Noriyuki Yamamoto

Functional role of prostacyclin receptor in rat dorsal root ganglion neurons.

Recent studies on prostanoids showed that some of prostanoid receptors are expressed in rat dorsal root ganglion (DRG) neurons. These facts suggest that prostanoid receptors might be involved in the excitation mechanism of DRG neurons. In the present study, PCR experiments revealed that one of prostanoid receptor, prostacyclin receptor (IP receptor) was expressed in L6 and S1 rat DRG neurons and that the expression of IP receptor was not changed in DRG neurons obtained from the cyclophosphamide (CYP)-induced cystitis rat. We examined the functional role of IP receptor agonist and other prostanoids by measuring cyclic AMP (cAMP) accumulation and substance P (SP) release in primary cultured DRG neurons. The pretreatment of DRG neurons with prostanoid agonists such as iloprost (IP), butaprost (EP(2)), misoprostol (EP(2-4)), PGE(2) (EP(1-4)) or PGD(2) (DP and CRTH2) sensitized the DRG neurons and hence potentiated the lys-bradykinin-induced SP release. The increase of SP release by lys-BK plus prostanoid agonists was proportion to cAMP accumulation. Iloprost was the most potent agonist to induce cAMP accumulation and SP release among prostanoid agonists evaluated in this study and its effect is mediated by IP receptor. Moreover, capsaicin-, ATP- and KCl-induced SP release was also enhanced by iloprost although iloprost did not change intracellular Ca(2+) and membrane depolarization induced by these chemical stimuli. These results strongly indicate that IP receptor play an important role in the sensitization of rat sensory neuron.

Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors

Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS.

Up-titration of vardenafil dose from 10 mg to 20 mg improved erectile function in men with spinal cord injury

Aim: Vardenafil is a highly selective phosphodiesterase type‐5 inhibitor for the treatment of erectile dysfunction (ED). Efficacy of vardenafil has been demonstrated in various ED populations, but that in Japanese patients with spinal cord injury (SCI) has not been assessed.

Establishment of the superoxide production assay with human monocytic cell line, U937, for the evaluation of Syk kinase inhibitors.

Protein tyrosine kinase Syk is known to play critical roles in the signal transduction from receptors for Fc portion of immunoglobulins (FcRs) and B cell receptor complex (BCR). Its importance was well studied in Fc epsilon RI-induced activation of mast cells; therefore, Syk inhibitors are expected to have anti-allergic effects and to be novel therapy for allergic diseases, such as asthma, allergic rhinitis and atopic dermatitis. We previously developed an enzyme assay of recombinant human Syk kinase for the high throughput screening. In order to evaluate the Syk kinase inhibitors in a human cell system, we have developed an assay with human monocytic cell line, U937, to monitor FcgammaRI-mediated superoxide production. We treated cells with IFN-gamma to enhance the expression of FcgammaRI and to obtain enough production of superoxide. Engagement of FcgammaRI stimulated superoxide production, which was accompanied with Syk phosphorylation. PMA, an activator of protein kinase C, also evoked superoxide production, but Syk was not phosphorylated. Moreover, the treatment of cells with antisense oligonucleotide against syk attenuated Syk protein expression and suppressed superoxide production induced by FcgammaRI-engagement, but not by PMA. These results confirm that Syk is involved in the signal transduction from FcgammaRI upstream of PKC in U937 cells and we can evaluate the efficacy and the selectivity of Syk inhibitors with this assay system.

Tetrahydro-naphthols as orally available TRPV1 inhibitors.

Starting from a naphthol-based lead series with low oral bioavailability, we have identified potent TRPV1 antagonists with oral bioavailability in rats. These compounds emerged from SAR studies aimed at replacing the leads phenol structure whilst maintaining potency. Compound rac-6a is an orally available TRPV1 antagonist with single-digit nanomolar activity. The enantiomers show a low eudismic ratio at the receptor level.