Host: The Japanese Society of Toxicology
Name : The 51st Annual Meeting of the Japanese Society of Toxicology
Date : July 03, 2024 - July 05, 2024
Degranulation of mast cells is caused by two distinct pathways: IgE-dependent and IgE-independent pathway. IgE-independent reaction is referred to as "pseudo-allergic" reaction, distinguishing it from IgE-dependent allergic reactions. In recent years, Mas-related G-protein coupled receptor X2 (MRGPRX2) has been identified as a crucial receptor for pseudo-allergic reactions. Dogs, commonly used in nonclinical safety studies, are known to be highly sensitive to pseudo-allergic reactions. However, the functional ortholog of human MRGPRX2 in dogs has not been identified, and the mechanism behind the high sensitivity in dogs remains unknown. Since there are rarely high-sensitive subjects which show severe pseudo-allergic reactions in humans, while research on human individual susceptibility differences is being investigated, little is known about the factors underlying human hypersensitivity. Therefore, elucidating the mechanism of high sensitivity in dogs would contribute to the advancement of research on hypersensitivity mechanisms in humans. Additionally, species differences in sensitivity to pseudo-allergic reactions make it challenging to extrapolate histamine release reactions observed in nonclinical studies to humans, highlighting the need to establish an assay system that can evaluate species differences.
The aim of this study was to identify the mechanism of sensitivity differences in MRGPRX2-related reactions and establish an assay system. First, we attempted to determine the functional ortholog in dogs. Through canine tissue analysis, canine MRGPRX2 was found to be expressed in limited tissues including the skin, and connective tissue-type mast cells residing in the skin expressed canine MRGPRX2. In evaluations using receptor-expressing cells, histamine-releasing agents activated canine MRGPRX2-expressing cells. Based on these results, canine MRGPRX2 was identified as the functional ortholog of human MRGPRX2. Next, we investigated the mechanism of high sensitivity in dogs from the perspective of receptor structure. The important residues involved in receptor binding were estimated by homology modeling and docking simulations, and reactivity of the mutant variants against MRGPRX2 ligands was evaluated with the variant-expressing cells. As a result, we identified residues M109 and F78 in human MRGPRX2 as contributing to high sensitivity to fluoroquinolones. This led to the first identification of gain-of-function variants in the transmembrane region of human MRGPRX2. Finally, we verified the usefulness of an assay system with MRGPRX2-expressing cells for evaluation of species differences using a launched drug, valemetostat, which induced histamine release in dogs during nonclinical safety studies. As a result, the sensitivity of human MRGPRX2-expressing cells to valemetostat was significantly lower compared to canine MRGPRX2-expressing cells, indicating the presence of a species difference in sensitivity to valemetostat-induced histamine release. Indeed, no increase in blood histamine levels was observed in clinical trials with valemetostat. Notably, the EC50 obtained in this study using MRGPRX2-expressing cells showed a correlation with the plasma concentration at which histamine release occurred in dogs, and it was also consistent with the results in clinical trials. This assay system would be useful for mechanistic analysis of histamine release reactions observed in nonclinical studies and extrapolation to humans.
