Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 97(7): 1033-1039, October 2002 In Vitro Chloroquine Resistance Modulation Study on Fresh Isolates of Brazilian Plasmodium falciparum: Intrinsic Antimalarial Activity of Phenothiazine Drugs Carla MS Menezes, Karin Kirchgatter*, Sílvia M Di Santi*, Carine Savalli**, Fabiola G Monteiro**, Gilberto A Paula**, Elizabeth I Ferreira/+ Faculdade de Ciências Farmacêuticas **Instituto de Matemática e Estatística, Universidade de São Paulo, Av. Prof. Lineu Prestes 580, Bl. 13, 05508-900 São Paulo, SP, Brasil *Divisão de Programas Especiais, Superintendência de Controle de Endemias, Phenothiazine drugs – fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine – were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming tosimulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum ma-laria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-foldserial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, thephenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodialactivity in concentrations lower than those described in the literature. In addition, IC estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increasein the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when usedin combination. Key words: antimalarial - chloroquine - multidrug resistance - phenothiazines - modulating agents - chemosensitizer agents The global situation of malaria is claiming attention we evaluated the potential effect of the phenothiazine more than ever. It has been estimated that about 300 mil- drugs – fluphenazine, chlorpromazine, methotrimeprazine lion acute clinical cases are reported each year, with at and trifluoperazine (Fig. 1) –, on modulating the chloro- least one million deaths (WHO 2000). Antimalarial drug quine resistance of Brazilian P. falciparum fresh isolates.
resistance is among the principal factors responsible for MATERIALS AND METHODS
this serious public health problem. Resistance has beenassociated with the parasite’s natural biological mecha- The drugs used were chloroquine diphosphate nisms in response to uncontrolled and unregulated drug (Fundação para o Remédio Popular), chlorpromazine hy- distribution programs, resulting in genetic mutations drochloride, fluphenazine dihydrochloride and me- thotrimeprazine maleate (Cristália Produtos Químicos e Many efforts have been made in the search for a new Farmacêuticos Ltda.), and trifluoperazine dihydrochloride and effective antimalarial agent. However, little success has been achieved and, therefore, the restoration of cur- Two fresh isolates of P. falciparum were used: Isolate rent available drugs becomes an important alternative.
1 (Sucen 198/94) and Isolate 2 (Sucen 206/94). Isolate 1 Combination therapy is particularly studied concerning was collected from a 37-year-old woman in her third infec- chloroquine (Fig. 1), although the major Plasmodium tion (6,600 asexual parasites per mm3) and Isolate 2 from falciparum strains and the emergent P. vivax are resis- a 21 year-old-man in his second infection (7,500 asexual tant, this drug remains the most important antimalarial parasites per mm3). The infections occurred in the North agent. Good pharmacokinectic features, easy use and low of Brazil and the individuals had not been submitted to cost are its principal properties (Ward & Bray 2001).
antimalarial treatment in the previous 28 days (Bruce- With the purpose of restoring chloroquine efficacy Chwatt 1986). Blood samples were collected after formal and considering the reported reversal effect of tricyclic drugs on resistant malaria (Bitonti et al. 1988, Basco & Le Assays were performed in microplates with 96 flat- Bras 1990, 1991, Kyle et al. 1990, 1993, Peters et al. 1990, bottomed wells. The culture medium was RPMI-1640, Basco & Le Bras 1992, Miki et al. 1992, Oduola et al. 1998), HEPES buffer, gentamicine sulphate, glucose, hypoxan-thine, sodium bicarbonate and human type A serum.
The statistical analysis was carried out using the soft- ware S-Plus, version 4.5, and the Microsoft Excel for Win-dows, version 5.0.
In vitro assays - The biological assays were carried
out based on the in vitro microtechnique for drug suscep- Corresponding author. Fax: +55-11-3815-4418. E-mail: tibility (Rieckmann et al. 1978). The microplates were ti- trated with two-fold serial dilutions of fluphenazine (0.06- 4 µg/l), chlorpromazine (3.75-240 µg/l), methotrimeprazine In Vitro Phenothiazine Antimalarial Activity• Carla MS Menezes et al.
Day 1980, Collett 1991). Linear and quadratic logistic mod- els and the log-log complement model were considered.
