EXPERIMENTAL
PARASITOLOGY
72,262-270 (1991)
Plasmodium falciparum: In Vitro Drug Interaction between Chloroquine and Enantiomers of Amlodipine LEONARDO
K. BASCO’ AND JACQUES LE BRAS
Centre National de Refireme pour d’Epidkmiologie Africaines et Tropicales,
la ChimiosensibilitC du Paludisme, Institut de Mkdecine et Laboratoire de Parasitologie, HGpital Bichat-Claude Bernard, 75018 Paris, France
BASCO, L. K., AND LE BRAS, J. 1991. Plasmodium falciparum: In vitro drug interaction between chloroquine and enantiomers of amlodipine. Experimental Parasitology 72, 262270. Both enantiomers of amlodipine, whose calcium antagonist action resides almost exclusively in the R( -) enantiomer, reversed chloroquine resistance in Plasmodium falciparum in vitro. R( -) enantiomer was slightly more effective than the S( +) enantiomer in potentiating chloroquine action against chloroquine-resistant strains of parasites. No potentiating effect was observed in chloroquine-sensitive parasites. Both enantiomers entered rapidly into parasitized erythrocytes to the same extent. Reversal of chloroquine resistance by the enantiomers of amlodipine was related to dose-dependent increase in the accumulation of chloroquine inside the erythrocytes parasitized by resistant parasites. These results suggest that the potentiating effect on chloroquine is independent of calcium metabolism of malaria parasites. 0 1991 Academic Press, Inc. INDEX DESCRIPTORS: Plasmodium falciparum; Malaria; Chloroquine; Calcium antagonist; Amlodipine; Enantiomers.
Chloroquine has played a key role in antimalarial chemotherapy since the end of World War II. It is characterized by excellent efficacy against susceptible Plasmodium falciparum and three other human malaria species, rapid action after oral administration, good tolerance, safety for young children and pregnant women, and suitability for both therapeutic and prophylactic uses, However, chloroquine resistance in P. falciparum, first reported in the early sixties in Southeast Asia and South America, is now well established in many areas of the endemic regions (WHO 1989). Only a few alternative drugs effective against chloroquine-resistant P. falciparum are currently available. Chloroquine resistance is thought to be due to an active efflux of the drug from resistant strains of parasites (Krogstad et al. 1987), leading to diminished accumulation ’ To whom correspondence should be addressed. 262 0014-4894/91
$3.00
Copyright 0 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved.
of chloroquine inside parasitized erythrocytes (Fitch 1970; Verdier et al. 1985). Recent works showed that this efflux process can be inhibited by structurally unrelated groups of drugs, collectively known as reversing agents, which include calcium channel blockers (Martin ef al. 1987; Tanabe et al. 1990), tricyclic antidepressants (Bitonti et al. 1988), and tricyclic antihistaminics (Peters et al. 1989), in rodent malaria in vivo and P. falciparum in vitro and in owl monkeys. Inhibition of calcium metabolism, the major known pharmacologic action of calcium channel blockers, was also reported in some tricyclic antidepressants (Levin and Weiss 1979; Reynolds and Claxton 1982) and antihistaminics (Murphy et al. 1983). However, the mechanism of reversal of chloroquine resistance is not yet clear (Ginsburg 1988; Warhurst 1988). In order to examine whether calcium metabolism is involved in the reversal of chloroquine resistance, we studied the in vitro drug interaction between chloroquine and the enantiomers of amlodipine. Amlodipine
POTENTIATIONOF
is a 1,Cdihydropyridine derivative of nifedipine (Fig. 1) whose calcium channel blocking action resides almost exclusively in the R( -) enantiomer (Arrowsmith et al. 1986). Unlike nifedipine, it is characterized by a long plasma half-life (35 hr) in man (Stopher et al. 1988). It is prescribed for the once daily treatment of angina and hypertension (Webster et al. 1987; Heber et al. 1989). We report here that both enantiomers of amlodipine reversed chloroquine resistance in P. fulcipurum in vitro, adding further evidence against the hypothesis that calcium transport is implicated in the reversal of drug resistance in falciparum malaria. MATERIALSANDMETHODS Three culture-adapted strains of P. falciparum were used: the highly chloroquine-resistant strain FCM 29/ Cameroon (50% inhibitory concentration, I&,, for chloroquine = 820 nM), the moderately chloroquineresistant strain FCM 6mhailand (IC,, = 430 nM), and the chloroquine-sensitive strain L-l/Gabon (IC,, = 22 r&f). Strain FCM 29/Cameroon was cloned by the dilution method (Rosario 1981). In addition, three chloroquine-resistant (isolate l/Gabon, isolate Z/Gabon, isolate 3/Cameroon, IC,,s = 17&180 nM) and two chloroquine-sensitive (isolate 4iIvory Coast, isolate 5/ Cameroon, ICs,s = 15 and 20 nM) wild African strains of P. fulciparum, freshly isolated from symptomatic nonimmune patients, were used. The culture-adapted strains were maintained in the suspension of type A human erythrocytes (8% hematocrit) in RPM1 1640 medium (GIBCO), 10% human serum, 25 m&f Hepes (Sigma Chemical Co.), and 25 n&f NaHCO, at 37°C in 5% O,, 5% CO,, 90% N,, and 95% humidity (Trager and Jensen 1976). Venous blood samples from malaria-infected patients were washed
