The Development of a Simple and Fast Bioassay for Potency Testing of LPS for Bronchial Challenge Testing

A.J. Lexmond, I. Yilmaz, G.W. Clarke, C.P. Page, B.F. Forbes

Bronchial provocation with lipopolysaccharide (LPS) has been developed into a clinical challenge model to investigate the effect of novel anti-inflammatory drugs under development for the treatment of diseases such as asthma and COPD that are associated with neutrophil recruitment into the lung. This model has been used for early proof of concept and dose ranging studies in both healthy volunteers and in smoking subjects. However, surprisingly there is no standardised method of LPS delivery to the lungs and several issues have to be addressed before it can be validated as a model to be used more effectively in drug development. These issues mostly relate to a lack of knowledge on the exact dose of LPS that is administered to the lungs in terms of amount and potency (endotoxin strength).
We have investigated whether a bioassay based on the human monocytic cell line (Mono Mac 6) can be developed into a simple and fast tool to determine LPS potency. We investigated various cell densities, LPS concentrations, and incubation times in a 96-well plate format to establish a dose-response relationship between LPS concentration and IL-6 release from the Mono Mac 6 cells. We found a linear dose-response relationship between 0.05–1 ng/mL LPS and IL-6 release at a cell density of 0.5*105 cells/mL and an incubation time of 4 h. The techniques are very simple and the assay can be performed within one day, which with further optimisation may be further shortened to a few hours. In conclusion, we have developed a bioassay that relates Mono Mac 6 IL-6 release to LPS concentration. Ongoing work focuses on reducing the variation between replicate measurements and testing the robustness of the assay using LPS from different strains.

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