Barbara M. Kotecka

Mutations in Plasmodium falciparum Cytochrome b That Are Associated with Atovaquone Resistance Are Located at a Putative Drug-Binding Site

ABSTRACT Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc1 (CYTbc1) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25- to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYTb.

Simple In Vitro Assay for Determining the Sensitivity of Plasmodium vivax Isolates from Fresh Human Blood to Antimalarials in Areas where P. vivax Is Endemic

ABSTRACT The aim of this study was to develop a simple, field-practical, and effective in vitro method for determining the sensitivity of fresh erythrocytic Plasmodium vivax isolates to a range of antimalarials. The method used is a modification of the standard World Health Organization (WHO) microtest for determination of P. falciparum drug sensitivity. The WHO method was modified by removing leukocytes and using a growth medium supplemented with AB+ serum. We successfully carried out 34 in vitro drug assays on 39 P. vivax isolates collected from the Mae Sod malaria clinic, Tak Province, Thailand. The mean percentage of parasites maturing to schizonts (six or more merozoites) in control wells was 66.5% ± 5.9% (standard deviation). This level of growth in the control wells enabled rapid microscopic determination (5 min per isolate per drug) of the MICs of chloroquine, dihydroartemisinin, WR238605 (tafenoquine), and sulfadoxine. P. vivax was relatively sensitive to chloroquine (MIC = 160 ng/ml, 50% inhibitory concentration [IC50] = 49.8 ng/ml) and dihydroartemisinin (MIC = 0.5 ng/ml, IC50 = 0.47 ng/ml). The poor response of P. vivax to both tafenoquine (MIC = 14,000 ng/ml, IC50 = 9,739 ng/ml) and sulfadoxine (MIC = 500,000 ng/ml, IC50 = 249,000 ng/ml) was due to the slow action of these drugs and the innate resistance of P. vivax to sulfadoxine. The in vitro assay developed in our study should be useful both for assessing the antimalarial sensitivity of P. vivax populations and for screening new antimalarials in the absence of long-term P. vivax cultures.

First Assessment in Humans of the Safety, Tolerability, Pharmacokinetics, and Ex Vivo Pharmacodynamic Antimalarial Activity of the New Artemisinin Derivative Artemisone

ABSTRACT 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 (t1/2) 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 artemisones short t1/2, 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 artemisinins, artemisones t1/2 is well suited for artemisinin-based combination therapy for the treatment of P. falciparum malaria.

Inhibition of Taurocholate and Ouabain Transport in Isolated Rat Hepatocytes by Cyclosporin A

The use of cyclosporin A in transplantation procedures has been reported to cause hepatotoxicity as evidenced by elevated serum bilirubin and bile salt levels. However, these biochemical abnormalities could also result from interference with hepatic transport processes. This possibility was investigated in the present study in which the effect of cyclosporin A on transport processes was examined in isolated rat liver cells. Taurocholate, ouabain, and alpha-aminoisobutyric acid were selected as compounds known to enter liver cells by distinct active transport systems and cadmium was selected as a substance taken up by a combination of simple and facilitated diffusion. Cyclosporin A was found to cause a dose-related inhibition of both taurocholate and ouabain uptake. On the other hand, the uptake of alpha-aminoisobutyric acid and of cadmium were unaffected by cyclosporin A. These findings indicate a substrate-specific effect of cyclosporin A rather than a general effect on cellular transport. Efflux of taurocholate from preloaded hepatocytes was also inhibited by cyclosporin A. Cyclosporin A caused a decrease in maximum velocity for ouabain uptake with no change in Km. Kinetic analysis for both uptake and efflux of taurocholate showed an unchanged maximum velocity and an increased Km. The data indicate that the ability of liver cells to take up and release bile acids is impaired in the presence of cyclosporin A. These findings provide a possible explanation for the finding of increased serum bile acids during cyclosporin A therapy and suggest that hepatic clearance of other compounds could also be impaired.

Evaluation of artemisone combinations in Aotus monkeys infected with Plasmodium falciparum.

ABSTRACT Artemisone (single oral dose, 10 mg/kg of body weight) cured nonimmune Aotus monkeys of their Plasmodium falciparum infections when combined with mefloquine (single oral dose, 5 and 10 mg/kg but not 2.5 mg/kg). In combination with amodiaquine (20 mg/kg/day), artemisone (10 mg/kg/day) given orally for 3 days cured all infected monkeys. Three days of treatment with artemisone (30 mg/kg/day) and clindamycin (100 mg/kg/day) was also curative.

Plasma chloroquine concentrations in young and older malaria patients treated with chloroquine

Plasma chloroquine (CQ) concentrations were measured by bioassay in young (0-4 years, n = 9) and older (5-60 years, n = 21) patients from Vanuatu infected with malaria following treatment with 25 mg/kg CQ over 3 days. CQ concentrations in young children tended to be lower than in older patients at days 2, 3, 4 and 7 after onset of treatment, with no drug present in two young children on day 3 and in one child on day 7. The greater difficulty experienced by young children to ingest all of their prescribed medication could have contributed to the lower CQ concentrations observed in the younger age group. The possibility that sub-therapeutic CQ concentrations are responsible for treatment failures in young children should be considered in areas where a high degree of CQ resistance has not yet been established. In such areas, the presence or prevalence of CQ-resistant infections should not be based on treatment failures observed in young children unless it can be confirmed that adequate blood CQ concentrations were achieved after treatment.

Chloroquine bioassay of plasma specimens obtained from soldiers on chloroquine plus doxycycline for malaria prophylaxis

In this study we describe the application of a bioassay for measuring chloroquine equivalent concentrations in plasma samples obtained from soldiers on chloroquine (300 mg weekly) and doxycycline (50 or 100 mg daily) for malaria prophylaxis. Chloroquine, its principal metabolite monodesethylchloroquine and doxycycline were also measured by high performance liquid chromatography (HPLC). Physiological concentrations of doxycycline did not interfere with the measurement of chloroquine equivalent concentrations. The correlation between bioassay and HPLC was rs = 0.88, with a median bioassay/HPLC chloroquine concentration ration of 1.1 (range 0.6-2.4, n = 26). The bioassay is a valuable method, particularly under field conditions, for measuring chloroquine concentrations and can be very helpful in distinguishing drug failures from either poor compliance or inadequate drug absorption.

In vivo-in vitro model to assess chloroquine activity in monkeys.

The bioassay technique and high-performance liquid chromatography (HPLC) method were used to establish chloroquine (CQ) concentration equivalents in serum samples collected from Aotus and Saimiri monkeys after administration of CQ. The results indicate some differences in CQ metabolism between the two simian species. They also indicate a strong positive relationship (rs = 0.96) between data obtained by the bioassay and HPLC technique. The findings suggest that the use of the bioassay in these small primates, using only a fraction of the serum required for HPLC analysis, should provide a useful mean for obtaining preliminary information about the degree and duration of serum antimalarial activity of promising experimental drugs. Apart from reducing the number of monkeys required for drug evaluation, this in vivo-in vitro model should also decrease the overall cost and duration of developing new antimalarial agents.