MATERIALS AND METHODS
Study design. This was a single-centre, double-blind, randomized, placebo-controlled trial conducted between 23 November 2015 and 8 June 2016 at the CTC Clinical Trial Consultants Phase I unit in Uppsala, Sweden. Study code IRL790C001, EudraCT No 2015-003586-29.
The study comprised two parts.
Part 1 was designed as a partial cross-over single-ascending-dose (SAD) study with ascending dose levels of mesdopetam and one food interaction cohort. Sixteen (16) subjects were included in one of two cohorts, eight subjects in each cohort (six active treatment and two placebo), and the cohorts were dosed in a zig-zag manner (5 mg-40 mg-160 mg and 15 mg-80 mg-food/80 mg) with a wash-out period of at least one week between doses. Following a screening period of maximum 28 days, subjects were confined to the research clinic from the evening before each dosing (Day -1) until 24 hours post dose (Days 1-2) (food interaction group subjects until at least eight hours after administration (Day 1)). A follow-up visit was performed 5-10 days after last dose.
Part 2 examined multiple-ascending-dose (MAD) cohorts with study treatment once daily for 10 consecutive days. Twenty-four (24) subjects were included, twelve subjects in each dose cohort (nine on active treatment, three on placebo), and the cohorts were dosed with 40 mg and 80 mg, respectively. Following a screening period of maximum 28 days, subjects were confined to the research clinic from the evening before first dosing (Day -1) and during the four first administration days (Day 1-4). Following a safety and tolerability review before leaving the clinic on Day 4, the subjects visited the clinic daily for administration of doses 5-9 under medical surveillance (Days 5-9). In the evening of Day 9 the subjects were confined to the research clinic again until 24 hours after administration of dose 10 (Days 10-11). A follow-up visit was performed 5-10 days after the last dose.
This study was approved by the Ethics Review Board in Uppsala, Sweden and conducted in compliance with the Declaration of Helsinki and the International Conference on Harmonization (ICH) Guidelines for Good Clinical Practice (GCP). All subjects provided written, informed written consent before participating in any study procedures.
Treatments. Mesdopetam was provided as 5 mg, 20 mg and 40 mg (free base) hard gelatine capsules. Placebo was provided formulated as hard gelatine capsules identical in appearance to the mesdopetam capsules.
Subjects. Healthy male subjects 18-50 years of age, weighing at least 50 kg, with a body mass index between 18 and 30 kg/m2 were eligible for the study. Good health was determined by medical history, physical examination, vital signs, electrocardiogram (ECG), and laboratory tests at screening. Main exclusion criteria included: history of any clinically significant disease which, in the opinion of the Investigator, could either put the subject at risk because of participation in the study, or influence the results or the subject’s ability to participate in the study; use of any prescribed or non-prescribed medication or herbal supplements within two weeks prior to the first administration of study treatment; administration of another new chemical entity or has participated in any other clinical study that included drug treatment within three months of the first administration of study treatment; regular use of tobacco products, and/or history of drug or alcohol abuse.
Primary objectives and endpoints (safety and tolerability). In both the SAD and MAD part of the study, safety assessments included: frequency, seriousness and intensity of Adverse Events (AEs), physical examination, 12-lead ECGs, Columbia Severity Suicidal Rating Scale (C-SSRS), vital sign measurements, laboratory measurements (clinical chemistry, hematology, and urinalysis). The SAD part also included telemetry. AEs were collected from signing the informed consent form (ICF) until the follow-up assessments. Serious Adverse Events (SAEs) spontaneously reported by a subject to the Investigator after the follow-up assessment, were to be handled in the same manner as SAEs that occurred during the study.
Secondary objectives and endpoints (pharmacokinetic parameters). In the SAD part, blood samples for measurement of plasma concentrations of mesdopetam and its metabolites IRL902 and IRL872 were collected at predetermined time points: pre‑dose, 20, 40 minutes, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours after intake of mesdopetam (up to 8 hours only in the food interaction cohort). In the MAD part, blood samples were collected pre-dose, 20, 40 minutes, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours after the first and last dose of mesdopetam. A 48‑hour sample was also taken after the first dose, and on Day 4 sampling was done pre-dose, 1, 2 and 3 hours post dose.
Urine was collected for measurement of the concentrations of mesdopetam and its metabolites in the single-dose cohorts (Part 1) at the predetermined interval 0 to 24 hours post dose.
Bioanalytical methods. Plasma samples were analyzed for mesdopetam and the metabolites IRL872 and IRL902 using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) performed by the National Veterinary Institute, Department of Chemistry, Environment and Feed Hygiene Section of Chemical Analysis, Uppsala, Sweden. A deuterated analogue of each molecule were used as internal standards. The method was validated according to EMA guidelines (7) and the bioanalytical experiments performed in accordance with the OECD Principles of Good Laboratory Practice (GLP). The validated concentration range was 6.00 to 6000 nmol/L for mesdopetam, and 3.00 to 1000 and 1.00 to 1000 nmol/L for IRL872 and IRL902 respectively.
Urine samples were analyzed for mesdopetam by ultra-performance liquid chromatography-mass spectrometry (UPLC‑MS) at MetaSafe AB, Södertälje, Sweden. One analytical run with the samples from dose cohort 80 mg (without food) of the SAD part was done. The samples were diluted 10-fold with 0.1% formic acid and centrifuged before analysis. Calibration samples and quality control samples were prepared in placebo urine and treated as the unknown samples. Deuterated mesdopetam was used as internal standard. The regression coefficient r2for the calibration curve, 2 to 12 µmol/L, was 0.9999. The accuracy of two quality controls of 2.5 and 5 µmol/L mesdopetam was 98 and 102%.
Statistical analysis. No formal sample size calculation was performed for this study and thereby no hypothesis testing. The sample size was considered sufficient to provide adequate information for the study objectives.
The Full Analysis Set (FAS), comprising all subjects who received randomized study treatment and have available data, has been used for the safety and tolerability assessments. Evaluations have been done according to actual treatment regardless of randomization.
The Per Protocol (PP) analysis set comprises data from all randomized subjects who have received study treatment and have evaluable PK parameter data with no major protocol deviations with an impact on the PK data. All protocol violations were presented and discussed at the clean file meeting. The PP set has been used for presentation of PK endpoints.
All statistical calculations were performed using the SAS® program (Version 90.4, SAS Institute Inc., Cary, NC, USA). The statistical analyses include descriptive statistics reflecting the explorative nature of the study.
Pharmacokinetic calculations. The pharmacokinetic parameters were calculated by non-compartmental analysis (NCA) using the software Phoenix WinNonlin® version 6.3 or later (Pharsight Corporation, U.S.A.). Plasma concentration values below the lower limit of quantitation and missing values (e.g. no blood sample collected, or no value obtained at analysis) were excluded from the NCA. Actual time-points for blood sampling were used in the calculations of the individual parameters, while nominal time points were denoted for summary statistics. Dose proportionality of AUC and Cmax was estimated using a non-linear power model. As an estimate of the accumulation ratio (AR) the quotient of the AUC0-24h for the first and the last dose for each subject was calculated.