Study population
All subjects provided signed and dated informed consent prior to screening. Local Ethics Review Committees provided approval for both studies (Microdose Intravenous Study: Independent Ethics Committee of the Foundation “Evaluation of the Ethics of Biomedical Research”, Assen, The Netherlands; Single Oral Dose Study: Medische Ethische ToetsingsCommissie, Stichting Beoordeling Ethik Biomedisch Onderzoek, Assen, The Netherlands) and they were conducted in accordance with Good Clinical Practice and the guiding principles of the 2008 Declaration of Helsinki[31].
Microdose intravenous study
Healthy subjects aged 18–50 years with a body mass index of 18.5–29.9 kg/m2 were eligible. Subjects had to be non-smokers or ex-smokers for a minimum of 6 months prior to screening and with a smoking history of <5 pack years. Exclusion criteria included positive testing for hepatitis B surface antigen, hepatitis C antibody and human immunodeficiency virus. Subjects unwilling to abstain from red wine, Seville oranges, grapefruit or grapefruit products 7 days prior to dosing were also ineligible.
Single oral dose study
Healthy subjects aged 18–65 years with a body mass index of 18.5–29.9 kg/m2 and a smoking history as described for the Microdose Intravenous Study were eligible. Key exclusion criteria were as for the Microdose Intravenous Study.
Study design
Microdose intravenous study
This was an open-label, single-dose study conducted from 21 January 2010 to 18 February 2010 at PRA International, Zuidlaren, The Netherlands (GlaxoSmithKline protocol: CC4114041; Clinicaltrials.gov identifier: NCT01086462). Subjects received a single intravenous infusion of approximately 10 kBq [14C]-GSK2239633 100 μg over 15 minutes. Subjects attended a screening visit within 30 days prior to receiving the first dose of study medication. Subjects were admitted to the clinical unit on Day −1 and remained there until 48 hours post-dose. As this was the first time GSK2239633 100 μg had been administered to humans, dosing was staggered so that only one subject received the study medication on Day 1. As the study medication was well-tolerated, the remaining subjects were dosed the following day in a staggered dosing schedule (20-minute interval between dosing of the subjects). Subjects received a follow-up telephone call 4–10 days after the last dose of study medication.
Single oral dose study
This was a randomised, double-blind, placebo-controlled, cross-over, single ascending-dose study conducted from 29 March 2011 to 1 July 2011 at PRA International, Zuidlaren, the Netherlands (GlaxoSmithKline protocol: CC4114660; Clinicaltrials.gov identifier: NCT01371812). Subjects completed a screening visit within 28 days prior to receiving the first dose of study medication. Subjects were admitted to the clinical unit the day before each dosing session for baseline assessments that included a physical examination and clinical laboratory tests. Single ascending oral doses of GSK2239633 or placebo were administered to two interlocking and alternating cohorts (Additional file1: Figure S1) (Cohort 1 and Cohort 2), each of which consisted of 12 male subjects randomised to receive either active or placebo (eight active: four placebo). The randomisation schedule was generated prior to the start of the study using validated internal software. Subjects from Cohort 1 underwent four dosing sessions; the starting dose of GSK2239633 was 150 mg, followed by 600 mg, 1200 mg and 1200 mg after eating the standard United States Food and Drug Administration (FDA) high fat/high caloric meal to assess any food effect. Subjects from Cohort 2 underwent three dosing sessions; the starting dose of GSK2239633 was 300 mg, followed by 900 mg and 1500 mg. As this was a first-time-in-human study, dosing was staggered over 2 days so that on Day 1 one subject received GSK2239633 and one subject received placebo at each dosing session in both cohorts (with the exception of the food effect dosing session for Cohort 1). On Day 2, the remaining subjects were dosed provided GSK2239633 was well-tolerated on Day 1. Subjects fasted for 10 hours before each dosing session, except those in last period of Cohort 1 who ate 30 minutes prior to administration of GSK2239633 1200 mg. For both cohorts, no food was permitted up to 4 hours after administration of study medication. During a 2-hour period (1 hour pre-dose until 1 hour post-dose), no water was allowed with the exception of that taken with the study medication (240–300 mL). After receiving randomised treatment, subjects underwent a period of observation and assessments for 3 days. They returned to the clinical unit, following a washout period of approximately 14 days, to receive their next randomised dose of study medication, with additional 3-day inpatient assessments. Subjects returned for a follow-up visit 10–14 days after their last dose of study medication.
