Central Role of Heme Oxygenase in Reversing Bromine Morbidity and Mortality

描述(申请人提供)：溴(BR2)是一种卤素，用作水消毒剂，用于漂白纤维，在抗癫痫药物、染料、阻燃剂、杀虫剂、钻井液和汽油添加剂的制造中吸入高浓度时，如在运输事故、集装箱之间的转移或恐怖行为中可能发生，BR2已导致呼吸衰竭死亡。很少有发表的研究评估吸入BR2的急性和慢性后遗症；治疗仍然是有症状的，没有有效的对策存在。我们令人兴奋和非常新颖的初步数据表明，与野生型对照相比，过度表达人类形式的血红素加氧酶(HO)-1基因和蛋白(HHO-1BAC)的小鼠在BR2暴露后返回室内空气中的死亡率显著低于野生型对照小鼠。相比之下，缺乏HO-1基因(HO-1-/-)的小鼠在暴露于BR2后表现出明显的死亡率增加。我们还利用高通量筛选确定了五个化合物，它们是人类HO-1基因的有效诱导物。这项应用的目标是：(1)确定HO-1在保护小鼠免受BR2损伤中的作用，以及(2)测试这些化合物在BR2暴露后系统地给药，上调HO-1，降低死亡率和肺损伤的有效性。我们将HHO-1 BAC、HO-1-/-小鼠及其野生型仔鼠暴露于环境舱中的BR2(600ppm，30分钟)，并将它们放回室内空气中，测量两周的死亡率和肺损伤。SA#2：(I)我们将人呼吸道Clara细胞样细胞(H441)的融合单层暴露于BR2，并将它们放回室内空气中；然后，我们将测试通过高通量筛选确定的五种化合物在增加HO-1和减少BR2诱导的细胞坏死和凋亡方面的有效性。(Ii)我们将对这些化合物进行分析，以确定与肌肉注射剂量相关的具有最佳药代动力学特性的化合物。(3)用体外细胞保护作用最好的化合物对HHO-1 BAC和野生型小鼠进行肌肉注射，分别于注射后2、24、72天测定肺组织中HHO-1基因的表达和活性。(Iv)我们将HHO-1 BAC、HO-1-/-小鼠及其野生型仔鼠(均为C57BL/6背景)暴露于BR2(600ppm，30分钟)，将它们放回室内空气中，并从暴露后2小时开始肌肉注射该化合物。我们将测量所有组的死亡率以及肺损伤指数和肺hHO1活性14天，如SA#1所述。

DESCRIPTION (provided by applicant): Bromine (Br2) is a halogen used as a water disinfectant, for bleaching fibers, in the manufacture of antiepileptic drugs, dyestuffs, flame retardants, insecticides, drilling fluids, and gasoline additives When inhaled at higher concentrations, as may occur during transportation accidents, transfers among containers or acts of terrorisms, Br2 has caused death from respiratory failure. There are very few published studies evaluating acute and chronic sequelae of Br2 inhalation; treatment remains symptomatic and no effective countermeasures exist. Our exciting and highly novel preliminary data show that mice overexpressing the human form of the heme oxygenase (HO)-1 gene and protein (hHO-1 BAC) exhibit significantly lower mortality when returned to room air post Br2 exposure as compared to their wild-type littermate controls. In contrast, mice deficient in the HO-1 gene (HO-1-/-), display markedly increased mortality following Br2 exposure. We have also identified five compounds that are potent inducers of the human HO-1 gene using high-throughput screening. The goals of this application are: (1) to establish the role of HO-1 in protecting mice from Br2 induced injury and (2) test the efficacy of these compounds, administered in mice systemically, post Br2 exposure, to upregulate HO-1, and decrease mortality and lung injury. SA #1. We will expose hHO-1 BAC, HO-1-/- mice and their wild-type littermates to Br2 (600 ppm for 30 min) in environmental chambers, and return them to room air and measure mortality and lung injury for two weeks. SA #2: (i) We will expose confluent monolayers of human airway Clara-cell like cells (H441) to Br2 and return them to room air; we will then test the efficacy of the five compounds identified by high throughput screening, to increase HO-1 and decrease Br2 induced cellular necrosis and apoptosis. (ii) We will profile these compounds to determine those with the best pharmacokinetic properties related to intramuscular dosing. (iii) We will inject hHO-1 BAC and wild-type mice intramuscularly with the compound that exhibits the best cytoprotective effects in vitro and measure lung mRNA and hHO-1 activity at two, 24 and 72 post injection. (iv) We will expose hHO-1 BAC, HO- 1-/- mice and their wild-type littermates (all in C57BL/6 background) to Br2 (600 ppm for 30 min), return them to room air and inject them intramuscularly starting at 2 h post-exposure with this compound. We will measure mortality in all groups and indices of lung injury and lung hHO1 activity for 14 d as described in SA #1.