CH300C
CH3 H
1. Chemical structure of amlodipine. Asterisk denotes asymmetric center. FIG.
CHLOROQUINE
263
by centrifugation three times with RPM1 1640 medium and used immediately without prior adaptation to culture conditions. Twofold serial dilutions of chloroquine sulfate (Specia) were prepared in sterile distilled water to give final concentrations ranging from 12.5 to 1600 nM. Optically pure R( - ) and S( + ) enantiomers of amlodipine maleate (Pfizer Central Research, U.K.) were dissolved in sterile distilled water. Dilutions of chloroquine, with each concentration in triplicate, were distributed in 24-well flat-bottom tissue culture plates (Becton Dickinson Co.) and tested with fixed concentrations of enantiomers of amlodipine ranging from 0.1 to5@f. The semimicro in vitro drug sensitivity test (Le Bras and Deloron 1983) was carried out with a suspension (700 pl per well) containing parasitized erythrocytes (hematocrit, 2.5%; parasitemia, O.l-l.O%), RPM1 1640 medium, and 10% human serum. The plates were incubated for 42 hr under the same atmospheric condition as in the in vitro method of culture. [3H]Hypoxanthine (1 &i/well) (sp act = 4.3 Ci/mmol, Amersham, France) was added 18 hr after initial incubation to assess parasite growth. The probit of chloroquine activity was plotted against the logarithm of chloroquine concentration, and 50 and 90% inhibitory concentrations (IC, and IC,) were determined by linear regression analysis (Grab and Wemsdorfer 1983). Regression lines were compared by analysis of variance and covariance. “Potentiation” was defined for individual fixed concentrations of amlodipine as statistically significant diminution of the value of IC,, for chloroquine. Based on our laboratory data on chloroquine sensitivity of wild strains, the cutoff point for in vitro chloroquine resistance was set at IC,,, of 90-120 nM. In the experiments on the combination of chloroquine and amlodipine, diminution of I&, for chloroquine below 70 nM was considered to be “total reversal” of chloroquine resistance and the values of IC,, between 70 and 120 n&f as “partial reversal” of drug resistance. The values of IC,, corresponding to total reversal of drug resistance are similar to those of chloroquine-sensitive strains. The overall interaction between chloroquine and amlodipine at different potentiating concentrations was analyzed by isobolograms (Berenbaum 1989). “Synergy” was defined solely on the basis of the curves obtained by isobologram analysis. [3H]Chloroquine (sp act 0.7 Ci/mmol, Atomic Energy Commission, France) uptake into the erythrocytes after a 3-hr incubation of parasite suspension (hematocrit, 2.5%; parasitemia, 15-21%) with or without amlodipine was measured in the resistant clone FCM 29. The 3-hr incubation corresponds to the time required in vitro for amlodipine to reach maximal binding to calcium channels (Burges et al. 1987). Following rapid separation of parasite suspension into red cell
264
BASCO
AND
pellet and supematant culture medium by centrifugation (14,000 rpm x 1 min), the pellet was digested with 1 ml of 0.5 N quatemary ammonium hydroxide in toluene (Soluene 350, Packard) and isopropyl alcohol (v/v, 1:l) and bleached with 50 pJ of 33% H,Or. Radioactivity in the aliquots was measured by the Wallac 1410 (Pharmacia) liquid scintillation counter. Similar methods were used to study the uptake of [‘H]R( -)- and [‘H]S( +)-amlodipine (sp act of both enantiomers = 84.6 Ci/mmol, Amersham, U.K.) into infected and uninfected erythrocytes. Cellular uptake of amlodipine was determined at 5,30,90, and 180 mitt of incubation. The mean count per minute (cpm) per microliter of cell pellet was compared by t test. A signiticance level of 95% was used for all statistical analysis. AU experiments were done three times on separate days. RESULTS
Enantiomers of amlodipine had the same antimalarial activity in each of the three culture-adapted strains, giving the following overall mean It&s for both enantiomers (&SD): 7.0 + 0.8 p.M (n = 11) for resistant strain FCM 29, 5.1 + 0.5 fl (n = 3) for resistant strain FCM 6, and 11.4 + 1.4 l&f (n = 8) for sensitive strain L-l (Table I). Antimalarial activity of amlodipine alone was approximately 10 and 500 times less potent than chloroquine alone against the resistant and sensitive strains, respectively. In the experiments on the combination of chloroquine and enantiomers of amlodipine, potentiation of chloroquine was observed at or above 0.6 l& of both enantio-
LE BRAS
mers (Table II). Partial reversal of chloroquine resistance was observed at 2.5 pJ4 of ( - ) enantiomer against the strains FCM 29 and FCM 6 and at the same concentration of (+) enantiomer against the strain FCM 6. Total reversal of chloroquine resistance was obtained at 3.8 pM of both enantiomers against the strain FCM 6; 2.5 p&f of ( +) enantiomer potentiated chloroquine action against the strain FCM 29 but did not reverse chloroquine resistance. Based on ICsO values, (-) enantiomer was 1.2 to 3.7 times more effective than ( + ) enantiomer at all potentiating concentrations used against the two culture-adapted chloroquine-resistant strains. Chloroquine resistance was totally reversed in three resistant wild African strains at the concentration of 2.5 pJ4 of both enantiomers (Table III). Isobolograms depict a synergistic action of the drug combination against the two culture-adapted resistant strains FCM 29 and FCM 6 (Fig. 2). Up to 5 pJ4 of enantiomers did not modify the antimalarial activity of chloroquine against the chloroquine-sensitive strain L1. Its overall I&, value (2 SD) for chloroquine, with or without amlodipine, was 22.3 * 2.4 nM (n = 12). Enantiomers (2.5 r-LM) tested against two chloroquine-sensitive wild strains (isolate 4/ivory Coast, isolate S/Cameroon, n = 3) had no effect on chlo-
TABLE I Mean 50 and 90% Inhibitory Concentrations for the Enantiomers of Amlodipine Strains of Plusmodium faiciparum Antimalarial ( +)-Amlodipine
against Culture-Adapted
activity (Mb
( - )-Amlodipine
DL-Amlodipine
Strains”
GO
GCI
GO
Go
Go
Go
FCM 29 FCM 6 L-l
7.0 5.1 12.3
21.7 17.1 29.2
7.0 5.6 10.5
21.0 20.4 27.5
6.7 4.7 ND’
17.6 17.0 ND
0 Mean IC,, and IC, values for chloroquine: 821 and 1519 n&f for FCM 29 (resistant), 429 and 899 nMfor FCM 6 (resistant), and 22 and 70 nM for L-l (sensitive). b Results are mean values of 50 and 90% inhibitory concentrations (ICY,,and IC,) determined on 3 separate days. Twofold dilutions of amlodipine (0.6-80 p&f) were tested in triplicate in each experiment. ’ Not determined.
POTENTIATION
OF CHLOROQUINE
265
TABLE II In Vitro Drug Interaction between Chloroquine and Enantiomers of Amlodipine against Two Culture-Adapted Chloroquine-Resistant Strains of Plasmodium falcipurum Chloroquine activity (n&f) Amlodipine (PM (+ ) Enantiomer 0” 0.6 1.2 2.5 3.8 (-) Enantiomer w 0.6 1.2 2.5 3.8
Strain FCM 29
Strain FCM 6
G3
Go
IGO
IGO
821.2 k 14.2 650.7 * 24.0 494.2 f 10.6 283.6 + 36.7 NDb
1519.4 + 4.8 1428.2 k 8.7 1395.8 e 14.9 1036.4 If: 28.6 ND
429.2 f 12.0 ND 229.2 k 11.1 85.9 * 2.1 43.9 -+ 1.4
899.0 + 9.5 ND 580.5 + 16.5 366.0 f 4.3 290.7 ‘-c 21.7
821.2 f 14.2 527.2 2 24.8 351.9 -+ 41.4 75.8 f 3.5 ND
1519.4 + 1408.2 f 1027.7 2 369.7 f ND
429.2 f ND 189.1 f 70.5 f 24.7 f
899.0 f ND 449.5 2 238.8 ” 107.9 2
4.8 7.3 15.2 10.7
12.0 3.5 9.5 3.1
9.5 5.4 16.1 6.2
Note. Mean IC,, and IC, (*SD) for chloroquine (in nM) in the presence of fixed concentrations of enantiomers of amlodipine (in l&) added to eight serial dilutions of chloroquine, with each chloroquine concentration in triplicate. 0 Chloroquine alone. b Not determined.
