ABSTRACTS / Bone 42 (2008) S17–S110
atypical apoptosis. The present study investigated the potential
Zol-resistance developed by osteosarcoma cells after prolonged
treatment. After 4 to 8 weeks of culture in the presence of 1 μM
Zol, the effects of high dose Zol (10 to 100 μM) were compared
between the untreated rat (OSRGA, ROS) and human (MG63,
Saos2) osteosarcoma cells and Zol-pretreated cells in terms of
cell proliferation, cell cycle analysis, migration assay and
cytoskeleton organization. Long-term treatment with 1 μM Zol
reduced the sensitivity of osteosarcoma cells to high concentra-
tions of Zol as measured by XTT assay, cell cycle analysis and
cell migration assay. The molecular mechanisms involved in
this reduced-Zol sensitivity were then analyzed. XTT assays
demonstrated that the Zol-resistant cells were always sensitive
to conventional anti-cancer agents such as methotrexate,
mafosfamide and doxorubicin and that the resistance process
was not associated with the multidrug resistance (MDR) pheno-
type. However, similar experiments performed in the presence
of clodronate and pamidronate revealed that this drug resistance
was restricted to the nitrogen containing bisphosphonates. This
resistance was also correlated with a higher transcript level and
enzymatic activity of farnesyl diphosphate synthase (FPPS), the
molecular target of Zol, in resistant cell lines. To demonstrate
the involvement of FPPS in the Zol-resistance mechanism, the
Zol-resistant cells were transfected with a siRNA for FPPS.
Inhibition of FPPS activity was then assessed indirectly, by
Western blot analysis of the unprenylated form of the small
GTPase Rap1A. The transfection of Zol-resistant cells with
FPPS siRNA strongly increased their sensitivity to Zol. This
study demonstrates the induction of metabolic resistance after
prolonged Zol treatment of osteosarcoma cells, probably by
selecting clones overexpressing FPPS. This study confirms the
therapeutic potential of Zol for the treatment of bone malignant
pathologies but reveals the possibility that the treatment
regimen may be important in terms of duration and dose to
avoid the development of drug metabolic resistance.
doi:10.1016/j.bone.2007.12.202
193
Mandibular osteonecrosis induced by bisphosphonates—
Clinical case reports
Victor P. Palarie
Oral Medicine and Surgery, Medical University, Chisinau,
Republic of Moldova
Malignant myeloma is a neoplasm characterized clinically
by osteolytic bone lesions, monoclonal immunoglobulin
detectable in the serum and/or urine, renal insufficiency, and
amyloidosis. Bone involvements are manifested in the most
patients at the time of diagnosis as lytic lesions, osteoporosis,
and fractures. Oncologists usually use prophylactic bispho-
sphonate therapy in these cases. Recent reports have shown an
increasing association between the use of bisphosphonates and
osteonecrosis of the mandible. We treated 4 patients with
malignant melanoma, who received chronic bisphosphonate
therapy and developed osteomyelitis of the mandible in 1 week
S105
after a tooth extraction. All were men, with middle age of
55 years old. After melanoma was diagnosed, treatment with
zoledronic acid and prednisone was initiated. After approxi-
mately 3 months, they developed a periodontics of lower mo-
lars, which were removed. The extraction socket never healed,
and the patients underwent biopsy of the exposed bone, which
was osteomyelitis. They were treated with antibiotics, without
resolution of the exposed bone. During that interval, the teeth
adjacent to the nonhealing socket also became loose and were
removed. 4 months later, the patients complained of jaw pain
and numbness of inferior lip — Vincent symptom. On physical
examination, patients had large areas of exposed and seemingly
nonvital bone in the mandible. There were purulent exudate
within the open wounds and numerous extraoral fistulas. All
inferior teeth became mobile. There were no lesions present in
the maxilla, and all of the maxillary teeth were stable. Pano-
ramic radiographs showed areas of patchy radiolucency within
the mandible, widened periodontal ligament spaces around the
mandibular dentition, and periosteal bone deposition along the
inferior border of the mandible. Local wound care was initiated,
and patients had a good response with near resolution of the
infection and decrease in pain. Reparative granulation tissue in
intervening sites along the open wounds was noted during a 3-
month period. Patients stopped bisphosphonate therapy. Defects
of mandible were augmented with autogenously bone from
tibia. The rehabilitation period was favorable to place implants
for dental rehabilitation.
