Mitsuhiro Ino

Differential nociceptive responses in mice lacking the alpha(1B) subunit of N-type Ca(2+) channels.

The role of N-type Ca(2+) channels in nociceptive transmission was examined in genetically engineered mice lacking the alpha(1B) subunit of N-type channels and in their heterozygote and wild-type littermates. In alpha(1B)-deficient mice, N-type channel activities in dorsal root ganglion neurons and spinal synaptoneurosomes were eliminated without compensation by other types of voltage-dependent Ca(2+) channels. The alpha(1B)-deficient mice showed a diminution in the phase 2 nociceptive responses more extensively than in the phase 1 nociceptive responses of the formalin test. The alpha(1B)-deficient mice exhibited significantly increased thermal nociceptive thresholds in the hot plate test, but failed to increase mechanical nociceptive thresholds in the tail pinch test. These results suggest a crucial role of N-type channels in nociceptive transmission, especially for persistent pain like phase 2 of the formalin test and for nociception induced by thermal stimuli.

Functional disorders of the sympathetic nervous system in mice lacking the α1B subunit (Cav 2.2) of N-type calcium channels

N-type voltage-dependent Ca2+ channels (VDCCs), predominantly localized in the nervous system, have been considered to play an essential role in a variety of neuronal functions, including neurotransmitter release at sympathetic nerve terminals. As a direct approach to elucidating the physiological significance of N-type VDCCs, we have generated mice genetically deficient in the α1B subunit (Cav 2.2). The α1B-deficient null mice, surprisingly, have a normal life span and are free from apparent behavioral defects. A complete and selective elimination of N-type currents, sensitive to ω-conotoxin GVIA, was observed without significant changes in the activity of other VDCC types in neuronal preparations of mutant mice. The baroreflex response, mediated by the sympathetic nervous system, was markedly reduced after bilateral carotid occlusion. In isolated left atria prepared from N-type-deficient mice, the positive inotropic responses to electrical sympathetic neuronal stimulation were dramatically decreased compared with those of normal mice. In contrast, parasympathetic nervous activity in the mutant mice was nearly identical to that of wild-type mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct evidence that N-type VDCCs are indispensable for the function of the sympathetic nervous system in circulatory regulation and indicate that N-type VDCC-deficient mice will be a useful model for studying disorders attributable to sympathetic nerve dysfunction.

Ataxia and peripheral nerve hypomyelination in ADAM22-deficient mice

BackgroundADAM22 is a member of the ADAM gene family, but the fact that it is expressed only in the nervous systems makes it unique. ADAM22s sequence similarity to other ADAMs suggests it to be an integrin binder and thus to have a role in cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM22, we employed gene targeting to generate ADAM22 knockout mice.ResultsADAM22-deficient mice were produced in a good accordance with the Mendelian ratio and appeared normal at birth. After one week, severe ataxia was observed, and all homozygotes died before weaning, probably due to convulsions. No major histological abnormalities were detected in the cerebral cortex or cerebellum of the homozygous mutants; however, marked hypomyelination of the peripheral nerves was observed.ConclusionThe results of our study demonstrate that ADAM22 is closely involved in the correct functioning of the nervous system. Further analysis of ADAM22 will provide clues to understanding the mechanisms of human diseases such as epileptic seizures and peripheral neuropathy.

Ca2+ Channel α1B Subunit (CaV 2.2) Knockout Mouse Reveals a Predominant Role of N-Type Channels in the Sympathetic Regulation of the Circulatory System

N-type voltage-dependent Ca(2+) channels (VDCCs), predominantly localized in the nervous system, have been proposed to play vital roles in a variety of neuronal functions such as neurotransmitter release at sympathetic nerve terminals. To directly approach the elucidation of the physiological significance of N-type VDCCs in the autonomic nervous system, alpha(1B) subunit (Ca(V) 2.2)-deficient mice were generated, in which peripheral neurons showed a complete and selective elimination of N-type channel currents sensitive to omega-conotoxin GVIA (the peptide toxin from the fish-hunting cone snail Conus geographus), without a significant effect on the activity of other VDCC types. In isolated left atria prepared from N-type-deficient mice, the positive inotropic response mediated by the sympathetic nervous system was dramatically decreased, whereas the negative inotropic response mediated by parasympathetic neurons was nearly intact compared with those of normal mice. The baroreflex response to bilateral carotid occlusion was markedly reduced in the mutant mice. Interestingly, the mutant mice showed sustained elevation of heart rate and blood pressure. These results provide direct in vivo evidence for an essential role of N-type VDCCs in maintaining the normal function of the sympathetic nervous system in circulatory regulation, demonstrating a potential of N-type VDCC-deficient mice as a useful model for studying disorders attributable to sympathetic nerve dysfunction.

