALN treatment. However, the
setting of controlled trials can be regarded as ‘‘in vivo
human laboratory’’, not necessarily reflecting real life:
Patients are pre-selected on the basis of adherence poten-
tial and lack of exclusion criteria (i.e. upper GI com-
plaints), are carefully monitored (including pill count) and
have easy access to the medical supporting team. There-
fore, we undertook to study the BMD response in a large,
unselected clinical sample of patients, who received ALN
treatment.
Methods: The study sample consisted of women who: (a)
had a BMD testing by DXA (either Hologic QDR 4500A or
Norland XR26) within 1 year prior to, or up to 3 months
following initiation of daily Fosamax 10 mg tablets; and (b)
were re-measured, using the same DXA machine, after a
mean treatment period of 405 days (1.1 years). The analysis
included 500 consecutive patients attending the Hadassah
Osteoporosis Center, who fulfilled these criteria. Their mean
age was 64 years and 93% were postmenopausal. Scans that
were either technically inadequate or included artifacts were
excluded.
Results: Mean T score at baseline (using manufacturers’
database) was �3.2 at both the spine and the FN. Ninety
three percent of the patients were osteoporotic (T score
<�2.5 in at least one site). At the follow-up visit, mean
BMD increased by 4.9% at the spine and by 3.1% at the
FN. Using a least significant change value for the lumbar
spine of >2.8% (based on a CV value of 1.0%), BMD
increased in 68% of the patients, remained unchanged in
29% and further deteriorated in only 3%. Similar BMD
changes were observed when the patients were stratified
on the basis of menopausal status, previous anti-osteopo-
rosis therapy, long-term glucocorticoid therapy or con-
comitant thyroxine therapy. There was a modest, but
ABSTRACTS / Bone 34 (2004) S46–S99S94
significant inverse correlation between the absolute
change in BMD and the baseline BMD at both skeletal
sites (r = �0.2).
Conclusions: Daily ALN treatment, in a large group of
unselected female patients with osteoporosis, yielded a
positive densitometric response, which was similar to that
observed in well-controlled clinical trials. The data of the
present investigation lend support to the consistent and
predictable favourable response to ALN therapy.
81
Screening for Osteoporosis in the General Population of
Postmenopausal Women
Maja Kozlevcar Zivec,* Rok Hren,y Marko Demsarz
*Physis, d.o.o., Ljubljana, SloveniayInstitute of Mathematics, Physics, and Mechanics,
University of Ljubljana, Ljubljana, SloveniazMedicus, d.o.o., Ljubljana, Slovenia
Early identification of postmenopausal women with oste-
oporosis by means of bone mineral density (BMD) mea-
surement is a prerequisite for reducing the incidence of
osteoporotic fractures. Objective of our study is to assess
simple decision rules that could enhance identifying
patients with high risk of fracture in the general population
of postmenopausal women. In the local women health
magazine, we invited to the DEXA measurement of
lumbar spine (L1–L4) and hip all women who were
postmenopausal for at least 5 years, had body mass index
(BMI) less than 26 kg/m2, and have never been diagnosed
with osteoporosis. Between February and May 2002, 201
women were enrolled in the study; 133 women (66%)
were identified as osteoporotic and 46 (23%) as osteo-
penic. Approximately 29% of all women (58 women)
suffered from previous low-trauma fracture; within this
fracture-group, 74% of patients were diagnosed with
osteoporosis and 24% had osteopenia. The fracture status
of 58 women was as follows: 53 of women suffered the
wrist fracture, 3 the hip fracture, and 6 vertebral fracture; 6
women had multiple fractures. None of the women en-
rolled in the study were treated for osteoporosis; about half
of the patients were given NSAIDs, while the other half
received no treatment. Results of our study suggest that
three simple decision rules provide efficient guidance for
BMD measurement referrals even among general popula-
tion of postmenopausal women that would otherwise rarely
see their GP. This is of importance since this study
corroborates and expands our earlier study in which these
decision rules proved to work well in the setting of
primary health care. In spite of the fact that women
enrolled in our study were at relatively high risk of fracture
(90% had either osteopenia or osteoporosis), they were left
undiagnosed and untreated. These rules should be thus
recognized as an effective tool for identifying patients with
osteoporosis in the general population that are still active
and unaware of their disease.