roquine action (I(&, for chloroquine with or without amlodipine = 14.9 + 2.1 and 20.1 f 1.9 ti, respectively). The first series of experiments on the uptake of chloroquine in the erythrocytes after a 3-hr incubation showed a significant increase of chloroquine (80 nM) in the cell pellet with 2.5 uJ4 of enantiomers of amlodipine (Fig. 3a). In the second series of experiments using 40 IN of tritium-labeled
chloroquine, a further significant increase in chloroquine uptake was observed at the concentration of 5 ~JJMof enantiomers (Fig. 3b). At both concentrations of amlodipine, a significantly higher uptake of chloroquine was observed in infected erythrocytes with (-) enantiomer, at the ratio of 1.2 to 1.4, than with (+) enantiomer of amlodipine. Uptake of enantiomers into infected or uninfected erythrocytes occurred in the
TABLE HI Drug Interaction between Chloroquine and Enantiomers of Amlodipine against Three Chloroquine-Resistant Wild Strains of Plasmodium falciparum I&, for chloroquine (nkf) Drugs=
Isolate lb
Isolate 2
Isolate 3
Chloroquine alone (CQ) CQ + (+ ) amlodipine CQ + (-) amlodipine
177.5 f 21.5 54.2 -t 1.9 45.5 + 9.7
171.8 -+ 11.6 56.3 2 1.4 68.3 2 2.9
173.7 k 19.4 47.7 +- 8.7 34.9 * 3.5
Note. Results are mean I&, (?SD) in nA4. In vitro chloroquine-resistance level is I& 90-120 nM. a 2.5 p,M of enantiomers of amlodipine was added to eight serial dilutions of chloroquine, with each concentration in triplicate. b Isolates 1 and 2 are imported malaria cases from Gabon (a couple infected at the same time). Isolate 3 is from Cameroon. All isolates were obtained from symptomatic, nonimmune travelers upon returning to France.
266
BASCO AND LE BRAS 200
2
1
a
0.8
'i 0.6 z z Y -a E 'C M r;:
0.4
0.2 0
URBC 0.2
0.4
0.6
0.8
0.2
0.4
0.6
0.8
(+)2.5
(-)2.5
1.0 200
Fractional IC.50 amlodipine
0
PRBC
1.0
Fractional IC50 amlodipine FIG. 2. Isobolograms of in vitro drug interaction between enantiomers of amlodipine and chloroquine against the highly chloroquine-resistant strain FCM 29Kameroon (a) and the moderately chloroquineresistant strain FCM 6/Thailand (b). The concentration response data are given in Tables 1 and 2. Each point in the isobolograms was obtained by dividing the fixed amlodipine concentration by KS0 of its intrinsic activity (abscissa) and IC,, for chloroquine plus amlodipine by IC,, for chloroquine alone (ordinate). Results are means of simultaneous assays in triplicate. Concave curves depict synergistic interaction between the drugs.
first 5 min and a constant level was maintained for up to 3 hr (Fig. 4). There was no statistical difference in the accumulation of ( + ) and ( - ) enantiomers in the infected (or uninfected) erythrocytes. A significantly higher accumulation of enantiomers was as-
-I
URBC pRBc (+)2.5(-)2.5 (+)5 (-)5 FIG. 3. Effects of enantiomers of amlodipine on chloroquine accumulation (uRBC-uninfected RBC, pRBC-parasitized RBC, (+) and (-) denote enantiomers, 2.5 and 5 denote concentrations of enantiomers in pJ4). 80 nM of tritium-labeled chloroquine (16% parasitemia, 95% ringkrophozoite) (a) or 40 nM of cNoroquine (21% parasitemia, 88% ringkrophozoite) (b) was used. Results are means of triplicate determinations. Bars denote *SD.
sociated with infected erythrocytes pared to uninfected erythrocytes.
as com-
DISCUSSION
Amlodipine alone possesses a weak antimalarial activity, within a similar micromolar range as other reversing agents. A weak base effect was hypothesized to be the mechanism of action of 4-aminoquinoline and amino alcohol groups of antimalarial drugs, leading to the passage of these weakly basic drugs into the parasite’s vacuole along the pH gradient and their accumulation in this organelle (Homewood ef
POTENTIATION
5
30 Time
90
OF CHLOROQUINE
180
(minutes)
4. Accumulation of amlodipine (3 nM) inside the erythrocytes: q l, uninfected erythrocytes plus (+) enantiomer; 0, uninfected erythrocytes plus (-) enantiomer; k2, infected erythrocytes (15% parasitemia, 98% ringkrophozoite) plus (+) enantiomer; m, infected erythrocytes plus (-) enantiomer. Results are means of triplicate determinations. Bars denote 2 SD. FIG.
