Drug plasma stability study of multidrug resistance inhibitors by LC-MS/MS analysis

The Multidrug Resistance (MDR), considered in this study, can be defined as the acquired resistance to the action of chemotherapeutic drugs, not correlated either by their chemical structures or by action mechanisms. The classic MDR is associated with the over-expression of Pglycoprotein (P-gp), an extrusion pump, expressed in many tissues, that eject a variety of antineoplastic drugs from the cells, lowering their concentrations below the necessary for anticancer action. In order to limit this effect the modulation or inhibition of the functions of P-gp with a new class of substances named MDR inhibitors is necessary. The aim of my thesis is the study of the chemical stability of a series of active compounds on MDR. The studied compounds show many common structural characteristics, they are constituted by two lateral aromatic moieties linked to a basic nitrogen atom by two alkylic chains of different length. The aromatic rings used in this series of compounds were: the trimethoxycinnamic residue, common to all the molecules, while the other aromatic portion can be constituted by a structure between the carboxiantracenic or the trimethoxybenzoic acids. Unfortunately the studied compounds contain ester groups in their structure that may be susceptible to hydrolysis by the plasma enzymes, modifying their bioavailability. Therefore, stability tests were carried out for all the new MDR inhibitors in phosphate-buffered solutions (PBS) and human plasma. The PBS solution was included in this study to evaluate the spontaneous hydrolysis suffered to the studied compounds by pH or he ionic strength of the physiological solution. The experiments of Drug Stability were carried out by the addition of each analyte to 100 μL of PBS or human plasma samples (1 microM final concentration). The obtained solutions were incubated at 37°C for different times (0, 30, 60 and 120 min) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The LC-MS/MS methods are considered the best choice for the quantitative determination of drugs and their metabolites in biological matrices in terms of sensitivity and specificity. In order to follow the degradation profiles of these MDR inhibitors, it was monitored the variation of the analyte concentration to the different incubation times. Furthermore, to verify that the degradation of compound was due to a hydrolysis mechanism by the esterase enzymes, the related alcohols were searched. This investigation is operated through MS/MS experiments such as Precursor Ion Scan and Multiple Reaction Monitoring.