The antimalarial drug mefloquine has promising antischistosomal properties killing haematophagous adult schistosomes as well as schistosomula. The mode of action and involved drug targets of mefloquine in Schistosoma mansoni schistosomula are unknown. In order to identify mefloquine-binding proteins and thus potential drug targets, mefloquine affinity chromatography with S. mansoni schistosomula crude extracts was performed. We found one specific mefloquine-binding protein that was identified by mass spectrometry as the glycolytic enzyme enolase (Q27877). Enolase activity assays were performed on schistosomula crude extracts and on the recombinant enolase Q27877 expressed in Escherichia coli. In schistosomula crude extracts enolase activity was inhibited by mefloquine and by the enolase inhibitor sodium fluoride, while activity of the recombinant enolase was not affected. In contrast to enolase from crude extracts, recombinant Q27877 did not bind to mefloquine-agarose. Using isothermal microcalorimetry, we next investigated the metabolic inhibition of mefloquine and 3 known glycolytic inhibitors in Schistosoma spp., namely sodium fluoride, 3-bromopyruvate and menadione on schistosomula in the presence or absence of glucose. We found that in the presence of glucose, schistosomula were less affected by mefloquine, sodium fluoride and 3-bromopyruvate, whereas glucose had no protective effect when schistosomula had been exposed to menadione. These results suggest a potential role of mefloquine as an inhibitor of glycolysis, at least in stages where other targets like haem degradation are not relevant.

During the last 2 decades there have been numerous reports of the emergence of mefloquine resistance in Southeast Asia and nearly 50% resistance is reported in Thailand. A World Health Organization report (2001) considers mefloquine as an important component of ACT (artesunate+mefloquine) which is the first line of treatment for the control of uncomplicated/multi-drug resistant (MDR) Plasmodium falciparum malaria. In view of the emergence of resistance towards this drug, it is proposed to develop new drug combinations to prolong the protective life of this drug. Prior studies have suggested that mefloquine resistance can be overcome by a variety of agents such as ketoconazole, cyproheptadine, penfluridol, Icajine and NP30. The present investigation reports that clarithromycin (CLTR), a new macrolide, being a potent inhibitor of Cyt. P450 3A4, can exert significant resistance reversal action against mefloquine resistance of plasmodia. Experiments were carried out to find out the curative dose of CLTR against multi-drug resistant P. yoelii nigeriensis. Mefloquine (MFQ) and clarithromycin (CLTR) combinations have been used for the treatment of this MDR parasite. Different dose combinations of these two drugs were given to the infected mice on day 0 (prophylactic) and day 1 with established infection (therapeutic) to see the combined effect of these combinations against the MDR malaria infection. With a dose of 32 mg/kg MFQ and 225 mg/kg CLTR, 100% cure was observed, while in single drug groups, treated with MFQ or CLTR, the cure was zero and 40% respectively. Therapeutically, MFQ and CLTR combinations 32+300 mg/kg doses cleared the established parasitaemia on day 10. Single treatment with MFQ or CLTR showed considerable suppression of parasitaemia on day 14 but neither was curative. Follow-up of therapeutically treated mice showed enhanced anti-malarial action as reflected by their 100% clearance of parasitaemia. The present study reveals that CLTR is a useful antibiotic to be used as companion drug with mefloquine in order to overcome mefloquine resistance in plasmodia.

There is a pressing need to develop novel anti-schistosomal drugs, as current treatment relies largely on praziquantel (PZQ). To further strengthen current evidence of the anti-schistosomal properties of mefloquine (MQ), we studied the temporal effect of this compound in vitro and in vivo, and examined alterations on the tegumental surface of schistosomula and adults of S. mansoni by means of scanning electron microscopy (SEM). Schistosomula and adults were each incubated in vitro using MQ over a wide concentration range (1–100 μg/ml). In addition, mice infected with adult S. mansoni were treated with a single oral dose of 400 mg/kg MQ, and worms were recovered 24, 48, 72, 96 and 120 h following treatment. MQ showed a rapid onset of action on schistosomula in vitro; 100 and 75 μg/ml of MQ killed schistosomula immediately; the minimal lethal and effective concentrations of MQ on schistosomula after 1 h were 25 and 5 μg/ml, respectively. Adult worms incubated with 100 and 10 μg/ml of MQ were dead after 1 h and 24 h of incubation, respectively. A hepatic shift of adult schistosomes was observed in mice already 24 h after treatment, and 120 h following treatment >98% of all worms had translocated to the liver. SEM observations revealed extensive tegumental destruction, including blebbing, shrinking and sloughing, particularly following in vitro incubation and on the tegument of female worms.

