Koh Kawasaki

Discovery of selective PDE4B inhibitors

In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.

Chronic forced swim stress of rats increases frontal cortical 5-HT2 receptors and the wet-dog shakes they mediate, but not frontal cortical β-adrenoceptors

We studied the effects of chronic forced swim stress on 5-HT2 receptors and beta-adrenoceptors in the rat frontal cortex. The number of 5-HT2 receptors was increased immediately after the last chronic stress, but not after an acute stress. In vivo, the number of wet-dog shakes induced by a 5-HT2 receptor agonist, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), was increased 24 h after the last chronic stress. However, the concentrations of 5-HT and 5-hydroxyindole acetic acid (5-HIAA), measured by high pressure liquid chromatography (HPLC), were not altered by this stress. Binding sites for [3H]CGP-12177, i.e., beta-adrenoceptor sites, were unchanged after both the acute and the chronic stress. These results suggest that, in the rat, the chronic forced swim stress increases the number of frontal cortical 5-HT2 receptors and the number of wet-dog shakes mediated by these receptors, while the number of frontal cortical beta-adrenoceptors is not increased by this treatment.

Demonstration of elevation and localization of Rho-kinase activity in the brain of a rat model of cerebral infarction

Evidence that Rho-kinase is involved in cerebral infarction has accumulated. However, it is uncertain whether Rho-kinase is activated in the brain parenchyma in cerebral infarction. To answer this question, we measured Rho-kinase activity in the brain in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere. At 6 h after injection, increase of activating transcription factor 3 (ATF3) and c-Fos was found in the ipsilateral hemisphere, suggesting that neuronal damage occurs. At 0.5, 3, and 6 h after injection of laurate, Rho-kinase activity in extracts of the cerebral hemispheres was measured by an ELISA method. Rho-kinase activity in extracts of the ipsilateral hemisphere was significantly increased compared with that in extracts of the contralateral hemisphere at 3 and 6 h but not 0.5 h after injection of laurate. Next, localization of Rho-kinase activity was evaluated by immunohistochemical analysis in sections of cortex and hippocampus including infarct area 6 h after injection of laurate. Staining for phosphorylation of myosin-binding subunit (phospho-MBS) and myosin light chain (phospho-MLC), substrates of Rho-kinase, was elevated in neuron and blood vessel, respectively, in ipsilateral cerebral sections, compared with those in contralateral cerebral sections. These findings indicate that Rho-kinase is activated in neuronal and vascular cells in a rat cerebral infarction model, and suggest that Rho-kinase could be an important target in the treatment of cerebral infarction.

Amelioration of endothelial damage/dysfunction is a possible mechanism for the neuroprotective effects of Rho-kinase inhibitors against ischemic brain damage

We investigated the neuroprotective effects of fasudils active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, in a rat stroke model in which endothelial damage and subsequent thrombotic occlusion were selectively induced in perforating arteries. By examining the effects on the endothelial damage/dysfunction, we thought to explore the mechanism of Rho-kinase inhibitors. Hydroxyfasudil (10mg/kg, i.p., once daily for 3 days) significantly improved neurological functions and reduced the size of the infarct area produced by internal carotid artery injection of sodium laurate in a rat cerebral microthrombosis model. Treatment with fasudil or hydroxyfasudil concentration-dependently inhibited tumor necrosis factor alpha-induced tissue factor expression on the surface of cultured human umbilical vein endothelial cells. They also inhibited thrombin-induced endothelial hyperpermeability. The present findings suggest that hydroxyfasudil is efficacious in preventing brain damage associated with cerebral ischemia, and is partially responsible for fasudils cytoprotective potential. The results also suggest that the therapeutic benefits against ischemic injury of Rho-kinase inhibitors are attributed, at least in part, to activity upon endothelial damage/dysfunction.

Antivasospastic effects of hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage.

We investigated the anti-vasospastic potential of fasudils active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage (SAH) and also its effect on hemorheological abnormalities following cerebral ischemia. Chronic cerebral vasospasm was produced using a two-hemorrhage canine model. On day 7, angiographic vasospasm was observed in all animals, and intravenous administration of hydroxyfasudil (3 mg·kg-1·30 min-1) significantly reversed the vasospasm (predose diameter of the basilar artery, 57.9% ± 2.0% of the baseline before the injection of blood; postdose diameter, 64.5% ± 1.9%). The viscosity of whole blood was significantly increased 24 h after 1 h middle cerebral artery occlusion in rats. Hydroxyfasudil (3 and 10 mg/kg, i.p.) significantly decreased blood viscosity. The specificity of hydroxyfasudil was examined against a panel of 17 protein kinases using ELISA analysis. Hydroxyfasudil inhibited Rho-kinase α and β at a concentration of 10 μM by 97.6% and 97.7%, respectively. No other protein kinase was inhibited with 10 μM hydroxyfasudil by over 40%. The present results indicate hydroxyfasudil is a selective inhibitor of Rho-kinase. The results also suggest that hydroxyfasudil contributes to the potency of fasudil to prevent cerebral vasospasm and hyperviscosity and suggest the potential utility of hydroxyfasudil as a therapeutic agent for patients with SAH.

