EXPERIMENTAL PARASITOLOGY 74, 134-142 (1992)

Plasmodium Resistance ATSUSHI

chabaudi: Association of Reversal of Chloroquine with Increased Accumulation of Chloroquine in Resistant Parasites

MIKI,* KAZUYUKI TANABE,?’

TETSUO NAKAYAMA,* AND KAYO OHSAWA*

CHE KIRYON,*

*Department of Medical Zoology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545, Japan; and iLaboratory of Biology, Osaka Institute of Technology, Ohmiya, Asahi-ku, Osaka 535, Japan MIKI, A., TANABE, K., NAKAYAMA, T., KIRYON, C., AND OHSAWA, K. 1992. Plasmodium chabaudi: Association of reversal of chloroquine resistance with increased accumulation of chloroquine in resistant parasites. Experimental Parasitology 74, 134-142. The effects of tricyclic antidepressants, desipramine and imipramine, and phenothiazines, chlorpromazine and trifluoperazine. on chloroquine (CQ)-resistant and CQ-sensitive lines of P. chabaudi were examined in Go. In mice that received daily injections of these drugs the growth of CQ-resistant and CQ-sensitive parasites was unaffected or affected very slightly, if at all. A combination of CQ and each drug suppressed the growth of CQ-resistant parasites in a dose-dependent manner. In addition, in CQ-sensitive parasites each drug also increased the susceptibility to CQ. Measurements of CQ levels by high-performance liquid chromatography showed that CQ accumulated in sensitive parasites to more than twice the level in resistant parasites at 2 to 4 hr after an injection of CQ. Verapamil and desipramine substantially increased CQ levels in both CQ-resistant and CQ-sensitive parasites. These results suggest that not only Ca’+ antagonists but tricyclic antidepressants revcrsc CQ resistance in CQ-resistant parasites and enhance the inhibitory effect in sensitive parasites by increasing CQ levels in those parasites. The effects of Ca’+ antagonists, tricyclic antidepressants, and phenothiazines on a pyrimethamine-resistant line of P. chabaudi were also studied. None of the Ca’+ antagonists (verapamil, nicardipine, and diltiazem) affected the growth of the parasite in combination with 20 mg/kg pyrimethamine. Tricyclic antidepressants and phenothiazines suppressed pyrimethamine-resistant parasites to some extent. However, the extent of this suppression was less pronounced as compared with that of suppression of CQ resistance by the same drugs. 0 1992 Academic Press. Inc INDEX DESCRIPTORS AND ABBREVIATIONS: Plasmodium chabaudi; Malaria; Ca’+ antagonists; Chloroquine (CQ); Pyrimethamine (Pyr); Antidepressants: Phenothiazines; Desipramine (Des); Imipramine (Imi); Chlorpromazine (CPZ); Trifluoperazine (TFP); Verapamil (Ver); Nicardipine (Nit); Diltiazem (Dil); Drug resistance; Carboxymethyl cellulose (CMC); High-performance liquid chromatography (HPLC); Hepes-buffered saline (HBS); Red blood cells (RBCs); Infected red blood cells (IRBCs).

and therapeutic purposes in endemic areas. Thus, much effort to develop and to exploit new effective antimalarials has been devoted for the last three decades. On the other hand, considering the low cost and the effectiveness of CQ against CQsensitive strains of P. falciparum, a new treatment strategy has begun that is aimed to reverse CQ resistance by drugs that are apparently unrelated to antimalarials. Those drugs that have been reported to

Resurgence of malaria in many tropical and subtropical countries partly results from the spreading of drug-resistant strains of Plasmodium falciparum. Among a wide variety of antimalarial drugs, resistance to chloroquine (CQ) is a serious matter because CQ is widely used for prophylactic ’ To whom correspondence

should be addressed. I34

0014-4894/92 $3.00 Copyright All rights

B 1992 by Academic Press, Inc. of reproduction in any form rcrerved.

