Steve A. Ward

A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino‐acid change K76T from resistant strains leads to wild‐type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ‐resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side‐chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug–protein interactions involving this mutant residue.

Chloroquine resistance before and after its withdrawal in Kenya

BackgroundThe spread of resistance to chloroquine (CQ) led to its withdrawal from use in most countries in sub-Saharan Africa in the 1990s. In Malawi, this withdrawal was followed by a rapid reduction in the frequency of resistance to the point where the drug is now considered to be effective once again, just nine years after its withdrawal. In this report, the polymorphisms of markers associated with CQ-resistance against Plasmodium falciparum isolates from coastal Kenya (Kilifi) were investigated, from 1993, prior to the withdrawal of CQ, to 2006, seven years after its withdrawal. Changes to those that occurred in the dihydrofolate reductase gene (dhfr) that confers resistance to the replacement drug, pyrimethamine/sulphadoxine were also compared.MethodsMutations associated with CQ resistance, at codons 76 of pfcrt, at 86 of pfmdr1, and at codons 51, 59 and 164 of dhfr were analysed using PCR-restriction enzyme methods. In total, 406, 240 and 323 isolates were genotyped for pfcrt-76, pfmdr1-86 and dhfr, respectively.ResultsFrom 1993 to 2006, the frequency of the pfcrt-76 mutant significantly decreased from around 95% to 60%, while the frequency of pfmdr1-86 did not decline, remaining around 75%. Though the frequency of dhfr mutants was already high (around 80%) at the start of the study, this frequency increased to above 95% during the study period. Mutation at codon 164 of dhf r was analysed in 2006 samples, and none of them had this mutation.ConclusionIn accord with the study in Malawi, a reduction in resistance to CQ following official withdrawal in 1999 was found, but unlike Malawi, the decline of resistance to CQ in Kilifi was much slower. It is estimated that, at current rates of decline, it will take 13 more years for the clinical efficacy of CQ to be restored in Kilifi. In addition, CQ resistance was declining before the drugs official withdrawal, suggesting that, prior to the official ban, the use of CQ had decreased, probably due to its poor clinical effectiveness.

Low Levels of Pyrazinamide and Ethambutol in Children with Tuberculosis and Impact of Age, Nutritional Status, and Human Immunodeficiency Virus Infection

ABSTRACT Recent pharmacokinetic studies that included children found that serum drug levels were low compared to those of adults for whom the same dosages were used. This study aimed to characterize the pharmacokinetics of pyrazinamide and ethambutol in Malawian children and to examine the impact of age, nutritional status, and human immunodeficiency virus (HIV) infection. We conducted a pharmacokinetic study of children treated for tuberculosis with thrice-weekly pyrazinamide (n = 27; mean age, 5.7 years) and of a separate group of children treated with thrice-weekly ethambutol (n = 18; mean age, 5.5 years) as portions of tablets according to national guidelines. Malnutrition and HIV infection were common in both groups. Blood samples were taken just prior to oral administration of the first dose, and subsequent samples were taken at intervals of 2, 3, 4, 7, 24, and 48 h after drug administration. Serum drug levels were low in all children for both drugs; in almost all cases, the maximum concentration of the drug in serum (Cmax) failed to reach the MIC for Mycobacterium tuberculosis. The Cmax of pyrazinamide was significantly lower in younger children (<5 years) than in older children. The Cmax of pyrazinamide was also lower for HIV-infected children and children with severe malnutrition, but these differences did not reach statistical significance. No differences were found for ethambutol in relation to age, HIV infection, or malnutrition, but the Cmax was <2 mg/liter in all cases. Studies of pharmacokinetic parameters and clinical outcomes obtained by using higher dosages of drugs for treatment of childhood tuberculosis are needed, and recommended dosages may need to be increased.

Rapid chloroquine efflux phenotype in both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum: A correlation of chloroquine sensitivity with energy-dependent drug accumulation

Recent reports suggest that lower levels of chloroquine accumulation in chloroquine-resistant isolates of Plasmodium falciparum are achieved by energy-dependent chloroquine efflux from resistant parasites. In support of this argument, a rapid chloroquine efflux phenotype has been observed in some chloroquine-resistant isolates of P. falciparum. In this study, no relationship was found between chloroquine sensitivity and the rate of [3H]chloroquine efflux from four isolates of P. falciparum with a greater than 10-fold range in sensitivity to chloroquine. All the isolates tested displayed the rapid efflux phenotype, irrespective of sensitivity. However, chloroquine sensitivity of these isolates was correlated with energy-dependent rate of drug accumulation into these parasites. Verapamil and a variety of other compounds reverse chloroquine resistance. The reversal mechanism is assumed to result from competition between verapamil and chloroquine for efflux protein translocation sites, thus causing an increase in steady-state accumulation of chloroquine and hence a return to sensitivity. Verapamil accumulation at a steady-state is increased by chloroquine, possibly indicating competition for efflux of the two substrates. Increases in steady-state verapamil concentrations caused by chloroquine were identical in sensitive and resistant strains, suggesting that similar capacity efflux pumps may exist in these isolates. These data suggest that differences in steady-state chloroquine accumulation seen in these isolates can be attributed to changes in the chloroquine concentrating mechanism rather than the efflux pump. It seems likely that chloroquine resistance generally in P. falciparum, results at least in part from a change in the drug concentrating mechanism and that changes in efflux rates per se are insufficient to explain chloroquine resistance.

