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

CAP：用 NOD1 抑制剂治疗小鼠胰腺炎模型

• 批准号: 8745577
• 负责人: Warren Strober
• 金额: $ 22.36万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8745577
• 关键词: Acinar Cell; Agonist; Amylases; Animals; Bacteria; Blood; Blood Circulation; CCL2 gene; Catheters; Cell Wall; Cells; Cholecystokinin Receptor; Cooperative Research and Development Agreement; Development; Disease; Dose; Family member; Gastrointestinal tract structure; Goals; Host Defense; Hour; Human; Immune response; Inflammation; Inflammatory; Injection of therapeutic agent; Interleukin-6; Intravenous; Laboratories; Libraries; Ligands; Measures; Medical Research; Modeling; Monitor; Mus; NF-kappa B; Organism; Pancreas; Pancreatitis; Peptidoglycan; Production; Protocols documentation; Reporter; Research Institute; STAT3 gene; Serum; Signal Transduction; Testing; United States National Institutes of Health; chemokine; cytokine; gut microflora; in vitro testing; inhibitor/antagonist; interest; pathogen; prevent; response; screening; sensor; trafficking; transcription factor

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.

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，我们表明该分子还通过其对共生生物的反应参与了非传染性胰腺炎的诱导。特别是，我们首先表明，高剂量雨蛙素（一种胆囊收缩素受体激动剂）给药诱发的胰腺炎取决于肠道微生物群对 NOD1 的刺激。然后，我们使用胰腺炎模型分析了 NOD1 活性，其中后者是通过同时施用低剂量的雨蛙素（本身不会诱发胰腺炎）和 FK156 诱导的，FK156 是一种 NOD1 激活剂，模拟突破粘膜屏障的肠道细菌的作用。我们发现这种“低剂量”的雨蛙素胰腺炎依赖于腺泡细胞产生趋化因子 MCP-1 和 CCR2+ 炎症细胞的胰腺内流入。此外，我们确定 MCP-1 的产生涉及转录因子 NF-κB 和 STAT3 的激活，每个因子都需要互补的 NOD1 和雨蛙蛋白信号传导。因此，这些研究证实，肠道共生体通过胰腺腺泡细胞中的 NOD1 信号传导实现非感染性胰腺炎症。 鉴于上述研究表明 NOD1 可能是诱导炎症状态（在本例中为胰腺炎）的一个因素，因此尝试鉴定可能预防实验性胰腺炎的 NOD1 抑制剂并最终用作治疗 NOD1 依赖性人类炎症性疾病的药物变得很有意义。 最近，Correa 等人 (Correa RG Chem Biol 2011, 18:825-832) 采用了对包含超过 300,000 种化合物的 NIH 文库进行高通量筛选，以使用包含 NF-kappaB 报告构建体的 HEK 细胞来鉴定此类 NOD1 抑制剂。 利用这种方法，一种名为 Nodinitib-1 (ML130) 的 2-氨基苯并咪唑化合物已被鉴定为一种有效且特异性的 NOD1 抑制剂，其通过干扰 NOD1 进行细胞内运输的能力来发挥作用。 然而，应该指出的是，这种抑制作用仅通过体外测试得到证实，并未在整个动物中得到证实。 我们已与上述筛选研究的赞助商 Sanford-Burnham 医学研究所签订了 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，并且已将足够的 M130 发送给我们进行适当的研究。 后者包括给予高剂量雨蛙素或低剂量雨蛙素加NOD1配体以及ML130，以确定后者是否可以分别预防高剂量和低剂量雨蛙素胰腺炎。 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了雨蛙素-胰腺炎，其中通过每小时间隔（X7）静脉注射雨蛙素诱导胰腺炎，然后在 8 小时评估胰腺炎。 ML130 预防此类胰腺炎发展的能力是在平行研究中确定的，其中在初次注射雨蛙素和注射一小时雨蛙素时向小鼠共同施用雨蛙素和 ML-30。 我们发现，根据停止给予雨蛙素一小时后循环中的血清淀粉酶水平和IL-6水平评估，ML130确实抑制了胰腺炎的发展。 这种抑制具有统计学意义。