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、漂白纤维、用于制造抗癫痫药物、染料、阻燃剂、杀虫剂、钻井液和汽油添加剂。当吸入较高浓度时，如在运输事故、集装箱之间转移或恐怖活动中可能发生的情况，Br2 会导致呼吸衰竭死亡。很少有发表的研究评估吸入 Br2 的急性和慢性后遗症；治疗仍然是对症治疗，没有有效的对策。我们令人兴奋且高度新颖的初步数据表明，与野生型同窝对照小鼠相比，过度表达人类血红素加氧酶 (HO)-1 基因和蛋白 (hHO-1 BAC) 的小鼠在 Br2 暴露后返回室内空气时表现出显着较低的死亡率。相比之下，缺乏 HO-1 基因 (HO-1-/-) 的小鼠在接触 Br2 后死亡率显着增加。我们还通过高通量筛选鉴定了五种作为人类 HO-1 基因有效诱导剂的化合物。本申请的目标是：(1) 确定 HO-1 在保护小鼠免受 Br2 诱导损伤中的作用，以及 (2) 测试这些化合物在接触 Br2 后全身给药的小鼠上调 HO-1 并降低死亡率和肺损伤的功效。 SA＃1。我们将在环境室中将 hHO-1 BAC、HO-1-/- 小鼠及其野生型同窝小鼠暴露于 Br2（600 ppm，30 分钟），然后将它们放回室内空气中，并测量死亡率和肺损伤两周。 SA #2：(i) 我们将人类气道 Clara 细胞样细胞 (H441) 的汇合单层暴露于 Br2 中，并将其放回室内空气中；然后，我们将测试通过高通量筛选确定的五种化合物增加 HO-1 并减少 Br2 诱导的细胞坏死和凋亡的功效。 (ii) 我们将对这些化合物进行分析，以确定那些具有与肌内给药相关的最佳药代动力学特性的化合物。 (iii)我们将在体外表现出最佳细胞保护作用的化合物肌肉注射hHO-1 BAC和野生型小鼠，并在注射后2、24和72时测量肺mRNA和hHO-1活性。 (iv) 我们将 hHO-1 BAC、HO- 1-/- 小鼠及其野生型同窝小鼠（均在 C57BL/6 背景中）暴露于 Br2（600 ppm，持续 30 分钟），将它们放回室内空气中，并在暴露该化合物后 2 小时开始肌内注射。我们将测量所有组的死亡率以及肺损伤指数和肺 hHO1 活性，持续 14 天，如 SA #1 中所述。

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.