Increased accumulation of chloroquine and desethylchloroquine in homozygous sickle cells

Bulletin of the World Health Organization, 65 (1): 51-55 (1987) © World Health Organization 1987 Increased accumulation of chloroquine and desethylchloroquine in homozygous sickle cells A. U. ORJIH' The effect ofhaemoglobin genotype on the level ofchloroquine in the erythrocytes of homozygous sickle-cell (SS), normal (AA), and heterozygous (AS) subjects was investi- gated in vivo and in vitro. Two hours after a single oral dose of chloroquine its level in plasma was consistently lower in SS than in AA subjects. In contrast, its level in the erythrocytes was higher in SS than in AA subjects. Desethylchloroquine, a metabolite of chloroquine, was detected only in the erythrocytes ofSS blood but was present in both the plasma and erythrocytes ofAA blood. For the in vitro test, a 5% suspension of erythro- cytes was incubated for I hour with a 2.06 ,mol/l solution of chloroquine. The mean chloroquine distribution ratio (tsmol chloroquine per kg erythrocytes: tmol chloroquine per litre medium) was 31.0, 3.5, and 2. 7for SS, AA, and AS erythrocytes, respectively. The results of the study indicate that haemoglobin genotype appears to influence the level of chloroquine in erythrocytes. Identification of chloroquine-resistant malaria in vivo requires evidence of intake of the drug and its absorption by the infected case. This is best achieved by determining the concentration of chloroquine in blood (1). Currently, however, there is no consensus as to which component of blood should be analysed; some workers have determined the level of chloro- quine in serum (2), while others have reported the level in whole blood (3). The concentrations of chloroquine in these components differ, and it is therefore difficult to compare data reported by different investigators and to interpret the possible relation between drug concentration in a given component and clinical effect. In general, however, in healthy individuals the ratio of the level of chloroquine in erythrocytes to that in plasma rarely exceeds 6, but the ratio for those who are diseased or have other special conditions has not yet been adequately determined. In areas where malaria is endemic those with sickle- cell disease regularly use antimalarial drugs, and this paper reports the distribution of chloroquine and its metabolite desethylchloroquine in the blood of sub- jects with sickle-cell anaemia (SS) and sickle-cell trait (AS) as well as normal individuals (AA). Senior Lecturer in Immunology and Parasitology, Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria. MATERIALS AND METHODS Accumulation of chloroquine in erythrocytes was determined after both in vivo and in vitro exposure to the drug. The prior consent of all subjects was obtained. In vivo test Vital data on the participants in the study are shown in Table 1. All nine subjects lived in Nigeria. Subject A was hospitalized with sickle-cell crisis, whereas the others were in good health. A few minutes before administration of chloro- quine, 2 ml of venous blood was collected from eight of the participants (subject A was excluded). The blood samples were stored refrigerated in heparinized plastic centrifuge tubes for about 2 hours before processing. The subjects (except A and H) received oral doses of 1 or 2 tablets of chloroquine phosphate, each tablet containing 150 mg chloroquine base; subjects H and A received 1 and 2 doses, respectively, of 100 mg chloroquine syrup. Two hours after drug intake a second sample of venous blood was taken from each subject for analysis. Test for malaria. Thin and thick smears of all blood samples collected were made on glass slides. After air-drying, the smears were treated with Giemsa 4750 51- 52 A. U. ORJIH Table 1. Vital data on the in vivo study subjects Blood Erythrocyte Chloroquine dose Subject Age (years) Sex Weight (kg) genotype' volume fraction (mg base) A 4 F 12 SS 0.24 2xlOd B 24 M 75 SS 0.26 1x 300 C 9 F 26 SS 0.27 1 x 150 D _b M 73 SS -c 1 x300 E 19 M 52 AA 0.38 1x300 F 17 F 53 AA 0.36 1x300 G 35 F 75 AA 0.35 1 x300 H 3.5 M 18 AS 0.33 1x100 43 M 63 AS 0.44 1 x300 SS = subject with sickle-cell anaemia; AA = normal subject; AS = subject with sickle-cell