P‐Glycoprotein preferentially effluxes anthracyclines containing free basic versus charged amine

Abstract

The multidrug resistant (MDR) tumor phenotype, characterized by a decreased cellular drug accumulation is achieved by ATP-dependent extrusions of drugs from cells by P-glycoprotein (P-gp) and/or by multidrug resistance protein (MRP1). Despite the huge amount of research that has been performed on the mechanisms of P-gpmediated efflux of drug, it is not yet known what the molecular parameters are required for a molecule to be recognized and pumped out by P-gp. Anthracyclines are weak bases and, depending on the pH, can exist either in the neutral or in the positively charged form. The aim of the work reported here was to determine which molecular form is actively pumped out by P-gp (the neutral form, the protonated form, or both), and if both, the relative efficiencies of pumping. We used spectrofluorometric methods to determine the efflux of anthracyclines in K562/Adr cells, at different intracellular and extracellular pH levels. Using 3 H -deamino, 3 H -hydroxyl doxorubicin (OH-DOX), which is permanently neutral, we first verified that our methodologies were accurate and that the P-gp-mediated efflux of OH-DOX would not depend on the pH being in the range 6.6±8.4. The P-gp-mediated efflux of daunorubicin (DNR) and 3 H -hydroxy-4-amino (WP608) was determined at different pH values. These two drugs were chosen because: (a) the lipophilicity of the neutral forms of these two molecules is so similar that any difference in the P-gp-mediated efflux cannot be assigned to lipohilicity variation, and (b) their pK a values are different (8.4 and 7.7 for DNR and WP608, respectively), which makes it easy to obtain a large variation in the proportions of the neutral and positively charged forms. Our data show that both forms are recognized by Pgp but the neutral form is pumped about three times more efficiently than the charged form. This is corroborated by results showing the active efflux (checked at pH i 7.3 only) of five other anthracycline containing a basic center. We interpret these data to mean that: (a) the positive charge of anthracycline is not a necessary requirement for P-gp recognition, but that (b) the presence of a protonable basic nitrogen facilitates the processing of these compounds by MDR efflux system.

Localization of Multidrug Transporter Substrates within Model Membranes

“…However, it is the uncharged form of the drug which is transported via passive diffusion across the membranes (59), and it has also been shown that it is transported better by PGP than charged doxorubicin (21). Our data show a very similar location profile of neutral doxorubicin ( Figure 5B) compared to that for data acquired at pH 7.4 (see the Supporting Information).…”

Lessons from patient-derived xenografts for better in vitro modeling of human cancer

“…For weakly basic therapeutics such as mitoxantrone, doxorubicin, and topotecan, the acidic extracellular environment in the tumor results in an accumulation of the charged species in the extracellular space and greatly decreases their efficacy [255,256]. Furthermore, the acidic microenvironment can also enhance the activity of known drug transporters such as p-glycoproteins and confer an additional mechanism of drug resistance through increased efflux [257,258]. However, the acidic tumor environment can also be exploited for therapeutic purposes.…”

Epirubicin and meglumine γ‐linolenic acid

“…This would be expected to cause a shift toward the uncharged moiety of the drug, increasing binding to efflux transporters and reducing cellular drug concentration. Further confirmatory evidence comes from the work of Frezard et al, who suggested that the uncharged form of anthracycline (favored in alkaline conditions) is pumped out of cells more efficiently through P‐gp effluxors 36. Therefore, it seems that drug uptake and cytotoxicity are favored if an alkaline outside/acid inside pH gradient is present.…”