Celecoxib Derivative: Host Cell-Directed Inhibitors of Intracellular Pathogens

项目摘要 AR-12是一种IND批准的环氧合酶-2抑制剂衍生药物，主要通过上调表达来影响病原体宿主细胞。 调节自噬。在体外，AR-12对几种细菌菌株显示出广谱的效果，包括 鼠伤寒沙门氏菌(S.typhimurium)、图拉氏镰刀菌F.tularensis(Schu S4，LVS)和新氏镰刀菌F.novicida。在体内，静脉注射AR-12。减少的器官 细菌是未经治疗的对照组的10倍，但不能防止由于伤寒造成的宿主死亡。AR-12 由于药物的疏水性，体内应用的浓度受到限制。要克服 溶解性问题，我们建议将AR-12包裹在缩醛葡聚糖(Ac-DEX)颗粒中，被动地 瞄准宿主细胞。AC-DEX是一种酸敏感聚合物，具有可调的释放动力学，将药物释放到 吞噬细胞的吞噬小体，由于存在较低的pH。R21部分有3个目标 求婚。第一个目标是制造和表征两种类型的Ac-DEX颗粒，它们包裹了AR-DEX- 12：1)静脉注射用纳米粒(NP)。或者IP。适合被动靶向巨噬细胞的理想大小的注射 (500-1,000 nm)；2)理想尺寸(5-15m)的多孔微粒(PMP)，适合经鼻吸入(I.N.) 以及对鼻腔相关淋巴组织(NALT)的穿透。这些颗粒将被制造、成像 通过扫描电子显微镜观察，并通过荧光光谱对载药量进行表征。 第二个目标是评估最小抑菌浓度(MIC)和最小杀菌浓度 无囊化AR-12对药物敏感和多药耐药M。 结核分枝杆菌(MDR TB)和包裹的AR-12抗结核杆菌、图拉氏杆菌(Schu S4)和鼠伤寒沙门氏菌 人类巨噬细胞。巨噬细胞将被感染三种细菌中的一种，并用AR-12治疗。细胞 将对相关细菌和CFU进行评估，以确定MIC和MBC。MIC和MBC 为图拉氏丝孢子虫确定的将在AIM 3中用于体内治疗，并给予微囊化AR-12 静脉注射。和IP。带着NPs和I.N.穿着PMP。将使用剂量研究来确定最佳路线和剂量 (MIC，MBC，10xMBC)通过评估器官CFU，当给药的时间和频率与 传统的抗生素疗法。最佳的治疗路线和剂量将用于深入的治疗评估 器官CFU、组织病理学、血液细胞因子水平和存活率。迈向R33的里程碑 赠款的一部分将是1)将AR-12包裹在Ac-DEX颗粒中；2)减少巨噬细胞相关 用AR-12治疗结核分枝杆菌；3)用AR-12提高存活率和降低器官细菌负荷 在活体内治疗图拉氏丝虫。这笔赠款的R33阶段也有3个具体目标。目标4和目标5是为了 分别以一种方式评价包裹的AR-12对鼠伤寒沙门氏菌和结核杆菌的体内治疗作用 类似于目的3。目的6是评价微囊化AR-12的免疫毒性，并对微囊化的进展 AR-12向IND批准。这些研究将有助于开发和表征新的广谱 抗生素和以宿主细胞为目标的递送平台。

Project Summary AR-12 is an IND approved, COX-2 inhibitor derived drug that affects pathogen host cells by primarily up- regulating autophagy. In vitro, AR-12 has shown broad spectrum efficacy on several bacterial strains including S. typhimurium, F. tularensis (Schu S4, LVS), and F. novicida. In vivo, AR-12 given i.v. reduced organ bacterial 10-fold compared to untreated controls, but did not prevent host death due to typhoid fever. AR-12 concentrations were limited with in vivo application because the of the drug's hydrophobicity. To overcome solubility issues, we propose encapsulating AR-12 in acetalated dextran (Ac-DEX) particles that passively target the host cell. Ac-DEX is an acid sensitive polymer with tunable release kinetics that will release drug in the phagocyte's phagosome, due to the lower pH present. There are 3 aims for the R21 portion of this proposal. The first aim is to manufacture and characterize two types of Ac-DEX particles that encapsulate AR- 12: 1) a nanoparticle (NP) for i.v. or i.p. injection that is at the ideal size for passively targeting macrophages (500-1,000 nm); 2) a porous microparticle (PMP) that is ideal size (5-15 m) for inhalation via the nose (i.n.) and penetration to the nasal associated lymphoid tissue (NALT). These particles will be manufactured, imaged through scanning electron microscopy, and characterized for drug loading through fluorescence spectroscopy. The second aim is to evaluate the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of unencapsulated AR-12 against drug susceptible and multidrug resistant M. tuberculosis (MDR TB) and encapsulated AR-12 against TB, F. tularensis (Schu S4), and S. typhimurium in human macrophages. Macrophages will be infected with one of three bacteria and treated with AR-12. Cell associated bacteria and CFUs will be evaluated to determine both the MIC and MBC. The MIC and MBC determined for F. tularensis will be used in Aim 3 for in vivo treatment with encapsulated AR-12 administered i.v. and i.p. with NPs, and i.n. with PMPs. A dosing study will be used to identify the best route and dosage (MIC, MBC, 10xMBC) by evaluating organ CFUs when administered at a comparable timing and frequency to traditional antibiotic regimens. The optimum route and dose will be used for an in depth treatment evaluation of organ CFUs, histopathology, blood cytokine levels and survival. The milestones for progress to the R33 portion of the grant will be 1) encapsulation of AR-12 in Ac-DEX particles; 2) reduced macrophage associated M. tuberculosis with AR-12 treatment; 3) Increased survival and decreased organ bacterial load with AR-12 treatment of F. tularensis, in vivo. The R33 phase of this grant also has 3 specific aims. Aim 4 and 5 are to evaluate encapsulated AR-12 treatment in vivo against S. typhimurium, and TB, respectively in a manner similar to Aim 3. Aim 6 is to evaluate the immunotoxicity of encapsulated AR-12 and to progress encapsulated AR-12 towards IND approval. These studies will help to develop and characterize a new broad spectrum antibiotic and delivery platform that targets host cells.