Nanoparticles for co-delivery of osimertinib and selumetinib to overcome osimertinib-acquired resistance in non-small cell lung cancer

Abstract

Osimertinib (OSI) is the first FDA-approved third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). It can be used for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutation and for patients who are resistant to first-generation EGFR TKIs due to T790M resistance mutation. However, patients treated with OSI ultimately develop acquired resistance, which prevents its long-term benefit for patients. Therefore, the development of effective strategies to overcome OSI resistance will address a significant clinical challenge and benefit patients by prolonging their survival time. Our previous studies indicated that combination therapy was a promising strategy for overcoming OSI resistance. In this study, we developed nanoparticle (NP) formulations for co-delivery of osimertinib (OSI) and selumetinib (SEL) to treat OSI-resistant NSCLC effectively. We conjugated SEL with PEG through a reactive oxygen species (ROS)-responsive linker to generate polyethylene glycol (PEG)-SEL conjugate prodrug (PEG-S-SEL). Due to the amphiphilic nature of PEG-S-SEL, it can self-assemble in an aqueous solution to form micelle NP and serve as a delivery carrier for OSI. The ROS-responsive linker can facilitate the release of drugs in the tumor microenvironment with elevated ROS levels. OSI and SEL combination NP can overcome OSI resistance by simultaneously inhibiting both EGFR and mitogen-activated protein kinase (MEK), thus effectively inducing apoptosis in OSI-resistant NSCLC cells and inhibiting OSI-resistant tumors in vivo. In conclusion, the OSI+SEL NP combination therapy showed promising anticancer efficacy and demonstrated potential for treating NSCLC patients with OSI acquired resistance. STATEMENT OF SIGNIFICANCE: Osimertinib (OSI) is the first FDA-approved third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. It has been successfully used for treating non-small cell lung cancer (NSCLC) patients with activating EGFR mutation. However, patients treated with OSI ultimately develop acquired resistance. This study developed OSI and selumetinib (SEL) co-delivering nanoparticles to overcome OSI-acquired resistance in NSCLC. PEG-SEL conjugate functions as reactive oxygen species (ROS)-responsive prodrug and forms micelle nanoparticles through self-assembly to deliver OSI. The combination NP can simultaneously inhibit EGFR and mitogen-activated protein kinase (MEK), thus effectively inducing apoptosis in OSI-resistant NSCLC cells. In summary, the OSI and SEL nanoparticle combination therapy showed promising anticancer efficacy and demonstrated potential for treating NSCLC patients with OSI acquired resistance.

Keywords: Acquired resistance; Non-small Cell Lung Cancer; Osimertinib; Prodrug; Reactive Oxygen Species; Selumetinib.

Figure 1.. Synthesis and characterization of PEG-S-SEL conjugate.

(A) Synthesis procedure. 1. EDCI, HOBT, DMF; 2. DCC, DMAP, DMF (B) TLC and (C) ¹H-NMR characterization of PEG-S-SEL conjugate. (D) ROS triggered release of SEL from conjugate. Results are mean ± SD (n=3). ***, P < 0.001. (E) ROS-responsive drug release mechanism.

Figure 2.. (A)

(A) Drug concentration, (B) Particle size, and (C) Zeta potential of different NPs. (D) Fluorescence image of PC-9/AR cells incubated with coumarin-6-labeled NPs for 3 hours. (E) Cellular uptake of NPs by PC-9/AR cells were determined by flow cytometry. Data are presented as the mean ± SD, n=3.

Figure 3.. In vitro anticancer efficacy against OSI-resistant PC-9/AR NSCLC cells.

(A) Cell viability of PC-9/AR cells were determined with the MTT assay after treatment with different formulations for 48 hours. The numbers in the graphs are combination indices. (B) Calcein-AM/PI staining. Cells treated with different formulations for 48 hours and stained with Calcein-AM/PI to detect living (green) and dead (red) cells. (C) Inhibition of colony formation by OSI+SEL combination therapy. Cells were treated with different NP formulations and free drugs for a total of 7 days. Cell colonies were stained with crystal violet and photographed. Crystal violet in each groups were also dissolved by methanol and the absorption at 560 nm were determined. Results are mean ± SD (n=3). ***, P < 0.001

Figure 4.

(A) Cell apoptosis was determined with flow cytometry. PC-9/AR were treated with OSI+SEL NP and other controls for 48 hours. The OSI +SEL NP induced significant increase of percentage of early apoptotic cells (Annexin V-FITC⁺/PI⁻) and late apoptotic cells (Annexin V-FITC⁺/PI⁺). Results are mean ± SD (n=3). ***, P < 0.001. (B) Western Blotting. PC-9/AR cells were exposed to the tested free drugs or NPs for 48 h and then harvested for Western blotting to detect cleavage of PARP and caspase-3. CF, cleaved form.

Figure 5.. In vivo anticancer studies with PC-9/AR xenograft tumor model.

(A) Change of tumor size in tumor-bearing nude mice received intravenous injection of different formulations. At the end of the study, isolated tumors were photographed (B) and weighted (C). Body weight of PC-9/AR tumor-bearing mice during the course of study (D). Results are mean ± SD (n=6). **, P < 0.01; ***, P < 0.001, compared with control and monotherapy groups.

Scheme 1.

Nanoparticle for co-delivery of Selumetinib (SEL) and Osimertinib (OSI) to overcome drug resistance in NSCLC though synergistic effects on mEGFR and MEK.