Measurement of adherence, drug concentrations and the effectiveness of artemether-lumefantrine, chlorproguanil-dapsone or sulphadoxine-pyrimethamine in the treatment of uncomplicated malaria in Malawi

BackgroundSulphadoxine-pyrimethamine (SP) is the only single dose therapy for uncomplicated malaria, but there is widespread resistance. At the time of this study, artemether-lumefantrine (AL) and chlorproguanil-dapsone (CPD), both multi-dose regimes, were considered possible alternatives to SP in Malawi. The aim of this study was to investigate the impact of poor adherence on the effectiveness of AL and CPD.MethodsChildren ≥12 months and adults with uncomplicated malaria were randomized to receive AL, CPD or SP. Adherence was measured using a questionnaire and electronic monitoring devices, MEMS™, pill bottles that recorded the date and time of opening. Day-7 plasma dapsone or lumefantrine concentrations were measured to examine their relationship with adherence and clinical response.Results841 patients were recruited. The day-28 adequate clinical and parasitological response (ACPR) rates, using intention to treat analysis (missing data treated as failure), were AL 85.2%, CPD 63.7% and SP 50%. ACPR rates for AL were higher than CPD or SP on days 28 and 42 (p ≤ 0.002 for all comparisons). CPD was more effective than SP on day-28 (p = 0.01), but not day-42.Very high adherence was reported using the questionnaire, 100% for AL treated patients and 99.2% for the CPD group. Only three CPD participants admitted missing any doses. 164/181 (90.6%) of CPD treated patients took all their doses out of the MEMS™ container and they were more likely to have a day-28 ACPR than those who did not take all their medication out of the container, p = 0.024. Only 7/87 (8%) AL treated patients did not take all of their doses out of their MEMS™ container and none had treatment failure.Median day-7 dapsone concentrations were higher in CPD treated patients with ACPR than in treatment failures, p = 0.012. There were no differences in day-7 dapsone or lumefantrine concentrations between those who took all their doses from the MEMS™ container and those who did not. A day-7 lumefantrine concentration reported to be predictive of AL treatment failure in Thailand was not useful in this population; only one of 16 participants with a concentration below this threshold (175 ng/ml) had treatment failure.ConclusionThis study provides reassurance of the effectiveness of AL, even with unsupervised dosing, as it is rolled out across sub-Saharan Africa. Self-reported adherence appears to be an unreliable measure of adherence in this population.

Variability in the metabolism of proguanil to the active metabolite cycloguanil in healthy Kenyan adults

Extensive metabolizers (EM) and poor metabolizers (PM) of the malaria chemoprophylactic drug proguanil have been identified by measuring the proguanil/cycloguanil ratio in urine following a single dose of the pro-drug. The pharmacokinetic characteristics of proguanil were similar in 8 EM and 8 PM subjects, but there were significant differences between the 2 groups with respect to cycloguanil pharmacokinetics. In none of the PM subjects could cycloguanil be detected in whole blood samples at any time after proguanil dosage. Plasma cycloguanil was measureable in only 2 of 8 PM subjects, despite an analytical sensitivity in the high-performance liquid chromatographic assay of 1 ng/ml cycloguanil. A comparatively high proportion of Black Kenyan adults appear to metabolize proguanil poorly, possibly because they lack the specific mixed function oxidase which will accept proguanil as substrate.

Population pharmacokinetics of artesunate and dihydroartemisinin following intra-rectal dosing of artesunate in malaria patients