Dosing and sample collection
Microdose intravenous study
Subjects were dosed over 15 minutes with: 10 μg/mL GSK2239633 (14C-labelled) in a saline solution for infusion (0.9% w/v sodium chloride solution) containing 10% w/v (2-hydroxypropyl)-beta-cyclodextrin.
Blood samples for pharmacokinetic analysis of plasma total radioactivity and GSK2239633 were collected at screening, Day −1, pre-dose and at 5, 10, 15, 20, 30, 45 minutes and 1, 1.5, 1.75, 2.25, 3.25, 4.25, 6.25, 8.25, 12.25, 16.25, 18.25, 24.25, 30.25, 36.25 and 48.25 hours from start of the infusion. Urine samples were collected prior to dosing and then until 24 hours after the infusion ended.
Single oral dose study
Subjects were dosed with GSK2239633 as a capsule formulation (Swedish orange coloured opaque hard gelatin capsules) with a unit dosage strength of 150 mg. Subjects received between one and 10 capsules (depending on the dose level), which were swallowed with 240 mL of water (or up to 300 mL of water in total for the higher number of capsules). Subjects randomised to placebo in each dosing session received the same number of capsules as those randomised to active treatment for the same dosing session.
Blood samples were drawn for pharmacokinetic analysis pre-dose and at 5, 15 and 30 minutes and 1, 2, 3, 4, 8, 10, 24 and 48 hours post-dose. For pharmacodynamic analysis, blood was collected pre-dose and at 1, 4 and 24 hours post-dose. The pharmacodynamic analysis was only conducted for subjects in the fasted condition; no analysis was performed for the fed cohort. An aliquot of urine was collected pre-dose; after dosing, all urine was collected and pooled during a 24-hour interval.
Pharmacokinetic analysis
Microdose intravenous study
The primary endpoints were maximum observed concentration (Cmax), area under the concentration-time curve from time 0 to last measurable concentration (AUC0–t), AUC from time 0 extrapolated to infinity (AUC0–∞) and terminal half-life (t½) of GSK2239633 and [14C]-radioactivity, apparent clearance (CL) and volume of distribution at steady state (Vss) of GSK2239633 and cumulative urinary excretion of total radioactivity for 24 hours post-dose. Total radioactivity was measured directly by accelerator mass spectrometry. Plasma GSK2239633 concentrations were determined using an internally validated analytical method by accelerator mass spectrometry (further details provided as Additional file1 material).
Urine radioactivity levels were measured by liquid scintillation counting with an external standardisation method. The lower limit of quantification (LLQ) was 0.98 pg/mL for the plasma assay and 10 pg GSK2239633 equiv/mL for the total plasma radioactivity assay. The LLQ for GSK2239633 in urine was 5 μg GSK2239633 equiv. Pharmacokinetic parameters for each subject were derived from plasma GSK2239633 concentration-time profiles by non-compartmental analysis using WinNonlin Professional Edition Version 5.2 or above (Pharsight Corporation, Mountain View, USA). Maximum observed concentration, time to Cmax (tmax), AUC from time 0 to 48 hours post-dose (AUC0–48), AUC0–t, AUC0–∞, CL, t½, volume of distribution during terminal elimination phase (Vd) and Vss were determined.
Single oral dose study
Blood concentrations of GSK2239633 were determined by an internally validated analytical method based on extraction from a dried blood spot disc by addition of methanol, followed by high performance liquid chromatography/tandem mass spectrometry. The LLQ of the assay was 10 ng/mL (further details provided as Additional file1 material). Analysis and derivation of pharmacokinetic parameters were conducted as for the Microdose Intravenous Study.
