Study design and objective
This Phase 2a, randomized, open-label, parallel-group study was conducted from 11th September 2018 to 6th November 2019 across seven study centres in Benin (Cotonou), Burkina Faso (Banfora and Nanoro), Gabon (Libreville and Lambaréné), Kenya (Kisumu), and Uganda (Kampala). The primary objective was to show the contribution of artefenomel to the clinical and parasitological activity of artefenomel and ferroquine in combination by analysing the exposure–response of artefenomel in patients with uncomplicated falciparum malaria. To achieve its objective, some patients would receive a sub-therapeutic dosing regimen, and this was considered in the design by using dedicated risk minimization activities, i.e., selection of a patient population at low risk of severe malaria, hospitalization for at least 48 h or longer based upon the investigator’s judgment, and administration of rescue therapy as soon as there was evidence of treatment failure or systematically on Day 29. The study protocol is provided as Additional file 1.
Treatment
Investigational products were ferroquine 100 mg capsules (Sanofi, France) and artefenomel 300/400/600 mg granules formulation (Sanofi, France) presented in a sachet with alpha tocopherol polyethylene glycol 1000 succinate formulation and sucrose. Dose selection aimed to characterize the anti-malarial contribution of artefenomel to the combination. Artefenomel has been evaluated to doses up to 1200 mg with no safety concerns, and a wide range of doses (0–1000 mg) was selected to evaluate the exposure–response. The 400 mg ferroquine dose was selected as a sub-therapeutic dose so as not to mask the contribution of artefenomel. Details of the dose selection methods are in Additional file 2.
Randomization
Patients were randomized centrally using interactive response technology in a ratio of 1:1:1:1 to 400 mg ferroquine alone, or 400 mg ferroquine plus artefenomel 300, 600, or 1000 mg. The administration of treatments was open label as a single oral dose on Day 0 and was directly observed. Ferroquine was administered in a fasted condition. Artefenomel was prepared as a suspension in sterile water and given approximately 15 min after ferroquine. If vomiting occurred after ferroquine, the patient was not re-dosed, artefenomel was not administered, and the patient received rescue therapy. If the artefenomel dose was vomited within 5 min of administration, the patient was re-dosed. Vomiting within 5–35 min of artefenomel administration did not prompt redosing, but any remaining drug was given. Rescue anti-malarial therapy as per local recommendations was administered to patients before Day 28 if clinically indicated, if the ferroquine dose was vomited, or at Day 29 if not given previously.
Patients
To evaluate exposure–response, a sub-therapeutic dosing regimen was to be administered. Thus, the study population was selected to be at low risk of severe malaria. Eligible participants were aged 14 to 69 years, body weight 35–95 kg, of either sex, presenting with microscopically confirmed uncomplicated P. falciparum malaria (≥ 3000 to ≤ 50,000 parasites/µL blood) plus fever or a history of fever in the previous 24 h. All participants were required to use effective contraception and pregnant or lactating women were excluded. Key exclusion criteria were severe malaria [33], mixed Plasmodium infection, clinically important medical conditions, severe vomiting or diarrhoea, severe malnutrition [34], splenectomy, known hypersensitivity to study medications, positive test for viral hepatitis, clinically relevant laboratory abnormalities, including aspartate aminotransferase (AST) > 2 times the upper limit of normal (xULN), alanine aminotransferase (ALT) > 2xULN, or total bilirubin > 1.5xULN, or Fridericia-corrected QTc (QTcF) > 450 ms. Full inclusion and exclusion criteria are detailed in the protocol (Additional file 1).
Procedures
Screening procedures included physical examination, medical history, 12-lead electrocardiogram (ECG), vital signs, clinical laboratory tests, viral hepatitis serology, and a pregnancy test. Patients were hospitalized for at least the first 48 h following treatment administration and longer if malaria symptoms or parasitaemia persisted. Patients were followed up on Days 3, 4, 5, 6, 7, 10, 14, 21 and 28. All patients received definitive approved anti-malarial treatment on Day 29 if they had not already received rescue therapy.
