Sickle cell vaso-occlusion causes activation of iNKT cells that is decreased by the adenosine A2A receptor agonist regadenoson

Abstract

Adenosine A2A receptor (A2AR) agonists reduce invariant natural killer T (iNKT) cell activation and decrease inflammation in sickle cell disease (SCD) mice. We conducted a phase 1 trial of the A2AR agonist regadenoson in adults with SCD. The target dose was 1.44 μg/kg/h. iNKT cell activation was evaluated using antibodies targeting the p65 subunit of nuclear factor-κB (phospho-NF-κB p65), interferon-γ (IFN-γ), and A2AR. Regadenoson was administered to 27 adults with SCD. We examined 21 patients at steady state and 6 during painful vaso-occlusive crises (pVOC). iNKT cell activation was also measured in 14 African-American controls. During pVOC, the fraction of iNKT cells demonstrating increased phospho-NF-κB p65 and A2AR expression was significantly higher compared with controls (P < .01) and steady-state patients (P < .05). IFN-γ expression was also significantly higher compared with controls (P = .02). After a 24-hour infusion of regadenoson during pVOC, phospho-NF-κB p65 activation in iNKT cells decreased compared to baseline by a median of 48% (P = .03) to levels similar to controls and steady-state SCD. No toxicities were identified. Infusional regadenoson administered to adults with SCD at 1.44 μg/kg/h during pVOC decreases activation of iNKT cells without toxicity.

Trial registration: ClinicalTrials.gov NCT01085201.

Figures

Figure 1

Phospho-NF-κB p65, A2AR, and IFN-γ expression in iNKT cells of controls and patients in steady state and pVOC (stages 2 and 3, respectively) at time 0 (preinfusion). (A) Flow cytometry plot of iNKT cells from controls and patients in steady state and pVOC at time 0. (B) Phospho-NF-κB p65, A2AR, and IFN-γ expression in iNKT cells compared with CD3+ non-iNKT cells at time 0. Solid peaks represent expression in iNKT cells and dashed peaks represent expression in CD3+ non-iNKT cells. Activation gate is defined as increased expression relative to CD3+ non-iNKT cells. Percent of iNKT cells in the activation gate is reported. (C) iNKT cell inflammatory marker data at time 0 for controls (white boxes), patients in steady state (stage 2, hatched boxes), and pVOC (stage 3, dark gray boxes). Lower end of box denotes 25% of data, upper end of box denotes 75% of data, and median is the line within the box. Error bars are the lowest and highest data points within 1.5 times the interquartile range, and circles represent outliers.

Figure 2

Phospho-NF-κB p65 expression in iNKT cells in subjects who received a 1.44 μg/kg/h regadenoson infusion for 24 hours with a 6-hour observation period when no drug was infused. (A-B) Phospho-NF-κB p65 expression in iNKT cells in a participant examined during pVOC (stage 3). (A) Flow cytometry and iNKT cell gates in serial blood samples taken at the indicated times. Regadenoson was infused starting at time 0 and ending at 24 hours. (B) Phospho-NF-κB p65 expression in iNKT cells compared with CD3+ non-iNKT cells from a pVOC patient. Solid peak represents phospho-NF-κB p65 in iNKT cells before regadenoson. Dashed peak represents phospho-NFκB expression in CD3+ non-iNKT cells, and the shaded area is phospho-NF-κB p65 expression in iNKT at time points after regadenoson infusion. NF-κB activation gate is defined as increased expression of phospho-NF-κB p65 relative to CD3+ non-iNKT cells. Percent iNKT cells in the activation gate at time points after regadenoson infusion is reported. (C) Percent change in iNKT cell phospho-NF-κB p65 before and after a 24-hour infusion of regadenoson in patients in steady state (stage 2, hatched box) and pVOC (stage 3, dark gray box). (D) Phospho-NF-κB p65 expression in iNKT cells from African-American controls (white box), steady-state patients (stage 2, hatched box), and pVOC participants before and immediately after a 24-hour regadenoson infusion (stage 3, dark gray boxes).

Figure 3

Pharmacokinetics of regadenoson infused for 12 hours during stage 1. Plasma concentrations of regadenoson were measured at 0, 1, 6, 12, and 18 hours. Dose level 0 was 0.24 μg/kg/h (white boxes), dose level 1 was 0.60 μg/kg/h (hatched boxes), and dose level 2 was 1.44 μg/kg/h (dark gray boxes).