al. 1972; Yayon et al. 1985; Krogstad
and Schlesinger 1986). Amlodipine is a weak base with a pK, 8.6 (verapamil pK, 8.7). The lysosomotropic property of calcium channel blockers was observed (Ranganathan and Jackson 1984), and this may be responsible for their antimalarial action and may explain the identical IC,, obtained with the enantiomers of amlodipine against a given strain of parasite. Desipramine (pK, 10.2), another reversing agent, was also shown to have a lysosomotropic action (Honegger et al. 1983). It seems, however, that the basicity of a drug is not correlated with its antimalarial activity, and the basis of antimalarial action of reversing agents alone still remains unclear. Based on ICso values in the isobologram analysis, ( - ) enantiomer of amlodipine is 1.2 to 1.8 times more effective than ( + ) enantiomer at all potentiating concentrations, except for 2.5 lo& of enantiomers against FCM 29 where ( - ) enantiomer was almost 4 times as effective as the ( + ) enantiomer. Chloroquine uptake into parasitized erythrocytes was 1.2 to 1.4 times higher with ( -)
267
enantiomer. Although the greater difference shown in isobolograms of FCM 29 at 2.5 pM refers to the values of IC,, for chloroquine and is not directly comparable to the chloroquine uptake experiments that use subinhibitory concentrations of chloroquine, the results of the two experiments are in good agreement in showing that (- ) enantiomer is a more effective reversing agent than ( +) enantiomer. Total reversal of chloroquine resistance was observed at 2.5 to 3.8 pM. A similar range of concentration of other reversing agents is required to potentiate chloroquine action against P. falciparum in vitro. This may suggest that there is a common underlying mechanism of reversal of chloroquine resistance. An active efflux of drugs may be involved, as in the multidrug-resistant (mdr) cancer cells (Krogstad et al. 1987). In resistant cancer cells, an increased amount of P-glycoprotein, which is thought to function as an efflux pump, is produced by the amplification of a mdr gene (Riordan 1985; Cornwell et al. 1987; Safa et al. 1987). In both cancer cells (Tsuruo et al. 1982) and malaria parasites (Martin et al. 1987), drug resistance can be reversed with calcium channel blockers. Malarial mdr genes were also detected (Foote et al. 1989; Wilson et al. 1989). Based on this close analogy, it may be hypothesized that drug-resistant falciparum malaria overexpresses a chloroquine eftlux pump and that the reversing agents inhibit this pump. However, it remains to be established whether the genetic mechanism of chloroquine resistance can be attributed to the malarial mdr genes so far identified (Foote et al. 1990; Wellems et al. 1990) and whether a protein similar to P-glycoprotein is present in falciparum malaria parasite. Verapamil also displays an enantioselective action on calcium channels. Its Lisomer is 10 times more effective than the o-isomer (Echizen et al. 1985). Despite this difference, both enantiomers of verapamil were shown to be equally effective in re-
268
BASCO AND LE BRAS
versing chloroquine resistance in P. falci(Ye and Dyke 1988)and in increasing intracellular vincristine concentration in cancer cells (Gruber et al. 1988). Amlodipine exhibits an even more marked stereospecificity in the calcium channel blocking activity; its (-) enantiomer being 1000times more active than (+) enantiomer (Arrowsmith et al. 1986). This is the largest enantiomeric ratio so far reported for the 1,6dihydropyridines. The enantiomers accumulate inside the infected erythrocytes to the same extent and exert the same intrinsic antimalarial activity against P. falciparum. Both potentiate chloroquine action against resistant strains, with (-) enantiomer being slightly more effective. These findings may imply that there is a stereospecific difference at the site of action (putative malarial P-glycoprotein) of the enantiomers. This slight but statistically significant difference in the potentiating action of the enantiomers cannot be accounted for by the calcium antagonist action, which is negligible in the (+ ) enanComer. Furthermore, our kinetic studies showed that both enantiomers accumulate inside the parasitized erythrocytes within 5 min and a constant level is maintained for up to 3 hr. Amlodipine is therefore present inside the parasitized erythrocytes at the moment when resistant parasites begin to expel chloroquine 4 min after exposure to the drug (Krogstad ef al. 1987) and inhibits chloroquine efflux. Amlodipine is a slowacting calcium channel blocker whose action on the calcium channel appears gradually and reaches its maximal inhibition after 3 hr in vitro. Its kinetic property on the calcium channel is thus not compatible with the rapid efflux of chloroquine from resistant P. falciparum. In addition, in vitro studies on isolated vascular smooth muscle and cardiac muscle showed that the calcium channel blocking action is observed at low nanomolar concentration of (-) enantiomer (Burges et al.
parum
1987). Intrinsic antimalarial activity and reversal of chloroquine resistance are observed in the micromolar range, setting apart the two probably distinct actions of amlodipine-calcium antagonism and reversal of chloroquine resistance-by approximately three orders of magnitude. We conclude that the results of this study provide additional evidence against the hypothesis that the chloroquine potentiating action of calcium channel blockers is mediated by their calcium antagonist properties. ACKNOWLEDGMENTS We are gratehi to Dr. R. A. Burges of Pfizer Central Research for supplying amlodipine, Dr. F. Verdier of INSERM U13, Paris, for useful discussion, and Dr. F. Sauniere for his encouragement. REFERENCES ARROWSMITH, J. E, CAMPBELL, S. F., CROSS, P. E., STUBBS, J. K., BURGES, R. A., GARDINER, G. G., AND BLACKBURN, K. J. 1986. Long-acting dihydropyridine calcium antagonists. 1. 2-Alkoxymethyl derivatives incorporating basic substituents. Journal of Medicinal Chemistry 29, 1696-1702. BERENBAUM, M. C. 1989. What is synergy? Pharmacological Reviews 41, 93-141. BITONTI, A. J., SJOERDSMA, A., MCCANN, P. P., KYLE, D. E., ODUOLA, A. M. J., ROSSAN, R. N., MILHOUS, W. K., AND DAVIDSON, D. E., JR. 1988. Reversal of chloroquine resistance in malaria parasite Plasmodium falciparum by desipramine. Science 242, 1301-1303. BURGES, R. A., GARDINER, D. G., GWILT, M., HIGGINS, A. J., BLACKBURN, K. J., CAMPBELL, S. F., CROSS, P. E., AND STUBBS, J. K. 1987. Calcium channel blocking properties of amlodipine in vascular smooth muscle and cardiac muscle in vitro: Evidence for voltage modulation of vascular dihydropyridine receptors. Journal of Cardiovascular Pharmacology 9, 110-l 19. CORNWELL, M. M., PASTAN, I., AND GOTTESMAN, M. M. 1987. Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein. Journal of Biological Chemistry 262, 2166-2170. ECHIZEN, H., BRECHT, T., NIEDERGESASS, S., VoGELGESANG, B., AND EICHELBAUM, M. 1985. The effect of dextro-, levo-, and racemic verapamil on atrioventricular conduction in humans. American Heart Journal 109, 210-217. FITCH, C. D. 1970. Plasmodium falciparum in owl
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TRAGER, W., AND JENSEN, J. B. 1976. Human malaria parasites in continuous culture. Science 193, 673675. TSURUO, T., IIDA, H., TSUKAGOSHI, S., AND SAKURAI, Y. 1982. Increased accumulation of vincristine and adriamycin in drug-resistant P388 tumor cells following incubation with calcium antagonists and calmodulin inhibitors. Cancer Research 42, 47304733. VERDIER, F., LE BRAS, J., CLAVIER, F., HATIN, I., AND BLAYO, M. C. 1985. Chloroquine uptake by
270
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Plasmodium falciparum-infected human erythrocytes during in vitro culture and its relationship to chloroquine resistance. Antimicrobial Agents and Chemotherapy 27, 561-564. WARHURST, D. C. 1988. Mechanism of chloroquine resistance in malaria. Parasitology Today 4, 211213. WEBSTER, .I., ROBB, 0. J., JEFFERS, T. A., SCOTT, A. K., PETRIE, J. C., AND TOWLER, H. M. 1987. Once daily amlodipine in the treatment of mild to moderate hypertension. British Journal of Clinical Pharmacology 24,713-719. WELLEMS, T. E., PANTON, L. J., GLUZMAN, I. Y., ROSARIO, V. E., GWADZ, R. W., WALKER-JONAH, A., AND KROGSTAD, D. J. 1990. Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross. Nature (London) 345, 253-255. WILSON, C. M., SERRANO, A. E., WASLEY, A., Bo-
LE BRAS
GENSCHUTZ, M. P., SHANKAR, A. H., AND WIRTH, D. F. 1989. Amplification of a gene related to mammalian mdr genes in drug-resistant Plasmodium falciparum. Science 244, 1184-l 186. World Health Organization 1989. World malaria situation: 1986-1987. Weekly Epidemiological Records 64,241-256. YAYON, A., CABANTCHIK, Z. I., AND GINSBURG, H. 1985. Susceptibility of human malaria parasites to chloroquine is pH dependent. Proceedings of the National Academy of Sciences USA 82, 2784-2788. YE, Z., AND DYKE, K. V. 1988. Reversal of chloroquine resistance in falciparum malaria independent of calcium channels. Biochemical and Biophysical Research Communications 155, 47U81. Received October