82
Early Use of Bisphosphonates in Congenital
Pseudarthrosis of the Tibia
David G: Little; Sukhdeep Dulai, Michael Bellemore,
Paul Williams, Julie Briody. Childrens Hospital Westmead.
Purpose: Congenital Pseudarthrosis of the Tibia (CPT) is an
uncommon but serious disorder. Half of the affected individ-
uals have Neurofibromatosis (NF-1). Previous studies have
shown that osteoclast number and osteoclast surface are
increased five-fold in CPT and there is increased expression
of RANKL. This and the osteopenia and fragility of the
disorder led us to the utilisation of bisphosphonates in CPT.
Method: This is an observational report of 9 children
treated with bisphosphonates for CPT. Each family gave
signed informed consent on an individual basis to a protocol
approved by the Hospital Pharmacy committee and Ethics
Committee approval to study the cohort was given. Most
patients received 2–3 doses of pamidronate 1 mg/kg in the
months around their operation for CPT. Radiographic out-
comes were used but in one unrodded case, longitudinal
DXA data was also available.
Results: Five of the nine patients had a diagnosis of
Neurofibromatosis. Patients ranged in age from 2 weeks
to 12 years at the time of presentation. Seven of the
patients had fibular involvement. Six of the patients were
treated with Pamidronate, two were treated with Zoledronic
acid and one was treated with both drugs at different times.
All the patients but one had standard surgical intervention
for their pseudarthrosis. In the remaining patient,
bisphosphonates were used to help build bone stock and
delay surgical intervention. In two of the patients, the
pseudarthrosis did not heal after the initial course of
bisphosphonates and surgical intervention. In these two
patients, a second surgical intervention was undertaken
including the implantation of OP-1 (BMP-7) at the site of
the pseudarthrosis. At the time of review, seven patients
were found to be clinically and radiologically united. One
patient developed a deep infection during the course of
treatment, resulting in an amputation. The final patient (who
did not have surgery) has shown increasing bone stock
around the site of the pseudarthrosis.
In the case followed longitudinally with DXA, significant
gains in BMC andBMDwere noted in the entire limb (Fig. 1).
Conclusion: Bisphosphonate therapy can increase bone
density and content in the limb affected by CPT. This and
autograft can lead to union in a majority of cases, while in
recalcitrant cases an additional anabolic therapy may be
required. We now use bisphosphonates routinely in this
group to maintain bone stock after fracture and to prolong
the anabolic response to bone grafting or distraction osteo-
genesis. Addition of OP-1 (BMP-7) has resulted in union in
two recalcitrant cases.
Significance: Bisphosphonates may significantly assist in
CPT by maintaining bone stock, improving internal fixation
and prolonging the anabolic response to bone grafting. Such
Fig. 1. Longitudinal DXA Aug 99 to Feb 03. This patient had significant gains in mineral content in the left lower limb with bisphosphonate therapy.
ABSTRACTS / Bone 34 (2004) S46–S99 S95
adjunctive therapy may improve the prospects of a func-
tional extremity for these children.
83
High Efficacy of Osteoporosis Treatment by the Use of
Fosamax and Vitamin D: A Case Report
Wojciech Pluskiewicz,* Bogna Drozdzowska,y
Dariusz Karasek*
*Silesian School of Medicine, Katowice, Poland,
Metabolic Bone Diseases UnitySilesian School of Medicine, Katowice, Poland, Dept. of
Pathology
A 57 year old postmenopausal woman was examined.
Menopause occurred at the age of 50 years, and one forearm
fracture was noted. No other risk factors for osteoporosis
were noted. She was evaluated towards osteoporosis using
Lunar DPX-L machine. Table presents the results of bone
measurements (Fig. 1).
