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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 97, Num. 7, 2002, pp. 1033-1039
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Mem Inst Oswaldo Cruz, Rio de
Janeiro, Vol. 97(7), October
2002, pp. 1033-1039
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, São Paulo, SP, Brasil
+Corresponding
author. Fax: +55-11-3815-4418. E-mail: hajudan@usp.br
Financial support:
CNPq
Received 8 March 2002
Accepted 15 August 2002
Code Number: oc02230
Phenothiazine drugs fluphenazine,
chlorpromazine, methotrimeprazine and trifluoperazine were evaluated
as modulating agents against Brazilian chloroquine-resistant fresh isolates
of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine
was employed at the concentration used for sensitive falciparum malaria
treatment and anti-psychotic therapeutic concentrations of the phenothiazine
drugs were adopted in two-fold serial dilutions. The in vitro microtechnique
for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine
modulating effect was not observed. However, all the drugs demonstrated intrinsic
antiplasmodial activity in concentrations lower than those described in the
literature. In addition, IC50 estimates have been shown to be inferior
to the usual anti-psychotic therapeutic concentrations. Statistical analysis
also suggested an increase in the parasitaemia rate or, even, a predominant
antiparasitic effect of phenothiazine over chloroquine when used in combination.
Key words: antimalarial - chloroquine
- multidrug resistance - phenothiazines - modulating agents - chemosensitizer
agents
The global situation of malaria is
claiming attention more than ever. It has been estimated that about 300 million
acute clinical cases are reported each year, with at least one million deaths
(WHO 2000). Antimalarial drug resistance is among the principal factors responsible
for this serious public health problem. Resistance has been associated with
the parasite's natural biological mechanisms in response to uncontrolled and
unregulated drug distribution programs, resulting in genetic mutations
(Wellems & Plowe 2001).
Many efforts have been made in the
search for a new and effective antimalarial agent. However, little success has
been achieved and, therefore, the restoration of current available drugs becomes
an important alternative. Combination therapy is particularly studied concerning
chloroquine (Fig. 1), although the
major Plasmodium falciparum strains and the emergent P. vivax
are resistant, this drug remains the most important antimalarial agent. Good
pharmacokinectic features, easy use and low cost are its principal properties
(Ward & Bray 2001).
With the purpose of restoring chloroquine
efficacy and considering the reported reversal effect of tricyclic drugs on
resistant malaria (Bitonti et al. 1988, Basco & Le Bras 1990, 1991, Kyle
et al. 1990, 1993, Peters et al. 1990, Basco & Le Bras 1992, Miki et al.
1992, Oduola et al. 1998), we evaluated the potential effect of the phenothiazine
drugs fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine
(Fig. 1) , on modulating the
chloroquine resistance of Brazilian P. falciparum fresh isolates.
MATERIALS AND METHODS
The drugs used were chloroquine diphosphate
(Fundação para o Remédio Popular), chlorpromazine hydrochloride,
fluphenazine dihydrochloride and me-thotrimeprazine maleate (Cristália
Produtos Químicos e Farmacêuticos Ltda.), and trifluoperazine dihydrochloride
(SmithKline Beecham).
Two fresh isolates of P. falciparum
were used: Isolate 1 (Sucen 198/94) and Isolate 2 (Sucen 206/94). Isolate 1
was collected from a 37-year-old woman in her third infection (6,600 asexual
parasites per mm3) and Isolate 2 from a 21 year-old-man in his second
infection (7,500 asexual parasites per mm3). The infections occurred
in the North of Brazil and the individuals had not been submitted to antimalarial
treatment in the previous 28 days (Bruce-Chwatt 1986). Blood samples were collected
after formal consent from the patients.
Assays were performed in microplates
with 96 flat-bottomed wells. The culture medium was RPMI-1640, HEPES buffer,
gentamicine sulphate, glucose, hypoxanthine, sodium bicarbonate and human type
A serum.
The statistical analysis was carried
out using the software S-Plus, version 4.5, and the Microsoft Excel for Windows,
version 5.0.
