Molecular and cellular mechanism of ONJ related to osteoclast inhibition

描述（由申请人提供）：本项目的长期目标是确定颌骨骨坏死（ONJ）的病理机制，并制定预防和治疗方法。ONJ是一种口腔并发症，一些患者接受含氮双膦酸盐（BP）治疗，最近接受人源化抗RANKL抗体治疗。确诊的临床ONJ病例的特征是口腔粘膜的异常伤口愈合促进了部分坏死的颌骨的长期暴露。与对破骨细胞的公认药理作用不同，BP对口腔粘膜的作用尚未完全阐明。大量研究表明，BP影响肠溃疡的愈合、腹膜炎的发展和 外周血先天性和获得性免疫。给药途径（分别为PO、IP和IV）允许BP在快速吸附至骨骼或排泄之前直接暴露于这些组织。然而，影响口腔粘膜的"游离" BP的来源尚未确定。为了阐明ONJ相关的口腔粘膜异常的机制，我们假设预先吸附在颌骨上的BP可以被破骨细胞去除并瞬时释放到口腔粘膜组织。我们设想，释放的BP的量可能取决于破骨细胞生成的程度，定位在牙槽骨界面的口腔粘膜的表面;并且当达到临界浓度时，释放的BP可能直接影响口腔粘膜驻留细胞，并对口腔粘膜伤口的愈合产生不利影响。为了解决这一假设，这个探索性的R21项目提出了新的BP为基础的探针的合成和应用，能够提供一个F rster共振能量转移（FRET）淬灭报告功能的破骨细胞，其中探针将产生一个荧光信号，只有在响应破骨细胞衍生的组织蛋白酶K（CatK）的活动，导致发射器和淬灭分离。由桥接荧光染料和猝灭剂的短CatK可切割肽组成的新型分子探针将通过USC开发的独特BP接头化学连接到生物活性或非活性BP支架上。沉默的BP-CatK-FRET探针将被强烈地吸附到骨或合成磷酸钙上，但骨钙素CatK将去除猝灭剂并激活荧光信号。破骨细胞的BP-CatK-FRET激活将使用合成的磷灰石包被板和小鼠破骨细胞在体外表征（将荧光信号与吸收陷窝面积、Rap1a异戊二烯化和破骨细胞凋亡相关联）。所提出的探针也将是体内评价的有力工具，以建立破骨细胞动员的BP和口腔粘膜驻留细胞之间的关键联系。它们也将有助于评估传统BP药物在确定ONJ风险中的作用，并检查未来的替代治疗方法。在实施该项目时，McKenna（南加州大学）和Nishimura（加州大学洛杉矶分校）的研究团队将利用他们在BP探针设计和化学（南加州大学）以及骨生物学模型（加州大学洛杉矶分校）方面的互补优势。[下划线：订正段落]

DESCRIPTION (provided by applicant): The long-term objectives of this project are to determine the pathological mechanism of osteonecrosis of the jaw (ONJ) and to develop preventive and therapeutic modalities. ONJ is an oral complication experienced by some patients treated with nitrogen-containing bisphosphonates (BPs) and more recently with humanized anti- RANKL antibody. Confirmed clinical ONJ cases are characterized by the prolonged exposure of partially necrotic jawbone facilitated by abnormal wound healing of oral mucosa. Unlike the well-established pharmacological effect on osteoclasts, the role of BP on oral mucosa has not been fully elucidated. A number of studies have demonstrated that BP affects the healing of intestinal ulceration, the development of peritonitis, and the activation of peripheral blood innate and acquired immunity. The administration routes (PO, IP and IV, respectively) allow the direct exposure of BP to these tissues, prior to rapid adsorption of BP to bone or excretion. However, the source of "free" BP affecting the oral mucosa has not been identified. To elucidate the mechanism of oral mucosa abnormality associated with ONJ, we have hypothesized that BP pre-adsorbed on the jawbone can be removed by osteoclasts and transiently released to the oral mucosa tissue. We envision that the amount of released BP may depend on the degree of osteoclastogenesis localized at the surface of alveolar bone interfacing oral mucosa; and upon reaching a critical concentration, the released BP may directly affect oral mucosa resident cells and adversely influence the healing of oral mucosa wound. To address this hypothesis, this exploratory R21 project proposes the synthesis and application of novel BP-based probes capable of delivering a F�rster resonance energy transfer (FRET)-quenched reporter function to osteoclasts, where the probes will generate a fluorescent signal only in response to osteoclast-derived cathepsin K (CatK) activity which causes separation of emitter and quencher. Novel molecular probes consisting of a short CatK- cleavable peptide bridging a fluorescent dye and a quencher will be attached to a biologically active or inactive BP scaffold through unique BP-linker chemistry developed at USC. The silent BP-CatK-FRET probe will be strongly adsorbed to bone or synthetic calcium phosphate, but osteoclastic CatK will remove the quencher and activate the fluorescent signal. BP-CatK-FRET activation by osteoclasts will be characterized in vitro using synthetic apatite-coated plates and mouse osteoclasts (correlating fluorescent signal with resorption pit area, Rap1a prenylation and osteoclast apoptosis). The proposed probes will also be powerful tools for in vivo evaluation to establish the critical link between BP mobilized by osteoclasts and oral mucosa resident cells. They will also be useful to assess the role of legacy BP drugs in determining ONJ risk and to examine future replacement therapy approaches. In implementing this project, the research teams of McKenna (USC) and Nishimura (UCLA) will leverage their complementary strengths in BP probe design and chemistry (USC) and expertise in bone biology models (UCLA). [Underline: revised paragraphs]