Repeated administration of milnacipran induces rapid desensitization of somatodendritic 5-HT1A autoreceptors but not postsynaptic 5-HT1A receptors

The effects of the repeated administration of milnacipran, a serotonin (5-HT)-noradrenaline reuptake inhibitor (SNRI), on the functional status of somatodendritic 5-HT1A receptors, and postsynaptic 5-HT1A receptors were explored using electrophysiological approaches in rats. In-vitro electrophysiological recordings in the dorsal raphe nucleus showed that 5-HT inhibited the firing of serotonergic neurones, and the selective 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), reversed the inhibitory effect of 5-HT. The potency of 5-HT to inhibit the firing of serotonergic neurones was slightly attenuated after 3 days of treatment with milnacipran (30 mg/kg, p.o., twice daily), and significantly attenuated after 7 or 14 days treatment at the same dose. The tricyclic antidepressant, imipramine, did not significantly modify the inhibitory effect of 5-HT. After 7 days treatment at 30 mg/kg, p.o., once daily, milnacipran reduced the potency of 5-HT to inhibit the firing of serotonergic neurones, whereas the selective serotonin reuptake inhibitors, fluvoxamine and fluoxetine (60 and 30 mg/kg, p.o., once daily, respectively), did not modify it under these conditions. Treatment with milnacipran (30 mg/kg, p.o., twice daily) for 14 days did not change the inhibition of the CA1 field potential in rat hippocampal slices by 5-HT. These data suggest that somatodendritic 5-HT1A receptors, but not postsynaptic 5-HT1A receptors, rapidly desensitize in response to the repeated administration of milnacipran.

Pleiotropic effects of the rho-kinase inhibitor fasudil after subarachnoid hemorrhage: a review of preclinical and clinical studies.

There is growing evidence that Rho-kinase contributes to cardiovascular disease, which has made Rho-kinase a target for the treatment of human diseases. To date, the only Rho-kinase inhibitor employed clinically in humans is fasudil, which has been used for the prevention of cerebral vasospasm and subsequent ischemic injury after surgery for subarachnoid hemorrhage (SAH). A number of pathological processes, in particular hemodynamic dysfunctions and inflammatory reactions, are thought to be related in the pathogenesis of delayed cerebral vasospasm and subsequent ischemic injury after SAH. This review focuses on fasudils pleiotropic therapeutic effects: amelioration of hemodynamic dysfunction and inflammation, and discusses in detail the clinical studies on fasudil administered after the occurrence of SAH.

Fasudil protects cultured N1E-115 cells against lysophosphatidic acid-induced neurite retraction through inhibition of Rho-kinase

The aim of this study was to investigate the possible effects of the Rho-kinase inhibitor, fasudil, on the lysophosphatidic acid (LPA)-induced neurite retraction in N1E-115 cells. In cultured N1E-115 cells, LPA produced a marked increase in the population of rounded cells. Fasudil or hydroxyfasudil, an active metabolite of fasudil, blocked cell rounding in a concentration-dependent manner at levels between 1 and 10 μM, with IC₅₀ values of 1.7 or 1.6 μM, respectively. Fasudil or hydroxyfasudil concentration-dependently inhibited phosphorylation of the myosin binding subunit of myosin phosphatase in N1E-115 cells. These results indicate that Rho-kinase is essential for LPA-induced neurite retraction in N1E-115 cells and that inactivation of Rho-kinase by a Rho-kinase inhibitor, such as fasudil, eliminates cell rounding and promotes neurite outgrowth, thus improving neurological function in the brain damage.

Thrombomodulin alfa attenuates the procoagulant effect and cytotoxicity of extracellular histones through the promotion of protein C activation.

INTRODUCTION Extracellular histones are reported to increase thrombin generation in the plasma and induce endothelial cell death in vitro. These effects of histones were suggested to involve histone-induced inhibition of TM-dependent activated protein C (APC) generation. Therefore, we hypothesized that TM alfa, a recombinant human soluble TM, attenuates these effects of histones by promoting the generation of APC. In the present study, we investigated the effects of TM alfa on the histone-induced decrease in APC generation, an increase in thrombin generation, and endothelial cell death in vitro. METHODS APC generation was investigated using a chromogenic substrate based assay. Thrombin generation in plasma was studied by using a calibrated automated thrombogram method. Histone cleavage was detected by western blot analysis. Histone-induced endothelial cell death was evaluated by the trypan blue exclusion test. RESULTS Histones decreased APC generation and increased thrombin generation in the presence of endothelial cells. TM alfa increased APC generation and decreased thrombin generation in the presence of histones and endothelial cells. TM alfa with thrombin and protein C cleaved histone H3, and attenuated histone-induced endothelial cell death. Antithrombin, an endogenous thrombin inhibitor, and gabexate mesilate, a synthetic protease inhibitor, inhibited thrombin generation, decreased APC generation, and did not have any effect on histone H3 cleavage or histone-induced endothelial cell death. CONCLUSIONS TM alfa attenuated the histone-induced increase in thrombin generation and endothelial cell death by promoting APC generation in vitro.