Small Molecules Promote Tendon Regeneration by Targeting Endogenous Stem Cells

摘要 肌腱和韧带损伤在美国是一个严重的医疗负担，花费超过300亿美元。 每年。肌腱损伤经常导致物理性能较差的疤痕样组织-然而， 再生疗法至今仍存在。最近，我们已经确定并表征了血管周围（CD 146+） 肌腱干/祖细胞（TSCs）通过FAK和ERK 1/2在肌腱愈合中发挥重要作用 发信号。 在我们的初步研究中，我们从FAK和ERK 1/2激动剂文库中筛选小分子， 鉴定了氧化震颤素M（Oxo-M）和马来酸PPBP（4-PPBP），其刺激TSC向再生方向发展， 肌腱愈合Oxo-M和4-PPBP最初被开发用于治疗神经元疾病，但从未被用于治疗神经元疾病。 在肌肉骨骼系统中进行了测试。在体外，Oxo-M和4-PPBP的组合诱导了显著的 参与肌腱修复的肌腱相关基因的表达增加。Oxo-M和4-PPBP显示 在10倍工作剂量下无细胞毒性。Western印迹和SiRNA敲除（KD）证实FAK和 ERK 1/2信号传导调节氧代-M&4-PMBP诱导的TSC的腱形成分化。体内，直接 将Oxo-M和4-PPBP局部递送到完全横断的大鼠髌腱（PT）上显著改善了 肌腱愈合，组织学上观察到密集重组的胶原纤维，功能上观察到 拉伸强度显著提高。这一过程是由一个快速但短暂的增加， 数量的内源性TSCs经历肌腱分化。此外，Oxo-M和4-PPBP特异性地 通过毒蕈碱乙酰胆碱受体（AChR）和σ1受体（σ 1 R）靶向CD 146 + TSC 途径，对其他类型的肌腱细胞的影响最小。这些发现表明了一种新颖的， Oxo-M和4-PPBP的组合通过特异性靶向肌腱愈合的有希望的活性 内源性TSC。 这项STTR赠款的总体目标是开发一种可靠有效的基于小分子的 通过瞬时激活内源性再生途径的肌腱损伤的再生治疗 TSC和修复肌腱撕裂。STTR第一阶段赠款的首要目标是优化 本发明涉及Oxo-M和4-PPBP（2种化合物）的组合，并确定其组合的可药用性。的2 第一阶段STTR的目标是获得可靠的概念验证和初步的安全数据，以建立 创新技术的技术价值、可行性和商业潜力。

Abstract Tendon and ligament injuries represent an acute healthcare burden in the United States, costing >$30 billion annually. Tendon injuries frequently result in scar-like tissue with inferior physical properties - however no regenerative therapy exists to date. Recently, we have identified and characterized perivascular (CD146+) tendon stem/progenitor cells (TSCs) that play an essential role in tendon healing via FAK and ERK1/2 signaling. In our preliminary study, we screened small molecules from a library of FAK and ERK1/2 agonists and identified Oxotremorine M (Oxo-M) and PPBP maleate (4-PPBP) that stimulated TSCs toward regenerative tendon healing. Oxo-M and 4-PPBP were originally developed for treating neuronal diseases but have never been tested in the musculoskeletal system. In vitro, a combination of Oxo-M and 4-PPBP induced significant increases in the expression of tendon-related genes involved in tendon repair. Oxo-M and 4-PPBP showed no cytotoxicity up to 10X working doses. Western blot and siRNA knockdown (KD) confirmed that FAK and ERK1/2 signaling regulate Oxo -M & 4-PPBP-induced tenogenic differentiation of TSCs. In vivo, direct topical delivery of Oxo-M and 4-PPBP onto full-transected rat patellar tendons (PT) significantly improved tendon healing, as observed histologically as densely reorganized collagen fibrils, and functionally as significantly enhanced tensile strength. This process was guided by a rapid but transient increase in the number endogenous TSCs undergoing tenogenic differentiation. In addition, Oxo-M and 4-PPBP specifically targeted CD146+ TSCs through muscarinic acetylcholine receptors (AChRs) and σ1 receptor (σ1R) pathways, with minimal effect on other types of tendon cells. These findings demonstrate a novel and promising activity of the combination of Oxo-M and 4-PPBP in tendon healing by specifically targeting endogenous TSCs. The overall objectives of this STTR grant are to develop a reliable and effective small molecule-based regenerative therapy for tendon injuries by transiently activating regenerative pathways of endogenous TSCs and repair tendon tears. The overarching goal of the STTR phase I grant is to optimize the combination of Oxo-M and 4-PPBP, the 2 compounds and determine their drugability in combination. The 2 aims of the phase I STTR are to obtain robust proof-of-concept and preliminary safety data to establish technical merit, feasibility, and commercial potential of the innovative technology.