Analysis of drug transport kinetics in multidrug-resistant cells using a novel coumarin-vinblastine compound

Abstract

We have synthesized an analogue of vinblastine wherein a coumarin molecule is attached to the C17 of the vindoline moiety via a succinimide bridge (cou-VBL). cou-VBL exhibits fluorescence similar to that exhibited by coumarin. Chinese hamster ovary fibroblasts (LR73 cells) that exhibit an IC50 for vinblastine (VBL) of about 100 nM in growth inhibition 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 similar to their VBL resistance. A large change in the quantum efficiency of cou-VBL fluorescence accompanies efflux from intact cells, and comparison between cou-VBL and [3H]VBL efflux from the MDR35 cells reveals that the transport kinetics of the fluorescent analogue is very similar (if not identical) to that exhibited by [3H]VBL. Thus, similar to continuous monitoring of fluorescence (CMF) studies performed with the naturally fluorescent chemotherapeutic doxorubicin (Roepe, 1992), cou-VBL may be used in CMF studies aimed at rigorously defining the kinetics of VBL efflux from multidrug-resistant (MDR) cells. Initial data suggest the following: (1) A single exponential term approximates efflux from sensitive cells, whereas two exponentials are required to fit efflux from MDR35 cells. (2) The faster MDR35 term is virtually identical to the single term for the sensitive cells, whereas the other defines a process that is 5-20 times slower than passive diffusion, depending on the drug concentration. (3) The slower component has a much steeper and nearly linear dependence on drug concentration, whereas the fast passive diffusion component becomes asymptotic near 0.3 pM exchangeable drug/microgram of cell protein.