Charlotte A. Lanteri

Dihydroartemisinin-piperaquine failure associated with a triple mutant including kelch13 C580Y in Cambodia: an observational cohort study

BACKGROUND Dihydroartemisinin-piperaquine has been adopted as first-line artemisinin combination therapy (ACT) for multidrug-resistant Plasmodium falciparum malaria in Cambodia because of few remaining alternatives. We aimed to assess the efficacy of standard 3 day dihydroartemisinin-piperaquine treatment of uncomplicated P falciparum malaria, with and without the addition of primaquine, focusing on the factors involved in drug resistance. METHODS In this observational cohort study, we assessed 107 adults aged 18-65 years presenting to Anlong Veng District Hospital, Oddar Meanchey Province, Cambodia, with uncomplicated P falciparum or mixed P falciparum/Plasmodium vivax infection of between 1000 and 200,000 parasites per μL of blood, and participating in a randomised clinical trial in which all had received dihydroartemisinin-piperaquine for 3 days, after which they had been randomly allocated to receive either primaquine or no primaquine. The trial was halted early due to poor dihydroartemisinin-piperaquine efficacy, and we assessed day 42 PCR-corrected therapeutic efficacy (proportion of patients with recurrence at 42 days) and evidence of drug resistance from the initial cohort. We did analyses on both the intention to treat (ITT), modified ITT (withdrawals, losses to follow-up, and those with secondary outcomes [eg, new non-recrudescent malaria infection] were censored on the last day of follow-up), and per-protocol populations of the original trial. The original trial was registered with ClinicalTrials.gov, number NCT01280162. FINDINGS Between Dec 10, 2012, and Feb 18, 2014, we had enrolled 107 patients in the original trial. Enrolment was voluntarily halted on Feb 16, 2014, before reaching planned enrolment (n=150) because of poor efficacy. We had randomly allocated 50 patients to primaquine and 51 patients to no primaquine groups. PCR-adjusted Kaplan-Meier risk of P falciparum 42 day recrudescence was 54% (95% CI 45-63) in the modified ITT analysis population. We found two kelch13 propeller gene mutations associated with artemisinin resistance--a non-synonymous Cys580Tyr substitution in 70 (65%) of 107 participants, an Arg539Thr substitution in 33 (31%), and a wild-type parasite in four (4%). Unlike Arg539Thr, Cys580Tyr was accompanied by two other mutations associated with extended parasite clearance (MAL10:688956 and MAL13:1718319). This combination triple mutation was associated with a 5·4 times greater risk of treatment failure (hazard ratio 5·4 [95% CI 2·4-12]; p<0·0001) and higher piperaquine 50% inhibitory concentration (triple mutant 34 nM [28-41]; non-triple mutant 24 nM [1-27]; p=0·003) than other infections had. The drug was well tolerated, with gastrointestinal symptoms being the most common complaints. INTERPRETATION The dramatic decline in efficacy of dihydroartemisinin-piperaquine compared with what was observed in a study at the same location in 2010 was strongly associated with a new triple mutation including the kelch13 Cys580Tyr substitution. 3 days of artemisinin as part of an artemisinin combination therapy regimen might be insufficient. Strict regulation and monitoring of antimalarial use, along with non-pharmacological approaches to malaria resistance containment, must be integral parts of the public health response to rapidly accelerating drug resistance in the region. FUNDING Armed Forces Health Surveillance Center/Global Emerging Infections Surveillance and Response System, Military Infectious Disease Research Program, National Institute of Allergy and Infectious Diseases, and American Society of Tropical Medicine and Hygiene/Burroughs Wellcome Fund.

The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei

ABSTRACT Human African trypanosomiasis (HAT) is a fatal tropical disease caused by infection with protozoans of the species Trypanosoma brucei gambiense and T. b. rhodesiense. An oral prodrug, DB289, is a promising new therapy undergoing phase III clinical trials for early-stage HAT. DB289 is metabolically converted to the active trypanocidal diamidine DB75 [2,5-bis(4-amidinophenyl)furan]. We previously determined that DB75 inhibits yeast mitochondrial function (C. A. Lanteri, B. L. Trumpower, R. R. Tidwell, and S. R. Meshnick, Antimicrob. Agent Chemother. 48:3968-3974, 2004). The purpose of this study was to investigate if DB75 targets the mitochondrion of T. b. brucei bloodstream forms. DB75 rapidly accumulates within the mitochondria of living trypanosomes, as indicated by the fluorescent colocalization of DB75 with a mitochondrion-specific dye. Fluorescence-activated cell sorting analysis of rhodamine 123-stained living trypanosomes shows that DB75 and other trypanocidal diamidines (pentamidine and diminazene) collapse the mitochondrial membrane potential. DB75 inhibits ATP hydrolysis within T. brucei mitochondria and appears to inhibit the oligomycin-sensitive F1F0-ATPase and perhaps other ATPases. DB75 is most likely not an inhibitor of electron transport within trypanosome mitochondria, since DB75 fails to inhibit mitochondrial respiration when glycerol-3-phosphate is used as the respiratory substrate. However, DB75 inhibits whole-cell respiration (50% inhibitory concentration, 20 μM) at drug concentrations and incubation durations that also result in the dissipation of the mitochondrial membrane potential. Taken together, these findings suggest that the mitochondrion is a target of the trypanocidal action of DB75.