According to these, coincident and separated, parallel and concurrent (one or two intercepts) lines were fitted. The10% significance level was adopted for the likelihood ra-tio statistic which, in this case, corresponds to the differ- ence between two goodness-of-fit statistics. In addition, the 50% inhibitory concentration, IC , of each drug and its combination with chloroquine was estimated.
The susceptibility of the isolates to chloroquine, phe- nothiazine drugs and their combinations can be seen in the descriptive analysis lines (Figs 2 to 6 show the linescalculated for Isolate 1). A decrease in the parasitaemiarate can be observed along the drug concentrations, mark- The inferential analysis suggested the concurrent lines (two intercepts) as the best fitted model for fluphenazine,chlorpromazine (at the 5% significance level), andmethotrimeprazine for Isolate 1. The coincident lines werefitted to trifluoperazine in the same isolate (Table I). For Isolate 2 the coincident lines model was the best fit for all the phenothiazines, with the exception of methotrime-prazine. The concurrent lines model (two intercepts) wasthe best fit for this drug (Table II).
The IC estimates are presented in the natural loga- Fig. 1: chemical structures of the phenothiazines and chloroquine.
Parasitaemia rate
(5-320 µg/l), and trifluoperazine (0.31-20 µg/l). In each se-ries, the intermediate value corresponded to the usual anti- ln(concentration)
psychotic therapeutic concentration (Clarke 1986, Benetet al. 1996). Chloroquine (30 µg/l) was added along the series (Tracy & Webster 1996). The phenothiazine series were also assayed alone with the purpose of evaluating the intrinsic antiplasmodial effect. The chloroquine sus-ceptibility was tested in a range from 3.75 to 240 µg/l.
A 10% haematocrit solution of infected blood was added to the plates. These were incubated according to the candle jar method (Trager & Jensen 1976) at 37°C for Parasitaemia rate
40 h (Isolate 1) and for 46 h (Isolate 2). Schizonts withthree or more nuclei in 200 parasites were counted.
Statistical analysis - Lines for the parasitaemia rate – number of parasites in each concentration/number of para- ln(concentration)
sites in the control – as a function of the chloroquine andphenothiazine drug concentrations and the respective Fig. 2: ratios of the number of Plasmodium falciparum parasites in combinations with chloroquine were constructed and sub- drug-treated cultures to the number in control cultures (parasitaemia mitted to descriptive and inferential analyses (Breslow & rate) after exposure to chloroquine (A: Isolate 1; B: Isolate 2).
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 97(7), October 2002 Parasitaemia rate
Parasitaemia rate
Parasitaemia rate
Parasitaemia rate
Fig. 3: ratios of the number of Plasmodium falciparum parasites in Fig. 4: ratios of the number of Plasmodium falciparum parasites in drug-treated cultures to the number in control cultures (parasitaemia drug-treated cultures to the number in control cultures (parasitaemia rate) after exposure to fluphenazine (▲) and fluphenazine plus rate) after exposure to chlorpromazine (▲) and chlorpromazine chloroquine (■) (A: Isolate 1; B: Isolate 2).
plus chloroquine (■) (A: Isolate 1; B: Isolate 2).
Fitted linear logistic model for the phenothiazine drugs and their combinations with chloroquine (Isolate 1, Sucen 198/94) In Vitro Phenothiazine Antimalarial Activity• Carla MS Menezes et al.
Parasitaemia rate
Parasitaemia rate 0,2
Parasitaemia rate
Fig. 5: ratios of the number of Plasmodium falciparum parasites in Fig. 6: ratios of the number of Plasmodium falciparum parasites in drug-treated cultures to the number in control cultures (parasitaemia drug-treated cultures to the number in control cultures (parasitaemia rate) after exposure to methotrimeprazine (▲) and metho- rate) after exposure to trifluoperazine (▲) and trifluoperazine plus trimeprazine plus chloroquine (■) (A: Isolate 1; B: Isolate 2).
chloroquine (■) (A: Isolate 1; B: Isolate 2).
