Study design
This was an open label, randomized, single dose, parallel-group and non-confirmatory study in healthy volunteers. It was conducted at Nucleus Network in Melbourne, Australia. The primary objective was to investigate the pharmacokinetic interaction potential between KAF156 and piperaquine in healthy subjects. Secondary objectives were to investigate the safety and tolerability of KAF156 and piperaquine alone and when co-administered in healthy subjects, and to investigate the potential effects on electrocardiogram (ECG) intervals (QT, PR, QRS) when KAF156 and piperaquine were given alone and in combination.
Treatments and follow-up schedule
The proposed doses were 800–1280 mg for KAF156 and piperaquine, respectively. The KAF156 dose of 800 mg (KAF156 base equivalent) was given in the form of eight 100 mg strength tablets. Piperaquine was administered as tetraphosphate tetrahydrate. The piperaquine dose of 1280 mg (piperaquine tetraphosphate equivalent) was given in the form of four 320 mg strength tablets. The study consisted of a screening period of up to 26 days (Day − 28 to − 3), a baseline on Day -1, followed by a single dose treatment in 3 parallel treatment arms on Day 1, and a study completion evaluation. The total duration for each subject to complete the study including baseline without screening was approximately 61 days.
Given the known food effect for piperaquine (~ threefold increased exposure) and its QTc liability [
20], all doses were given fasting. Subjects were admitted to the study site the night prior to dosing (approximately 12 h) in each arm for baseline evaluations. Eligible subjects fasted (i.e., no food or liquid except for water) for at least 10 h prior to administration of study drug on Day 1 and continued to fast for at least 4 h thereafter. No fluid intake apart from the fluid given at the time of drug intake was allowed from 2 h before until 2 h after dosing. Lunch and dinner was served approximately 4 and 8 h post dosing, respectively.
Subjects were domiciled at the site from baseline until 48 h post-dose. Subjects then returned to the site at the follow-up visits detailed in the assessment schedule, for up to a 61-day period, to undergo safety evaluations and PK sampling. Study completion evaluation was conducted after the last PK sampling on Day 11 for KAF156 or Day 61 for arms containing piperaquine.
Subjects
The study population comprised healthy males, aged 18–45 years of age and in good health as determined by past medical history, physical examination, vital signs, ECG, and laboratory tests at screening. Subjects weighed at least 50 kg to participate in the study, with a body mass index (BMI) within the range of 18–30 kg/m2. The study was open to female subjects of non-childbearing potential, but none were recruited.
Exclusion criteria included use of other investigational drugs at the time of enrollment, or within five half-lives or within 30 days of enrollment; a history of clinically significant ECG abnormalities, screening/baseline Fridericia’s formula corrected QT interval (QTcF) elevation (> 430 ms for males, > 440 ms for females); women of child-bearing potential; smokers or smokeless tobacco users who were unwilling or unable to refrain from tobacco use during confinement to the clinical research centre and during required study visits/evaluations; haemoglobin levels below 12.0 g/dL at baseline; significant illness which did not resolve within 2 weeks prior to initial dosing; active disease; infections or conditions which may alter drug pharmacokinetics; renal or hepatic dysfunction; a history of drug or alcohol abuse within the 12 months prior to dosing, or clinical/laboratory evidence of such abuse.
A total of approximately 72 (n = 24/arm) subjects were planned to be enrolled and randomly assigned 1:1:1 into one of the three treatment arms:
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Arm I, 24 subjects received a single morning dose of 800 mg KAF156 + a single dose of 1280 mg piperaquine (KAF156 + PPQ).
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Arm II, 24 subjects received a single morning dose of 800 mg KAF156 (800 mg KAF156).
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Arm III, 24 subjects received a single morning dose of 1280 mg piperaquine (1280 mg PPQ).
Safety assessment
Any potential relationship of drug exposure parameters to changes in ECG parameters was assessed. Safety assessments consisted of collecting all adverse events (AEs) and serious adverse events (SAEs), with their severity and relationship to study drug. They included regular monitoring of haematology, blood chemistry and urine performed at study centre and regular assessment of vital signs, physical condition, body weight and 12-lead electrocardiograms (ECG). Triplicate ECG assessments were performed at pre-dose and 2, 4, 8, 12, 24, and 48 h post-dose, while single ECGs were performed at the later time points (72 and 96 h and the end of study).
Pharmacokinetic (PK) assessment
Plasma concentrations were determined at pre-dose and then at 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 48, 72, 96, 144, 192, and 240 h for both KAF156 and piperaquine. Additional samples were taken at 336, 504, 672, 1008 and 1440 h post dose for piperaquine. Venous blood samples were collected either through an indwelling catheter or by venipuncture into K2EDTA-containing polyethylene tubes followed by gentle mixing and centrifugation between 2 and 8 °C for 10 min at approximately 1500 g. Tubes were stored on wet ice or cryoblock until centrifuged (within 60 min). Immediately after centrifugation, the supernatant plasma was transferred into 1.8 mL polypropylene screw-cap tubes which were placed in dry ice. The tubes were kept frozen at or below − 70 °C until bioanalysis. Plasma concentrations of KAF156 and piperaquine were determined by a validated liquid chromatography-tandem mass spectrometry (LC–MS/MS) method; the Lower Limit of Quantification (LLOQ) is 5 and 0.5 ng/mL for KAF156, and piperaquine, respectively.