Roles for the Trypanosoma brucei P2 transporter in DB75 uptake and resistance

A novel trypanocide, 2,5-bis(4-amidinophenyl)furan (DB75), in its prodrug amidoxime-derivative form, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), is in trials as the first orally administered drug for human African trypanosomiasis. DB75 is a diamidine. Resistance to some diamidines correlates to loss of uptake via the P2 aminopurine transporter. We show here that uptake of DB75 into Trypanosoma brucei also occurs principally via the P2 transporter. Uptake of tritiated DB75 occurred via a high-affinity (Km app, 3.2 μM) carriermediated route that was inhibited by adenosine, adenine, and pentamidine, all known substrates of the P2 transporter. Trypanosomes lacking the TbAT1 gene that encodes the P2 transporter demonstrated an 11-fold reduction in sensitivity to DB75 when measured under controlled in vitro conditions. These knockout cells were also less sensitive to DB75 than wild-type cells in mice. Initial uptake rates of DB75 into the Δtbat1 knockout cell line were greatly reduced compared with rates in wild-type cells. A trypanosome cell line selected in vitro for DB75 resistance was shown to have lost P2-mediated DB75 uptake. The TbAT1 gene was mapped to chromosome V of the T. brucei genome and the DB75-resistant parasites were shown to have deleted both alleles of this gene. Fluorescence microscopy of DB75-treated trypanosomes revealed that DB75 fluorescence localizes rapidly within the DNA-containing organelles of wild-type trypanosomes, whereas no fluorescence was observed in Δtbat1-null parasites or in the parasites selected for resistance to DB75.

New chimeric antimalarials with 4-aminoquinoline moiety linked to a tetraoxane skeleton.

The synthesis of the chimeric molecules consisting of two pharmacophores, tetraoxane and 7-chloro-4-aminoquinoline, is reported. The tetraoxanes 2, 4, and 8 show relatively potent in vitro antimalarial activities, with IC90 values for the Plasmodium falciparum strain W2 of 2.26, 12.44, and 10.74 nM, respectively. In addition, two compounds, 2 and 4, cured mice in a modified Thompson test for antimalarial blood stage activity, with a minimum curative dose of 80 mg/kg, a minimum active dose of 20 mg/kg/day, and a maximum tolerated dose of >960 mg/kg.

DB75, a novel trypanocidal agent, disrupts mitochondrial function in Saccharomyces cerevisiae

ABSTRACT The aromatic diamidines represent a class of compounds with broad-spectrum antimicrobial activity; however, their development is hindered by a lack of understanding of their mechanism of antimicrobial action. DB75 [2,5-bis(4-amidinophenyl)furan] is a trypanocidal aromatic diamidine that was originally developed as a structural analogue of the antitrypanosomal agent pentamidine. DB289, a novel orally active prodrug of DB75, is undergoing phase IIb clinical trials for early-stage human African trypanosomiasis, Pneumocystis jiroveci carinii pneumonia, and malaria. The purpose of this study was to investigate mechanisms of action of DB75 using Saccharomyces cerevisiae as a model organism. The results of this investigation suggest that DB75 inhibits mitochondrial function. Yeast cells relying upon mitochondrial metabolism for energy production are especially sensitive to DB75. DB75 localizes (by fluorescence) within the mitochondria of living yeast cells and collapses the mitochondrial membrane potential in isolated yeast mitochondria. Furthermore, addition of DB75 to yeast cells or isolated rat liver mitochondria results in immediate uncoupling of oxidative phosphorylation and subsequent inhibition of respiration. We conclude that the mitochondrion is a cellular target of DB75 in yeast cells and anticipate that the results of this study will aid in the target-based design of new antimicrobial aromatic diamidines.

