Treatment of Murine Cerebral Malaria by Artemisone in Combination with Conventional Antimalarial Drugs: Antiplasmodial Effects and Immune Responses

The decreasing effectiveness of antimalarial therapy due to drug resistance necessitates constant efforts to develop new drugs. Artemisinin derivatives are the most recent drugs that have been introduced and are considered the first line of treatment, but there are already indications of Plasmodium falciparum resistance to artemisinins. Consequently, drug combinations are recommended for prevention of the induction of resistance. The research here demonstrates the effects of novel combinations of the new artemisinin derivative, artemisone, a recently described 10-alkylamino artemisinin derivative with improved antimalarial activity and reduced neurotoxicity. We here investigate its ability to kill P. falciparum in a high-throughput in vitro assay and to protect mice against lethal cerebral malaria caused by Plasmodium berghei ANKA when used alone or in combination with established antimalarial drugs. Artemisone effects against P. falciparum in vitro were synergistic with halofantrine and mefloquine, and additive with 25 other drugs, including chloroquine and doxycycline. The concentrations of artemisone combinations that were toxic against THP-1 cells in vitro were much higher than their effective antimalarial concentration. Artemisone, mefloquine, chloroquine, or piperaquine given individually mostly protected mice against cerebral malaria caused by P. berghei ANKA but did not prevent parasite recrudescence. Combinations of artemisone with any of the other three drugs did completely cure most mice of malaria. The combination of artemisone and chloroquine decreased the ratio of proinflammatory (gamma interferon, tumor necrosis factor) to anti-inflammatory (interleukin 10 [IL-10], IL-4) cytokines in the plasma of P. berghei-infected mice. Thus, artemisone in combinations with other antimalarial drugs might have a dual action, both killing parasites and limiting the potentially deleterious host inflammatory response.     

Comparative Ex Vivo Activity of Novel Endoperoxides in Multidrug-Resistant Plasmodium falciparum and P. vivax

The declining efficacy of artemisinin derivatives against Plasmodium falciparum highlights the urgent need to identify alternative highly potent compounds for the treatment of malaria. In Papua Indonesia, where multidrug resistance has been documented against both P. falciparum and P. vivax malaria, comparative ex vivo antimalarial activity against Plasmodium isolates was assessed for the artemisinin derivatives artesunate (AS) and dihydroartemisinin (DHA), the synthetic peroxides OZ277 and OZ439, the semisynthetic 10-alkylaminoartemisinin derivatives artemisone and artemiside, and the conventional antimalarial drugs chloroquine (CQ), amodiaquine (AQ), and piperaquine (PIP). Ex vivo drug susceptibility was assessed in 46 field isolates (25 P. falciparum and 21 P. vivax). The novel endoperoxide compounds exhibited potent ex vivo activity against both species, but significant differences in intrinsic activity were observed. Compared to AS and its active metabolite DHA, all the novel compounds showed lower or equal 50% inhibitory concentrations (IC(50)s) in both species (median IC(50)s between 1.9 and 3.6 nM in P. falciparum and 0.7 and 4.6 nM in P. vivax). The antiplasmodial activity of novel endoperoxides showed different cross-susceptibility patterns in the two Plasmodium species: whereas their ex vivo activity correlated positively with CQ, PIP, AS, and DHA in P. falciparum, the same was not apparent in P. vivax. The current study demonstrates for the first time potent activity of novel endoperoxides against drug-resistant P. vivax. The high activity against drug-resistant strains of both Plasmodium species confirms these compounds to be promising candidates for future artemisinin-based combination therapy (ACT) regimens in regions of coendemicity.     

Ex vivo drug susceptibility of ferroquine against chloroquine-resistant isolates of Plasmodium falciparum and P. vivax

Ferroquine (FQ; SSR97193), a ferrocene-containing 4-aminoquinoline derivate, has potent in vitro efficacy against chloroquine (CQ)-resistant Plasmodium falciparum and CQ-sensitive P. vivax. In the current study, ex vivo FQ activity was tested in multidrug-resistant P. falciparum and P. vivax field isolates using a schizont maturation assay. Although FQ showed excellent activity against CQ-sensitive and -resistant P. falciparum and P. vivax (median 50% inhibitory concentrations [IC(50)s], 9.6 nM and 18.8 nM, respectively), there was significant cross-susceptibility with the quinoline-based drugs chloroquine, amodiaquine, and piperaquine (for P. falciparum, r = 0.546 to 0.700, P < 0.001; for P. vivax, r = 0.677 to 0.821, P < 0.001). The observed ex vivo cross-susceptibility is likely to reflect similar mechanisms of drug uptake/efflux and modes of drug action of this drug class. However, the potent activity of FQ against resistant isolates of both P. falciparum and P. vivax highlights a promising role for FQ as a lead antimalarial against CQ-resistant Plasmodium and a useful partner drug for artemisinin-based combination therapy.     

