2.1 Study Design
This was a Phase I, first-in-human study, conducted at a single site in Germany in healthy male volunteers (NCT02068690), which was split into two parts. The first part was a partially randomised, placebo-controlled, single-blind, single-rising dose (SRD) component. The partially randomised design was utilised to maintain, for safety reasons, an initial treatment sequence of active–placebo–active–active 1 h apart in the first cohort of each dose level, ensuring an interval of at least 2 h between the first and second active dose of each dose level, which was expected to be sufficient to detect relevant acute effects of BI 425809 whilst protecting patient safety. This part aimed to assess the safety and tolerability of single doses of BI 425809, ranging from 0.5 to 150 mg. The secondary aim was to investigate the pharmacokinetic profile of BI 425809, including dose proportionality. The second part of the trial was a randomised, open-label, three-way cross-over bioavailability/food effect (BA/FE) component involving three treatment periods. Part 2 aimed to assess the relative bioavailability of BI 425809 as a tablet or oral solution in the fasted state, and the relative bioavailability of the tablet in the fed versus fasted state.
2.1.1 Part 1: SRD Component Design
Eight volunteers were randomised to a single dose of BI 425809 as solution or placebo at each intended dose level: 0.5, 1, 2, 5, 10, 25, 50, 100 and 150 mg, administered in a fasted state. Within each dose group, six subjects received the active drug and two received placebo. For safety reasons, each dose group was split into cohorts of four subjects each. The randomisation was conducted using a computerised random number generator and was performed in blocks of four. This part of the study was conducted across 3 visits [Visit 1: screening; Visit 2: drug administration and plasma sampling (on Day 1 of each visit)], followed by continuous medical care of a physician or the medical staff for 34 h after administration and adequate safety monitoring up to Day 9 of each visit; and Visit 3: end-of-study examination].
For pharmacokinetic assessment, 2.7 mL of venous blood samples were taken at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 24, 34, 48, 72, 96, 120, 144, 168 and 192 h using an indwelling cannula. All blood samples were centrifuged at 2000–4000 g at 4–8 °C for at least 10 min, with intermittent storage on ice. The plasma obtained was split into two cryotubes, which were frozen immediately (no later than 60 min after blood sampling). Both aliquots contained at least 0.5 mL of plasma (one for the analysis and the other was used as an analytical back-up sample). The plasma samples were then stored frozen, in upright position at − 20 °C or below until analysis. Urine was collected 0–4, 4–8, 8–12 and 12–24 h and then at 24-h intervals up to 192 h to determine the concentration of BI 425809 excreted.
2.1.2 Part 2: BA/FE Component Design
A separate group of 12 healthy volunteers was randomised (using a computerised random number generator) to receive (1) BI 425809 25 mg as a tablet in the fasting state (reference treatment, R), (2) BI 425809 25 mg as a tablet after a standardised high-fat, high-calorie meal (test treatment, T1) and (3) BI 425809 25 mg as solution in the fasting state (test treatment, T2); subjects were thereby allocated to one of six possible treatment sequences: R/T1/T2, R/T2/T1, T1/T2/R, T1/R/T2, T2/T1/R or T2/R/T1. This part of the study was conducted across five visits [Visit 1: screening; Visits 2, 3 and 4: drug administration and plasma sampling (on Day 1 of each visit), followed by pharmacokinetic collection and safety monitoring up to Day 9 of each visit (each treatment was separated by a washout phase of at least 14 days); and Visit 5: end-of-study examination].
BI 425809 was administered after an overnight fast of at least 10 h for the fasting assessments and 30 min after a high-calorie, high-fat breakfast for the fed assessments. Blood samples for pharmacokinetic assessment were taken at the same time points as in Part 1, after treatment administration.
Both parts of the study were approved by the Independent Ethics Committee at the Human Pharmacology Centre of Boehringer Ingelheim Pharma GmbH and Co. KG, Ingelheim, Germany, according to national and international regulations. The study was conducted in accordance with the International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Guideline for Good Clinical Practice and local legislation, in accordance with the principles of the Declaration of Helsinki [
10,
11]. All subjects provided written, informed consent before procedures were performed.