Synthesis and structure-activity relationships of pyrazolo[1,5-a]pyridine derivatives: potent and orally active antagonists of corticotropin-releasing factor 1 receptor.

Design, synthesis, and structure-activity relationships of a series of 3-dialkylamino-7-phenyl pyrazolo[1,5-a]pyridines (I) as selective antagonists of the corticotropin-releasing factor 1 (CRF(1)) receptor are described. The most prominent compound to emerge from this work, 46 (E2508), exhibits potent in vitro activity, excellent drug-like properties, and robust oral efficacy in animal models of stress-related disorders. It has advanced into clinical trials.

Expression analysis of P/Q-type Ca2+ channel α1A subunit mRNA in olfactory mitral cell in N-type Ca2+ channel α1B subunit gene-deficient mice

Abstract N-type and P/Q-type Ca 2+ channels play an important role in the processing of olfactory information. However, N-type Ca 2+ channel α 1B -deficient mice show normal behavior, presumably owing to compensation by other Ca 2+ channels. P/Q-type Ca 2+ channel α 1A mRNA was expressed at a higher level in olfactory bulb of homozygous α 1B -deficient mice than wild-type or heterozygous mice. LacZ expression in olfactory mitral cells of homozygous α 1B -deficient x α 1A 1.5-lacZ mice, carrying a 1.5-kb 5′-upstream fragment of the α 1A gene fused to the lacZ reporter gene, was increased compared to that in wild-type or heterozygous mice. Therefore, a possible explanation for the normal behavior of α 1B -deficient mice is compensation by the α 1A gene and that the 1.5-kb 5′-upstream region of this gene contains an enhancer cis -element for compensation in olfactory mitral cells.

Design, synthesis, and structure-activity relationships of novel pyrazolo[5,1-b]thiazole derivatives as potent and orally active corticotropin-releasing factor 1 receptor antagonists.

This paper describes the design, synthesis, and structure-activity relationships of a novel series of 7-dialkylamino-3-phenyl-6-methoxy pyrazolo[5,1-b]thiazole derivatives for use as selective antagonists of the corticotropin-releasing factor 1 (CRF(1)) receptor. The most promising compound, N-butyl-3-[4-(ethoxymethyl)-2,6-dimethoxyphenyl]-6-methoxy-N-(tetrahydro-2H-pyran-4-yl)pyrazolo[5,1-b][1,3]thiazole-7-amine (6t), showed high affinity (IC(50) = 70 nM) and functional antagonism (IC(50) = 7.1 nM) for the human CRF(1) receptor as well as dose-dependent inhibition of the CRF-induced increase in the plasma adrenocorticotropic hormone (ACTH) concentration at a dose of 30 mg/kg (po). Further, in the light/dark test in mice, the compound 6t showed anxiolytic activity at a dose of 30 mg/kg (po).

Selective corticotropin-releasing factor 1 receptor antagonist E2508 reduces restraint stress-induced defecation and visceral pain in rat models

N-Cyclopropylmethyl-7-(2,6-dimethoxy-4-methoxymethylphenyl)-2-ethyl-N-(tetrahydro-2H-pyran-4-ylmethyl)pyrazolo[1,5-a]pyridin-3-amine tosylate (E2508) is a newly discovered selective corticotropin-releasing factor 1 receptor antagonist. Here, we investigated the effects of E2508 on wrap restraint stress-induced defecation and visceral pain in rats. Oral pretreatment with E2508 dose-dependently decreased stool weights after 20min wrap restraint stress and significant effects were observed at doses of 30 and 100mg/kg. However, E2508 did not affect basal defecation at doses up to 100mg/kg. In contrast, alosetron, a 5-HT3 receptor antagonist, decreased both wrap restraint stress-induced and basal stool output at a dose of 0.1mg/kg. In a rat visceral pain model, subcutaneous injections of both E2508 (0.01 and 0.1mg/kg) and alosetron (0.001 and 0.01mg/kg) significantly decreased the number of abdominal muscle contractions induced by colonic distention, suggesting these drugs reduced visceral pain. Together, these results demonstrate E2508 has the potential to be an effective therapy for the treatment of irritable bowel syndrome with a lower risk of adverse events such as constipation compared with the current clinically used 5-HT3 receptor antagonist.

