(T1230-07-38) Polymeric Chloroquine as an Oral Chemopreventive Agent for Colorectal Cancer

Purpose: Despite significant progress in clinical management of colorectal cancer (CRC), it remains the second leading cause of cancer related deaths and has become a heavy financial burden and global challenge [1]. Therefore, prevention strategies are crucial in reducing CRC risk and CRC related deaths. Metastasis to the distal organs remains the main culprit in CRC-associated patient deaths. Approximately, 22% of CRCs are metastatic at initial diagnosis, and about 70% of patients will eventually develop metastatic relapse [2]. Patients with metastatic CRC face poor prognosis in general, with a relative 5‐year survival rate of 14%, compared to 71% and 90% in those with regional and localized CRC in the United States [2]. Inflammation plays a key role in CRC progression and in this regard, chronic colonic inflammation leads to a two-fold increase in the risk of developing colorectal carcinoma in patients suffering from Inflammatory Bowel Disease (IBD). Hydroxyhloroquine (HCQ) is an antimalarial and anti-inflammatory agent with potential use in cancer treatment [3]. The objective of this study is to test if an oral formulation of polymeric chloroquine (PCQ), previously developed in our lab and shown to exhibit strong antimigration and anti-metastasis effect in various cancers, can be used as an oral chemopreventive agent in CRC [4].

Methods: PCQ was synthesized using RAFT polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with methacryloylated hydroxychloroquine (HCQ-MA). The polymer was characterized using several analytical methods such as Gel Permeation Chromatography (GPC), Proton Nuclear Magnetic Resonance (1H NMR), Dyanamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). In vitro studies were performed using CRC cell lines namely Caco-2, HCT116 and SW620. The cell viability and anti-proliferation effect of PCQ was determined using Cell Titer Blue (CTB) and IncuCyte^® cell count proliferation assay. In vitro antimigration effect of PCQ was determined using scratch wound healing assay and transwell cell migration assay. In vitro anti invasion effect of PCQ was shown using Matrigel coated transwell cell invasion assay. The uptake of PCQ in Caco-2 cells was determined using previously validated LC/MS/MS method. Autophagy inhibition effect of PCQ was seen in-vitro using western blot.

Results: The number average molecular weight of PCQ was determined to be about 11.5 kDa. Polymerization of HPMA and HCQ-MA was confirmed and HCQ content of about 9 mol% was determined using 1H NMR. The average hydrodynamic diameter and zeta potential was found to be 26 ± 18 nm and 5 ± 4 mV using DLS. TEM showed spherical morphology and confirmed size of PCQ nanoparticles. IC50 of PCQ for Caco-2, HCT116, and SW620 was > 800µM, 300µM, and 610µM respectively whereas of IC50 of HCQ was 130µM, 50µM, and 80µM respectively. HCQ showed strong anti-proliferation effect towards all CRC cell lines whereas PCQ showed preferential anti-proliferation effect towards more aggressive CRC cell line. The uptake of PCQ was determined to be about 8ng/cell. Accumulation of autophagy related proteins namely LC3 I, LC3 II and P62 showed that PCQ had autophagy inhibition activity. PCQ inhibited wound closure in HCT116 cell line as much as HCQ after 72 hours. The transwell cell migration and invasion assay showed that PCQ inhibited cell migration and invasion in vitro.

Conclusion: PCQ forms almost spherical nanoparticles when dissolved in water with slightly positive charge. It shows preferential cytotoxicity and anti-proliferation effect towards more aggressive cancer cell line. PCQ is uptaken by Caco-2 cells in vitro. It shows anti migration, anti invasion and autophagy inhibition effect in vitro. All these results demonstrate its potential to be used as an oral chemopreventive agent for colorectal cancer.

References: 1] Xi, Y. and P. Xu, Global colorectal cancer burden in 2020 and projections to 2040. Translational Oncology, 2021. 14(10): p. 101174.
2] Howlader N, Noone AM, Krapcho M, Miller D, Brest A, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2016, National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2016/, based on November 2018 SEER data submission, posted to the SEER web site, April 2019.
3] dos Reis Neto, E.T., et al., Revisiting hydroxychloroquine and chloroquine for patients with chronic immunity-mediated inflammatory rheumatic diseases. Advances in Rheumatology, 2020. 60(1): p. 32.
4] Yu, F., et al., Chloroquine-Containing HPMA Copolymers as Polymeric Inhibitors of Cancer Cell Migration Mediated by the CXCR4/SDF-1 Chemokine Axis. ACS Macro Lett, 2016. 5(3): p. 342-345.

Acknowledgments: This work was supported by the NIH grant R01DK124095.

Figure 1: PCQ synthesis scheme: HPMA and CQ-MA (4:1) were dissolved in solvent (DMSO: Dioxane (1:1)) along with chain transfer agent and AIBN. The reaction gives maximum conversion at 24 h (All ratios are wt/wt).



Figure 2: Cell proliferation of A) Caco-2 and B) SW620 cells after treatment



Figure 3: Transwell migration in HCT116 cells using 20% FBS as chemoattractant after 24 hours (Scale bar = 200 μm).