trait. Age not known but subject was an adult. c Not determined. dSubject A was hospitalized and received the first dose of chloroquine on admission; the second dose was given 10 hours later, 2 hours before the test blood sample was collected. stain and examined microscopically for para- sitaemia. Detection ofchloroquine and desethylchloroquine. The concentrations of chloroquine and desethyl- chloroquine in the blood samples were determined by the Centres for Disease Control (CDC), Atlanta, GA, USA using high-performance liquid chroma- tography (HPLC) and a fluorescence method (4). Blood samples were first centrifuged at 2500 g for 20 minutes, and 0.1 ml of the plasma layer was carefully removed and absorbed on to a strip of thick filter- paper (4). The residual plasma in the tube and the blood leukocytes were then carefully removed and discarded, and 0.1 ml of the erythrocyte pellet was smeared on another filter-paper. The smears were carefully dried in air at about 27 °C for 24 hours. After drying, the samples were packed in cellophane bags and forwarded to CDC for analysis. In vitro test Samples of blood from 18 blood donors aged 17-42 years who were resident in the USA were used for the in vitro chloroquine accumulation test. The distri- bution of haemoglobin genotypes was as follows: 8 SS, 5 AA, and 5 AS. Donors of SS genotype had sickle-cell anaemia and were patients at either the Saint Louis University Medical Center or Saint Louis City Hospital. Analysis of haemoglobin in the blood samples by electrophoresis indicated that a few of the SS patients had recently received blood transfusions, and this was confirmed by examination of their hospital records. The AA and AS donors were in good health, and there were two males in each of the three genotype groups. Heparinized venous blood from each subject was depleted of leukocytes and platelets as described previously (5). The erythrocytes were then washed ( x 4) by centrifugation and resuspension in a standard medium (containing sodium chloride (68 mmol/l), potassium chloride (4.8 mmol/l), mag- nesium sulfate (1.2 mmol/l), and disodium hydrogen phosphate (50 mmol/l)) that was adjusted to pH 7.4 with hydrochloric acid. A 50/o suspension of the washed erythrocytes in the standard medium was then incubated with a solution of radiolabelled chloroquine (2.06 MmolUl)' in plastic tubes at 37 °C for 1 hour. Subsequently, the erythrocyte pellet was precipitated from the medium by centrifugation and the concentration of chloroquine in each fraction determined radiochemically (6). RESULTS Parasitology Because malarial parasitaemia increases the uptake of chloroquine by infected erythrocytes (7), all blood samples were examined for malarial infection: a From New England Nuclear Corporation, Boston, MA, USA. CHLOROQUINE AND DESETHYLCHLOROQUINE IN SICKLE CELLS 53 Giemsa-stained thick and thin smears were negative in all instances. In vivo levels ofchloroquine and desethylchloroquine in blood Before drug administration. Chloroquine and desethylchloroquine were not detected in the plasma and erythrocytes of samples of blood from AA and AS subjects collected before administration of chloroquine; the detection limit of the method was 5 tg/l. The plasma of SS subjects B and C contained neither chloroquine nor its metabolite; however, the erythrocytes of subject B contained chloroquine (14 Ag/l) and desethylchloroquine (39 iLg/l). Subject B had been taking chloroquine as a malaria prophy- lactic but had changed to pyrimethamine several months before the present study. In contrast, subject C had not taken chloroquine for at least 6 months prior to the study, but exhibited chloroquine in erythrocytes (20 ug/l) without any detectable meta- bolite. The blood of subject D had no trace of either chloroquine or desethylchloroquine prior to com- mencement of the study, while subject A was already receiving chloroquine when the study began. After drug administration. The concentrations of chloroquine and desethylchloroquine, respectively, in the plasma and erythrocytes of blood samples collected 2 hours after the administration of chloro- quine phosphate are shown in Tables 2 and 3. Except for subject A who received multiple doses of chloro- quine, mean levels