Binding of Dihydroartemisinin to Hemoglobin H: Role in Drug Accumulation and Host-Induced Antimalarial Ineffectiveness of a-Thalassemic Erythrocytes

Dihydroartemisinin and other artemisinin derivatives are relatively ineffective against Plasmodium falciparum infecting a-thalassemic erythrocytes, namely hemoglobin (Hb) H or HbH/Hb Constant Spring erythrocytes, as compared with those infecting genetically normal erythrocytes. The variant erythrocytes accumulate radiolabeled dihydroartemisinin to a much higher extent than the normal ones, and the accumulated drug was retained after extensive washing, in contrast to the drug in normal erythrocytes which was mostly removed. At initial drug concentration of 1 mM, most (82–88%) of the drug was found in the cytosol fraction of both variant and normal erythrocytes. Binding of the drug to hemoglobins accounted for 40–70% of the total uptake. Hb H accounted for 10.9 6 2.7% and 12.4 6 6.2% of total protein in HbH and HbH/Hb Constant Spring erythrocytes. HbH bound with 28.7 6 6.7% of the drug, whereas HbH/Hb Constant Spring erythrocytes bound with 21.8 6 8.3% of the drug. Binding experiments showed that Hb H had 5–7 times the drug-binding capacity of Hb A. For Hb H, the maximum binding capacity (Bmax) 5 1.67 6 0.17 mol/mol Hb, and the dissociation constant (Kd) 5 66 6 17 mM, and for Hb A, Bmax 5 0.74 6 0.18 mol/mol Hb and Kd 5 224 6 15 mM. It is concluded that preferential binding of dihydroartemisinin to Hb H over Hb A accounts partly for the higher accumulation capacity of the a-thalassemic erythrocytes, which leads to its antimalarial ineffectiveness. The artemisinins form a group of antimalarials derived from Artemisia annua, an herbal plant long used in China for the treatment of fevers (United Nations Development Program et al., 1997; Klayman, 1993). They are sesquiterpenoids with an endoperoxide essential for antimalarial activity. DHART is more active than artemisinin against Plasmodium falciparum, and is probably the metabolically active form of the derivatives already in use or in advanced stages of development. With the threat of multidrug-resistant malaria on the rise, the artemisinins, which have proven to be effective against parasites resistant to chloroquine and other drugs, will be playing an increasing role in antimalarial chemotherapy. Although no resistance to these drugs has been reported so far from the field, it is important to understand factors that may contribute to the development of resistance and that may reduce the efficacy of the drugs in the future. We have shown previously that the genetic type of the host erythrocytes can influence the efficacy of artemisinin derivatives against P. falciparum (Yuthavong et al., 1989; Kamchonwongpaisan et al., 1994). Parasites in culture infecting a-thalassemic erythrocytes, both of the genetic type a-thalassemia 1/a-thalassemia 2 (--/-a) and a-thalassemia1/Hb Constant Spring (--/aa), or of the phenotypes HbH and HbH/ HbCS respectively, are more resistant to the artemisinins than the same parasites infecting genetically normal erythrocytes. Resistance is therefore generated from the host, not the parasite, and is caused by the competition from the erythrocytes, which take up the drugs in large quantities, resulting in low medium concentration and low drug uptake of the parasite. Drug-binding sites may therefore be present in the variant erythrocytes and be responsible for the uptake. The search for such possible binding sites is important in the understanding of the apparent drug resistance of the parasite infecting a-thalassemic erythrocytes, and may yield information on the nature of the drug receptor. This article reports the results of the study on distribution and localization of dihydroartemisinin in a-thalassemic and normal erythrocytes. It was found that Hb H binds