Safety, tolerability, pharmacokinetics and pharmacodynamics of GSK2239633, a CC-chemokine receptor 4 antagonist, in healthy male subjects: results from an open-label and from a randomised study

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.

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.

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.

Microdose intravenous study

The primary endpoints were maximum observed concentration (C_max), area under the concentration-time curve from time 0 to last measurable concentration (AUC_0–t), AUC from time 0 extrapolated to infinity (AUC_0–∞) and terminal half-life (t_½) of GSK2239633 and [¹⁴C]-radioactivity, apparent clearance (CL) and volume of distribution at steady state (V_ss) 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 C_max (t_max), AUC from time 0 to 48 hours post-dose (AUC_0–48), AUC_0–t, AUC_0–∞, CL, t_½, volume of distribution during terminal elimination phase (Vd) and V_ss 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.

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.

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].

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 C_max, AUC_0–10 and AUC_0–t using the power model. Each parameter was log_e-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 log_e-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 C_max, AUC_0–10 and AUC_0–t after log_e-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, AUC_0–10 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.

Microdose intravenous study

Single oral dose study

Twenty-four male subjects were enrolled and completed the study. The population mean [range] age was 37.2 [20.0–65.0] years and mean [range] body mass index was 24.9 [19.8–29.1] kg/m² (Table 1). Twenty (83%) subjects were of Caucasian/European heritage, one (4%) subject was of Central/South Asian heritage, one (4%) subject was of South East Asian heritage and two (8%) subjects were of Arabic/North Africa heritage.

Microdose intravenous study

Single oral dose study

Microdose intravenous study

Adverse events were reported by three of the six (50%) study subjects. The events reported were abdominal discomfort (n = 1), diarrhoea (n = 1), rhinitis (n = 1) and headache (n = 1); all were mild, transient and had resolved at follow-up. The episode of diarrhoea was the only event judged to be possibly drug-related by the investigator. There were no clinically significant abnormalities in clinical laboratory results, physical exam, vital signs, 12-lead ECG parameters or continuous cardiac telemetry.

Single oral dose study

Single oral dose study

Dose–response curves for the relative increase in filamentous actin (F-actin) content of CD4⁺ CCR4⁺ cells in response to TARC showed that, although highly variable, CCR4 inhibition was evident from GSK2239633 150 mg to 1500 mg (Figure 1). For comparison, the results from in vitro studies assessing the effect of GSK2239633 (1–10 μM) on TARC-induced increases in F-actin content of CD4⁺ CCR4⁺ T-cells are also presented (Figure 2). At the GSK2239633 1500 mg dose, the calculated mean level of CCR4 inhibition equated to a predicted Ro of approximately 74% at 1 hour after dosing. Predicted Ro levels decreased over time following the blood pharmacokinetic profile, which showed an initial rapid peak in blood GSK2239633 exposure followed by a rapid decline to much lower levels 4–8 hours post-dose. The Ro estimates for each dose group at 1 and 4 hours post-dose are presented in Table 5. Receptor occupancy estimates at 4 hours post-dose were more variable than those at 1 hour post-dose and therefore, difficult to interpret. Although, this was not unexpected given the low blood exposure of GSK2239633 at the 4 hours post-dose time-point. Blood from placebo subjects, analysed in the same way and at the same time-points, did not show any shifts in the response curves, which would indicate CCR4 inhibition, when the post-dose curves were compared with the pre-dose curves. The placebo data also revealed the inherent variability in the technique as shown by the approximately 3-fold range in variability for the derived EC₅₀ of TARC (pre-dose = 0.34 nM; all data = 0.1–0.34 nM).