Liposomal Formulations of Metal-CX5461 complexes: Copper-CX5461 complexation mediates CX5461 degradation while Zinc-CX5461 formulations are suitable for development

Abstract

Copper (Cu²⁺) ions have facilitated the encapsulation of CX5461 within liposomes and the formulations showed promising anticancer potential as judged by in-vivo studies. In our attempt to translate this formulation into a product suitable for use in formal pre-clinical safety pharmacology studies, we uncovered a problem. In this report, studies have demonstrated that Cu mediated CX5461 encapsulation within DSPC/Chol (55:45 mol%) and DMPC/Chol (55:45 mol%) liposomes can lead to instability of CX5461. Chromatographic analysis revealed that CX5461 degradation increases as the incubation temperature increased from 40 °C to 60 °C. The change in CX5461 structure results in the emergence of new peaks detected by HPLC and this is associated with a reduction in the parent drug. The rate of CX5461 degradation was influenced by incubation time and liposome lipid composition. When encapsulated in the DSPC/Chol:Cu(CX5461) liposome formulation, just 1 h of incubation at 40 °C or 60 °C resulted in greater than 50 % CX5461 degradation. This degradation was associated with a 1.7-fold and a 3.8-fold decrease in the CX5461 to liposomal lipid ratio within 1 h at 40 °C and 60 °C, respectively. When the CX5461 formulation was prepared using DMPC/Chol liposomes, CX5461 was less prone to degradation. Significant decrease in the CX5461 to liposomal lipid ratio for this formulation required 6 h of incubation at 40 °C or 60 °C. Surprisingly, even after complete degradation of CX5461, the degraded compounds were therapeutically as active as parent CX5461 based on in-vitro cytotoxicity studies. This suggested that the biologically active portion of CX5461 was maintained even after Cu-mediated degradation. Regardless, the extent and rate of degradation preclude the pharmaceutical development of the copper formulation as the active ingredient instability poses significant (perhaps insurmountable) challenges for chemistry, manufacturing, and control documentation. To overcome this limitation, we shifted to zinc as an alternative metal to complex CX-5461. Preliminary data demonstrate that Zn(CX5461) enables efficient liposomal encapsulation while preserving chemical stability.