6 August 1990.
1990; accepted
with
revision
25
PARASITOLOGY
72,262-270 (1991)
Plasmodium falciparum: In Vitro Drug Interaction between Chloroquine and Enantiomers of Amlodipine LEONARDO
K. BASCO’ AND JACQUES LE BRAS
Centre National de Refireme pour d’Epidkmiologie Africaines et Tropicales,
la ChimiosensibilitC du Paludisme, Institut de Mkdecine et Laboratoire de Parasitologie, HGpital Bichat-Claude Bernard, 75018 Paris, France
BASCO, L. K., AND LE BRAS, J. 1991. Plasmodium falciparum: In vitro drug interaction between chloroquine and enantiomers of amlodipine. Experimental Parasitology 72, 262270. Both enantiomers of amlodipine, whose calcium antagonist action resides almost exclusively in the R( -) enantiomer, reversed chloroquine resistance in Plasmodium falciparum in vitro. R( -) enantiomer was slightly more effective than the S( +) enantiomer in potentiating chloroquine action against chloroquine-resistant strains of parasites. No potentiating effect was observed in chloroquine-sensitive parasites. Both enantiomers entered rapidly into parasitized erythrocytes to the same extent. Reversal of chloroquine resistance by the enantiomers of amlodipine was related to dose-dependent increase in the accumulation of chloroquine inside the erythrocytes parasitized by resistant parasites. These results suggest that the potentiating effect on chloroquine is independent of calcium metabolism of malaria parasites. 0 1991 Academic Press, Inc. INDEX DESCRIPTORS: Plasmodium falciparum; Malaria; Chloroquine; Calcium antagonist; Amlodipine; Enantiomers.
Chloroquine has played a key role in antimalarial chemotherapy since the end of World War II. It is characterized by excellent efficacy against susceptible Plasmodium falciparum and three other human malaria species, rapid action after oral administration, good tolerance, safety for young children and pregnant women, and suitability for both therapeutic and prophylactic uses, However, chloroquine resistance in P. falciparum, first reported in the early sixties in Southeast Asia and South America, is now well established in many areas of the endemic regions (WHO 1989). Only a few alternative drugs effective against chloroquine-resistant P. falciparum are currently available. Chloroquine resistance is thought to be due to an active efflux of the drug from resistant strains of parasites (Krogstad et al. 1987), leading to diminished accumulation ’ To whom correspondence should be addressed. 262 0014-4894/91
$3.00
Copyright 0 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved.
of chloroquine inside parasitized erythrocytes (Fitch 1970; Verdier et al. 1985). Recent works showed that this efflux process can be inhibited by structurally unrelated groups of drugs, collectively known as reversing agents, which include calcium channel blockers (Martin ef al. 1987; Tanabe et al. 1990), tricyclic antidepressants (Bitonti et al. 1988), and tricyclic antihistaminics (Peters et al. 1989), in rodent malaria in vivo and P. falciparum in vitro and in owl monkeys. Inhibition of calcium metabolism, the major known pharmacologic action of calcium channel blockers, was also reported in some tricyclic antidepressants (Levin and Weiss 1979; Reynolds and Claxton 1982) and antihistaminics (Murphy et al. 1983). However, the mechanism of reversal of chloroquine resistance is not yet clear (Ginsburg 1988; Warhurst 1988). In order to examine whether calcium metabolism is involved in the reversal of chloroquine resistance, we studied the in vitro drug interaction between chloroquine and the enantiomers of amlodipine. Amlodipine
POTENTIATIONOF
is a 1,Cdihydropyridine derivative of nifedipine (Fig. 1) whose calcium channel blocking action resides almost exclusively in the R( -) enantiomer (Arrowsmith et al. 1986). Unlike nifedipine, it is characterized by a long plasma half-life (35 hr) in man (Stopher et al. 1988). It is prescribed for the once daily treatment of angina and hypertension (Webster et al. 1987; Heber et al. 1989). We report here that both enantiomers of amlodipine reversed chloroquine resistance in P. fulcipurum in vitro, adding further evidence against the hypothesis that calcium transport is implicated in the reversal of drug resistance in falciparum malaria. MATERIALSANDMETHODS Three culture-adapted strains of P. falciparum were used: the highly chloroquine-resistant strain FCM 29/ Cameroon (50% inhibitory concentration, I&,, for chloroquine = 820 nM), the moderately chloroquineresistant strain FCM 6mhailand (IC,, = 430 nM), and the chloroquine-sensitive strain L-l/Gabon (IC,, = 22 r&f). Strain FCM 29/Cameroon was cloned by the dilution method (Rosario 1981). In addition, three chloroquine-resistant (isolate l/Gabon, isolate Z/Gabon, isolate 3/Cameroon, IC,,s = 17&180 nM) and two chloroquine-sensitive (isolate 4iIvory Coast, isolate 5/ Cameroon, ICs,s = 15 and 20 nM) wild African strains of P. fulciparum, freshly isolated from symptomatic nonimmune patients, were used. The culture-adapted strains were maintained in the suspension of type A human erythrocytes (8% hematocrit) in RPM1 1640 medium (GIBCO), 10% human serum, 25 m&f Hepes (Sigma Chemical Co.), and 25 n&f NaHCO, at 37°C in 5% O,, 5% CO,, 90% N,, and 95% humidity (Trager and Jensen 1976). Venous blood samples from malaria-infected patients were washed
