Celecoxib Derivative: Host Cell-Directed Inhibitors of Intracellular Pathogens

描述（由申请人提供）：AR-12是IND批准的COX-2抑制剂衍生药物，主要通过上调自噬来影响病原体宿主细胞。在体外，AR-12对包括鼠伤寒沙门氏菌、土拉螺旋体（Schu S4， LVS）和新螺旋体在内的几种细菌菌株显示出广谱疗效。在体内，与未经治疗的对照组相比，静脉注射AR-12使器官细菌减少了十倍，但并不能防止宿主因伤寒而死亡。由于药物的疏水性，AR-12浓度在体内应用时受到限制。为了克服溶解度问题，我们建议将AR-12封装在醋酸化葡聚糖（Ac-DEX）颗粒中，被动靶向宿主细胞。Ac-DEX是一种酸敏感聚合物，具有可调节的释放动力学，由于pH值较低，它将在吞噬细胞的吞噬体中释放药物。该提案的R21部分有三个目标。第一个目标是制造和表征两种类型的包裹AR- 12的Ac-DEX颗粒：1)用于静脉注射或口服注射的纳米颗粒（NP），其尺寸为被动靶向巨噬细胞的理想尺寸（500-1,000 nm）；2)多孔微粒（PMP），理想尺寸（5- 15mm），可通过鼻腔吸入并穿透鼻腔相关淋巴组织（NALT）。这些颗粒将被制造出来，通过扫描电子显微镜成像，并通过荧光光谱表征药物负载。第二个目的是评价未包封AR-12对药敏和耐多药结核分枝杆菌（MDR TB）的最小抑制浓度（MIC）和最小杀菌浓度（MBC），以及包封AR-12对TB、土拉菌（Schu S4）和鼠伤寒沙门氏菌（S. typhimurium）在人巨噬细胞中的作用。巨噬细胞将被三种细菌中的一种感染，然后用AR-12治疗。将评估细胞相关细菌和cfu，以确定MIC和MBC。为土拉螺旋体测定的MIC和MBC将在Aim 3中用于包裹AR-12的体内治疗，与NPs一起静脉注射，与pmp一起静脉注射。一项剂量研究将通过评估与传统抗生素方案在相当的时间和频率下给药时的器官cfu来确定最佳途径和剂量（MIC、MBC和10倍MBC）。最佳途径和剂量将用于器官cfu，组织病理学，血液细胞因子水平和生存的深入治疗评估。R33部分进展的里程碑将是：1)将AR-12封装在Ac-DEX颗粒中；2) AR-12治疗减少巨噬细胞相关结核分枝杆菌；3)在体内，AR-12治疗土拉菌可提高生存率，降低器官细菌负荷。该赠款的R33阶段也有3个具体目标。目的4和5是评估包封AR-12在体内对鼠伤寒沙门氏菌和结核病的治疗效果，方法与目的3类似。目的6是评估包封AR-12的免疫毒性，并推动包封AR-12获得IND批准。这些研究将有助于开发和表征一种新的广谱抗生素和靶向宿主细胞的给药平台。

DESCRIPTION (provided by applicant): AR-12 is an IND approved, COX-2 inhibitor derived drug that affects pathogen host cells by primarily upregulating 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 tenfold 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 three 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 mm) 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, and 10 x MBC) 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. PUBLIC HEALTH RELEVANCE: Therapies that target the host cell rather than the bacteria, which traditional antibiotics primarily focus, can help to combat pathogens that have drug resistance. AR-12 is an antibiotic that has shown broad spectrum activity against multiple bacterial pathogens by primarily effecting the infected host cell. We propose to further evaluate AR-12 in animal models against bacteria that cause salmonella, tularemia, drug susceptible tuberculosis and multi-drug resistant tuberculosis.