Before the therapy a decrease in spine BMD by 7% was
observed.
The therapy consisted of Fosamax (daily 10 mg) and 1000
UI of cholecalciferol was initiated after second visit. No
adverse events were noted. The percentage increase in spine
BMD was 34%, for neck 12%, for Wards 9% and a drop in
trochanter by 6% was observed.
Fig. 1.
Concluding, the therapy have shown an excellent response
expressed by BMD measurements (except for trochanter).
84
Preserving Hip Bone Mineral Density Following Stroke
Using Intravenous Zoledronic Acid: Findings from a 1
Year Randomised Controlled Trial
Kenneth Poole,*,y Elizabeth Warburton,y,z
Nigel Loveridge,*,y Collette Rose,*,y Jonathan Reeve,*,y
*MRC Bone Research Group, Cambridge, UKyUniversity of Cambridge, Cambridge UKzDepartment of Stroke Medicine, Cambridge UK
Hip fractures are a common complication of stroke [1]. Stroke
patients are prone to falls, particularly onto the hemiplegic side.
Hemiplegia and subsequent immobility predisposes patients to
disturbed bone physiology, resulting in reduced bone mineral
density at the affected hip. These factors result in a
substantial increase in hip fracture rates in both sexes and
across all age ranges after stroke [1]. A single injection of
intravenous bisphosphonate given soon after stroke has been
proposed as a way of preventing bone loss and reducing hip
fractures associated with this condition [2].
In this 1-year randomised double-blind controlled trial, a single
dose of 4mg zoledronic acid or placebo was administered
intravenously to 16 patients (6F, 10M) within 35 days of an
Fig. 1.
ABSTRACTS / Bone 34 (2004) S46–S99S96
acute stroke. Bone mineral density was measured in the
hemiplegic and unaffected total hip region at baseline, 6 and
12 months using a Lunar Prodigy bone densitometer. All
patients received oral calcium (1g) and vitamin D (800iu)
supplementation.
After 1 year, patients in the placebo group had a signifi-
cantly greater reduction in bone mineral density in both hips
than those in the zoledronic acid group (hemiplegic side p =
0.0036, unaffected side p = 0.0089; repeated measures
ANOVA, BMD at 12 months versus baseline). In keeping
with previous studies, the mean percentage decrease in
BMD at the hemiplegic hip with placebo was 10.2% at 12
months, with a 6.0% decrease at the unaffected hip. In
contrast, there was no decrease in bone density at 12 months
in the zoledronic acid treated group (see Fig. 1).
Asymptomatic hypocalcaemia and hypophosphataemia oc-
curred more commonly following the zoledronic acid infu-
sion than following placebo, and the treatment was
generally well tolerated. These preliminary findings are
from the first 16 patients to complete the one year study.
We conclude that a single injection of zoledronic acid given
within 35 days may prevent the reduction in bone mineral
density seen after acute stroke.
References
[1] Kanis J, Oden A, Johnell O. Acute and long-term increase in fracture
risk after hospitalization for stroke. Stroke 2001;32(3):702–6.
[2] Poole, KE, Reeve J,Warburton EA. Falls, fractures, and osteoporosis
after stroke: time to think about protection? Stroke 2002;33(5):1432–6.
85
Intravenous Pamidronate Treatment in Children with
Osteogenesis Imperfecta Increases Cancellous Bone
Volume but Does not Alter Mineralization Density of
Bone Matrix
P: Roschger ,* F. Rauch,y M. Weber,z N. Fratzl-Zelman,*
F.H. Glorieux,y P. Fratzl,§ K. Klaushofer*
*Ludwig Boltzmann-Institute of Osteology, 4th Med. Dept.,
Hanusch Hospital & Traumatology Center Meidling,
Vienna, Austria
yGenetics Unit, Shriners Hospital for Children, and McGill
University, Montreal, Quebec, CanadazErich Schmid Institute of Materials Science, Austrian
Academy of Sciences and Institute of Metal Physics,
University of Leoben, Leoben, Austria§Max Planck Institute of Colloids and Interfaces, Dept.
