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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, November 2007, p. 4090-4097, Vol. 51, No. 11
0066-4804/07/$08.00+0 doi:10.1128/AAC.00486-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
International Health Division, Menzies School of Health Research and Charles Darwin University, Darwin, Northern Territory, Australia,1 Centre for Vaccinology & Tropical Medicine, Nuffield Department of Clinical Medicine, Churchill Hospital, Oxford, United Kingdom,2 National Institute of Health Research and Development, Ministry of Health, Jakarta, Indonesia,3 Menzies School of Health Research-National Institute of Health Research and Development Malaria Research Program, Timika, Indonesia,4 District Health Office, Timika, Papua, Indonesia,5 Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand,6 Public Health and Malaria Control Department, PT Freeport Indonesia, Tembagapura, Papua, Indonesia,7 International SOS, Tembagapura, Papua, Indonesia,8 Directorate General of Disease Control and Environmental Health, Ministry of Health, Jakarta, Indonesia9
Received 10 April 2007/ Returned for modification 8 July 2007/ Accepted 2 September 2007
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dihydroartemisinin-piperaquine (DHP) is a novel fixed-dose combination that is gaining a reputation as an important addition to the artemisinin combination therapy pharmacopoeia, particularly in areas with high levels of antimalarial drug resistance. The antimalarial activity of piperaquine has been recognized since the 1960s, although its widespread use in clinical practice has been restricted mainly to the People's Republic of China, where it replaced chloroquine in the national malaria control program in 1978 (4), and more recently Vietnam. In the last 5 years there has been an evolving body of evidence demonstrating excellent efficacy of DHP against multidrug-resistant strains of P. falciparum (1, 5, 7, 12, 15, 19, 22) and P. vivax (7, 15).
In Papua, Indonesia, where drug resistance has emerged in both P. falciparum and P. vivax (16), we have recently conducted two clinical drug studies with DHP. In both studies, the combination was found to be well tolerated and more effective at reducing recurrent malaria within 42 days than the comparator treatment arms (amodiaquine plus artesunate or artemether-lumefantrine) (7, 15). In this paper we have pooled these studies and incorporated measures of plasma piperaquine concentrations to assess the clinical and pharmacological parameters associated with the therapeutic response.
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MATERIALS AND METHODS |
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Study design. These prospective open-label randomized comparative trials administered DHP for the treatment of infections with P. falciparum or P. vivax in children and adults with uncomplicated symptomatic malaria. The studies were based on the 2003 WHO in vivo antimalarial drug sensitivity protocol, modified to include mixed infections and any level of parasitemia. Patients were followed for 42 days using a standardized drug efficacy record form.
Patients. Patients with slide-confirmed malaria (P. falciparum, P. vivax, or mixed infections) and fever or a history of fever during the 48 h preceding presentation to the outpatient clinic were eligible for enrolment. Pregnant or lactating women and children under 5 kg were excluded, as were patients with WHO danger signs or signs of severity, a parasitemia of >4% of infected red blood cells, or concomitant disease requiring hospital admission.
Study procedures. After enrolment a standardized data sheet was completed recording demographic information, details of symptoms and their duration, and the history of previous antimalarial medication. Clinical examination findings were documented, including the axillary temperature, which was measured using a digital thermometer. Venous blood was taken for blood film, hematocrit, hemoglobin, and white cell count. Parasite counts were determined on Giemsa-stained thick films as the number of parasites per 200 white blood cells, and peripheral parasitemia was calculated assuming a white cell count of 7,300 µl–1. Hemoglobin level was measured using a battery-operated portable photometer (Hb201+; HemoCue, Angelholm, Sweden). Blood spots on filter paper (Whatman; BFC 1802) were also collected on day 0.
DHP (Artekin; Holley Pharmaceutical Co., People's Republic of China; containing 40 mg dihydroartemisinin and 320 mg piperaquine) was administered as a weight-per-dose regimen of 2.25 and 18 mg/kg of body weight per dose of dihydroartemisinin and piperaquine, respectively, rounded up to the nearest half tablet. Children under 9 kg were dosed with a suspension made by crushing one tablet in 5 ml of water (i.e., 8 mg DHA and 64 mg piperaquine per ml). Tablets for both studies came from the same batch, which was manufactured in February 2004, with an expiry date of February 2007. All doses were supervised and administered on admission and 24 and 48 h after admission. When drug administration was observed and vomiting occurred within 60 min, administration of the full dose was repeated. Primaquine (0.3 mg of base/kg of body weight for 14 days) was administered unsupervised to those individuals with P. vivax infection or mixed infection.
