Weak Base Permeability Characteristics Influence the Intracellular Sequestration Site in the Multidrug-resistant Human Leukemic Cell Line HL-60

Abstract

A number of organelles contained within mammalian cells have been implicated in the selective sequestration of chemical entities including drug molecules. Specifically, weakly basic molecules have been shown to selectively associate with either the mitochondrial compartment or lysosomes; however, the structural basis for this differentiation has not been understood. To investigate this, we have identified a series of seven weakly basic compounds, all with pK a near neutrality, which have different sequestration sites within the multidrug-resistant HL-60 human leukemic cell line. Three of the compounds were selectively sequestered into the mitochondria of the cells, whereas the remainder were predominantly localized within lysosomes. Using specific chemical inhibitors to disrupt either mitochondrial or lysosomal accumulation capacity, we demonstrated that accumulation of these compounds into respective organelles are not competitive processes. Comparison of the permeability characteristics of these compounds as a function of pH revealed striking differences that correlate with the intracellular sequestration site. Only those compounds with significantly reduced permeability in the ionized state relative to the un-ionized state had the capacity to accumulate within lysosomes. Alternatively, those compounds with relatively pH-insensitive permeability selectively accumulated into mitochondria. Using novel quantitative assays for assaying drug accumulation into subcellular organelles, we demonstrated a correlation between these permeability characteristics and the lysosomal versus mitochondrial accumulation capacity of these compounds. Together, these results suggest that the selective accumulations of weakly basic compounds in either lysosomes and mitochondria occur via exclusive pathways governed by a unique permeability parameter.

Quantitative modeling of selective lysosomal targeting for drug design

“…Dramatically, the model no longer predicts accumulation of weak bases in lysosomes but instead predicts accumulation by mitochondria if pK a ≥ 6. This is in accordance with experimental findings for rhodamine 123 and rhodamine 6G (Duvvuri et al 2004). …”

“…Accumulation of chloroquine in lysosomes "several hundred-fold" has been observed (De Duve et al 1974). Quinacrine, another bivalent base accumulating in lysosomes (Duvvuri et al 2004) has a log K OW of 4.79 and pK a -values at 10.2 and 8.2 (Hansch et al 1995, Newton andKluza 1978). The properties of both compounds are in the predicted optimum range.…”

“…It was shown that the ratio of membrane permeabilities in the dissociated and neutral state strongly influences lysosomal accumulation (Duvvuri et al 2004). We therefore generated a simulation analogous to Figure 2 but assuming equal permeability of ion and neutral molecule (Fig.…”

“…But also if the site of action of a drug is not lysosomal, side-effects associated with unintentional lysosomal accumulation may occur. Recently, sequestration of anticancer drugs in acidic intracellular organelles has been studied as a mechanism determining the cell typeselectivity of candidate anticancer agents (Duvvuri et al 2004, Duvvuri and. Lysosomal drug sequestration can also underlie rapid clearance of anticancer drugs from intrinsically drug resistant cancer cells (Chen and Rosania 2006).…”

A Cell-Based Molecular Transport Simulator for Pharmacokinetic Prediction and Cheminformatic Exploration

“…Plugging Eq 18 , Eq 19 and Eq 20 into Eq 4 , Eq 5, and Eq 6 to get Eq 21 , Eq 22, and Eq 23. The membrane permeability P 23 can be estimated using: [24] D is the diffusion coefficient which is about 10 −14 m 2 / s for organic molecules in biomembranes. K is the partition coefficient, and approximates K ow .…”