Abstract 1729: Novel liposomal topotecan formulation has a lower IC50 than the free form on neuroblastoma cells
Authors: Gomes Paula Viza , Shen Stephanie , Hill Meghan , Flynn Kilkee , Marquez Mendi , Frolova Liliya , Kandel Jessica J. , Tartis Michaelann , Hernandez Sonia L. 
Published: September 2020

Introduction: Neuroblastomas (NB) are the most common solid tumor in children and infants, and are frequently resistant to all standard therapies. Patients are accordingly vulnerable to acute and long-term systemic effects of toxic chemotherapy. These drugs target rapidly dividing cells throughout the body, leading to severe side effects. One strategy to reduce off-target toxicities is to selectively increase drug uptake in the tumor. In pilot studies, we found that microbubbles containing liposomal doxorubicin, an agent that is effective against NB, and focused ultrasound (sonoporation) increased doxorubicin uptake in NB xenografts. However, NB is typically treated with multidrug chemotherapy, raising the importance of testing additional agents. In these studies, we evaluated a novel liposomal formulation of topotecan, an agent used in NB treatment which is both effective and associated with significant systemic toxicities. In vitro studies are needed to determine the IC50 (concentration of the drug needed to reduce the number of live cells by half). A required esterase cleavage at the delivery site can inhibit liposome encapsulated drug release. Therefore, we hypothesized that liposomal topotecan (2T-T) would have a higher IC50 than free topotecan.

Methods: We tested the effect of 2T-T on nine different NB cell lines:LA1-55n, LA-1-5S, LAN-5, NGP, SK-N-AS, BE2, SHEP, NBL-WN, and SH-SY5Y;five N-type (invasive), three S-type (noninvasive), seven MYCN-amplified (poor prognosis). We have tested free topotecan in 3 of these (LA-1-5S, NBL-WN, SH-SY5Y). Cells were plated in full RPMI at 80% confluence. 2T-T or topotecan (0 to 50 uM) were added to cells 24 hours later for 72 hours. Viable cells were estimated using a WST cell counting kit. 50uM empty liposomes and lysis buffer were used as controls. Experiments were performed in triplicate, with p≥0.05deemed significant (student t-test or ratio-paired t-test using PRISM).

Results: 2T-T had a mean IC50 of 0.37±0.58uM(mean R2=0.9±0.07).Empty liposomes caused no cytotoxicity in any cell line. We found no difference in IC50 according to S or N type (0.50±0.65vs 0.35±0.58(p=ns)). The mean IC50 of MYCN-amplified cells was 0.47±0.63,while that of non-MYCN-amplified was 0.031±0.029(n=2), suggesting no difference in cytotoxicity based on MYCN status. 2T-T had a 2 fold lower IC50 than free topotecan (1.14±1.19vs 0.48±0.82,p=0.046), suggesting liposomes did not inhibit topotecan release.

Conclusion: These findings suggest that liposomal topotecan (T2-T) has a lower IC50 than free topotecan in NB, and that MYCN amplification and phenotype do not modify 2T-T cytotoxicity. Our results suggest that liposomal encapsulation does not inhibit topotecan release, but could increase its cytotoxicity by increasing topotecan half-life. We predict that, in vivo 2T-T could reduce toxicities and side effects, warranting the investigation of 2T-T sonoporation in NB xenografts.

Citation Format: Paula Viza Gomes, Stephanie Shen, Meghan Hill, Kilkee Flynn, Mendi Marquez, Liliya Frolova, Jessica J. Kandel, Michaelann Tartis, Sonia L. Hernandez. Novel liposomal topotecan formulation has a lower IC50 than the free form on neuroblastoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1729.