REVERSAL OF CHLOROQUINE

date to reverse CQ resistance in resistant strains of P. fulciparum include certain Ca*’ antagonists and tricyclic antidepressants (Martin et al. 1987; Bitonti et al. 1988; Kyle et al. 1990). Among these drugs, desipramine is the only drug that has been proved to reverse CQ resistance in vivo (Bitonti et al. 1988). Whether other reversing agents exert their effects in vivo on CQresistant P. fulcipurum or not has not been studied. The major reason for this is obviously due to the limited availability of experimental hosts of the parasite, the Aotus and Saimiri monkeys. It is therefore of extreme importance to develop a rodent model for surveying drugs with the potential of reversing CQ resistance in vivo. Previous studies by us and other investigators have shown that certain Ca2+ antagonists, including verapamil and antihistaminics, reverse CQ resistance in vivo in resistant strains of P. chubuudi and P. berghei (Peters et al. 1989; Tanabe et al. 1990). However, many antidepressants and phenothiazines that are used clinically for psychosis remain to be tested for their reversing effects. Meanwhile, in the case of P. fulciparum Ca*+ antagonists and other agents that reverse CQ resistance have been reported to induce the accumulation of CQ in CQ-resistant parasites in vitro (Krogstad et al. 1987; Bitonti et al. 1988). However, it is unknown whether the accumulation of CQ occurs in vivo or not in CQresistant strains of both P. falciparum and rodent malaria species. In the present study, we studied the in vivo effects of tricyclic antidepressants and phenothiazines on CQ-resistant and CQ-sensitive lines of P. chabaudi and simultaneously measured CQ levels in these parasites. In addition, the effects of Ca2+ antagonists and other drugs on a pyrimethamine-resistant line of P. chubuudi were examined. MATERIALS

AND METHODS

1. Infection. Female ICR mice (Nippon SLC, Japan), 6 to 7 weeks old, were infected with either a

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135

CQ-resistant or a CQ-sensitive line of the AS strain of P. chabaudi. Procedures for infection of P. chabaudi were described (Tanabe et al. 1990). Briefly, blood was collected by cardiac puncture from infected mice and their RBCs were suspended in HBS (155 mM NaCl, 10 mM KCl, 1 nu’r4 MgS04, 25 mM HepesNaOH, pH 7.2) at a density of 5 x 10’ IRBCslml. Mice were inoculated intravenously with 0.1 ml of the suspension and parasitemias were monitored daily by counting numbers of IRBCs per 10,000 RBCs on tail blood smears stained with Giemsa. Previously, we showed that P. chabaudi grew logarithmically from Day 0 to Day 3 under these experimental conditions (Tanabe et a/. 1990). 2. Administration of drugs. Chloroquine diphosphate (CQ; Sigma Chemical Co.) was dissolved at desired concentrations in 0.9% (w/v) NaCl and 0.1 ml of the CQ solution was injected intraperitoneally into mice daily for 3 consecutive days from Day 0 of infection. Pyrimethamine (Pyr; Sigma Chemical Co.) was suspended in 0.5% (w/v) carboxymethyl cellulose (CMC) in 0.9% NaCl and 0.1 ml of the Pyr suspension was injected subcutaneously in mice daily for 3 consecutive days from Day 0 of injection. Desipramine (Des; Sigma Chemical Co.), imipramine (Imi), verapamil (Ver), nicardipine (Nit), and diltiazem (Dil) were dissolved at desired concentrations in distilled water. Chlorpromazine (CPZ) and trifluoperazine (TFP) were suspended at desired concentrations in 05% (w/v) CMC. Imi, Ver, Nit, Dil, CPZ, and TFP are the generous gifts of Nissho Co. Ltd. Four to six mice in each group were subcutaneously dosed daily with 0. l-ml solutions or suspensions containing one of these drugs for 3 consecutive days from Day 0 of infection. Maximum doses of the drugs were determined preliminarily to be those at which mice neither lost body weight nor showed abnormal lay of hair. Results were expressed as the mean of parasitemia 2 standard deviations of the mean (SDM) and difference in parasitemias was analyzed by the Student’s t test. Preliminary results showed that none of the daily intraperitoneal injections of 0.1 ml of 0.9% NaCl, daily subcutaneous injections of 0.1 ml of distilled water, and 0.5% CMC affected the course of the infection (data now shown). 3. Measurements of CQ levels by high-performance liquid chromatography (HEX). When parasitemia

reached 5 to 7%, infected mice were administered 6 mg/kg of CQ alone or in combination with 50 mg/kg of Ver or 100 mg/kg of Des. This dose of CQ was not the same as for the above-mentioned infections, because at parasitemias as high as 5 to 7%, a higher dose of CQ was required to suppress the growth of P. chabaudi (Ohsawa er al. 1991). At 1, 2, and 4 hr after drug administration, infected mice were anesthetized with chloroform and their bloods withdrawn by cardiac puncture with a heparinized syringe, followed by centrifugation at 900g for 5 min at 4°C. After aspirating the