The linearity ranges for KAF156 and PPQ are 1–5000 ng/mL, and 0.5–250 ng/mL, respectively. For the KAF156 assay, briefly, a 20.0 μL aliquot plasma sample was mixed with a 25.0 μL aliquot of the internal standard working solution [(M + 6)KAF156 500 ng/mL in 50% methanol]. A 200 μL aliquot of acetonitrile (ACN) was added to the mixture. Subsequently, the sample was centrifuged at 2000
g for 10 min at 10 °C. A 150 μL aliquot of each supernatant was evaporated to dryness under nitrogen at 45 °C. The sample was reconstituted in a 300 μL aliquot of MeOH–water–formic acid (FA) (10:89.9:0.1, v/v/v). A 3.00 μL aliquot of the sample was injected onto the LC–MS/MS system. The piperaquine method has already been described elsewhere [
21].
The accuracy and precision for both KAF156 and piperaquine were within acceptable limits for study validation. For calibration standards, both KAF156 (5.00, 10.0, 50.0, 250, 500, 1500, 4000 and 5000 ng/mL) and piperaquine (0.500, 1.00, 2.50, 10.0, 50.0, 100, 225 and 250 ng/mL) had bias within the acceptable range of ± 20.0% at the LLOQ and ± 15.0% at the other concentration levels. Similarly, the 3 levels of quality control samples for both KAF156 and piperaquine had bias within the acceptable range of ± 15% for at least 2/3 of the individual values.
The following PK parameters were determined using the actual recorded sampling times and non-compartmental method(s) with Phoenix WinNonlin (Version 6.4): Cmax, Tmax, AUClast, AUCinf, T1/2, Vz/F and CL/F for KAF156 and piperaquine from the plasma concentration–time data.
The linear trapezoidal rule was used for AUC calculation. Regression analysis of the terminal plasma elimination phase for the determination of T1/2 included at least three data points after Cmax. If the adjusted R2 value of the regression analysis of the terminal phase was to be less than 0.75, no values were to be reported for T1/2, AUCinf and CL/F. If extrapolated AUCinf was more than 20% for KAF156 or more than 40% for piperaquine (due to its long terminal half-life) AUCinf and related parameters were not included in statistical analysis.
PK/PD relationships, using the individual concentrations and QTcF changes from baseline at each time point, were explored to establish the relationship between QTcF and drug exposure.
Secondary variables
Analysis of ECG parameters over time points
A key secondary endpoint for this trial was change from baseline in QTcF. Bazett’s formula corrected QT interval (QTcB) analysis was limited to descriptive statistics and reported only as a summary table. The baseline was calculated from the pre-dose triplicate ECGs. At pre-dose and 2, 4, 8, 12, 24, and 48 h post-dose triplicate ECG assessments were collected. The mean of triplicate QTcF values was calculated and used for all subsequent calculations and statistical evaluations. The endpoint for this analysis was calculated by subtracting the mean of triplicated QTcF pre-dose from post-dose assessment for each subject at each time point.
For 72 and 96 h post-dose time points, only a single assessment was performed. This endpoint was obtained by subtracting the mean of triplicate pre-dose QTcF value from the post-dose QTcF value for each subject and time point.
The change from baseline for QTcF for each time point was analysed using a linear model with treatment as the fixed effect and baseline (baseline was taken as 0 h on Day 1) as covariate in the model separately. The difference in adjusted means along with the 90% two-sided CI was calculated for KAF156 800 mg + piperaquine 1280 mg vs. KAF156 800 mg, and KAF156 800 mg + piperaquine 1280 mg vs. piperaquine 1280 mg.
An arithmetic mean (± standard deviation (SD)) plot for change from baseline QT data (in Y axis) over several observed time points (in X axis) was performed for each treatment.
Analysis of maximal change in ECG parameters
A similar analysis was performed for maximal change from baseline for QTcF separately for KAF156 concentrations alone and in the presence of piperaquine, and for piperaquine concentrations alone and in the presence of KAF156, using a linear model with treatment as the fixed effect and concentration of KAF156 and piperaquine at maximal change as the covariate. The difference in adjusted means along with the 90% CI was calculated for KAF156 800 mg + piperaquine 1280 mg vs. KAF156 800 mg, and KAF156 800 mg + piperaquine 1280 mg vs. piperaquine 1280 mg. For the endpoint maximal change from baseline the same model was explored using AUCinf or Cmax as covariate in the model instead of plasma concentration.