|Ph.D Student||Katzir Hagar|
|Subject||Cellular Pharmacokinetics of Anticancer Drugs|
|Department||Department of Biology||Supervisor||Professor Gera Eytan|
|Full Thesis text|
The present research examines various aspects of the passive uptake of anticancer drugs into cells, multidrug resistance (MDR) mediated by ABC transporters and modulation (inhibition) of the latter.
Previously, hundreds of MDR modulators have been identified, most of them acting as competitive inhibitors of the ABC transporters. The mechanism used by a diverse group of surface active agents to modulate MDR has been studied in cell free systems. In the present study, conducted in living cells, agents belonging to the classes of detergents and excipients were shown to modulate MDR by inhibition of Pgp-mediated efflux similarly to “classical” MDR modulators. Anesthetics, on the other hand, modulated MDR by accelerating the transbilayer movement of drugs. No correlation was observed between modulation and membrane fluidizing characteristics.
Pharmacokinetics of anticancer drugs in a variety of presumably “sensitive” cells was studied using quantitative assays of rhodamines’ uptake and efflux. At quasi-equilibrium conditions, the rhodamines concentration in the cells’ cytoplasm turned out to be lower than their extracellular concentration. An efflux mechanism active in presumably sensitive cells was identified and shown to be capable of preventing uptake of even high concentrations of rhodamines. This mechanism is inhibited by NBD-Cl and various inhibitors of the ABCC family of transporters. This innate resistance is probably mediated by the basal levels of MRP1 expressed in “sensitive” cells. Uptake of rhodamines into mitochondria overcame their active efflux. This observation suggests that the amount of drugs taken up by cells is determined by competition between their active efflux by pumps and their binding to internal sinks and their rate of passive movement across the plasma membrane.
The role of the inner leaflet of the plasma membrane in passive and active transport of drugs across the plasma membrane was studied taking advantage of FRET from a TMA-DPH probe located in the plasma membrane to rhodamines. It turned out that the inner plasma membrane constitutes a kinetic compartment separate from the cytoplasm. Mitochondria proved capable of extracting the rhodamines from the inner leaflet compartment. Therefore, we suggest that the kinetic barrier between the plasma membrane and the cytoplasm is the diffusion of the rhodamines away from the membrane rather than their desorption from the membrane. Whereas P-glycoprotein appeared to pump its substrates from the inner leaflet of the plasma membrane, MRP1 seemed to pump its substrates from the cytoplasm.