Assessment of the Induction of Dormant Ring Stages in Plasmodium falciparum Parasites by Artemisone and Artemisone Entrapped in Pheroid Vesicles In Vitro

The in vitro antimalarial activities of artemisone and artemisone entrapped in Pheroid vesicles were compared, as was their ability to induce dormancy in Plasmodium falciparum. There was no increase in the activity of artemisone entrapped in Pheroid vesicles against multidrug-resistant P. falciparum lines. Artemisone induced the formation of dormant ring stages similar to dihydroartemisinin. Thus, the Pheroid delivery system neither improved the activity of artemisone nor prevented the induction of dormant rings.     

First assessment in humans of the safety, tolerability, pharmacokinetics, and ex vivo pharmacodynamic antimalarial activity of the new artemisinin derivative artemisone

In preclinical studies, artemisone (BAY 44-9585), a new artemisinin derivative, was shown to possess enhanced efficacy over artesunate, and it does not possess the neurotoxicity characteristic of the current artemisinins. In a phase I program with double-blind, randomized, placebo-controlled, single and multiple ascending oral-dose studies, we evaluated the safety, tolerability, pharmacokinetics, and ex vivo pharmacodynamic antimalarial activity of artemisone. Single doses (10, 20, 30, 40, and 80 mg) and multiple doses (40 and 80 mg daily for 3 days) of artemisone were administered orally to healthy subjects. Plasma concentrations of artemisone and its metabolites were measured by liquid chromatography/tandem mass spectrometry (LC/MS-MS). Artemisone was well tolerated, with no serious adverse events and no clinically relevant changes in laboratory and vital parameters. The pharmacokinetics of artemisone over the 10- to 80-mg range demonstrated dose linearity. After the single 80-mg dose, artemisone had a geometric mean maximum concentration of 140.2 ng/ml (range, 86.6 to 391.0), a short elimination half-life (t_1/2) of 2.79 h (range, 1.56 to 4.88), a high oral clearance of 284.1 liters/h (range, 106.7 to 546.7), and a large volume of distribution of 14.50 liters/kg (range, 3.21 to 51.58). Due to artemisone's short t_1/2, its pharmacokinetics were comparable after single and multiple dosing. Plasma samples taken after multiple dosing showed marked ex vivo pharmacodynamic antimalarial activities against two multidrug-resistant Plasmodium falciparum lines. Artemisone equivalent concentrations measured by bioassay revealed higher activity than artemisone measured by LC/MS-MS, confirming the presence of active metabolites. Comparable to those of other artemisinin's, artemisone's t_1/2 is well suited for artemisinin-based combination therapy for the treatment of P. falciparum malaria.     

Ferrocene-chloroquine analogues as antimalarial agents: in vitro activity of ferrochloroquine against 103 Gabonese isolates of Plasmodium falciparum

The in vitro activities of ferrochloroquine, chloroquine, quinine, mefloquine, halofantrine, amodiaquine, primaquine, atovaquone and artesunate were evaluated against Plasmodium falciparum isolates from children with uncomplicated malaria from Libreville (Gabon), using an isotopic, micro, drug susceptibility test. The IC(50) values for ferrochloroquine were in the range 0.43-30.9 nM and the geometric mean IC(50) for the 103 isolates was 10.8 nM (95% CI 8.6-13.5 nM), while the geometric means for chloroquine, quinine, mefloquine, amodiaquine and primaquine were 370 nM, 341 nM, 8.3 nM, 18.1 nM and 7.6 microM, respectively. Ferrochloroquine was active against P. falciparum isolates, 95% of which showed in vitro resistance to chloroquine. Weak positive significant correlations were observed between the responses to ferrochloroquine and that to chloroquine, amodiaquine and quinine, but too low to suggest cross-resistance. There was no significant correlation between the response to ferrochloroquine and those to mefloquine, halofantrine, primaquine, atovaquone or artesunate. Ferrochloroquine may be an important alternative drug for the treatment of chloroquine-resistant malaria.     