2.3 Subjects
2.3.1 Inclusion Criteria
Subjects included in this study were healthy male volunteers, 18–45 years of age, with a body mass index (BMI) of 18.5–29.9 kg/m2.
2.3.2 Exclusion Criteria
Subjects were excluded if they showed evidence of a concomitant disease upon examination, vital sign assessment, electrocardiograms (ECG) or laboratory test [follicle-stimulating hormone (FSH), luteinising hormone (LH) and haemoglobin values were to be strictly within the reference range]. Other exclusion criteria included gastrointestinal, hepatic, renal, respiratory, cardiovascular, metabolic, immunological or hormonal disorders; diseases of the central nervous system (such as epilepsy), other neurological disorders or psychiatric disorders; history of relevant orthostatic hypotension, fainting spells or blackouts; chronic or relevant acute infections; history of relevant allergy/hypersensitivity (including allergy to the trial medication or its excipients) or evidence or history of macular degeneration or any abnormal finding in colour discrimination test. Subjects were also excluded if they had a history of surgery of the gastrointestinal tract that could have interfered with kinetics of the study drug, had taken drugs with a long half-life (more than 24 h) within 30 days or less than 10 half-lives before study drug administration or had use of any drug within the previous 10 days that may have influenced the study results or prolonged QT interval. Additional exclusion criteria included drug abuse, excessive alcohol intake (> 20 g/day), use of tobacco (> 10 cigarettes, three cigars/pipes per day) or inability to refrain from smoking on study days and blood donation of > 100 mL within 30 days before the study.
2.4 Study Endpoints and Assessments
The primary endpoint was the frequency [n (%)] of subjects with drug-related adverse events (AEs) following single-rising doses, as determined by the investigator. An AE was defined as any untoward medical occurrence, including an exacerbation of a pre-existing condition, in a subject who received the study drug. The event did not have to have a causal relationship with the treatment. The intensity of AEs were graded as mild (awareness of symptom[s] which were easily tolerated), moderate (enough discomfort to cause interference with usual activity) or severe (incapacitating/causing inability to work or to perform usual activities).
AEs included clinically relevant findings from safety laboratory tests (chemistry; haematology; enzymes and hormones including LH, FSH, testosterone and sex hormone-binding protein), neurological examination, 12-lead ECGs, vital signs (blood pressure), oxygen saturation (SpO2), physical examinations (occurrence of findings), visual tests (colour discrimination, Amsler grid and visual acuity), and Bond and Lader [
8] and Bowdle [
9] visual analogue scales (VAS) for possible psychedelic effects. Safety assessments were carried out at several time points through Days 1–9 and spontaneous AEs (including details of the time of onset, the end time and the intensity of these events) were recorded throughout the study. Subjects were kept under close medical surveillance until at least 34 h following drug administration and were allowed to leave the trial site after formal assessment and confirmation of their fitness by the investigator.
Secondary endpoints included the pharmacokinetic parameters of maximum plasma concentration (C
max) and area under the concentration–time curve from time zero extrapolated to infinity (AUC0–∞) of BI 425809. Other pharmacokinetic parameters examined were AUC from time zero to the last measurable concentration (AUC0–tz
), AUC from time interval t
1 to t
2 (AUC
t1–t2), time to C
max (t
max), terminal half-life (t
½), fraction eliminated in urine from time point t
1 to t
2 \( (f{\text{e}}_{{t_{1} {-}t_{2} }} ) \), amount of analyte in urine over the time interval t
1–t
2 \( (A{\text{e}}_{{t_{1} {-}t_{2} }} ) \), apparent clearance after extravascular administration (CL/F) and apparent volume of distribution during the terminal phase after extravascular administration (V
z/F).
2.5 Bioanalytical Assay
Concentration of BI 425809 in both plasma and urine were analysed at BI Pharmaceuticals, Inc., Ridgefield, CT, USA, using a validated liquid chromatography tandem mass spectrometry method.
