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Antimicrobial Agents and Chemotherapy, November 2000, p. 3107-3111, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Gametocytocidal Activity and Synergistic
Interactions of Riboflavin with Standard Antimalarial Drugs against
Growth of Plasmodium falciparum In Vitro
Thomas
Akompong,1,*
Saliha
Eksi,2
Kim
Williamson,2 and
Kasturi
Haldar1
Departments of Pathology and Microbiology-Immunology,
Northwestern University Medical School, Chicago, Illinois
60611-3008,1 and Department of Biology,
Loyola University of Chicago, Chicago, Illinois
606262
Received 20 June 2000/Returned for modification 2 August
2000/Accepted 24 August 2000
 |
ABSTRACT |
Our previous studies have shown that riboflavin has activity
against Plasmodium falciparum asexual-stage parasites in
vitro. In the present study we examine the gametocytocidal activity of riboflavin and the interaction of riboflavin with some commonly used
antimalarial drugs against the asexual forms of P. falciparum in vitro. The addition of riboflavin to P. falciparum cultures killed gametocytes at all stages, even those
at late stages (III to V), which are not affected by many of the
commonly used antimalarials. Combinations of riboflavin with
mefloquine, pyrimethamine, and quinine showed a marked potentiation of
the activities of these drugs against asexual-stage parasites in vitro.
The combination of riboflavin with artemisinin was additive, while that
with chloroquine was mildly antagonistic. High doses of riboflavin are
used clinically to treat several inborn errors of metabolism with no
adverse side effects. Its efficacy in combination with standard
antimalarial drugs in treating and preventing the transmission of
P. falciparum malaria can therefore be evaluated in humans.
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INTRODUCTION |
During intraerythrocytic development
of malaria parasites, both asexual and sexual parasites are produced.
When the extracellular merozoites invade erythrocytes, most of the
resulting intracellular parasites develop in the asexual cycle, which
comprises three distinct morphological stages known as rings,
trophozoites, and schizonts. In the human-malaria species
Plasmodium falciparum, this cycle is completed in 48 h.
A small proportion of parasites, however, differentiate into
sexual-stage parasites, gametocytes that are required for transmission
of the disease by the mosquito vector. There are five distinct
morphological stages of gametocyte development designated stages I to
V. The complete maturation of P. falciparum gametocytes
after merozoite invasion takes 10 to 12 days.
The mainstay of malaria management is chemotherapy with antimalarial
drugs. Due to the continued appearance of parasites resistant to
first-line antimalarial drugs, the therapeutic value of most antimalarials currently in use has been greatly diminished. The difficulty of managing malaria is further compounded by the fact that
antimalarial drugs commonly used in countries where malaria is endemic
(such as chloroquine, quinine, sulfadoxine-pyrimethamine [SP], and
mefloquine) are not effective against the sexual forms of P. falciparum (6, 14, 25). Antifolate drugs such as pyrimethamine and the sulfa drugs, as well as SP, have been reported to
raise the proportion of gametocytes in treated patients (4). Treatment of malaria with SP alone results in elevated levels of
gametocytes that may increase the potential for malaria transmission from individuals already cured of clinical symptoms (33).
Combinations of antimalarial drugs may be used for two purposes: (i) to
enhance activity in the treatment of individual infections and (ii) to
delay the appearance of resistance to one or both the associated drugs
when they are to be used widely in an area where malaria is endemic
(34). To prevent or reduce malaria transmission, there is a
need for safe and inexpensive gametocytocidal drugs that can be used in
combination with first-line drugs against asexual-stage parasites.
Researchers have previously demonstrated the antimalarial activity of
riboflavin against asexual-stage P. falciparum in vitro (1). In the present study, the effect of riboflavin on the sexual-stage parasites and of combinations with standard antimalarial drugs against the asexual-stage parasites (P. falciparum
strains) were examined in vitro. Our results show that riboflavin is
effective against sexual-stage parasites and potentiates the activity
of mefloquine, pyrimethamine, and quinine. Thus, riboflavin used in
combination with these drugs could prevent the spread of resistant parasites and also lower malaria transmission by lowering gametocytogenesis.
