CAP: Treatment of a Murine Model of Pancreatitis with a NOD1 Inhibitor

细胞内传感器NOD1与多种病原菌相关，具有重要的寄主防御功能。在本研究之前的研究中，我们表明该分子还通过对共生生物的反应参与了非感染性胰腺炎的诱导。特别是，我们首先证明了大剂量蓝蛋白(一种胆囊素受体激动剂)引起的胰腺炎依赖于肠道微生物区系对NOD1的刺激。然后，我们使用胰腺炎模型分析了这种NOD1活性，在胰腺炎模型中，后者是由同时给予低剂量的雨蛋白(本身不会导致胰腺炎)和FK156诱导的，FK156是一种NOD1的激活剂，模拟了突破粘膜屏障的肠道细菌的影响。我们发现，这种“低剂量”的蓝绿色胰腺炎依赖于腺泡细胞产生趋化因子MCP-1和CCR2炎症细胞在胰腺内的流入。此外，我们发现MCP-1的产生涉及转录因子NF-954；B和STAT3的激活，每个转录因子都需要互补的NOD1和蓝蛋白信号。因此，这些研究证实，肠道共生通过胰腺腺泡细胞中的NOD1信号使非感染性胰腺炎症成为可能。 鉴于上述研究表明，NOD1可以是一种诱导炎症状态(在本案例中为胰腺炎)的因子，因此，人们有兴趣尝试寻找可以预防实验性胰腺炎的NOD1抑制剂，从而最终找到一种用于治疗NOD1依赖型人类炎症性疾病的药物。最近，Correa等人(Correa RG Chem Biol 2011，18：825-832)采用高通量筛选NIH文库，该文库包含300,000个化合物，使用包含NF-kappaB报告结构的HEK细胞来鉴定这种NOD1抑制剂。利用这种方法，一种命名为NodInitib-1(ML130)的2-氨基苯并咪唑化合物已被确定为一种有效和特异的NOD1抑制剂，其作用是通过干扰NOD1执行细胞内转运的能力来发挥作用。然而，应该注意的是，这种抑制只通过体外测试才得到证实，而不是在整个动物身上。 我们已经与上述筛查研究的赞助商Sanford-Burnham医学研究所签订了M-CRADA，以获得ML130，用于ML130预防实验性胰腺炎的能力研究。这份M-CRADA现已全面执行，并已将足够的M130送到我们进行适当的研究。后者包括大剂量雨蛋白或小剂量雨蛋白加NOD1配体与ML130联合应用，以确定后者是否能预防大剂量和小剂量雨蛋白胰腺炎。为了促进这些研究，我们利用永久植入静脉导管的小鼠，以便我们可以通过测量血液淀粉酶和MCP-1(以及其他细胞因子和趋化因子)的水平来监测ML130对胰腺炎发展的影响。 在到目前为止进行的研究中，我们使用了一种标准的方案来诱导雨蛙素-胰腺炎，在该方案中，每小时静脉注射雨蛙素诱导胰腺炎(X7)，然后在8小时评估胰腺炎。ML130预防这种胰腺炎的能力是在平行研究中确定的，在这些平行研究中，小鼠在初始蓝蛋白注射和一小时蓝蛋白注射时联合给予雨蛋白和ML-30。我们发现ML130确实抑制了胰腺炎的发展，这是通过停止给药一小时后血清淀粉酶水平和循环中IL-6水平来评估的。这种抑制作用具有统计学意义。

The intracellular sensor NOD1 has important host defense functions relating to a variety of pathogens. In studies antecedent to the present study we showed that this molecule also participated in the induction of a non-infectious pancreatitis via its response to commensal organisms. In particular, we showed first that pancreatitis induced by high-dose cerulein (a cholecystokinin receptor agonist) administration depends on NOD1 stimulation by gut microflora. We then analyzed this NOD1 activity using a model of pancreatitis wherein the latter is induced by the simultaneous administration of low-dose of cerulein (that does not itself induce pancreatitis) and FK156, an activator of NOD1 that mimics the effect of gut bacteria that have breached the mucosal barrier. We found that such "low-dose" cerulein pancreatitis was dependent on acinar cell production of the chemokine MCP-1 and the intra-pancreatic influx of CCR2+ inflammatory cells. Moreover, we established that MCP-1 production involved activation of the transcription factors NF-κB and STAT3, each requiring complementary NOD1 and cerulein signaling. These studies thus established that gut commensals enable non-infectious pancreatic inflammation via NOD1 signaling in pancreatic acinar cells. In the light of the above studies showing that NOD1 can be a factor in the induction of an inflammatory state (in this case pancreatitis), it became of interest to try to identify inhibitors of NOD1 that could conceivable prevent experimental pancreatitis and thus ultimately find use as an agent that would be use of treatment of NOD1-dependent human inflammatory disease. Recently, Correa et al (Correa RG Chem Biol 2011, 18:825-832)employed high through-put screening of an NIH library containing >300,000 compounds to identify such NOD1 inhibitors using HEK cells containing an NF-kappaB reporter construct. Using this approach, a 2-aminobenzimidazole compound designated Nodinitib-1 (ML130) has been identified as a potent and specific NOD1 inhibitor that acts by disturbing the ability of NOD1 to perform intra-cellular trafficking. It should be noted, however, that such inhibition has only been demonstrated by in vitro testing not in the whole animal. We have entered into an M-CRADA with the Sanford-Burnham Medical Research Institute the sponsors of the above described screening study to obtain ML130 for use in studies of the ability of ML130 to prevent experimental pancreatitis. This M-CRADA is now fully executed and sufficient M130 has been sent to us for appropriate studies. The latter consists of administration of high dose cerulein or low dose cerulein plus NOD1 ligand together with ML130 to determine if the latter can prevent high dose and low dose cerulein pancreatitis respectively. To facilitate these studies we utilize mice with permanently implaced intravenous catheters so that we can monitor the effects of ML130 on pancreatitis development by measuring blood levels of amylase and MCP-1 (as well as other cytokines and chemokines). In studies conducted so far, we have induced cerulein-pancreatitis using a standard protocol in which pancreatitis is induced by IV administration of cerulein at hourly intervals (X7) followed by assessment of pancreatitis at 8 hours. The capacity of ML130 to prevent development of such pancreatitis was determined in parallel studies in which mice were co-administered cerulein and ML-30 at the initial cerulein injection and at the one hour cerulein injection. We found that ML130 did inhibit inhibit pancreatitis development as assessed by serum amylase levels and IL-6 levels in the circulation at one hour after cessation of cerulein administration. This inhibition was statistlcally significant.