(T1430-01-03) Exploring the Potential of Inhaled Nano Therapy for Malignant Pleural Mesothelioma

Purpose: Malignant Mesothelioma (MM) is a rare malignancy affecting the mesothelial lining around major organs. Out of different subtypes of MM, Malignant Pleural Mesothelioma (MPM) is the most prevalent subtype with around 70-75% occurrence in all patients. While current therapies such as surgical resection, chemotherapy (Cisplatin + Pemetrexed) and immunotherapy (Nivolumab + Ipilimumab) are able to inhibit tumor growth, the chances of tumor relapse are pretty high with poor life expectancy. One reason behind lack of efficacy of these therapies is failure to focus on novel molecular mechanisms. Moreover, poor localization of therapy in the target organ is also a drawback. Therefore, there is a need to develop a localized therapy for MPM with prime focus on novel molecular pathway. Recently, a novel molecular pathway SPHK1/S1P has been evaluated for its role against different cancers. Studies have shown elevated levels of S1P In cancer cells, promoting tumor growth and proliferation. Interestingly, a small molecule Carmofur (anti-neoplastic agent, initially developed as a prodrug of 5-fluorouracil) has been found to bind irreversibly to Acid Ceramidase, an enzyme which indirectly catalyzes S1P. However, Carmofur’s low aqueous solubility and severe neurotoxicity further limits its applications. Therefore, in this present study, we developed an optimized biodegradable polymeric nanoparticle for local delivery of carmofur to the lungs, thus increasing its therapeutic efficacy at reduced dose.

Methods: Carmofur loaded PLGA (Poly-Lactic-Co-Glycolic Acid) polymeric nanoparticles (Carmofur-NPs) were prepared using a full factorial DoE model with varied concentrations of Poly Vinyl Alcohol (PVA) as an external phase using conventional double emulsion/solvent evaporation technique. Target profile of optimized NPs included drug loading >1.5%, and particle size < 200 nm with uniform polydispersity index (PDI ≤0.1). Formulations were analyzed for their physicochemical properties and drug loading. In-vitro release and stability of Carmofur-NPs were also conducted. Ability of nanoparticles to achieve deep lung deposition was evaluated using Next Gen Impactor (NGI). Carmofur-NPs were tested for their therapeutic efficacy via preliminary cytotoxicity in immortalized and patient-derived cell lines. Potential to inhibit tumor reoccurrence and efficacy against 3D-cell based model was also assessed in MPM cell lines. Other potential mechanistic pathways for Carmofur were discovered using cell cycle analysis and multiple in-vitro kits for testing apoptosis.

Results: DoE model rendered 4 formulations with varied PVA concentrations (Fig. 1A) Out of all the prepared formulations, F2 was considered as an optimized formulation with 1.8% drug loading (Fig. 1B) and particle size of 182.2±8.3 nm with PDI of 0.1±0.01 (Fig. 1C). In-vitro release revealed Higuchi kinetics as best fit release kinetic model (Fig. 1D &E). Optimized formulation showed excellent aerosolization potential with fine particle fraction (FPF) of 81.4±2.9% and mass median aerodynamic diameter (MMAD) of 2.6±0.05 µm (Fig. 1G). Cell viability results revealed significantly higher cytotoxicity of Carmofur-NPs as compared to plain carmofur (Figs. 2A-F, and Fig. 2G) in all MPM cell lines after 72 hours treatment. Moreover, colony inhibition potential of carmofur-NPs in H2452 and ROB cells showed no colony formation at different Carmofur-NPs concentrations (Figs. 2H & 2I). Further, the quantification of colonies showed significant reduction in % colony growth in case of carmofur-NPs when compared to control (Fig. 2J & 2K). Moreover, carmofur-NPs were found to be efficacious against 3D spheroids mimicking in-vivo tumor formation. The shrinkage in the tumor size was clearly observed in 3D tumor models of MSTO and ROB respectively. The quantification of tumor volume after Day 12 clearly indicated enhanced therapeutic efficacy of carmofur-NPs. Fluorescent images of Live and dead cells also demonstrated dead cells (red fluorescence) at the core of the tumor for carmofur-NPs (Fig. 3E&F). Lastly, it was found that percentage of apoptotic cells present in Carmofur-NPs treated H2452 cells were remarkably higher than other treatments within 24 hours (Fig. 3L), thus demonstrating its ability to completely eradicate tumor and to be developed as a standalone therapy for MPM.

Conclusion: This study demonstrates the efficacy of inhalable carmofur loaded nanoparticles through extensive phenotypic and mechanistic assays. Optimized nano carriers portrayed higher loading of carmofur with deep lung deposition, thus overcoming the challenges associated with carmofur. Results depicted enhanced therapeutic efficacy of carmofur in immortalized as well as patient derived MPM cell lines via different phenotypic assays. This study lays a solid groundwork for carmofur loaded carriers to be developed as a standalone therapy to treat MPM patients and also objectify the relevance of inhalable therapy as a novel treatment for lung cancers. We will also test the molecular markers such as AC and S1P with Carmofur-NPs to evaluate effect on their levels when treated with Carmofur. Keywords: Malignant Pleural Mesothelioma, Carmofur, Acid Ceramidase, Inhalation

Figure 1. (A) Different Carmofur-NP formulation based on DoE model. Graphical representation of (B) Drug Loading and (C) Particle Size. (D) In-vitro release of Carmofur-NP through dialysis method in Simulated Lung Fluid. (E) Higuchi Kinetics represent best fit model of in-vitro release. In vitro aerodynamic performance of Carmofur-NP. (F) In-vitro aerodynamic deposition of Carmofur-NP in Next-Generation ImpactorTM (NGI) stages (G) % cumulative deposition of Carmofur-NP in NGI. (H) Aerosolization properties of Carmofur-NP complex. Data represent mean ± SD (n = 3).

Figure 2. Cytotoxicity Assay of Carmofur in immortalized cell lines namely (A) MSTO, (B) H28 and (C) H2452 and patient derived cell lines (D) YOU, (E) ORT and (F) ROB. (G) Tabular representation of IC50 values of Carmofur and Carmofur-NPs in different MPM cell lines. Colony inhibition assay was performed in (H) H2452 and (I) ROB cell lines for 48 hours. Quantification of colonies treated with different treatment groups in (J) H2452 and (K) ROB. Data represent mean ± SD (n = 3).

Figure 3. 3D tumor simulation studies. Effect of treatment on the growth of the tumor in 3D spheroids study of (A) MSTO and (B) ROB in therapeutic model. Images demonstrate the spheroids on day 0 to day 12 after single dose treatment. (C) and (D) Represents spheroids volume vs time (days) plot in MSTO and ROB respectively. Spheroids were treated on day 0 (3rd day of growing spheroids). Data represent mean ± SEM (n = 8). Qualitative and quantitative assessment of anti-tumorigenic activity of Carmofur and Carmofur-NPs. (E) and (F) Fluorescent images of spheroids been subjected to live-dead assay on day 12 in MSTO and ROB respectively. Data represents mean ± SD (n=8), **p < 0.01, ***p < 0.001,****p < 0.0001 shows comparison between different treatment groups. Scalebar = 200𝛍m. Detection of apoptosis using Annexin V and Dead Cell Assay in H2452. (G) Represents total % apoptotic H2452 cells due to exposure to different concentration of Carmofur and Carmofur-NPs for 24 hours. Data represents mean ± SD (n=3).