Intraerythrocytic malaria parasites ingest the cytosol of their host cell and digest it inside their acid food vacuoles. Acidified (pH 4–5·5, 37 °C) human red blood cell lysates were used to simulate this process, measuring the denaturation of haemoglobin (Hb) and the release of iron, in the absence or presence of exogenous protease. Spontaneous Hb denaturation and appearance of non-heme iron were observed upon lysate acidification, their rates decreasing with increasing pH, and increasing in the presence of protease. Both processes were inhibited by the quinoline-containing anti-malarial drugs (QCDs) chloroquine, quinine, mefloquine and amodiaquine at concentrations well below those expected in the acidic food vacuole of the parasite. Spectrophotometric analysis indicated that chloroquine complexes with heme in acid-denatured haemoglobin. Other weak bases as well as verapamil and diltiazem, known to reverse the resistance of malarial parasites to chloroquine, were without efifect indicating that the action of QCDs is specific. Based on our previous results and the present report, we suggest that iron release in acidified lysates is mediated through the formation of ferryl (Fe(IV)) radicals. QCDs possibly complex with this radical, as they do with heme, and prevent its contact with an adjacent heme molecule which is required for ring opening and iron release. These results may suggest that one of the anti-malarial effects of QCDs is to deprive the parasite of an adequate iron supply. Addition of iron to cultures of Plasmodium falciparum was expected to circumvent the deprivation of iron and reduce the anti-malarial eflfect of QCDs. However, adding iron as penetrating fructose or nitrilotriacetate complexes did not alter the parasite's susceptibility to chloroquine. Ascorbate markedly increased the release of iron in acidified lysates, and this effect was not reduced by chloroquine. Ascorbate was found to decrease parasite susceptibility to chloroquine, suggesting that iron deprivation may be an important factor in the anti-malarial action of QCDs.

The present report deals with the status of hepatic and splenic glutathione-S-transferase (GST) activities in mice during experimental infection with Plasmodium yoelii nigeriensis and subsequent treatment of infected mice with mefloquine (Mf) and menadione (Md). The infection caused significant decline in the hepatic and splenic glutathione-S-transferase (GST) activities of albino and db/+ mice. The decline was observed in the levels of both cytosolic and microsomal GST(s) of liver and spleen in both types of mice. Intraperitoneal administration of mefloquine at a dose of 5 mg/kg and menadione at a dose of 100 mg/kg, twice daily from day 1 p.i. (day 0) until day 10, caused restoration in the levels of hepatic as well as splenic GST(s), albeit to varying degrees. Mf was able to suppress parasitaemia by day 5 in the case of albino mice and by day 3 in the case of db/+ mice but was unable to cure both types of mice completely. On the other hand, Md caused a delay in maturation of infection in both cases, but could not cure the mice.

Clinical data on the use of artesunate combined with mefloquine in a variety of treatment regimens and parasite loads in Thailand were modelled on the basis of experimentally determined pharmacokinetic data. The model assumed no pharmacodynamic interaction between artesunate and mefloquine, but that the parasites were already resistant to mefloquine. Predictions of the model accorded well with the data. In particular, in accordance with clinical observations, the model showed that monotherapy with either drug failed to cure at moderate parasitaemia, yet such patients could be treated effectively with the combination of 3 days of artesunate + mefloquine. For high levels of parasitaemia, 5 days of artesunate+mefloquine were needed. Simulations were also performed for situations of lower resistance to mefloquine and for the immune human populations found in Africa. The importance of mathematical modelling of combination therapy is borne out by this study and suggests its wider application for other drug combinations.

Treatment protocols for the chemotherapy of malaria are usually acquired through clinical trials. Once pharmacokinetic and pharmacodynamic information becomes available, it is possible to use mathematical modelling for testing these protocols and, possibly, for improving them. In this report the case of monotherapy by mefloquine is analysed. Published pharmacokinetic and clinical results are used to derive the essential model parameters such as kill rate, parasite growth rates, drug sensitivity and the pharmacokinetic parameters. Good agreement is obtained between clinical results and simulated parasite numbers using the derived parameters. It is demonstrated that the 2 exponential kinetics of mefloquine elimination can be reduced to an operational single exponent for pharmacodynamic modelling by educated choice of sampling times of plasma drug concentration. It is deduced that a second drug dose, at a properly chosen time-interval, results in radical cure even when resistant parasites are present and at maximal parasite growth rates such as those found in non-immune patients. Finally, a table is provided for guiding the optimal choice of dosing intervals under different values of population pharmacokinetics, drug resistance and individual immunity parameters.