Biomaterials, Potsdam, Germany
Osteogenesis Imperfecta (OI) is characterized by increased
bone fragility and low bone mass. Recent studies (1,2) have
shown that cyclical intravenous pamidronate increases bone
mass and improves the clinical course in children and
adolescents with OI. Iliac bone histomorphometry indicates
a marked gain in cortical width and cancellous bone volume
within the first few years of treatment (2). However, it is
unknown whether pamidronate has an effect on bone
material quality. This is an important question, because OI
bone is characterized by extreme brittleness on the material
level, in association with hypermineralized matrix and
disturbed collagen structure.
In this study we used quantitative backscattered electron
imaging (3, 4) to measure the effect of pamidronate on the
bone mineralization density distribution (BMDD) of OI
bone. Paired iliac bone biopsies from sixteen patients (age
4 to 18 years) with OI types I, III and IV were studied before
and after treatment with 4-monthly cycles of intravenous
pamidronate (yearly cumulative dose 9 mg/kg). The treat-
ment period ranged from 1.2 to 3.4 years. Results in the 10
to 15 year old patients (n = 5) were also compared to those
of a healthy control group (n = 6). From the BMDD we
derived the mean calcium concentration (CaMean), the most
frequent calcium concentration (CaPeak) and the width of
the calcium concentration distribution (CaWidth), which
describes the variety of mineralization density found in
the bone matrix.
At baseline, CaMean and CaPeak were significantly higher
in OI bone compared to healthy controls (+9.5% and +8.9%,
respectively, p = 0.0002 in both cases). Ca width was similar
in OI and control bone. These results were in agreement
with a previous study (5). Pamidronate treatment did not
ABSTRACTS / Bone 34 (2004) S46–S99 S97
show significant effects on any BMDD parameter in OI
patients (p > 0.05, paired t-tests). This observation is in
sharp contrast to the effect of bisphosphonates in postmen-
opausal osteoporosis, where the antiresorptive treatment
moderately increases the CaMean and CaPeak, but mark-
edly reduces CaWidth (4).
In conclusion, these data demonstrate that the antiresorptive
treatment of pamidronate does not further increase the degree
of mineralization in the already hypermineralized bone matrix
of OI. Thus the increase in bone mass seen during treatment is
exclusively due to an increase in the volume of mineralized
bone and not to changes in the material bone density.
References
1) Rauch, et al. Bone Miner Res 2003;18:610–14.
2) Rauch, et al. J Clin Invest 2002;110:1293–9.
3) Roschger, et al. Bone 1998;23:319–26.
4) Roschger, et al. Bone 2001;29:185–91.
5) Boyde, et al. Calcif Tissue Int 1999;64:185–90.
86
Safety and Pharmacokinetic Analysis of Zoledronic Acid
in a Multicentre Randomised Trial of Post-Transplant
Thalidomide Maintenance Therapy for Multiple
Myeloma
�������������Andrew Spencer,* Andrew Roberts,y Michael Bailey,z
Horst Schran,§ Kevin Lynchb
*Myeloma Research Group, The Alfred Hospital,
Melbourne, AustraliayBMT Service, Royal Melbourne Hospital, Parkeville,
AustraliazDepartment of Epidemiology and Preventive Medicine,
Monash University, Melbourne, Australia§Novartis Pharmaceuticals, New Jersey, USAbNovartis Pharmaceuticals, Sydney, Australia
Since May 2002 the Australasian Leukaemia and Lympho-
ma Group (ALLG) has been conducting a randomised trial
of thalidomide maintenance therapy in patients with Multi-
ple Myeloma undergoing an initial autologous stem cell
transplant (ASCT). Patients had received prior bisphospho-
nate therapy, had baseline serum creatinine (Cr) values
within 2� upper limit of normal and were conditioned with
melphalan 200mg/m2. Those showing no disease progres-
sion at 6 weeks post-ASCT were randomised to zoledronic
acid (ZA) 4mg IV by 15 minute infusion Q4 weekly and
alternate day prednisolone 50mg, with (ARM 1) or without
(ARM 2) thalidomide (200mg daily for a maximum of 12
months). Cr was measured prior to each ZA infusion and the
ZA was withheld based on previously used criteria. Addi-
tionally, ZA Cmax and AUC (0–48) were determined for
the day 1 and day 29 ZA infusions in a subgroup of patients.