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MATERIALS AND METHODS |
Materials.
RPMI 1640 medium was from GIBCO/BRL, and
A+ human serum was from Gemini Biological Products
(Calabasas, Calif.). Artemisinin, chloroquine diphosphate salt,
pyrimethamine, quinine hydrochloride, and riboflavin were from Sigma
(St. Louis, Mo.). Mefloquine was a gift from Dennis E. Kyle (Walter
Reed Army Institute of Research, Washington, D.C.).
Parasite cultivation and treatment with riboflavin.
The
chloroquine-resistant FCB strain of P. falciparum was
synchronized with 5% sorbitol and cultivated under standard conditions (13, 32). In the "riboflavin-pulse" experiments,
riboflavin was added to the culture for 2 h and then subsequently
washed out, and the parasites were cultured in the absence of
riboflavin for 3 to 4 h before riboflavin was reintroduced for
2 h. The reintroduction and washing procedure were repeated three
times a day for 2 days. The control parasites were treated with or
without riboflavin for 48 h, with medium changes after 24 h.
Thin blood smears were Giemsa stained to determine parasitemia.
Culturing of gametocytes.
P. falciparum gametocytes
(strain 3D7) were cultured as described by Ifediba and Vanderberg
(16). Briefly, asexual-parasite cultures were diluted to a
0.2% parasitemia and 6% hematocrit with fresh erythrocytes (day 0).
On day 3 the cultures were diluted from a 6% to a 3% hematocrit with
RPMI-10% serum and then maintained for the next 18 days with daily
medium changes. After day 0, no erythrocytes were added to the
cultures. Giemsa-stained slides of the cultures were prepared daily to
monitor parasitemia and gametocytogenesis.
Drug combination test.
The antiparasite activities of
riboflavin and standard antimalarial drugs were assessed by
hypoxanthine incorporation into parasite DNA, essentially as described
by Lauer et al. (21). Briefly, parasite suspensions at a 1 to 2% parasitemia and a 1% hematocrit were dispensed into 96-well
plates. To determine the effect of one drug on the dose response of the
other, 2 concentrations of the test drug were added in triplicate.
[3H]hypoxanthine (0.5 µCi/well) was added, and the
samples were incubated under culture conditions overnight. The cells
were harvested to glass fiber filters and liquid scintillation cocktail
was added and counted to determine cell-associated
[3H]hypoxanthine. A and B represent the drugs used;
IC50 represents the 50% inhibitory concentration. The
results were expressed as the sums of the fractional inhibitory
concentrations (sum FIC) (5, 7), which were defined as
Sum FIC values of <1 indicate synergism; values equal to 1 indicate addition; and values of >1 indicate antagonism.
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RESULTS AND DISCUSSION |
Gametocytocidal activity of riboflavin.
Several studies have
shown that higher gametocyte densities lead to increased transmission
(11, 23, 28, 31). The antimalarial drugs commonly used to
treat blood-stage infections of P. falciparum have little or
no activity against mature gametocytes. Hence, malaria may be
transmitted from patients who have been successfully cured of
asexual-stage infections; thus, drugs that are effective against both
asexual- and sexual-stage parasites will be invaluable in our efforts
to manage malaria caused by P. falciparum. We have previously shown that riboflavin, which is effective against
asexual-stage parasites in vitro, has a profound effect on hemoglobin
metabolism by the parasite (1). Since gametocytogenesis
results in complete digestion of host hemoglobin, we examined the
effects of riboflavin on gametocytogenesis.
Previous studies have demonstrated differences in the metabolisms of
early- and late-stage gametocytes (
20). Thus, a time
course
study was done to determine the effect of riboflavin on
the entire
spectrum of gametocytogenesis. An aliquot was removed
once daily from a
P. falciparum culture from days 1 to 10 after
invasion and
exposed to 100 µM riboflavin for the rest of the
experiment. Daily
blood smears of all cultures were made to determine
the number and
stage of gametocytes. As shown in Table
1, the
addition of riboflavin to cultures
resulted in killing of gametocytes
at all stages. The effect of
riboflavin on gametocytes at each
of the different stages was similar.
