Celecoxib Derivative: Host Cell-Directed Inhibitors of Intracellular Pathogens

项目摘要 AR-12是IND批准的考克斯-2抑制剂衍生药物，其主要通过上调- 调节自噬。在体外，AR-12对几种细菌菌株显示出广谱功效，包括 S.鼠伤寒沙门氏菌（F. typhimurium）; tularensis（Schu S4，LVS），和F. novicida。在体内，AR-12静脉内给药减少了器官 细菌的10倍相比，未经处理的对照，但并没有防止宿主死亡，由于伤寒。AR-12 由于药物的疏水性，体内应用的浓度受到限制。克服 溶解性问题，我们提出将AR-12封装在缩醛化葡聚糖（Ac-DEX）颗粒中， 靶向宿主细胞Ac-DEX是一种酸敏感聚合物，具有可调的释放动力学， 吞噬细胞的吞噬体，由于存在较低的pH值。R21部分有3个目标 提议第一个目的是制造和表征包封AR的两种类型的Ac-DEX颗粒。 12：1）用于i. v.或i. p.注射的纳米颗粒（NP），其具有用于被动靶向巨噬细胞的理想尺寸 （500- 1，000 nm）; 2）多孔微粒（PMP），其是通过鼻（i.n.）吸入的理想尺寸（5-15 μ m）; 并渗透至鼻相关淋巴组织（NALT）。这些粒子将被制造，成像 通过扫描电子显微镜，并通过荧光光谱表征药物负载。 第二个目的是评价最低抑菌浓度（MIC）和最低杀菌浓度（MIC）。 未包封的AR-12对药物敏感和多药耐药M的浓度（MBC）。 结核分枝杆菌（MDR TB）和抗TB的包封AR-12，F. tularensis（Schu S4）和S.鼠伤寒 人类巨噬细胞。巨噬细胞将感染三种细菌之一，并用AR-12治疗。细胞 将评估相关细菌和CFU以确定MIC和MBC。的MIC和MBC 确定为F。土拉热菌将用于目标3中的体内治疗，其中施用包封的AR-12 用NP i. v.和i. p.，和i.n.与PMP将使用给药研究确定最佳途径和剂量 (MIC，MBC，10 xMBC），通过评价器官CFU， 传统的抗生素疗法。最佳途径和剂量将用于深入治疗评价 器官CFU、组织病理学、血液细胞因子水平和存活率。R33进展的里程碑 资助的一部分将是1）AR-12在Ac-DEX颗粒中的包封; 2）减少巨噬细胞相关的 M. AR-12治疗的肺结核; 3）AR-12增加的存活率和降低的器官细菌负荷 治疗F.土拉热，在体内。该补助金的R33阶段也有三个具体目标。目标4和5是 评价针对S.鼠伤寒沙门氏菌和结核杆菌， 类似于目标3。目的6评价微囊化AR-12的免疫毒性， AR-12申请IND批准。这些研究将有助于开发和表征一种新的广谱 靶向宿主细胞的抗生素和递送平台。

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.