Heat enhanced molecular delivery to growth plates for targeted bone lengthening

描述（由申请人提供）：线性生长缺陷有多种病因，从受伤和疾病到遗传性骨病。骨伸长障碍是特别具有挑战性的治疗，因为相对不能调节分子输送到生长的骨骼。成功的临床干预的主要障碍是缺乏对生长中的骨骼的分子递送。成功的临床干预的主要障碍是缺乏将治疗剂靶向生长板软骨的方法，生长板软骨不具有穿透性血液供应。现有的肢体延长手术涉及侵入性手术或药物治疗，迄今为止仅部分有效。申请人产生的数据显示，局部加热增加了体内生长板软骨中的分子摄取，表明加热可能是增强骨延长药物递送的非侵入性且廉价的替代方案。长期目标是确定生长板中温度增强骨伸长的生理机制。解决问题的建议的总体目标是确定热是否增加全身性生长调节剂的骨延长作用。胰岛素样生长因子（IGF）-I是一种有效的刺激因子， 线性生长和儿童用药方案的一部分。中心假设，基于强有力的初步数据，并在两个特定的目标下，使用动态体内多光子显微镜测试，是热局部传递全身IGF-I到生长板，以促进添加剂骨延长。具体目标1使用体内软骨成像来确定剂量，时间和温度，最大限度地提高IGF-I摄取5周龄小鼠胫骨生长板。将注射荧光标记的IGF-I和大小替代示踪剂，以定量生长板中的分子摄取和清除。将使用真实的时间成像来确定定时注射后生长板和周围脉管系统中标记分子的递送速率和总体积。将在不同剂量和温度下测量血管和基质的渗透性。具体目标2使用一种新的肢体加热模型来确定生长板中热增强的IGF-I摄取是否会导致单侧肢体延长，通过量化可热处理的胫骨中IGF-I活化的生长速率和生物标志物进行分析。注射IGF-I及其受体拮抗剂将明确显示热增强的摄取是否增加骨长度。IGF-I缺陷生长激素受体敲除小鼠将用于测试热在疾病模型中的药物靶向功效。其基本原理是促进基于热的药物靶向方法的设计，以使用非侵入性技术在特定骨骼部位增加长度。该项目是创新的，通过使用多光子成像来评估生长板生理在体内细胞水平，在动态的方式不可能与其他方法。这一贡献是重要的，因为它可以产生变革性的发现，机械地链接热，骨延长，血管进入生长板。这些结果可以从根本上改变医生治疗一系列生长板疾病的方法，通过减少高剂量全身性药物的数量，毒性和成本，导致新的治疗方法具有更好的结果。

DESCRIPTION (provided by applicant): Linear growth deficiencies have multiple etiologies, ranging from injury and illness to genetic bone disease. Bone elongation disorders are particularly challenging to treat because of the relative inability to regulate molecular delivery o the growing skeleton. The primary obstacle to successful clinical intervention is lack of molecular delivery to the growing skeleton. The primary obstacle to successful clinical intervention is lack of methods for targeting therapeutics to growth plate cartilage, which does not have a penetrating blood supply. Existing procedures for limb lengthening involve invasive surgery or drug regimens, which are to date only partially effective. Data generated by the applicant show that localized heating increases molecular uptake in growth plate cartilage in vivo, suggesting that heat could be a noninvasive and inexpensive alternative for augmenting delivery of bone-lengthening drugs. The long-term goal is to identify physiological mechanisms underlying temperature-enhanced bone elongation in the growth plate. The overall objective of the problem-solving proposal is to determine whether heat augments the bone-lengthening effects of systemic growth regulators. Insulin-like growth factor (IGF)-I is a potent stimulator of linear growth and part of a drug regimen used in children. The central hypothesis, based on strong preliminary data and tested under two specific aims using dynamic in vivo multiphoton microscopy, is that heat localizes delivery of systemic IGF-I into growth plates to promote additive bone lengthening. Specific Aim 1 uses in vivo cartilage imaging to determine dose, timing, and temperatures that maximize IGF-I uptake in 5-week-old mouse tibial growth plates. Injections of fluorescently labeled IGF-I and size proxy tracers will be given to quantify molecular uptake and clearance in the growth plate. Real time imaging will be used to determine delivery rate and total volume of labeled molecules in the growth plate and surrounding vasculature after timed injections. Permeability of the vessels and matrix will be measured at different doses and temperatures. Specific Aim 2 uses a novel limb heating model to determine if heat-enhanced IGF-I uptake in growth plates causes unilateral limb lengthening, analyzed by quantifying growth rate and biomarkers of IGF-I activation in heat-treatable tibiae. Injections of IGF-I and its receptor antagonist will explicitly show if heat-enhanced uptake increases bone length. IGF-I deficient growth hormone receptor knockout mice will be used to test the drug-targeting efficacy of heat in a disease model. The rationale is to facilitate design of heat based drug-targeting approaches to enhance length at specific skeletal sites using noninvasive techniques. This project is innovative by using multiphoton imaging to assess growth plate physiology at the cellular level in vivo, in a dynamic way not possible with other methodologies. This contribution is significant because it can yield transformative findings that mechanistically link heat, bone lengthening, and vascular access to growth plates. Such results could fundamentally shift the approach that physicians take in treating a spectrum of growth plate disorders, leading to new therapies with better outcomes by reducing amount, toxicity and costs of high-dose systemic pharmaceuticals.