New quinoline di-Mannich base compounds with greater antimalarial activity than chloroquine, amodiaquine, or pyronaridine.

We have compared the ex vivo antimalarial activity of 12 new quinoline di-Mannich base compounds containing the 7-dichloroquinoline or 7-trifluoromethylquinoline nucleus with amodiaquine, chloroquine, and pyronaridine using the Saimiri-bioassay model. Each compound was administered orally (30 mg/kg of body weight) to three or more noninfected Saimiri sciureus monkeys, and serum samples were collected at various times after drug administration and serially diluted with drug-free (control) serum. In vitro activity against the multidrug-resistant K1 isolate of Plasmodium falciparum was determined in serum samples by measuring the maximum inhibitory dilution at which the treated monkey serum inhibited schizont maturation in vitro. Of the 12 Mannich bases tested, 8 were associated with levels of ex vivo antimalarial activity in serum greater than those of amodiaquine, chloroquine, or pyronaridine 1 to 7 days after drug administration. Further studies were carried out with four of these compounds, and the results showed that the areas under the serum drug concentration-time curves for the four compounds were between 7- and 26-fold greater than that obtained for pyronaridine. Activity against four multidrug-resistant strains of P. falciparum was also much greater in serum samples collected from monkeys after administration of these four compounds than in serum samples collected after administration of pyronaridine or chloroquine. These findings suggest that these four quinoline Mannich base compounds possess a very marked and prolonged antimalarial activity and that further studies should be performed to determine their value as antimalarial drugs.     

In vitro activities of antibiotics against Plasmodium falciparum are inhibited by iron

The in vitro activities of cyclines (tetracycline, doxycycline, minocycline, oxytetracycline, and rolitetracycline), macrolides (erythromycin, spiramycin, roxithromycin, and lincomycin), quinolones (norfloxacin and ofloxacin), rifampin, thiamphenicol, tobramycin, metronidazole, vancomycin, phosphomycin, and cephalosporins (cephalexin, cefaclor, cefamandole, cefuroxime, ceftriazone, cefotaxime, and cefoxitin) were evaluated on Plasmodium falciparum clones, using an isotopic, micro-drug susceptibility test. Only tetracyclines, macrolides, quinolones, and rifampin demonstrated in vitro activity against P. falciparum, which increased after a prolonged exposure (96 or 144 h). In the presence of iron (FeCl(3)), only the activities of tetracyclines and norfloxacin were decreased. Their in vitro activity against intraerythrocytic stages of multidrug-resistant P. falciparum and their efficacy in vivo favor the use of antibiotics as antimalarial drugs. However, due to their slow antimalarial action and to the fact that they act better after prolonged contact, they probably need to be administered in conjunction with a rapidly acting antimalarial drug, such as a short course of chloroquine or quinine.     

Potentiation of chloroquine activity against Plasmodium falciparum by the peroxidase-hydrogen peroxide system.

In this study, we examined the potential interactions between antimalarial (chloroquine, quinine, and mefloquine) and oxidant reagents. The data indicate that their effects enhance those of one another in vitro. The viability of Plasmodium falciparum in culture was assessed by [3H]hypoxanthine incorporation during 24 h of incubation in the presence of lactoperoxidase, glucose-glucose oxidase, hydrogen peroxide, chloroquine, quinine, and mefloquine, either alone or in combination. At subinhibitory concentrations, a significant inhibition was produced by the following combinations: lactoperoxidase plus hydrogen peroxide, lactoperoxidase plus glucose-glucose oxidase, lactoperoxidase plus hydrogen peroxide or glucose-glucose oxidase plus chloroquine or quinine but not with mefloquine. Deletion of any component from the system markedly decreased the toxic effect on P. falciparum. This toxic effect was not inhibited by catalase. These results indicate that the peroxidase-hydrogen peroxide system and antimalarial drugs can potentiate each other to inhibit the growth of P. falciparum.     