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buffy coat, the pellet was diluted to 50% cell suspension in HBS and layered onto 600 ~1 of silicon E (Shinetsu Chemical Co., Tokyo, Japan) in a microtube and sedimented at 9000g for 1 min in a microfuge (MR15A, Tomy Seiko, Japan) at 4°C. After aspirating the supernatant and silicon oil, the pellet was washed in ice-cooled HBS by centrifugation for 5 min at 900g three times and stored at -80°C. Uninfected mice were also injected with CQ and their bloods were processed similarly. Preliminary experiments showed that little CQ leaked out from IRBCs during these procedures. The frozen samples were thawed, diluted in distilled water, sonicated, and alkalinized with 1 N NaOH. CQ was extracted by adding dichloromethane at pH 11. The organic phase was reextracted with 0.1 N HCI, and the hydrochloric extract was subjected to assay for CQ levels (Bergqvist and Frisk-Holmberg 1980; Verdier et al. 1985). For measurement by HPLC, samples were introduced into a Waters 510 injector with a p,Bondapak C,, reversed-phase column and separated in a mixture of 3 vol of 10mM NaH,PO, in 100 mM NaCIO, and I vol of acetonitrile. The fluorescence of CQ was detected on a Hitachi 6SO-1OLC fluorescence detector, under alkaline conditions, by adding 50 mM sodium borate/NaOH at pH 9.2 with settings of excitation at 328 nm and emission at 386 nm. Results were expressed as concentrations of CQ per 10s IRBCs. At this time, concentrations of CQ in uninfected RBCs in infected blood were subtracted after calculating parasitemias of infected blood and CQ levels of uninfected RBCs in normal blood.

RESULTS

1. Effects of tricyclic antidepressants and phenothiazines on CQ-resistant and CQsensitive P. chabaudi. Figure 1 illustrates the effects of tricyclic antidepressants, Des and Imi, on the growth of CQ-resistant P. chabaudi. Des and Imi, when administered alone, did not affect the growth of the parasite (Figs. la and lc). However, a combination of 3 mg/kg of CQ with either Des or Imi substantially suppressed the parasite growth in a dose-dependent manner (P < 0.01 for 10 to 100 mg/kg of Des and Imi) (Figs. lb and Id). Since CQ at this dose does not inhibit CQ-resistant P. chabaudi (Tanabe et al. 1990), these results indicate reversal of CQ resistance by Des and Imi. Similarly, coadministration of CQ with a phenothiazine, CPZ or TFP, inhibited the growth of resistant parasites in a dosedependent manner (P < 0.01) (Figs. 2b and

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FIG. 1. Reversal of chloroquine (CQ) resistance by tricyclic antidepressants in CQ-resistant P. chubaudi. Mice were treated daily with desipramine (a, b) or imipramine (c, d) at 0 (e), 3 (m), 10 (O), 30 (A), or 100 (0) mg/kg in combination with (b, d) or without (a, c) 3 mg/kg of CQ. Bars represent SDM that are shown wherever the range of SDM is greater than a symbol.

2d). Although CPZ and TFP alone at a high dose slightly suppressed the growth (P > 0.05) (Figs. 2a and 2c), the suppression was very marginal compared with that by coadministration with CQ. Effects of various drugs on CQ-sensitive parasites are listed in Fig. 3. In the case of sensitive parasites, a dose of CQ was reduced to 2 mg/kg at which dose the parasites grew well whereas they did not at 3 mg/kg (Tanabe et al. 1990). A combination of CQ with each drug significantly suppressed the parasite growth (P < 0.01 for 100mg/kg of Des and Imi, 30 mglkg of CPZ, 10 mg/kg of TFP) (Figs. 3b and 3d). However, all of the drugs, when administered alone, did not affect the parasite growth (P > 0.05) (Figs. 3a and 3~). Thus, the above results indicate that the tricyclic antidepressants and phenothiazines not only re-

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OF CHLOROQUINE

137

RESISTANCE

4~ b

341 a

0

1

2

3 Days

0

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3 Days

FIG. 2. Reversal of chloroquine (CQ) resistance by phenothiazines in CQ-resistant P. chabaudi. Mice were treated daily with chlorpromazine (a, b) at 0 (0) 3 (Cl), 10 (A), or 30 (0) mg/kg or trifluoperazine (c, d) at 0 (O), 3 (Cl), or 10 (0) mg/kg in combination with (b, d) or without (a, c) 3 mg/kg of CQ. Bars represent SDM that are shown wherever the range of SDM is greater than a symbol.