Fitted linear logistic model for the phenothiazine drugs and their combinations with chloroquine (Isolate 2, Sucen 206/94) Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 97(7), October 2002 Estimated median inhibitory concentrations (IC ) of chloroquine, the phenothiazine drugs and the respective combinations on a: Log 50% inhibitory concentration; b: same values (coincident lines model) DISCUSSION
trimeprazine, and trifluoperazine. Other tricyclic com- Many hypotheses have been advanced to explain the pounds, e.g., desipramine, cyproheptadine, and their ana- P. falciparum resistance to chloroquine. The reversal of logues (Bitonti et al. 1988, Basco & Le Bras 1990, Peters et chloroquine resistance by verapamil suggested a similar al. 1990, Basco et al. 1991) were early reported as modulat- mammalian tumor cells multidrug-resistant phenotype in ing agents on resistant malaria. Previous studies with malaria. Furthermore, many different drugs and com- chlorpromazine, trifluoperazine and promethazine have pounds have been assayed for their modulating effect in also demonstrated the in vitro and in vivo reversal of chlo- restoring antimalarial drugs effectiveness (Ward & Bray roquine resistance (Kyle et al. 1990, 1993, Basco & Le Bras 1992, Miki et al. 1992, Oduola et al. 1998).
In our study, a series of drugs reported as modulat- However, different results were observed in our study.
ing agents in resistant malaria and/or neoplastic The phenothiazines drugs did not modulate chloroquine multidrug-resistant cell lines were evaluated in Brazil- resistance. Similar behavior was observed for verapamil ian chloroquine-resistant fresh isolates of P. (Menezes et al. unpublished data), imipramine (Menezes falciparum. The in vitro microtechnique (Rieckmann et et al. 1997) and most of the other modulating agents as- al. 1978) was chosen as it matches closely with sessed. These results corroborated recent observations scintillometric measurements (Le Bras et al. 1984), and when Brazilian chloroquine-resistant strains demonstrated it presents great feasibility in field studies (Yang et al.
a lesser degree of susceptibility to verapamil when com- 1997, Philipps et al. 1998, Arez et al. 1999, Warsame et pared to strains from Africa and Southeast Asia (Mehlotra al. 1999). The chloroquine IC estimates indicated that the isolates were resistant, according to WHO state- It is also important to note that, in our study, all the ments (Bruce-Chwatt 1986). Moreover, these concen- phenothiazine drugs demonstrated intrinsic antiplasmodial trations are among those reported in previous studies effects at concentrations inferior to those described as on chloroquine P. falciparum resistance reversal sub-inhibitory in modulating the antimalarial resistance (Krogstad et al. 1987, Martin et al. 1987, Bitonti & Mc (as example, 625 nM, that corresponds to 199.30 µg/l, for Cann 1989, Basco & Le Bras 1990, Kyle et al. 1990).
chlorpromazine) (Kyle et al. 1990, Basco & Le Bras 1992).
With the purpose of simulating therapeutic schedules, However, in spite of the common phenothiazine chemical a particular drug combination was adopted. The interme- structure (Fig. 1), distinct behaviors were observed in the diate concentration in the two-fold serial dilutions corre- combinations with chloroquine. For Isolate 1, the concur- sponded to the usual therapeutic concentration of the rent lines model (two intercepts) was the best fit for me- assessed modulating agent. A fixed concentration of chlo- thotrimeprazine, fluphenazine and chlorpromazine while roquine (30 µg/l) was employed in the combinations. This the coincident lines model was the best model for triflu- concentration is capable of clearance of parasitaemia in operazine. The latter model was also the best fit for all sensitive P. falciparum infections (Tracy & Webster 1996).
drugs except methotrimeprazine, in the case of Isolate 2.
Once the modulating effect was observed, chloroquine The distinct fitted statistical lines models may be related would return its effectiveness. A fixed concentration of to peculiarities of the isolates. To date, these results are chloroquine was also used to determine the antimalarial response modification index throughout the combination Inhibition of falcipain and the interaction with heme of modulating agents (Kyle et al. 1990, Oduola et al. 1998).
moiety (ferriprotoporfyrin IX) have been associated to This study reports the evaluation of the phenothiaz- the antiplasmodial activity of phenothiazine compounds ine drugs: fluphenazine, chlorpromazine, metho- (Panijpan & Kantakanit 1983, Domínguez et al. 1997). Dur- In Vitro Phenothiazine Antimalarial Activity• Carla MS Menezes et al.
ing the intraerythrocytic stage, inside the food vacuole, Basco LK, Ringwald P, Le Bras J 1991. Chloroquine-potentiat- specific parasite proteases, such as falcipain, degrade the ing action of antihistaminics in Plasmodium falciparum in host hemoglobin in order to generate amino acids for pro- vitro. Ann Trop Med Parasitol 85: 223-228.