Univariate analysis was conducted using student t-tests and
chi-square tests for equal proportion. Multivariate analysis
was conducted using a repeat measures analysis of variance.
Differences in Cr between arms over time were determined
by fitting an interaction between dose and arm, with dose
treated as a linear predictor. To-date 57 patients (ARM 1 =
26; ARM 2 = 31) have been randomised to maintenance
therapy and 21 of 24 patients have enrolled in the PK study
with analysis completed in 10 patients (ARM 1 = 6; ARM 2 =
4). The arms were well matched for gender, age, pre-ASCT
B2microglobulin and baseline Cr.Median number of doses of
ZA administered are 7.5 and 6, for ARM1 and ARM 2,
respectively. Two patients, both in ARM 2 (no thalidomide),
have had ZAwithheld following Cr rises of 50micromol/L to
120micromol/L and 80micromol/L to 130micromol/L after 5
and 11 doses of ZA, respectively. Multivariate analysis
demonstrated that higher Cr levels were associated with
male gender (p < .001) and pre-ASCT B2microglobulin
>4mg/L (p = .002). Furthermore, the relationship between
Cr and dose was significantly different between the two
arms with the thalidomide arm increasing at a reduced
rate per ZA dose (0.1micromol/L vs 1.0 micromol/L, p =
.01). The pharmacokinetic profile of Zometa in the 2
groups was similar and predictable from prior knowledge
of the drug, with peak levels falling rapidly such that
very low levels were detectable after 24 and 48 hours.
Peak drug concentrations and overall drug exposure were
similar between the two arms in the study, both at day 1
and day 29 (p = NS, t-test), and there was no suggestion
of accumulation or changed pharmacokinetic profile after
the second Zometa dose. We conclude that there is no
evidence for a pharmacokinetic interaction between ZA
and thalidomide. Furthermore, based on serial measure-
ments of renal function ZA was found to be safe in this
previously treated population irrespective of concomitant
thalidomide administration.
87
Feeding Tube Administration of Bisphosphonates for
Treating Osteoporosis in Institutionalized Patients with
Developmental Disabilities
S: Bobo Tanner,* H.M. Taylory
*Vanderbilt University, Nashville, Tennessee, USAyCloverbottom Developmental Disabilities Center,
Nashville, Tennessee, USA
Purpose and Background: Treating institutionalized
patients with osteoporosis can pose special challenges
including route of administration of medications as well as
monitoring the benefits of the treatment. The purpose of this
analysis was to review the safety and efficacy of the use of
enteral bisphosphonates in developmentally disabled osteo-
porosis patients who receive their nutrition and medications
primarily through feeding tubes. Absorption of oral
bisphosphonates occurs in both the stomach and the upper
small bowel. The bioavailability of oral bisphosphonates is
low and decreases when the drug is given with meals as well
as certain liquids other than plain water. In addition oral
bisphosphonates have been associated with erosive esoph-
ABSTRACTS / Bone 34 (2004) S46–S99S98
agitis. Many of these patients are unable to participate in a
spine and hip DXA scans and therefore peripheral devices
such as heel ultrasound devices are used to generate T
scores. Monitoring the response to treatment is therefore
also difficult although bone turnover markers such as urine
N-telopeptide values can be collected. We reviewed the
incidence of side effects and efficacy of bisphosphonates
delivered through feeding tubes in this special population of
institutionalized patients with developmental disabilities.
