AAC Accepted Manuscript Posted Online 14 December 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.01605-15 Copyright © 2015 Nyunt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
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Artemether-Lumefantrine Pharmacokinetics and Clinical Response is Minimally Altered in Ugandan
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Pregnant Women Treated for Uncomplicated Plasmodium Falciparum Malaria
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Myaing M. Nyunt,a Vy K. Nguyen,b Richard Kajubi,c Liusheng Huang,b Joshua Ssebuliba,c Sylvia Kiconco,c
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Moses W. Mwima,c Jane Achan,c Francesca Aweeka,b Sunil Parikh,d# Norah Mwebazac
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Institute for Global Health, University of Maryland Baltimore School of Medicine, Baltimore, Maryland, USAa,
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Department of Clinical Pharmacy, University of California – San Francisco, San Francisco, California, USAb,
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Infectious Disease Research Collaboration, Makerere University, Kampala, Ugandac, Yale School of Public
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Health, New Haven, Connecticut, USAd
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M.M.N. and V.K.N. contributed equally to this work.
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Running Head: Artemether-lumefantrine pharmacokinetics in pregnancy
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# Address correspondence to: Sunil Parikh,
[email protected] and Norah Mwebaza
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(
[email protected])
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ABSTRACT
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Artemether-lumefantrine is a first-line regimen for the treatment of uncomplicated malaria during the second
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and third trimesters of pregnancy. Previous studies have reported changes in the pharmacokinetics and clinical
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outcomes following treatment with artemether-lumefantrine in pregnant populations, however results are
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inconclusive. We conducted a study in rural Uganda to compare the pharmacokinetics of artemether-
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lumefantrine, as well as treatment responses, between 30 pregnant women and 30 non-pregnant adults with
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uncomplicated Plasmodium falciparum malaria. All participants were HIV-uninfected and treated with a six-
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dose regimen of artemether-lumefantrine and monitored clinically for 42 days. The pharmacokinetics of
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artemether, its metabolite, dihydroartemisinin, and lumefantrine were evaluated for 21 days following
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treatment. We found no significant differences in the overall pharmacokinetics of artemether,
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dihydroartemisinin, or lumefantrine in a direct comparison of pregnant women to non-pregnant adults, except
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for a statistically significant but small difference in the terminal elimination half-life of both dihydroartemisinin
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and lumefantrine. There were seven PCR-confirmed reinfections (5 pregnant and 2 non-pregnant participants).
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The observation of a shorter terminal half-life for lumefantrine may have contributed to a higher frequency of
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reinfection or a shorter post-treatment prophylactic period in pregnant women versus non-pregnant adults.
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While the comparable overall pharmacokinetic exposure is reassuring, studies are needed to further optimize
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antimalarial efficacy in pregnant women, particularly in high transmission settings and in the era of emerging
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drug resistance.
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INTRODUCTION
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Malaria, the most important parasitic disease globally, carries a high burden of morbidity and mortality,
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particularly in children and pregnant women residing in sub-Saharan Africa (1-3), where an estimated 12.4
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million pregnancies are exposed to malaria annually (4). Malaria parasitemia during pregnancy, with or without
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clinically symptomatic illness, places the women at a higher risk of severe maternal anemia (5). Placental
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sequestration of P. falciparum parasites (6, 7) is strongly associated with adverse pregnancy outcomes,
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including intrauterine growth restriction and/or prematurity resulting in a low birth weight of the newborn or
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spontaneous abortion (8-11).
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A fixed-dose oral formulation of artemether plus lumefantrine (AL), an artemisinin-based combination therapy
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(ACT), is a first-line regimen for uncomplicated P. falciparum malaria in pregnant women in the second or third
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trimester (12). Artemether is rapidly converted to its active metabolite dihydroartemisinin (DHA) (13, 14). Both
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compounds have potent antimalarial activity and are responsible for rapidly reducing parasite biomass (15-17).
