Objective. We recently developed an ex vivo cellular model of pannus, the aberrant overgrowth of human synovial tissue. This study was undertaken to use that model to investigate the role of prostaglandin E 2 (PGE 2 ) and its receptor subtypes in the development of pannus growth and osteoclast activity in rheumatoid arthritis (RA).Methods. Inflammatory cells that infiltrated pannus from patients with RA were collected without enzyme digestion and designated synovial tissue-derived inflammatory cells. Their single-cell suspensions were cultured in medium alone to observe an aberrant overgrowth of inflammatory tissue in vitro. Levels of cytokines produced in culture supernatants were measured using enzyme-linked immunosorbent assay kits. Osteoclast activity was assessed by the development of resorption pits in calcium phosphate-coated slides.Results. Primary culture of the synovial tissuederived inflammatory cells resulted in spontaneous reconstruction of inflammatory tissue in vitro within 4 weeks, during which tumor necrosis factor ␣, PGE 2 , macrophage colony-stimulating factor, and matrix metalloproteinase 9 were produced in the supernatant. This aberrant overgrowth was inhibited by antirheumatic drugs including methotrexate and infliximab. On calcium phosphate-coated slides, synovial tissue-derived inflammatory cells showed numerous resorption pits. In the presence of inhibitors of endogenous prostanoid production such as indomethacin and NS398, exogenous PGE 1 and EP4-specific agonists significantly inhibited all these activities of synovial tissue-derived inflammatory cells in a dose-dependent manner. Addition of indomethacin, NS398, or EP4-specific antagonist resulted in the enhancement of these cells' activities. EP2-specific agonist had a partial effect, while EP1-and EP3-specific agonists had no significant effects.Conclusion. These results suggest that endogenous PGE 2 produced in rheumatoid synovium negatively regulates aberrant synovial overgrowth and the development of osteoclast activity via EP4.Rheumatoid arthritis (RA) is a progressive, systemic inflammatory disorder that is characterized by a chronic synovitis which leads to pannus formation and joint destruction. Pannus tissue is composed mainly of activated macrophages, T cells, and proliferating fibroblast-like synoviocytes (FLS) (1-3). Both cytokine networks and cell-cell interaction contribute to the development of pannus tissue. Previous studies indicated that both peripheral blood and synovial monocyte/ macrophages had the ability to serve as osteoclast precursors and developed osteoclastic activity when cocultured with either CD14Ϫ synovial cells or FLS in the presence of macrophage colony-stimulating factor (M-CSF) (4). Cultured synovial fibroblasts and mesen-

Using rat peritoneal neutrophils, the complete nucleotide sequence of rat macrophage inflammatory protein-2 (MIP-2) mRNA including 5'untranslated region (UTR) and 3'UTR was determined (GenBank Accession number, AB060092). It was found that the MIP-2 mRNA has a 70 bp 5'UTR, a 303 bp coding region and a 728 bp 3'UTR which contains adenylate/uridylate (AU)-rich areas defined as AU-rich elements (AREs). Site-directed mutagenesis studies using the tetracycline-sensitive transactivator protein-expressing rat basophilic leukemia cells (RBL-2H3-TO cells) revealed that MIP-2 mRNA mutants which lack the 3'UTR are more stable than MIP-2-wild-type (wt) mRNA. A MIP-2 mRNA mutant in which some mutations were introduced to the ARE was also stable. The stability of MIP-2 mRNA was low in untreated RBL-2H3-TO cells, but it increased in the antigen-stimulated immunoglobulin E (IgE)-sensitized cells. The antigen-induced MIP-2 mRNA stabilization was counteracted by the highly specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and the MAPK/ERK kinase (MEK-1) inhibitor PD98059. These findings indicate that ARE is the cis-element which mediates the rapid decay of MIP-2 mRNA, and the antigen stimulation stabilizes MIP-2 mRNA and the p38 MAPK and p44/42 MAPK pathways are involved in the antigen-induced stabilization of MIP-2 mRNA.

Acromegaly is a disease caused by the oversecretion of growth hormone. It is currently treated by intravenous injection with cyclic peptide drugs that activate somatostatin receptor subtype 2 (SSTR2). Here, novel nonpeptidic, small-molecule, and orally active SSTR2 agonists were identified from a hit compound (13). Pharmacophore studies enabled scaffold hopping to obtain a unique 3,4,5-trisubstituted pyridine motif. Further optimization conferred potent SSTR2 agonistic activity and metabolic stability. Several compounds were evaluated and these showed good oral pharmacokinetic profiles in rats, and one representative compound (25) showed highly potent inhibition of growth hormone secretion induced by growth hormone-releasing hormone in rats. Based on these results, 25 was identified as a promising lead for further optimization. A structure–activity relationship (SAR) study and the metabolic stability data for this compound are also described.

