(M1530-01-06) Combination Therapy of BRD4/PI3K Dual Inhibitor and Novel Tubulin Inhibitor Efficiently Treats Chemo-Resistant Pancreatic Cancer

Purpose: Treatment of pancreatic cancer is challenging due to the development of chemoresistance by PI3K and BRD4 signaling pathways, which play key roles in tumor growth, cancer stem cell (CSCs) proliferation, and tumorigenesis. In addition, inefficient drug delivery to the pancreatic tumor and drug-induced systemic toxicity reduces the benefits of chemotherapy. PI3K/AKT inhibition plays a crucial role in this phenomenon as a downstream effector of oncogenic KRAS. A transcription factor, MYC, has induced tumor growth and promotes DNA replication and transcription. Further, dual-activity inhibition of PI3K and BRD4 promotes MYC degradation. However, pharmacologically targeting MYC is challenging due to the need for a direct protein target and severe toxicity due to the pleiotropic role of MYC in the biological system. We recently synthesized a novel microtubule polymerization inhibitor CH-2-102, which showed anti-tumor efficacy in pancreatic cancer cells. CH-2-102 targets the colchicine binding site. In addition, we synthesized MDP5, a new BRD4-PI3K dual inhibitor with robust antiproliferative properties. As epidermal growth factor receptor (EGFR) is overexpressed in 90% of pancreatic tumors, we designed a nanoformulation decorated with EGFR targeting peptide AAEYLRK-DSPE-PEG system, which will stabilize the free form of drug as well as target the pancreatic cancer cells. It will also have a fluorescent dye, TAMRA, to detect uptake at the tumor site. Herein, we propose combining a novel microtubule inhibitor CH-2-102 and a novel BRD4/PI3K dual inhibitor MDP5 loaded in EGFR-targeted nanoparticles (NPs) to treat orthotopic pancreatic tumor-bearing mice.

Methods: Cytotoxic effects of ONC201, CH-2-102, and their combination were determined using MTT assay in MIA PaCa-2 cells. One drug treatment varied in concentrations for combination treatment, keeping the other constant; synergistic effects were evaluated by determining the combination index (CI). The apoptotic activity of CH-2-102 and MDP5 with their combinations was confirmed by cell apoptosis assay. Also, the cell cycle arrest for these drugs was evaluated by cell cycle assay. To increase the drug loading of CH-2-102 into liposomes, we synthesized a lipid conjugate (LDC) of CH-2-102 and confirmed the structure by ¹H NMR. We have also synthesized TAMRA-EGFR-DSPE-PEG through click chemistry and envisioned the targeted delivery of loaded drugs in pancreatic cancer cells due to overexpression of EGFR. Each step has been characterized using ¹H NMR, FTIR, and Mass Spectroscopy. We have also formulated CH-2-102 LDC and MDP5 into the liposomal system using cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and DSPE-mPEG2000 in optimal ratios as backbone structural framework. We have characterized this formulation by determining the size and effective drug loading.

Results: CH-2-102 and MDP5 showed effective dose-dependent cell killing with synergistic effects at lower concentrations showing a combination index (CI) of less than 1. The IC₅₀ values for these compounds were 15 nM and 5 μM, respectively. CH-2-102 LDC had no significant interference as the IC₅₀ value was 25 nM and reproduced synergism with MDP5. The apoptotic activity of these drug combinations established a trend where the combination approach showed increased apoptosis compared to single-drug therapy. We performed the cell apoptosis for CH-2-102 and MDP5 with their combination on MIA PaCa-2 cells. We observed an increase in the early and late-stage apoptosis for the combination treatment (14%). Also, apoptosis for CH-2-102 was higher (11%) than MDP5 (8.6%), indicating these drugs are potent in killing pancreatic cancer cells. We also performed a cell cycle analysis and observed cell cycle arrest in the G2/M phase when MIA PaCa-2 cells were treated with CH-2-102, indicating inhibition through mitosis. Conversely, MDP5 arrested the cells in the G1 phase, while the combinational approach showed G2/M phase cell cycle arrest. We synthesized TAMRA-EGFR-DSPE-PEG and TAMRA-Scramble-DSPE-PEG, where there was a clear differentiation between scramble and EFGR confirmed by NMR. We determined the particle size distribution of liposomes loaded with CH-2-102 LDC and MDP5 by a Malvern Particle sizer, with the mean particle size of 98.99 ±1.6 nm and 96.15 ±1.2 nm, respectively. HPLC-UV analyses showed effective loading of CH-2-102 LDC and MDP5 with 4.4% w/w and 4.8% w/w in the liposomes, respectively, with an encapsulation efficiency of 92% and 96%, respectively.

Conclusion: The combinational approach has proved to be an effective treatment strategy. Our data has shown effective dose-dependent cell apoptosis of MIA PaCa-2 cells with CH-2-102 and MDP5. The cell cycle analysis reports the G2/M phase cell arrest for CH-2-102, which indicates mitosis catastrophe of tubulin polymerization inhibitors; on the other hand, MDP5 arrested the cells in the G1 phase, indicating apoptotic mechanism. The lipid conjugation of CH-2-102 increased the molecule's lipophilicity, allowing better encapsulation into the liposomal system. Both the drugs were comfortably loaded into liposomes and showed a uniform particle size distribution. Lastly, we have confirmed the synthesis of EGFR and scrambled TAMRA-DSPE-PEG for an effective targeting approach.