KMN-159, a novel EP4 receptor selective agonist, stimulates osteoblastic differentiation in cultured whole rat bone marrow.

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.