Eliapixant is a selective P2X3 receptor antagonist for the treatment of disorders associated with hypersensitive nerve fibers

Abstract

ATP-dependent P2X3 receptors play a crucial role in the sensitization of nerve fibers and pathological pain pathways. They are also involved in pathways triggering cough and may contribute to the pathophysiology of endometriosis and overactive bladder. However, despite the strong therapeutic rationale for targeting P2X3 receptors, preliminary antagonists have been hampered by off-target effects, including severe taste disturbances associated with blocking the P2X2/3 receptor heterotrimer. Here we present a P2X3 receptor antagonist, eliapixant (BAY 1817080), which is both highly potent and selective for P2X3 over other P2X subtypes in vitro, including P2X2/3. We show that eliapixant reduces inflammatory pain in relevant animal models. We also provide the first in vivo experimental evidence that P2X3 antagonism reduces neurogenic inflammation, a phenomenon hypothesised to contribute to several diseases, including endometriosis. To test whether eliapixant could help treat endometriosis, we confirmed P2X3 expression on nerve fibers innervating human endometriotic lesions. We then demonstrate that eliapixant reduces vaginal hyperalgesia in an animal model of endometriosis-associated dyspareunia, even beyond treatment cessation. Our findings indicate that P2X3 antagonism could alleviate pain, including non-menstrual pelvic pain, and modify the underlying disease pathophysiology in women with endometriosis. Eliapixant is currently under clinical development for the treatment of disorders associated with hypersensitive nerve fibers.

Figure 1

Eliapixant (BAY 1817080) is a potent antagonist of P2X3 in vitro. (A) Representative manual patch clamp traces for human P2X3 (left panel), human P2X2/3 (middle panel), and representative whole-cell patch-clamp concentration–response curves for eliapixant using recombinant cell lines expressing human P2X3 or P2X2/3. Responses to an EC₈₀ concentration of α,β-meATP agonist (10 µM for hP2X3 receptors and 30 μM for human P2X2/3 receptors) are shown (human P2X3, n = 3–6 cells; P2X2/3, n = 3–10 cells). Data are expressed as the mean ± SEM. (B) Representative manual patch clamp traces for eliapixant against native rat dorsal root ganglion (DRG, L6–L4) neurons showing predominantly P2X3-like currents (left panel), and rat nodose ganglion (NDG) currents reflecting a majority of P2X2/3 channels (middle panel), dissected from female Sprague Dawley rats. Representative whole-cell patch-clamp concentration–response curves are shown in the right panel. Responses to the α,β-meATP agonist (10 µM) were measured (DRG, n = 3–6 cells; NDG, n = 3–5 cells). Data are expressed as the mean ± SEM. (C) Representative concentration–response curves for eliapixant against recombinant 1321N1 cell lines expressing human P2X3 homotrimers and human P2X2/3 heterotrimers in a fluorescence imaging plate reader (FLIPR) calcium-flux assay. Responses normalized to an EC₈₀ concentration of the agonist, α,β-meATP, based on the increase in peak relative fluorescence units (RFU) are shown (mean ± standard error of the mean [SEM], n = 3). Representative FLIPR traces for human P2X3 and human P2X2/3 3 are shown in supplementary Fig. 2. (D) Representative concentration–response curves for eliapixant against recombinant HEK T-REx cell lines expressing rat P2X3 and rat P2X2/3 in a FLIPR calcium-flux assay (mean ± SEM, n = 3). Representative FLIPR traces for rat P2X3 and rat P2X2/3 are shown in supplementary Fig. 2.

Figure 2

Eliapixant (BAY 1817080) reverses hyperalgesia in rodent models of inflammatory pain. (A) Dose-dependent efficacy of eliapixant on Complete Freund’s Adjuvant (CFA)-induced mechanical hyperalgesia in male Sprague Dawley rats (n = 8 per experimental group). All rats were orally administered with the test substances 24 h after CFA injection into one hind paw. Increasing pressure was applied to the hind paw until a behavioral response (paw withdrawal) was observed. The pressure at which the behavioral response occurred was recorded as the “Paw Withdrawal Threshold” (PWT), and was measured at 2, 4, and 6 h after test substance dosing. The mean ± standard deviation (SD) of the behavioral responses for the groups are shown; *P < 0.05, ***P < 0.001, ****P < 0.0001 versus vehicle (0.5% carboxymethylcellulose [CMC]:Tween 80), Dunnett’s post hoc test. (B) Dose-dependent efficacy of eliapixant on CFA-induced hyperalgesia in female C57BL/6 mice (n = 9 per experimental group). Compounds were administered orally, twice daily, starting one hour before CFA injection. Mechanical hyperalgesia was assessed using von Frey filaments, which were used to stimulate the hind paw. The strength of the von Frey filament used to stimulate the paw was expressed in [g], and the threshold was recorded when a response of the animal was observed. Response thresholds were determined 48 h after CFA injection. The mean ± SD of the behavioral responses for the groups are shown; *P < 0.05, **P < 0.001 versus vehicle (0.5% CMC:Tween 80), Dunnett’s post hoc test.

Figure 3

Eliapixant (BAY 1817080) shows efficacy in a rat model of uterine neurogenic inflammation. Female Sprague Dawley rats in pro-estrus phase were treated with either a bolus intravenous injection of eliapixant (0.7 mg/kg), which was followed by an intravenous infusion of 0.2 mg/kg eliapixant or the vehicle (saline) (n = 10 or 12, respectively). Inflammation of the uterus (induced by mustard oil) in rats results in blue dye extravasation (or blue dots) on the ventral and dorsal side of the abdominal region. The individual number of dots in the skin and the mean ± standard deviation for each group is shown; ** P < 0.01 versus the mustard oil/vehicle group, Mann–Whitney U test.

Figure 4

P2X3 is expressed on nerve fibers in human endometriotic lesions. Endometrial tissue samples were stained with antibodies for P2X3 and for the neuroendocrine marker, PGP9.5. Nerve fibers are stained green using PGP9.5 as a marker protein. P2X3 is stained red. The merged image shows the overlay of the two fluorescence signals showing P2X3 receptor expression (red) and the expression of PGP9.5 (green) which overlap spatially in the nerve fibers (yellow). Immunofluorescence images were obtained under a tenfold magnification.

Figure 5

Eliapixant (BAY 1817080) shows efficacy in a rat model of dyspareunia. (A) In female Sprague Dawley rats, the visceromotor response (VMR) to vaginal distension (inflated vaginal balloon) was measured by counting the number of abdominal muscle contractions in conscious animals as an objective measure of vaginal sensitivity (individual records of VMR response to vaginal distension of one rat after vehicle and eliapixant, week six post-implantation). (B) Eliapixant (15 mg/kg) or the vehicle (0.5% carboxymethylcellulose [CMC]:Tween 80; 5 mL/kg) were dosed orally, twice daily in female rats (n = 15/17 per experimental group), during two consecutive weeks, from week four to week five post-implantation of the uterine horn pieces. VMR/vaginal distension tests were performed at week five (on-drug) and week six (off-drug) post-implantation when animals were in the pro-estrus phase. The individual area under the curve (AUC, determined from a plot of the cumulative number of abdominal contractions against vaginal distension volume for each animal) and the mean ± SD for each group is shown. One outlier was removed from the eliapixant treatment group at week five (Grubb’s test); ^#P < 0.05, ^##P < 0.01 versus vehicle (0.5% CMC:Tween 80), Mann–Whitney U test.