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

细胞内传感器NOD1对多种病原体具有重要的宿主防御功能。在本研究之前的研究中，我们发现该分子也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，我们首先表明，高剂量给药蓝蛋白（一种胆囊收缩素受体激动剂）诱导的胰腺炎依赖于肠道菌群对NOD1的刺激。然后，我们使用胰腺炎模型分析了NOD1的活性，其中后者是由同时给予低剂量的蓝蛋白（其本身不会诱发胰腺炎）和FK156诱导的，FK156是NOD1的激活剂，模仿突破粘膜屏障的肠道细菌的作用。我们发现这种“低剂量”的毛蛋白胰腺炎依赖于腺泡细胞产生趋化因子MCP-1和胰腺内CCR2+炎症细胞的内流。此外，我们确定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补充NOD1和cerulein信号。因此，这些研究证实，肠道共生体通过胰腺腺泡细胞中的NOD1信号传导使非感染性胰腺炎症发生。

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 inhibiting the ability of NOD1 to induce 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 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. In addition, we have conducted additional studies in which we explored the potential of ML130 to inhibit cerulein-pancreatitis when given in various doses; we found that even when ML140 was given at a single dose at the initiation of the pancreatitis by cerulein injection it was effective in inhibition of the pancreatitis.