Low-dose endotoxin inhalation in healthy volunteers--a challenge model for early clinical drug development

Abstract

Background: Inhalation of endotoxin (LPS) induces a predominantly neutrophilic airway inflammation and has been used as model to test the anti-inflammatory activity of novel drugs. In the past, a dose exceeding 15-50 μg was generally needed to induce a sufficient inflammatory response. For human studies, regulatory authorities in some countries now request the use of GMP-grade LPS, which is of limited availability. It was therefore the aim of this study to test the effect and reproducibility of a low-dose LPS challenge (20,000 E.U.; 2 μg) using a flow- and volume-controlled inhalation technique to increase LPS deposition.

Methods: Two to four weeks after a baseline sputum induction, 12 non-smoking healthy volunteers inhaled LPS on three occasions, separated by at least 4 weeks. To modulate the inflammatory effect of LPS, a 5-day PDE4 inhibitor (Roflumilast) treatment preceded the last challenge. Six hours after each LPS inhalation, sputum induction was performed.

Results: The low-dose LPS inhalation was well tolerated and increased the mean percentage of sputum neutrophils from 25% to 72%. After the second LPS challenge, 62% neutrophils and an increased percentage of monocytes were observed. The LPS induced influx of neutrophils and the cumulative inflammatory response compared with baseline were reproducible. Treatment with Roflumilast for 5 days did not have a significant effect on sputum composition.

Conclusion: The controlled inhalation of 2 μg GMP-grade LPS is sufficient to induce a significant neutrophilic airway inflammation in healthy volunteers. Repeated low-dose LPS challenges potentially result in a small shift of the neutrophil/monocyte ratio; however, the cumulative response is reproducible, enabling the use of this model for "proof-of-concept" studies for anti-inflammatory compounds during early drug development.

Trial registration: ClinicalTrials.gov NCT01400568.

Figures

Figure 1

Study design. LPS: inhalation of 2 μg (20,000 E.U.) nebulized Lipopolysaccharide. Treat: Oral administration (500 μg/day) of the PDE-4 inhibitor Roflumilast. FACS: Flow cytometry of sputum cells was performed. In a separate study performed >56 days after the end of the LPS challenge trial, 11 subjects underwent a follow-up sputum induction. (Visits 4, 6, and 9 refer to phone calls done 24 h after the respective challenges).

Figure 2

Procedures performed on challenge days LPS 1, LPS 2 and LPS Tx (Figure 1). FEV1 = lung function measurement, EBT = exhaled breath temperature, BT = body temperature, LPS = low dose lipopolysaccharide challenge (20,000 E.U.), 1 blood samples were not taken at visit 3 (LPS1). Lung function was also monitored by a portable AM1 detector hourly for 6 h, as well as 9, 11, 13, and 24 h after LPS challenge. Lung function results and the subject’s symptoms at 24 h were assessed by phone call.

Figure 3

Sputum neutrophils (left) and the sum of sputum monocytes and small macrophages (right). Individual data and mean values of percent sputum leukocytes are displayed. BL: baseline, LPS 1: first LPS challenge, LPS 2: second LPS challenge at least 4 weeks after LPS 1, LPS Tx: third LPS challenge at least 4 weeks after LPS 2 and after 5 days of treatment with Roflumilast (500 μg /day). For statistical details please refer to Table 2. ** p < 0.01, *** p < 0.001 compared with baseline; # p < 0.05 compared with LPS 1.

Figure 4

Concentrations of IL-8 and MPO in sputum supernatants. Individual data and mean values of log transformed values are displayed. BL: baseline, LPS 1: first LPS challenge, LPS: second LPS challenge at least 4 weeks after LPS 1, LPS Tx: third LPS challenge at least 4 weeks after LPS 2 and after 5 days of treatment with roflumilast (500 μg /day). For statistical details please refer to Table 2. *** p < 0.001 compared with baseline.

Figure 5

Reproducibility of the low dose LPS induced inflammatory response (LPS 1 vs. LPS 2). Top row (A, B): Change in the percentage of sputum neutrophils compared with baseline. Bottom row (C, D): Change in the percentage of the sum of neutrophils, monocytes and small macrophages compared with baseline. On the left (A, C) Bland-Altman plots with lines indicating the mean and 2*SD of the differences and on the right (B, D) the respective correlations with the line of identity.