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ABSTRACTS / Bone 42 (2008) S17–S110 S105

atypical apoptosis. The present study investigated the potential after a tooth extraction. All were men, with middle age of
Zol-resistance developed by osteosarcoma cells after prolonged 55 years old. After melanoma was diagnosed, treatment with
treatment. After 4 to 8 weeks of culture in the presence of 1 μM zoledronic acid and prednisone was initiated. After approxi-
Zol, the effects of high dose Zol (10 to 100 μM) were compared mately 3 months, they developed a periodontics of lower mo-
between the untreated rat (OSRGA, ROS) and human (MG63, lars, which were removed. The extraction socket never healed,
Saos2) osteosarcoma cells and Zol-pretreated cells in terms of and the patients underwent biopsy of the exposed bone, which
cell proliferation, cell cycle analysis, migration assay and was osteomyelitis. They were treated with antibiotics, without
cytoskeleton organization. Long-term treatment with 1 μM Zol resolution of the exposed bone. During that interval, the teeth
reduced the sensitivity of osteosarcoma cells to high concentra- adjacent to the nonhealing socket also became loose and were
tions of Zol as measured by XTT assay, cell cycle analysis and removed. 4 months later, the patients complained of jaw pain
cell migration assay. The molecular mechanisms involved in and numbness of inferior lip — Vincent symptom. On physical
this reduced-Zol sensitivity were then analyzed. XTT assays examination, patients had large areas of exposed and seemingly
demonstrated that the Zol-resistant cells were always sensitive nonvital bone in the mandible. There were purulent exudate
to conventional anti-cancer agents such as methotrexate, within the open wounds and numerous extraoral fistulas. All
mafosfamide and doxorubicin and that the resistance process inferior teeth became mobile. There were no lesions present in
was not associated with the multidrug resistance (MDR) pheno- the maxilla, and all of the maxillary teeth were stable. Pano-
type. However, similar experiments performed in the presence ramic radiographs showed areas of patchy radiolucency within
of clodronate and pamidronate revealed that this drug resistance the mandible, widened periodontal ligament spaces around the
was restricted to the nitrogen containing bisphosphonates. This mandibular dentition, and periosteal bone deposition along the
resistance was also correlated with a higher transcript level and inferior border of the mandible. Local wound care was initiated,
enzymatic activity of farnesyl diphosphate synthase (FPPS), the and patients had a good response with near resolution of the
molecular target of Zol, in resistant cell lines. To demonstrate infection and decrease in pain. Reparative granulation tissue in
the involvement of FPPS in the Zol-resistance mechanism, the intervening sites along the open wounds was noted during a 3-
Zol-resistant cells were transfected with a siRNA for FPPS. month period. Patients stopped bisphosphonate therapy. Defects
Inhibition of FPPS activity was then assessed indirectly, by of mandible were augmented with autogenously bone from
Western blot analysis of the unprenylated form of the small tibia. The rehabilitation period was favorable to place implants
GTPase Rap1A. The transfection of Zol-resistant cells with for dental rehabilitation.
FPPS siRNA strongly increased their sensitivity to Zol. This
study demonstrates the induction of metabolic resistance after doi:10.1016/j.bone.2007.12.203
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 194
pathologies but reveals the possibility that the treatment Preclinical renal safety profile of ibandronate
regimen may be important in terms of duration and dose to T. Pfister a, E. Atzpodien a, F. Bauss b
a
avoid the development of drug metabolic resistance. Preclinical Research and Development, F. Hoffmann-La
Roche Ltd, Basel, Switzerland
b
doi:10.1016/j.bone.2007.12.202 Roche Diagnostics GmbH, Pharma Research Penzberg,
Penzberg, Germany

