Novel lipid-based nanoparticles containing saponins for targeted delivery and enhanced efficacy of chemotherapeutics in neuroblastoma

• Applicant: Dr. Roger Gilabert-Oriol
• Subject Area: Pharmacy
• Term: from 2015 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 276307910

• Final Report Year: 2018

Final Report Abstract

Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system and represents the most common solid tumor of infancy. In patients with high-risk disease, the 5-year event free survival remains less than 50%. Therefore, new treatment strategies must be considered. This research project focussed on the establishment of novel strategies to increase the therapeutic efficacy of chemotherapeutic agents against neuroblastoma. First, a liposomal formulation of topotecan was developed. Liposomes were prepared by extrusion, loaded with topotecan by pH gradient and copper-drug complexation, and fully characterized in vitro. Pharmacokinetic and efficacy studies revealed modest increases in the life span of mice bearing neuroblastoma. In order to further increase the efficacy of the formulation, liposomal topotecan was applied to cells in combination with saponins (SO1861). Unexpectedly, SO1861 failed to enhance cytotoxicity. Similar results were obtained for a panel of chemotherapeutic drugs supplemented with SO1861. Subsequently, a series of experiments to understand the reasons why neither topotecan nor liposomal topotecan were enhanced by SO1861 were conducted. Fluorescence microscopy studies evidenced that liposomes did not directly interact with cancer cells and that topotecan was released from liposomes soon after administration. To investigate drug release in a more accurate way and in real time, a novel in vitro method based on fluorescence de-quenching and an automated microscopy imaging platform was developed. This assay confirmed that topotecan was released from the liposomes during the first hours of incubation in serum. Extending the retention time may contribute to increase the therapeutic efficacy of liposomal topotecan and in turn allow the coadministration of SO1861 that will enhance the cytotoxicity by preventing drug degradation in the lysosomes. Although compounds capable of forming ternary complexes with topotecan and copper were successfully loaded into liposomes, no extension in retention time was observed for liposomal topotecan. In the future, other compounds to extend retention time will be investigated. Furthermore, a new methodology for conjugation of antibodies to liposomes was developed. This new method has several technical and biological advantages, including improvements in reproducibility, preparation time and overall yield, and it will be used in future studies with dinutuximab. Finally, xenograft mouse models of neuroblastoma were developed with luciferasetransfected LAN-1 neuroblastoma cells on NRG mice and athymic nude mice. These models will be employed to assess therapeutic efficacy of liposomal topotecan containing either SO1861 or dinutuximab. Although the liposomal formulation of topotecan developed here achieved only modest improvements in therapeutic efficacy, with additional experiments the retention time of the formulation could be substantially increased and the formulation may then have the potential to be developed as a pre-clinical and clinical product with augmented therapeutic efficacy.

Publications

• Augmenting the efficacy of immunotoxins and other targeted protein toxins by endosomal escape enhancers. Toxins (2016) 8(7):200-27
  H. Fuchs, A. Weng, R. Gilabert-Oriol
  
• A simple passive equilibration method for loading carboplatin into preformed liposomes incubated with ethanol as a temperature dependent permeability enhancer. Journal of Controlled Release (2017) 252(1):50-61
  M. Wehbe, A. Malhotra, M. Anantha, J. Roosendaal, A.W.Y. Leung, D. Plackett, K. Edwards, R. Gilabert-Oriol, M.B. Bally
  
• In vitro assay for measuring real time topotecan release from liposomes: Release kinetics and cellular internalization. Drug Delivery and Translational Research (2017) 7(4):544-557
  R. Gilabert-Oriol, L. Chernov, M. Anantha, W.H. Dragowska, M.B. Bally
  
• Optimization of liposomal topotecan for use in treating neuroblastoma. Cancer Medicine (2017) 6(6):1240-1254
  L. Chernov, R.J. Deyell, M. Anantha, N. Dos Santos, R. Gilabert-Oriol, M.B. Bally
  
• Unique therapeutic properties and preparation methodology of multivalent rituximab-lipid nanoparticles. European Journal of Pharmaceutics and Biopharmaceutics (2017) 117:256-269
  J. Popov, R. Gilabert-Oriol, M.B. Bally