In vitro assays - The
biological assays were carried out based on the in vitro microtechnique for
drug susceptibility (Rieckmann et al. 1978). The microplates were titrated with
two-fold serial dilutions of fluphenazine (0.06-4 µg/l), chlorpromazine
(3.75-240 µg/l), methotrimeprazine (5-320 µg/l), and trifluoperazine
(0.31-20 µg/l). In each series, the intermediate value corresponded to
the usual anti-psychotic therapeutic concentration (Clarke 1986, Benet et 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 susceptibility
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 40 h (Isolate 1) and
for 46 h (Isolate 2). Schizonts with three or more nuclei in 200 parasites were
counted.
Statistical analysis - Lines
for the parasitaemia rate number of parasites in each concentration/number
of parasites in the control as a function of the chloroquine and phenothiazine
drug concentrations and the respective combinations with chloroquine were constructed
and submitted to descriptive and inferential analyses (Breslow & Day 1980,
Collett 1991). Linear and quadratic logistic models and the log-log complement
model were considered. According to these, coincident and separated, parallel
and concurrent (one or two intercepts) lines were fitted. The 10% significance
level was adopted for the likelihood ratio statistic which, in this case, corresponds
to the difference between two goodness-of-fit statistics. In addition, the 50%
inhibitory concentration, IC50, of each drug and its combination
with chloroquine was estimated.
RESULTS
The susceptibility of the isolates
to chloroquine, phenothiazine drugs and their combinations can be seen in the
descriptive analysis lines (Figs 2, 3,
4, 5,
6 show the lines calculated for Isolate
1). A decrease in the parasitaemia rate can be observed along the drug concentrations,
markedly for phenothiazines.
The inferential analysis suggested
the concurrent lines (two intercepts) as the best fitted model for fluphenazine,
chlorpromazine (at the 5% significance level), and methotrimeprazine for Isolate
1. The coincident lines were fitted 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) was the best fit for this drug (Table
II).
The IC50 estimates are
presented in the natural logarithm form in Table
III.
DISCUSSION
Many hypotheses have been advanced
to explain the P. falciparum resistance to chloroquine. The reversal
of chloroquine resistance by verapamil suggested a similar mammalian tumor cells
multidrug-resistant phenotype in malaria. Furthermore, many different drugs
and compounds have been assayed for their modulating effect in restoring antimalarial
drugs effectiveness (Ward & Bray 2001).
In our study, a series of drugs reported
as modulating agents in resistant malaria and/or neoplastic multidrug-resistant
cell lines were evaluated in Brazilian chloroquine-resistant fresh isolates
of P. falciparum. The in vitro microtechnique (Rieckmann et al. 1978)
was chosen as it matches closely with scintillometric measurements (Le Bras
et al. 1984), and it presents great feasibility in field studies (Yang et al.
1997, Philipps et al. 1998, Arez et al. 1999, Warsame et al. 1999). The chloroquine
IC50 estimates indicated that the isolates were resistant, according
to WHO statements (Bruce-Chwatt 1986). Moreover, these concentrations are among
those reported in previous studies on chloroquine P. falciparum resistance
reversal (Krogstad et al. 1987, Martin et al. 1987, Bitonti & Mc Cann 1989,
Basco & Le Bras 1990, Kyle et al. 1990).
With the purpose of simulating therapeutic
schedules, a particular drug combination was adopted. The intermediate concentration
in the two-fold serial dilutions corresponded to the usual therapeutic concentration
of the assessed modulating agent. A fixed concentration of chloroquine (30 µg/l)
was employed in the combinations. This concentration is capable of clearance
of parasitaemia in sensitive P. falciparum infections (Tracy &
Webster 1996). Oncethe modulating effect was observed, chloroquine would return
its effectiveness. A fixed concentration of chloroquine was also used to determine
the antimalarial response modification index throughout the combination of modulating
agents (Kyle et al. 1990, Oduola et al. 1998).
