This study identified and evaluated the malaria transmission-blocking activities of several tricyclic compounds after screening a drug library against parasites with a disrupted gene encoding a putative ABC transporter. Although our initial goal was to investigate whether PfABCG2 played a role in drug transport and resistance in gametocytes of P. falciparum parasites, identification of this group of transmission-blocking drugs may have a significant impact on malaria control and eradication if results from clinical field trials confirm our laboratory observations. The sexual stages inhibited by ketotifen do not contribute to the increase of parasite numbers in the human host; with combinations including drugs against asexual stages, the risk of selecting parasites resistant to ketotifen can be minimized. Although we have good evidence showing that PfABCG2 plays a role in the parasite response to ketotifen, identification of the drug target(s) and the mechanism of action in blocking oocyst development in mosquitoes require additional investigation.
In addition to ketotifen, our study also identified several other promising tricyclic compounds, including cyproheptadine, protryptyline, and MLS000708402-02, which were highly active in blocking P. yoelii nigeriensis
N67 oocyst formation in the mosquito midgut. MLS000708402-02 has not been tested in humans but may represent a new lead for a potential therapeutic agent. Ketotifen, cyproheptadine, and protryptyline, on the other hand, all have been approved for use in humans. Ketotifen and cyproheptadine are antihistamines that act as a 5-HT2 receptor antagonist in humans (38
), and protryptyline is an antidepressant. Ketotifen is used to relieve irritation associated with seasonal allergies and to prevent asthma attacks (38
). It can be administered orally or topically with generally mild side effects. Clinical results have shown no serious signs or symptoms after ingestion of up to 20 mg of ketotifen fumarate (38
). Cyproheptadine is also used to relieve mild allergy symptoms as well as to treat posttraumatic stress disorder (39
) and shows few side effects, although the dosage is limited to 12 mg per day for children and 32 mg per day for adults. The efficacy of these related tricyclic compounds may provide structural activity data for designing additional and better transmission-blocking drugs.
Because ketotifen has been approved for human use, its pharmacokinetics (PK) and pharmacodynamics (PD) have been extensively studied. Ketotifen has been extensively tested in animals for safety and toxicity (38
). Large doses have been tested in rats and rabbits with minimum side effects, including during pregnancy. Ketotifen is well absorbed after oral administration, with peak plasma drug concentrations within 2 to 4 h. Peak plasma drug concentrations after multiple oral doses of 1 mg twice daily were 1.92 mg/liter in adults and 3.25 mg/liter in children (41
). The drug is 75% protein bound and is metabolized in the liver to the inactive form, ketotifen-N
-glucoronide, and active norketotifen. The terminal elimination half-life is 2 to 27 h, with a mean half-life of 12 h. Approximately 60 to 70% of a dose of ketotifen is eliminated in the urine within 48 h, with the remainder excreted in the feces; ~50% is recovered as ketotifen-N
-glucuronide and ~10% as norketotifen (38
). In a recent study evaluating antimalarial activity, both ketotifen and its metabolite norketotifen were shown to be active against schizonts and liver-stage Plasmodium berghei
parasites, and the plasma drug concentration was maintained above the IC50
for approximately 8 h for ketotifen and over 24 h for norketotifen (38
). The form of ketotifen metabolite(s) that is active against the sexual stages of the malaria parasite is still unknown.
Ketotifen, cyproheptadine, and other antihistamines have been shown to be effective in reversing CQ resistance in P. falciparum
) and in P. yoelii nigeriensis
). These compounds have also been tested for reducing gametocytemia, although no obvious effect on patient gametocytemia was observed (49
). It was not clear, however, whether the gametocytes in some of the previous studies were still capable of infecting mosquitoes or if the oral dose used (0.0125 to 0.25 mg/kg) was sufficient to eliminate gametocytes from the blood circulation. Our observations of significant reductions in gametocyte counts against in vitro
-cultured P. falciparum
gametocytes and more than 50% reductions in oocyst counts in mosquitoes after treatment with 5 μM ketotifen suggested that doses as low as 0.1 mg/kg body weight should have some effect on gametocyte and/or oocyst formation in vivo
. Indeed, treatment of P. yoelii
parasites with a single dose of 0.1 mg/kg reduced oocyst count from 27% (1 h) to 81% (24 h) (). Ketotifen showed slightly higher potency in blocking oocyst formation than PQ at 5 mg/kg and 10 mg/kg in mice (), which suggests that ketotifen may act more effectively on mosquito stages or the process of fertilization. In addition, ketotifen appeared to affect gamete production; treatment of P. falciparum
gametocytes with ketotifen greatly reduced the numbers of exflagellation centers (). Because the 3D7 line is sensitive to CQ, it is difficult to test whether disruption of pfabcg2
will affect the ability of ketotifen and other antihistamines in reversing CQ resistance. Future studies using a CQ-resistant parasite with disrupted pfabcg2
may provide insights on how these compounds reverse CQ resistance.
Interestingly, ART was more active than ketotifen in inhibiting oocyst formation after in vitro
treatment of P. falciparum
gametocytes, whereas ketotifen appeared to be more potent against P. yoelii
(; ). The discrepancies could have been due to species differences in responses to the drugs or, more likely, to a higher rate of ART removal from the body (half-life, ~1 h) and the loss of activity against gametocytes shortly after administration to the animal. Thus, the ART exposure time was likely longer in vitro
, enhancing the effectiveness of ART. In one study, artesunate was found to reduce gametocyte infectivity of patient blood dramatically, but it could not abolish the infectivity completely (51
). More recently, ART and derivatives were shown to have some activities against exflagellation and to reduce the number of oocysts (52
). These observations suggest that even if ART has some activity against gametocytes, addition of another drug that can kill gametocytes and/or mosquito stages to ACT may be necessary to completely block malaria transmission. Our preliminary results showed that a combination of low-dose ketotifen and ART was more effective in blocking oocyst formation in mosquitoes than either drug alone ( and ).