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Lumefantrine is absorbed more slowly, which can be impacted by multiple factors including food intake (18),
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and has a longer active terminal half-life which helps to eradicate residual parasites and is critical in protecting
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the host against recurrent infection (19, 20).
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Both artemether and lumefantrine undergo cytochrome P450 (CYP) metabolism (14) with final systemic
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clearance via uridine glucuronosyltransferase (UGT) enzymes (21, 22), both of which may be altered by
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malaria infection (23, 24) and pregnancy-related physiologic changes (25). Induction of these pathways may
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lead to a reduction in AL pharmacokinetic (PK) exposure during pregnancy (26, 27), thereby increasing the risk
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of reinfection, treatment failure (recrudescence) or selective pressures for the development of drug resistance
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(20, 28, 29). However, drug dosing strategies for pregnant women are typically not guided by these and other
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physiologic changes in pregnancy, and are the same as those for non-pregnant adults (30, 31). AL PK has
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been studied in pregnant women from Thailand, Uganda, and Tanzania (26, 27, 32-37). These studies
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reported a variable magnitude of PK changes but all suggested that artemether, DHA, and lumefantrine PK
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parameters may be reduced in pregnant women compared to non-pregnant adults. The impact of this 3
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reduction in PK exposure on clinical outcomes for AL is unclear; a study in Thailand suggested low
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concentrations were associated with sub-optimal cure rates, while the high overall cure rates in Africa
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precluded the ability to test for PK-recrudescence associations. While potential determinants of malaria
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treatment outcomes are many, it is largely agreed that lumefantrine exposure is a critical determinant (19, 38,
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39). It has been suggested that lumefantrine concentration should be kept above a certain threshold on day 7,
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often cited as 175 ng/mL or 280 ng/ml, to minimize the risk of treatment failure (34, 38, 40). However, more
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definitive exposure-response studies are needed in pregnant women in sub-Saharan Africa, where malaria
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transmission patterns and intensity differ. To fill this knowledge gap, we conducted a prospective intensive PK
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clinical study with extended PK sampling and follow-up to directly compare artemether, DHA, and lumefantrine
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PK and treatment outcomes between HIV-uninfected pregnant women and non-pregnant adults in Tororo,
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Uganda, where P. falciparum transmission is high and holoendemic (41).
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MATERIALS AND METHODS
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Study design. A prospective single-center open-label clinical PK cohort study was conducted to compare AL
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PK and treatment outcomes between the pregnant and non-pregnant adults treated for uncomplicated P.
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falciparum malaria. Inclusion criteria included a diagnosis of uncomplicated malaria (defined as a fever
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(tympanic temperature ≥ 38⁰C or history of fever within the past 24 hours) and microscopy-confirmed P.
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falciparum mono-infection), age 16 years or older, and confirmed pregnancy of 12 to 38 weeks gestational age.
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Exclusion criteria included severe malaria, as defined by the World Health Organization (WHO) (42), or
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significant other illnesses (such as HIV and TB), hemoglobin (Hb) less than 7.0 g/dL, concurrent use of
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medications with potential interactions with the study drugs, and antimalarial drug treatment within two weeks
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prior to study enrollment. HIV negative status was confirmed by two rapid diagnostic tests. The study was
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independently approved by the Uganda National Council for Science and Technology, Makerere University
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School of Medicine Research and Ethics Committee, Yale University Human Investigations Committee, and
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University of California, San Francisco, Committee on Human Research, and registered on ClinicalTrials.gov
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(NCT01717885).
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Clinical procedures. Participants were recruited from Tororo District Hospital and referral clinics from January
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2013 to February 2014. Informed consent was obtained in the participant’s local language, as appropriate. For
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women, pregnancy was confirmed by urine test and gestational age was specified by last menstrual period,
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clinical examination, and ultrasound. All participants were provided an insecticide-treated bednet at time of
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enrollment and all pregnant women received two doses of sulfadoxine/pyrimethamine at 16–24 weeks and 28–
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36 weeks of gestation, following Ugandan guidelines. Day 0 was designated as the first day of AL treatment.
