Y-27632 2017

As A General Information;

http://www.cosmobio.co.jp/product/detail/y-27632-dihydrochloride-enz.asp?entry_id=16716

Y27632はROCK/ Rho-kinaseを ATPと拮抗的に阻害する。

細胞においては10-30μM程度の濃度でよく使用される。
ROCK/Rho-kinase活性の阻害により
基質のミオシン調節軽鎖(MRLC)のリン酸化は抑制され、

また別の基質であるMYPT1のリン酸化が抑制される
ことによりMYPT1をサブユニットとする
ミオシンフォスファターゼが活性化し、
MRLCの脱リン酸化が亢進する。


これによりミオシンIIのATPase活性の低下、
脱重合が起こるため、細胞内のミオシンIIの機能は低下する。
ストレスファイバーの崩壊など、
アクチン細胞骨格の大きな変化が見られる。

ROCK/Rho-kinaseの基質は他にもあるものの、多くの細胞において上記の変化は目立つ。

IL-23 production was elevated by lipoteichoic acid (LTA), 
which increased the activation of RhoA in association with increased the nuclear translocation of NF-kB
and its DNA-binding activity.

Pretreatment of RA macrophages with the pharmacological inhibitors exoenzyme C3 (RhoA),
Y27632 (Rho-kinase) or BAY11-7082 (NF-kB)
inhibited IL-23 production by LTA.

Inhibition of the RhoA/Rho-kinase pathway by these drugs attenuated NF-kB activation.


Cilostazol suppressed the TLR2-mediated activation of RhoA,
decreased NF-kB activity with down-regulated IL-23 production, and these effects were reversed by RpcAMPS,
as an inhibitor of cAMP-dependent protein kinase.




ML7 とML9 はそれぞれミオシン軽鎖キナーゼ(MLCK)をATPと拮抗的に阻害する。


しかしその細胞への使用には大いに注意が必要である。
MLCKによるリン酸化部位を擬似リン酸化型に変えた
変異MRLCを発現する細胞では、
Y27632で処理をしてもその変異MRLCは影響を受けずに
機能し続けるので,ストレスファイバーに変化はないが、
ML7で処理をするとストレスファイバーは崩壊する。


これはML7がMRLCのリン酸化を介さずにアクチン細胞骨格に大きな作用をするということを示している。

GPCR Modulators: Statin Is Inverse Agonistic !!

3. The HMG CoA Pathway & Rho

Statins are well known inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase—the crucial rate limiting step in hepatic cholesterol biosynthesis.

Independently of the cholesterol lowering effect,
the inhibition of HMG-CoA leads to reduced synthesis of isoprenoid intermediaries—farnesyl pyrophosphate (FPP) and geranylgeranyl
pyrophosphate (GGPP)  (Figure 2) [32].

 

These precursors of cholesterol biosynthesis are essential for post-translational modification and prenylation of certain small GTPase proteins, include the Ras and Rho superfamilies [33].

Prenylation of these proteins facilitates their intracellular trafficking and covalent attachment to the lipid membrane, which is often essential for biological function.

These small GTPases cycle between an inactive GDP bound form found in the cytosol in association with guanine dissociation inhibitor (Rho GDI), and an active GTP bound form usually associated with the cell membrane (Figure 3).

When Rho proteins are released from GDIs, they can insert into the cell membrane where they are activated by guanine nucleotide exchange factors (GEFs), and this initiates interaction with membrane effector proteins such as Rho kinase (ROCK).

The Rho family of proteins is implicated in many key intracellular events and signalling pathways, including regulation of the actin cytoskeleton, cell adhesion, cell to cell interaction, and cell-cycle progression.

This topic is comprehensively reviewed by Burridge and colleagues [34].

It is thought that inhibition of the Rho pathway by statins is one of the major mechanisms via which statins affect cell physiology.

 

5.2. Rho Inhibition

 

Probably the most important pleiotropic effect of statins is the inhibition of small GTPases, including the Rho family of proteins [77].

 

The Rho pathway is responsible for various integral intracellular processes and for the interaction between cells and their environment.

 

Three subfamilies constitute the Rho superfamily—Rho, Rac, and Cdc42.

Regulation of the actin cytoskeleton, microtubule dynamics, vesicle trafficking, cell polarity and cell-cycle progression are all under the control of the Rho proteins (Figure 4).

This topic is comprehensively reviewed by Burridge et al. [34].

 

The Rho proteins contain a lipophilic isoprenoid group, which permits their attachment to the cell membrane and is generally essential for biological function.

By inhibiting the conversion of HMG-CoA to L-mevalonic acid, statins inhibit the synthesis of isoprenoid intermediaries, including FFP and GGPP.