CH300C
CH3 H
1. Chemical structure of amlodipine. Asterisk denotes asymmetric center. FIG.
CHLOROQUINE
263
by centrifugation three times with RPM1 1640 medium and used immediately without prior adaptation to culture conditions. Twofold serial dilutions of chloroquine sulfate (Specia) were prepared in sterile distilled water to give final concentrations ranging from 12.5 to 1600 nM. Optically pure R( - ) and S( + ) enantiomers of amlodipine maleate (Pfizer Central Research, U.K.) were dissolved in sterile distilled water. Dilutions of chloroquine, with each concentration in triplicate, were distributed in 24-well flat-bottom tissue culture plates (Becton Dickinson Co.) and tested with fixed concentrations of enantiomers of amlodipine ranging from 0.1 to5@f. The semimicro in vitro drug sensitivity test (Le Bras and Deloron 1983) was carried out with a suspension (700 pl per well) containing parasitized erythrocytes (hematocrit, 2.5%; parasitemia, O.l-l.O%), RPM1 1640 medium, and 10% human serum. The plates were incubated for 42 hr under the same atmospheric condition as in the in vitro method of culture. [3H]Hypoxanthine (1 &i/well) (sp act = 4.3 Ci/mmol, Amersham, France) was added 18 hr after initial incubation to assess parasite growth. The probit of chloroquine activity was plotted against the logarithm of chloroquine concentration, and 50 and 90% inhibitory concentrations (IC, and IC,) were determined by linear regression analysis (Grab and Wemsdorfer 1983). Regression lines were compared by analysis of variance and covariance. “Potentiation” was defined for individual fixed concentrations of amlodipine as statistically significant diminution of the value of IC,, for chloroquine. Based on our laboratory data on chloroquine sensitivity of wild strains, the cutoff point for in vitro chloroquine resistance was set at IC,,, of 90-120 nM. In the experiments on the combination of chloroquine and amlodipine, diminution of I&, for chloroquine below 70 nM was considered to be “total reversal” of chloroquine resistance and the values of IC,, between 70 and 120 n&f as “partial reversal” of drug resistance. The values of IC,, corresponding to total reversal of drug resistance are similar to those of chloroquine-sensitive strains. The overall interaction between chloroquine and amlodipine at different potentiating concentrations was analyzed by isobolograms (Berenbaum 1989). “Synergy” was defined solely on the basis of the curves obtained by isobologram analysis. [3H]Chloroquine (sp act 0.7 Ci/mmol, Atomic Energy Commission, France) uptake into the erythrocytes after a 3-hr incubation of parasite suspension (hematocrit, 2.5%; parasitemia, 15-21%) with or without amlodipine was measured in the resistant clone FCM 29. The 3-hr incubation corresponds to the time required in vitro for amlodipine to reach maximal binding to calcium channels (Burges et al. 1987). Following rapid separation of parasite suspension into red cell
264
BASCO
AND
pellet and supematant culture medium by centrifugation (14,000 rpm x 1 min), the pellet was digested with 1 ml of 0.5 N quatemary ammonium hydroxide in toluene (Soluene 350, Packard) and isopropyl alcohol (v/v, 1:l) and bleached with 50 pJ of 33% H,Or. Radioactivity in the aliquots was measured by the Wallac 1410 (Pharmacia) liquid scintillation counter. Similar methods were used to study the uptake of [‘H]R( -)- and [‘H]S( +)-amlodipine (sp act of both enantiomers = 84.6 Ci/mmol, Amersham, U.K.) into infected and uninfected erythrocytes. Cellular uptake of amlodipine was determined at 5,30,90, and 180 mitt of incubation. The mean count per minute (cpm) per microliter of cell pellet was compared by t test. A signiticance level of 95% was used for all statistical analysis. AU experiments were done three times on separate days. RESULTS
Enantiomers of amlodipine had the same antimalarial activity in each of the three culture-adapted strains, giving the following overall mean It&s for both enantiomers (&SD): 7.0 + 0.8 p.M (n = 11) for resistant strain FCM 29, 5.1 + 0.5 fl (n = 3) for resistant strain FCM 6, and 11.4 + 1.4 l&f (n = 8) for sensitive strain L-l (Table I). Antimalarial activity of amlodipine alone was approximately 10 and 500 times less potent than chloroquine alone against the resistant and sensitive strains, respectively. In the experiments on the combination of chloroquine and enantiomers of amlodipine, potentiation of chloroquine was observed at or above 0.6 l& of both enantio-
LE BRAS
mers (Table II). Partial reversal of chloroquine resistance was observed at 2.5 pJ4 of ( - ) enantiomer against the strains FCM 29 and FCM 6 and at the same concentration of (+) enantiomer against the strain FCM 6. Total reversal of chloroquine resistance was obtained at 3.8 pM of both enantiomers against the strain FCM 6; 2.5 p&f of ( +) enantiomer potentiated chloroquine action against the strain FCM 29 but did not reverse chloroquine resistance. Based on ICsO values, (-) enantiomer was 1.2 to 3.7 times more effective than ( + ) enantiomer at all potentiating concentrations used against the two culture-adapted chloroquine-resistant strains. Chloroquine resistance was totally reversed in three resistant wild African strains at the concentration of 2.5 pJ4 of both enantiomers (Table III). Isobolograms depict a synergistic action of the drug combination against the two culture-adapted resistant strains FCM 29 and FCM 6 (Fig. 2). Up to 5 pJ4 of enantiomers did not modify the antimalarial activity of chloroquine against the chloroquine-sensitive strain L1. Its overall I&, value (2 SD) for chloroquine, with or without amlodipine, was 22.3 * 2.4 nM (n = 12). Enantiomers (2.5 r-LM) tested against two chloroquine-sensitive wild strains (isolate 4/ivory Coast, isolate S/Cameroon, n = 3) had no effect on chlo-
TABLE I Mean 50 and 90% Inhibitory Concentrations for the Enantiomers of Amlodipine Strains of Plusmodium faiciparum Antimalarial ( +)-Amlodipine
against Culture-Adapted
activity (Mb
( - )-Amlodipine
DL-Amlodipine
Strains”
GO
GCI
GO
Go
Go
Go
FCM 29 FCM 6 L-l
7.0 5.1 12.3
21.7 17.1 29.2
7.0 5.6 10.5
21.0 20.4 27.5
6.7 4.7 ND’
17.6 17.0 ND
0 Mean IC,, and IC, values for chloroquine: 821 and 1519 n&f for FCM 29 (resistant), 429 and 899 nMfor FCM 6 (resistant), and 22 and 70 nM for L-l (sensitive). b Results are mean values of 50 and 90% inhibitory concentrations (ICY,,and IC,) determined on 3 separate days. Twofold dilutions of amlodipine (0.6-80 p&f) were tested in triplicate in each experiment. ’ Not determined.
