Elution of Tumoricidal Doses of Bortezomib from a Resorbable Cement Carrier

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1 Elution of Tumoricidal Doses of Bortezomib from a Resorbable Cement Carrier Matthew Allen, Vet MB, PhD, Brittani Jones, BS. The Ohio State University, Columbus, OH, USA. Disclosures: M. Allen: 5; Johnson and Johnson. B. Jones: None. Introduction: Systemic chemotherapy, local radiation, and surgical management are commonly used in the treatment of primary and secondary (metastatic) tumors of the skeleton. Unfortunately, these treatments are often ineffective in completely eliminating tumors that are large, highly vascularized or that invade local soft tissues or neurovascular structures. Systemic delivery of chemotherapy also runs a risk of undesirable systemic toxicity that may be dose limiting in some patients. We have been exploring an alternative, potentially less invasive approach in which the tumor is debulked with a minimally invasive (potentially percutaneous) procedure, and the resulting bone defect is packed with a resorbable calcium phosphate cement containing the chemotherapy agent. In preliminary in-vitro studies we have demonstrated successful elution of a proteasome inhibitor, bortezomib, from calcium phosphate cement. The specific aim of the current study was to build on this earlier work by determining whether bortezomib eluted from the cement is biologically active and capable of modulating the growth of tumor cell lines in vitro. We hypothesized that bortezomib-loaded bone cement would demonstrate dose-dependent activity against cells lines representative of both a primary bone cancer, osteosarcoma, and a secondary cancer, breast cancer, that commonly affects the skeleton. Methods: Drugs: Bortezomib (Velcade; Millenium Pharmaceuticals) was provided as sterile powder for injection. Stock solutions of the drug were prepared by dissolving the powder in dimethyl sulfoxide, and serial dilutions were made using normal saline. These dilute drug stocks were added to cells to determine the sensitivity of the tumor cell lines to bortezomib (0 to 10 microgram/ml) in the culture medium. Cement beads: Bortezomib-loaded cement was prepared by incorporating the drug into calcium phosphate cement (Callos Inject; Acumed, Inc.). The cement paste was then transferred into a silicon bead mold in order to fabricate 4mm-diameter beads for cell culture experiments. Based on data from the earlier elution studies, beads were manufactured with bortezomib concentrations ranging from microgram/gram of cement up to 200 µg/gram. Negative control samples (i.e. calcium phosphate cement with no drug) were fabricated by incorporating an equivalent volume of saline into the final cement mix. Tumor cells: The Abrams canine osteosarcoma (OSA) cell line and the MDA-MB-231 human breast cancer cell line were maintained in Dulbecco's Modification of Eagles Medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotics. Cells were routinely sub-cultured at approximately 80-90% confluence. For the experiments described below, cells were seeded at 50,000 cells per well in 24-well plates and allowed to settle for 24 hours. They were then treated with bortezomib administered either directly into the culture medium or as a drug-eluting cement bead. Cell viability: Cell viability was determined by MTT assays performed 24, 48, 72 and 96 hours after drug was added. At each time point, MTT activity in treated cells was expressed as a percentage of that seen in untreated cells. Each experiment was performed with N=4 samples at each dose and time, and the experiments were repeated to ensure consistency of the biological effect. Data Analysis: Within each experiment, the effects of bortezomib dose were determined by one-way analysis of variance, with Tukey post-hoc testing as appropriate. A significance level of p<0.05 was used throughout. Results: Direct addition of bortezomib into the culture medium resulted in statistically significant reductions in viable cell numbers are concentrations greater than or equal to 0.1 microgram/ml. When bortezomib-loaded resorbable cement beads were used, there was a significant decrease in cell number at a threshold of 12.5 microgram/g in both cell lines (Figure 1). The maximum inhibitory effect was evident within 48 hours of drug addition, and exposure for longer than 48 hours did not result in greater cell death. Discussion: Our data confirm that bortezomib released from calcium phosphate cement is biologically active and capable of killing two tumor cell lines of direct relevance to orthopedic oncology. We are actively working to expand this study to include other primary and secondary bone tumors, including Ewing sarcoma, renal carcinoma and prostate cancer. Bortezomib was selected for this application because studies in myeloma patients (1), mouse models of myeloma (2) and cell culture (3) have suggested that in addition to having potent anti-myeloma activity, bortezomib stimulates osteoblastic activity and inhibits osteoclastic activity within myeloma lesions. Data from our cell culture experiments have been used to design a preclinical animal study that will quantify the effects of bortezomib-loaded bone cement on bone turnover in vivo. Encouraging findings from this animal study will provide a strong rationale for evaluating this drug-cement combination in patients with inoperable bone tumors. The calcium phosphate cement that was used in this study is FDA approved as a bone graft substitute, and bortezomib is FDA-approved for the treatment of multiple myeloma. As a result, we would anticipate that the combination of these therapies would have a relatively quick transition into clinic trials in patients with primary or secondary bone cancers.

2 Significance: Calcium phosphate cement shows promise as a resorbable carrier for delivering tumoricidal doses of bortezomib. Percutaneous delivery of the drug-loaded cement into bone defects resulting from surgical excision of primary or secondary bone tumors offers a simple and potentially effective approach to controlling the re-growth of residual tumor and stimulating bone repair within the defect. Acknowledgments: Bortezomib was provided by Johnson & Johnson. Calcium phosphate cement was provided by Acumed. References: 1.Uy GL et al. Clin Lymphoma Myeloma 7: , Garrett IR et al. J Clin Invest 2003;111: , 2003.

3 3. Hongming H and Jian H. Leuk Res 33: ,

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5 ORS 2014 Annual Meeting Poster No: 1098

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