Safety and tolerability assessments
Microdose and single dose studies
The primary endpoints of the Single Oral Dose Study were adverse events and clinically relevant changes in safety parameters. Adverse events were recorded throughout both studies. For each event, the potential causal relationship with the study drug was assessed by the investigator. Other safety assessments in both studies included clinical laboratory tests (chemistry, haematology, urinalysis), vital signs, 12-lead electrocardiogram (ECG) and continuous cardiac telemetry.
Pharmacodynamic analysis
Single oral dose study
Blood samples (9 volumes) were collected into a 3.8% sodium citrate solution (1 volume) and incubated for 15 minutes at room temperature with saturating concentrations of fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD4 antibody and non-inactivating phycoerythrin (PE)-conjugated mouse anti-human CCR4 antibody (BD Biosciences, Oxford, United Kingdom), or appropriate isotype control antibodies. The samples were then incubated for 30 minutes at 37°C. For pre-clinical studies, antagonists or vehicle were added at the beginning of this incubation. Following this, the blood cells were incubated for 15 seconds with varying concentrations of TARC (PeproTech EC, London, United Kingdom) before addition of 10 volumes of fluorescence-activated cell sorting (FACS) lysing solution (BD Biosciences, Oxford, United Kingdom). After 30 minutes, the blood cell suspension was centrifuged (500 g for 5 minutes) and resuspended in fresh FACS lysing solution for further 10 minutes to ensure complete red blood cell lysis. The cell suspensions were centrifuged (500 g for 5 minutes) again and washed twice by resuspending in phosphate buffered saline (PBS) solution and centrifuging at 500 g for 5 minutes. After incubating the cell suspensions for 15 minutes with lysophosphatidylcholine (100 μg/mL) and Alexa fluor 647 phalloidin (0.075 units/mL), the cells were recovered by centrifugation at 500 g for 5 minutes and resuspended in PBS. The F-actin content of the CD4+ CCR4+ lymphocytes in each sample was determined on a FACSCantoII flow cytometer by measuring the mean Alexa fluor 647 fluorescence intensity of 1,000 cells. This was expressed as a fraction of the Alexa fluor 647 fluorescence intensity of the CCR4– lymphocytes in the same sample. The fractional occupancy of CCR4 (Ro) was then estimated by determining the dose-ratio (DR) from the change in effective concentration giving 50% of the maximal response (EC50) of the TARC concentration-response curve before and after dosing with GSK2239633 and using the formula Ro = (DR – 1)/DR[32].
Statistical analysis
Microdose intravenous study
No formal sample size estimation was performed. As this was an exploratory study, no formal statistical hypotheses for safety, tolerability or pharmacokinetics were tested.
Single oral dose study
No statistical analysis was done to determine the sample size. There was no statistical analysis of safety parameters.
Dose proportionality was primarily evaluated based on Cmax, AUC0–10 and AUC0–t using the power model. Each parameter was loge-transformed prior to analysis. Additionally, a mixed model was fitted to the dose-normalised pharmacokinetic parameters to compare each dose with the reference dose (GSK2239633 150 mg). The data were loge-transformed prior to analysis and the results were then back-transformed to calculate ratios between the doses. Food effect was assessed by performing a statistical analysis of Cmax, AUC0–10 and AUC0–t after loge-transformation of the data. An analysis of variance (ANOVA) model was fitted along with 90% confidence intervals (CIs) by a mixed effects model, with fed/fasted condition as a fixed effect and subject as a random effect. Using data obtained in the Microdose Intravenous Study it was possible to make an estimation of GSK2239633 bioavailability following oral administration. For that, AUC0–10 was used as a comparison.
For the pharmacodynamic analysis, population estimates of the parameters, such as EC50, were derived using non-linear mixed effects models in NonMEM Version 7 (ICON Development Solutions, PA, USA) for all profiles generated. Analysis of the entire individual pharmacodynamic and pharmacokinetic datasets was conducted to derive mean EC50 estimates pre-dose and in the presence of GSK2239633 (each subject acted as their own control as their pre-dose data was compared with their post-dose data). Although not a direct method for formal calculation of Ro, this DR was used to give an estimate of Ro as described above.