POTENTIATION
OF CHLOROQUINE
265
TABLE II In Vitro Drug Interaction between Chloroquine and Enantiomers of Amlodipine against Two Culture-Adapted Chloroquine-Resistant Strains of Plasmodium falcipurum Chloroquine activity (n&f) Amlodipine (PM (+ ) Enantiomer 0” 0.6 1.2 2.5 3.8 (-) Enantiomer w 0.6 1.2 2.5 3.8
Strain FCM 29
Strain FCM 6
G3
Go
IGO
IGO
821.2 k 14.2 650.7 * 24.0 494.2 f 10.6 283.6 + 36.7 NDb
1519.4 + 4.8 1428.2 k 8.7 1395.8 e 14.9 1036.4 If: 28.6 ND
429.2 f 12.0 ND 229.2 k 11.1 85.9 * 2.1 43.9 -+ 1.4
899.0 + 9.5 ND 580.5 + 16.5 366.0 f 4.3 290.7 ‘-c 21.7
821.2 f 14.2 527.2 2 24.8 351.9 -+ 41.4 75.8 f 3.5 ND
1519.4 + 1408.2 f 1027.7 2 369.7 f ND
429.2 f ND 189.1 f 70.5 f 24.7 f
899.0 f ND 449.5 2 238.8 ” 107.9 2
4.8 7.3 15.2 10.7
12.0 3.5 9.5 3.1
9.5 5.4 16.1 6.2
Note. Mean IC,, and IC, (*SD) for chloroquine (in nM) in the presence of fixed concentrations of enantiomers of amlodipine (in l&) added to eight serial dilutions of chloroquine, with each chloroquine concentration in triplicate. 0 Chloroquine alone. b Not determined.
roquine action (I(&, for chloroquine with or without amlodipine = 14.9 + 2.1 and 20.1 f 1.9 ti, respectively). The first series of experiments on the uptake of chloroquine in the erythrocytes after a 3-hr incubation showed a significant increase of chloroquine (80 nM) in the cell pellet with 2.5 uJ4 of enantiomers of amlodipine (Fig. 3a). In the second series of experiments using 40 IN of tritium-labeled
chloroquine, a further significant increase in chloroquine uptake was observed at the concentration of 5 ~JJMof enantiomers (Fig. 3b). At both concentrations of amlodipine, a significantly higher uptake of chloroquine was observed in infected erythrocytes with (-) enantiomer, at the ratio of 1.2 to 1.4, than with (+) enantiomer of amlodipine. Uptake of enantiomers into infected or uninfected erythrocytes occurred in the
TABLE HI Drug Interaction between Chloroquine and Enantiomers of Amlodipine against Three Chloroquine-Resistant Wild Strains of Plasmodium falciparum I&, for chloroquine (nkf) Drugs=
Isolate lb
Isolate 2
Isolate 3
Chloroquine alone (CQ) CQ + (+ ) amlodipine CQ + (-) amlodipine
177.5 f 21.5 54.2 -t 1.9 45.5 + 9.7
171.8 -+ 11.6 56.3 2 1.4 68.3 2 2.9
173.7 k 19.4 47.7 +- 8.7 34.9 * 3.5
Note. Results are mean I&, (?SD) in nA4. In vitro chloroquine-resistance level is I& 90-120 nM. a 2.5 p,M of enantiomers of amlodipine was added to eight serial dilutions of chloroquine, with each concentration in triplicate. b Isolates 1 and 2 are imported malaria cases from Gabon (a couple infected at the same time). Isolate 3 is from Cameroon. All isolates were obtained from symptomatic, nonimmune travelers upon returning to France.
266
BASCO AND LE BRAS 200
2
1
a
0.8
'i 0.6 z z Y -a E 'C M r;:
0.4
0.2 0
URBC 0.2
0.4
0.6
0.8
0.2
0.4
0.6
0.8
(+)2.5
(-)2.5
1.0 200
Fractional IC.50 amlodipine
0
PRBC
1.0
Fractional IC50 amlodipine FIG. 2. Isobolograms of in vitro drug interaction between enantiomers of amlodipine and chloroquine against the highly chloroquine-resistant strain FCM 29Kameroon (a) and the moderately chloroquineresistant strain FCM 6/Thailand (b). The concentration response data are given in Tables 1 and 2. Each point in the isobolograms was obtained by dividing the fixed amlodipine concentration by KS0 of its intrinsic activity (abscissa) and IC,, for chloroquine plus amlodipine by IC,, for chloroquine alone (ordinate). Results are means of simultaneous assays in triplicate. Concave curves depict synergistic interaction between the drugs.
first 5 min and a constant level was maintained for up to 3 hr (Fig. 4). There was no statistical difference in the accumulation of ( + ) and ( - ) enantiomers in the infected (or uninfected) erythrocytes. A significantly higher accumulation of enantiomers was as-
-I
URBC pRBc (+)2.5(-)2.5 (+)5 (-)5 FIG. 3. Effects of enantiomers of amlodipine on chloroquine accumulation (uRBC-uninfected RBC, pRBC-parasitized RBC, (+) and (-) denote enantiomers, 2.5 and 5 denote concentrations of enantiomers in pJ4). 80 nM of tritium-labeled chloroquine (16% parasitemia, 95% ringkrophozoite) (a) or 40 nM of cNoroquine (21% parasitemia, 88% ringkrophozoite) (b) was used. Results are means of triplicate determinations. Bars denote *SD.
sociated with infected erythrocytes pared to uninfected erythrocytes.
as com-
DISCUSSION
Amlodipine alone possesses a weak antimalarial activity, within a similar micromolar range as other reversing agents. A weak base effect was hypothesized to be the mechanism of action of 4-aminoquinoline and amino alcohol groups of antimalarial drugs, leading to the passage of these weakly basic drugs into the parasite’s vacuole along the pH gradient and their accumulation in this organelle (Homewood ef
POTENTIATION
5
30 Time
90
OF CHLOROQUINE
180
(minutes)
4. Accumulation of amlodipine (3 nM) inside the erythrocytes: q l, uninfected erythrocytes plus (+) enantiomer; 0, uninfected erythrocytes plus (-) enantiomer; k2, infected erythrocytes (15% parasitemia, 98% ringkrophozoite) plus (+) enantiomer; m, infected erythrocytes plus (-) enantiomer. Results are means of triplicate determinations. Bars denote 2 SD. FIG.
