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

基本信息

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
项目类别：
财政年份：
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

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

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项目摘要

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具有与多种病原体有关的重要宿主防御功能。在本研究的前期研究中，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，我们首先表明，高剂量cerulein（胆囊动蛋白受体激动剂）诱导的胰腺炎取决于肠道微生物的NOD1刺激。然后，我们使用胰腺炎模型分析了这种NOD1活性，其中，其中的一种由低剂量的cerulein（本身并非诱导胰腺炎）和FK156诱导，而NOD1的激活剂是模拟粘膜障碍的肠道效果的NOD1激活剂。我们发现，这种“低剂量” Cerulein胰腺炎取决于CCR2+炎性细胞的趋化因子MCP-1和胰腺内流入的腺泡细胞的产生。此外，我们确定MCP-1的产生涉及转录因子NF-κB和STAT3的激活，每个转录因子都需要互补的NOD1和Cerulein信号传导。因此，这些研究确定了肠道分子能够通过胰腺腺泡细胞中的NOD1信号传导非感染性胰腺炎症。鉴于上述研究表明，NOD1可能是诱导炎症状态的一个因素（在本例中为胰腺炎），试图鉴定NOD1的抑制剂可能可以想象可以预见实验性胰腺炎，从而最终发现使用NOD1依赖性人类炎症性疾病的药物。最近，Correa等人（Correa RG Chem Biol 2011，18：825-832）使用含有NF-kappab Reporter构建体的HEK细胞来识别含有> 300,000种化合物的NIH文库的高贯穿筛选。使用这种方法，已将2-氨基苯甲酰二咪唑化合物指定的Nodinitib-1（ML130）被确定为一种有效的特异性NOD1抑制剂，该抑制剂通过干扰NOD1执行细胞内运输的能力而起作用。但是，应该指出的是，这种抑制仅通过在整个动物的体外测试中得到证明。我们已经与桑福德·伯纳姆医学研究所（Sanford-Burnham Medical Research Institute）一起进入了M-Crada，上述筛查研究的赞助商获得了ML130，以用于ML130预防实验性胰腺炎的能力研究。现在，该M-Crada已完全执行，并已将足够的M130发送给我们进行适当的研究。后者包括给药高剂量的Cerulein或低剂量的Cerulein加NOD1配体以及ML130，以确定后者是否可以预防高剂量和低剂量的Cerulein胰腺炎。为了促进这些研究，我们利用具有永久性静脉内导管的小鼠，因此我们可以通过测量淀粉酶和MCP-1的血液水平（以及其他细胞因子和趋化因子和趋化因子）来监测ML130对胰腺炎发育的影响。在到目前为止进行的研究中，我们使用标准方案诱导了谷蛋白酶胰腺炎，在该标准方案中，胰腺炎是通过小时静脉内静脉注射的静脉炎诱导的，然后以小时的时间间隔（x7）诱导胰腺炎，然后在8小时时评估胰腺炎。在平行研究中确定了ML130防止这种胰腺炎发育的能力，在该研究中，小鼠在初次注射cerulein注射和一小时的Cerulein注射时是co固定的Cerulein和ML-30。我们发现，ML130确实抑制了抑制胰腺淀粉酶水平的抑制性胰腺炎发育，并在停止Cerulein施用后一小时通过血清淀粉酶水平和IL-6水平评估。这种抑制作用具有统计学意义。