Methods: We did a retrospective chart review of the 9
patients (5 male, 4 female) at a state run developmental
disabilities center who are receiving risedronate or alendr-
onate via a percutaneous feeding tube (G tube) for the
treatment of osteoporosis. All patients had received a
quantitative ultrasound of the calcaneous to quantify bone
density as a T-score. Six of the patients had a history of a
previous fracture. 4 of the patients had baseline urine N-
telopeptide levels done prior to therapy and again at
intervals after initiation of therapy.
Results: The average age of these patient was 44 (30–54)
and average T-score was �3.2 (�2.3 to �4.5). Five patients
were on alendronate 70mg/week and 4 were on risedronate
35mg/week for an average of 18 months (11–25). There
were no fractures during the time of treatment. One patient
died because of abdominal adhesions and intestinal obstruc-
tion felt to be unrelated to the bisphosphonate therapy. One
patient experienced an increase in alkaline phosphotase after
8 months of therapy to 241 U/L (22% Intestinal, 28%
hepatic, 50% bone) that remained elevated after the
bisphosphonate was discontinued for 4 months and ulti-
mately therapy was re-started. Of the 4 patients with a
baseline and follow-up urine N-telopeptide level there was
an average drop of 38% (0–71%) after an average of 15
months (8–19mos.) of bisphosphonate therapy.
Conclusion: Bisphosphonate therapy via feeding tube in
developmentally disabled patients appears to be a generally
well tolerated means of delivering the medication. Further-
more bone turnover markers declined after treatment indi-
cating a therapeutic effect. Larger studies for longer time
frames are necessary to confirm this data.
88
Three Years AlendronAtE Therapy of Pediatric Patients
With Osteogenesis Imperfecta
Vaclav Vyskocil. Bone Disease Center, Charles University
Hospital Pilsen Czech Republic.
Osteogenesis imperfecta (OI) is a rare crippling disorder
leading to fractures and bone deformities. Previously
employed therapy with calcitonin, calcium and vitamin
D resulted in minimal increase in bone mineral density
(BMD) and didn’t reduce the incidence of fractures. As
there have been reports on promising effect of pamidr-
onate treatment in children with OI, we intitiated therapy
with another bisphosphonate - alendronate - in a group
of children with OI.
Parental and patients’ consent was obtained prior to this
treatment. We observed 30 children with OI, mean age 13.7
years. Alendronate was administered orally for one year:
children aged 4–10 years received 5 mg/day, children above
10 years of age were given 10 mg/day. Prior to the onset of
therapy, spinal BMD Z-score (measured by DXA) was
below �2.1 in all children and all patients had more than
2 fractures/year. After one year of therapy, we observed
significant increase in spinal BMD (+14.5%, p < 0.05).
After another 2 years of therapy, there was yet further
increase in spinal BMD (+6.3%). The changes in BMD
were related to the mobility status of the patients, as
immobilized patients had a less apparent increase in
BMD. The interpretation of BMD measurements in hip
and forearm was not possible due to multiple bone defor-
mities. The values of bone markers (S-osteocalcin, S-pro-
collagen III, U-deoxypyridinoline crosslinks) decreased
significantly (p < 0.05) after one year of therapy. In the
following 2 years there were no further significant changes
in these parameters with the exception of U-deoxypyridino-
line. In the course of the first year of alendronate therapy we
observed just one fracture in the entire group, this as a result
of skiing accident in a patient with OI type Sillence I. We
didn’ t observe any adverse effect of alendronate therapy
regarding gastrointestinal tract or biochemical changes. The
X-rays of long bones didn’t reveal any negative effect of
alendronate on metaphysis or growth cartilage. In conclu-
sion, alendronate seems to be a safe and effective drug in
pediatric patients with OI.
