Comparison of different lots of endotoxin and evaluation of in vivo potency over time in the experimental human endotoxemia model

Abstract

The experimental human endotoxemia model is used to study the systemic inflammatory response in vivo. The previously used lot of endotoxin, which was used for over a decade, is no longer approved for human use and a new Good Manufacturing Practices-grade batch has become available. We compared the inflammatory response induced by either bolus or continuous administration of either the previously used lot #1188844 or new lots of endotoxin (#94332B1 and #94332B4). Compared with lot #1188844, bolus administration of lot #94332B1 induced a more pronounced systemic inflammatory response including higher plasma levels of pro-inflammatory cytokines and more pronounced clinical signs of inflammation. In contrast, continuous infusion of lot #94332B4 resulted in a slightly less pronounced inflammatory response compared with lot #1188844. Furthermore, we evaluated whether lot #1188844 displayed in vivo potency loss by reviewing inflammatory parameters obtained from 17 endotoxemia studies performed in our centre between 2007 and 2016. Despite inter-study variability in endotoxemia-induced effects on temperature, heart rate, symptoms, and leukocyte counts, the magnitude of these effects did not decrease over time. In conclusion, although all lots of endotoxin induce a pronounced inflammatory response, the magnitude differs between lots. We observed no potency loss of endotoxin over time.

Keywords: Endotoxemia; lipopolysaccharide; potency; systemic inflammation.

Conflict of interest statement

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.

Plasma cytokine/chemokine levels upon bolus administration of endotoxin of lot #1188844 (n = 10) and lot #94332B1 (n = 8). Plasma concentrations (pg/ml) of TNF-α, IL-6, IL-8, IL-10, MCP-1, MIP-1α, and MIP-1β are depicted over time. At t = 0 endotoxin was administered at a dose of 2 ng/kg. Data are expressed as mean ± SEM. Differences between groups were evaluated using two-way ANOVA on log transformed data and interaction term (time × group) P-values are displayed.

Figure 2.

Plasma cytokine/chemokine levels upon continuous administration of endotoxin of lot #1188844 (n = 10) and lot #94332B4 (n = 20). Plasma concentrations (pg/ml) of TNF-α, IL-6, IL-8, IL-10, MCP-1, MIP-1α, and MIP-1β are depicted over time. At t = 0 endotoxin was administered at a dose of 1 ng/kg, followed by a continuous infusion of 1 ng/kg/h for 3 h, as indicated by the grey bar. Data are expressed as mean ± SEM. Differences between groups were evaluated using two-way ANOVA on log transformed data and interaction term (time × group) P-values are displayed.

Figure 3.

Clinical parameters of systemic inflammation upon bolus administration endotoxin of lot #1188844 (n = 10) and lot #94332B1 (n = 8). Changes over time of (a) symptoms (arbitrary units), (b) temperature (°C), (c) heart rate (beats per min [bpm]), and (d) mean arterial pressure (mmHg) are depicted. At t = 0 endotoxin was administered at a bolus dose of 2 ng/kg. Data are expressed as mean ± SEM. Differences between groups were evaluated using two-way ANOVA and interaction term (time × group) P-values are displayed.

Figure 4.

Clinical parameters of systemic inflammation upon continuous administration of endotoxin of lot #1188844 (n = 10) and lot #94332B4 (n = 20). Changes over time of (a) symptoms (arbitrary units), (b) temperature (°C), (c) heart rate (beats per min [bpm]), and (d) mean arterial pressure (mmHg) are depicted. At t = 0 endotoxin was administered at a dose of 1 ng/kg, followed by a continuous infusion of 1 ng/kg/h for 3 h, as indicated by the grey bar. Data are expressed as mean ± SEM. Differences between groups were evaluated using two-way ANOVA and interaction term (time × group) P-values are displayed.

Figure 5.

Circulating neutrophil (a), monocyte (b), and lymphocyte (c) numbers upon bolus administration of endotoxin of lot #1188844 (n = 10) and #94332B1 (n = 8). At t = 0 endotoxin was administered at a bolus dose of 2 ng/kg. Data are expressed as mean ± SEM. Differences between groups were evaluated using two-way ANOVA and interaction term (time × group)P-values are displayed.

Figure 6.

Circulating neutrophil (a), monocyte (b), and lymphocyte (c) numbers upon continuous administration of endotoxin of lot #1188844 (n = 10) and #94332B4 (n = 20). At t = 0 endotoxin was administered at a dose of 1 ng/kg, followed by a continuous infusion of 1 ng/kg/h for 3 h, as indicated by the grey bar. Data are expressed as mean ± SEM. Differences between groups were evaluated using two-way ANOVA and interaction term (time × group) P-values are displayed.

Figure 7.

In vivo potency of endotoxin administration of lot #1188844 over time. Clinical parameters of systemic inflammation induced by bolus administration of 2 ng/kg lot #1188844 in studies performed in our center from 2007 to 2016 (in black) and novel lot #94332B1 in 2017 (in red) are depicted. Maximum temperature increase (Δ temperature) (a), peak heart rate (b), peak leukocytes (c), and peak symptoms (d).In vitro potency as determined by LAL tests between 2007 and 2015 for lot #1188844, and for the #94332B1 lot in 2017 (e). Data are expressed as mean±SEM. Linear regression analysis was performed, and the slopes (R) with 95% confidential intervals andP-values are reported.