Project 2: PK/PD requirements for mucolytic therapeutic agents in vitro and in vivo

项目2的重点是假设：(1)高度浓缩(脱水)的粘液产生呼吸道 粘液黏附/斑块推动粘液阻塞性肺疾病的进展；以及，(2)清除 这些塞子/斑块将在治疗上对黏膜阻塞性表型患者有用。近期 数据表明，一旦发生粘液抑制，粘液斑块可能发生改变，从而使 他们是“永久性的”，对补液疗法有抵抗力。我们的生物物理分析表明粘蛋白 在这种情况下，减少分子量(MW)将有效地清除粘液，而不需要再水化。此外， 项目2假设，通过生成疾病特异性模型，有效地模拟 我们疾病人群的细胞外粘蛋白/粘液靶标，将产生有用的信息来指导 与药物剂量的选择、给药频率、主动车辆的要求有关的临床项目 (例如，高渗盐水)，并产生关于“偏离目标”的氧化还原效应的数据。与tPPG合作 研究人员发现，在慢性阻塞性肺疾病(和慢性阻塞性肺疾病)中，MUC5B是阻塞呼吸道的主要粘蛋白。 相反，我们已经确定MUC5AC是哮喘患者的主要呼吸道粘蛋白。 因此，我们已经产生了适当的细胞培养、小动物和绵羊模型来模拟粘蛋白 每个疾病实体的显性表型。利用这些工具，项目2将测量和比较 两种不同但互补的硫醇降解剂的药效学和药代动力学特性 临床候选人。P2176是一种具有快速还原活性的二硫醇化合物(KCAT)，而一硫醇化合物 P2114具有较慢的内源还原(KCAT)活性。这两个分子都有共同的特征，旨在保持 呼吸道表面上的分子和这两个分子的硫醇都被醋酸酯基团“封顶” 被呼吸道表面的酯酶裂解，释放出活性化合物。的相对有效性/特性 每个分子将在：(1)模拟细胞外MUC5AC/MUC5B的体外HBE培养中进行测试 CF/COPD(IL-1β治疗)和哮喘(IL-13治疗)的比率；(2)CF/COPD(βENaC)小鼠模型， 和哮喘(治疗室内尘螨)，以测量气雾剂给药后粘液塞的清除情况；以及， 大动物绵羊模型，包括野生型、猪蛔虫暴露，以及HNE/CF172治疗绵羊 测量药效学和药代动力学参数。基于这些数据，和GLP毒理学 研究，铅分子剂量/剂量频率，将选择一种载体。重要的是，项目2还将 利用从项目1/项目2研究以及临床项目3和4获得的信息来研究备份 作为领跑者改进的化合物在母体硫醇化合物中被识别/实施 底盘。因此，2B项目的首要目标是确定最佳的药物特性，平衡率 粘蛋白硫醇的减少、活性持续时间和安全性，以开发一种新的粘液清除助剂 专注于降低粘蛋白分子量的药剂，可单独提供或与保湿剂联合使用。

Project 2 has focused on the hypotheses that: (1) hyperconcentrated (dehydrated) mucus produces airway mucus adhesion/plaques that drive the progression of muco-obstructive lung diseases; and, (2) that clearance of these plugs/plaques will be therapeutically useful in patients with the muco-obstructive phenotype. Recent data have indicated that once mucostasis has occurred, modification of mucus plaques may occur that make them “permanent” and resistant to rehydration therapies. Our biophysical analyses suggest that mucin molecular weight (MW) reduction will be effective in clearing mucus in this setting without rehydration. Further, Project 2 hypothesizes that by the generation of disease specific models, that effectively mimic the extracellular mucins/mucus targets of our disease populations, will produce useful information to guide the clinical projects with respect to selection of drug doses, dosing frequency, requirement for an active vehicle (e.g., hypertonic saline) and generate data on “off target” redox effects. In collaboration with tPPG investigators, we have established that in CF (and COPD) MUC5B is the dominant mucin obstructing airways. In contrast, we have identified that MUC5AC is the dominant airway mucin in subjects with asthma. Accordingly, we have generated appropriate cell culture, small animal, and sheep models that mimic the mucin dominance phenotype for each disease entity. Utilizing these tools, Project 2 will measure and compare the pharmacodynamics and pharmacokinetic properties of two different, but complementary, thiol reducing agent clinical candidates. P2176 is a di-thiol compound with rapid reductive activity (Kcat) whereas the mono-thiol P2114 has a slower intrinsic reductive (Kcat) activity. Both molecules share features designed to retain the molecules on the airway surface and both molecules have their thiols “capped” by acetate groups that are cleaved by esterases on airway surfaces to liberate active compounds. The relative effectiveness/properties of each molecule will be tested in: (1) in vitro HBE cultures designed to mimic the extracellular MUC5AC/MUC5B ratios of CF/COPD (Il-1β treated) and asthma (IL-13 treated); (2) mouse models of CF/COPD (βENaC) mice, and asthma (house dust mite treated) to measure clearance of mucus plugs after aerosol drug delivery; and, large animal sheep models, including wild-type, Ascaris suum exposed, and HNE/CF172 treated sheep to measure pharmacodynamics and pharmacokinetic parameters. Based on these data, and GLP toxicology studies, a lead molecule doses/dosing frequency, and a vehicle will be selected. Importantly, Project 2 will also utilize information gained from Project 1/Project 2 studies and clinical Projects 3 and 4 to study backup compounds as improvements in the frontrunner become identified/implemented in the parent thiol compound chassis. Thus the overarching goal of the 2b Project is to identify the optimal drug properties, balancing rate of mucin thiol reduction, duration of activity, and safety, to develop a new class of muco-clearance assisting agents that focus on mucin MW reduction that can be delivered alone or in combination with hydrating agents.