This study reports the evaluation
of the phenothiazine drugs: fluphenazine, chlorpromazine, metho-trimeprazine,
and trifluoperazine. Other tricyclic compounds, e.g., desipramine, cyproheptadine,
and their analogues (Bitonti et al. 1988, Basco & Le Bras 1990, Peters et
al. 1990, Basco et al. 1991) were early reported as modulating agents on resistant
malaria.Previous studieswithchlorpromazine, trifluoperazine and promethazine
have also demonstrated the in vitro and in vivo reversal of chloroquine resistance
(Kyle et al. 1990, 1993, Basco & Le Bras 1992, Miki et al. 1992, Oduola
etal. 1998).
However, different results were observed
in our study. The phenothiazines drugs did not modulate chloroquine resistance.
Similar behavior was observed for verapamil (Menezes et al. unpublished data),
imipramine (Menezes et al. 1997) and most of the other modulating agents assessed.
These results corroborated recent observations when Brazilian chloroquine-resistant
strains demonstrated a lesser degree of susceptibility to verapamil when compared
to strains from Africa and Southeast Asia (Mehlotra et al. 2001).
It is also important to note that,
in our study, all the phenothiazine drugs demonstrated intrinsic antiplasmodial
effects at concentrations inferior to those described as sub-inhibitory in modulating
the antimalarial resistance (as example, 625 nM, that corresponds to 199.30
µg/l, for chlorpromazine) (Kyle et al. 1990, Basco & Le Bras 1992).
However, in spite of the common phenothiazine chemical structure (Fig.
1), distinct behaviors were observed in the combinations with chloroquine.
For Isolate 1, the concurrent lines model (two intercepts) was the best fit
for me-thotrimeprazine, fluphenazine and chlorpromazine while the coincident
lines model was the best model for trifluoperazine. The latter model was also
the best fit for all drugs except methotrimeprazine, in the case of Isolate
2. The distinct fitted statistical lines models may be related to peculiarities
of the isolates. To date, these results are most interesting.
Inhibition of falcipain and the interaction
with heme moiety (ferriprotoporfyrin IX) have been associated to the antiplasmodial
activity of phenothiazine compounds (Panijpan & Kantakanit 1983, Domínguez
et al. 1997). During the intraerythrocytic stage, inside the food vacuole, specific
parasite proteases, such as falcipain, degrade the host hemoglobin in order
to generate amino acids for protein synthesis. The resulting heme is detoxified
by a polymerization process to form the malarial pigment, hemozoin. Thus, inhibition
of falcipain would precede the interference in the heme polymerization process,
the most accepted hypothesis regarding the mechanism of chloroquine action (Padmanaban
& Rangarajan 2000).
Our results should be contemplated
taking into account the phases in which chloroquine and phenothiazine compounds
are believed to interfere with the Plasmodium hemoglobin metabolism.
Inhibition of falcipain and/or a stronger interaction of the phenothiazine drugs
with heme moiety compared to that of chloroquine could be responsible for the
predominant antiplasmodial effects of these compounds over chloroquine. This
hypothesis may be supported by the coincident lines model. Conversely, the competition
for the heme moiety could explain the concurrent lines model. In this situation,
the addition of chloroquine caused an increase in the parasitaemia rate for
most phenothiazine concentrations.
Although further assays are needed
to check the proposed mechanistic hypothesis, we believe our findings are important
to demonstrate the antiplasmodial effect of phenothiazine drugs at concentrations
lower than those employed in anti-psychotic therapy, as observed by the estimated
IC50 values. In addition, those concentrations showed to be lower
than those previously related to antimalarial activity. Falcipain inhibition
as well as the interaction with heme moiety (Panijpan & Kantakanit 1983,
Domínguez et al. 1997) and other antiplasmodial activities of phenothiazines,
such as calmodulin antagonism (Scheibel et al. 1987), membrane stabilization
(Kristiansen & Jepsen 1985), disturbance in mitochondrial function (Geary
et al. 1986) and the inhibition of glutathione reductase (Luond et al. 1998)
were all observed in higher and, in general, toxic concentrations.
In resume, the results here presented
corroborate previous findings in considering phenothiazine derivatives as interesting
lead compounds in the design of new antimalarial agents (Vennerstrom et al.1995,
Atamna et al. 1996, Domínguez et al. 1997).
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Copyright 2002 Instituto Oswaldo
Cruz - Fiocruz
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