Nanomedicine of Hedgehog and AKT/ERK Dual Inhibitors for Pancreatic Cancer

项目总结 吉西他滨(GEM)是一种一线药物，由于其代谢速度快，给药效率低，疗效有限。 到促结缔组织增生性胰腺肿瘤部位。Hedgehog(HH)信号激活胰腺星状细胞(PSCs) 在肿瘤干细胞(CSCs)的结缔组织形成和增殖中起关键作用。KRAS是 KRAS主要发生在胰腺癌(PC)中，然而KRAS仍然是一个困难的靶点。由于抑制了 MTORC1/2增加ERK的磷酸化，我们建议GEM与ONC201联合治疗，即 AKT/ERK双重抑制剂可有效治疗PC。ONC201抑制细胞增殖并诱导肿瘤坏死因子相关 凋亡诱导配体(TRAIL)介导细胞凋亡。此外，我们还采取了基质耗尽战略。 通过顺序给予HH抑制剂MDB5，以降低药物输送到肿瘤部位的物理屏障。 虽然Sonic Hedgehog(Shh)缺陷肿瘤的间质含量减少，但这种肿瘤具有侵袭性 血管增多和转移潜能增加。因此，通过抑制HH途径减少促结缔组织发育 允许高效地将加载到EGFR靶向NPs中的ONC201和GEM运送到胰腺肿瘤部位。我们 已经确定了一种使用临床上可行的抑制剂的有效组合治疗策略，这可以是 适用于具有不同KRAS突变的PDAC肿瘤。在我们的初步研究中，(I)与免费创业板相比， Mpeg-co-PCC-g-GEM-g-DC纳米粒可使GEM在原位肿瘤中的积聚增加2.5倍。控制创业板 释放到肿瘤内，我们合成了mpeg-co-P(Asp)-g-DC-S-S-GEM，GEM载量为14%(w/w)。 经L-谷胱甘肽孵育后，90%的宝石从聚合物中释放出来。创业板与创业板的结合 ONC201在体外杀伤耐药PC细胞和更有效地抑制体内肿瘤生长方面显示出协同作用 而不是他们的单一疗法。我们还合成了2-氯-N1-[4-氯-3-(2-吡啶基)苯基]-N4，N4-双(2- 1，4-苯二甲酰胺(MDB5)对HH配体和CSC标志物的抑制作用更强 比vismodegib更有效。通过在其表面修饰EGFR来制备和优化靶向NPs 不同配基密度下的结合肽Ge11。MDB5负载Ge11-NPs进入PC机的系统给药 荷瘤小鼠在给药后4h肿瘤内的药物浓度高于 无针对性的NPs。因此，我们假设加载MDB5的NPs的顺序给药将增加 GEM和ONC201通过逆转耐药对PC的协同抑制作用 通过促结缔组织增生症和CSC增殖更有效。我们的具体目标是：1)评估ONC201的影响 GEM与GEM联合在体内外耐GEM PC细胞中的应用II)靶向性氧化还原敏感性的发展 MDB5、ONC201和GEM的纳米药物，以及III)MDB5、ONC201和GEM的纳米颗粒递送 联合应用于原位、PDX和自发KPC小鼠模型。长远的影响是发展小说 利用MDB5多功能纳米药物降低结缔组织增生症诱导的PC化疗耐药性的策略， GEM和ONC201。

PROJECT SUMMARY Gemcitabine (GEM), a frontline drug, shows limited efficacy due to its rapid metabolism and inefficient delivery to the desmoplastic pancreatic tumor site. Hedgehog (Hh) signaling activates pancreatic stellate cells (PSCs) and plays a critical role in the formation of desmoplasia and proliferation of cancer stem cells (CSCs). KRAS is predominantly mutated in pancreatic cancer (PC), yet KRAS remains a difficult target. Since inhibition of mTORC1/2 increases ERK phosphorylation, we propose combination therapy of GEM with ONC201, which is an AKT/ERK dual inhibitor to effectively treat PC. ONC201 inhibits cell proliferation and induces TNF-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Further, we have adopted a stroma depletion strategy by sequentially administrating Hh inhibitor MDB5 for reducing physical barrier of drug delivery to the tumor site. While sonic hedgehog (Shh)-deficient tumors have reduced stromal content, such tumors are aggressive with increased vascularity and metastatic potential. Therefore, reduction of desmoplasia by inhibiting Hh pathway will allow efficient delivery of ONC201 and GEM loaded into EGFR targeted NPs to the pancreatic tumor site. We have identified an effective combinatorial treatment strategy using clinically viable inhibitors, which can be applied to PDAC tumors with different KRAS mutations. In our preliminary studies, (i) compared to free GEM, mPEG-co-PCC-g-GEM-g-DC NPs increased GEM accumulation in orthotopic tumor by 2.5-fold. To control GEM release into the tumor, we synthesized mPEG-co-P(Asp)-g-DC-S-S-GEM with GEM payload of 14% w/w. There was 90% GEM release from the polymer upon incubation with L-glutathione (GSH). Combination of GEM with ONC201 showed synergy in killing resistant PC cells in vitro and reduced tumor growth in vivo more effectively than their monotherapies. We also synthesized 2-chloro-N1-[4-chloro-3-(2-pyridinyl) phenyl]-N4, N4-bis(2- pyridinylmethyl)-1,4-benzenedicarboxamide (MDB5), which inhibited Hh ligands and CSC markers more efficiently than vismodegib. Targeted NPs were prepared and optimized by decorating their surface with EGFR binding peptide GE11 at different ligand density. Systemic administration of MDB5 loaded GE11-NPs into PC tumor bearing mice resulted in higher drug concentration in the tumor at 4h post administration compared to non-targeted NPs. Therefore, we hypothesize that sequential administration of MDB5 loaded NPs will increase GEM and ONC201 delivery to the tumor and result in synergistic inhibition of PC by reversing resistance induced by desmoplasia and CSC proliferation more efficiently. Our specific aims are to i) assess the effects of ONC201 and GEM combination in GEM resistant PC cells in vitro and in vivo, ii) development of targeted redox sensitive nanomedicine of MDB5, ONC201 and GEM, and iii) nanoparticulate delivery of MDB5, ONC201, and GEM combination in orthotopic, PDX and spontaneous KPC mouse models. Long-term impact is to develop novel strategies to reduce desmoplasia-induced chemoresistance in PC using multifunctional nanomedicine of MDB5, GEM and ONC201.