Hence proper subcellular localisation and trafficking of these GTPase proteins is inhibited, with significant
functional consequences.

 Importantly, post-translationally immature forms of G-proteins may maintain partial function [79] [80], and interfere with the activity of mature membrane-anchored proteins.

 By inhibiting Rho and its downstream effector proteins including Rho kinase (ROCK), statins are likely to
affect the contractile properties of the conventional outflow pathway.

 

 Cells of the conventional pathway possess a contractile tone which is regulated through Rho signaling [81].

 ROCK phosphorylates and inhibits the myosin- binding subunit of myosin light chain (MLC) phosphatase.

 This action increases MLC phosphorylation and
myosin contractility, hence driving the formation of stress fibres and focal adhesion [34].

 

Early work on specific inhibitors of the Rho pathway has shown that Rho inhibition results in relaxation of the contractile tone of cells of the aqueous outflow pathway in vitro and ex vivo [78] [82] [83].

 This increases aqueous outflow and reduces intraocular pressure.

 

Indeed Rho kinase inhibitors have been shown to be potent agents in lowering intraocular pressure, and are undergoing phase 2 and 3 clinical trials [31] [84].

 This effect of Rho inhibition in lowering the O. Pokrovskaya et al. 130

 

More recent studies in this field have demonstrated the potential beneficial effect of combination therapy with a statin plus a Rho kinase inhibitor [86].

 Rho inhibitors also affect the actin cytoskeleton, and cellular morphology of the aqueous outflow pathway.

In vitro work has shown decreases in actin stress fibres and focal adhesions in cultured porcine and human TM cells [83].

 

Treatment of monkey Schlemm’s canal (SC) cells with a Rho inhibitor increases the number of giant vacuoles within cell, and decreases the expression of certain cytoskeletal proteins (ZO-1 and claudin-5) [87].

 This leads to morphological changes—cell rounding and detachment of cells from each other, as well as wider
paracellular spaces [78] [88].

 In cultured cells of the SC, Rho inhibition results in increased permeation in vitro, which facilitates aqueous drainage [88].

 Ex vivo perfusion experiments, where porcine, monkey, cow and cadaver eyes are perfused with an aqueous humour substitute, have shown that perfusion with a Rho inhibitor increases the conventional outflow facility [78] [83] [88] [89].

 

5.3. Rac and Reactive Oxygen Species (ROS)

 

One of the 3 key members of the Rho family is Rac1—this important GTPase protein is responsible for cytoskeletal
remodelling—specifically the formation of lamellipodia and membrane ruffles [90].

Lamellipodia are actin-rich cellular protrusions, essential for cell migration, and play an important role in the invasion and metastases of cancer cells [91].

 

Furthermore, Rac1 binds to p67phox which leads to activation of the NADPH oxidase
system and generation of ROS [92].

 

The presence of high concentrations of ROS can overwhelm the cell’s natural defence mechanisms and lead to programmed cell death. However the role of ROS in cell physiology is
more complex thanthat and more recent studies have shown that in some scenarios, ROS (in small doses) promote
cell survival—contrary to the traditional view that they are solely destructive molecules [93] [94].

 

ROS have also been shown to act as signalling molecules in their own right [95]. In smooth muscle and heart cells, it
has been shown that by inhibiting the prenylation and subsequent activation of Rac1, statins inactivate NADPH
oxidase and hence reduce angiotensin-II-induced ROS production [50] [96]. Our own research group has previously
demonstrated evidence of oxidative stress and mitochondrial dysfunction in lamina cribrosa cells from
the optic nerve heads of glaucoma donors, compared to normal donors [97].

 

 

Furthermore, our group has shown
that up to 50% of POAG patients have a pathogenic mitochondrial DNA mutation, which may lead to mitochondrial respiratory dysfunction and subsequent predisposition to oxidative stress in TM, LC and RGC [98].

 

Increased levels of ROS have been found in the aqueous humour of glaucoma patients [99] [100]. Glutathione is
a tripeptide found in the eye and other tissues, and is a key element of the protective mechanism of the eye
against oxidative stress [101]. Altered glutathione levels have been reported in the aqueous humour of glaucoma
patients [102], and abnormally low levels of glutathione have even been demonstrated in the serum of glaucoma
patients [101]. ROS affect the cellularity of the trabecular meshwork, and may cause endothelial dysfunction—
O. Pokrovskaya et al. 132
which would contribute to impaired aqueous outflow and higher IOP [103].

 

Mitochondrial dysfunction in LC and RG cells allows the build-up of ROS, and may lead to increased susceptibility to cell death and impaired
repair mechanisms [97] [104].

