Molecular characterization of eluxadoline as a potential ligand targeting mu-delta opioid receptor heteromers

Abstract

Eluxadoline, an orally active mixed μ opioid receptor (μOR) agonist δ opioid receptor (δOR) antagonist developed for the treatment of diarrhea-predominant irritable bowel syndrome, normalizes gastrointestinal (GI) transit and defecation under conditions of novel environment stress or post-inflammatory altered GI function. Furthermore, compared to loperamide, which is used to treat non-specific diarrhea, the effects of eluxadoline on GI transit occur over a wider dosage range. However, the mechanisms of action of eluxadoline are unclear. In this study, we compared the ability of eluxadoline and loperamide to activate G-protein- and β-arrestin-mediated signaling at μOR homomers or μOR-δOR heteromers in heterologous cells. We also examined the ability of both compounds to reduce castor oil induced diarrhea in wild type (WT) and mice lacking δOR. We find that eluxadoline is more potent than loperamide in eliciting G-protein activity and β-arrestin recruitment in μOR expressing cells. However, in cells expressing μOR-δOR heteromers, the potency of eluxadoline is higher, but its maximal effect is lower than that of loperamide. Moreover, in these cells the signaling mediated by eluxadoline but not loperamide is reduced by μOR-δOR heteromer-selective antibodies. We find that in castor oil-induced diarrhea eluxadoline is more efficacious compared to loperamide in WT mice, and δOR appears to play a role in this process. Taken together these results indicate that eluxadoline behaves as a potent μOR agonist in the absence of δOR, while in the presence of δOR eluxadoline's effects are mediated through the μOR-δOR heteromer.

Keywords: Anti-diarrhea; Eluxadoline; Irritable bowel syndrome; Loperamide; μOR-δOR heteromer.

Fig. 1

Effect of DAMGO, loperamide and eluxadoline on G-protein activation. (A–C) Membranes (10 μg) from spinal cords of WT, μOR^−/− and δOR^−/− mice were subjected to a [³⁵S]GTPγS binding assay using DAMGO (A), loperamide (B), and eluxadoline (C) (0–10 μM final concentration) as described in Section 2. (D–G) Membranes (20 μg) from the ileum of WT mice were subjected to a [³⁵S]GTPγS binding assay using DAMGO (D and G), loperamide (E and G), and eluxadoline (F and G) (0–10 μM final concentration) in the presence or absence of TIPPψ (10 nM final concentration) as described in Section 2. (G) Represents E_max (% of basal) obtained with 10 μM final concentration of DAMGO (±10 nM final concentration of TIPPψ), eluxadoline or loperamide. Basal values determined in the absence of the agonist were taken as 100%. Results are the mean ± S.E.M. n= 3–9. n.d., Not determined. *p < 0.05; **p < 0.01, Dunnett’s test.

Fig. 2

Effect of DAMGO, loperamide and eluxadoline on β-arrestin recruitment. Cells (5000/well) expressing either μ^βgalOR (A–C) or μ^βgalOR-δOR (D–F) were treated with either DAMGO (A and D), loperamide (B and E), eluxadoline (C and F) (0–10 μM final concentration) in the absence or presence of the δOR antagonist, TIPPψ (10 nM final concentration) for 60 min at 37 °C and β-arrestin recruitment was measured as described in Section 2. Results are the mean ± S.E.M. n = 4–12. *p < 0.05, **p < 0.01, vs. absence of TIPPψ, t-test.

Fig. 3

Effect of μOR-δOR heteromer-selective antibody on eluxadoline-mediated signaling. Cells (5000 cells) expressing either μ^βgalOR (A) or μ^βgalOR-δOR (B) were treated with either deltorphin II (Delt II), loperamide or eluxadoline (1 μM final concentration) in the absence or presence of antibodies (Ab, 1 μg/well) to either μOR, μOR-δOR heteromer or CB1R-AT1R heteromer for 60 min at 37 °C and β-arrestin recruitment was measured as described in Section 2. Results are the mean ± S.E.M. n = 4. *p < 0.05, **p < 0.01, vs. no Ab treatment for each group, Dunnett’s test.

Fig. 4

Effect of loperamide and eluxadoline on castor oil-induced diarrhea. Diarrhea was induced by oral administration of castor oil (0.6 ml/mouse) in WT (A) or δOR^−/− (B) mice. Stools were scored for diarrhea (0 = normal; 1 = wet and irregular shape; or 2 = shapeless) for 4 has described in Section 2. Loperamide and eluxadoline (5 or 10 mg/kg) were administered orally 15 min before the castor oil administration. Naltrexone (10 mg/kg, i.p.) was administered 20 min before loperamide or eluxadoline administration. Results are the mean ± S.E.M. n = 3–6. **p < 0.01, vs. vehicle control; ##p < 0.01, vs. castor oil alone; $$p < 0.01, vs. castor oil + loperamide (10 mg/kg, p.o.); ††p < 0.01, vs. castor oil + eluxadoline (10 mg/kg, p.o), Student–Newman–Keuls test.

Fig. 5

Effect of loperamide and eluxadoline on castor oil-induced diarrhea (fecal output). Diarrhea was induced by oral administration of castor oil (0.6 ml/mouse) in WT (A) or δOR^−/− (B) mice. Stools were collected and weighed during 4 h as described in Section 2. Loperamide and eluxadoline (5 or 10 mg/kg) were administered orally 15 min before the castor oil administration. Naltrexone (10 mg/kg, i.p.) was administered 20 min before loperamide or eluxadoline administration. Results are the mean ± S.E.M. n = 3–6. **p < 0.01, vs. vehicle control; ##p < 0.01, vs. castor oil alone; $$p < 0.01, vs. castor oil + loperamide (10 mg/kg, p.o.); ††p < 0.01, vs. castor oil + eluxadoline (10 mg/kg, p.o), Student–Newman–Keuls test.

Fig. 6

Effect of loperamide and eluxadoline on castor oil-induced body weight change. Diarrhea was induced by oral administration of castor oil (0.6 ml/mouse) in WT (A) or δOR^−/− (B) mice. Body weight was measured before and 4 h after castor oil administration. Loperamide and eluxadoline (5 or 10 mg/kg) were administered orally 15 min before the castor oil administration. Naltrexone (10 mg/kg, i.p.) was administered 20 min before loperamide or eluxadoline administration. Results are the mean ± S.E.M. n = 3–6. **p < 0.01, vs. vehicle control; ##p < 0.01, vs. castor oil alone; $$p < 0.01, vs. castor-oil + loperamide (10 mg/kg, p.o.); †p < 0.05, ††p < 0.01, vs. castor oil + eluxadoline (10 mg/kg, p.o), Student–Newman–Keuls test.

Fig. 7

Effect of chronic treatment of loperamide and eluxadoline on body weight (A) and on receptor expression levels in ileal longitudinal muscle (B). Mice were treated with loperamide or eluxadoline (10 mg/kg, p.o., once a day for 5 days), or with 0.5% methylcellulose (0.1 ml/10 g; vehicle). (A) Body weight was measured immediately before the daily administration. Results are the mean ± S.E.M. n= 6–7. (B) On 6th day, ileum was collected (3–4 mice/sample). Membranes (10 μg) from mouse ileal longitudinal muscle (containing the myenteric plexus) were subjected to an ELISA assay in as described in Section 2. Tissues from 3 to 4 individual animals were pooled and collected as one sample. ELISA was performed in triplicate. Results are the mean ± S.E.M. n = 5.