89
Early Fracture Reduction with Risedronate
N :B: Watts,y C. Roux,* S. Horlait z
*Hopital Cochin, Paris, FranceyUniversity of Cincinnati, Cincinnati, OH, USAzProcter & Gamble Pharmaceuticals, Neuilly-sur-Seine,
France
Postmenopausal osteoporotic women with pre-existing or
new incident vertebral fractures are at high risk for
future fracture, so prompt treatment is warranted. Risedr-
onate has been shown to reduce the incidence of
radiographically-defined vertebral fractures by approxi-
mately two-thirds within one year. We have examined
the effects of risedronate treatment on the time course of
the reduction in the risk of clinical vertebral fractures
(i.e., symptomatic fractures), on the risk of moderate-to-
severe radiographic vertebral fractures, and on height. In
2442 postmenopausal women with prevalent vertebral
fractures from the Vertebral Efficacy with Risedronate
Therapy (VERT) studies who received either risedronate
5 mg or placebo, daily risedronate reduced the risk of
clinical vertebral fractures within 6 months (RR = 0.08,
95% CI 0.01–0.63, p < 0.01), and by 69% at one year
(RR 0.31, 95% CI 0.12, 0.78, p = 0.009). The clinical
vertebral fracture incidence for 3, 6, 9 and 12 months
Fig. 1. Effect of risedronate on clinical vertebral fractures.
ABSTRACTS / Bone 34 (2004) S46–S99 S99
were 0.3, 1.0, 1.6 and 1.7% for placebo vs 0.08, 0.1, 0.3
and 0.6% for risedronate-treated patients respectively
(Fig. 1). Clinical vertebral fractures represented 20% of
all radiographically-defined vertebral fractures. At one
year, risedronate also reduced the risk of moderate-to-
severe radiographically-defined vertebral fractures by
71% (RR 0.29 95% CI 16, 54). Height loss was
attenuated with treatment, most notably in patients who
experienced new vertebral fractures, with a median
difference of 0.73 cm compared with subjects receiving
placebo (p = 0.005).
In conclusion, risedronate reduces the risk of clinical verte-
bral fractures in postmenopausal women with osteoporosis
within six months of commencing treatment, demonstrating
rapid fracture efficacy.
90
Treatment Discontinuation Effects on Bone Turnover
and BMD with Risedronate
Nelson Watts,* W.P. Olszynski,y C.D. McKeever,z
Andreas Grauer,§ Arkadi Chines§
*University of Cincinnati, OH, USA
yMidtown Medical Center, Saskatoon, CanadazMcKeever Orthopedic Clinic, Houston, TX§Procter & Gamble Pharmaceuticals, Mason, OH
Treatment with risedronate is safe and effective through 7
years. It is unknown how long the effects of treatment
would persist after therapy is stopped. It might not be
desirable to have prolonged suppression of bone turnover
after discontinuation of bisphosphonate (BP) treatment.
Patients received risedronate 5 mg daily (N = 398) or
placebo (N = 361) for 3 years during the VERT-NA study
and continued to receive calcium 1000 mg daily and
vitamin D for another year while risedronate was discon-
tinued. Patients were assessed at the end of the 4th year. In
the absence of risedronate treatment, urinary NTX/creati-
nine increased significantly, from a median of 30.3 nMol/
uMol at the end of 3 years of treatment (p < 0.05 vs
placebo) to 50.9 nMol/uMol after 1 year off treatment (n.s.
vs placebo). Bone alkaline phosphatase returned to pre-
treatment levels after stopping treatment and was no differ-
ent fromplacebo. Lumbar spine BMD decreased by 0.9% in
the year off treatment but remained 4.4% higher than
baseline (p V 0.001) and higher than placebo (p <
0.001). Similar results were seen at the femoral neck and
trochanter.
In conclusion, the effects of 3 years of risedronate
treatment on bone turnover begin resolving within 12
months after stopping treatment. BMD decreases, but
remains higher than in placebo patients. Risedronate
appears to differ from some other nitrogen-containing
BPs both in early onset of effect (as shown previously)
and in reversibility of effect (shown here) possibly due
to differences in binding affinity to bone or in skeletal
retention, or both. This may become clinically important,
for example, if other therapeutic options are being
considered.