 

By reducing the production of ROS in ocular tissues, statins may help to reduce ROS-induced damage and glaucoma progression.

癌代謝静止効果を有するジェネリック薬とその活用

たとえば

http://www.google.co.jp/patents/WO2010044404A1?cl=ja

縮合複素環誘導体及びその医薬用途
WO 2010044404 A1

要約書

血漿尿酸値異常に起因する疾患等の予防又は治療薬として有用な化合物を提供する。 本発明は、キサンチンオキシダーゼ阻害活性を有し、血漿尿酸値異常に起因する疾患の予防又は治療薬として有用な、下記式（Ｉ）で表される縮合複素環誘導体、そのプロドラッグ、又はその塩等に関する。式（Ｉ）中、Ｔはトリフルオロメチル、ニトロ又はシアノ；環Ｑはヘテロアリール；Ｘ^１及びＸ^２は独立してＣＨ又はＮ；環Ｕはアリール又はヘテロアリール；ｍは０～２の整数；ｎは０～３の整数；Ｒ^１は水酸基、アミノ又はＣ_１－６アルキル；Ｒ^２は、Ｃ_１－６アルキル、Ｃ_１－６アルコキシＣ_１－６アルキル等である。

特許請求の範囲(21)

1. 式
   

   

   〔式中、
   Ｔはトリフルオロメチル、ニトロ又はシアノ；
   環Ｑは５又は６員環ヘテロアリール；
   Ｘ^１及びＸ^２は独立して、ＣＨ又はＮ；
   環ＵはＣ_６アリール又は５又は６員環ヘテロアリール；
   ｍは０～２の整数；
   ｎは０～３の整数；
   Ｒ^１は水酸基、ハロゲン原子、アミノ又はＣ_１－６アルキル（ｍが２であるとき、２つのＲ^１は異なっていてもよい）；
   Ｒ^２は、
   （ｉ）環Ｑ内の炭素原子に結合している場合は、（１）～（１１）：
   　（１）ハロゲン原子；
   　（２）水酸基；
   　（３）シアノ；
   　（４）ニトロ；
   　（５）カルボキシ；
   　（６）カルバモイル；
   　（７）アミノ；
   　（８）それぞれ独立して、置換基群αから選択される任意の基を有していてもよいＣ_１－６アルキル、Ｃ_２－６アルケニル又はＣ_１－６アルコキシ；
   　（９）Ｃ_２－６アルキニル、Ｃ_１－６アルキルスルホニル、モノ（ジ）Ｃ_１－６アルキルスルファモイル、Ｃ_２－７アシル、Ｃ_１－６アルコキシカルボニル、Ｃ_１－６アルコキシカルボニルオキシ、モノ（ジ）Ｃ_１－６アルキルアミノ、モノ（ジ）Ｃ_１－６アルコキシＣ_１－６アルキルアミノ、Ｃ_１－６アルコキシＣ_１－６アルキル（Ｃ_１－６アルキル）アミノ、Ｃ_２－７アシルアミノ、Ｃ_１－６アルコキシカルボニルアミノ、Ｃ_１－６アルコキシカルボニル（Ｃ_１－６アルキル）アミノ、モノ（ジ）Ｃ_１－６アルキルカルバモイル、モノ（ジ）Ｃ_１－６アルコキシＣ_１－６アルキルカルバモイル、Ｃ_１－６アルコキシＣ_１－６アルキル（Ｃ_１－６アルキル）カルバモイル、モノ（ジ）Ｃ_１－６アルキルアミノカルボニルアミノ、Ｃ_１－６アルキルスルホニルアミノ又はＣ_１－６アルキルチオ；
   　（１０）Ｃ_３－８シクロアルキル、３～８員環ヘテロシクロアルキル、Ｃ_５－８シクロアルケニル又は５～８員環ヘテロシクロアルケニル；
   　（１１）Ｃ_６アリール、Ｃ_６アリールオキシ、Ｃ_６アリールカルボニル、５又は６員環ヘテロアリール、５又は６員環ヘテロアリールオキシ、５又は６員環ヘテロアリールカルボニル、Ｃ_６アリールアミノ、Ｃ_６アリール（Ｃ_１－６アルキル）アミノ、５又は６員環ヘテロアリールアミノ又は５又は６員環ヘテロアリール（Ｃ_１－６アルキル）アミノ；の何れかであり、
   （ｉｉ）　環Ｑの窒素原子に結合している場合は、（１２）～（１５）：
   　（１２）それぞれ独立して、置換基群αから選択される任意の基を有していてもよいＣ_１－６アルキル又はＣ_２－６アルケニル；
   　（１３）Ｃ_２－６アルキニル、Ｃ_１－６アルキルスルホニル、モノ（ジ）Ｃ_１－６アルキルスルファモイル、Ｃ_２－７アシル、Ｃ_１－６アルコキシカルボニル又はモノ（ジ）Ｃ_１－６アルキルカルバモイル；
   　（１４）Ｃ_３－８シクロアルキル又は３～８員環ヘテロシクロアルキル；
   　（１５）Ｃ_６アリール、５又は６員環ヘテロアリール、Ｃ_６アリールカルボニル又は５又は６員環ヘテロアリールカルボニル；の何れかであり；
   （ｎが２又は３であるとき、Ｒ^２はそれぞれ異なっていてもよく、更に、２つのＲ^２が、環Ｑ内の隣り合った原子に結合して存在し、それぞれＣ_１－６アルコキシを有していてもよいＣ_１－６アルキルである場合は、結合する環Ｑ内の原子と共に、５～８員環を形成してもよい。）；
   　　　置換基群αは、フッ素原子、水酸基、アミノ、カルボキシ、Ｃ_１－６アルコキシ、モノ（ジ）Ｃ_１－６アルキルアミノ、モノ（ジ）Ｃ_１－６アルコキシＣ_１－６アルキルアミノ、Ｃ_１－６アルコキシＣ_１－６アルキル（Ｃ_１－６アルキル）アミノ、Ｃ_１－６アルコキシカルボニルアミノ、Ｃ_２－７アシル、Ｃ_１－６アルコキシカルボニル、モノ（ジ）Ｃ_１－６アルキルカルバモイル、モノ（ジ）Ｃ_１－６アルコキシＣ_１－６アルキルカルバモイル、Ｃ_１－６アルコキシＣ_１－６アルキル（Ｃ_１－６アルキル）カルバモイル、Ｃ_１－６アルキルスルホニルアミノ、Ｃ_２－７アシルアミノ、Ｃ_１－６アルコキシカルボニルアミノ、Ｃ_３－８シクロアルキル、３～８員環ヘテロシクロアルキル、Ｃ_６アリール及び５又は６員環ヘテロアリールである〕で表される縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
2. Ｔがシアノである請求項１記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
3. Ｘ^１がＣＨである請求項１又は２記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
4. Ｘ^２がＣＨである請求項１～３の何れかに記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
5. 環Ｑがピリジン、ピリミジン、ピラジン、チアゾール、イミダゾール、ピラゾール、オキサゾール、イソチアゾール、イソオキサゾール、チオフェン、フラン又はピロール環である請求項１～４の何れかに記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
6. 環Ｑがピリジン、チオフェン又はピロール環である請求項５記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
7. 環Ｕがベンゼン、ピリジン、チアゾール、ピラゾール又はチオフェン環である請求項１～６の何れかに記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
8. ｍが０であるか、又はｍが１であり、かつ、環Ｕが下記式：
   