Ex Vivo Activity of Endoperoxide Antimalarials,Including Artemisone and Arterolane,against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

Novel synthetic endoperoxides are being evaluated as new components of artemisinin combination therapies (ACTs) to treat artemisinin-resistant Plasmodium falciparum malaria. We conducted blinded ex vivo activity testing of fully synthetic (OZ78 and OZ277) and semisynthetic (artemisone, artemiside, artesunate, and dihydroartemisinin) endoperoxides in the histidine-rich protein 2 enzyme-linked immunosorbent assay against 200 P. falciparum isolates from areas of artemisinin-resistant malaria in western and northern Cambodia in 2009 and 2010. The order of potency and geometric mean (GM) 50% inhibitory concentrations (IC₅₀s) were as follows: artemisone (2.40 nM) > artesunate (8.49 nM) > dihydroartemisinin (11.26 nM) > artemiside (15.28 nM) > OZ277 (31.25 nM) > OZ78 (755.27 nM). Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC₅₀ susceptibility ratios of <2.0. All isolates had P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 (pfmdr1) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC₅₀s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance reported in the literature and pfmdr1 mutations should be examined for their combined contributions to emerging ACT resistance.     

Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique.

A rapid, semiautomated microdilution method was developed for measuring the activity of potential antimalarial drugs against cultured intraerythrocytic asexual forms of the human malaria parasite Plasmodium falciparum. Microtitration plates were used to prepare serial dilutions of the compounds to be tested. Parasites, obtained from continuous stock cultures, were subcultured in these plates for 42 h. Inhibition of uptake of a radiolabeled nucleic acid precursor by the parasites served as the indicator of antimalarial activity. Results of repeated measurements of activity with chloroquine, quinine, and the investigational new drug mefloquine demonstrated that the method is sensitive and precise. Several additional antimalarial drugs and compounds of interest were tested in vitro, and the results were consistent with available in vivo data. The use of P. falciparum isolates with known susceptibility to antimalarial drugs also permitted evaluation of the cross-resistance potential of each compound tested. The applications and expectations of this new test system within a drug development program are discussed.     

Determinants of in vitro drug susceptibility testing of Plasmodium vivax

In Papua, Indonesia, the antimalarial susceptibility of Plasmodium vivax (n = 216) and P. falciparum (n = 277) was assessed using a modified schizont maturation assay for chloroquine, amodiaquine, artesunate, lumefantrine, mefloquine, and piperaquine. The most effective antimalarial against P. vivax and P. falciparum was artesunate, with geometric mean 50% inhibitory concentrations (IC50s) (95% confidence intervals [CI]) of 1.31 nM (1.07 to 1.59) and 0.64 nM (0.53 to 0.79), respectively. In contrast, the geometric mean chloroquine IC50 for P. vivax was 295 nM (227 to 384) compared to only 47.4 nM (42.2 to 53.3) for P. falciparum. Two factors were found to significantly influence the in vitro drug response of P. vivax: the initial stage of the parasite and the duration of the assay. Isolates of P. vivax initially at the trophozoite stage had significantly higher chloroquine IC50s (478 nM [95% CI, 316 to 722]) than those initially at the ring stage (84.7 nM [95% CI, 45.7 to 157]; P < 0.001). Synchronous isolates of P. vivax and P. falciparum which reached the target of 40% schizonts in the control wells within 30 h had significantly higher geometric mean chloroquine IC50s (435 nM [95% CI, 169 to 1,118] and 55.9 nM [95% CI, 48 to 64.9], respectively) than isolates that took more than 30 h (39.9 nM [14.6 to 110.4] and 36.9 nM [31.2 to 43.7]; P < 0.005). The results demonstrate the marked stage-specific activity of chloroquine with P. vivax and suggest that susceptibility to chloroquine may be associated with variable growth rates. These findings have important implications for the phenotypic and downstream genetic characterization of P. vivax.     

The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand

On the western border of Thailand, Plasmodium falciparum has become resistant to almost all antimalarial agents. The molecular basis of resistance in these parasite populations has not been well characterized. This study assessed genetic polymorphisms in the pfmdr1 gene in 54 parasites collected from the western border of Thailand to determine the relationship of pfmdr1 copy number and codon mutations with parasite sensitivities to mefloquine, chloroquine, halofantrine, quinine, and artesunate assessed in vitro. A point mutation at codon 86 (resulting in a change of Asn to Tyr) was associated with a significantly lower 50% inhibitory concentration (IC(50)) of mefloquine (median, 9 ng/ml versus 52.4 ng/ml; P = 0.003). Overall 35% of the isolates (19 of 54) had an increase in pfmdr1 copy number, and all 19 carried the wild-type allele at codon 86. Increased pfmdr1 copy number was associated with higher IC(50)s of mefloquine (P = 0.04) and artesunate (P = 0.005), independent of polymorphism at codon 86. The relationship between pfmdr1 and resistance to structurally distinct antimalarial agents confirms the presence of a true multidrug-resistant phenotype.     