Selective corticotropin-releasing factor 1 receptor antagonist E2508 has potent antidepressant-like and anxiolytic-like properties in rodent models

Corticotropin-releasing factor (CRF) is a hormone secreted by the hypothalamus in response to stress, and CRF antagonists may be effective for the treatment of stress-related disorders including major depressive and anxiety disorders. Here, we investigated the in vivo pharmacological profile of N-cyclopropylmethyl-7-(2,6-dimethoxy-4-methoxymethylphenyl)-2-ethyl-N-(tetrahydro-2H-pyran-4-ylmethyl)pyrazolo[1,5-a]pyridin-3-amine tosylate (E2508), a recently synthesized, orally active CRF1 receptor antagonist. Oral administration of a single dose of E2508 (3 or 10mg/kg), but not fluoxetine (30mg/kg), a selective serotonin reuptake inhibitor (SSRI), significantly shortened immobility time in rats in the forced swim test. E2508 (10, 30, or 100mg/kg) also showed an antidepressant-like effect in the forced swim test in mice, with no sedative or muscle relaxant effects for doses up to 100mg/kg. Moreover, E2508 (5 or 20mg/kg) significantly reduced anxiety-like behavior in the rat defensive burying test. Diazepam, a benzodiazepine anxiolytic agent, also showed an anxiolytic effect in the defensive burying test at the same dose that induced a muscle relaxant effect in mice. Administration of E2508 (30mg/kg) for 14 consecutive days did not affect sexual behavior. By contrast, fluoxetine (30mg/kg) administration for ≥7 consecutive days decreased sexual behavior. These results indicate that E2508 has both potent antidepressant-like and anxiolytic-like effects in rodent models, and is well tolerated compared with a commonly prescribed therapeutic SSRI or benzodiazepine.

Abstract 1413: Eribulin caused re-modeling of tumor vasculature altering gene expression profiling in angiogenesis and Epithelial Mesenchymal Transition (EMT) signaling pathway of host cells within human breast cancer cell (BCC) xenografts in nude mice.

Objective: Eribulin mesylate (ERI) is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. The objective of this study was to examine the effect of ERI on tumor vasculature with immunohistchemical (IHC) analysis and gene expression profiling (GEP) in normal host cells, such as endothelial cells and vascular mural cells within tumor microenvironments in human BCC xenograft models Methods: Anti-tumor activity of ERI was examined at doses of 1.5 and 3.0 mg/kg, i.v. at day 1, in human BCC MX-1, MDA-MB-231 and MDA-MB-453 sc xenografts in nude mice. For IHC and GEP analysis, tumor tissues were collected at day 4 and day 8. IHC analysis was performed using mouse CD31 antibody to stain endothelial cells. Microvessel density (MVD) and vessel perimeter were determined by using Aperio Image Scope. GEP analysis for mouse host and human tumor cells within tumor tissues was done by using mouse and human TaqMan Low Density Arrays (TLDAs) consisting of a set of 92 genes related to angiogenesis, metastasis/EMT and cell differentiation signal pathways. Results shows % of non-treatment group (NT). Results: ERI showed significant anti-tumor activity against all three human BCC xenografts in a dose dependent manner. IHC analysis showed that ERI altered morphology of tumor vasculature day 8 after treatments and increased number of vessels with small size of perimeter ( 300um) in both MX-1 and MDA-MB-231 xenograft models (p Conclusions: ERI induced re-modeling of tumor vasculature in human BCC xenograft models. GEP related to angiogenesis and EMT/metastasis pathway was significantly affected with ERI treatment in host cells under tumor microenvironments. ERI might cause remodeling of tumor vasculature by regulating GEP in host cells. Further investigation may be warranted to examine if the activity of ERI against host cells in tumor tissues contributed to anti-tumor activity of ERI. Citation Format: Junji Matsui, Osamu Toyama, Mitsuhiro Ino, Taro Semba, Mai Uesugi, Hiroki Muto, Judith L. Oestreicher, Kentaro Takahashi, Kentaro Matsuura, Yoshiaki Sato, Taisuke Uehara, Takayuki Kimura, Hideki Watanabe, Yoichi Ozawa, Makoto Asano, Yusuke Adachi, Ken Aoshima, Yasuhiro Funahashi. Eribulin caused re-modeling of tumor vasculature altering gene expression profiling in angiogenesis and Epithelial Mesenchymal Transition (EMT) signaling pathway of host cells within human breast cancer cell (BCC) xenografts in nude mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2013-1413