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Breast cancer patients are at high risk for bone metastasis. Metastatic bone disease is a major clinical problem that leads to a reduction in mobility, increased risk of pathologic fracture, severe bone pain, and other skeletal-related events. The transcription factor Gli2 drives expression of parathyroid hormone-related protein (PTHrP), which activates osteoclast-mediated bone destruction, and previous studies showed that Gli2 genetic repression in bone-metastatic tumor cells significantly reduces tumor-induced bone destruction. Small molecule inhibitors of Gli2 have been identified; however, the lipophilicity and poor pharmacokinetic profile of these compounds have precluded their success in vivo . In this study, we designed a bone-targeted nanoparticle (BTNP) comprising an amphiphilic diblock copolymer of poly[(propylene sulfide)- block -(alendronate acrylamide- co - N , N -dimethylacrylamide)] [PPS- b -P(Aln- co -DMA)] to encapsulate and preferentially deliver a small molecule Gli2 inhibitor, GANT58, to bone-associated tumors. The mol % of the bisphosphonate Aln in the hydrophilic polymer block was varied in order to optimize BTNP targeting to tumor-associated bone by a combination of nonspecific tumor accumulation (presumably through the enhanced permeation and retention effect) and active bone binding. Although 100% functionalization with Aln created BTNPs with strong bone binding, these BTNPs had highly negative zeta-potential, resulting in shorter circulation time, greater liver uptake, and less distribution to metastatic tumors in bone. However, 10 mol % of Aln in the hydrophilic block generated a formulation with a favorable balance of systemic pharmacokinetics and bone binding, providing the highest bone/liver biodistribution ratio among formulations tested. In an intracardiac tumor cell injection model of breast cancer bone metastasis, treatment with the lead candidate GANT58-BTNP formulation decreased tumor-associated bone lesion area 3-fold and increased bone volume fraction in the tibiae of the mice 2.5-fold. Aln conferred bone targeting to the GANT58-BTNPs, which increased GANT58 concentration in the tumor-associated bone relative to untargeted NPs, and also provided benefit through the direct antiresorptive therapeutic function of Aln. The dual benefit of the Aln in the BTNPs was supported by the observations that drug-free Aln-containing BTNPs improved bone volume fraction in bone-tumor-bearing mice, while GANT58-BTNPs created better therapeutic outcomes than both unloaded BTNPs and GANT58-loaded untargeted NPs. These findings suggest GANT58-BTNPs have potential to potently inhibit tumor-driven osteoclast activation and resultant bone destruction in patients with bone-associated tumor metastases.

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide) 135 -b-poly[(oligoethylene glycol) 9 methyl ether acrylate] 17 (PPS 135 -b-POEGA 17 ). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p<.01) and lesion number by 38% (p<.05) and resulted in a 2.5-fold increase in trabecular bone volume (p<.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro, nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation., (Published by Elsevier B.V.)