Yasutaka Takase

Antipruritic effect of the topical phosphodiesterase 4 inhibitor E6005 ameliorates skin lesions in a mouse atopic dermatitis model.

Phosphodiesterase (PDE) 4 inhibition is a well-known anti-inflammatory mechanism, but the development of PDE4 inhibitors has been hampered by side effects such as nausea and emesis. Local delivery of a PDE4 inhibitor to the site of inflammation may overcome these issues. The purpose of this study was to assess the therapeutic potential of E6005 (methyl 4-[({3-[6,7-dimethoxy-2-(methylamino)quinazolin-4-yl]phenyl}amino)carbonyl]benzoate), a novel PDE4 inhibitor developed as a topical agent for atopic dermatitis (AD). E6005 potently and selectively inhibited human PDE4 activity with an IC50 of 2.8 nM and suppressed the production of various cytokines from human lymphocytes and monocytes with IC50 values ranging from 0.49 to 3.1 nM. In mice models, the topical application of E6005 produced an immediate antipruritic effect as well as an anti-inflammatory effect with reduced expression of cytokines/adhesion molecules. On the basis of these observed effects, topical E6005 ameliorated the appearance of atopic dermatitis-like skin lesions in two types of AD models, hapten- and mite-elicited models, exhibiting inhibitory effects comparable to that of tacrolimus. The use of 14C-labeled E6005 showed rapid clearance from the blood and low distribution to the brain, contributing to the low emetic potential of this compound. These results suggest that E6005 may be a promising novel therapeutic agent with antipruritic activity for the treatment of AD.

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment.

Section Review Cardiovascular & Renal: Phosphodiesterase 5 inhibitors in development for cardiovascular therapy

Phosphodiesterase 5 (PDE 5), which is actually a family of PDE isozymes, hydrolyses cGMP with a high degree of selectivity and is insensitive to calmodulin (CaM). PDE 5 has been identified in the aorta, mesenteric or pulmonary arteries and saphenous vein, suggesting that it plays an important role in the regulation of cGMP levels in vascular smooth muscle, much like stimulators of cGMP synthesis, such as endothelium-derived relaxing factor (EDRF) and atrial natriuretic peptide (ANP). Several selective and potent PDE 5 inhibitors, which can be used to determine the pharmacological effects on cardiovascular diseases, have been synthesised by various pharmaceutical companies and are currently being studied. Results have shown that these PDE 5 inhibitors dilate the large epicardial coronary artery in conscious pigs and selectively lower the pulmonary arterial pressure in animal models of congestive heart failure or pulmonary hypertension, suggesting that these inhibitors may be useful as a therapy for heart f...

Determination of a novel and potent cyclic GMP phosphodiesterase inhibitor, 4-{[3,4-(methylenedioxy)-benzyl]amino}-6,7,8-trimethoxyquinazoline, in dog plasma by high-performance liquid chromatography

A simple and accurate method for determining levels of 4-([3,4-(methylenedioxy)benzyl]amino)-6,7,8-trimethoxyquinazoline, a novel cyclic GMP phosphodiesterase inhibitor, in dog plasma was developed. This method involves extraction with diethyl ether and reversed-phase high-performance liquid chromatography with ultraviolet detection. Regression analysis showed that the method was linear over the range 1.0-1000 ng/ml. The method was employed for the analysis of plasma samples in a preliminary pharmacokinetic study in beagle dogs.

Abstract 1377: Chemical probe-based approach clarifies binding of receptor tyrosine kinases (RTKs) to lenvatinib in preclinical models

