Danirixin formulation
The structure of danirixin free base is shown in Fig. 1. Immediate release (IR) tablets contained danirixin free base and standard excipients including mannitol, microcrystalline cellulose, HPMC, croscarmellose sodium and magnesium stearate.
Study designs and study subjects
Clinical studies were conducted in accordance with the Declaration of Helskini. Prior to study initiation approvals from appropriate regulatory authorities and ethics committee/institutional review boards were obtained. Written, informed consent was obtained from participating subjects prior to the performance of any study related assessments and procedures.
Study 1, FTIH (GSK Protocol CX3112483, ClinicalTrials.gov identifier NCT01209052, reviewed and approved by the Brent Medical Ethics Committee, Harrow, UK)
The first time in human study was a single-center, randomized, double-blind, placebo-controlled, adaptive design comprising five cohorts. Two cohorts of 15 healthy non-smoking male subjects aged 18 to 65 years were randomized to receive single escalating oral doses of danirixin. Cohort 1 received doses of 10 mg, 50 mg and 200 mg; Cohort 2 received doses of 25 mg, 100 mg (as a single tablet) and 100 mg (as 2 × 50 mg tablets), as well as placebo. An interlocking design allowed a period of at least 7 days to elapse between dosing in one cohort and administration of a higher dose in the other cohort. Subjects in Cohorts 3 and 4 received repeat doses of danirixin or placebo once daily for 14 days in a parallel-group design. Fourteen subjects in each cohort were randomized 5:2 to receive danirixin 50 mg (Cohort 3) or 200 mg (Cohort 4) or placebo. Cohort 5 consisted of 15 subjects. Ten subjects were randomized to receive danirixin 400 mg and five subjects received placebo as a single dose. The schematic for the study design of the FTIH study is depicted in Fig. 2.
Study 2, Fed versus Fasted, with Omeprazole (GSK Protocol CX3113722, ClinicalTrials.gov identifier NCT01209104, reviewed and approved by the Western Institutional Review Board, Olympia, WA, USA)
Study 2 was conducted at a single center in two parallel parts as depicted in Fig. 3. Cohort 1 was a partially randomized, open-label, single-dose, crossover study in 16 healthy male and female adult subjects between 40 and 64 years of age. In treatment periods 1 and 2, subjects were randomized to two single dose treatment regimens in a crossover fashion, in the fed (after a high-fat meal) and fasted states. In treatment period 3, subjects were administered omeprazole once daily for 5 days. On the fifth day, all subjects received danirixin 100 mg together with the final dose of omeprazole.
Cohort 2 was a single-dose, double-blind, parallel group investigation in 16 healthy elderly individuals between 65 and 80 years of age. Subjects were randomized 3:1 to receive danirixin 100 mg or placebo in the fasting state.
Safety
For each subject, adverse events and serious adverse events were collected from the start of first dosing until the final follow-up contact. Safety was monitored by the measurement of ECGs, vital signs, clinical laboratory assessments (clinical chemistry, hematology, and urinalysis), and assessment of adverse events.
Determination of danirixin concentrations in whole blood and pharmacokinetic assessment
Whole blood concentrations of danirixin free base were determined using a validated analytical method based on extraction from a dried blood spot [9], with a 3 mm disc being punched from a 0.015 mL sample on Whatman FTA™ cards. Danirixin was extracted using methanol (0.1 mL) containing isotopically labeled [2H7]-danirixin (racemic version of danirixin) at a concentration of 50 ng/mL as an internal standard. The extraction tubes were shaken for 1 h at ambient temperature, prior to the supernatant being transferred to clean tubes. The supernatant (5 μL) was injected onto a high-performance liquid chromatography system utilizing a Thermo Scientific Hypersil Gold C18 (5 μm packing 50 × 4.6 mm) column (supplied by Thermo Scientific, Hempstead, UK), eluted using an isocratic composition: 55:45 (v/v) of aqueous 10 mM ammonium formate containing 0.1 % formic acid (A) and acetonitrile (B). Danirixin free base has a retention time of approximately 1 min and was detected using tandem mass spectrometry on a Sciex 5000 (Applied Biosystems, Warrington, UK) using Turbolon Spray in positive polarity mode. Mass transitions monitored were 84 and 89 from precursor ions of 442 and 449 for danirixin and the internal standard. The assay had a linear dynamic range of 5 – 1000 ng/mL and quantification was performed using peak area ratios with 1/×2 weighted linear regression. Assay quality control samples were 15 ng/mL (low), 200 ng/mL (medium) and 800 ng/mL (high). The average within-run precision (%CV) was 9.9, 7.2 and 9.0 respectively, for the low, mid and high quality controls for study CX3112483 and 18.9, 5.9 and 5.2, respectively, for the low, mid and high quality controls for study CX3113722.
Pharmacokinetic parameters were determined from the blood concentration-time data for danirixin. The pharmacokinetic parameters were calculated based on actual sampling times using standard non-compartmental analysis in WinNonlin Professional, Version 5.2. The area under the blood concentration-time curve (AUC) was determined from the time of dosing to the last quantifiable concentration (AUC(0-t)) using the lin – log trapezoidal rule. The apparent terminal elimination half-life (t1/2) was obtained as the ratio of ln2/λz, where λz is the terminal phase rate constant, estimated by linear regression through the log transformed terminal data. AUC(0-t) was extrapolated to infinity (AUC(0-∞)) as the sum of AUC(0-t) and Ct/λz, where Ct is the last observed blood concentration and λz is the terminal phase rate constant.
