A Novel Therapeutic Peptide to Treat P. aeruginosa Infection in Cystic Fibrosis Patients

项目摘要 我们的目标是开发6 K-F17作为抗微生物剂，可与妥布霉素一起用于治疗 治疗囊性纤维化（CF）患者的慢性耐药铜绿假单胞菌感染。CF 影响全球超过70，000人1。即使使用目前的抗生素，80-95%的CF患者将 最终死于慢性细菌感染引起的呼吸衰竭3。因此，有一个 开发用于CF患者中的慢性耐药性感染的新抗菌剂的显著未满足的需求。 囊性纤维化是一种由囊性纤维化跨膜传导调节因子突变引起的遗传性疾病 （CFTR）基因，其编码氯离子转运蛋白4。受损的跨上皮氯离子转运导致 脱水的气道分泌物和缺乏气道粘液清除5，6.因此，患有CF的人 易患慢性生物膜细菌感染、支气管扩张和呼吸衰竭6，7特别是， 铜绿假单胞菌的慢性感染已被证明会导致更快的肺功能下降， 生命和过早死亡8 -11。铜绿假单胞菌的慢性感染特别具有挑战性， 菌株不断适应环境挑战，包括形成粘液样生物膜。这些 生物膜保护细菌免受自然宿主防御机制的影响，使它们对小的 分子抗生素5，12-18。 已经通过使用阳离子聚合物在开发生物膜感染的治疗方面取得了进展。 抗菌肽（CAPs）。CAP天然存在于从植物到微生物的各种生物体中。 人的免疫系统，并构成先天免疫系统的主要组成部分19 -21。重要的是，这些天然CAP 通过破坏细菌的细胞膜来杀死细菌--实际上是通过在细胞膜上沉积和积累来“炸毁"细菌。 他们的膜-而不是通过针对特定的细菌蛋白质或生化途径。这 这一机制阻碍了细菌对CAP的耐药性的出现。最近，我们的团队开发了一种新的 一种名为6 K-F17的肽，可以破坏铜绿假单胞菌产生的生物膜，并使用一种新的 膜结合机制。由于其独特的作用机制，6 K-F17与 抗生素，如妥布霉素，以杀死铜绿假单胞菌的耐药性菌株。 在这项工作的基础上，本提案的目标是产生最佳剂量、药代动力学和 小鼠中6 K-F17的毒理学特征。具体目标是：1）生产足够量的6 K-F17， 标准化分析和效力测定; 2）确定6 K-F17消除P的最佳有效剂量。 铜绿假单胞菌感染的小鼠的急性毒理学特征;和3）在正常小鼠中建立6 K-F17的初步急性毒理学特征。 这些研究的成功完成将推动一种令人兴奋的抗微生物剂用于治疗 CF患者中的耐药铜绿假单胞菌和潜在的其他耐药肺部感染。

Project Summary Our objective is to develop 6K-F17 as an antimicrobial agent that can be prescribed with tobramycin for the treatment of chronic, antibiotic-resistant Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients. CF affects over 70,000 people worldwide1. Even with the current array of antibiotics, 80-95% of CF patients will ultimately succumb to respiratory failure brought on by chronic bacterial infections3. As a result, there is a significant unmet need to develop new antimicrobials for chronic, antibiotic-resistant infections in CF patients. CF is a genetic disease arising from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a chloride ion transporter4. Impaired trans-epithelial chloride transport leads to dehydrated airway secretions and a lack of airway mucus clearance5, 6. As a result, individuals with CF are prone to chronic, biofilm-based bacterial infections, bronchiectasis and respiratory failure6, 7In particular, chronic infection with P. aeruginosa has been shown to lead to more rapid lung function decline, a lower quality of life, and premature death8-11. Chronic infection with P. aeruginosa is particularly challenging to treat as this strain continually adapts to environmental challenges, including the formation of a mucus-like biofilms. These biofilms protect bacteria from natural host defense mechanisms and make them more resistant to small molecule antibiotics 5, 12-18. Progress toward developing treatments for biofilm infections has been made through the use of cationic antimicrobial peptides (CAPs). CAPs are found naturally in a wide variety of organisms ranging from plants to humans and constitute a major component of the innate immune system19-21. Importantly, these natural CAPs kill bacteria by destroying their membranes – in effect ‘blowing up’ the bacteria by lodging and accumulating in their membranes - rather than by targeting a specific bacterial protein or biochemical pathway. This mechanism impedes the advent of bacterial resistance to CAPs. Recently, our team developed a novel peptide, called 6K-F17, that can disrupt biofilms created by P. aeruginosa and kill the bacteria using a novel mechanism of membrane association. Due to its unique mechanism of action, 6K-F17 synergizes with antibiotics, such as tobramycin, to kill antibiotic-resistant strains of P. aeruginosa. Building from this work, the objective of this proposal is to generate the optimal dose, pharmacokinetics and toxicology profiles for 6K-F17 in mice. The specific aims are to: 1) produce sufficient amounts of 6K-F17 and standardize analytical and potency assays; 2) determine the optimal effective dose of 6K-F17 to eliminate P. aeruginosa infection in mice; and 3) establish a preliminary acute toxicology profile for 6K-F17 in normal mice. Successful completion of these studies will advance an exciting antimicrobial agent for the treatment of antibiotic-resistant P. aeruginosa, and potentially other antibiotic-resistant lung infections, in CF patients.