al. 1972; Yayon et al. 1985; Krogstad
and Schlesinger 1986). Amlodipine is a weak base with a pK, 8.6 (verapamil pK, 8.7). The lysosomotropic property of calcium channel blockers was observed (Ranganathan and Jackson 1984), and this may be responsible for their antimalarial action and may explain the identical IC,, obtained with the enantiomers of amlodipine against a given strain of parasite. Desipramine (pK, 10.2), another reversing agent, was also shown to have a lysosomotropic action (Honegger et al. 1983). It seems, however, that the basicity of a drug is not correlated with its antimalarial activity, and the basis of antimalarial action of reversing agents alone still remains unclear. Based on ICso values in the isobologram analysis, ( - ) enantiomer of amlodipine is 1.2 to 1.8 times more effective than ( + ) enantiomer at all potentiating concentrations, except for 2.5 lo& of enantiomers against FCM 29 where ( - ) enantiomer was almost 4 times as effective as the ( + ) enantiomer. Chloroquine uptake into parasitized erythrocytes was 1.2 to 1.4 times higher with ( -)
267
enantiomer. Although the greater difference shown in isobolograms of FCM 29 at 2.5 pM refers to the values of IC,, for chloroquine and is not directly comparable to the chloroquine uptake experiments that use subinhibitory concentrations of chloroquine, the results of the two experiments are in good agreement in showing that (- ) enantiomer is a more effective reversing agent than ( +) enantiomer. Total reversal of chloroquine resistance was observed at 2.5 to 3.8 pM. A similar range of concentration of other reversing agents is required to potentiate chloroquine action against P. falciparum in vitro. This may suggest that there is a common underlying mechanism of reversal of chloroquine resistance. An active efflux of drugs may be involved, as in the multidrug-resistant (mdr) cancer cells (Krogstad et al. 1987). In resistant cancer cells, an increased amount of P-glycoprotein, which is thought to function as an efflux pump, is produced by the amplification of a mdr gene (Riordan 1985; Cornwell et al. 1987; Safa et al. 1987). In both cancer cells (Tsuruo et al. 1982) and malaria parasites (Martin et al. 1987), drug resistance can be reversed with calcium channel blockers. Malarial mdr genes were also detected (Foote et al. 1989; Wilson et al. 1989). Based on this close analogy, it may be hypothesized that drug-resistant falciparum malaria overexpresses a chloroquine eftlux pump and that the reversing agents inhibit this pump. However, it remains to be established whether the genetic mechanism of chloroquine resistance can be attributed to the malarial mdr genes so far identified (Foote et al. 1990; Wellems et al. 1990) and whether a protein similar to P-glycoprotein is present in falciparum malaria parasite. Verapamil also displays an enantioselective action on calcium channels. Its Lisomer is 10 times more effective than the o-isomer (Echizen et al. 1985). Despite this difference, both enantiomers of verapamil were shown to be equally effective in re-
268
BASCO AND LE BRAS
versing chloroquine resistance in P. falci(Ye and Dyke 1988)and in increasing intracellular vincristine concentration in cancer cells (Gruber et al. 1988). Amlodipine exhibits an even more marked stereospecificity in the calcium channel blocking activity; its (-) enantiomer being 1000times more active than (+) enantiomer (Arrowsmith et al. 1986). This is the largest enantiomeric ratio so far reported for the 1,6dihydropyridines. The enantiomers accumulate inside the infected erythrocytes to the same extent and exert the same intrinsic antimalarial activity against P. falciparum. Both potentiate chloroquine action against resistant strains, with (-) enantiomer being slightly more effective. These findings may imply that there is a stereospecific difference at the site of action (putative malarial P-glycoprotein) of the enantiomers. This slight but statistically significant difference in the potentiating action of the enantiomers cannot be accounted for by the calcium antagonist action, which is negligible in the (+ ) enanComer. Furthermore, our kinetic studies showed that both enantiomers accumulate inside the parasitized erythrocytes within 5 min and a constant level is maintained for up to 3 hr. Amlodipine is therefore present inside the parasitized erythrocytes at the moment when resistant parasites begin to expel chloroquine 4 min after exposure to the drug (Krogstad ef al. 1987) and inhibits chloroquine efflux. Amlodipine is a slowacting calcium channel blocker whose action on the calcium channel appears gradually and reaches its maximal inhibition after 3 hr in vitro. Its kinetic property on the calcium channel is thus not compatible with the rapid efflux of chloroquine from resistant P. falciparum. In addition, in vitro studies on isolated vascular smooth muscle and cardiac muscle showed that the calcium channel blocking action is observed at low nanomolar concentration of (-) enantiomer (Burges et al.
parum
1987). Intrinsic antimalarial activity and reversal of chloroquine resistance are observed in the micromolar range, setting apart the two probably distinct actions of amlodipine-calcium antagonism and reversal of chloroquine resistance-by approximately three orders of magnitude. We conclude that the results of this study provide additional evidence against the hypothesis that the chloroquine potentiating action of calcium channel blockers is mediated by their calcium antagonist properties. ACKNOWLEDGMENTS We are gratehi to Dr. R. A. Burges of Pfizer Central Research for supplying amlodipine, Dr. F. Verdier of INSERM U13, Paris, for useful discussion, and Dr. F. Sauniere for his encouragement. REFERENCES ARROWSMITH, J. E, CAMPBELL, S. F., CROSS, P. E., STUBBS, J. K., BURGES, R. A., GARDINER, G. G., AND BLACKBURN, K. J. 1986. Long-acting dihydropyridine calcium antagonists. 1. 2-Alkoxymethyl derivatives incorporating basic substituents. Journal of Medicinal Chemistry 29, 1696-1702. BERENBAUM, M. C. 1989. What is synergy? Pharmacological Reviews 41, 93-141. BITONTI, A. J., SJOERDSMA, A., MCCANN, P. P., KYLE, D. E., ODUOLA, A. M. J., ROSSAN, R. N., MILHOUS, W. K., AND DAVIDSON, D. E., JR. 1988. Reversal of chloroquine resistance in malaria parasite Plasmodium falciparum by desipramine. Science 242, 1301-1303. BURGES, R. A., GARDINER, D. G., GWILT, M., HIGGINS, A. J., BLACKBURN, K. J., CAMPBELL, S. F., CROSS, P. E., AND STUBBS, J. K. 1987. Calcium channel blocking properties of amlodipine in vascular smooth muscle and cardiac muscle in vitro: Evidence for voltage modulation of vascular dihydropyridine receptors. Journal of Cardiovascular Pharmacology 9, 110-l 19. CORNWELL, M. M., PASTAN, I., AND GOTTESMAN, M. M. 1987. Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein. Journal of Biological Chemistry 262, 2166-2170. ECHIZEN, H., BRECHT, T., NIEDERGESASS, S., VoGELGESANG, B., AND EICHELBAUM, M. 1985. The effect of dextro-, levo-, and racemic verapamil on atrioventricular conduction in humans. American Heart Journal 109, 210-217. FITCH, C. D. 1970. Plasmodium falciparum in owl
POTENTIATION
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CHLOROQUINE
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phosphodiesterase.
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1990; accepted
with
revision
25