of chloroquine in plasma were consistently lower for SS (47.8 ± 14.0 gg/l) than for AA (84.5 ± 15.2 ,g/l) subjects. Conversely the mean concentration of chloroquine in the erythrocytes was higher in SS subjects (1614.3 ± 709.6 1&g/1) than in AA subjects (375.2± 115.2 ytg/l). The concentration of desethylchloroquine in erythrocytes of AA subjects was 3-4 times higher than that in plasma; however, for SS subjects desethylchloroquine was detected only in erythro- cytes. The level of desethylchloroquine 2 hours after taking chloroquine was similar in samples of blood from both SS and AA subjects, suggesting that there was no genotype-dependent difference in chloroquine metabolism. Table 2. In vivo distribution of chloroquine in blood of study subjects Chloroquine concentration (,tg/1) in: Blood Distribution Subject genotypea Plasma Erythrocytes ratio b A SS 179.5 5057.5 28.2 B SS 40.0 2431.0 60.8 C SS 39.5 1148.0 29.1 D SS 64.0 1264.0 19.8 E AA 102.0 454.5 4.5 F AA 76.0 243.0 3.2 G AA 75.5 428.0 5.7 H AS 72.0 228.0 3.2 I AS 93.0 - e SS= subject with sickle-cell anaemia; AA = normal subject; AS= subject with sickle-cell trait. b Ratio of the concentration of chloroquine in erythrocytes to that in plasma. c Not available. Table 3. In vivo distribution of desethylchloroquine in blood of study subjects Desethylchloroquine concentration (gg/l) in: Blood Distribution Subject genotype ° Plasma Erythrocytes ratio A SS 96.5 3968.0 41.1 B SS <5 260.6 65.1-260.5b C SS <5 127.5 31.9-127.5b D SS <5 153.0 38.3-1530b E AA 26.5 115.5 4.4 F AA 23.5 74.0 3.1 G AA 31.5 116.0 3.7 H AS 40.0 103.5 2.6 I AS 32.5 -c - a SS= subject with sickle-cell anaemia; AA = normal subject; AS= subject with sickle-cell trait. b Detection limit is 5 ,g/l. The range given refers to a maxi- mum and minimum undetectable concentration of desethyl- chloroquine in plasma of 4 and 1 jg/l, respectively. c Not available. A. U. ORJIH Table 4. In vitro accumulation of chloroquine by a 5% suspension (wfv) of erythrocytes in 2.06 ;tmol/l chloro- quine solution Distribution ratiob Blood genotypea No. of samples Mean Range SS 8 31.0 6.0-65.6 AA 5 3.5 3.2-3.7 AS 5 2.7 2.2-3.4 e SS = subject with sickle-cell anaemia; AA = normal subject; AS= subject with sickle-cell trait. b smol chloroquine per kg erythrocytes :,mol chloroquine per litre medium. In vitro test Erythrocytes were treated for 1 hour with a solution containing chloroquine (2.06 zmol/l) and the distribution of the drug in the erythrocytes and the medium was determined. The results are summarized in Table 4. The mean concentration of chloroquine in the erythrocytes was 21.9 ± 9.5 gmol/kg for SS, 6.2± 0.5 gmol/kg for AA, and 4.7 ± 0.8 smol/kg for AS subjects, while the corre- sponding mean chloroquine distribution ratios were 31.0± 19.9, 3.5 ±0.3, and 2.7 ±0.5. DISCUSSION Variability in the level of chloroquine in plasma is common (8), but the reason for this is not clearly understood; incorrect intake or malabsorption are, nevertheless, suspected when the level of plasma chloroquine is lower than normal (9). The findings reported here indicate that genetic factors can also influence the distribution of chloroquine in blood, e.g., erythrocytes from SS individuals accumulated a higher amount of chloroquine in vivo than those from AA individuals. In contrast, the level of chloroquine in the plasma was lower in SS subjects than in AA subjects. The apparently high level of chloroquine in one of the SS patients (plasma: 179.5 ug/l) arose because several large doses of the drug were administered during the illness; however, the drug distribution ratio indicates that the plasma chloro- quine level was low. The erythrocytes of SS subjects accumulated a greater level of chloroquine in vitro than those of AA subjects. Uptake of chloroquine by AS subjects was comparable to that of AA, and this may also be the case in vivo. In the absence of malarial parasitaemia, the distri- bution ratio of chloroquine in erythrocytes to that in plasma rarely exceeds 6 for AA and AS subjects. For SS subjects, on the other hand, the distribution ratio can be as high as 157 (5), but the reason for the high variability within the SS group is not yet known. Previously, it was reported that, unlike AA and AS