OR30-4 Parathyroid Hormone-related Peptide Antagonist and Inverse Agonist Reduce Breast Cancer Metastasis to Bone and Bone Loss

Abstract

Bone is the most frequent site for breast cancer metastasis. Metastatic cancer cells secrete parathyroid-hormone related peptide (PTHrP) that stimulates bone turnover, releasing growth factors and creating a cavity for tumor growth. Current treatments include bisphosphonates and the RANK Ligand inhibitors that have limited efficacy. PTHrP antagonists have failed clinically due to rapid drug turnover and kidney side effects causing hypocalcemia. We synthesized two novel PTHrP antagonists that are fused to an inert bacterial collagen binding domain (CBD) of colG collagenase from Clostridium histolyticum. The collagen binding activity directs PTH analogs to the bone matrix where it blocks PTHrP action. PTH(7-33)-CBD is an amino truncated PTHrP antagonist fused to the CBD that we previously showed reduced metastatic tumor burden in bone and prevented bone destruction. We now compare PTH(7-33)-CBD to [W2]PTH(1-33)-CBD, an inverse agonist at the PTH/PTHrP receptor. In vitro neither drug induced PTH receptor activation in SaOS-2 osteosarcoma cells as measured by cAMP accumulation. Both compounds partially antagonized PTH(1-34) agonist-induced cAMP accumulation. Importantly, [W2]PTH(1-33)-CBD reduced agonist-stimulated cAMP accumulation by >95% (vs. 71% for PTH(7-33)-CBD) exceeding that reported previously for any PTH antagonist. Both compounds induced apoptosis of MDA-MB-231 breast cancer cells in vitro. In vivo testing was performed with an established mouse model of breast cancer bone metastasis using a bone-trophic variant of MDA-MB-231 breast cancer cells expressing luciferase (MDA-MB-231-BM/luc+). 24 hours after drug administration (1000 µg/kg administered IP), MDA-MB-231-BM/luc+ cells were injected intratibially into nude mice. PTH(7-33)-CBD significantly reduced tumor burden in bone (P<0.05) compared to both vehicle control (VC) (weeks 4-8), and to control PTH(7-34) antagonist that lacks the CBD (weeks 6-7). [W2]PTH(1-33)-CBD showed a trend towards decrease in tumor burden but did not reach statistical significance. Both PTH(7-33)-CBD and [W2]PTH(1-33)-CBD significantly reduced osteolytic lesions (P<0.05) compared to VC and PTH(7-34) weeks 3-8 and weeks 6-8, respectively. Drug treatments did not significantly affect animal weight nor alter serum calcium, alkaline phosphatase or N-terminal propeptide of Type 1 procollagen (PN1P) bone formation marker. Tartrate resistant acid phosphatase (TRAP), a marker of bone removal, was significantly reduced by treatments. In summary, a new PTHrP inverse agonist, [W2]PTH(1-33)-CBD, partially suppressed basal signaling of the PTH/PTHrP receptor and demonstrated a higher degree of antagonist activity in-vitro than any PTH antagonist published to date. It is expected that the superior antagonist activity of [W2]PTH(1-33)-CBD will provide significant tumor protection against breast cancer metastasis to bone.