Background: Lenvatinib mesilate (lenvatinib) is the selective inhibitor of VEGFR1-3, and other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib inhibits angiogenesis through the inhibition of VEGFR2 and FGFR1 and tumor proliferation through the inhibition of FGFR1 and RET in preclinical thyroid cancer models. Chemical probe-based binding protein isolation is a powerful tool to demonstrate the direct compound-protein interactions even in complicated biological materials such as intact cells and their robust protein extracts. The purpose of this study is to assess the interaction of lenvatinib with RTKs by using lenvatinib derivative probe as an analysis of mode of action in preclinical models. Methods: Lenvatinib chemical probe was designed based on a result of co-crystal structure of lenvatinib and VEGFR2. A linker moiety was introduced onto free space of quinoline core (position-7) and its inhibitory activities against RTKs were confirmed by cell-free enzyme assay system. Lenvatinib chemical probe was immobilized onto the monolithic affinity matrices and the binding proteins were isolated from the extracts of the human umbilical vein endothelial cells (HUVEC), the human differentiated thyroid cancer cell lines, RO82-W-1 and TPC-1. SDS-PAGE and western blotting (WB) with drug competition assay were performed to assess the binding RTKs of lenvatinib. Results: The chemical probe showed acceptable inhibitory activities against RTKs for affinity isolation. In the isolated protein sample from HUVEC, VEGFR2 and FGFR1 were detected by WB and each protein band was clearly reduced by lenvatinib competition in a dose-dependent manner. It indicates lenvatinib specifically interacts with VEGFR2 and FGFR1 even in such complicated protein mixture. In the sample from RO82-W-1, WB analysis showed that lenvatinib chemical probe efficiently isolated PDGFRα and FGFR1. Those protein bands were dramatically decreased by drug competition, similar to the experiment with HUVEC. The chemical probe-based approach was subjected to the protein extract from TPC-1, which expresses aberrant RET fusion kinase derived from CCDC6-RET gene rearrangement. WB analysis showed that CCDC6-RET was detected as binding protein of lenvatinib probe. Conclusions: The chemical probe-based approach proved the interaction of lenvatinib with VEGFR2, FGFR1, PDGFRα and RET fusion kinase in the complicated biological materials from HUVEC and thyroid cancer cell lines. These results supported that lenvatinib is likely to possess a unique anti-tumor activity, in addition to the anti-angiogenic activity, through the interaction with these RTKs. Citation Format: Takayuki Kimura, Noboru Yamamoto, Hiroshi Kamiyama, Megumi Ikemori Kawada, Akihiko Yamamoto, Yoshihiko Kotake, Yasutaka Takase, Yasuhiro Funahashi, Yoshiya Oda. Chemical probe-based approach clarifies binding of receptor tyrosine kinases (RTKs) to lenvatinib in preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1377. doi:10.1158/1538-7445.AM2015-1377

Application of phosphodiesterase 4 inhibitor for atopic dermatitis.

キーワード:アトピー性皮膚炎,痒み,phosphodiesterase 4,E6005 エーザイ株式会社 筑波研究所(〒300 -2635 茨城県つくば市東光台 5 -1 -3) E-mail: n3 ishii@hhc.eisai.co.jp 原稿受領日:2014 年 7 月 2 日,依頼原稿 Title: Application of phosphodiesterase 4 inhibitor for atopic dermatitis Author: Naoto Ishii, Hisatsugu Wakita, Kazuki Miyazaki, Yasutaka Takase, Osamu Asano, Kazutomi Kusano, Manabu Shirato 要約:日本皮膚科学会によるとアトピー性皮膚炎 (AD)の定義は「増悪と寛解を繰り返す,痒みを伴う 湿疹を主病変とする慢性に経過する疾患」とされてお り,今なお患者数が増大する傾向にある.ADでは重 度な痒みを伴うことが特徴であり,既存薬では十分な 痒み抑制作用が得られているとは言えず,痒みのコン トロールが治療の課題の一つと考えられている.そこ で改めて AD病態を振り返り,治療薬開発の現状を纏 めた.その中で phosphodiesterase 4(PDE4)阻害薬 に注目し,E6005 を題材として PDE4 阻害薬の AD適 応を目指した取り組みを紹介する.E6005 は無細胞 PDE活性測定系において選択的な PDE4 阻害作用を 示し,ヒト末梢血リンパ球・単球からのサイトカイン 産生を抑制したことから,PDE4 阻害に基づく E6005 の抗炎症作用を確認できた.ハプテン誘発接触皮膚炎 型マウスモデルにおいて,E6005 を連続塗布すると有 意な皮膚炎抑制効果が得られ,かつ皮疹部におけるサ イトカイン・接着分子の発現抑制効果が認められた. さらに ADマウスモデルである NC/Ngaマウスに E6005 を連続塗布すると AD様皮膚炎抑制効果が得ら れたほか,単回塗布による即時的な掻破行動抑制効果 も認められた.PDE4 阻害作用に基づく嘔吐誘発に関 してキシラジン・ケタミン麻酔覚醒モデルを用いて検 討したところ,E6005 は第一世代 PDE4 阻害薬シロミ ラストと比較して嘔吐誘発性が低いことが分かり,治 療濃度域の広さが認められた.E6005 は血液中で速や かに代謝され,中枢神経系への分布が非常に少ないこ とから嘔吐誘発性の低下に繋がった可能性がある.こ れらの結果より,E6005 は全身的暴露を最小限に抑え た局所投与型薬剤として,抗炎症作用のみならず痒み 抑制作用を併せ持つアトピー性皮膚炎治療薬として期 待される.