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Six doses of 80 mg artemether and 480 mg lumefantrine (four tablets of Coartem®, Novartis Pharma AG,
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Basel, Switzerland), with 200 ml of milk to provide adequate fat to enhance and control for lumefantrine
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absorption (18), were administered over a dosing schedule adjusted to permit the timing of the 6th dose to
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occur in the morning of Day 3 to allow intensive PK blood collection during the day. Dosing was provided to
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assure the first 2 doses were administered on the day of diagnosis, followed by dosing intervals of no more
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than 16 hours for the doses 3 and 4. Doses 5 and 6 were 12 hours apart. A full dose was repeated if vomiting
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occurred within 30 minutes. Enrolled participants were clinically evaluated in the study clinic on study days 0,
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1, 2, 3, 4, 8, 14, 21, 28, and 42 with active and passive surveillance (Figure 1). Participants were given study
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contact information for emergency and encouraged to come to clinic (open seven days a week) anytime they
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felt unwell.
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The scheme of blood collection for a various laboratory analyses is displayed in Figure 1. At each visit, blood
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was collected for thin and thick malaria blood smears, and on filter papers for parasite genotyping. Complete
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blood count and liver function tests were assessed on study days 0, 14, and 28. Blood was collected for
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lumefantrine PK analysis on day 8, instead of day 7 which is typically reported in the published literature, since
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AL dosing in our study was extended to day 3 instead of day 2 to allow for intensive blood sampling to occur
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during the day. Thus, day 8 results will be referred to as “day 7” from this point forward to permit a convenient
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comparison to prior studies. Blood for PK analyses was collected (Figure 1) on study days 0 (prior to first
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dose), before and after the last (6th) dose on day 3 at 0, 0.5, 1, 2, 3, 4, 8, and 24 hours, and on study days 7,
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14 and 21. Both capillary and venous blood were simultaneously collected at 2 and 24 hours post-last dose
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and on day 7 to assess the correlation between the capillary and venous plasma lumefantrine concentrations
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(37, 43), and only capillary blood was collected at later time points. Only participants who completed the full 5
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dosing regimen were included in the intensive PK study. For PK analyses, 200-500 µL of blood was collected
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in a K3EDTA-coated tube, immediately placed on ice and centrifuged at 800 g for 10 minutes at 4 ⁰C. Plasma
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was separated and split into aliquots and stored at -70 ⁰C until analysis.
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Laboratory methods. Giemsa-stained thick and thin blood smears were evaluated to quantify parasite density
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and speciation, respectively. Parasite densities were calculated by counting the number of asexual parasites
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per 200 leukocytes, assuming a leukocyte count of 8,000/µL. A blood smear was declared negative when no
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asexual parasites were seen under 100 high-power fields. Dried blood spots collected on filter paper were
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used to distinguish recrudescence from new infections for all recurrent episodes of malaria using methods
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previously described (44). DNA was isolated from blood spots, and samples were genotyped in a step-wise
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fashion with use of 6 polymorphic markers (merozoite surface protein (MSP)-1, MSP-2, and 4 microsatellites).
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If, for any of the 6 loci, an allele was not shared between consecutive episodes of parasitemia, the episode
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was classified as a new infection. If at least 1 allele was shared at all 6 loci, the episode was classified as a
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recrudescence.
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Assay analysis of artemether, DHA, and lumefantrine. Plasma concentrations of the study drugs were
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determined using high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), as
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previously described (45, 46). Artemether and DHA concentrations were quantified from 50 µl plasma samples,
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with the calibration range between 0.5-200 ng/ml, and the lower limit of quantification (LLOQ) of 0.5 ng/ml for
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both artemether and DHA (46). The coefficient of variation (CV%) for quality control (QC) samples was