with the This work was supported by National Institutes of Health International Collaborations in Infectious Disease Research Grant U01-AI35827, a United States Agency for International Development Cooperative Development Research Grant TA-U01-C09–060, and a Senior Research Fellowship (P.W.) from Thailand Research Fund. ABBREVIATIONS: DHART, dihydroartemisinin; Hb, hemoglobin; HbH, a-thalassemia 1/a-thalassemia 2; HbH/HbCS, a-thalassemia 1/hemoglobin Constant Spring; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 0026-895X/98/030492-05$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:492–496 (1998). 492 at A PE T Jornals on Sptem er 7, 2017 m oharm .aspeurnals.org D ow nladed from drug with much higher avidity than Hb A and that the former accounts for a significant portion of the drug taken up. Experimental Procedures Sample preparations. About 15 ml of venous blood from a-thalassemic patients (both HbH and HbH/HbCS phenotypes) and normal individuals was collected with citrate-phosphate-dextrose as anticoagulant. Whole blood was centrifuged at 800 3 g, at 4° for 15 min, after which plasma and the buffy coat were removed. The packed erythrocytes were washed twice with culture medium composed of RPMI 1640 supplemented with 25 mM HEPES, pH 7.4, 0.2% NaHCO3, and 40 mg/ml gentamicin. The erythrocytes were then resuspended in an equal volume of culture medium, and cell numbers were counted by an automated cell counter (Technicon, Bayer Diagnostics, Tarrytown, NY). For DHART inhibition assay, the packed erythrocytes were resuspended in a 10% human-serum-supplemented culture medium. DHART inhibition assay. The antimalarial activity of DHART was measured against P. falciparum infecting normal and a-thalassemic erythrocytes using the [H] hypoxanthine incorporation method of Desjardins et al. (1979). Aliquots (25 ml) of serially diluted DHART in dimethylsulfoxide were pipetted into a microtitration plate containing 96 flat-bottomed wells. Parasitized erythrocyte suspension (200 ml) containing 1.5% hematocrit with 0.5% parasitemia were added. After 24-hr incubation in a candle jar at 37°, 25 ml of [H]hypoxanthine (0.5 mCi, specific activity 20–30 Ci/mmol; Amersham, Paisley, UK) were added into each well and the plate was reincubated under the same condition for 18 hr. Using a cell harvester (Nunc, Roskilde, Denmark), the cell suspension was aspirated through glass filter paper (no. 934-AH; Whatman, Maidstone, UK), and washed with distilled water. The disks were dried and placed in toluene-based scintillation fluid for counting in a b-counter (LS1801; Beckman Instruments, Palo Alto, CA). IC50 values were evaluated from the sigmoidal graph of percent [H]hypoxanthine incorporation versus log of drug concentration. [C]dihydroartemisinin accumulation. Aliquots (140 ml) of 50% red blood cell suspension were incubated with 560 ml of 1.25 mM [C]DHART [specific activity 12.1 mCi/mmol; final concentration, 1.0 mM in 0.1% dimethylsulfoxide (a kind gift from Dr. Kenneth H. Davis, Jr., Chemistry and Life Sciences Division, Research Triangle Institute, NC)] in 1.5-ml microcentrifuge tube at 37° for 2 hr (Kamchonwongpaisan et al., 1994). Cells were pelleted by centrifugation at 10,000 3 g for 5 min. The packed erythrocytes were washed with 1 ml of culture medium three times and were then incubated with 700 ml of 2% sodium dodecyl sulfate solution at 60° for 1 hr. Solutions were bleached with 400 ml of 15% hydrogen peroxide at 60° for 12 hr. Four milliliters of Triton-based liquid scintillation fluid was added and the radioactivity was determined. To study drug retention, 70 ml of [C]DHART-labeled packed erythrocytes were incubated in 1 ml of culture medium in triplicates for a further 3 hr, and the amounts of radioactivity remaining within the cells were measured hourly as described above for