Optimizing selective in vivo inhibition of pancreatic tumor JAK2/STAT3 signaling

描述（由申请人提供）：胰腺癌是美国第四大癌症死亡原因，5年生存率低于5%。胰腺癌对细胞毒性治疗具有耐药性，其深刻的免疫抑制性质使患者对免疫治疗无反应。这些障碍突出表明迫切需要针对这种疾病的新的治疗策略。胰腺癌的一个主要中介是转录-3 （STAT3）信号传导和激活因子蛋白。STAT3在大多数人类胰腺癌标本中被激活（磷酸化为pSTAT3），并与活化的K-Ras和Pdx合作，驱动胰腺导管腺癌的发生和进展，正如在小鼠模型中所证明的那样因此，STAT3信号在调节细胞增殖、凋亡、侵袭、血管生成和免疫抑制中发挥作用，是一个有吸引力的靶标。在体外和小鼠肿瘤模型中，STAT3抑制可促进人胰腺癌细胞系的凋亡，并且STAT3是与细胞存活相关的多种上游酪氨酸激酶的汇聚点。因此，在大多数患者中观察到，STAT3靶向治疗可能优于其他方法来克服治疗耐药。最后，STAT3在正常胰腺或其他分化组织中不活跃，也不需要条件敲除小鼠的胰腺发育或稳态。基于这些证据，STAT3是胰腺癌重要的临床相关靶点。然而，直接和特异性抑制STAT3蛋白的抑制剂的开发一直具有挑战性。这是由于STAT3-STAT3同二聚化及其下游效应所需的STAT3 Src同源2结构域内的phospho-Tyr705位点的疏水性。我们的团队之前描述了一种新的先导化合物FLLL32，它直接与STAT3 SH2结构域相互作用，抑制磷酸化和DNA结合，并诱导胰腺和其他肿瘤细胞系的caspase依赖性凋亡。虽然FLLL32是有效的和STAT3特异性的，但它的疏水性损害了生物利用度。我们随后开发了第二代磷酸盐前药FLLL100P。药代动力学（PK）研究表明，FLLL100P具有优越的溶解度，对活性FLLL100的快速去磷酸化，与FLLL32相比，其全身暴露量高10倍。重要的是，活性FLLL100代谢物保留了STAT3的特异性和与FLLL32相似的效力。在本文中，我们将系统地定义胰腺肿瘤中FLLL100P PK与STAT3信号通路（PD）调节之间的体内关系。这些研究将有助于制定剂量方案，以达到体内胰腺肿瘤中FLLL100P的浓度和暴露时间，从而最佳地抑制STAT3信号。这是以前没有完成的，因此对这些药物的进一步发展至关重要。所得数据对于指导进一步的结构或配方修改以进一步增强其活性并最终将其用于胰腺癌患者的临床评估具有重要价值。

DESCRIPTION (provided by applicant): Pancreatic cancer is the fourth leading cause of cancer death in the United States with a grim 5-year survival rate of less than 5%. Pancreatic cancers are resistant to cytotoxic therapies, and their profound immunosuppressive nature renders patients unresponsive to immunologic therapies. These barriers highlight the urgent need for new therapeutic strategies for this disease. One principal mediator in pancreatic cancer is the Signal-Transducer and Activator of Transcription-3 (STAT3) protein. STAT3 is activated (phosphorylated to pSTAT3) in most human pancreatic cancer specimens and cooperates with activated K-Ras and Pdx to drive initiation and progression of pancreatic ductal adenocarcinoma, as has been demonstrated in mouse models.6 STAT3 signaling is therefore an attractive target due to its role in regulating cell proliferation, apoptosis, invasion, angiogeness and immune suppression. STAT3 inhibition is known to promote apoptosis of human pancreatic cancer cell lines in vitro and in mouse tumor models, and it serves as a convergence point for multiple upstream tyrosine kinases relevant to cell survival. STAT3 targeting may therefore be superior to other approaches for overcoming therapy resistance that is observed in most patients. Finally, STAT3 is inactive in normal pancreas or other differentiated tissues and is not required for pancreatic development or homeostasis as shown by conditional knockout mice. Based on this evidence, STAT3 is an important and clinically relevant target for pancreatic cancer. However, development of inhibitors to directly and specifically inhibit STAT3 protein has been challenging. This is due to the hydrophobic nature of the phospho-Tyr705 site within the STAT3 Src homology 2 domain required for STAT3-STAT3 homodimerization and its downstream effects. Our group previously described a novel lead compound, FLLL32, that directly interacts with the STAT3 SH2 domain, inhibits phosphorylation and DNA binding, and induces caspase-dependent apoptosis of pancreatic and other tumor cell lines. Although FLLL32 was potent and STAT3 specific, its hydrophobic nature was a detriment to bioavailability. We subsequently developed a second generation phosphate pro-drug, FLLL100P. Pharmacokinetic (PK) studies revealed FLLL100P has superior solubility, rapid de-phosphorylation to active FLLL100, and 10-fold higher systemic exposure compared to FLLL32. Importantly, the active FLLL100 metabolite retains STAT3 specificity and similar potency to FLLL32. In this proposal, we will systematically define the in vivo relationship between FLLL100P PK and modulation of STAT3 signaling (PD) in pancreatic tumors. These studies will enable development of dose regimens to achieve FLLL100P concentrations and durations of exposure within pancreatic tumors in vivo for optimal suppression of STAT3 signaling. This has not been accomplished previously, and is therefore essential for movement of these agents further in development. Resulting data will be valuable for guiding further structural or formulation modifications to further enhance their activities and ultimately move them into clinical evaluation in patients with pancreatic cancer.