Khatwa, Rina (2010) Tumour delivery of anticancer agents via colloidal carriers constructed with P-glcoprotein inhibitory excipients. (PhD thesis), Kingston University, .
Abstract
The poor aqueous-solubility of many anticancer drugs limits their use as therapeutic agents. PacIitaxel, for example, is extremely useful for many refractory cancers yet its poor aqueous-solubility «1IJg/mL) requires solubilisation in Cremophor® EL, the latter which presents many clinical side-effects. To overcome such issues, micellar vehicles constructed from generally regarded as safe (GRAS) surfactants, such as poloxamer P105, that can not only solubilise but enhance the action of chemotherapeutic agents due to their inherent P-glycoprotein (Pgp) inhibitory activity has been investigated. Whilst poloxamer surfactants form thermodynamically stable hydrophilic micelles at high concentrations, they are prone to dissociation upon dilution especially in the case of intravenous injections. Micelles were stabilised to prevent premature drug release via three methods (i) cross-linking di-acetylene bonds in tricosadiynoic acid which was initiated by UV irradiation in the presence of classical micelles formed from sodium dodecyl sulphate (SOS), (ii) generation of an interpenetrating polymer network within and around poloxamer P105 micelles by free radical generation using N,N-diethylacrylamide (NNOEA), and (iii) physical adsorption of a pre-formed, hydrophilic polymer, .polyacrylamíde (10kOa) onto poloxamer P105, P103, L64 and F127 micelles: This final method of stabilisation produced superior results with respect to control over particle size, formulation homogeneity, and shelf-life stability hence, drug release from the stabilised system was further investigated. Thermal differential scanning calorimetry (OSC) analysis suggested that a blend of poloxamer P105 and polyacrylamide were miscible resulting in a change in glass transition (Tg) from -71°C to -30°C. Photon correlation spectroscopy (PCS) also confirmed an interaction as particulate size increased from 18nm ± 0.62nm (non-coated micelles) to 22nm ± 1.36nm (after coating; P<0.05) with low sample polydispersity values. The physlco-chemtcal stability of coated micelles containing poorly water-soluble molecule, dye Oil Red O (ORO) or anticancer drug, methotrexate, demonstrated an inverse relationship with polyacrylamide coating with respect to drug release. Thus, an inCrease in the coating level reduced methotrexate/dye release, which suggested overall formulation intactness. Chemosensitivity studies (via MTT assay) on breast cancer cell lines showed that MCF-7 cells were far more sensitive to free methotrexate (lCso of 23nM ± 3nM) than drug resistant MCF-7/AOR cells (ICso>200nM) at 48h. In the presence of poloxamer P105 and methotrexate, MCF-7/AOR cells became sensitised, attaining a significantly lower ICso of 85nM ± 12nM (P<0.05) at 48h, while exposure of poloxamer alone at concentrations up to 15001JM, which is well above the critical micelle concentration (CMC) of,P105 demonstrated little to no signs of cell toxicity at 48h. These results demonstrate Pgp inhibitory activity bought about by poloxamer P105. Further investigations led to comparison of in vitro data obtained for poloxamer P105 in contrast to other poloxamers which were either reported to exhibit Pgp activity (poloxamer L64), to have unknown Pgp activity (poloxamer P103) or no Pgp activity (poloxamer F127). Results demonstrated that all the poloxamers tested exhibited Pgp inhibitory activity since a decrease in IC50 values were recorded between free methotrexate and poloxamer in the presence of methotrexate. However, while the resistant cells showed little sensitivity to P105 exposure alone at high concentrations all the other polexamera indicated cell sensitivity at concentrations just above their CMC values. Stabilised, coated poloxamer P105 micellar formulations containing methotrexate show great potential in the treatment of refractory tumours as developed by this study.
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