Dioxotriazine derivatives as a new class of P2X3 receptor antagonists: Identification of a lead and initial SAR studies.

P2X3 receptor is an ATP-gated ion channel, mainly localized on peripheral sensory neurons. Currently, several clinical trials are being conducted with P2X3 receptor antagonists for the treatment of chronic pain or cough. To identify a P2X3 lead compound, we reexamined the HTS evaluation compounds and selected dioxotriazine derivatives from which we identified a hit compound. As a result of the hit-to-lead SAR, we obtained lead compound 1 which had a moderate inhibitory effect on P2X3 receptors (IC50, 128 nM). Further improvement of the potency and PK profiles of this lead compound finally led to the selected compound 74 (P2X3 IC50, 16.1 nM; P2X2/3 IC50, 2931 nM), which demonstrated a strong analgesic effect against allodynia on oral administration in the rat partial sciatic nerve ligation model of neuropathic pain (ED50, 3.1 mg/kg).

Pyrrolinone derivatives as a new class of P2X3 receptor antagonists. Part 3: Structure-activity relationships of pyrropyrazolone derivatives.

The P2X3 receptor is an attractive target for the treatment of pain and chronic coughing, and thus P2X3 antagonists have been developed as new therapeutic drugs. We previously reported selective P2X3 receptor antagonists by derivatization of hit compound 1. As a result, we identified hit compound 3, the structure of which was similar to hit compound 1. On the basis of SAR studies of hit compound 1, we modified hit compound 3 and compound 42 was identified as having analgesic efficacy by oral administration.

Discovery of NR2B-selective antagonists via scaffold hopping and pharmacokinetic profile optimization.

Selective N-methyl-d-aspartate receptor subunit 2B (NR2B) antagonists show potential as analgesic drugs, and do not cause side effects associated with non-selective N-methyl-d-aspartate (NMDA) antagonists. Using a scaffold-hopping approach, we previously identified isoxazole derivative 4 as a potent selective NR2B antagonist. In this study, further scaffold hopping of isoxazole derivative 4 and optimization of its pharmacokinetic profile led to the discovery of the orally bioavailable compound 6v. In a rat study of analgesia, 6v demonstrated analgesic effects against neuropathic pain.

Pyrrolinone derivatives as a new class of P2X3 receptor antagonists Part 2: Discovery of orally bioavailable compounds.

Some P2X3 receptor antagonists have been developed as new therapeutic drugs for pain. We discovered a novel chemotype of P2X3 receptor antagonists with a pyrrolinone skeleton. Because of SAR studies to improve bioavailability of lead compound 2, compound (R)-24 was identified, which showed an analgesic effect against neuropathic pain by oral administration. We constructed a human P2X3 homology model as a template for the zebrafish P2X4 receptor, which agreed with SAR studies of pyrrolinone derivatives.

Discovery of orally bioavailable cyclohexanol-based NR2B-selective NMDA receptor antagonists with analgesic activity utilizing a scaffold hopping approach.

NR2B subunit containing N-methyl-d-aspartate (NMDA) receptor is an attractive target for chronic pain due to its involvement in disease states and its limited distribution in the central nervous system. Cyclohexanol-based leads 6a and 6c were identified as potent NR2B-selective NMDA antagonists utilizing a scaffold hopping approach. Further optimization of this series through replacement of the amide in the leads with an isoxazole and efforts to optimize the pharmacokinetic profiles led to the discovery of orally available brain penetrants 7k and 7l, which demonstrated analgesic activity in the mouse formalin test at early and late phases.

Incidence and risk factors for medial tibial stress syndrome and tibial stress fracture in high school runners.

