Thomas A. Owen, Chandni Patel, Shanqiao Wei, Chi S. Ho, Kaylah Birmingham, Samuel Sanchez, Natalie Chung, Alexa Cahill, James P. O'Malley, Stephen D. Barrett, María Inés Morano

The selective EP₄ receptor agonist KMN-159 synthesized and developed by Cayman scientists induces osteogenesis in rat bone marrow cells and promotes healing in a rat calvarial defect model.

Highlights

• Because stimulation of the prostaglandin receptor EP₄ mediates bone formation by osteoblasts and resorption of existing bone by osteoclasts, an EP₄ agonist with the pharmacological characteristics desirable in a local therapeutic agent was developed to stimulate bone formation in local applications.
• EP receptor-specific agonists and antagonists were used to demonstrate that KMN-159 stimulates alkaline phosphatase enzyme activity in rat bone marrow in an EP₄-dependent manner.
• A short exposure to KMN-159 significantly enhanced the osteogenesis in rat bone marrow stem cells and was effective regardless of sex or age of the donor rat, stimulating these cells to secrete the factors necessary for regulating osteoclastogenesis.
• Ultimately, the osteogenic biological activity of KMN-159 was demonstrated by incorporating it into a calcium phosphate cement and applying it in a demineralized bone matrix to a bone defect made in rat calvaria where the healing rate was equivalent to that of BMP-2.

Abstract

KMN-159 is the lead compound from a series of novel difluorolactam prostanoid EP₄ receptor agonists aimed at inducing local bone formation while avoiding the inherent side effects of systemic EP₄ activation. KMN-159 is a potent, selective small molecule possessing pharmacokinetic properties amenable to local administration. Unfractionated rat bone marrow cells (BMCs) were treated once at plating with escalating doses of KMN-159 (1 pM to 10 μM). The resulting elevated alkaline phosphatase (ALP) levels measured 9 days post-dose are consistent with increased osteoblastic differentiation and exposure to KMN-159 at low nanomolar concentrations for as little as 30 min was sufficient to induce complete osteoblast differentiation of the BMCs from both sexes and regardless of age. ALP induction was blocked by an EP₄ receptor antagonist but not by EP₁ or EP₂ receptor antagonists and was not induced by EP₂ or EP₃ receptor agonists. Addition of BMCs to plates coated with KMN-159 24 days earlier resulted in ALP activation, highlighting the chemical stability of the compound. The expression of phenotype markers such as ALP, type I collagen, and osteocalcin was significantly elevated throughout the osteoblastic differentiation timecourse initiated by KMN-159 stimulation. An increased number of tartrate-resistant acid phosphatase-positive cells was observed KMN-159 or PGE₂ treated BMCs but only in the presence of exogenous receptor activator of nuclear factor kappa-Β ligand (RANKL). No change in the number of adipocytes was observed. KMN-159 also increased bone healing in a rat calvarial defect model with a healing rate equivalent to recombinant human bone morphogenetic protein-2. Our studies show that KMN-159 is able to stimulate osteoblastic differentiation with a very short time of exposure, supporting its potential as a therapeutic candidate for augmenting bone mass.

Read this paper

Learn about Cayman’s Medicinal Chemistry & Structural Biology Services