ANALYSIS OF DRUG TRANSPORT KINETICS IN MULTIDRUG-RESISTANT CELLS USING A NOVEL COUMARIN-VINBLASTINE COMPOUND

We have synthesized an analogue of vinblastine wherein a coumarin mole cule is attached to the C17 of the vindoline moiety via a succinimide bridge (cou-VBL). cou-VBL exhibits fluorescence similar to that exhibi ted by coumarin. Chinese hamster ovary fibroblasts (LR73 cells) that e xhibit an IC50 for vinblastine (VBL) of about 100 nM in growth inhibit ion assays are similarly sensitive to the cou-VBL compound. LR73 cells transfected with the mu MDR 3 gene that were subsequently selected on vinblastine (MDR35 cells) exhibit resistance to cou-VBL that is simil ar to their VBL resistance. A large change in the quantum efficiency o f cou-VBL fluorescence accompanies efflux from intact cells, and compa rison between cou-VBL and [H-3]VBL efflux from the MDR35 cells reveals that the transport kinetics of the fluorescent analogue is very simil ar (if not identical) to that exhibited by [H-3]VBL. Thus, similar to continuous monitoring of fluorescence (CMF) studies performed with the naturally fluorescent chemotherapeutic doxorubicin (Roepe, 1999), cou -VBL may be used in CMF studies aimed at rigorously defining the kinet ics of VBL efflux from multidrug-resistant (MDR) cells. Initial data s uggest the following: (1) A single exponential term approximates efflu x from sensitive cells, whereas two exponentials are required to fit e fflux from MDR35 cells. (2) The faster MDR35 term is virtually identic al to the single term for the sensitive cells, whereas the other defin es a process that is 5-20 times slower than passive diffusion, dependi ng on the drug concentration. (3) The slower component has a much stee per and nearly linear dependence on drug concentration, whereas the fa st passive diffusion component becomes asymptotic near 0.3 pM exchange able drug/mu g of cell protein.