PURPOSE: Medial tibial stress syndrome (MTSS) and tibial stress fracture (SF) are common lower leg disorders in runners. A prospective study was done to identify the incidence of MTSS and SF in high school runners and to determine risk factors. METHODS: A total of 230 runners participating in high school running teams were evaluated. All runners aged 15 years as first grade of high school were involved in the study. They were followed up for 3 years. The measured items included height, weight, body mass index (BMI), range of hip and ankle motion, straight leg raising (SLR), intercondylar and intermalleolar interval, Q-angle, navicular drop test, hip abductor strength and physical conditioning. Each runner was followed for 3 years to report occurrence of MTSS and SF. RESULTS: A total number of 102 MTSS (0.29 athlete exposures) and 21 SF (0.06 athlete exposures) were identified. In females, BMI significantly increased the risk of MTSS after adjustment for the other variables in this study (adjusted odds ratio, 0.51; 95 % confidence interval, 0.31-0.86). Increased internal rotation of the hip significantly increased the risk of MTSS (adjusted odds ratio, 0.91; 95 % confidence interval, 0.85-0.99). In males, limited SLR also significantly increased the risk of SF with adjustment for the other variables in this study (adjusted odds ratio, 1.38; 95 % confidence interval, 1.04-1.83). CONCLUSION: A significant relationship was found between BMI, internal hip rotation angle and MTSS in females, and between limited SLR and SF in males. LEVEL OF EVIDENCE: Prospective cohort study, Level II.

An engineered tetra-valent antibody fully activates the Tie2 receptor with comparable potency to its natural ligand angiopoietin-1.

Activation of the tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) receptor by angiopoietin-1 (Ang1) is critical for vascular stabilization: it promotes survival signal transduction via auto-phosphorylation and reduces vascular permeability by strengthening tight junctions between endothelial cells. Thus, Tie2/Ang1 signaling is a promising therapeutic target for vascular diseases. However, in vivo use of existing Tie2 signaling modulators, such as recombinant Ang1, is restricted by limitations in manufacturability and stability. Here, we present a novel engineered tetra-valent agonistic antibody, ASP4021, which can specifically and fully activate the Tie2 receptor in an equivalent manner to Ang1. ASP4021 induced Tie2 self-phosphorylation and inhibited apoptosis in a human primary endothelial cell line. Additionally, single administration of ASP4021 significantly suppressed mustard-oil-induced vascular permeability in rats. ASP4021 may thus be a potential therapeutic candidate for diseases associated with vascular weakness such as diabetic retinopathy, diabetic macular edema and critical limb ischemia.

Identification and characterization of Xenopus laevis homologs of mammalian TRAF6 and its binding protein TIFA.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) transduces signals from members of the TNFR superfamily and the Toll/IL-1R family, leading to activation of transcription factors such as NFkappaB and AP-1. Genetic disruption of the TRAF6 gene in mice results in various developmental abnormalities during embryogenesis, including osteopetrosis, failure of neural tube closure, defective formation of skin appendices, absence of lymph nodes, and absence of mature thymic epithelial cells. To clarify the effect of TRAF6 in development, we previously identified a TRAF-interacting protein with a forkhead-associated domain (TIFA), which binds and activates TRAF6 upon extracellular stimulation. To understand the physiological roles of TRAF6 and TIFA in early development, we studied these genes in Xenopus laevis. Here, we describe identification of X. laevis homologs of mammalian TRAF6 (XTRAF6) and TIFA (XTIFA). As was the case for the mammalian homologs, overexpression of XTRAF6 or XTIFA activated NFkappaB, whereas XTIFA carrying a mutation that abolishes XTRAF6 binding failed to activate NFkappaB, suggesting that XTIFA activates NFkappaB by binding to XTRAF6. XTIFA and XTRAF6 mRNAs were expressed at similar levels in zygotes from the neurula stage and then increased. Whole-mount in situ hybridization revealed that XTRAF6 mRNA was expressed in the head region and neural tube during the neurula stage, and the expression expanded to the pharyngeal apparatus during the tailbud stage. This localization is consistent with the defective neural tube closure and abnormal thymus organogenesis observed in TRAF6-deficient mice. Our results suggest possible cooperation between XTRAF6 and XTIFA during embryogenesis.

Stachyflin and acetylstachyflin, novel anti-influenza A virus substances, produced by Stachybotrys sp. RF-7260. I. Isolation, structure elucidation and biological activities.

Two novel compounds, stachyflin and acetylstachyflin, have been isolated by solid-state fermentation of Stachybotrys sp. RF-7260. The structures of both metabolites, determined by detailed NMR analyses and X-ray crystallographic analysis, are novel with a pentacyclic moiety including cis-fused decalin. The absolute stereochemistry of stachyflins was determined by circular dichroism analysis. Stachyflin showed antiviral activity against influenza A virus (H1N1) in vitro with an IC50 value of 0.003 microM. Acetylstachyflin was about 77-fold less active than stachyflin.