Patients were examined daily after enrolment until they became afebrile and aparasitemic. At each visit a blood smear was taken and a symptom questionnaire completed. Patients were then seen weekly for 6 weeks. At each clinic appointment a full physical examination was performed, the symptom questionnaire was completed, and blood was taken to check for parasite count and hemoglobin. Blood spots on filter paper were also collected on the day of failure. On days 7 and 28, 10 ml of blood was collected from patients who agreed to venipuncture. Samples were spun down within 2 h and stored at –80°C until processing. Plasma piperaquine concentrations were determined by high-performance liquid chromatography with UV detection (11); the lower level of quantification was 2.5 ng/ml, with an interassay coefficient of variation of 8.4% at 20 ng/ml.
Statistical analysis.
Data were double entered and validated using EpiData 3.02 software (EpiData Association, Odense, Denmark), and analysis was performed using SPSS for Windows (version 15; SPSS Inc., Chicago, IL). The Mann-Whitney U test, Kruskal-Wallis method, or Spearman's rank correlation was used for nonparametric comparisons, and Student's t test or one-way analysis of variance was used for parametric comparisons. Proportions were examined using 
2 with Yates' correction or Fisher's exact test. The influence of the date of enrolment on treatment efficacy was investigated by categorizing patients according to four 4-month periods over the 16-month period of the study.
Efficacy end points were assessed using survival analysis. All patients meeting the enrolment criteria were included in the evaluable population. Results for anyone failing to complete follow-up were censored on the last day of follow-up and were regarded as not representing a treatment failure. In patients with P. falciparum alone or mixed infections in both the initial and recurrent parasitemia, reinfections and recrudescent infections were determined by PCR according to polymorphisms in MSP-1, MSP-2, and GLURP as described previously (3). Failure rates were defined by the cumulative incidence at day 42 calculated by the Kaplan-Meier method. The risks of treatment failure were compared by the Mantel-Haenszel log rank test, and the hazard ratio (HR) was presented. In the multivariable analysis any variables found to be associated significantly with the dependent variable in univariate analysis were entered into a Cox regression model and the model was constructed using all factors.
Since the relationship of the therapeutic response and plasma concentrations of antimalarial drugs is nonlinear (23), plasma piperaquine concentrations on day 7 were dichotomized using the receiver operator curve, and the optimal cutoff was defined as the maximum value of Youden's index (sensitivity plus specificity minus 1). The terminal elimination half-life was derived from the elimination rate constant, calculated from plasma piperaquine concentrations on day 7 and day 28 by log-linear interpolation.
Ethics. The study was approved by the ethics committee of the National Institute of Health Research and Development, Indonesian Ministry of Health (Jakarta, Indonesia), the ethics committee of the Menzies School of Health Research (Darwin, Australia), and the Oxford Tropical Research Ethics Committee (United Kingdom). Written informed consent was obtained from adult patients and parents of enrolled children. The trials were registered with the clinical trial website (http://www.clinicaltrials.gov/ct) as NCT 00157833 and NCT 00157885.
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RESULTS |
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Clinical risk factors for treatment failure. Four univariate factors on admission were associated with treatment failure: the species of the initial infection, being an indigenous Papuan (HR = 4.7; 95% CI: 1.7 to 13; P = 0.003), having diarrhea at presentation (HR = 1.9; 95% CI: 1.1 to 3.1; P = 0.014), and prior treatment for malaria within the preceding month (HR = 1.7; 95% CI: 1.0 to 2.9; P = 0.039). The overall cumulative risk of failure was 10.1% (95% CI: 6.2 to 14.0%) in patients presenting initially with P. falciparum infections, compared to 19.4% (95% CI: 12 to 26.8%) in patients infected with P. vivax and 27.3% (95% CI: 16.2 to 38.5%) in those with mixed infections; the overall P value was <0.001. In a multivariable model including all patients enrolled, after stratifying by the initial species of infection, the only significant risk factor for treatment failure was Papuan ethnicity (adjusted hazards ratio [AHR] = 5.0; 95% CI: 1.8 to 14; P = 0.002). The risk of recurrent parasitemia with P. vivax was 7.3% (95% CI: 4.4 to 10.2%) in children under 5 years old, compared to 17.0% (95% CI: 7.6 to 26.4%) in older children and adults (HR = 2.5; 95% CI: 1.2 to 5.1; P = 0.012). There was no significant difference in the cumulative risk of reappearance for P. falciparum between age groups. The risk of failure did not differ with sex, vomiting of medication, or baseline parasitemia. And there was not a significant difference or discernible trend in either the cumulative risk of treatment failure at day 42 or the speed of parasite clearance between patients recruited at the start and end of the study period.
