Selective inhibition of p38 mitogen activated protein kinase with the pyridinyl

宿主炎症介质的过度释放直接参与了脓毒症和脓毒性休克的发病机制。丝裂原活化蛋白激酶（MAPK）是丝氨酸/苏氨酸细胞内信号蛋白，在这种炎症反应中发挥核心作用。这些MAPK被磷酸化激活，并介导基因表达和响应细胞外信号的其他细胞功能。哺乳动物细胞中的三种主要MAPK是p38、细胞外信号调节激酶（ERK）和c-jun N-末端激酶（JNK）。这些MAPK中的每一个调节响应于脂多糖（LPS）的炎症介质产生，脂多糖是与革兰氏阴性细菌性肺炎和脓毒症的发病机制密切相关的细菌毒素。这些介质包括细胞因子、趋化因子、一氧化氮、活性氧和前列腺素代谢物。基于大量数据支持MAPK在炎症中的关键作用，对这些蛋白质的选择性抑制剂的治疗应用越来越感兴趣。然而，这些药物在脓毒症中的应用主要是在仅采用LPS攻击的体内模型中进行测试的。由于许多介质MAPKs调节参与保护性先天免疫或宿主防御反应以及炎症损伤，它们的抑制在细菌感染期间可能是有害的。我们用炭疽杆菌致死毒素（LeTx）进行的一项研究的数据支持MAPK抑制可能有风险和益处的可能性。LeTx的致死因子（LF）组分是锌蛋白酶，其切割并灭活促分裂原活化蛋白激酶（MAPKK）家族的几个不同成员及其下游靶标，包括p38。我们发现，当LeTx以亚致死剂量给予致死LPS攻击的动物时，炎性细胞因子和一氧化氮释放减少，血流动力学功能和存活率改善。然而，在用肠内E.大肠杆菌，同时也减少细胞因子和一氧化氮的释放，降低生存。这些发现表明，这些MAPK的非选择性抑制可能对活细菌有害。然而，选择性抑制这些MAPK可能仍然是有益的。<br><br>SB 203580 4-（4-flurophenyl）-2-（4-methylsulfinylphenyl）-5-（4-pyridyl）imidazole是一种吡啶基咪唑，在体外和体内炎症模型中广泛用作p38的选择性抑制剂。该试剂竞争p38上的ATP结合位点并抑制其磷酸化和活化。在体内模型中，通过几种不同的途径以及在炎症刺激之前和之后给予该药剂已显示出抑制细胞因子和一氧化氮释放、白细胞运输和器官损伤。该试剂和相关试剂也已显示可改善LPS攻击模型中的存活率。尽管这些研究的结果为在炎症状态（如脓毒症）期间使用p38抑制剂提供了主要依据，但广泛的文献检索仅显示了一份已发表的报告（12），该报告评估了该药物或相关药物在采用活细菌挑战的脓毒症动物模型中的作用。这是一项在接受盲肠结扎和穿孔（CLP）的小鼠中进行的非常有限的研究，未纳入抗生素支持或微生物评估。然而，该研究的结果确实表明，当其给药在脓毒症发作后延迟12小时时，SB 203580的有效性增加。<br><br>我们以前建立了E.在这种情况下，抗生素和液体被证明可以协同提高生存率，因为它们被认为在临床上是如此。该模型目前正用于测试SB 203580的效果。在最初的实验中，我们发现，以前在非常有限的研究中报告的这种药剂的剂量是有益的，实际上在小鼠模型中细菌攻击前1小时或细菌攻击后1小时给药时似乎是有害的。延迟治疗12小时不会导致有害影响，但也不是有益的。将SB 203580的剂量减少3 log剂量导致该药剂的有益效果，但仅当其以非对称方式给予时。后来的治疗没有任何好处。在随后的研究中，我们发现，尽管高剂量和低剂量的SB 203580都能改善超声心动图测量的血流动力学功能，但高剂量但现在低剂量使E.大肠杆菌相关的炎性肺损伤。总体而言，这些研究表明，抑制p38的药物可能对细菌感染或有细菌感染风险的患者产生不利影响。

Excessive release of host inflammatory mediators contributes directly to the pathogenesis of sepsis and septic shock. Mitogen-activated protein kinases (MAPKs) are serine/threonine intracellular signaling proteins that have a central role in this inflammatory response. These MAPKs are activated by phosphorylation and mediate gene expression and other cellular functions in response to extracellular signals. Three major MAPKs in mammalian cells are p38, extracellular signal-regulated kinase (ERK), and c-jun N-terminal kinase (JNK). Each of these MAPKs regulates inflammatory mediator production in response to lipopolysaccharide (LPS), a bacterial toxin closely associated with the pathogenesis of gram-negative bacterial pneumonia and sepsis. These mediators include cytokines, chemokines, nitric oxide, reactive oxygen species, and prostaglandin metabolites. Based on substantial data supporting a pivotal role for MAPKs in inflammation, there is growing interest in the therapeutic application of selective inhibitors of these proteins. However, the application of such agents in sepsis has been primarily tested in in vivo models employing LPS challenge only. Since many of the mediators MAPKs regulate participate in protective innate immune or host defense responses as well as inflammatory injury, their inhibition could be harmful during bacterial infection. Data from a study we conducted with Bacillus anthracis lethal toxin (LeTx) supports the possibility that there may be risks as well as benefits with MAPK inhibition. The lethal factor (LF) component of LeTx is