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Randomized Controlled Trial
. 2015 Dec;80(6):1324-36.
doi: 10.1111/bcp.12724. Epub 2015 Oct 2.

The effect of a selective CXCR2 antagonist (AZD5069) on human blood neutrophil count and innate immune functions

Stipo Jurcevic  1 , Charles Humfrey  2 , Mohib Uddin  3 , Steve Warrington  4 , Bengt Larsson  3 , Christina Keen  3
Affiliations
Randomized Controlled Trial

The effect of a selective CXCR2 antagonist (AZD5069) on human blood neutrophil count and innate immune functions

Stipo Jurcevic et al. Br J Clin Pharmacol. 2015 Dec.

Abstract

Aims: The aim of the present study was to investigate whether selective antagonism of the cysteine-X-cysteine chemokine receptor-2 (CXCR2) receptor has any adverse effects on the key innate effector functions of human neutrophils for defence against microbial pathogens.

Methods: In a double-blind, crossover study, 30 healthy volunteers were randomized to treatment with the CXCR2 antagonist AZD5069 (100 mg) or placebo, twice daily orally for 6 days. The peripheral blood neutrophil count was assessed at baseline, daily during treatment and in response to exercise challenge and subcutaneous injection of granulocyte-colony stimulating factor (G-CSF). Neutrophil function was evaluated by phagocytosis of Escherichia coli and by the oxidative burst response to E. coli.

Results: AZD5069 treatment reversibly reduced circulating neutrophil count from baseline by a mean [standard deviation (SD)] of -1.67 (0.67) ×10(9) l(-1) vs. 0.19 (0.78) ×10(9) l(-1) for placebo on day 2, returning to baseline by day 7 after the last dose. Despite low counts on day 4, a 10-min exercise challenge increased absolute blood neutrophil count, but the effect with AZD5069 was smaller and not sustained, compared with placebo treatment. Subcutaneous G-CSF on day 5 caused a substantial increase in blood neutrophil count in both placebo- and AZD5069-treated subjects. Superoxide anion production in E. coli-stimulated neutrophils and phagocytosis of E. coli were unaffected by AZD5069 (P = 0.375, P = 0.721, respectively vs. baseline, Day 4). AZD5069 was well tolerated.

Conclusions: CXCR2 antagonism did not appear adversely to affect the mobilization of neutrophils from bone marrow into the peripheral circulation, phagocytosis or the oxidative burst response to bacterial pathogens. This supports the potential of CXCR2 antagonists as a treatment option for diseases in which neutrophils play a pathological role.

Keywords: COPD; CXCR2 antagonist; asthma; neutrophil recruitment; oxidative burst; phagocytosis.

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Figures

Figure 1
Figure 1
Study plan. Following screening and enrolment, AZD5069 or placebo were administered orally bid for 6 d, followed by a washout period of at least 21 d and then a second 6‐d treatment period. Subjects were randomized to one of two treatment sequences, AB or BA, where A was 100 mg AZD5069 bid and B was matching placebo bid. A two‐way crossover design was chosen to counter any inter‐individual variation in neutrophil counts in human volunteers. PK, pharmacokinetic
Figure 2
Figure 2
Arithmetic mean (± standard deviation) morning predose circulating neutrophil count vs. time profiles for subjects receiving AZD5069 and placebo. The lowest mean predose neutrophil value was observed prior to the day 2 morning dose (1.10 × 109 l–1). G‐CSF, granulocyte‐colony stimulating factor. formula image AZD5069 100 mg bid, formula image Placebo bid
Figure 3
Figure 3
Increases in peripheral blood neutrophil counts (A – percentage change, B – absolute numbers) over time in response to exercise challenge in human volunteers. Subjects were administered either AZD5069 (100 mg bid) or placebo orally (bid) prior to physical exercise. Peripheral blood samples were collected at the indicated time points. Each point is presented as mean ± standard deviation of n = 28 subjects per arm. formula image AZD5069 100 mg bid formula image Placebo bid
Figure 4
Figure 4
Increases in peripheral blood neutrophil counts (A – percentage change, B – absolute numbers) over time in response to granulocyte‐colony stimulating factor (G‐CSF) stimulation in vivo. Subjects were administered either AZD5069 (100 mg bid) or placebo orally (bid) following stimulation with G‐CSF given by subcutaneous injection (300 µg). Peripheral blood samples were collected at the indicated time points. The data represent mean ± standard deviation (SD) of n = 28 subjects per arm. formula image AZD5069 100 mg bid formula image Placebo bid
Figure 5
Figure 5
Neutrophil phagocytosis in the presence of AZD5069. Neutrophils were incubated with fluorescein‐labelled Escherichia coli at 0°C as a negative control or at 37°C to stimulate phagocytosis. The number of ingested/phagocytosed fluorescein–E. coli bacteria is measured as mean fluorescence intensity (MFI) values. A. The data are shown as mean and 95% confidence interval of MFI values for all subjects at baseline, in the presence of AZD5069 or placebo (day 4 of treatment) and at follow up (day 14). formula image AZD5069, formula image Placebo. B. Representative flow cytometry graphs for negative control (incubation with fluorescein–E. coli at 0°C) and phagocytosis of fluorescein–E. coli at 37°C are shown
Figure 6
Figure 6
Neutrophil oxidative burst in the presence of AZD5069. Neutrophils were incubated with Escherichia coli to stimulate the oxidative burst or were left unstimulated as a control. The oxidative burst is measured quantitatively by flow cytometric detection of green fluorescence generated by the oxidation of the fluorogenic substrate dihydrorhodamine (DHR) 123. A. The data are shown as mean and 95% confidence interval of mean fluorescence intensity (MFI) values for all subjects at baseline, in the presence of AZD5069 or placebo (day 4 of treatment) and at follow‐up (day 14). formula image AZD5069, formula image Placebo. B. Representative flow cytometry graphs for control (no stimulation) and the oxidative burst in the presence of E. coli stimulation are shown
Figure 7
Figure 7
Neutrophil activation marker expression in the presence of AZD5069 or placebo. The cell surface expression of CD11b (A), CD62L (B) and CD16 (C) are presented as mean fluorescence intensity (MFI) units (mean and 95% confidence interval) at pretreatment baseline (day 1), treatment day 4 and day 14/follow‐up. formula image AZD5069, formula image Placebo. Representative flow cytometry histograms for each of the neutrophil cell surface markers are shown (D). The general appearance of these flow cytometry histograms was not affected by the treatment or the time point in the study.
Figure 8
Figure 8
Changes in serum concentrations of the CXCR2 ligands A interleukin 8 (IL‐8), B growth‐related oncogene α (GROα), C epithelial‐derived neutrophil‐activating protein 78 (ENA‐78) and D granulocyte‐colony stimulating factor (G‐CSF) observed during treatment with AZD5069, compared with placebo, in human volunteers. The baseline, day 1 pretreatment concentrations and post‐treatment day14/follow‐up concentrations are also shown for each analyte. Subjects were administered either AZD5069 (100 mg bid) or placebo orally (bid). Peripheral blood samples were collected for mediator analysis at the indicated time points. The data represent the mean ± standard deviation of n = 28 subjects per arm. A. formula image AZD5069 (100 mg bid), formula image Placebo. B. formula image AZD5069 (100 mg bid), formula image Placebo. C. formula image AZD5069 (100 mg bid), formula image Placebo. D. formula image AZD5069 (100 mg bid), formula image Placebo
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