Independent of stage, a decrease
in gametocytes was observed 4 days
after the addition of riboflavin.
The maximum effect on
gametocytogenesis occurred 7 days after
the introduction of riboflavin.
Thus, when riboflavin was initially
added on day 5 to the culture
(mostly stage I gametocytes), gametocytes
were no longer observed by
day 15 (Table
1). Addition of riboflavin
to the culture (stage II and
III gametocytes) on day 10 resulted
in a 57% reduction in gametocytes
on day 15 and an 88% reduction
on day 17. No gametocytes remained on
day 18 (Table
1). At these
stages (II and III), during the first 2 days
of exposure to riboflavin,
the gametocytes continued to mature. But
after that the number
of gametocytes began to decrease, and
morphologically abnormal
gametocytes were observed in the culture (data
not shown). These
data demonstrate that riboflavin was effective
against both immature
and mature
P. falciparum gametocytes
in vitro.
Artemisinin and its derivatives are also effective against early-stage
gametocytes as well as asexual parasites. They have
been shown to
reduce total parasitemia and gametocyte load in
clinical trials
(
25,
33). Administration of artemisinin derivatives
in
combination with mefloquine to treat malaria on the Thai-Burmese
border
resulted in the reduction of malaria transmission by 50%
from 1994 to
1996 (
25). Since riboflavin is effective against
both
immature and mature gametocytes in vitro, it may also have
the
potential to block gametocytogenesis in vivo, which may lead
to a
reduction in
P. falciparum transmission from treated
patients.
Combination of riboflavin with standard antimalarial drugs.
Riboflavin has a short half-life in animals and humans (2 to 6 h)
(17, 18). Since micromolar concentrations are required to
kill asexual and sexual parasites in vitro, it may not be suitable for
use as a single agent to treat malaria. It may be useful, however, when
combined with standard antimalarial drugs to treat drug-resistant
malaria. Antimalarial-drug combinations may slow the emergence of
drug-resistant strains and prolong the effectiveness of each drug in
the treatment of infections. Several drug combinations have been tested
in vitro and in vivo for their use in malaria chemotherapy (7-9,
25-27, 30, 33, 34).
Mefloquine, SP, and quinine remain first-line drugs for treatment of
malaria in areas of endemicity. However, declining efficacy
due to the
emergence of resistance to these drugs has prompted
the search for
suitable combination partners to treat resistant
parasites (
25,
33). In addition to the problem of drug resistance,
these drugs
have no significant activity against
P. falciparum gametocytes. As shown in Table
2 and Fig.
1C to E, riboflavin
interacts
synergistically with mefloquine, pyrimethamine, and
quinine in vitro.
This suggests that riboflavin might be a suitable
combination partner
in vivo against the asexual-stage parasites
and also provide
gametocytocidal activity necessary to prevent
or reduce transmission
from drug-treated individuals.
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TABLE 2.
In vitro efficacies of riboflavin in combination with
standard antimalarial drugs against P. falciparum clone FCB
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FIG. 1.
Isobolograms of the interactions of riboflavin with
artemisinin (addition), chloroquine (weak antagonism), mefloquine
(synergism), pyrimethamine (synergism), and quinine (synergism). The
axes represent normalized FICs.
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|
Due to the relatively high recrudescence rates observed when
artemisinin and its derivatives are used clinically to treat
P. falciparum infections (
22,
34), artemisinin compounds
are
used in combination with other drugs (in particular, those with
a
long half-life) for improved action against asexual-stage parasites.
The in vitro antimalarial additive activity of riboflavin and
artemisinin shown in Fig.