Ketotifen appeared to have some effect on relapse of P. cynomolgi
in rhesus monkeys, although the results were not conclusive. The first relapse peak seen in the control monkeys did not appear in the monkeys treated with ketotifen, but the second peak at day 45 appeared in all the groups, which was duplicated in a second independent experiment. There are several potential explanations for the disappearance of the first relapse peak in the ketotifen-treated group. First, the first relapse peak in the control group could have come from residual parasites that were not totally cleared by quinine treatment, which was unlikely, and additional treatment with ketotifen killed those residual parasites in the blood because ketotifen also has some activity against asexual stages. Second, assuming all the blood forms were killed by quinine treatment, ketotifen might be active against more mature or “active” stages (late trophozoites and schizonts) but not the stage still dormant in the liver (53
), which might prevent the appearance of the first relapse peak but not the second peak. It would be interesting to investigate whether a second ketotifen treatment at day 35 or a longer ketotifen treatment period prevented the second relapse peak at day 45. The standard regimen of PQ for treating Plasmodium vivax
relapse in humans is a daily dose of 15 mg/day for 2 weeks. In a study of mass treatment to eliminate malaria in Cambodian villages, a dose of 9 mg per adult was given at 10-day intervals for up to 6 months (7
). Ketotifen may prevent further relapses if a similar treatment regimen is given. Indeed, ketotifen and its metabolite, norketotifen, have recently been shown to be active against the liver stage of P. berghei
; no evidence of live parasites was detected after treatment with both drugs at 160 mg/kg/day for 3 days (40
The mechanism of how ketotifen, cyproheptadine, and other TCA drugs block P. falciparum
oocyst formation in mosquitoes requires further investigation. Many ABC transporters, including ABCG2, have been shown to play a role in drug transport as well as other transport functions (54
). Genetic modification of genes encoding a drug target or a drug transporter has widely been employed to demonstrate the involvement of a gene in modulating drug susceptibility of malaria parasites (18
). In addition to showing that disruption of pfabcg2
changed the parasite response to ketotifen in asexual stages, we also showed that reintroduction of pfabcg2
could partially reverse this differential effect. The partial restoration of sensitivity could be due to differences in gene expression level and/or protein localization of the endogenous gene and reintroduced episomal copies. Indeed, the green fluorescent protein signal was present throughout the transfected parasites (data not shown), instead of the expected membrane localization. In addition, the rescue construct also encoded a C-terminal GFP tag fused to the protein, which may have perturbed its transporter function. The same reasons could also be used to explain our failure to restore gametocyte production in the pfabcg2
-disrupted parasite C11 (four attempts [data not shown]), although we could not rule out the possibility that the loss of gametocyte production was due to a changes in another gene(s) in the genome during parasite transformation and selection. Interaction of ketotifen with an ABC transporter has been reported; ketotifen was found to restore the sensitivity of P-glycoprotein-overexpressing multidrug-resistant MCF-7/adr cells to doxorubicin, mitoxantrone, VP-16, and vinblastine (59
). In theory, resistance to this drug may develop due to mutations in pfabcg2
; however, since the gene appears to play almost no role in asexual stages, the pressure for selecting resistant mutations will be minimal. Gametocytes, gametes, and ookinetes do not replicate (except that the male gametocyte that produces eight gametes), and the chance of selecting drug-resistant parasites from sexual stages, the intended target of the drug, should be greatly reduced. We cannot rule out the possibility of a related cellular process that is rendered essential by the drug treatment, as seen in screening yeast either (60
). Although the normal asexual stage of growth of the parasites with a disrupted pfabcg2
gene suggests that the gene is not essential during blood-stage development, the change in the IC50
to ketotifen does not prove that PfABCG2 transports the drug, and more studies are necessary to answer this question. Overexpression of an ABC transporter generally makes a cell more resistant to a drug; however, the opposite has also been reported (61
). ABCG2 has also been shown to contribute to cell survival in an oxygen-poor environment by reducing the accumulation of toxic heme metabolites (55
). A large amount of heme is produced during gametocyte maturation, and a potential biologic function of PfABCG2 could be in extruding heme that accumulates during gametocyte development. Although more studies are necessary to elucidate the functional roles of PfABCG2 in parasite development and drug responses, the identification and testing of this group of urgently needed drugs represent significant contributions in malaria research and disease control.
Various high-throughput methods have been developed for screening antimalarial drugs against asexual stages (19
) and liver stages (67
). More recently, assays for screening gametocytocidal drugs have been reported in response to the urgent need for drugs to block malaria transmission; however, the throughput of these assays was still relatively low (12
). High-throughput drug screening requires a method to obtain large numbers of parasites, which is still the limiting step for assays using gametocytes or mosquito stages as screening targets. Methods to increase the yield of gametocyte production are necessary to improve the throughput level of drug assays for testing a large number of compounds against this important sexual stage. Our approach of screening parasites with a disrupted gene that was expressed at higher levels in gametocytes or mosquito stages than in asexual stages may represent a novel, productive approach to search for drugs that can preferentially inhibit nonerythrocytic stages of malaria parasites.