Cytochrome P450 in Reperfusion Injury

描述（由申请人提供）：在该提案中，我们提出了涉及心肌缺血/再灌注（I/R）损伤中细胞色素P450单加氧酶（CYP）的新数据。我们已经表明，用氯霉素或磺胺苯吡唑等化合物抑制心肌缺血和再灌注后的组织损伤可以减少。重要的是，用氯霉素或磺胺苯吡唑预处理可使梗死面积减少70%。在离体灌注心脏模型中，这些药物即使在缺血后给药也是有益的。在目标1中，我们将扩展现有的初步研究，目的如下：确定在离体灌注心脏模型中的最小有效剂量;确定给药前延迟多长时间仍具有保护作用;以及评估磺胺苯吡唑（迄今为止确定的最有效的先导化合物）在麻醉家兔局部缺血和再灌注模型中的疗效。由于我们已经证明具有心脏保护作用的几种β-内酰胺酶抑制剂已经安全地用于人类的其他目的，因此希望这些临床前研究将为随后的临床试验奠定基础。我们还将确定心脏中CYP的上调是否会增加I/R损伤。在目标2中，我们试图了解这些CYP抑制剂的心脏保护机制，因为这将深入了解CYP在缺血/再灌注后引起心肌损伤的作用。为此，我们将确定在心脏中被磺胺苯吡唑和氯霉素抑制的类花生四烯酸异构体，并将考虑两种可能的损伤机制-产生过量的活性氧，以及花生四烯酸代谢失调，从而产生血管活性和心脏毒性的类花生四烯酸。在目的3中，我们考虑了CYP对SR Ca +2 ATP酶和线粒体KATP通道的潜在影响。基于抗凋亡蛋白ARC（具有CARD结构域的凋亡抑制物）和Bcl-xL抑制大鼠肝微粒体中的CYP活性，而促凋亡蛋白tBid刺激大鼠肝微粒体中的CYP活性的发现，我们讨论CYP在凋亡中的潜在作用。该建议将细胞色素P450单加氧酶确定为以前被低估的心肌再灌注损伤因素，并为新的治疗方法奠定了基础

DESCRIPTION (provided by applicant): In this proposal, we present new data implicating cytochrome P450 monooxygenases (CYP) in myocardial ischemia/reperfusion (I/R) injury. We have shown that inhibition of CYP with compounds such as chloramphenicol or sulfaphenazole can reduce tissue injury following myocardial ischemia and reperfusion. Importantly, infarct size is reduced by up to 70 percent by pretreatment with chloramphenicol or sulfaphenazole. In the isolated perfused heart model, these drugs are beneficial even when administered after ischemia. In Aim 1 we will extend the existing preliminary studies with the following objectives: to establish the minimum effective dose in the isolated perfused heart model; to establish how long a delay before drug administration is still protective; and to assess efficacy of sulfaphenazole (the most potent lead compound identified thus far) in a model of regional ischemia and reperfusion in the anesthetized rabbit. Since several of the CYP inhibitors we have shown to be cardioprotective are already safely used in humans for other purposes, it is hoped that these preclinical studies will lay the groundwork for a subsequent clinical trial. We will also establish whether upregulation of CYPs in the heart increases I/R injury. In Aim 2, we seek to understand the mechanism of cardioprotection by these CYP inhibitors, as this will yield insights into the role of CYPs in causing myocardial damage after ischemia/reperfusion. To this end, we will identify the CYP isoforms in heart that are inhibited by sulfaphenazole and chloramphenicol, and will consider the two likely mechanisms of injury-production of excessive reactive oxygen species, and the dysregulated metabolism of arachidonic acid to vasoactive and cardiotoxic eicosanoids. In Aim 3 we consider the potential effect of CYPs on the SR Ca+2 ATPase and the mitochondrial KATP channel. We address the potential role of CYPs in apoptosis, based on the finding that the anti-apoptotic proteins ARC (apoptosis repressor with CARD domain) and Bcl-xL inhibit CYP activity, while pro-apoptotic tBid stimulates CYP activity in rat liver microsomes. This proposal identifies cytochrome P450 monooxygenases as a previously underestimated factor in myocardial reperfusion injury, and establishes the basis for a novel therapeutic approach