Elucidating Mechanisms of Toxicity Using Human Primary Cell Systems

Elucidating Mechanisms of Toxicity Using Human Primary Cell Systems

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Purpose: Our goal is to identify in vitro assays that can predict adverse effects in humans and help elucidate mechanisms of toxicity. We performed large scale screening of approved drugs and experimental chemicals across panels of human primary cell based assays and co-cultures to identify assay endpoints that may be associated with particular adverse drug reactions (ADRs). MEK inhibitors are associated with skin rash ADR, and the MEK inhibitors trametinib, pimasertib and selumetinib were found to share a number of in vitro activities. Some activities are related to increased risk of infection (inhibition of T cell responses and reduction in acute infl ammatory endpoints, TNFa, IL-1a and IL-8) while others are associated with chronic skin reactions (increased levels of the adhesion molecule VCAM-1 in a human primary cell fi broblast model of wound healing, the BioMAP HDF3CGF system). Interestingly, increased VCAM-1 is also observed with other drug classes reported to exhibit skin rash side effects: p38 MAPK inhibitors, human interferon beta, and the IL-1beta receptor antagonist, anakinra.

Methods: To characterize the signaling pathways regulating VCAM by these different drug classes, an analysis of the effects of PD-184352 (MEKi), BIRB-796 (p38 MAPKi), and anakinra on the protein phosphorylation states of various intracellular signaling kinases was performed at multiple time points. Results: Treatment with PD-184352 resulted in decreased levels of p-ERK (Thr185/Tyr187), and p-p38 (Thr180/Tyr182), but increased levels of p-MEK1 (Ser222) and p-STAT1 (Tyr707). BIRB-796 treatment led to decreased levels of phosphorylated p-p38 (Thr180/Tyr182) but increased levels of p-c-Jun (Ser73). Exposure to anakinra resulted in increased levels of p-HSP27 (Ser78) at 5 hr, but decreased p-AKT (Ser473) and p-TSC2 (Ser939) at 24 hr, demonstrating that the three different mechanisms are clearly distinguished by phosphomapping analysis.

Conclusions: We have identified VCAM-1 in a human primary fibroblast model of wound healing as a sentinel assay that may be useful for predicting skin side effects. Phosphomapping analysis in this system was able to distinguish compounds with different target mechanisms.