Gametocytocidal activity and synergistic interactions of riboflavin with standard antimalarial drugs against growth of Plasmodium falciparum in vitro

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.     

Ligands of the peripheral benzodiazepine receptor are potent inhibitors of Plasmodium falciparum and Toxoplasma gondii in vitro

The increase in resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands the development of new antimalarial agents. In this quest, we have found that ligands to the peripheral benzodiazepine receptor such as flurazepam, an agonist of the benzodiazepine family, and PK11195, an antagonist derived from isoquinoline, were active against Plasmodium falciparum. These two compounds effectively and rapidly inhibited parasite growth in vitro, irrespective of parasite resistance to chloroquine and mefloquine. Treatment with both drugs induced a sharp and consistent decline in parasitemia, a complete inhibition of parasite replication, and the destruction of parasites within the host red blood cells. Using electron microscopy, we showed that dramatic morphological changes, involving swollen endoplasmic reticulum and the reduction of hemozoin, were consistent with parasite death. The potent activities of flurazepam and PK11195 were also evaluated for antagonist or synergistic effects with currently used antimalarial drugs such as chloroquine and mefloquine. Moreover, flurazepam was found to be active against Toxoplasma gondii, another member of the phylum Apicomplexa. Taken together, our results indicated that benzodiazepines could be considered promising candidates in the treatment of both malaria and toxoplasmosis.     

In vivo and in vitro antimalarial properties of azithromycin-chloroquine combinations that include the resistance reversal agent amlodipine

Evidence of emerging Plasmodium falciparum resistance to artemisinin-based combination therapies, documented in western Cambodia, underscores the continuing need to identify new antimalarial combinations. Given recent reports of the resurgence of chloroquine-sensitive P. falciparum parasites in Malawi, after the enforced and prolonged withdrawal of this drug, and indications of a possible synergistic interaction with the macrolide azithromycin, we sought to further characterize chloroquine-azithromycin combinations for their in vitro and in vivo antimalarial properties. In vitro 96-h susceptibility testing of chloroquine-azithromycin combinations showed mostly additive interactions against freshly cultured P. falciparum field isolates obtained from Mali. Some evidence of synergy, however, was apparent at the fractional 90% inhibitory concentration level. Additional in vitro testing highlighted the resistance reversal properties of amlodipine for both chloroquine and quinine. In vivo experiments, using the Peters 4-day suppressive test in a P. yoelii mouse model, revealed up to 99.9% suppression of parasitemia following treatment with chloroquine-azithromycin plus the R enantiomer of amlodipine. This enantiomer was chosen because it does not manifest the cardiac toxicities observed with the racemic mixture. Pharmacokinetic/pharmacodynamic analyses in this rodent model and subsequent extrapolation to a 65-kg adult led to the estimation that 1.8 g daily of R-amlodipine would be required to achieve similar efficacy in humans, for whom this is likely an unsafe dose. While these data discount amlodipine as an additional partner for chloroquine-based combination therapy, our studies continue to support azithromycin as a safe and effective addition to antimalarial combination therapies.     

New ways of treating and preventing malaria: mefloquine (Lariam)

Mefloquine--a quinolinemethanol--opens new aspects in the therapy and prophylaxis of malaria. Since this drug will be soon available in Austria, a review of the pharmacological and clinical data is given. Preliminary results of studies dealing with resistance induction against mefloquine showed that Plasmodium falciparum can develop resistance to this substance in vivo as well as in vitro. Hence, some guidelines are given, in particular with regard to the use of mefloquine as a prophylactic drug. It should be used only in malaria endemic zones in which highly chloroquine resistant Plasmodium falciparum is prevalent and the duration of prophylactic administration should be limited to 3 months in order to prevent the development of resistance. However, mefloquine has proved to be a valuable new drug, well tolerated in clinical trials and with pharmacological properties that make its use simple and safe.     