Blood samples for parasite assessments were taken at screening, every 6 h until 36 h post-dose, at hours 48, 72, 96, 120, 144, and 168 post-dose, on Days 10, 14, 21, and 28, and at any time if clinically indicated. Giemsa-stained thick and thin blood films were prepared, and parasites identified and enumerated independently by two trained microscopists using standard procedures [35]. Parasite polymerase chain reaction (PCR) genotyping to differentiate recrudescence from re-infection was done centrally by the Swiss Tropical and Public Health Institute following any positive parasite assessment after initial parasite clearance, as per published recommendations [36]. Based on the P. falciparum marker genes msp1, msp2 and glurp, new infection was assumed when all the alleles in parasites from the post-treatment sample were different from those in the baseline sample, for one or more loci tested. Recrudescence was defined as at least one allele at each locus common to paired samples from baseline and at recurrence [36].
Adverse events were assessed throughout the study. Additional post-treatment safety assessments were 12-lead ECGs, vital signs, haematology, and clinical laboratory tests (Additional file 1).
Blood samples were taken pre- and post-dose for PK assessments of artefenomel (12 sample time points) and ferroquine (11 sample time points) (Additional file 1). PK samples were analysed using liquid chromatography tandem mass spectroscopy (LC-MS-MS). Artefenomel concentrations were determined at Swiss BioQuant (Basel, Switzerland) with a lower limit of quantification (LLQ) of 1 ng/mL and ferroquine samples at Covance (Salt Lake City, USA) with an LLQ of 5 ng/mL. Where anti-malarial rescue therapy was administered before Day 28, blood samples were taken for artefenomel and ferroquine PK assessments and parasite assessments.
Endpoints
The primary efficacy endpoint was PCR-adjusted adequate clinical and parasitological response (ACPR) at Day 28, defined as the absence of parasitaemia without previous treatment failure or rescue therapy, adjusted for re-infection using PCR genotyping [35].
Secondary efficacy endpoints were Day 28 ACPR unadjusted for re-infection (crude ACPR); parasitaemia at baseline then every 6 h during the first 36 h post-dose, then at 48 h and every 24 h until Day 7; parasite clearance time; time to parasitaemia re-emergence, recrudescence, or reinfection; time elapsed below the LLQ of parasitaemia; fever clearance time; observed parasite reduction ratio at 24, 48 and 72 h post-dose (observed PRR24, PRR48, and PRR72); parasite clearance rate; and time to parasite reduction by 50% (PC50) and 99% (PC99) of baseline parasitaemia, time for parasitaemia to reduce by 50% (TPC50) and 90% (TPC90) independent of baseline parasitaemia, and the estimated parasite reduction ratio at 24, 48 and 72 h post-dose (estimated PRR24, PRR48, and PRR72) (Additional file 1).
Safety endpoints were the frequency, severity, and causality of all adverse events coded using MedDRA (version 22.0), the frequency of serious adverse events, clinically important changes in clinical laboratory data, ECGs, vital signs, or physical examination. Adverse events of special interest were pregnancy, symptomatic overdose, increase in ALT ≥ 3xULN (or ≥ 2 × the baseline value if baseline ALT was ≥ ULN), QTcF ≥ 500 ms, or QTcF prolongation > 60 ms from baseline.
Pharmacokinetic assessments were secondary endpoints, but also supported the primary analysis evaluating the exposure–response for artefenomel (Additional files 3 and 4). The following individual patient exposures for artefenomel in plasma and ferroquine and desmethyl-ferroquine in blood were estimated: maximal observed concentration (Cmax), concentration at Day 7 post-dose (Cd7), area under the concentration–time curve from time 0 to infinity (AUC0–∞) for artefenomel and ferroquine, and AUC from time 0 to Day 28 (AUC0–d28) for ferroquine and desmethyl-ferroquine only.
