Two complementary fluorimetric assays for the determination of aminoquinoline binding and uptake by human erythrocytes in vitro

Abstract

a b s t r a c tWe provide two simple low-cost and low-tech procedures to measure with good precision and accuracy the binding and internalization into human erythrocytes of chloroquine and other aminoquinolines. The methods are based on the high fluorescence of the quinoline ring and are complementary. Method A evaluates residual drugs in the supernatants of treated erythrocytes, whereas method B quantifies the total uptake by whole cells and the fraction bound to the membranes. Drug uptake is dose dependent and related to the number of erythrocytes. These assays could be useful when studying the cell interaction of quinoline-type compounds not available in the radioactive form.Ó 2008 Elsevier Inc. All rights reserved.Chloroquine (CQ) 1 has been one of the most successful antimalarial drugs due to its specificity, safety, and stability, but it is now largely ineffective in most malaria endemic areas because of the emergence of Plasmodium falciparum (Pf) resistance. Artemisin-based combination therapy is currently recommended as first-line treatment for uncomplicated malaria, but there is a continuous necessity of new drugs or new drug combinations to increase efficacy and delay the onset of resistance [1,2].We recently synthesized new quinolizidinyl and quinolizidinylalkyl derivatives of 4-aminoquinolines in which a terminal bulky bicyclic basic moiety was introduced to prevent the metabolic oxidation that limits the usefulness of quinoline compounds. Leads that are highly effective in vitro against several drug-resistant strains of Pf and in vivo in the murine Plasmodium berghei model were obtained [3]. Two of these compounds, named AM1 and AP4b, were selected for further characterization (Fig. 1). Preliminary pharmacokinetic data indicate that more than 60% of compounds are bound to the corpuscular blood fraction. This is consistent with what has been described previously for CQ [4].Since the early 1970s, the capacity of red blood cells (RBC) uninfected or infected by different strains of Pf to bind CQ or other aminoquinoline antimalarial drugs has been evaluated using 3 H-or 14 C-labeled compounds. The uptake of the drug by RBC was quantified by counting the radioactivity that is associated with the cell pellet or that disappeared from the incubation medium after centrifugation [5][6][7]. To avoid the laborious synthesis and risky use of radiolabeled compounds, we developed two simple, low-cost, and reproducible methods based on the high fluorescence of the quinoline ring to evaluate binding/internalization of the quinolines to human RBC.Method A measures the residual fluorescence in the supernatants of drug-treated RBC, whereas method B was optimized to differentiate and quantify the amount of compound bound to RBC membranes or internalized by RBC. The experiments were done using fresh RBC from healthy donors. Cells were washed three times with 5 mM cold phosphate-buffered saline (PBS)-glucose and incubated with the drugs at 37°C in the optimized conditions described below.In preliminary experiments, we dete...

Novel Antimalarial Aminoquinolines: Heme Binding and Effects on Normal or Plasmodium falciparum -Parasitized Human Erythrocytes

“…The uptake of the 4-aminoquinoline compounds by RBC was determined by a fluorimetric method described recently (2). Briefly, human RBC at 15% Htc in PBS-5 mM glucose were incubated at 37°C with serial dilutions (10 to 50 M) of the test compounds using CQ as a reference.…”

A method for determining supernatant-free spectra generated from fluorescently stained cells in suspension

“…While the reference spectra F W (k) of unbound fluorochromes can be easily acquired using cell-free solutions, the assessment of their F B (k) counterparts is usually difficult. An often used approach for the determination of the content and/or spectral properties of the cell-bound fluorochromes is based on collecting the cells by centrifugation and recording the fluorescence from the supernatant, which is then subtracted from the total fluorescence of the cell suspension; see for example [6][7][8]. Though this approach seems to be feasible at first glance, it bears a problem caused by the scattering of both the excitation and emission light, which can significantly affect the intensity of fluorescence measured in turbid cell suspensions [9][10][11].…”