versed resistance to CQ in CQ-resistant parasites but increased the susceptibility to CQ in CQ-sensitive parasites. 2. Effects of Ca2+ antagonists, tricyclic antidepressants, and phenothiazines on Pyr-resistant P. chabaudi. The CQ-

resistant line, but not the CQ-sensitive line, of P. chabaudi used in this study is also resistant to Pyr at 20 mg/kg (data not shown, Rosario 1976). Hence, whether various agents that reverse CQ resistance reverse Pyr resistance or not was examined. Results are shown in Fig. 4. None of the Ca2+ antagonists (Vet-, Nit, and Dil) in combination with Pyr significantly affected the parasite growth (P > 0.05). On the other hand, a combination of Pyr with Des, Imi, CPZ, or TFP suppressed the parasite

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FIG. 3. Effects of tricyclic antidepressants and phenothiazines on the growth of chloroquine (CQ)sensitive P. c/&au&. Mice were treated daily with (a, b) 100 mg/kg of desipramine (0), imipramine (Ki), or none (0) or with (c, d) 30 mg/kg of chlorpromazine (0) 10 mg/kg of trifluoperazine (O), or none (0) in combination with (b, d) or without (a, c) 2 mg/kg of CQ. Bars represent SDM that are shown wherever the range of SDM is greater than a symbol.

growth to some extent (P < 0.01). However, the extent of this suppression was less marked than that of suppression of resistance to CQ by the same reversing agents (cf. Figs. 1 and 2). 3. Measurements

of CQ levels by HPLC.

Accumulation of CQ in CQ-sensitive and in CQ-resistant P. chabaudi measured by HPLC is shown in Fig. 5. CQ accumulated extensively in RBCs infected with CQsensitive parasites at 1 hr and continued to accumulate steadily afterwards. In contrast, the accumulation of CQ in RBCs infected with CQ-resistant parasites was low at 1 hr with 38% reduction as compared with the accumulation in the sensitive

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MIKI ET AL.

b

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ioi ( , , i,J ( , , 1

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2 Days

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1

2

3 Days

FIG. 4. Effects of Ca”

antagonists, tricyclic antidepressants, and phenothiazines on the growth of pyrimethamine (Pyr)-resistant P. chubaudi. Mice were treated daily with 20 mgikg of Pyr in combination with (a) 50 mg/kg of Ca’+ antagonists; verapamil (O), nicardipine (A), diltiazem (O), or none (a), or in combination with (b) 100 mg/kg of desipramine (W), 100 mgikg of imipramine (Cl), 30 mg/kg of chlorpromazine (0), IO mg/kg of trifluoperazine (a), or none (0). Bars represent SDM that are shown wherever the range of SDM is greater than a symbol.

group and CQ levels in the resistant group did not increase after 1 hr. In contrast to IRBCs, little CQ accumulated in uninfected RBC.

0

1

4

2 hr

FIG. 5. Accumulation of chloroquine (CQ) in P. chabaudi. CQ was injected at 0 hr into mice infected

with CQ-sensitive (0) or CQ-resistant parasites (0) or into normal mice (Cl). Variations of values at each point are within 5% of the mean.

CQ levels in RBCs infected with CQresistant parasites with or without coadministration of Ver or Des are shown in Fig. 6. Although an increase in cell-associated CQ was less evident at I hr, Ver and Des greatly stimulated the CQ accumulation at 2 and 4 hr: the CQ levels in drug-treated groups being 2.Zfold of untreated levels at 4 hr (Fig. 6). Effects of Ver and Des on the accumulation of CQ in RBCs infected with CQsensitive parasites are shown in Fig. 7. Ver stimulated the accumulation of CQ and this stimulation was evident at 2 to 4 hr, during which the rate of CQ accumulation was relatively slow in the Ver-untreated group (Fig. 7a). An increase in the accumulation was also induced by Des, although the increase was notable only at 4 hr (Fig. 7b). On the other hand, neither Ver nor Des stimulated the CQ accumulation in uninfected RBCs (not shown). These results indicate that the increased accumulation of CQ by Ver and Des primarily occurred within parasites.