193 Bisphosphonates as a class share the kidney as primary target


Mandibular osteonecrosis induced by bisphosphonates— of systemic toxicity. However, unlike other intravenous (i.v.)
Clinical case reports bisphosphonates no severe renal adverse events occurred
Victor P. Palarie neither in clinical studies with ibandronate nor in patients post
Oral Medicine and Surgery, Medical University, Chisinau, marketing. Thus, the dose levels of concern for renal safety in
Republic of Moldova humans are unknown. Here we present preclinical data from
single dose studies, repeated intermittent dose studies, and renal
Malignant myeloma is a neoplasm characterized clinically tissue kinetics using 14C-labelled ibandronate characterizing the
by osteolytic bone lesions, monoclonal immunoglobulin renal effects of ibandronate and provide evidence for its
detectable in the serum and/or urine, renal insufficiency, and favourable renal safety profile. Based on single dose experi-
amyloidosis. Bone involvements are manifested in the most ments, the minimally nephrotoxic dose varied between 1 and
patients at the time of diagnosis as lytic lesions, osteoporosis, 3 mg/kg in rats and dogs corresponding to about 8 to 25 times
and fractures. Oncologists usually use prophylactic bispho- the highest clinical dose of 6 mg. Typical findings were
sphonate therapy in these cases. Recent reports have shown an degenerative changes with or without single cell necroses in the
increasing association between the use of bisphosphonates and proximal convoluted tubules (PCT). Despite a general dose
osteonecrosis of the mandible. We treated 4 patients with dependency the findings were restricted to the PCT and up to a
malignant melanoma, who received chronic bisphosphonate dose of 20 mg/kg the severity was of a degree that remained
therapy and developed osteomyelitis of the mandible in 1 week 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 inhibition was determined after incubation with free or loaded
proteins). When administered repeatedly the risk of cumulative DXR. All the cell lines were able to selectively incorporate both
renal damage is expected to be related to the residual tissue DXR loaded NPs and free DXR. Selective nuclear uptake of the
concentration i.e. the amount of bisphosphonate remaining in drug, typical of sensitive cells, was observed after 30’ of expo-
the kidney from the previous dose. The terminal tissue half-life sure. The cell lines were sensitive to free DXR, as confirmed by
of ibandronate was determined as 24 days which is about growth inhibition after exposition to the free DXR. NPs-loaded
consistent with the treatment interval of 3–4 weeks in the DXR was effective at the equivalent concentration as free DXR.
oncology indication. Modeling of tissue concentrations predicts In conclusion, NPs loaded with DXR were able to inhibit cell
that there should be no relevant accumulation in the kidney proliferation in vitro at the same conditions as free DXR.
when ibandronate is given every 3 weeks. This was experi-
mentally confirmed in a controlled 25-week study of repeated doi:10.1016/j.bone.2007.12.205
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 196
a high safety margin based on single doses but also a very low In vivo effects of zoledronic acid on peripheral γ/δ
risk of cumulative renal toxicity when given intermittently due T lymphocytes in early breast cancer patients
to a favourable short renal tissue half-life. Daniele Santini, Federico Martini, Maria Elisabetta Fratto, Sara
Dr. Thomas Pfister; Salary; Employed at F. Hoffmann-La Galluzzo, Bruno Vincenzi, Chiara Agrati, Federica Turchi,
Roche Ltd, pre-clinical research and development. Paola Piacentini, Laura Rocci, Giuseppe Tonini, Fabrizio Poccia
Medical Oncology, University Campus Bio-Medico, Rome, Italy
doi:10.1016/j.bone.2007.12.204 Infectious Diseases, National Institute for Lazzaro Spallanzani,
Rome, Italy

195 Introduction: Amino-bisphosphonates are potent activators


Antineoplastic activity of bone-targeted nanoparticles of human γ/δ T cells both in vitro and in vivo. We conducted an
loaded with doxorubicin observational perspective study aimed to evaluate immunomo-
Manuela Salerno a, Sofia Avnet a, Caterina Fotia a, Elisabetta dulating properties of a single-dose of zoledronic acid on γ/δ T
Cenni a, Donatella Granchi a, Francesco Castelli c, Dorotea cells in a selected patient subset.
Micieli c, Rosario Pignatello c, Armando Giunti a,b, Material and methods: 23 disease-free breast cancer
Nicola Baldini a,b patients with diagnosis of osteopenia during inhibitor aromatase
a
Pathophysiology Lab, Istituti Ortopedici Rizzoli, Bologna, Italy treatment received a single-dose (4 mg) of zoledronic acid.
b
Department of Orthopaedic Surgery, Istituti Ortopedici Rizzoli, Blood samples were obtained before ZA infusion, and 7, 28, 56,
Bologna, Italy 90 and 180 days after. Kinetics of different γ/δ T cells subsets
c
Department of Pharmaceutical Sciences, University of Catania, were determined by flow cytometry, as well as γ/δ T cells in
Catania, Italy vitro response to phosphoantigens.
Results: A significant decrease of Naïve Vδ2 T lymphocytes
Bone is the third most common site of incidence of metastasis after 180 days (p b 0,01) and a significant progressive decrease
from different primary tumors, and bone cancer is associated of Central Memory subset after 28 (p b 0,05), 90 (p b 0,01) and
with a high mortality rate. Current therapeutic strategies are 180 days (p b 0,01) were observed. ZA induced also a significant
unfortunately not effective on the progression of the tumor, and reduction of Effector Memory Vδ2 T lymphocytes after 56
cause a range of systemic side effects. Biocompatible nanocar- (p b 0,01) and 90 days (p b 0,05). Moreover, we observed that
riers are being investigated as a new therapeutic tool that is able patients could be divided in two groups, according to γ/δ T
to transport anticancer drugs to specific targets meanwhile lymphocyte kinetics: “responder patients” showing a significant
reducing the systemic toxicity associated with free drug decrease of γ/δ T lymphocytes total number, and “non-
distribution through the body. The aim of this study was to test responder patients” without any significant modulation of γ/δ
the effectiveness of nanoparticles (NPs) engineered to feature a T lymphocytes. By comparing the two different populations
high affinity to bone and loaded with doxorubicin (DXR). NPs observed during the in vivo study, no significant differences in
were obtained by binding alendronate to PLGA, characterized term of IFN-gamma response by Vdelta2 T cells were recorded
for their chemical–physical properties and biocompatibility, and after in vitro phosphoantigen stimulation.
then loaded with DXR. The in vitro study was performed on six Conclusions: We describe here for the first time a long-
human cell lines, considered as representative of the spectrum of lasting activation of effector subsets of γ/δ T lymphocytes in
bone tumors, including osteosarcoma (Saos-2, U2 OS), renal healthy patients after a single dose of ZA. Our results highlight
adenocarcinoma (ACHN), breast adenocarcinoma (MDA-MB- the need to further investigate the clinical significance of the
231, MCF7), and neuroblastoma (SH-SY5Y). To analyze the immunomodulating properties of aminobisphosphonates.
uptake of the NPs, the cells were observed by confocal
microscopy after incubation with the free or loaded DXR. To doi:10.1016/j.bone.2007.12.206
analyze the antineoplastic effects, the percentage of growth