comparison with the initial unwashed cells. [C]dihydroartemisinin distribution within red blood cells. One volume (70 ml) of packed [C]DHART-labeled erythrocytes was mixed with half a volume of lysis buffer (10 mM TriszHCl, 1 mM EDTA, pH 8.8), and the cell suspension was then freeze-thawed to lyse the intact erythrocytes. The membrane fraction was separated by centrifugation at 10,000 3 g for 15 min. Radioactivity in 105 ml of hemolysate was measured, and the amount of drug was calculated. The membrane fraction was washed five times with a buffer containing 1 mM EDTA and 0.2 mM phenylmethylsulfonyl fluoride in 5 mM TriszHCl, pH 7.6, and then incubated with 500 ml of 2% sodium dodecyl sulfate solution at 60° for 1 hr. Four milliliters of Tritonbased liquid scintillation fluid was added and radioactivity was determined for calculation of the amount of the drug in the membrane fraction. Hemoglobin typing by cellulose acetate gel-electrophoresis. Two microliters of hemolysate from [C]DHART-labeled erythrocytes was electrophoresed on a cellulose acetate plate (cellogel; Chemetron, Milano, Italy) in Tris-glycine buffer, pH 8.6, at 280 V for 30 min. The cellulose acetate plate was stained with Ponceau S solution and destained with 5% acetic acid. Cellogel was dehydrated and dried, and the percentages of hemoglobin types were quantified using a densitometer (eDC; Helena, Beaumont, TX). Total hemoglobin concentrations were assayed by the cyanmethemoglobin method (Brown, 1988). Hemoglobin binding capacity. Hemolysates (10–20 ml) from normal and thalassemic red blood cells was separated on a cellulose acetate plate as described above. Each lane was cut into areas containing band at origin, Hb A, Hb A2, Hb H, Hb CS and area(s) with no Hb band. Corresponding areas from the same sample were pooled and eluted with 5 ml of distilled water by shaking overnight at room temperature. The radioactivity was determined after bleaching with 15% hydrogen peroxide in Triton-based liquid scintillation fluid. Drug-binding capacity of each Hb was calculated as moles of [C]drug per mole of Hb. Hemoglobin isolation by carboxy methyl cellulose chromatography. Hemolysates, prepared from drug-free erythrocytes using the freeze-thaw technique as described above, were dialyzed in bisTris buffer (0.03 M bis-Tris, pH 6.1, with 0.01% potassium cyanide) at 4° for 12 hr. The dialyzed hemolysates were loaded onto a carboxy methyl cellulose column (1 3 20 cm, CM-52 cellulose; Whatman), and washed with 1–2 column volumes of bis-Tris buffer at a flow rate of 50 ml/hr, followed by 800 ml of salt gradient (between 0.030 and 0.065 M sodium chloride in bis-Tris buffer) (Schroeder and Huisman, 1980). Ten-milliliter fractions of the effluent were collected. Conductance and absorption at 280 and 415 nm were measured. Fractions from the same peak of Hb were pooled, dialyzed in 10 mM phosphate buffer, pH 7.4, and concentrated. Hb concentrations were assayed by the cyanmethemoglobin method. Binding constant measurements. Binding constants of DHART with Hb A and Hb H were measured by dialysis technique (Kabat and Mayer, 1961). The isolated hemoglobin was diluted to 10 mM with 10 mM phosphate buffer, pH 7.4, and 1 ml aliquots were placed in dialysis tubes (16 mm in diameter, retaining protein of molecular mass $ 12,000 Da; Sigma, St. Louis, MO). Each tube was incubated in 1 ml of [C]DHART (varying from 1 3 10 M to 5 3 10 M) in the same buffer at 37° for 20 hr. Then 500 ml of the solutions within and outside the tube was collected, and bleached with 500 ml of 15% hydrogen peroxide. Four milliliters of Tritonbased liquid scintillation fluid was added, and radioactivity was measured. The concentrations of bound and free drugs were calculated and the binding curves were evaluated using the program ENZFITTER (Cambridge Biosoft, Northwich, UK).