1A, and Table
2 suggest that riboflavin
and
artemisinin or artemisinin's derivatives could be used together
in
vivo to more effectively reduce transmission of malaria caused
by
P. falciparum. However, since both riboflavin and
artemisinin
have short half-lives, a third drug with a longer half-life
may
also be needed to effectively clear asexual parasites. The
interaction
of riboflavin with chloroquine was mildly antagonistic
against
asexual-stage parasites. This suggests that chloroquine may not
be a suitable combination partner for riboflavin in
vivo.
Inhibition of parasite growth by short pulses of riboflavin.
The concentration of riboflavin required to inhibit asexual- and
sexual-stage parasites (25 to 100 µM) can be achieved in vivo only by
administration of high doses of riboflavin (50 to 400 mg/day). When
high doses of riboflavin are administered orally to humans, the maximum
concentration in serum is reached by 2 h and then declines to
basal levels 4 to 6 h later (18). We therefore examined
whether short pulses of riboflavin similar to what may occur in vivo
after administration of high-dose riboflavin were effective in killing
malaria parasites in vitro. These experiments were done by treating
infected cultures with or without riboflavin for 2 h, once, twice,
or three times a day (t.i.d.) for a total of 2 days. The effect of
treatment on parasite growth was measured by examining Giemsa-stained
blood smears. As shown in Fig. 2, administration of riboflavin (50 or 100 µM) (t.i.d.) inhibited parasite growth by 80 to 92%. The administration of 25 µM riboflavin t.i.d., on the other hand, resulted in a 45% inhibition (Table 3). Administration of 50 µM riboflavin
twice a day resulted in only a 26% inhibition, while administration of
50 or 100 µM riboflavin once a day had no significant effect (Table 3
and data not shown). These results suggest that a brief elevation of
riboflavin concentration in vitro, similar to the increase in the serum
concentration of riboflavin that occurs after the administration of
high oral doses in vivo, is effective in killing asexual-stage P. falciparum. However, riboflavin may bind tightly to many proteins
in the cell as a cofactor for many oxidative reactions. Thus, the
fluctuations in its concentration after the administration of high
doses in vivo may be less severe than those caused by our in vitro
washing procedure, a difference that could affect its antimalarial
activity in vivo. The data are consistent with the hypothesis that the administration of high doses of riboflavin several times a day may be
effective in killing P. falciparum in vivo. However,
riboflavin binds serum proteins, and its uptake in vivo exhibits
saturable kinetics. It is therefore possible that the administration of high doses in vivo may not produce sufficient concentrations to kill
P. falciparum in one generation, as occurs in vitro. The need to administer riboflavin three times a day may hamper its development as an antimalarial because of problems with compliance. However, multiple dosing is not unusual in malaria chemotherapy; for
example, the standard regimen of quinine and tetracycline to treat
uncomplicated malaria is given in multiple doses (quinine every 8 h for 3 days with tetracycline every 6 h for 7 days)
(34).
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FIG. 2.
Effect of short pulses of riboflavin on parasite growth.
Ring-stage parasites were cultured with 50 or 100 µM riboflavin for
2 h t.i.d. or continuously incubated for 48 h. Parasitemia
was determined by counting the number of parasites per 1,000 erythrocytes.
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The data presented here strongly suggest that riboflavin could be an
effective agent in treating and blocking the transmission
of malaria
caused by
P. falciparum. High doses of riboflavin are
used
to treat patients with several inborn errors of metabolism
(
2,
3,
10,
12,
15,
19,
24,
29), in some cases
up to 2 years, with no
adverse side effects. Since the safety
of riboflavin is well
established, it should be possible in clinical
trials to test its
efficacy in combination with standard antimalarials
against malaria
caused by
P. falciparum.
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ACKNOWLEDGMENTS |
We were supported by NIH grant AI39071 and Burroughs Wellcome New
Initiatives in Malaria Awards (to K.H.) and NIH grant AI40592 (to
K.W.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611-3008. Phone: (312) 503-1443. Fax: (312) 503-0281. E-mail: t-akompong{at}nwu.edu.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