Somatostatin analogues (SSAs) are indicated for use in acromegaly and gastroenteropancreatic neuroendocrine tumors (GEP NETs). However, SSAs are administered as injections which can be a challenge for patients and healthcare providers. ONO-5788 is a novel low molecular weight orally administered molecule with potent and selective agonistic effects at the human SST2 subtype. ONO-5788 could offer the advantages of a more convenient route of administration for patients with acromegaly and other indications and also a preferential safety profile compared to injections. In order to support clinical evaluation of ONO-5788, a series of non-clinical experiments were undertaken. Findings: In vitro, ONO-5788 and its active metabolite, ONO-ST1-641, has a greater agonistic effect on human SST2 compared to other somatostatin receptor subtypes, with EC50 values of 0.11 and 0.016 nmol/L, respectively. There is no difference in mean EC50 values between human SST2 and rat SST2. ONO-5788 and ONO-ST1-641 significantly inhibits growth hormone-releasing hormone (GHRH)-induced GH secretion from primary cultured rat pituitary cells. In vivo studies in rats confirmed that a single oral dose of ONO-5788 resulted in systemic exposure of ONO-5788 and ONO-ST1-641 that increased in a dose dependent manner. Orally administrated ONO-5788 significantly inhibited both GHRH-induced GH secretion and basal GH secretion at all the doses tested (≥0.1 and ≥0.03 mg/kg, respectively). ONO-5788 and ONO-ST1-641 administrated intravenously significantly inhibited GHRH-induced GH secretion with effect on maximum inhibition of GH secretion similar to octreotide (ONO-5788 98.0%, ONO-ST1-641 98.6%, octreotide 98.7%, respectively). Plasma concentrations of ONO-5788 and ONO-ST1-641 required to supress serum GH level to a clinically meaningful degree in acromegaly patients were estimated to be 21.3 and 1.29 ng/mL. In experiments evaluating the effects on insulin and glucagon secretion in anaesthetised rats, ONO-5788, ONO-ST1-641, octreotide and pasireotide significantly inhibited glucagon secretion, but ONO-5788 and ONO-ST1-641 had no significant impact on insulin secretion at doses up to 10-fold higher than the dose required for either of these compounds to almost maximally inhibit GH secretion. In contrast, pasireotide and octreotide showed a significant inhibition of insulin secretion at equivalent doses of both ONO-5788 and ONO-ST1-641. A standard battery of preclinical safety and toxicology studies provided adequate evidence of safety to support commencement of a Phase I study in healthy volunteers to measure the safety, pharmacokinetics, and pharmacodynamics of ONO-5788. Conclusion: Orally administered ONO-5788 demonstrates potency as a SST2 agonist and has the potential to be a new treatment option for patients with acromegaly and NET.

Purpose: The verification of efficacy of new drug candidates in primate models may address the translational gap between non-clinical and clinical studies and support the prediction of efficacy in humans. Given this, and to support the evaluation of novel SST2 agonists, a GHRH/arginine-induced GH hypersecretion model was established in the monkey. This model was used to evaluate the effect of both octreotide and ONO-ST-468, a novel and potent selective SST2 agonist. Methods: Male cynomolgus monkeys (3-6 years old) were anesthetized with ketamine and maintained with constant infusion of propofol. Octreotide (0.23, 0.47 or 0.70 μg/0.88mL/kg/h), ONO-ST-468 (0.35 or 1.05 μg/1.1mL/kg/h) and saline were continuously administrated by intravenous (i.v.) infusion 2 hours before and after GH hypersecretion (n=7-9 in each group). GH hypersecretion was induced by i.v. bolus administration of GHRH (“time zero”), followed immediately by a 30 minutes i.v. infusion of arginine. Blood samples were collected for 2 hours after GHRH/arginine treatment and the plasma GH level was measured by ELISA. Inhibitory rates of GH hypersecretion were calculated by the area under the curve (AUC0-2h). Results: As a result of examinations for several experimental conditions, excessive GH secretion was induced by i.v. bolus administration of GHRH (30 μg/0.5mL/kg) and the i.v. infusion of arginine (5 g/17.9mL/kg/h). GH AUC0-2h was approximately 72 ng·h/mL in the vehicle group. Octreotide inhibited GHRH/arginine-induced GH hypersecretion in a dose dependent manner. The inhibitory rates (AUC0-2h) by octreotide were 18% (low dose), 59% (medium dose) and 83% (high dose), respectively. The plasma concentration of octreotide in high dose group was 1.76 ng/mL at 2 hours after GHRH/arginine treatment, being similar to effective plasma concentration of octreotide in acromegaly patient (approximately 1 ng/mL). ONO-ST-468 also inhibited GHRH/arginine-induced GH hypersecretion in a dose dependent manner. The inhibitory rates (AUC0-2h) by ONO-ST-468 were 70% (low dose) and 94% (high dose), respectively, demonstrating that the maximum inhibitory effect of ONO-ST-468 was similar to that of octreotide and has the potential to suppress GH hypersecretion in acromegaly patients. Conclusion: Octreotide and ONO-ST-468, a novel small molecule SST2 agonist, suppressed excessive GH secretion in a newly established GHRH/arginine-induced GH hypersecretion model in the monkey.