Discovery of Novel Nontuberculous Inhibitors

项目总结/摘要 非结核分枝杆菌（NTM）是广泛存在于环境中的病原体， 进行性肺部疾病以及皮肤和软组织、中枢神经系统和播散性感染。 NTM感染率在全球范围内不断上升，并已成为全球重要的人类病原体。的 引起这些感染的特定病原体是以下两种之一的一部分：i）分枝杆菌 （M.（ii）鸟分枝杆菌（Mycobacterium avium，M. avium）复合体。分枝脓肿 复合体包括亚种M。M.P. M. tagliense和M.博莱蒂，已经成为一个重要的 全球威胁导致结构性肺患者肺部感染数量增加 慢性阻塞性肺病、支气管扩张和囊性纤维化（CF）等疾病。特别 值得关注的是，与MABSC病原体共感染的CF患者通常无法治疗，尽管多年来一直在治疗。 联合治疗导致60-70%的治疗失败。因此，具有强效抗NTM活性的新型抗NTM剂 对MABSC菌株的活性，可以缩短处理时间，降低耐药率， 强烈需要有效性。 我们发现了一系列新的吲哚-2-甲酰胺类化合物， 对临床感兴趣的各种缓慢和快速生长的NTM的浓度（MIC）值为0.0039 - 8 µg/ml， 包括M. M.P. M. tagliense，M. bolletii和M.龟类。此外，IC在体外有效， 在小鼠中，证明了它们的安全性和有效性。然而，IC是亲脂性的，并且水溶性差 导致口服吸收差。为了避免这种不良的药代动力学性质，我们建议设计 新型氨基酸类IC类似物，开发安全有效的载药脂质体 来配制难溶的IC。将评价新的类似物的抗分枝杆菌活性和活性。 化合物将经历一系列ADMETox测定以确定推定的PK/PD作用。IC-和IC-模拟- 负载的脂质体将被设计用于吸入给药 通过巨噬细胞，NTM病原体驻留在感染的患者体内。这将通过以下方式实现： IC合成、载药脂质体的制备以及药物释放和巨噬细胞的表征 摄取。脂质体将使用薄膜水合法生产，随后使用IC加载， 被动诱捕法将通过31 P NMR监测脂质体的层状性，并通过 低温透射电镜将进行药物释放研究，以确保释放治疗IC浓度 （浓度高于MIC值）。最后，具有优化药物释放的IC负载脂质体将受到 巨噬细胞摄取研究。这将使用荧光标记的IC负载脂质体进行， 表达GFP的巨噬细胞以可视化摄取和TEM。

Project Summary/Abstract Non-tuberculous mycobacteria (NTM) are widespread pathogens found in the environment and cause progressive lung disease as well as skin and soft tissue, central nervous system and disseminated infections. NTM infections rates are rising globally and have emerged as important human pathogens globally. The specific pathogens responsible for these infections are part of one of two species, i) Mycobacterium abscessus (M. abscessus) complex (MABSC) and ii) Mycobacterium avium (M. avium) complex. The M. abscessus complex comprises the subspecies M. abscessus, M. massiliense and M. bolletii, has emerged as a significant global threat causing an increasing number of pulmonary infections among patients with structural lung disease such as chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis (CF). Of particular concern, patients with CF co-infected with an MABSC pathogen are often untreatable despite years of combination therapy resulting in 60-70% treatment failures. Therefore, novel anti-NTM agents with potent activity against MABSC strains that can shorten treatment time, decrease resistance rates with improved efficacy are strongly needed. We have discovered a novel series of indole-2-carboxamides (IC) that have potent minimum inhibitor concentration (MIC) values of 0.0039 - 8 µg/ml against various slow- and fast-growing NTM of clinical interest, including M. abscessus, M. massiliense, M. bolletii and M. chelonae. In addition, ICs are effective in vitro and in mice, demonstrating their safe and effective profile. However, ICs are lipophilic and are poorly water soluble resulting in poor oral absorption. To circumvent this poor pharmacokinetic property, we propose to design novel amino-acid based IC analogs and develop drug-loaded liposomes, which are a safe and effective option to formulate poorly soluble ICs. Novel analogs will be evaluated for antimycobacterial activity and active compounds will undergo a series of ADMETox assays to determine putative PK/PD action. IC- and IC-analog- loaded liposomes will be designed for inhalation administration and be engineered to be preferentially taken up by macrophages, where NTM pathogens reside in infected patients. This will be accomplished through synthesis of ICs, production of drug-loaded liposomes and characterization of drug release and macrophage uptake. Liposomes will be produced using thin film hydration method with subsequent IC-loading using passive trapping methods. The lamellarity of the liposomes will be monitored by 31P NMR and confirmed with cryo-TEM. Drug-release studies will be performed to ensure therapeutic IC concentrations are released (concentrations above MIC values). Finally, IC-loaded liposomes with optimized drug-release will be subjected to macrophage uptake studies. This will be performed using a fluorescent labeled IC loaded liposome and macrophages expressing GFP to visualize uptake and TEM.