Abstracts from the Workshop: What is New in Bisphosphonates?

www.elsevier.com/locate/bone

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


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


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


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-


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).


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


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.


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


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


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


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


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.


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


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.


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.


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


‘‘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.


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.


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


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.


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.


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


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.


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.


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


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


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


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.


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.


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.


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


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-


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.


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


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


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


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%


(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


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,


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


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.


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


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


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;


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)


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


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.


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.


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


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-


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.


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.

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Abstracts from the Workshop: What is New in Bisphosphonates?

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