Development of Gallium-Based Therapies for Pulmonary Mycobacterial Infections

描述(由申请人提供)： 结核病(TB)是由结核分枝杆菌(M.TB)引起的全球性健康问题。对药物敏感的结核病的治疗需要使用多种可能有毒的抗生素进行数月的治疗。耐多药结核分枝杆菌的出现和流行使结核病的治疗变得更加困难。这项建议的重点是开发一种新的治疗方法，针对结核分枝杆菌铁(Fe)的获取，这一过程对其导致 疾病。硝酸镓是FDA批准的治疗恶性肿瘤高钙血症的药物。它通过与铁竞争摄取/利用来干扰细胞的铁代谢。我们发现，GA还减少结核分枝杆菌铁的获取，抑制体外细胞外和人巨噬细胞内生长的药物敏感和耐药结核分枝杆菌菌株的生长。GA在感染结核分枝杆菌的小鼠模型中也显示了疗效。间歇性全身注射硝酸镓几天，对人体的毒性最小。然而，结核病的治疗可能需要更持久的治疗方案。改善GA对感染巨噬细胞的靶向性、延长给药间隔或避免全身给药的方法将提高GA的治疗指数。近年来，在向肺部的气雾剂给药以及纳米颗粒的细胞靶向方面取得了相当大的进展，以实现这些目标。因此，我们推测，通过气雾剂或纳米颗粒向肺部输送镓可以被证明是一种新颖的、耐受性良好的、有效的结核病治疗方法，它将通过扰乱结核分枝杆菌铁的代谢而发挥作用。为了验证上述假设，我们建议实现以下特定目标：1.确定在体外对细胞外和细胞内结核分枝杆菌具有最大效力的形式，包括MDR-TB和XDR-TB，目标是获得先导化合物。2.开发两种铅镓化合物的配方，使其能够吸入给药治疗肺结核病，并测定它们在小鼠肺结核病模型中的生物分布、药代动力学、毒性和疗效。3.开发两种铅镓化合物的纳米制剂，使其能够用于治疗肺结核病，并测定它们在小鼠肺结核病模型中的生物分布、药代动力学、毒性和疗效。4.确定铅镓制剂对结核分枝杆菌的作用机制，出现耐药性的可能性，以及其他结核病药物对镓抗分枝杆菌活性的影响。研究设计和方法：人类单核细胞来源的巨噬细胞和小鼠结核病模型将与完全毒力的结核分枝杆菌菌株一起使用。这项工作将着重于：结核分枝杆菌对铁和镓的获取：镓的生物分布、药代动力学、毒性；以及镓抗菌活性的潜在细菌靶标。所采用的实验方法包括：开发适合于气雾剂或纳米颗粒介导的镓制剂；放射性标记的铁和镓的细菌摄取；结核分枝杆菌的体外和体内生长；小鼠肺结核病模型；组织中镓的定量。对退伍军人医疗保健的潜在影响：退伍军人患肺结核的风险很高。这项工作可能导致开发新的结核病治疗方法，使退伍军人受益。

DESCRIPTION (provided by applicant): Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is a global health problem. Treatment of drug-susceptible TB involves months of therapy with multiple potentially toxic antibiotics. Treatment of TB has become even more difficult by the emergence and increasing prevalence of multi-drug resistant M.tb. This proposal focuses on the development of a novel therapy that targets M.tb iron (Fe) acquisition, a process that is critical to its ability to cause disease. Ga(NO3)3 is a FDA- approved drug for treating hypercalcemia of malignancy. It interferes with cellular Fe metabolism by competing with Fe for uptake/use. We find that Ga also decreases M.tb Fe acquisition, inhibits growth of both drug-susceptible and drug-resistant M.tb strains growing in vitro extracellularly and within human macrophages. Ga also demonstrated efficacy in murine models of M.tb infection. Intermittent systemic delivery of Ga(NO3)3 for a few days has shown minimal toxicity in humans. However, treatment of TB would likely require more sustained treatment regimens. Ways to improve targeting of Ga to infected macrophages, extend dosing intervals, or avoid systemic administration would improve Ga's therapeutic index. There have been considerable advances in recent years in the aerosol delivery of drugs to the lung, as well as cellular targeting of nanoparticles to accomplish these goals. Therefore, we hypothesize that Ga delivery by aerosol or nanoparticle to the lung could prove to be novel, well-tolerated, and effective therapies for TB that would work via disruption of M.tb Fe metabolism. In order to test the above hypothesis we propose to accomplish the following specific aims: 1. Identify forms of Ga that exhibit maximal in vitro potency against extracellular and intracellular M.tb, including MDR- and XDR-TB, with the goal of arriving at lead compounds. 2. Develop formulations of two lead Ga compounds that will allow them to be administered by inhalation for the treatment of pulmonary TB and determine their biodistribution, pharmacokinetics, toxicity and efficacy in murine models of pulmonary TB. 3. Develop nanoparticle formulations of two lead Ga compounds that will allow them to be administered for the treatment of pulmonary TB and determine their biodistribution, pharmacokinetics, toxicity and efficacy in murine models of pulmonary TB. 4. Determine mechanisms of action of the lead Ga formulations against M.tb, potential for emergence of resistance, and effect of the presence of other TB drugs on Ga anti-mycobacterial activity. RESEARCH DESIGN AND METHODOLOGY: Human monocyte-derived macrophages and murine models of TB will be employed along with fully virulent strains of M.tb. The work will emphasize: Fe and Ga acquisition by M.tb: Ga biodistribution, pharmacokinetics, toxicity; and potential bacterial target for Ga antimicrobial activity. Experimental methods employed include: development of Ga formulations suitable for aerosol or nanoparticle-mediated delivery; bacterial uptake of radiolabeled Fe and Ga, growth of M.tb in vitro and in vivo, murine models of pulmonary TB, quantitation of Ga in tissues. POTENTIAL IMPACT ON VETERAN HEALTH CARE: Veterans are at high risk for pulmonary TB. This work could lead to development of novel treatments for TB that would benefit veterans.