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

细胞内传感器NOD1具有与多种病原体相关的重要宿主防御功能。 在本研究之前的研究中，我们发现这种分子也通过其对肠道微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，胰腺炎诱导的高剂量雨蛙肽（胆囊收缩素受体激动剂）管理依赖于NOD1刺激肠道微生物。然后，我们使用胰腺炎模型分析了这种NOD1活性，其中后者是通过同时施用低剂量的雨蛙肽（其本身不诱导胰腺炎）和FK156诱导的，FK156是NOD1的激活剂，其模拟已经突破粘膜屏障的肠道细菌的作用。我们发现这种"低剂量"雨蛙肽胰腺炎依赖于趋化因子MCP-1的腺泡细胞产生和胰腺内CCR2+炎性细胞的流入。此外，我们确定MCP-1的产生涉及转录因子NF-κ B和STAT 3的激活，每个转录因子都需要互补的NOD 1和雨蛙素信号传导。因此，这些研究证实，肠道炎症通过胰腺腺泡细胞中的NOD1信号传导而使非感染性胰腺炎症成为可能。 基于这些发现，我们推测，胰腺腺泡细胞的初始破裂的各种刺激（在这种情况下雨蛙素）导致改变肠道通透性和肠道细菌进入循环。 然后，这种细菌刺激腺泡细胞中的NOD1，并诱导趋化因子产生，趋化因子产生将炎性细胞吸引到胰腺中并引发胰腺炎症 鉴于上述研究表明NOD 1可以是诱导炎性状态（在这种情况下是胰腺炎）的因素，尝试鉴定NOD 1的抑制剂变得令人感兴趣，所述抑制剂可以预防实验性胰腺炎并因此最终发现用作治疗NOD 1依赖性人类炎性疾病的药剂。 最近，Correa等人（Correa RG Chem Biol 2011，18：825 - 832）采用含有> 300，000种化合物的NIH文库的高通量筛选，以使用含有NF-κ B报道构建体的HEK细胞鉴定此类NOD1抑制剂。 使用这种方法，命名为Nodinitib-1（ML130）的2-氨基苯并咪唑化合物已被鉴定为有效且特异的NOD 1抑制剂，其通过抑制NOD 1诱导细胞内运输的能力而起作用。 然而，应该注意的是，这种抑制作用仅通过体外试验而不是在整个动物中得到证实。 我们已与上述筛选研究的申办者Sanford-Burnham Medical Research Institute签订了M-CRADA，以获得ML130用于研究ML130预防实验性胰腺炎的能力。 该M-CRADA现已完全执行，并已向我们发送足够的M130进行适当的研究。 后者包括给予高剂量雨蛙肽或低剂量雨蛙肽加NOD 1配体以及ML 130，以确定后者是否可以分别预防高剂量和低剂量雨蛙肽胰腺炎。 为了促进这些研究，我们迄今为止使用了永久植入静脉导管的小鼠，以便我们可以通过测量淀粉酶和MCP-1（以及其他细胞因子和趋化因子）的血液水平来监测ML130对胰腺炎发展的影响。 在迄今为止进行的研究中，我们使用标准方案诱导雨蛙肽-胰腺炎，其中胰腺炎通过每小时（X7）IV施用雨蛙肽诱导，随后在8小时评估胰腺炎。在平行研究中测定了ML 130预防这种胰腺炎发展的能力，其中在初始雨蛙肽注射和1小时雨蛙肽注射时向小鼠共施用雨蛙肽和ML-30。 我们发现，ML130确实抑制胰腺炎的发展，如通过血清淀粉酶水平和循环中IL-6水平在停止雨蛙肽给药后1小时所评估的。 这种抑制作用具有统计学意义。 此外，我们还进行了其他研究，探索了ML130以不同剂量给药时抑制雨蛙肽-胰腺炎的潜力;我们发现，即使在胰腺炎开始时通过雨蛙肽注射单次给药ML140，也能有效抑制胰腺炎。 使用永久植入静脉导管的小鼠有助于这些研究;然而，这些小鼠价格昂贵且不易获得。 这妨碍了我们认为必要的全面研究。

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-kappaB 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. On the basis of these findings we postulate that initial dysruption of pancreatic acinar cells by a variety of stimuli (in this case cerulein) leads to altered gut permeability and the entry of gut bacteria into the circulation. Such bacteria then stimulates NOD1 in acinar cells and the induction of chemokine production that draws inflammatory cells into the pancreas and initiates the pancreatic inflammation 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 have so far utilized 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. The use of mice with permanently implaced intravenous catheters facilitates these studies; however, these mice are expensive and not readily available. This has impeded the full panoply of studies we think are necessary.