A Novel Thioredoxin Mimetic Prodrug for Prevention of Bronchopulmonary Dysplasia

描述（由申请人提供）：Radikal Therapeutics（RTX）正在开发一种针对早产儿的新型药物疗法，该疗法针对硫氧还蛋白（Trx）的发育缺陷，硫氧还蛋白是一种充当细胞还原剂状态的中枢调节剂的蛋白质。Trx在胎儿肺中的表达水平太低，不能充分对抗子宫外生命的氧化还原应激，因此在极端早产中相对缺乏。肺泡内Trx水平的降低被认为是极低出生体重（VLBW）早产儿发生新生儿呼吸窘迫综合征（RDS）的重要原因。我们目前对RDS及其演变为支气管肺发育不良（BPD）的理解与肺实质内过量超氧化物产生的损伤机制一致。使这种超氧化物产生增加的是抗氧化蛋白质的发育缺陷，如超氧化物歧化酶和Trx，它们在早期胎龄时数量不足。我们的创新是基于一种新的代理商，R-908，一个Trx模拟（R-901）的前药，取代缺陷的Trx蛋白和恢复细胞内氧化还原状态的管理。R-901是一种富含巯基的三肽，与天然保守的Trx基序非常相似，并表现出非凡的效力：R- 901在保护培养细胞免受氧化应激方面的效力是N-乙酰半胱氨酸的450倍。在小鼠卵清蛋白模型的肺部炎症模型，R-901减少组织损伤，减少中性粒细胞浸润，减弱组织氧化，阻断促炎细胞因子的表达和核转位的NF-？B，减少抗炎细胞质蛋白I的降解？B，并恢复还原型和氧化型谷胱甘肽的平衡。在急性Cl 2吸入诱导的氧化还原应激的小鼠LD 60模型中，损伤后给予R-901消除了所有死亡率。为了克服R-901的游离巯基的货架不稳定性，我们发明了一种稳定的二硫代酯， 前药（R-908），其在体内释放R-901。现在将在临床相关的BPD大鼠幼仔模型中对R-908进行评价。目的#1：确定R-908在新生大鼠中的药代动力学（PK）。我们将在2日龄大鼠幼仔中确定R-908及其代谢产物R-901的血浆和组织PK特征。目标二：确定R-908可减轻新生大鼠BPD高氧模型中肺血管和肺泡结构的变化; 2日龄大鼠幼仔将接受10天的高氧。R-908将在广泛的剂量范围内给药3周，在该模型系统中，这一时期的特征是进行性肺纤维化、肺动脉高压（PAH）和肺泡发育不良。将分析尸检时采集的肺组织的肺血管结构和生长、肺泡化、脂质过氧化（丙二醛）、还原型和氧化型谷胱甘肽（GSH、GSSG）、过氧亚硝酸盐形成（3-硝基酪氨酸）、聚（ADP-核糖）形成、纤维化（胶原蛋白Mason三色染色）、R-908和R-901水平以及促炎基因表达。将对心脏进行PAH证据的形态测定分析（如右心室（RV）肥大所示）。

DESCRIPTION (provided by applicant): Radikal Therapeutics (RTX) is developing a novel pharmaceutical therapy for prematurity that targets a developmental deficiency in thioredoxin (Trx), a protein that serves as a central regulator of cellular reductant status. Trx is expressed t levels in the fetal lung too low to adequately counter the redox stress of extrauterine life, and i thus relatively deficient in extreme prematurity. The low level of Trx in alveoli is thought to pla a major role in the susceptibility of very low birthweight (VLBW) premature infants to neonatal respiratory distress syndrome (RDS). Our current understanding of RDS and its evolution into bronchopulmonary dysplasia (BPD) is consistent with a mechanism of injury produced by an excess of superoxide within the lung parenchyma. Compounding this increase in superoxide production is the developmental deficiency of anti-oxidant proteins, such as superoxide dismutase and Trx, that are present in insufficient quantity at an early gestational age. Our innovation is based upon the administration of a novel agent, R-908, a prodrug of a Trx mimetic (R-901) that substitutes for the deficient Trx protein and restores intracellular redox status. R-901 is a thiol-rich tripeptide closely analogous to the native conserved Trx motif and exhibits extraordinary potency: R- 901 is 450-fold more potent than N-acetyl cysteine in the protection of cultured cells from oxidant stress. In a murine ovalbumin model of pulmonary inflammation model, R-901 reduced histologic injury, diminished neutrophil infiltration, attenuated tissue oxidation, blocked pro-inflammatory cytokine expression and nuclear translocation of NF-?B, diminished the degradation of the anti-inflammatory cytoplasmic protein I?B¿, and restored the balance of reduced and oxidized forms of glutathione. In a murine LD60 model of redox stress, induced by acute Cl2 inhalation, post-insult administration of R-901 eliminated all mortality. To overcome shelf instability of the free thiol groups of R-901, we have invented a stable dithioester prodrug (R-908) that releases R-901 in vivo. R-908 will now undergo evaluation in a clinically-relevant rat pup model of BPD. Aim #1: Define the pharmacokinetics (PK) of R-908 in newborn rats. We will define the plasma and tissue PK profile of R-908, and its metabolite R-901, in 2-day old rat pups. Aim #2: Establish that R-908 attenuates changes of pulmonary vascular and alveolar structure in a hyperoxic model of BPD in neonatal rats; 2-day old rat pups will be subjected to hyperoxia for 10 days. R-908 will be administered over a broad dose range for 3 weeks, a period characterized in this model system by progressive lung fibrosis, pulmonary arterial hypertension (PAH), and hypoalveolarization. Lung tissue taken at necropsy will be analyzed for pulmonary vascular structure and growth, alveolarization, lipid peroxidation (malondialdehyde), reduced and oxidized glutathione (GSH, GSSG), peroxynitrite formation (3-nitrotyrosine), poly(ADP-ribose) formation, fibrosis (Mason Trichrome staining for collagen), R-908 and R-901 levels, and pro-inflammatory gene expression. The heart will undergo morphometric analysis for evidence of PAH (as shown by right ventricular (RV) hypertrophy).