Background Intra-rectal artesunate has been developed as a potentially life-saving treatment of severe malaria in rural village settings where administration of parenteral antimalarial drugs is not possible. We studied the population pharmacokinetics of intra-rectal artesunate and the relationship with parasitological responses in patients with moderately severe falciparum malaria. Methods and Findings Adults and children in Africa and Southeast Asia with moderately severe malaria were recruited in two Phase II studies (12 adults from Southeast Asia and 11 children from Africa) with intensive sampling protocols, and three Phase III studies (44 children from Southeast Asia, and 86 children and 26 adults from Africa) with sparse sampling. All patients received 10 mg/kg artesunate as a single intra-rectal dose of suppositories. Venous blood samples were taken during a period of 24 h following dosing. Plasma artesunate and dihydroartemisinin (DHA, the main biologically active metabolite) concentrations were measured by high-performance liquid chromatography with electrochemical detection. The pharmacokinetic properties of DHA were determined using nonlinear mixed-effects modelling. Artesunate is rapidly hydrolysed in vivo to DHA, and this contributes the majority of antimalarial activity. For DHA, a one-compartment model assuming complete conversion from artesunate and first-order appearance and elimination kinetics gave the best fit to the data. The mean population estimate of apparent clearance (CL/F) was 2.64 (l/kg/h) with 66% inter-individual variability. The apparent volume of distribution (V/F) was 2.75 (l/kg) with 96% inter-individual variability. The estimated DHA population mean elimination half-life was 43 min. Gender was associated with increased mean CL/F by 1.14 (95% CI: 0.36–1.92) (l/kg/h) for a male compared with a female, and weight was positively associated with V/F. Larger V/Fs were observed for the patients requiring early rescue treatment compared with the remainder, independent of any confounders. No associations between the parasitological responses and the posterior individual estimates of V/F, CL/F, and AUC0–6h were observed. Conclusions The pharmacokinetic properties of DHA were affected only by gender and body weight. Patients with the lowest area under the DHA concentration curve did not have slower parasite clearance, suggesting that rectal artesunate is well absorbed in most patients with moderately severe malaria. However, a number of modelling assumptions were required due to the large intra- and inter-individual variability of the DHA concentrations.

Chemosensitization of Plasmodium falciparum by Probenecid In Vitro

ABSTRACT Resistance to drugs can result from changes in drug transport, and this resistance can sometimes be overcome by a second drug that modifies the transport mechanisms of the cell. This strategy has been exploited to partly reverse resistance to chloroquine in Plasmodium falciparum. Studies with human tumor cells have shown that probenecid can reverse resistance to the antifolate methotrexate, but the potential for reversal of antifolate resistance has not been studied in P. falciparum. In the present study we tested the ability of probenecid to reverse antifolate resistance in P. falciparum in vitro. Probenecid, at concentrations that had no effect on parasite viability alone (50 μM), was shown to increase the sensitivity of a highly resistant parasite isolate to the antifolates pyrimethamine, sulfadoxine, chlorcycloguanil, and dapsone by seven-, five-, three-, and threefold, respectively. The equivalent effects against an antifolate-sensitive isolate were activity enhancements of approximately 3-, 6-, 1.2-, and 19-fold, respectively. Probenecid decreased the level of uptake of radiolabeled folic acid, suggesting a transport-based mechanism linked to folate salvage. When probenecid was tested with chloroquine, it chemosensitized the resistant isolate to chloroquine (i.e., enhanced the activity of chloroquine). This enhancement of activity was associated with increased levels of chloroquine accumulation. In conclusion, we have shown that probenecid can chemosensitize malaria parasites to antifolate compounds via a mechanism linked to reduced folate uptake. Notably, this effect is observed in both folate-sensitive and -resistant parasites. In contrast to the activities of antifolate compounds, the effect of probenecid on chloroquine sensitivity was selective for chloroquine-resistant parasites (patent P407595GB [W. P. Thompson & Co., Liverpool, United Kingdom] has been filed to protect this intellectual property).

Selective determination, in plasma, of artemether and its major metabolite, dihydroartemisinin, by high-performance liquid chromatography with ultraviolet detection

A sensitive and selective reversed-phase high-performance liquid chromatographic method for the determination of artemether and its major metabolite dihydroartemisinin in plasma has been developed. It involves extraction of plasma with dichloromethane, solid-phase separation of the two analytes and acid decomposition prior to chromatography on a C18 Spherisorb column with a mobile phase of acetonitrile-water (50:50, v/v). Run time is 30 min. The assay satisfies all of the criteria required for use in clinical pharmacokinetic studies.

The safety and kinetics of intramuscular quinine in Malawian children with moderately severe falciparum malaria

The safety and kinetics of intramuscular quinine (10 mg salt/kg every 8 h for 3 doses) were assessed in Malawian children suffering from uncomplicated falciparum malaria, who were unable to take oral antimalarial drugs. Treatment was completed with oral pyrimethamine-sulfadoxine. The mean (+/- SD) peak plasma quinine concentration after the first injection was 9.0 (+/- 2.3) micrograms/ml, at 1.1 (+/- 0.7) h. Mean plasma concentrations increased further after the second and third doses to a maximum of 11.5 (+/- 2.6) micrograms/ml at 16.1 (+/- 3.2) h. No hypotension, hypoglycaemia or electrocardiographic abnormalities developed during quinine treatment. These results provide further evidence for the safety of intramuscular quinine in children with moderately severe malaria. Plasma concentrations of alpha 1-acid glycoprotein (AGP) were higher, and the degree of protein binding of quinine was greater, in acute malaria than in convalescence. There was a significant correlation between AGP concentration and the fraction of plasma quinine bound to plasma protein. These findings suggest a role for AGP in the binding of quinine in plasma in vivo and are of interest since unbound quinine is responsible for both the efficacy and toxicity of the drug.