Dose of DHP. The mean total dose of dihydroartemisinin administered was 6.75 mg/kg (95% CI: 6.68 to 6.82 mg/kg; range: 4.62 to 9.23 mg/kg) and that of piperaquine was 54.0 mg/kg (95% CI: 53.4 to 54.6 mg/kg; range: 36.9 to 73.9 mg/kg) (Fig. 2a). Neither the dose of dihydroartemisinin nor that of piperaquine correlated with the speed of clearance of peripheral parasitemia. The mean dose of piperaquine administered to children under 5 years old was 55.4 mg/kg (95% CI: 52.8 to 58.1 mg/kg) compared to 56.9 mg/kg (95% CI: 55.9 to 57.9 mg/kg) in children aged 5 to 15 years and 51.9 mg/kg (95% CI: 51.6 to 52.2 mg/kg) in adults (overall P < 0.001). The spread of dosing was greatest in children under 5 years old (standard deviation [SD] = 12.7 mg/kg) compared to children aged 5 to 15 years (SD = 6.8 mg/kg) and adults (SD = 2.8 mg/kg) (Fig. 2b). Using Youden's index the best cutoff for the total piperaquine dose predicting treatment failure was 48 mg/kg, with 46% (42/92) of children under 5 years of age receiving a dose of piperaquine below this level compared to 8.1% (14/173) of children aged 5 to 14 years and 12.9% (39/263) of adults (overall P < 0.001). The overall risk of failure was 31% (95% CI: 20 to 42%) in patients administered less than 48 mg/kg compared to 9.0% (95% CI: 6.1 to 11.9%) in those receiving higher doses (HR = 3.1; 95% CI: 1.9 to 5.2; P < 0.001).
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The mean plasma piperaquine concentration was 46.6 ng/ml (95% CI: 43.3 to 49.8 ng/ml) on day 7 and 16.8 ng/ml (95% CI: 15.1 to 18.6 ng/ml) on day 28. Age and the plasma concentration of piperaquine on day 7 were positively correlated (rs = 0.289; P < 0.001) (Fig. 3), with day 7 concentrations 26% lower in children aged less than 15 years (mean = 37.1 ng/ml; 95% CI: 32.8 to 41.3 ng/ml) than in adults (mean = 50.4 ng/ml; 95% CI: 46.4 to 54.5 ng/ml; P < 0.001).
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In patients with recurrent parasitemia, the mean plasma piperaquine level on day 7 was 28.4 ng/ml (95% CI: 22.1 to 34.8 ng/ml), compared to 48.7 ng/ml (95% CI: 45.3 to 52.1 ng/ml) in those treated successfully (P < 0.001). The best cutoff for the day 7 piperaquine concentration predicting any treatment failure was 30 ng/ml, with levels below this observed in 38% (21/56) of children and 22% (31/140) of adults (relative risk [RR] = 1.69; 95% CI: 1.1 to 2.7; P = 0.04). In the 196 patients with available day 7 piperaquine concentrations, the overall the risk of failure was 36% (95% CI: 20 to 52%) in patients with concentrations below this level compared to 6.8% (95% CI: 1.9 to 12%) in those above (HR = 6.1; 95% CI: 2.4 to 15.5; P < 0.001) (Fig. 4). The risks of failure stratified by species are given in Table 2.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Artemisinin combination therapies achieve their antimalarial effect through an initial rapid reduction in parasite biomass, attributable to the short-acting but highly potent dihydroartemisinin, with the subsequent removal of the remaining parasites by the intrinsically less active but more slowly eliminated piperaquine. Overall cure rates depend upon there being sufficient partner drug to remove the residual parasite biomass left by the artemisinin derivative. Our venous sampling was pragmatic, but sparse and therefore did not allow us to define the full pharmacokinetic profile of piperaquine. However, as with other long-acting antimalarial drugs, the day 7 level may provide a useful surrogate marker of the area under the curve and time above the MIC (6, 18), both crucial determinants of the in vivo response to antimalarial drugs (23). Studies with both artemether-lumefantrine and mefloquine plus artesunate have shown that the day 7 concentrations of the long-acting partner drug are the primary predictors of subsequent treatment outcome (14, 18).
