A Nonprostanoid EP4 Receptor Selective Prostaglandin E2 Agonist Restores Bone Mass and Strength in Aged, Ovariectomized Rats

Pfizer Global Research and Development, Groton Laboratories, Groton, Connecticut, USA.
Journal of Bone and Mineral Research (Impact Factor: 6.83). 05/2006; 21(4):565-75. DOI: 10.1359/jbmr.051110
Source: PubMed

ABSTRACT CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia.
The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 (PGE2) agonist, CP432, could produce bone anabolic effects in aged, ovariectomized (OVX) rats with established osteopenia.
CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to 12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone resorption, and bone strength were determined.
Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432 completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3 mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test of femoral shaft compared with both sham and OVX controls.
CP432 completely restored trabecular and cortical bone mass and strength in established osteopenic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical surfaces.

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Available from: Hua Z Ke, Sep 16, 2014
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    • "EP2 and EP4 receptor knockout mice have been shown to have impaired fracture healing and impaired bone resorption.(18,19) An EP2-selective agonist induced bone healing in beagles(20); similarly, an EP4-selective agonist has been used in a rat model of bone repair with positive effects.(21,22) Recent work in our laboratory showed that another EP4-selective agonist accelerated the delayed fracture healing in aged mice and compensated for the reduced fracture healing observed in COX-2−/− mice.(23,24) "
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    ABSTRACT: As a downstream product of cyclooxygenase 2 (COX-2), prostaglandin E(2) (PGE(2)) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro-computed tomographic (µCT), and biomechanical measures. EP1(-/-) mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1(-/-) mice also had an earlier appearance of tartrate-resistant acid phosphatase (TRAcP)-positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1(-/-) mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE(2) receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing.
    Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 04/2011; 26(4):792-802. DOI:10.1002/jbmr.272 · 6.83 Impact Factor
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    • "This has led to the discovery of VEGF inhibitors as potential therapies in cancer treatment (Cardones and Banez, 2006; Ferrara, 2005). VEGF has been shown to promote bone growth (Peng et al., 2002; Young et al., 2002) probably by its eVects on angiogenesis (FilvaroV, 2003; Kent Leach et al., 2006; Kleinheinz et al., 2005; Peng et al., 2005). "
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    ABSTRACT: A major unmet need in the medical field today is the availability of suitable treatments for the ever-increasing incidence of osteoporosis and the treatment of bone deficit conditions. Although therapies exist which prevent bone loss, the options are extremely limited for patients once a substantial loss of skeletal bone mass has occurred. Patients who have reduced bone mass are predisposed to fractures and further morbidity. The FDA recently approved PTH (1-34) (Teriparatide) for the treatment of postmenopausal osteoporosis after both preclinical animal and clinical human studies indicated it induces bone formation. This is the only approved bone anabolic agent available but unfortunately it has limited use, it is relatively expensive and difficult to administer. Consequently, the discovery of low cost orally available bone anabolic agents is critical for the future treatment of bone loss conditions. The intricate process of bone formation is co-ordinated by the action of many different bone growth factors, some stored in bone matrix and others released into the bone microenvironment from surrounding cells. Although all these factors play important roles, the bone morphogenetic proteins (BMPs) clearly play a central role in both bone cartilage formation and repair. Recent research into the regulation of the BMP pathway has led to the discovery of a number of small molecular weight compounds as candidate bone anabolic agents. These agents may usher in a new wave of more innovative and versatile treatments for osteoporosis as well as orthopedic and dental indications.
    Current Topics in Developmental Biology 02/2007; 78:127-71. DOI:10.1016/S0070-2153(06)78004-8 · 4.68 Impact Factor
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    ABSTRACT: Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology, 2008. Clinically, the use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with impaired bone healing. NSAIDs inhibit arachidonic acid metabolism. Interruption of arachidonic acid metabolism during fracture healing results in cartilage-related defects of the fracture callus. Prostaglandin E2 (PGE2) is the major product of arachidonic acid metabolism in cartilage. Together, these observations suggest depression of PGE2 levels (and possibly, PGE2-mediated signaling events) in cartilage may be the mechanism by which NSAIDs inhibit bone healing. This prompted us to examine the role of PGE2 in chondrogenesis and chondrocyte hypertrophy—two processes involved in callus formation and removal during fracture repair. The hypothesis tested was comprised of three separate, but interconnected components: (i) expression of the EP receptors is maturationally-regulated, (ii) PGE2’s promotion of chondrogenesis and depression of chondrocyte hypertrophy are dependent upon activation of EP receptor signaling, and (iii) PGE2 inhibits chondrocyte hypertrophy by decreasing BMP signaling. Primary murine cell culture systems possessing the ability to undergo chondrogenesis and chondrocyte hypertrophy in vitro were used to study the effect of PGE2 on these processes. A murine limb bud-derived mesenchymal cell model known to undergo chondrogenesis in vitro was chosen to examine the role of PGE2 during chondrogenesis. As the cells became more chondrogenic, the expression levels of the PGE2 receptors EP1, EP2, and EP4 increased. Signaling by these receptors has previously been shown to induce chondrogenesis in other cell systems. Timing of increased expression of these receptors in our model coincided with the first effects of PGE2 on the expression of genes induced during chondrogenesis and cartilage matrix accumulation. These results suggest the initial limited effects exerted by PGE2 during murine chondrogenesis may be a consequence of low expression levels of its receptors. A primary murine sternal chondrocyte model previously shown to undergo chondrocyte hypertrophy in vitro was used in the second set of experiments. PGE2’s effects on chondrocyte maturational markers were gene-specific, with the biggest effect seen in type X collagen (col10A1) gene expression. The mechanism by which PGE2 inhibited type X collagen (col10A1) gene expression was examined further. Cultures were treated with or without BMP-2, a master regulator of chondrocyte maturation, in the presence or absence of PGE2. PGE2 treatment diminished the ability of BMP-2 to (i) phosphorylate its downstream effectors, Smads 1, 5, and 8, and (ii) stimulate col10A1 gene expression. Of the EP receptors, EP4 exhibited the highest expression when the effect of PGE2 on BMP-2-mediated Col10A1 expression was maximal. This receptor can activate adenylyl cyclase activity. Artificial enhancement of adenylyl cyclase activity by dibutrylyl cyclic AMP treatment impeded BMP-2’s ability to activate a BMP-Smad-specific Col10A1 promoter. Activation of Smad signaling has been shown to enhance Col10A1 promoter activity and gene transcription. Collectively, these findings imply activation of adenylyl cyclase activity by PGE2 may depress BMP-Smad signaling events; resulting in decreased col10A1 gene transcription. The results demonstrate activation of adenylyl cyclase signaling, as well as addition of PGE2, stimulates chondrogenesis, and the expression of the EP receptors changes as chondrogenesis progresses. The timing of the first PGE2 effects coincides with the up-regulation of the EP2 and EP4 receptors. Combined, these data imply the reason for the delayed response of the cells to PGE2 may be attributed to initial low levels of EP2 and EP4, the receptors PGE2 utilizes to activate adenylyl cyclase activity. The findings from the chondrocyte hypertrophy studies also strongly implicate the EP4 receptor and adenylyl cyclase activity in PGE2-induced effects on Col10A1 expression. They also suggest crosstalk between the PGE2 and BMP signaling pathways may work to regulate the timing of the transition from proliferation to hypertrophy in chondrocytes.
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