Abstract 2788: Antiproliferative schweinfurthins reveal a role of the trans-Golgi-network in mTOR-AKT signaling and cancer cell survival

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The schweinfurthin class of natural compounds showed selective and potent anti-proliferative activities in the NCI-60 cancer cell panel1,2). Recent work by Burgett et al3) suggested that schweinfurthins may bind to oxysterol-binding protein-related proteins (ORPs) for their anti-proliferative activity. However, the mechanism of the selectivity and potency is still unknown. Here we show that schweinfurthins preferentially inhibited cell proliferation of PTEN-deficient cancer cells across different cancer types, and specifically inhibited AKT phosphorylation in both PTEN wild-type and mutant cancer cells. Intracellularly, a fluorescent schweinfurthin probe localized within the endosome/lysosome structure. Treatment of cells with active schweinfurthins caused fragmentation of the trans-Golgi-network (TGN), a vesicular trafficking system, in a dose- and time-dependent manner, without impacting the cis, med or trans-Golgi matrix structures. An inactive trimethylated analog of schweinfurthins did not display this activity. Using biotinylated probes, we found that schweinfurthins bind to the sterol-binding domain of ORPs; the capacity of binding to ORPs was positively correlated with both TGN fragmentation activity and anti-proliferative activity. These results are consistent with the recent observation that knockdown of specific ORP proteins leads to TGN trafficking disruption4). The fragmentation of TGN by schweinfurthins resulted in specific inhibition of mTOR-AKT pathway signaling in two ways: 1) disruption of lipid raft organization at the plasma membrane, evidenced by diminished trafficking of ceramide, GM1 and sphingomyelin, which inhibited PDK1 phosphorylation; and 2) dislocation of mTOR from its active sites at endosomes and/or lysosomes. Furthermore, we found that PTEN-deficient DLBCL cancer cells were highly sensitive to schweinfurthin treatment in vitro (IC50 values < 10 nM). Normal human fibroblasts were 1000 times less sensitive than the DLBCL cells. Taken together, our results disclosed an important role of the TGN, which was specifically targeted by schweinfurthins, in mTOR-AKT signaling and in cancer cell survival, suggesting that analogs of schweinfurthin might offer promising novel therapeutics for treating mTOR-AKT signaling-addicted cancer cells such as PTEN-deficient DLBCL. 1. Beutler JA, et al. Cytotoxic geranyl stilbenes from Macaranga schweinfurthii. J Nat Prod., 1998, 61:1509-1512. 2. Yoder BJ, et al. Antiproliferative prenylated stilbenes and flavonoids from Macaranga alnifolia from the Madagascar rainforest. J Nat Prod., 2007, 70:342-346. 3. Burgett AW, et al. Natural products reveal cancer cell dependence on oxysterol-binding proteins. Nat Chem Biol., 2011, 7:639-647. 4. Du X, et al. A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking. J Cell Biol., 2011, 192:121-135. Citation Format: Xingfeng Bao, Naoko Hata, Kishan Agarwala, Zichun Wang, Winnie Lee, Lana Parent, Hongsheng Cheng, Dayong Qiu, Yongchun Shen, Bingfan Du, Wanjun Zheng, Nanding Zhao, Maarten Postema, Mary Woodall Jappe, Yasutaka Takase, Toshimitsu Uenaka, Kenichi Nomoto. Antiproliferative schweinfurthins reveal a role of the trans-Golgi-network in mTOR-AKT signaling and cancer cell survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2788. doi:10.1158/1538-7445.AM2014-2788