In the present study we show that two pharmacological parameters, the day 7 piperaquine concentration and the total mg/kg dose of piperaquine administered, were important determinants of treatment outcome. Children were more vulnerable to both inadequate dosing and low plasma drug concentrations. Even though the dose of DHP was administered on the basis of weight, this was carried out according to weight groups to the nearest half tablet, with a suspension used only for those weighing less than 9 kg. This strategy amounted to increments in dose of 160 mg of piperaquine and 20 mg of dihydroartemisinin, and the effects of such an increase resulted in a wide range in the mg/kg dose administered, particularly in young children (Fig. 2). Although this variability would be ameliorated by the wider use of pediatric syrup or suspensions, such formulations are not yet available for many antimalarials. Furthermore, in practice most rural clinics administer drugs according to age groups, with inevitable inaccuracies in dosing (21). These findings highlight the need for practical attention to, and reporting of, dosing practices, particularly in young children.
We were not able to define the relevance of inadequate dosing on plasma levels of piperaquine, since parents of only four children under 5 years of age agreed for their children to be bled. However, in older children the overall mean dose administered to children was actually 9% higher than that given to adults and yet the day 7 plasma piperaquine concentrations were 26% lower. This discrepancy could have arisen from higher clearance rates, which have been noted in children (9). Our estimate of the terminal elimination half-life of piperaquine was 396 h, similar to that observed previously (9), although no difference in half-life between adults and children aged 5 to 14 years was noted. An alternative explanation is that the low plasma concentrations of piperaquine are a consequence of inadequate drug absorption, to which children are particularly vulnerable, especially those presenting with diarrhea. Since coadministration with a high-fat meal increases the oral bioavailability of piperaquine by 120% (17), advocating that medication be taken with a glass of milk or a biscuit, as is currently recommended for artemether-lumefantrine, may help to improve piperaquine absorption.
Young children were 2.5-fold more likely to suffer a recurrent parasitemia with P. vivax than older children or adults. Although this difference was not apparent for P. falciparum infections, this may reflect the low failure rates with P. falciparum and the fact that only 16% of the patients recruited in our study were under 5 years of age. Hence our study may simply have been underpowered in this respect. The clinical implications of this may be more apparent in areas of high transmission, where nearly all of the patients seeking medical treatment are young children. Indeed cure rates for Rwanda suggest that the efficacy of DHP in this age group may be significantly lower than that observed in Asia (10).
Most formal pharmacokinetic studies of antimalarial drugs are conducted either with healthy volunteers or adults. Since pharmacokinetic profiles in children and pregnant women can be markedly different, the derivation of appropriate dosing recommendations necessitates that prospectively designed pharmacokinetic studies be conducted with the target population rather than extrapolated from studies with adults. Recently this has been highlighted in a study of the use of sulfadoxine-pyrimethamine which suggests that this widely deployed drug has systematically been underdosed in children for over 20 years (2). Our preliminary findings raise the possibility that a similar discrepancy may arise with DHP and that the currently recommended dose in children (2.25 mg/kg dihydroartemisinin and 18 mg/kg piperaquine) is suboptimal and accounts for approximately 20% of treatment failures. However, formal pharmacokinetic studies are needed to confirm our findings and define the relationship between the day 7 piperaquine concentration and the complete pharmacokinetic profile.
Concerns have been raised over the stability of the dihydroartemisinin component in the fixed-dose combination (8). Reassuringly, over the 16-month duration of the study, despite using the same batch of drugs there was no discernible change or temporal trend in the early or late treatment responses. Further studies are needed to confirm the clinical relevance of tablet stability, but in practice the DHP appears to retain excellent efficacy in a tropical rural setting.
In summary DHP is a well-tolerated and effective antimalarial that results in excellent treatment outcome both in curing initial infections with multidrug-resistant P. falciparum and P. vivax and preventing reinfection and relapse. Using current recommendations, health workers should aim to give a total of at least 48 mg/kg of piperaquine. Further studies are warranted to define the pharmacokinetic profile of piperaquine in children and determine whether increased dosing can optimize the efficacy of this important antimalarial combination regimen.
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ACKNOWLEDGMENTS |
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The study was funded by the Wellcome Trust-NHRMC (Wellcome Trust ICRG GR071614MA-NHMRC ICRG ID 283321). N.M.A. is supported by an NHMRC Practitioner Fellowship. R.N.P. is funded by a Wellcome Trust Career Development Award, affiliated to the Wellcome Trust, Mahidol University, Oxford Tropical Medicine Research Programme (074637).
We declare no conflict of interest.
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FOOTNOTES |
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Published ahead of print on 10 September 2007.
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This article has been cited by other articles:
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| Clin. Vaccine Immunol. | Clin. Microbiol. Rev. |
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30 ng/ml; diamonds, day 7 piperaquine concentration of <30 ng/ml. The overall P value for the difference between treatment groups at day 42 was 0.001.