a zinc protease that cleaves and inactivates several different members of the mitogen-activated protein kinase (MAPKKs) family and their downstream targets including p38. We showed that when LeTx was administered in sublethal doses to animals challenged with lethal LPS, inflammatory cytokine and nitric oxide release were reduced and hemodynamic function and survival improved. However, similar pretreatment in animals challenged with intratracheal E. coli, while also reducing cytokine and nitric oxide release, decreased survival. These findings suggested that nonselective inhibition of these MAPKs may be harmful with live bacteria. Selective inhibition of these MAPKs may still be beneficial however.<br><br>SB203580 4-(4-flurophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole is a pyridinyl imidazole that has been employed extensively as a selective inhibitor of p38, in both in vitro and in vivo models of inflammation. This agent competes for the ATP binding site on p38 and inhibits its phosphorylation and activation. In in vivo models, administration of this agent via several different routes, as well as both before and after inflammatory stimulus, has been shown to inhibit cytokine and nitric oxide release, leukocyte trafficking, and organ injury. This agent and a related one have also been shown to improve survival in LPS challenged models. Although the results of such studies have provided a major rational for the use of p38 inhibitors during states of inflammation such as sepsis, an extensive literature search has revealed only one published report (12) assessing the effects of this or related agents in an animal model of sepsis employing live bacterial challenge. This was a very limited study in mice undergoing cecal ligation and puncture (CLP) and did not incorporate antibiotic support or microbiological assessment. The results of this study did suggest however that the effectiveness of SB203580 was increased when its administration was delayed for 12 h after the onset of sepsis. <br><br>We previously developed a mouse model of E. coli pneumonia in which antibiotics and fluids were shown to synergistically improve survival as they are believed to do clinically. This model is now being used to test the effects of SB203580. In initial experiments we have found that doses of this agent previously reported to be beneficial in very limited studies, actually appeard harmful when administered either 1 h before or 1 h after bacterial challenge in the mouse model. Delaying treatment for 12 h is did not result in harmful effects but was also not beneficial. Reducing the dose of SB203580 3 logs doses resulted in beneficial effects with the agent, but only if it is given prophylactically. Later treatment had no benefit. In subsequent studies we have now found that although both high and low doses of SB203580 improve hemodynamic function measured echocardiographically, high but now low doses worsen E. coli associated inflammatory lung injury. Overall these studies suggest that agents inhibiting p38 may have detrimental effects in patients with or at risk of bacterial infection.