Therapeutic responses to different antimalarial drugs in vivax malaria

The therapeutic responses to the eight most widely used antimalarial drugs were assessed in 207 adult patients with Plasmodium vivax malaria. This parasite does not cause marked sequestration, so parasite clearance can be used as a direct measure of antimalarial activity. The activities of these drugs in descending order were artesunate, artemether, chloroquine, mefloquine, quinine, halofantrine, primaquine, and pyrimethamine-sulfadoxine (PS). Therapeutic responses to PS were poor; parasitemias did not clear in 5 of the 12 PS-treated patients, whereas all the other patients made an initial recovery. Of 166 patients monitored for > or =28 days, 35% had reappearance of vivax malaria 11 to 65 days later and 7% developed falciparum malaria 5 to 21 days after the start of treatment. There were no significant differences in the times taken for vivax malaria reappearance among the different groups except for those given mefloquine and chloroquine, in which all vivax malaria reappearances developed >28 days after treatment, suggesting suppression of the first relapse by these slowly eliminated drugs. There was no evidence of chloroquine resistance. The antimalarial drugs vary considerably in their intrinsic activities and stage specificities of action.     

Human malaria in immunocompromised mice: new in vivo model for chemotherapy studies

We have recently designed a new Plasmodium falciparum mouse model and documented its potential for the study of immune effector mechanisms. In order to determine its value for drug studies, we evaluated its response to existing antimalarial drugs compared to that observed in humans. Immunocompromised BXN (bg/bg xid/xid nu/nu) mice were infected with either the sensitive NF54 strain or the multiresistant T24 strain and then treated with chloroquine, quinine, mefloquine, or dihydroartemisinin. A parallelism was observed between previously reported human responses and P. falciparum-parasitized human red blood cell (huRBC)--BXN mouse responses to classical antimalarial drugs, measured in terms of speed of decrease in parasitemia and of morphological alterations of the parasites. Mice infected with the sensitive strain were successfully cured after treatment with either chloroquine or mefloquine. In contrast, mice infected with the multiresistant strain failed to be cured by chloroquine or quinine but thereafter responded to dihydroartemisinin treatment. The speed of parasite clearance and the morphological alterations induced differed for each drug and matched previously reported observations, hence stressing the relevance of the model. These data thus suggest that P. falciparum-huRBC-BXN mice can provide a valuable in vivo system and should be included in the short list of animals that can be used for the evaluation of P. falciparum responses to drugs.     

Influence of the Plasmodium falciparum P-glycoprotein homologue 1 (pfmdr1 gene product) on the antimalarial action of cyclosporin

BACKGROUND AND OBJECTIVES: The immunosuppressant cyclosporin A and a number of other cyclosporins have potent and selective antimalarial activity. Their exact mechanism of antimalarial action is unknown but the structure-activity relationships for malarial parasite inhibition and immunosuppression differ markedly. The 3'-keto derivative of cyclosporin D (valspodar) is particularly potent against the human malarial parasite Plasmodium falciparum in culture but causes negligible immunosuppression. Multidrug resistance in mammalian cancer cells, the result of overproduction of the P-glycoprotein, can be reversed by certain cyclosporins, particularly valspodar. We therefore investigated the possibility that the antimalarial target of cyclosporin might be a P-glycoprotein homologue. P. falciparum P-glycoprotein homologue 1 (Pgh1; the pfmdr1 gene product) is located in the digestive vacuole (DV) membrane of the parasite. Its function is unknown but it modulates the susceptibility of parasites to quinolines and related antimalarial drugs, including quinine, mefloquine, halofantrine and chloroquine, and to artemisinin. METHODS AND RESULTS: Here we demonstrate that (i) sequence polymorphisms in pfmdr1 altered the susceptibility of parasites to cyclosporin A and (ii) pfmdr1-overexpressing strains were slightly less susceptible to the drug. Furthermore, we found synergistic antimalarial interactions between cyclosporin A and quinine, mefloquine or halofantrine and antagonism between cyclosporin A and chloroquine. However, we were unable to detect a direct interaction between cyclosporin and Pgh1. CONCLUSIONS: The amino acid sequence and copy number of Pgh1 may influence cyclosporin susceptibility as a result of a direct interaction between the drug and the protein, or via indirect effects on the physiology of the DV.