Flow cytometric analysis of CD11b expression on blood neutrophils
CXCR2 ligand-induced cell surface expression of CD11b has been demonstrated to be a useful pharmacodynamic biomarker for the effects of CXCR2 antagonists [4]. To assess the pharmacodynamic effects of danirixin following oral administration, CXCL1-induced CD11b cell surface expression on blood neutrophils was determined with a whole blood assay as previously described [4, 10].
Statistical analyses
Study 1
The primary endpoint was safety and tolerability assessed by clinical monitoring of blood pressure, pulse rate, ECG, and clinical laboratory assessments as well as reporting of adverse events. Secondary endpoints included: 1) AUC (0-∞), AUC (0-t), maximum blood concentration (Cmax), time to maximum blood concentration (Tmax), and terminal half-life (t1/2). 2) ex vivo CXCL1-induced CD11b cell surface expression on peripheral blood neutrophils, and 3) the relationship between the blood concentration of danirixin and ex vivo CXCL-1-induced CD11b cell surface expression on peripheral blood neutrophils.
In the single dose cohorts, dose proportionality was calculated on AUC (0 to ∞) and Cmax for Cohorts 1 and 2 and repeated again with Cohorts 1, 2, and 5. The power model analysis was performed on loge-transformed AUC (0 to ∞) and Cmax for danirixin. For each of these parameters, a mixed effects model was fitted with loge (dose) as a fixed effect and individual subject intercept fitted as random effects. Estimates of the mean slope of loge (dose) were reported along with corresponding 90 % confidence intervals.
To evaluate the accumulation ratio and time invariance of the repeat dose cohorts, a statistical analysis was performed after a log transformation of the data from all active treatment groups. A mixed effect model was fitted with treatment group, day, and treatment group by day interaction as fixed effects and subject as a random effect. Day 14 was compared with Day 1 in order to estimate the accumulation ratio and time invariance ratios for each treatment group. The ratios were calculated by back-transforming the difference between the least squares means. Using the pooled estimate of variance, 90 % confidence intervals were calculated for the difference and then back-transformed.
A mixed effects model was used to analyze the ratio to baseline fractional increase from control CD11b values over time. The model included the same effect as mentioned above except for time (hours). Subject was fitted as a random effect. In the repeat dose cohorts, a mixed effects model was used to analyze the ratio to baseline fractional increase from control CD11b values (treatment group for all pre-treatment data was set to the same dummy value, regardless of the treatment the subject went on to receive). The model included the following fixed effects (effects were fitted as categorical: time (hours) and treatment. Treatment*time and time* baseline interactions were fitted. For each day, a separate mixed model was fitted with time.
Another mixed effects model was used to analyze the weighted mean (0–9 h) ratio to baseline fractional increase from control CD11b values. The model included the following fixed effects (effects were fitted as categorical): day and treatment. Treatment*day interaction was fitted.
In the single and repeat dose cohorts, adjusted geometric means for each treatment and time point were calculated along with 95 % CIs. Point estimates of the treatment differences (each danirixin dose vs. placebo) and their associated 95 % confidence intervals were also calculated (using the pooled estimate of variance) and were back-transformed to provide point estimates and 95 % confidence intervals for the ratios. The ratios were converted into percent values to give the percent inhibition of danirixin versus placebo and 95 % confidence intervals.
A Bayesian analysis was conducted to derive the posterior probability distributions for the ratio to baseline fractional increase from control CD11b values at 24 h. The probabilities of seeing the percent inhibition of danirixin versus placebo greater than 0 %, 50 % and greater than 60 % were derived from the frequentist analysis mixed effects model. A students T cumulative distribution function was used to obtain the probabilistic statements, assuming a non-informative prior.
Study 2
The pharmacokinetic parameters AUC (0 to 24 h), AUC (0 to ∞), and Cmax were derived from the blood concentration of study drug. To assess age and gender, the exposures from Cohorts 1 and 2 (both in the fasted state) were combined. Following loge-transformation, AUC (0 to ∞) and Cmax of study drug were separately analyzed using a mixed effects model with fixed effect terms age and gender. Age-gender interaction was fitted and explored. To estimate the food effect on the primary pharmacokinetic endpoint, data from Cohort 1 were used. Following loge-transformation, AUC (0 to ∞) and Cmax of study drug were separately analyzed using a mixed effects model with fixed effect terms for regimen and period. Subject was treated as a random effect in the model. The same method of analysis, as for the food effect analysis, was applied to estimate the repeat oral doses of omeprazole on the primary pharmacokinetic endpoints, except for the exclusion of period from the model. In the single dose cohorts, a mixed effects model was used to analyse the ratio to baseline fractional increase from control CD11b values. The model included the following fixed effects (effects were fitted as categorical): period (6 levels), time (hours) and treatment. Subject baseline and period baseline were fitted as continuous covariates. Treatment*time and time*period baseline interactions were fitted. Subject was fitted as a random effect.