Celecoxib Derivative: Host Cell-Directed Inhibitors of Intracellular Pathogens

描述(申请人提供)：AR-12是一种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、图拉沙门氏菌(Schu S4，LVS)和新城疫沙门氏菌在内的几种细菌菌株显示出广谱的疗效。在体内，静脉注射AR-12。与未经治疗的对照组相比，器官细菌减少了10倍，但并未防止因伤寒而导致的宿主死亡。由于药物的疏水性，Ar-12的浓度在体内应用受到限制。为了克服溶解性问题，我们建议将AR-12包裹在被动靶向宿主细胞的缩醛葡聚糖(Ac-DEX)颗粒中。AC-DEX是一种酸敏感聚合物，具有可调节的释放动力学，由于存在较低的pH，它将在吞噬细胞的吞噬小体中释放药物。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包裹AR-12的Ac-DEX颗粒：1)静脉注射用纳米颗粒(NP)。或者IP。适合被动靶向巨噬细胞(500-1,000 nm)的理想大小的注射剂；2)理想大小(5-15 mm)的多孔微粒(PMP)，适合经鼻腔吸入(I.N.)以及对鼻腔相关淋巴组织(NALT)的穿透。这些微粒将被制造出来，通过扫描电子显微镜成像，并通过荧光光谱表征药物载药量。第二个目的是评估未包裹的AR-12对药物敏感和耐多药结核分枝杆菌(MDR TB)的最低抑菌浓度(MIC)和最小杀菌浓度(MBC)，以及包裹AR-12对结核杆菌、图拉氏杆菌(Schu S4)和鼠伤寒沙门氏菌的最低抑菌浓度(MIC)和最小杀菌浓度(MBC)。巨噬细胞将被感染三种细菌中的一种，并用AR-12治疗。将对细胞相关细菌和CFU进行评估，以确定MIC和MBC。为图拉氏丝虫测定的MIC和MBC将用于AIM 3体内注射AR-12的治疗。和IP。带着NPs和I.N.穿着PMP。剂量研究将用于通过评估器官CFU来确定最佳路线和剂量(MIC、MBC和10倍MBC)，这些器官CFU的给药时间和频率与传统抗生素方案相当。最佳途径和剂量将用于对器官CFU、组织病理学、血液细胞因子水平和存活率进行深入治疗评估。进展到赠款的R33部分的里程碑将是1)将AR-12包裹在Ac-DEX颗粒中；2)通过AR-12治疗减少与巨噬细胞相关的结核分枝杆菌；3)通过AR-12治疗在活体内提高存活率和降低器官细菌负荷。这笔赠款的R33阶段也有3个具体目标。目的4和目的5是分别以类似于目的3的方式评价微囊化AR-12对鼠伤寒沙门氏菌和结核杆菌的体内治疗作用。目的6是评估微囊化AR-12的免疫毒性，并将微囊化AR-12推向IND批准。这些研究将有助于开发和表征一种新的针对宿主细胞的广谱抗生素和给药平台。 与公共卫生相关：针对宿主细胞而不是传统抗生素主要关注的细菌的疗法可以帮助对抗具有耐药性的病原体。AR-12是一种抗生素，主要作用于受感染的宿主细胞，对多种细菌病原体具有广谱活性。我们建议在动物模型中进一步评估AR-12对引起沙门氏菌、图拉热症、药物敏感性结核病和多药耐药结核病的细菌的作用。

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.