The use of manganese ion gradients in the preparation of anticancer drug formulations.

摘要

One goal of drug delivery systems is to increase the therapeutic index of an associated drug. Liposomes as lipid-based drug carriers have proven to be a versatile formulation technology for intravenous use, particularly when considering their use with anticancer drugs. Liposomes have the potential to increase the therapeutic index of a drug by altering the drug's pharmacokinetics and bio-distribution. It is understood for drug loaded liposomes that these attributes are often dictated by the materials used to prepared the liposomes, by the physical attributes of the resulting formulation and by the manner in which the drug is associated with carrier. This thesis characterises a novel method of encapsulating anticancer drugs within liposomes containing entrapped manganese. It is demonstrated that interactions between the transition metal manganese and doxorubicin, an anthracycline antibiotic and chemotherapeutic, are sufficient to promote drug loading in liposomes. These studies concluded that doxorubicin possesses co-ordination sites capable of complexing transition metals. This complexation reaction occurred at neutral pH and the loading reaction was not dependent on use of liposomes exhibiting or maintaining a transmembrane pH gradient. Unlike pH gradient based loading methods, which result in formation of a doxorubicin fibre bundles within the liposome core, doxorubicin-manganese complexation did not promote bundle formation as judged by cryo-electron microscopy. Studies assessing whether manganese complexation could promote encapsulation of another anticancer drug, topotecan, were completed. Although drug loading was not achieved through complexation, the studies confirmed that topotecan can be loaded into liposomes exhibiting a pH gradient. The stability of this formulation appeared to be dependent on the presence of sulfate as a counter ion and loading resulted in formation of precipitated structures within liposomes.; Given these initial data, it was suggested that manganese gradients could be used to encapsulate multiple drugs. This was demonstrated by pursuing development of a liposomal formulation containing two anticancer drugs, doxorubicin and vincristine. Doxorubicin loading was driven by complexation with manganese while vincristine was loaded using a pH gradient. The co-encapsulated doxorubicin/vincristine formulation exhibited pharmacokinetic attributes comparable to liposomal formulations containing only a single drug. Interestingly, the formulation containing both drugs proved to be less effective than liposomes containing only vincristine in the treatment of an established breast cancer model. These data were explained by in vitro studies suggesting that the co-formulated drugs exhibited antagonistic interactions when simultaneously added to tumour cells as free drugs.; Another advantage of achieving drug loading through manganese complexation was illustrated by characterizing drug loading in a novel thermosensitive liposomal formulation. Although efficacious when used in combination with mild heating, other investigators have shown that doxorubicin encapsulation achieved through use of transmembrane pH gradients (inside acid) was limited and the resulting doxorubicin loaded thermosensitive liposomes exhibited poor stability in vivo. It is demonstrated here that the limitation in drug loading capacity can be overcome through methods relying on metal-drug complexation. Drug loading limitations in these formulations may be dependent on the physical state in which doxorubicin exists within the liposomes. In total, the data presented provides new insights into the factors influencing drug encapsulation into liposomes containing manganese, an ion that can facilitate loading through a complexation reaction or via an ionophore mediated pH gradient formation.