The concentration of BI 425809 was determined over the range of 1.00–1000 nM using linear calibration standard curves (seven concentration levels) with 1/x
2
weighting. Drug concentration data identified below the lower limit of quantification (BLQ) were displayed as such and not replaced by zero at any time point (including the lag phase and predose values). For the non-compartmental analysis, concentration values identified as BLQ in the lag phase were set to zero. The lag phase was determined as the period between time zero and the first time point with a concentration above the quantification limit.
During the study, assay performance was assessed by back-calculation of calibration standards, tabulation of the standard curve fit function parameters and measurement of quality control samples. The quality control plasma samples were prepared in K3-ethylenediaminetetraacetic acid human plasma at three concentration levels and those for the urine samples were prepared in 0.05% Tween 20 human urine, also at three concentration levels. These samples were analysed to assess accuracy and precision of pharmacokinetic measurements.
The pharmacokinetic software used in this study was Phoenix WinNonlin Version 6.3 (for the non-compartmental analysis). Statistical analysis software, Version 9.2 was used to produce graphs and tables.
2.5.1 Criteria for Relative Bioavailability Investigation
For trials investigating relative bioavailability, the cross-over design is viewed favourable due to its efficiency: since each subject served as his own control, the comparison between the treatments was based on a comparison within subjects rather than between subjects. Thus, the inter-subject variability was removed from the comparison between treatments, resulting in a lower number of subjects required.
2.6 Statistical Analysis
2.6.1 Part 1: Determination of Sample Size
A total of 72 subjects were planned for inclusion in this component of the study. The planned sample size was not based on a power calculation; eight subjects per dose group (six on active treatment and two on placebo) is commonly used in SRD studies of this nature and is generally considered sufficient for the exploratory evaluation of single dose safety and pharmacokinetics [
12].
2.6.2 Part 1: SRD Statistical Analysis
All subjects from Part 1 who received active treatment of BI 425809 and had at least one observation for the endpoints of C
max, AUC0–∞, \( {\text{AUC}}_{{0{-}t_{\text{z}} }} \) and \( A{\text{e}}_{{t_{1} {-}t_{2} }} \) without important protocol violations were included for statistical analysis. C
max, AUC0–∞, \( {\text{AUC}}_{{0{-}t_{\text{z}} }} \) and \( A{\text{e}}_{{t_{1} {-}t_{2} }} \) were assessed for dose proportionality using a power model in which a regression model was applied to log-transformed data. The corresponding analysis of covariance model included the logarithm of the dose as a covariate. Analysis of safety, disposition, demographic data and baseline characteristics included all subjects who received at least one dose of the study drug.
2.6.3 Part 2: Determination of Sample Size
A total of 12 subjects were planned for inclusion in this component of the study, with all subjects receiving BI 425809 25 mg. With this sample size, sufficient precision [defined through the ratio of upper to lower confidence interval (CI) limit] in estimating the ratio of geometric Means (gMeans; test/reference) could be expected with 95% probability.
2.6.4 Part 2: BA/FE Statistical Analysis
All subjects from Part 2 of the study with at least one observation for at least one of the endpoints of AUC0–∞, \( {\text{AUC}}_{{0{-}t_{\text{z}} }} \) or C
max of BI 425809 were included for statistical analysis. Analysis of safety, disposition, demographic data and baseline characteristics included all subjects who received at least one dose of study drug. Relative bioavailability was based on an analysis of AUC0–∞, \( {\text{AUC}}_{{0{-}t_{\text{z}} }} \) or C
max parameters, using the transformed natural logarithms. The statistical model analysed variance on the logarithmic scale, while including sequence, subjects within sequence and period and treatment effects to account for potential variation. The difference between the expected means for test treatments and reference treatment was estimated by the difference in the corresponding least-squares means (point estimate), and their two-sided 90% CIs based on the t distribution were computed. These quantities were then back transformed to the original scale to give the point estimator gMean and interval estimates for the ratio between value-under-test and value-under-reference treatments.
2.6.5 Parts 1 and 2: Safety Endpoints
Safety data were analysed descriptively.