REVERSAL

OF CHLOROQUINE

- a zoo-

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139

RESISTANCE

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FIG. 6. Effects of verapamil and desipramine on the accumulation of chloroquine (CQ) in CQresistant P. chabaudi. Mice received 6 mglkg of CQ with (0) or without (0) 50 mg/kg of verapamil (a) or 100 mg/kg of desipramine (b). Variations of values at each point are within 5% of the mean.

DISCUSSION

Although the cellular and biochemical mechanism for resistance to CQ in resistant strains of P. fulciparum and rodent malaria

parasites has not been fully understood, it is generally believed that resistant parasites accumulate less CQ than do sensitive parasites. Earlier, Macomber et al. (1966) observed a reduced accumulation of [r4C]CQ b

1 300 i

0

1

2

4 hr

0

1

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2 hr

FIG. 7. Effects of verapamil and desipramine on the accumulation of chloroquine (CQ) in CQsensitive P. chubaudi. Mice received 6 mg/kg of CQ with (0) or without (0) 50 mg/kg of verapamil (a) or 100 mg/kg of desipramine (b). Variations of values at each point are within 5% of the means.

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in CQ-resistant P. berghei in infected mice. Similar results were obtained with CQresistant P. fulciparum by measuring the in vitro accumulation of [3H]CQ (Krogstad et al. 1987). In the present study, we measured total CQ in P. chabaudi by HPLC after in vivo administration of CQ. Results showed less accumulation of CQ in resistant parasites than in sensitive parasites. Furthermore, our measurements revealed that Ver and Des increased the CQ accumulation in CQ-resistant parasites. This is consistent with our previous and present findings that Ver and Des reverse CQ resistance in CQ-resistant P. chabaudi (Tanabe et al. 1990). Meanwhile, CQ-resistant strains of P. falciparum are shown to extrude CQ more extensively than do CQsensitive parasites in vitro (Krogstad et al. 1987). Ver and Des are also reported to inhibit the active extrusion of CQ from resistant strains of P. falciparum (Krogstad et al. 1987; Bitonti et al. 1988). Hence, it is very likely that Ver- and Des-induced reversal of malarial resistance to CQ is due to the inhibition of this drug export process, which leads to enhanced accumulation of CQ in resistant parasites. Ca2+ antagonists, tricyclic antidepressants, and phenothiazines tested in our previous (Tanabe et al. 1990) and present studies also increased the susceptibility to CQ in CQ-sensitive P. chabaudi. Consistently, Ver and Des enhanced the accumulation of CQ in sensitive parasites. These results are apparently in contrast to results obtained with CQ-sensitive strains of P. falciparum, in which these drugs neither increased the susceptibility to CQ nor inhibited the efflux of CQ from the parasite. The reason for this discrepancy between sensitive strains of P. chabaudi and P. falciparum remains unknown. In our study, a time course pattern of Ver- and Des-induced accumulation of CQ in CQ-sensitive parasites is rather different from that in CQ-resistant parasites. The accumulation of CQ started earlier in resistant parasites than in sensitive para-

ET AL.

sites. For example, at 2 hr Ver and Des increased CQ levels to 1.6- to 1.9-fold of controls in resistant parasites whereas the increases in sensitive parasites were 1.O- to 1.3-fold of controls (Figs. 6 and 7). Thus, the mode of action of Ver and Des may differ between CQ-sensitive and CQresistant P. chubaudi. In a CQ-sensitive strain of P. fulciparum the susceptibility to CQ is reportedly increased by cyproheptadine, an antihistaminic agent (Peters et al. 1989). It seems likely that certain agents that reverse resistance to CQ also induce the accumulation of CQ in some CQsensitive strains of P. falciparum and, as a consequence, increase the susceptibility to CQ, The above-mentioned different effects of Ca2+ antagonists and tricyclic antidepressants on CQ susceptible strains of P. chabaudi and P. falciparum might lead us to postulate that the mode of action of CQ and/or mechanism(s) of resistance to CQ may differ between the two parasite species. Clarification of a mechanism(s) of action of CQ on the parasite species is a prerequisite to justify the postulation. Nevertheless, the observed association of reversal of CQ resistance with increased accumulation of CQ in resistant P. chabaudi encourages us to employ the present rodent species for screening agents that reverse CQ resistance in resistant P. falciparum. This notion is supported by the observations that the reversing agents so far tested in CQ-resistant P. chabaudi have also the potential of reversing CQ resistance in resistant strains of P. falciparum in vitro and in vivo (Martin et al. 1987; Bitonti et al. 1988; Kyle et al. 1990). Therefore, we consider that the CQ-resistant P. chabaudi AS strain is useful for evaluating the potential of reversing agents prior to testing certain selected agents using the P. fulciparum primate hosts. A mechanism for reversal of malarial CQ resistance by Ca*+ antagonists and other reversing agents has been a matter of con-