Analysis populations
The safety population included all randomized patients who received one dose or a partial dose of the investigational drugs. The PK population was a sub-set of the safety population with at least one evaluable PK blood sample for either artefenomel or ferroquine. The microbiological intention-to-treat population (mITT) included all randomized patients who received the investigational drugs, had microscopically confirmed P. falciparum infection at baseline, and a post-baseline parasitological assessment. The per-protocol (PP) population was a sub-set of the mITT population who were evaluable for Day 28 ACPR with no major protocol violations. The pharmacokinetic/pharmacodynamic (PK/PD) efficacy population was the primary efficacy analysis population and included patients in both the PK and mITT populations who had at least one evaluable PK sample for both artefenomel and ferroquine. Thus, patients who vomited or who did not receive a complete dose of study drug were not excluded from the PK/PD efficacy population.
Sample size
Sample size was based on the estimated efficacy for artefenomel (0, 300, 600, and 1000 mg) plus ferroquine (400 mg) derived from clinical trial simulations assuming a parasitaemia > 3000 parasites/µL (Additional file 2). Based on an estimated PCR-adjusted ACPR of 72% for ferroquine alone and 81%, 91% and 97% in the three escalating ferroquine plus artefenomel arms, 30 patients per arm would be required to detect an exposure–response effect with artefenomel with ~ 90% power. Allowing for subject withdrawals, target sample size was 140 patients (35 per arm).
Statistical methods
For the primary efficacy analysis, data processing, PK parameter estimation and logistic regression analyses were conducted within R (3.5.1) combined with MONOLIX (MLX2019R1) and the IQR package (v1.1.1) developed by IntiQuan (Basel, Switzerland) to support the entire workflow of a PK and logistic regression analysis from estimations to simulations. For simulations, IQR uses the library SUNDIALS (v2.9.0) from Computation (USA) (Additional files 3 and 4).
The contribution of artefenomel exposure to the Day 28 PCR-adjusted ACPR of the combination was evaluated using logistic regression evaluating the exposure to artefenomel (AUC0–∞) and ferroquine/desmethyl-ferroquine (AUC0–d28) as covariates as well as baseline parasitaemia, age, body weight, sex, vomit status, and study centre (Additional file 4). Data exploration suggested that three study centres (Libreville, Lambaréné, and Cotonou) had lower efficacy (Day 28 PCR-adjusted ACPR ≤ 75%) compared to the other centres (≥ 85%). These sites were identified based on their efficacy data and no quality issues were identified in the data review of this study. The covariate ‘low efficacy study centre’ was created by grouping these three centres versus all other centres to identify any study centre effects.
The base model included ferroquine AUC0–d28 as the predictor variable and the contribution of each of the remaining potential covariates on the base model was first assessed in a univariate addition analysis. A backward elimination approach was then implemented including all significant covariates from the univariate addition analysis, plus artefenomel exposure. Model selection was based on Akaike Information Criterion. Odds ratio estimates, corresponding 95% two-sided Wald confidence intervals (CI) and P values were calculated for covariates. As a secondary efficacy analysis, the relationship between the estimated exposure of artefenomel and ferroquine and Day 28 crude ACPR was evaluated as described for Day 28 PCR-adjusted ACPR.
For other secondary efficacy outcomes and safety outcomes, statistical analysis was done using SAS (version 9.4). Day 28 PCR-adjusted and crude ACPR were summarized for the PP and mITT populations, with Clopper–Pearson 95% CI. All other secondary efficacy outcomes were evaluated in the mITT population. Parasite clearance parameters were calculated using the WorldWide Antimalarial Resistance Network parasite clearance estimator (WWARN PCE) based on the linear portion of the individual natural logarithm parasitaemia–time profiles [37]. The time to each parasite clearance endpoint, parasite re-emergence, recrudescence, re-infection, fever clearance, and elapsed time below the LLQ of parasitaemia were estimated using Kaplan–Meier analysis. There was no adjustment for multiplicity of comparisons in this exploratory study.
Pharmacokinetic analysis was performed using non-linear mixed effect modelling as implemented in Monolix (version 2019R1), applying previously developed population PK models (see Additional file 3).
Ethics
The study was conducted in accordance with the Declaration of Helsinki, Good Clinical Practice, and all applicable laws, rules, and regulations of the participating countries. Final approval by the relevant Independent Ethics Committees and, where relevant, local regulatory authorities, was obtained at each participating study centre before any patient was enrolled. All patients or their legal guardians provided informed consent and those under the age of legal majority provided assent.