Selective inhibition of p38 mitogen activated protein kinase with the pyridinyl

宿主炎症介质的过度释放直接导致脓毒症和感染性休克的发生。丝裂原活化蛋白激酶(MAPK)是细胞内丝氨酸/苏氨酸信号蛋白，在炎症反应中起核心作用。这些MAPK被磷酸化激活，并介导基因表达和其他细胞功能，以响应细胞外信号。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氨基末端激酶(JNK)。这些MAPK中的每一个都调节炎症介质的产生，以响应内毒素(LPS)，内毒素是一种与革兰氏阴性细菌性肺炎和脓毒症的发病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MAPKs在炎症中的关键作用，人们对这些蛋白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只使用内毒素激发的活体模型中进行测试。由于许多MAPK调节的介质参与保护性的先天免疫或宿主防御反应以及炎性损伤，它们的抑制在细菌感染过程中可能是有害的。我们对炭疽杆菌致死毒素(LeTx)进行的一项研究的数据支持了抑制MAPK可能既有好处也有风险的可能性。LeTx的致死因子(Lf)组分是一种锌蛋白酶，它能裂解和失活丝裂原激活蛋白激酶(MAPKKs)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性内毒素攻击的动物时，炎性细胞因子和一氧化氮的释放减少，血流动力学功能和存活率提高。然而，对气管内感染大肠杆菌的动物进行类似的预处理，虽然也减少了细胞因子和一氧化氮的释放，但降低了存活率。这些发现表明，对这些MAPK的非选择性抑制可能对活细菌有害。然而，选择性地抑制这些MAPK可能仍然是有益的。 SB203580 4-(4-flurophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole是一种吡啶基咪唑类化合物，在体外和体内炎症模型中被广泛用作p38的选择性抑制剂。该试剂竞争p38上的ATP结合位点，并抑制其磷酸化和激活。在体内模型中，通过几种不同的途径以及在炎症刺激前后给药，已经显示出抑制细胞因子和一氧化氮的释放、白细胞的运输和器官损伤。该制剂和一种相关制剂也被证明能提高内毒素攻击模型的存活率。尽管这类研究的结果为在炎症状态如脓毒症时使用p38抑制剂提供了主要的理由，但广泛的文献搜索显示，只有一篇已发表的报告(12)在使用活菌激发的脓毒症动物模型中评估了该药物或相关药物的效果。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，没有纳入抗生素支持或微生物学评估。然而，这项研究的结果确实表明，如果SB203580在脓毒症发生后延迟12小时给药，其疗效会增加。 我们之前开发了一个大肠杆菌肺炎的小鼠模型，在该模型中，抗生素和液体被证明可以协同提高存活率，正如他们被认为在临床上所做的那样。这个模型现在被用来测试SB203580的效果。在最初的实验中，我们发现这种制剂的剂量以前在非常有限的研究中被报道是有益的，但在小鼠模型中，无论是在细菌攻击前1小时还是之后1小时给药，实际上都是有害的。推迟治疗12小时并无不良影响，但也无益。减少SB203580 3对数剂量对该药物有有益影响，但前提是预防性给药。后来的治疗没有任何好处。在随后的研究中，我们现在发现，尽管预防剂量的SB203580对心脏功能的影响与超声心动图评估的相似，但它们对炎性细胞因子释放的影响是不同的。重要的是，虽然低剂量与细胞因子的早期释放增加和后来的减少有关，但高剂量会产生一致的减少。因此，高剂量但不是低剂量的SB203580的有害作用可能与宿主介质的差异表达有关，这些介质不仅对炎性组织损伤很重要，而且对宿主防御也很重要。其他研究正在调查SB203580对P38水平的影响。

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. 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. 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 while high and low prophylactic doses of SB203580 have similar effects on myocardial function as assessed by echocardiography, their effects on inflammatory cytokine release differed. Importantly, while low doses were associated with increased cytokine release early and decreases later, high doses produce uniform reductions. Thus, the harmful effects of high but not low doses of SB203580 may relate to differential expression of host mediators that are important not only for inflammatory tissue injury but also for host defense as well. Additional studies are ongoing investigating the effects of SB203580 on P38 levels.