I41

REVERSALOFCHLOROQUINERESISTANCE

troversy. Recent studies suggest similarities of reversal phenomena of drug resistance between P. fulciparum and mammalian neoplastic cells. In the latter, multidrug resistance (MDR) phenotypes are mediated by overexpression of a membrane protein called P-glycoprotein, encoded by the mdr gene (Gottesman and Pastan 1988). A variety of compounds such as Ca’+ antagonists and calmodulin inhibitors is believed to compete for the drug-binding site of the P-glycoprotein with anticancer drugs, thus leading to the intracellular accumulation of anticancer drugs. Several researchers have argued, in analogy with reversal of the MDR phenotype, that a malarial gene (pfmdr), equivalent to the mdr genes of mammalian tumor cells, is expressed at higher levels to confer parasite’s resistance to CQ (Wilson et al. 1989; Foote et al. 1989). Mutations of the pfmdr gene have also been suggested to be involved in resistance to CQ (Foote et al. 1990; Cowman 1991). On the other hand, experimental data from genetic crosses between CQresistant and CQ-sensitive clones of P. fulciparum have not shown any clear correlation in the progeny between phenotypic resistance to CQ and the presence of multiple copies of the pfmdr genes (Wellems et al. 1990). There is evidence that Ca*’ antagonists reverse resistance to quinoline-containing drugs other than CQ in a multidrug resistant strain of P. falciparum (Kyle et al. 1990). Here, Ca*+ antagonists did not reverse resistance to pyrimethamine in CQ (and Pyr)resistant P. chubaudi. This finding is not unexpected because pyrimethamine has a definitive target molecule, i.e., dihydrofolate reductase (Peterson et al. 1988). Although the parasite line used in this study showed resistance to both CQ and Pyr, phenotypic resistance to the two drugs can be segregated, indicating the presence of different genetic mechanisms for the resistance to the two drugs (Rosario 1976). Thus, reversal of malarial multidrug resis-

tance by Ca2+ antagonists is rather restricted to some, but not all, antimalarials. Meanwhile, in contrast to Ca*+ antagonists, tricyclic antidepressants and phenothiazines suppressed Pyr-resistant P. chabaudi to some extent. The reason for this remains unknown. These drugs might potentiate the antimalarial activity of Pyr in P. chabaudi by disturbing unknown enzymes linked directly or indirectly to folate metabolic pathways. However, the extent of suppression of Pyr resistance by tricyclic antidepressants and phenothiazines is less marked than that of suppression of CQ resistance by the same agents. Obviously, further experiments using Pyr-susceptible strains of P. chabaudi are required to address whether the suppression of resistance to Pyr by those drugs is truly “reversal” of Pyr resistance or not. We are currently examining this issue. ACKNOWLEDGMENTS We thank Professor S. Takada for his encouragement during this study, Dr. S. Nakamura of Nissho Co. Ltd. for suggestions and supplying various drugs, and Mr. Y. Mine of Kinki University School of Medicine for assisting with HPLC experiments. Thanks are also due to Dr. S. Doi for valuable discussions. This work was supported in part by a Grant-in-Aid from the Ministry of Science, Culture and Education, Japan (No. 02807044)and a research grant from Osaka Institute of Technology. REFERENCES BERGQVIST,Y., AND FRISK-H• LMBERG,M. 1980.Sensitive method for the determination of chloroquine and its metabolite desethylchloroquine in human plasma and urine by high performance liquid chromatography. Journal of Chromatography 221, 119127. BITONTI, A., SJOERSMA,A., MCCANN, P. P., KYLE, D. E., ODUOLA, A. M. J., ROSSAN, R. N., AND DAVIDSON,D. E., JR. 1988.Reversal of chloroquine resistance in malaria parasite Plasmodium falciparum by desipramine. Science 242, 1301-1303. COWMAN, A. F. 1991. The P-glycoprotein homologues of Plasmodium falciparum: Are they involved in chloroquine resistance? Parasitol. Today 7, 70-76.

FOOTE, S. J., THOMPSON, J. K., COWMAN, A. F., AND KEMP, D. J. 1989. Amplification of multidrug

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Received 6 June 1991; accepted with revision 11 October 1991