SUMMARYPurpose: To assess the pharmacology of perampanel and its antiseizure activity in preclinical models. Perampanel [2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl) benzonitrile] is a novel, orally active, prospective antiepileptic agent currently in development for refractory partial-onset seizures. ]Perampanel binding to rat forebrain membranes was not significantly displaced by glutamate or AMPA but was displaced by the noncompetitive AMPA receptor antagonists CP465022 (K i 11.2 ± 0.8 nM) and GYKI52466 (K i 12.4 ± 1 lM). In mice, perampanel showed protective effects against audiogenic, MES-induced, and PTZ-induced seizures (ED 50 s 0.47, 1.6, and 0.94 mg/kg, respectively). Perampanel also inhibited 6 Hz electroshock-induced seizures when administered alone or in combination with other antiepileptic drugs (AEDs). In amygdala-kindled rats, perampanel significantly increased afterdischarge threshold (p < 0.05 vs. vehicle), and significantly reduced motor seizure duration, afterdischarge duration, and seizure severity recorded at 50% higher intensity than afterdischarge threshold current (p < 0.05 for all measures vs. vehicle). Perampanel caused dosedependent motor impairment in both mice (TD 50 1.8 mg/ kg) and rats (TD 50 9.14 mg/kg), as determined by rotarod tests. In mice, the protective index (TD 50 in rotarod test/ ED 50 in seizure test) was 1.1, 3.8, and 1.9 for MES-induced, audiogenic, and PTZ-induced seizures, respectively. In rat, dog, and monkey, perampanel had a half-life of 1.67, 5.34, and 7.55 h and bioavailability of 46.1%, 53.5%, and 74.5%, respectively. Significance: These data suggest that perampanel is an orally active, noncompetitive, selective AMPA receptor antagonist with potential as a broad spectrum antiepileptic agent.

Mechanical loading is crucial for maintenance of bone integrity and architecture, and prostaglandins are an important mediator of mechanosensing. Cyclooxygenase-2 (COX-2), an inducible isoform of prostaglandin G/H synthase, is induced by mechanical loading-derived fluid shear stress in bone-forming cells such as osteoblasts and osteocytes. In this study, we investigated transcription factor and transcriptional regulatory elements responsible for the shear stress-induced COX-2 expression in osteoblastic MC3T3-E1 cells. When the cells were transfected with luciferase-reporter plasmids including the 5-flanking region of the murine cox-2 gene, the fluid shear stress increased the luciferase activities, consistent with the induction of COX-2 mRNA and protein expression. Deletion analysis of the promoter region revealed that the shear stress-induced luciferase responses were regulated by two regions, ؊172 to ؊100 base pair (bp) and ؊79 to ؊46 bp, of the cox-2 promoter, in which putative cis-elements of C/EBP ␤, AP-1, cAMP-response element-binding protein (CREB), and E box are included. Mutation of sites of C/EBP ␤, AP-1, and/or cAMP-response element decreased the shear stress-induced luciferase activities, whereas mutation of the E box did not affect the responses. In an electrophoretic mobility shift assay, shear stress enhanced nuclear extract binding to double-stranded oligonucleotide probes containing C/EBP ␤ and AP-1-binding motifs, and the bands of the complexes were supershifted by the addition of antibody specific for each regulator. Although the binding activity of CREB toward its probe was unaffected by shear stress, the phosphorylation of CREB was enhanced by the stress. These data suggest that C/EBP ␤, AP-1, and CREB play crucial roles in the shear stress-induced cox-2 expression in osteoblasts.Mechanical loading applied to the skeleton is crucial to the development and maintenance of bone integrity and architecture. A decrease in the mechanical loading due to prolonged immobilization or weightlessness in space reduces the bone formation rate, resulting in bone loss (1-3). On the other hand, an increase in mechanical loading causes a gain in bone density (4, 5). Thus, bone tissue is sensitive to mechanical stimulation. Mechanical loading on bone generates extracellular matrix deformation and fluid flow, and the mechanical stimuli are translated to mechanical signals such as mechanical strain and fluid shear stress, respectively (6). Evidence obtained from in vitro studies indicates that osteocytes embedded in the lacunae/ canaliculi system and osteoblasts and bone cells lining the bone surface are mechanosensors that detect load-derived mechanical stimuli (7,8). By these bone-forming cells, the mechanical stimuli are translated into cellular signaling factors.Mechanical stress induces the expressions of several kinds of proteins in bone-forming cells such as insulin-like growth factor-I and -II, transforming growth factor-␤, osteocalcin, osteopontin, c-Fos, nitric-oxide synthase, and cyclooxygenase-2 (COX-...

Differentiation of osteoclasts, the cells primarily responsible for bone resorption, is controlled by a variety of osteotropic hormones and cytokines. Of these factors, receptor activator of NF-κB (RANK) ligand (RANKL) has been recently cloned as an essential inducer of osteoclastogenesis in the presence of M-CSF. Here, we isolated a stroma-free population of monocyte/macrophage (M/Mφ)-like hemopoietic cells from mouse unfractionated bone cells that were capable of differentiating into mature osteoclasts by treatment with soluble RANKL (sRANKL) and M-CSF. However, the efficiency of osteoclast formation was low, suggesting the requirement for additional factors. The isolated M/Mφ-like hemopoietic cells expressed TGF-β and type I and II receptors of TGF-β. Therefore, we examined the effect of TGF-β on osteoclastogenesis. TGF-β with a combination of sRANKL and M-CSF promoted the differentiation of nearly all M/Mφ-like hemopoietic cells into cells of the osteoclast lineage. Neutralizing anti-TGF-β Ab abrogated the osteoclast generation. These TGF-β effects were also observed in cultures of unfractionated bone cells, and anti-TGF-β blocked the stimulatory effect of 1,25-dihydroxyvitamin D3. Translocation of NF-κB into nuclei induced by sRANKL in TGF-β-pretreated M/Mφ-like hemopoietic cells was greater than that in untreated cells, whereas TGF-β did not up-regulate the expression of RANK, the receptor of RANKL. Our findings suggest that TGF-β is an essential autocrine factor for osteoclastogenesis.