erythrocytes, SS erythrocytes have a high affinity for chloroquine, and this is probably due to the accumulation in SS erythrocytes of haemichrome, containing ferriprotoporphyrin IX (5, 10-12), a com- pound that has a high affinity for chloroquine (13). The detection of desethylchloroquine in blood 2 hours after oral administration of chloroquine phosphate is consistent with previous findings that metabolism of the drug commences shortly after its intake (14). The metabolite was detected both in the plasma and erythrocytes of AA and AS subjects but only in the erythrocytes of SS subjects, which bind desethylchloroquine even more avidly than chloro- quine. Non-specific analytical methods that do not distinguish chloroquine metabolites from the parent drug therefore afford even higher apparent levels of chloroquine in the plasma ofAA than SS individuals; for example, summation of the in vivo levels of chloroquine and desethylchloroquine in plasma in the present study gives levels of the drug 2 to 3 times higher in AA than in SS blood. It would therefore be advantageous to provide some haematological data when blood chloroquine levels are reported, particularly if they refer to plasma or serum. Blood platelets and leukocytes accumulate relatively large amounts of chloroquine (15), and excessive thrombocytosis or leukocytosis could there- fore alter the normal distribution of the drug in blood. It has also been reported that erythrocytes which are deficient in glucose-6-phosphate dehydro- genase have a high affinity for binding chloroquine (16). ACKNOWLEDGEMENTS Dr Frederick C. Churchill, Centres for Disease Control, Atlanta, GA, USA is thanked for the supply of filter-papers and for the analysis of chloroquine and desethylchloroquine levels. The in vitro test was conducted in the laboratories of Professor Coy D. Fitch, Department of Internal Medicine, St. Louis University School of Medicine, St. Louis, MO, USA. The study received financial support, in part, from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. 54 CHLOROQUINE AND DESETHYLCHLOROQUINE IN SICKLE CELLS 55 RtSUME ACCUMULATION ACCRUE DE LA CHLOROQUINE ET DE LA DESETHYLCHLOROQUINE DANS LES DREPANOCYTES HOMOZYGOTES Les taux plasmatiques de chloroquine varient conside- rablement d'un sujet A I'autre. Lors d'une etude de cette variabilite, nous avons determine la distribution, in vivo et in vitro, de la chloroquine dans le sang de sujets de genotypes differents. Lors de 1'etude in vivo, une dose de 4 a 6 mg de chloro- quine base par kg de poids corporel a e administree par voie orale a des volontaires presentant un genotype de l'hemoglobine avec anemie drepanocytaire (SS), avec trait dr6panocytaire (AS) ou normal (AA). Des echantillons de sang ont e preleves deux heures apres et on a mesure la concentration de chloroquine et de son metabolite, la desethylchloroquine, dans le plasma et les erythrocytes par chromatograhie liquide a haute pression. Le taux moyen de chloroquine dans le plasma etait plus faible chez les sujets SS (47,8 jtg/l) que chez les sujets AA (84,5 Ftg/l). En revanche, le taux moyen de chloroquine dans les erythrocytes des sujets SS (1614 sg/l) etait plus eleve que dans les erythro- cytes des sujets AA (375 gsg/l). Le quotient moyen de distri- bution (concentration de chloroquine par kg d'erythro- cytes/concentration par litre de plasma) etait de 36,6 pour les sujets SS et 4,5 pour les sujets AA. Le taux de desethyl- chloroquine dans les erythrocytes des sujets AA etait 3 a 4 fois superieur au taux plasmatique; par contre, chez les sujets SS on ne trouvait de desethylchloroquine que dans les erythrocytes. Le taux de desethylchloroquine dans le sang deux heures apres administration de chloroquine etait analogue chez les sujets SS et AA, ce qui tend a montrer qu'il n'y a pas de difference Miee au genotype en ce qui concerne le metabolisme de la chloroquine. Lors de l'etude in vitro, une suspension a 5% d'erythro- cytes laves provenant de 8 sujets SS, 5 sujets AA et 5 sujets AS a e incubee avec une solution de chloroquine radio- marqu&e (2,06 limol/l), et on a mesure la concentration de medicament dans les erythrocytes et dans la solution par une methode radiochimique. 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