   

   （式中、Ｒ^１aは水酸基；アミノ；又はＣ_１－６アルキルであり、Ａは縮合環との結合を、Ｂはカルボキシとの結合をそれぞれ表す）で表される何れかの環である、請求項７記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
   
9. ｍが０であるか、又はｍが１であり、かつＲ^１aが水酸基又はＣ_１－６アルキルである、請求項８記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
10. 環Ｕがチアゾール環である請求項８又は９記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
11. ｍが０であるか、又はｍが１；かつＲ^１aが水酸基であり、かつ環Ｕがピリジン環である請求項９記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
12. Ｒ^１aがメチルである、請求項１０記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
13. Ｒ^１aが水酸基である、請求項１１記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
14. ｎが０であるか、又はｎが１～３であり、かつＲ^２が環Ｑの炭素原子に結合するハロゲン原子；水酸基；それぞれフッ素原子、水酸基及びアミノから選択される任意の基を１～３個有していてもよいＣ_１－６アルキル又はＣ_１－６アルコキシ；Ｃ_１－６アルコキシＣ_１－６アルキル；もしくはＣ_１－６アルコキシＣ_１－６アルコキシ；又は環Ｑの窒素原子に結合する、フッ素原子、水酸基及びアミノから選択される任意の基を１～３個有していてもよいＣ_１－６アルキル；もしくはＣ_１－６アルコキシＣ_１－６アルキルである、請求項１～１３の何れかに記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
15. ｎが０であるか、又はｎが１～３であり、かつＲ^２が環Ｑの炭素原子に結合するハロゲン原子；水酸基；もしくはフッ素原子を１～３個有していてもよいＣ_１－６アルキル；又は環Ｑの窒素原子に結合する、フッ素原子を１～３個有していてもよいＣ_１－６アルキル；もしくはＣ_１－６アルコキシＣ_１－６アルキルである、請求項１４記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
16. キサンチンオキシダーゼ阻害薬である、請求項１～１５の何れかに記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
17. 請求項１～１５の何れかに記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩を有効成分として含有する医薬組成物。
18. 高尿酸血症、痛風結節、痛風関節炎、高尿酸血症による腎障害及び尿路結石から選択される疾患の予防又は治療用である、請求項１７記載の医薬組成物。
19. 高尿酸血症の予防又は治療用である、請求項１８記載の医薬組成物。
20. 血漿尿酸値低下薬である、請求項１７記載の医薬組成物。
21. 尿酸生成抑制薬である、請求項１７記載の医薬組成物。

