Peripherally Restricted, Highly Potent, Selective, Aqueous-Soluble EP2 Antagonist with Anti-Inflammatory Properties

Abstract

The prostaglandin E₂ receptor, EP2, plays an important role in physiology and in a variety of pathological conditions. Studies indicate that EP2 is pro-inflammatory in chronic peripheral and central nervous system disease and cancer models. Thus, targeting the EP2 receptor with small molecules could be a therapeutic strategy for treating inflammatory diseases and cancer. We recently reported a novel class of competitive antagonists of the EP2 receptor. However, earlier leads displayed low selectivity against the DP1 prostanoid receptor, moderate plasma half-life, and low aqueous solubility, which renders them suboptimal for testing in animal models of disease. We now report a novel compound TG8-69, which has suitable drug-like properties. We present synthesis, lead-optimization studies, pharmacological characterization, and anti-inflammatory properties of this compound that support its use in chronic peripheral inflammatory diseases, including rheumatoid arthritis, endometriosis, and cancer, in which EP2 appears to play a pathogenic role.

Keywords: EP2 antagonist; anti-inflammatory; aqueous-solubility; cytokines; lead-optimization; tetrazole.

Figure 1.

Earlier lead compound TG8–15 led to the synthesis of preclinical lead compound TG8–69.

Figure 2.

EP2 potency of TG8–69 and PF-04418948. (A) TG8–69 inhibited PGE₂-induced EP2 receptor activation in a concentration-dependent manner. Data were normalized to percent maximum response to PGE₂ in the absence of antagonist; data points represent mean ± SEM from one experiment run in triplicate, the concentration-response experiment was repeated four times in triplicate. (B) TG8–69 displayed competitive antagonism of the EP2 receptor as shown by Schild regression analysis with mean K_B = 48.5 nM and slope = 1.2. The Schild plots from four independent experiments are shown. (C) PF-04418948 similarly inhibited PGE₂-induced EP2 receptor activation in a concentration-dependent manner and this experiment was repeated three times (D). The Schild plots from three independent experiments are shown. The mean Schild K_B = 147 nM and slope = 1.5 was found for PF-04419848.

Figure 3.

(A) Inhibition of H³-PGE₂ binding to EP2 receptors by TG8–69. (B) TG8–69 showed >10,000 nM Schild potency in the TR-FRET assay for other Gas-coupled prostanoid receptors, DP1, EP4, and IP. The K_Bs are estimated from fold changes to agonist EC₅₀ caused by 10 uM compound. Agonists were PGE₂ for EP2 and EP4, BW245C for DP1, and iloprost for IP (n = 3–4 repeats in duplicate).

Figure 4.

TG8–69 displayed high stability in liver microsomes and low off-target inhibition of most CYP450 enzymes and potassium channel hERG. (A) TG8–69 displayed >60 minutes half-life in pooled liver microsomal fractions. 1 μM compound was incubated in 0.5 mg/mL liver microsomes for the indicated time and the TG8–69 levels were measured by LC-MS/MS. (B). Inhibition of ligand binding to various CYP450 enzymes and fiERG. TG8–69 (10 μM) showed < 30 % inhibition of six out of seven CYP450s tested, and no inhibition of hERG.

Figure 5:

Plasma pharmacokinetics of TG8–69. TG8–69 was dissolved in N-methyl-2-pyrrolidone (NMP) (5%), solutol-HS-15 (5%) and saline (90%) and injected to mice via tail-vein intravenous (i.v.) or oral gavage (p.o.) routes. The blood was harvested and the compound extracted and analyzed by LCMS/MS.

Figure 6.

Mean fold change in mRNA expression of cytokines in BV2-hEP2 cells upon TG8–69, Ono and LPS treatment. 200,000 cells/well were grown overnight and treated with vehicle or TG8–69 for 1 hr, then vehicle or ONO-AE1-259-01 (abbreviated to Ono in the Figure 6 and 7) for 1 hr, then vehicle or LPS for 2 hrs. Analyte mRNAs were measured by qRT-PCR. For statistical analysis, AACT values were used as they were normally distributed compared to fold changes. ANOVA-with Holm-Sidak multiple comparisons test for post-hoc analysis was used. P values were considered significant at *<0.05. Experimental repeats n = 7 for COX-2, IL-1β and IL-6; n = 5 for hEP2.

Figure 7:

Mean fold change in mRNA expression of cytokines in BV2-hEP2 cells upon TG8–69 and LPS treatment. Cells were grown overnight and treated with vehicle or TG8–69 (1 μM) for 1 hr and vehicle or LPS (100 ng/ml) for 2 hrs. Analyte mRNAs were measured by qRT-PCR. For statistical analysis, ΔΔCT values were used as they were normally distributed compared to fold changes. Student t-test was used to compare among the LPS treated and untreated groups. P values were found to be non-significant (>0.05) for all the comparisons. n = 3 experimental repeats.

Scheme 1.

Synthesis of EP2 antagonists (Detailed procedures are provided in SI).