A novel liposomal formulation of irinotecan comprising a combination of copper complexation and a transmembrane pH gradient to significantly improve irinotecan encapsulation and retention

A liposomal CPT-11 formulation was previously developed and approved for pancreatic cancer (Onivyde), but it carries a FDA black-box warning because it exacerbated CPT-11 associated gastrointestinal (GI) toxicities. An alternative liposomal irinotecan formulation, Irinophore C, reduced GI toxicity as judged by a validated animal model of irinotecan mediated early and late onset diarrhea. Irinophore C did not progress to clinical trials, partly because its development coincided with the approval of Onivyde. Nevertheless, an Irinophore C-like formulation remains of interest because of its potential to reduce GI toxicity and its potential to be developed as a combination product. Irinophore C was prepared using liposomes containing entrapped copper (Cu 2 ⁺). The divalent metal ionophore A23187 was used to exchange entrapped Cu 2 ⁺ for protons, creating a transmembrane pH gradient. Following addition of CPT-11 to the liposomes, it crossed into the liposome interior to achieve efficient (>98%) liposome association. The results were consistent with previous findings that CPT-11 can be loaded into liposomes with a transmembrane pH gradient (inside acid). The presence of copper, however, imparted unique properties. The resulting formulation retained CPT-11 better than liposomes loaded using a simple transmembrane pH gradient or a pH gradient generated with A23187 added to liposomes containing other divalent metal ions (e.g., Mg 2 ⁺, Mn 2 ⁺, Zn 2 ⁺). The data suggested improved rug retention arose due to Cu 2 ⁺ binding with CPT-11 and/or with lipids in the inner membrane of the liposomes. It was further suggested that Cu 2 ⁺ may mitigate GI toxicity. In this study it is argued that if Cu 2 ⁺ ions confer unique advantages to the liposomal CPT-11 formulation, then the original method used to prepare Irinophore C was not optimal. This is because A23187 significantly reduced the amount of copper retained in the liposomes. Here, we characterize an alternative method for CPT-11 encapsulation; a method that retains Cu 2 ⁺ within liposomes. In this formulation, A23187 is not required and the low internal pH is maintained with ammonium sulfate. The results show that CPT-11 can be efficiently loaded into these liposomes, achieving CPT-11 to liposomal-lipid ratios exceeding 0.6 (mol:mol). The resulting formulations exhibits significantly improved drug retention and cryo-transmission electron microscopy analysis suggests the morphology of the new formulation differs from Irinophore C. Efficacy studies in a syngeneic BALB/c CT26 colorectal cancer model demonstrated the therapeutic potential of the new formulation, but highlight a challenge when developing high CPT-11 to liposomal-lipid ratio formulations. Specifically, high drug to liposomal-lipid formulations utilize significantly lower lipid doses that are eliminated rapidly after administration. It is suggested that this new formulation is suitable for development in combination with other therapies, however the rapid elimination of the liposomes administered at low liposomal lipid dosed must be addressed.