Flow cytometric assessment of the cellular pharmacokinetics of fluorescent drugs

Abstract

Development of multidrug resistance (MDR) in cancer cells decreases net doxorubicin uptake as a result of either increased efflux, or decreased intracellular sequestration, or decreased membrane permeability. Kinetic parameters of drug uptake can distinguish among these forms of altered transport. Cellular uptake of fluorescent drugs was monitored by a flow cytometric assay using a rapid-injection system and analyzed with a three-compartment model in which rapid diffusion from extracellular fluid into the cell was followed by uptake into a nonexchangeable pool. In agreement with our recent studies of '*C-doxorubicin distribution (Dordal et al.: J Pharmacol Exp Ther 271:12861290, 1994), sequestration of doxorubicin was decreased 2.7-fold in P-glycoprotein-expressing SU-4R lymphoma cells compared to drug-sensitive SU-4 cells Malignant cells may become resistant to anticancer drugs by the overexpression of proteins that alter net cellular drug accumulation. Clinically significant resistance to anthracyclines, vinca alkaloids, and other natural products has been reported in association with increased expression of P-glycoprotein, the most widely studied of these proteins (9,ll). More recently, intracellular proteins have been described that are found in resistant cells that do not express P-glycoprotein but do exhibit a similar phenotype (4,16). P-glycoprotein is believed to act as a drug efflux pump, because cell lines expressing the protein lose intracellular drug much more quickly than their nonresistant progenitors when transferred to drug free medium.However, our recent studies of multidrug-resistant HL-60 and SU-4 subclones suggested that, in these cell lines, the most significant alteration in doxorubicin transport was decreased sequestration in a nonexchangeable compartment (7). The result was that proportionally more intracellular drug existed in exchangeable compartments, where it could freely diffuse from the cell. Drugresistant cells showed no evidence of active efflux (14.0 2 4.8 vs. 5.0 f 0.9 nl s-') without a change in membrane permeability or evidence of active efflux. In contrast, sequestration of the highly fluorescent dye rhodamine 123 was decreased 20-fold (17.1 f 8.3 vs. 0.9 f 0.8 nl s-'). Resistant

Mathematical and computational models of drug transport in tumours

“…The remaining two (a discrete, compartment-based, model and a continuum, PDE-based, model) are tailored to the specific problem of drug delivery from a single vessel to a homogeneous tumour cord, assuming radial symmetry. All were built on a binding model involving extracellular drug and free and bound intracellular drug, which extends those of [12] and [17] by allowing drug binding to be saturable and reversible.…”

“…This model is illustrated by the two-dimensional schematic in figure 2. Note that the model presented in (2.1)-(2.3) is an extension of those in [12] and [17], in which drug binding is non-saturable and nonreversible. It would be straightforward to modify the binding model in this way, or to account for Michaelis-Menten-type transport should this be required, as in [8,18,19], for example.…”

“…The precise nature of this relationship can be derived by noting that ð 17) in which n represents the unit normal pointing outwards from volume V i . Note that the direction of n varies over the surface of V i .…”

Intracellular Accumulation and Mechanism of Action of Doxorubicin in a Spatio-temporal Tumor Model

“…A generalized version of the three-compartment model for drug dynamics within the tumor presented in Dordal et al (1995) is considered. The first compartment represents the extracellular space in which cells move, the second corresponds to the intracellular fluid space (including the cell membrane) which is in direct equilibrium with the extracellular space, and the third is a non-exchangeable compartment that represents sequestered drug which is trapped, perhaps in the nucleus, where it begins to damage the cellular DNA directly triggering cell death.…”

“…DOX is an anthracycline antibiotic used in the treatment of a wide variety of malignancies such as breast, ovarian, sarcomas, lymphomas, and acute luekemias (Young et al, 1981). An abundance of experimental data exists on the cellular uptake, metabolism, and cytotoxity of doxorubicin (Demant & Friche, 1998;Degregorio et al, 1984;Svensson et al, 1995;Dordal et al, 1995;Gieseler et al, 1994;Andreoni et al, 1994;Paul et al, 1979). Although intracellular DOX accumulation varies from one cell line to another, most cells achieve intracellular steady-state levels within 2-8 hr.…”