(T1430-09-52) Encorafenib and Trametinib Co-Loaded Nanoparticles as a Therapy for Colon Cancer

Purpose: The combination of a BRAF V600E small molecule inhibitor (e.g., encorafenib or dabrafenib) and a MEK small molecule inhibitor (e.g., trametinib or binimetinib) are now options as second line therapy for metastatic CRC (mCRC) patients possessing BRAF V600E, providing a more effective blockade of the MAPK/ERK pathway than either drug alone. Poor water solubility, poor bioavailability, nonspecific distribution, and the development of resistance make it challenging for its emergence as a primary therapy. However, unlike the impressive response rates seen in patients with BRAF V600E metastatic melanoma treated with this combination, the overall survival for mCRC patients (with BRAF V600E) is still disappointingly low even when this combination is combined with antiEGFR therapy [1]. Thus, there is a need to improve the efficacy of the BRAF V600E targeting such that dual targeting RAS-RAF-MAPK pathway can accommodate further additions to the therapeutic regime without affecting safety. In this study we investigated the development and subsequent administration of a nanoparticle formulation co-loaded with the encorafenib and trametinib at a clinically relevant ratio and delivered at doses equating to maximum tolerated clinical doses in a murine xenograft CRC (HT-29: BRAF V600E) model.

Methods: We fabricated PLGA-based nanoparticles (NPs) co-loaded with encorafenib and trametinib (ET-NPs) using a nanoprecipitation method [2] for the treatment of CRC in a preclinical setting. The NPs were characterized (by dynamic light scatter (DLS) and transmission electron microscopy (TEM)) and their anticancer activity was investigated in vitro using HT-29; whilst cellular uptake of NPs was analyzed by confocal microscopy and flow cytometry. We performed IVIS imaging to ensure the in vivo tumor localization and biodistribution in animal model. In this study we investigated the development and subsequent administration of a nanoparticle formulation co-loaded with the encorafenib and trametinib at a clinically relevant ratio and delivered at doses equating to maximum tolerated clinical doses in a murine xenograft CRC (HT-29) model. Female nude mice were subcutaneously challenged with HT-29 cells (5 × 10⁶). Once the tumors reached ~ 200 mm³, the mice were grouped (n=5) and assigned to one of three treatment groups: DPBS (control), free ET (oral), and ET-NPs (IV). The soluble drugs, encorafenib (60 mg/kg) and trametinib (0.4 mg/kg), were given orally on day 0, 4, 8, and 12. The ET-NP formulation was administered IV (tail vein) on the same schedule.

Results: By encapsulating encorafenib and trametinib within PLGA NPs, we aimed to enhance their solubility, stability, and bioavailability while minimizing systemic toxicity. The ET-NPs had an average size about of 41.30 ± 3.07 nm, smooth surface morphology, optimal drug loading (E: 89.56 ± 6.77 µg/mg, T: 0.656 ± 0.24 µg/mg) (Figure 1A, B; Table 1). Cellular uptake study confirmed time-dependent uptake by HT-29 cells (Figure 1C-F). In vivo tumor localization and biodistribution of DiR-labelled ET-NPs in in vivo was confirmed by IVIS imaging system. Upon formulation of ET in to PEGylated polymeric NPs, tumor uptake of ET-NPs was significantly improved compared to free drug, and this was reflected in the improved the overall antitumor efficacy of ET-NPs in vivo (Figure 2).

Conclusion: This study aimed to identify a BRAF and MEK inhibitor combination that exhibits superior anti-tumor activity in a HT-29 (BRAF V600E) xenograft mouse model. Results indicated that the combination of the BRAF inhibitor encorafenib and the MEK inhibitor trametinib showed the significant anti-tumor activity in vitro and in vivo compared to the soluble combination. We anticipate that our findings will contribute to the development of more effective and tolerable therapies for CRC patients, ultimately improving clinical outcomes and quality of life. This combination is currently not used in patient treatment; thus, it deserves an opportunity to be included in clinical trials.

References: 1. Van Cutsem E, Huijberts S, Grothey A, Yaeger R, Cuyle PJ, Elez E, Fakih M, Montagut C, Peeters M, Yoshino T, Wasan H. Binimetinib, encorafenib, and cetuximab triplet therapy for patients with BRAF V600E–mutant metastatic colorectal cancer: safety lead-in results from the phase III BEACON colorectal cancer study. Journal of clinical oncology. 2019 Jun 6;37(17):1460.
2. Sen R, Ganguly S, Ganguly S, Debnath MC, Chakraborty S, Mukherjee B, Chattopadhyay D. Apigenin-loaded PLGA-DMSA nanoparticles: a novel strategy to treat melanoma lung metastasis. Molecular Pharmaceutics. 2021 Mar 29;18(5):1920-38.

Acknowledgements: The author would like to acknowledge use of The University of Iowa Central Microscopy Research Facility. We are grateful to all members of the research group for their continuous support and encouragement.

Funding: This work was supported by the Gastro-Intestinal Research Foundation (GIRF).
Notes: The authors declare no competing financial interest. All mouse experiments were done in accordance with The University of Iowa IACUC Protocol.
Conflict of interests: No conflict of interest to disclose.

Figure 1: Physicochemical characterization and cellular uptake of experimental ET-coloaded NPs (ET-NPs). Representative figures of: (A) particle size, (B) TEM images of ET-NPs (Magnification 150X, scale bar = 100 nm). (C) Flow cytometric distribution profiles of HT-29 cells after incubation with FITC-loaded ET-NPs over time (D) Representative bar diagram depicting mean fluorescence intensity versus time for HT-29 WT after incubation with FITC-loaded ET-NPs over time. Confocal microscopic images illustrating the cellular uptake of NPs by (E) HT-29 WT cells at 1h and 2h. (F) Bar diagrams depicting the FITC mean fluorescence intensity values at different treatment durations (1h and 2h). Green color corresponds to FITC-labeled experimental NPs, red color represents F-actin, and blue color indicates the nucleus stained with DAPI. Scale bars denote 10 μm.

Figure 2: In vivo antitumor efficacy of different treatment regimens. (A and B) Mean tumor volume recorded during the treatment period for HT-29 colon cancer xenograft mice model. (C) % reduction in tumor volume comparing the tumor volume on day 1 and day 13 of treatment. The negative values indicate that tumor volume increased compared to the initial volume. (D) Body weight recorded during the treatment period for the HT-29 colon cancer xenograft mice model. Data are plotted as mean ± SD (n = 3). One-way ANOVA and unpaired t-test were used for statistical analysis. *, < 0.0186: **, < 0.0070: ***, < 0.0006: non-significant (ns).

Tables: 1 Physicochemical Evaluation of Formulations: Data from the three independent experiments. E = encorafenib, T = trametinib