tein synthesis. The resulting heme is detoxified by a poly- Benet LZ, Øie S, Schwartz JB 1996. Design and optimization merization process to form the malarial pigment, hemozoin.
of dosage regimens pharmacokinetic data. In JG Hardman,LE Limbird, PB Molinoff, RW Ruddon, AG Gilmann (eds), Thus, inhibition of falcipain would precede the interfer- Goodman & Gilman’s: the Pharmacological Basis of Thera- ence in the heme polymerization process, the most ac- peutics, 9th ed., Pergamon-Press, New York, p. 1707-1792.
cepted hypothesis regarding the mechanism of chloro- Bitonti AJ, McCann PP 1989. Desipramine and cyprohepta- quine action (Padmanaban & Rangarajan 2000).
dine for reversal of chloroquine resistance in Plasmodium Our results should be contemplated taking into ac- falciparum. Lancet 2: 1282-1283.
count the phases in which chloroquine and phenothiaz- Bitonti AJ, Sjoerdsma A, Mccann PP, Kyle DE, Oduola AMJ, ine compounds are believed to interfere with the Plasmo- Rossan RN, Milhous WK, Davidson Jr DE 1988. Reversal dium hemoglobin metabolism. Inhibition of falcipain and/ of chloroquine resistance in malaria parasite Plasmodium or a stronger interaction of the phenothiazine drugs with falciparum by desipramine. Science 242: 1301-1303.
heme moiety compared to that of chloroquine could be Breslow NE, Day NE 1980. Statistical Methods in Cancer Re- responsible for the predominant antiplasmodial effects of search. 1. The Analysis of Case Control Studies, Interna-tional Agency for Research on Cancer, Lyon, 350 pp.
these compounds over chloroquine. This hypothesis may Bruce-Chwatt LJ 1986. Chemotherapy of Malaria, 2nd ed., be supported by the coincident lines model. Conversely, the competition for the heme moiety could explain the Clarke EC 1986. Isolation and Identification of Drugs in Phar- concurrent lines model. In this situation, the addition of maceuticals, Body Fluids and Post-mortem Material, 2nd chloroquine caused an increase in the parasitaemia rate ed., Pharmaceutical Press, London, 1223 pp.
for most phenothiazine concentrations.
Collett D 1991. Modelling Binary Data, Chapman & Hall, Lon- Although further assays are needed to check the pro- posed mechanistic hypothesis, we believe our findings Domínguez JN, López S, Charris J, Iarruso L, Lobo G, Semenov are important to demonstrate the antiplasmodial effect of A, Olson JE, Rosenthal PJ 1997. Synthesis and antimalarial phenothiazine drugs at concentrations lower than those effects of phenothiazine inhibitors of a Plasmodium employed in anti-psychotic therapy, as observed by the falciparum cysteine protease. J Med Chem 40: 2726-2732.
Geary TG, Divo AA, Jensen JB 1986. Effect of calmodulin estimated IC values. In addition, those concentrations inhibitors on viability and mitochondrial potential of Plas- showed to be lower than those previously related to anti- modium falciparum in culture. Antimicrob Ag Chemother malarial activity. Falcipain inhibition as well as the inter- action with heme moiety (Panijpan & Kantakanit 1983, Kristiansen JE, Jepsen S 1985. The susceptibility of Plasmo- Domínguez et al. 1997) and other antiplasmodial activities dium falciparum in vitro to chlorpromazine and the stereo- of phenothiazines, such as calmodulin antagonism isomeric compounds cis(Z)- and trans(E)-clopenthixol. Acta (Scheibel et al. 1987), membrane stabilization (Kristiansen Pathol Microb Immunol Scand (Section B) 93: 249-251.
& Jepsen 1985), disturbance in mitochondrial function Krogstad DJ, Gluzman IY, Kyle DE, Oduola AMJ, Martin SK, (Geary et al. 1986) and the inhibition of glutathione reduc- Milhous WK, Schlesinger PH 1987. Efflux of chloroquine tase (Luond et al. 1998) were all observed in higher and, in from Plasmodium falciparum: mechanism of chloroquine resistance. Science 238: 1283-1285.
In resume, the results here presented corroborate pre- Kyle DE, Milhous WK, Rossan RN 1993. Reversal of Plasmo- dium falciparum resistance to chloroquine in Panamanian vious findings in considering phenothiazine derivatives Aotus monkeys. Am J Trop Med Hyg 48: 126-133.
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