Preclinical MRGPRX2, Mrgprb2, and Mast Cell Data
We examined whether ISRs may be caused by activation of mast cells
through MRGPRX2. First, we found that HEK293 cells stably transfected
with MRGPRX2 showed massive calcium mobilization – a key component of
MRGPRX2-mediated intracellular signaling – after incubation with
elamipretide, as assessed by an increase in fluorescence intensity of
the calcium sensitive dye Fluo-4 (Figure 1A). Untransfected cells did
not demonstrate calcium mobilization. This was also observed in Chem-1
cells transfected with MRGPRX2 (Figure 2A), but not untransfected cells
(Figure 2B), confirming that the response was due to MRGPRX2 and that
elamipretide is an MRGPRX2 agonist. Quercetin, proposed to antagonize
MRGPRX2 signaling (Ding et al., 2019), inhibited elamipretide activation
of MRGPRX2 (Figure 2C). Neither M1 (Figure 2D) or M2 metabolites (Figure
2E) elicited calcium mobilization in MRGPRX2 cells, suggesting that only
the parent compound is responsible for mast cell activation. Using the
HEK293 cell line, we established that the half maximal effective
concentration (EC50) was 63 ± 13 µg/ml (Figure 1B), well
under the 80 mg/ml injection concentration used for human administration
and suggesting that elamipretide readily activates skin mast cells at
this concentration. Next, we found that elamipretide also activated
Mrgprb2, the mouse ortholog of MRGPRX2 (Figure 3), and triggered calcium
mobilization in wild type but not Mrgprb2 knockout primary mouse
peritoneal mast cells, demonstrating that mast cell activation was
Mrgprb2-specific (Figure 3B). Finally, we used the Evans Blue assay to
examine skin inflammation after in vivo administration of
elamipretide. Evans Blue is a dye that binds to albumin in the blood;
intravenous injection of Evans Blue thus labels albumin blue, and when
albumin-containing fluid escapes from the bloodstream and into tissues
after mast cell degranulation, the tissue becomes filled with dye. We
found that SC administration of elamipretide into wild type mouse
hindpaws triggered immediate tissue swelling and fluid extravasation,
reminiscent of ISRs, and that this was reduced by ~80%
in Mrgprb2 knockout mice, demonstrating that most of the reaction was
due to Mrgprb2 activation of mast cells (Figure 4).