Tackling bone metastatic breast cancer growth with novel bone-seeking matrix metalloproteinase-2 inhibitors

Background. Despite medical advances, currently there is no treatment for breast to bone metastasis. The progression of bone metastatic breast cancer is critically dependent on interactions with the surrounding microenvironment. Therefore, identifying the underpinning molecular mechanisms is vital for the development of new therapies. Rationale. Gene expression analysis and validation in human and murine specimens of bone metastases revealed matrix metalloproteinases, such as MMP-2, are highly expressed in the bone metastatic microenvironment. Genetic ablation of MMP-2 demonstrated the importance of this MMP in driving the growth of the osteolytic bone metastatic breast cancer by regulating the bioavailability of transforming growth factor β (TGFβ). These data support the rationale for the development of a highly specific MMP-2 inhibitor for the eradication of active bone metastatic breast cancer. Methods. We utilized a novel chemical approach to synthesize bone seeking MMP inhibitors (BMMPIs) on a bisphosphonic backbone, with specificity for MMP-2 in the nanomolar range (IC50=140 nM). In vitro, we tested the effect of BMMPIs at varying doses (1nM-100μM) on the viability of the major cellular components of the cancer-bone microenvironment, namely breast cancer cells (PyMT, 4T1), osteoblasts (MC3T3) and osteoclasts (primary monocytes and RAW 264.7). In vivo, mice were intratibially inoculated with either luciferase expressing 4T1 or PyMT (1x105) cells. Mice (n=10/group) then received vehicle, zoledronate (1 mg/kg) or BMMPIs (1 mg/kg). Tumor growth was determined via luminescence quantitation. Cancer induced bone disease was measured ex vivo by μCT, Xray and histomorphometry. MMP activity in vivo and ex vivo was determined via specific activatable MMP probes. Pharmacokinetic and pharmacodynamic studies were performed. Plasma and bone marrow supernatants were collected from PyMT-R221A tumor bearing mice treated with ML115 (5mg/Kg) at 0.25, 0.5, 1, 2, 4, 8, 24 hours and three weeks (n=3 mice/time point). Currently, we are investigating the BMMPIs ability to impact the metastatic process through an in vivo model of intracardiac inoculation. Results. BMMPIs significantly impacted the viability of breast cancer cells and osteoclasts in vitro (p<0.05) compared to control. In vivo, BMMPIs significantly reduced the growth of bone metastatic breast cancer compared to control and the standard of care bisphosphonate, zoledronate. MMP activity was also lower in the BMMPI treated groups (using tumor burden to normalize values). μCT/Xray/Histomorphometry analysis also illustrated the significant beneficial effects of the BMMPIs in reducing the size of osteolytic lesions (up to 80% by μCT; p<0.05). ML115 is rapidly cleared from the plasma and accumulates selectively in the bone marrow microenvironment over time. Conclusions. MMP-2 specific BMMPIs prevent bone metastatic breast cancer growth by impacting cancer cell viability and cancer induced osteolysis. Given that bisphosphonates are well tolerated in the clinical setting, we predict that BMMPIs could be translated to the clinical setting for the treatment and eradication of bone metastatic breast cancer.