doi:10.1016/j.bone.2007.12.203
194
Preclinical renal safety profile of ibandronate
T. Pfister a, E. Atzpodien a, F. Bauss b
a
Preclinical Research and Development, F. Hoffmann-La
Roche Ltd, Basel, Switzerland
b
Roche Diagnostics GmbH, Pharma Research Penzberg,
Penzberg, Germany
Bisphosphonates as a class share the kidney as primary target
of systemic toxicity. However, unlike other intravenous (i.v.)
bisphosphonates no severe renal adverse events occurred
neither in clinical studies with ibandronate nor in patients post
marketing. Thus, the dose levels of concern for renal safety in
humans are unknown. Here we present preclinical data from
single dose studies, repeated intermittent dose studies, and renal
tissue kinetics using 14C-labelled ibandronate characterizing the
renal effects of ibandronate and provide evidence for its
favourable renal safety profile. Based on single dose experi-
ments, the minimally nephrotoxic dose varied between 1 and
3 mg/kg in rats and dogs corresponding to about 8 to 25 times
the highest clinical dose of 6 mg. Typical findings were
degenerative changes with or without single cell necroses in the
proximal convoluted tubules (PCT). Despite a general dose
dependency the findings were restricted to the PCT and up to a
dose of 20 mg/kg the severity was of a degree that remained
undetectable by standard renal monitoring measures (e.g. serum
S106 ABSTRACTS / Bone 42 (2008) S17–S110
creatinine, serum urea, or urinary excretion of enzymes or
proteins). When administered repeatedly the risk of cumulative
renal damage is expected to be related to the residual tissue
concentration i.e. the amount of bisphosphonate remaining in
the kidney from the previous dose. The terminal tissue half-life
of ibandronate was determined as 24 days which is about
consistent with the treatment interval of 3–4 weeks in the
oncology indication. Modeling of tissue concentrations predicts
that there should be no relevant accumulation in the kidney
when ibandronate is given every 3 weeks. This was experi-
mentally confirmed in a controlled 25-week study of repeated
dosing every 3 weeks: Sub-clinical renal damage after a single
dose of 1 mg/kg ibandronate did not accumulated to clinically-
relevant nephrotoxicity. In conclusion, ibandronate has not only
a high safety margin based on single doses but also a very low
risk of cumulative renal toxicity when given intermittently due
to a favourable short renal tissue half-life.
Dr. Thomas Pfister; Salary; Employed at F. Hoffmann-La
Roche Ltd, pre-clinical research and development.
