Preclinical MRGPRX2, Mrgprb2, and Mast Cell Data
We examined whether ISRs may be caused by activation of mast cells
through MRGPRX2. 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 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 showed that the half maximal effective concentration
(EC50) was 63 ± 13µg/mL (Figure 1B), well under the
80mg/mL injection concentration used for human administration,
suggesting that this elamipretide concentration readily activates skin
mast cells. 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). Evans Blue
assay was used to examine skin inflammation after in vivoadministration of elamipretide because it binds to albumin in blood.
Intravenous Evans Blue labels albumin blue, and when albumin-containing
fluid escapes from the bloodstream and into tissues after mast cell
degranulation, tissue become dye filled. SC administration of
elamipretide into wild type mouse hind-paws triggered immediate tissue
swelling and fluid extravasation, reminiscent of ISRs, and was reduced
by ~80% in Mrgprb2 knockout mice, demonstrating a due
to Mrgprb2 activation of mast cells (Figure 4).