Development and application of novel analytical methods for molecularly targeted cancer therapeutics

A detailed understanding of correlations of drug levels with drug action is an important aspect of the pre-clinical, clinical development and routine use of anti-cancer drugs. In furtherance of this important aim, the research in this thesis sought to develop and apply novel, highly sensitive, analytical methods to examine and understand correlations of drug levels with drug resistance mechanisms, therapeutic efficacy and side effects. LC-MS/MS is a state of the art analytical technique used to characterise and quantify both bio-molecules and pharmaceutical agents. Based on LC-MS/MS techniques, novel sensitive analytical methods were developed for the quantification of 1) tyrosine kinase inhibitor anti-cancer drugs to examine the interaction of these agents with drug resistance mechanisms, and 2) the multiple myeloma drug thalidomide as a pilot study to identify potential correlations between serum drug levels and toxicity/efficacy. Acquired resistance to chemotherapeutics through the over-expression of ABC transport proteins has presented a significant clinical challenge. The up-regulation of ABC transporters such as P-glycoprotein (P-gp), Breast Cancer Resistant Protein (BCRP) and Multidrug Resistance-associated Protein (MRP-1) is an important cellular mechanism of resistance in vivo and in vitro. Using cell-line models and the developed LC-MS/MS quantification method it was established that dasatinib, an agent used in treatment of chronic myelogenous leukaemia, is a substrate of ABC transporters P-gp and BCRP but not of MRP-1, and that dasatinib does not inhibit these transporters at clinically relevant concentrations. Another Tyrosine Kinase Inhibitor (TKI) being used in breast cancer, Lapatinib, was shown to be an inhibitor of P-gp but not a substrate of this resistance mechanism. Levels of other TKIs were also found to vary in their uptake in primary models of brain cancer. Using LC-MS/MS, the circulating serum levels of thalidomide in multiple myeloma patients were studied. A clear correlation between dosing regime and serum level was seen; however, the research also unearthed evidence of a significant incidence of non-compliance which could have far reaching consequences for patient treatment. Overall, LC-MS/MS methods for the quantification of chemotherapeutic agents in complex biological matrices produced sensitive and accurate data which we successfully exploited to characterise resistance to new drugs and examine correlations between serum levels and treatment variables