Efficacy of Two versus Three-Day Regimens of Dihydroartemisinin-Piperaquine for Uncomplicated Malaria in Military Personnel in Northern Cambodia: An Open-Label Randomized Trial

Introduction Emerging antimalarial drug resistance in mobile populations remains a significant public health concern. We compared two regimens of dihydroartemisinin-piperaquine in military and civilians on the Thai-Cambodian border to evaluate national treatment policy. Methods Efficacy and safety of two and three-day regimens of dihydroartemisinin-piperaquine were compared as a nested open-label evaluation within a malaria cohort study in 222 otherwise healthy volunteers (18% malaria-infected at baseline). The first 80 volunteers with slide-confirmed Plasmodium falciparum or vivax malaria were randomized 1:1 to receive either regimen (total dose 360mg dihydroartemisinin and 2880mg piperaquine) and followed weekly for up to 6 months. The primary endpoint was malaria recurrence by day 42. Volunteers with vivax infection received primaquine at study discharge with six months follow-up. Results Eighty patients (60 vivax, 15 falciparum, and 5 mixed) were randomized to dihydroartemisinin-piperaquine. Intention-to-treat all-species efficacy at Day 42 was 85% for the two-day regimen (95% CI 69–94) and 90% for the three-day regimen (95% CI 75–97). PCR-adjusted falciparum efficacy was 75% in both groups with nearly half (45%) still parasitemic at Day 3. Plasma piperaquine levels were comparable to prior published reports, but on the day of recrudescence were below measurable in vitro piperaquine IC50 levels in all falciparum treatment failures. Conclusions In the brief period since introduction of dihydroartemisinin-piperaquine, there is early evidence suggesting declining efficacy relative to previous reports. Parasite IC50 levels in excess of plasma piperaquine levels seen only in treatment failures raises concern for clinically significant piperaquine resistance in Cambodia. These findings warrant improved monitoring of clinical outcomes and follow-up, given few available alternative drugs. Trial Registration ClinicalTrials.gov NCT01280162

Structure–activity relationships amongst 4-position quinoline methanol antimalarials that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum

Utilizing mefloquine as a scaffold, a next generation quinoline methanol (NGQM) library was constructed to identify early lead compounds that possess biological properties consistent with the target product profile for malaria chemoprophylaxis while reducing permeability across the blood-brain barrier. The library of 200 analogs resulted in compounds that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum. Herein we report selected chemotypes and the emerging structure-activity relationship for this library of quinoline methanols.

Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

ABSTRACT Cambodias first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure.

Randomized, Double-Blind, Placebo-Controlled Clinical Trial of a Two-Day Regimen of Dihydroartemisinin-Piperaquine for Malaria Prevention Halted for Concern over Prolonged Corrected QT Interval

ABSTRACT Dihydroartemisinin-piperaquine, the current first-line drug for uncomplicated malaria caused by Plasmodium falciparum and Plasmodium vivax in Cambodia, was previously shown to be of benefit as malaria chemoprophylaxis when administered as a monthly 3-day regimen. We sought to evaluate the protective efficacy of a compressed monthly 2-day treatment course in the Royal Cambodian Armed Forces. The safety and efficacy of a monthly 2-day dosing regimen of dihydroartemisinin-piperaquine were evaluated in a two-arm, randomized, double-blind, placebo-controlled cohort study with 2:1 treatment allocation. Healthy military volunteers in areas along the Thai-Cambodian border where there is a high risk of malaria were administered two consecutive daily doses of 180 mg dihydroartemisinin and 1,440 mg piperaquine within 30 min to 3 h of a meal once per month for a planned 4-month period with periodic electrocardiographic and pharmacokinetic assessment. The study was halted after only 6 weeks (69 of 231 projected volunteers enrolled) when four volunteers met a prespecified cardiac safety endpoint of QTcF (Fridericias formula for correct QT interval) prolongation of >500 ms. The pharmacodynamic effect on the surface electrocardiogram (ECG) peaked approximately 4 h after piperaquine dosing and lasted 4 to 8 h. Unblinded review by the data safety monitoring board revealed mean QTcF prolongation of 46 ms over placebo at the maximum concentration of drug in serum (Cmax) on day 2. Given that dihydroartemisinin-piperaquine is one of the few remaining effective antimalarial agents in Cambodia, compressed 2-day treatment courses of dihydroartemisinin-piperaquine are best avoided until the clinical significance of these findings are more thoroughly evaluated. Because ECG monitoring is often unavailable in areas where malaria is endemic, repolarization risk could be mitigated by using conventional 3-day regimens, fasting, and avoidance of repeated dosing or coadministration with other QT-prolonging medications. (This study has been registered at ClinicalTrials.gov under registration no. NCT01624337.)

Using amplicon deep sequencing to detect genetic signatures of Plasmodium vivax relapse

Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity-generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapse-reinfection epidemiology.