In Vitro Pharmacological Profile of a New Small Molecule Bradykinin B2 Receptor Antagonist

Abstract

We here report the discovery and early characterization of Compound 3, a representative of a novel class of small molecule bradykinin (BK) B 2 receptor antagonists, and its superior profile to the prior art B 2 receptor antagonists Compound 1 and Compound 2. Compound 3, Compound 2, and Compound 1 are highly potent antagonists of the human recombinant B 2 receptor (K b values 0.24, 0.95, and 1.24 nM, respectively, calcium mobilization assay). Compound 3 is more potent than the prior art compounds and icatibant in this assay (K b icatibant 2.81 nM). The compounds also potently inhibit BK-induced contraction of endogenous B 2 receptors in a human isolated umbilical vein bioassay. The potencies of Compound 3, Compound 2, Compound 1, and icatibant are (pA 2 values) 9.67, 9.02, 8.58, and 8.06 (i.e. 0.21, 0.95, 2.63, and 8.71 nM), respectively. Compound 3 and Compound 2 were further characterized. They inhibit BK-induced c-Fos signaling and internalization of recombinant human B 2 receptors in HEK293 cells, and do not antagonize the venous effects mediated by other G protein-coupled receptors in the umbilical vein model, including the bradykinin B 1 receptor. Antagonist potency of Compound 3 at cloned cynomolgus monkey, dog, rat, and mouse B 2 receptors revealed species selectivity, with a high antagonist potency for human and monkey B 2 receptors, but several hundred-fold lower potency for the other B 2 receptors. The in vitro off-target profile of Compound 3 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B 1 receptor. Compound 3 showed a lower intrinsic clearance in the microsomal stability assay than the prior art compounds. With an efflux ratio of 1.0 in the Caco-2 permeability assay Compound 3 is predicted to be not a substrate of efflux pumps. In conclusion, we discovered a novel chemical class of highly selective and very potent B 2 receptor antagonists, as exemplified by Compound 3. The compound showed excellent absorption in the Caco-2 assay, predictive of good oral bioavailability, and favourable metabolic stability in liver microsomes. Compound 3 has provided a significant stepping stone towards the discovery of the orally bioavailable B 2 antagonist PHA-022121, currently in phase 1 clinical development.

A Robust Bioassay of the Human Bradykinin B2 Receptor That Extends Molecular and Cellular Studies: The Isolated Umbilical Vein

“…The structure of selected small molecule antagonists developed since 1994 by various industrial organizations are illustrated in Figure 2. A few that have reached clinical trials have not been introduced in therapeutics (anatibant, fasitibant, possibly FK3657) [36]. Except the low potency phosphonium prototype WIN 64338 [66], significant structural commonalities ("chemotypes") can be found in this series, and this line of investigation has led to the definition of a minimal pharmacophore for small molecule B2R antagonists [67].…”

“…When given subcutaneously, the peptide icatibant aborts or limits attacks of HAE of type I and type II and also attacks in patients with normal C1 inhibitor (HAE-nC1 INH) [32,34]. An orally bioavailable class of B2R antagonists is currently developed [36] (see below) and the bioassay based on the human umbilical vein has a particular relevance in this context. Table 2.…”

A Systematic Review of Molecular Imaging Agents Targeting Bradykinin B1 and B2 Receptors

“…However, their development was discontinued due to the lack of efficacy. The hope is that new scaffolds will be able to find clinical utility in other disease settings [ 27 ].…”

In Vitro Modeling of Bradykinin-Mediated Angioedema States

“…C1-INH replenishment and various strategies to inhibit plasma kallikrein, FXII or fibrinolysis have also been or are being developed [ 27 ]. An orally bioavailable small molecule antagonist of the BK B 2 receptor is also at an early stage of development [ 28 ].…”