doi:10.1016/j.bone.2007.12.204
195
Antineoplastic activity of bone-targeted nanoparticles
loaded with doxorubicin
Manuela Salerno a, Sofia Avnet a, Caterina Fotia a, Elisabetta
Cenni a, Donatella Granchi a, Francesco Castelli c, Dorotea
Micieli c, Rosario Pignatello c, Armando Giunti a,b,
Nicola Baldini a,b
a
Pathophysiology Lab, Istituti Ortopedici Rizzoli, Bologna, Italy
b
Department of Orthopaedic Surgery, Istituti Ortopedici Rizzoli,
Bologna, Italy
c
Department of Pharmaceutical Sciences, University of Catania,
Catania, Italy
Bone is the third most common site of incidence of metastasis
from different primary tumors, and bone cancer is associated
with a high mortality rate. Current therapeutic strategies are
unfortunately not effective on the progression of the tumor, and
cause a range of systemic side effects. Biocompatible nanocar-
riers are being investigated as a new therapeutic tool that is able
to transport anticancer drugs to specific targets meanwhile
reducing the systemic toxicity associated with free drug
distribution through the body. The aim of this study was to test
the effectiveness of nanoparticles (NPs) engineered to feature a
high affinity to bone and loaded with doxorubicin (DXR). NPs
were obtained by binding alendronate to PLGA, characterized
for their chemical–physical properties and biocompatibility, and
then loaded with DXR. The in vitro study was performed on six
human cell lines, considered as representative of the spectrum of
bone tumors, including osteosarcoma (Saos-2, U2 OS), renal
adenocarcinoma (ACHN), breast adenocarcinoma (MDA-MB-
231, MCF7), and neuroblastoma (SH-SY5Y). To analyze the
uptake of the NPs, the cells were observed by confocal
microscopy after incubation with the free or loaded DXR. To
analyze the antineoplastic effects, the percentage of growth
inhibition was determined after incubation with free or loaded
DXR. All the cell lines were able to selectively incorporate both
DXR loaded NPs and free DXR. Selective nuclear uptake of the
drug, typical of sensitive cells, was observed after 30’ of expo-
sure. The cell lines were sensitive to free DXR, as confirmed by
growth inhibition after exposition to the free DXR. NPs-loaded
DXR was effective at the equivalent concentration as free DXR.
In conclusion, NPs loaded with DXR were able to inhibit cell
proliferation in vitro at the same conditions as free DXR.
doi:10.1016/j.bone.2007.12.205
196
In vivo effects of zoledronic acid on peripheral γ/δ
T lymphocytes in early breast cancer patients
Daniele Santini, Federico Martini, Maria Elisabetta Fratto, Sara
Galluzzo, Bruno Vincenzi, Chiara Agrati, Federica Turchi,
Paola Piacentini, Laura Rocci, Giuseppe Tonini, Fabrizio Poccia
Medical Oncology, University Campus Bio-Medico, Rome, Italy
Infectious Diseases, National Institute for Lazzaro Spallanzani,
Rome, Italy
Introduction: Amino-bisphosphonates are potent activators
of human γ/δ T cells both in vitro and in vivo. We conducted an
observational perspective study aimed to evaluate immunomo-
dulating properties of a single-dose of zoledronic acid on γ/δ T
cells in a selected patient subset.
Material and methods: 23 disease-free breast cancer
patients with diagnosis of osteopenia during inhibitor aromatase
treatment received a single-dose (4 mg) of zoledronic acid.
Blood samples were obtained before ZA infusion, and 7, 28, 56,
90 and 180 days after. Kinetics of different γ/δ T cells subsets
were determined by flow cytometry, as well as γ/δ T cells in
vitro response to phosphoantigens.
Results: A significant decrease of Naïve Vδ2 T lymphocytes
after 180 days (p b 0,01) and a significant progressive decrease
of Central Memory subset after 28 (p b 0,05), 90 (p b 0,01) and
180 days (p b 0,01) were observed. ZA induced also a significant
reduction of Effector Memory Vδ2 T lymphocytes after 56
(p b 0,01) and 90 days (p b 0,05). Moreover, we observed that
patients could be divided in two groups, according to γ/δ T
lymphocyte kinetics: “responder patients” showing a significant
decrease of γ/δ T lymphocytes total number, and “non-
responder patients” without any significant modulation of γ/δ
T lymphocytes. By comparing the two different populations
observed during the in vivo study, no significant differences in
term of IFN-gamma response by Vdelta2 T cells were recorded
after in vitro phosphoantigen stimulation.
Conclusions: We describe here for the first time a long-
lasting activation of effector subsets of γ/δ T lymphocytes in
healthy patients after a single dose of ZA. Our results highlight
the need to further investigate the clinical significance of the
immunomodulating properties of aminobisphosphonates.
doi:10.1016/j.bone.2007.12.206