明細書

縮合複素環誘導体及びその医薬用途 　本発明は、医薬品として有用な縮合複素環誘導体に関するものである。
　さらに詳しく述べれば、本発明は、キサンチンオキシダーゼ阻害活性を有し、血清尿酸値異常に起因する疾患の予防又は治療薬として有用な、縮合複素環誘導体もしくはそのプロドラッグ、又はその薬理学的に許容される塩に関するものである。 　尿酸はヒトにおける、プリン体代謝の最終産物である。多くの哺乳類では、ヒトと異なり、肝臓の尿酸酸化酵素（ウリカーゼ）により尿酸がさらにアラントインに分解され、腎臓より排泄される。ヒトにおける尿酸排泄の主な経路は腎臓であり、約２／３が尿中に排泄され、残りは糞便より排泄される。尿酸産生が過剰になったり、尿酸排泄が低下することにより高尿酸血症が起こる。高尿酸血症は、尿酸産生過剰型、尿酸排泄低下型及びその混合型に分類される。この高尿酸血症の分類は臨床上重要であり、治療薬の副作用軽減を考慮して、各分類における治療薬が選択されている（例えば、非特許文献１参照）。

縮合複素環誘導体及びその医薬用途
WO 2010044404 A1

 

 

 

要約書

 

 

血漿尿酸値異常に起因する疾患等の予防又は治療薬として有用な化合物を提供する。

 

 

本発明は、キサンチンオキシダーゼ阻害活性を有し、血漿尿酸値異常に起因する疾患の予防又は治療薬として有用な、下記式（Ｉ）で表される縮合複素環誘導体、そのプロドラッグ、又はその塩等に関する。式（Ｉ）中、Ｔはトリフルオロメチル、ニトロ又はシアノ；環Ｑはヘテロアリール；Ｘ^１及びＸ^２は独立してＣＨ又はＮ；環Ｕはアリール又はヘテロアリール；ｍは０～２の整数；ｎは０～３の整数；Ｒ^１は水酸基、アミノ又はＣ_１－６アルキル；Ｒ^２は、Ｃ_１－６アルキル、Ｃ_１－６アルコキシＣ_１－６アルキル等である。

 

 

 

特許請求の範囲(21)

1. 式
   

   

   
   
2.  
3. 〔式中、Ｔはトリフルオロメチル、ニトロ又はシアノ；
   環Ｑは５又は６員環ヘテロアリール；Ｘ^１及びＸ^２は独立して、ＣＨ又はＮ；環ＵはＣ_６アリール又は５又は６員環ヘテロアリール；
   ｍは０～２の整数；
   ｎは０～３の整数；
   Ｒ^１は水酸基、ハロゲン原子、アミノ又はＣ_１－６アルキル（ｍが２であるとき、２つのＲ^１は異なっていてもよい）；
   Ｒ^２は、
   （ｉ）環Ｑ内の炭素原子に結合している場合は、（１）～（１１）：
   　（１）ハロゲン原子；
   　（２）水酸基；
   　（３）シアノ；
   　（４）ニトロ；
   　（５）カルボキシ；
   　（６）カルバモイル；
   　（７）アミノ；
   　（８）それぞれ独立して、置換基群αから選択される任意の基を有していてもよいＣ_１－６アルキル、Ｃ_２－６アルケニル又はＣ_１－６アルコキシ；
   　（９）Ｃ_２－６アルキニル、Ｃ_１－６アルキルスルホニル、モノ（ジ）Ｃ_１－６アルキルスルファモイル、Ｃ_２－７アシル、Ｃ_１－６アルコキシカルボニル、Ｃ_１－６アルコキシカルボニルオキシ、モノ（ジ）Ｃ_１－６アルキルアミノ、モノ（ジ）Ｃ_１－６アルコキシＣ_１－６アルキルアミノ、Ｃ_１－６アルコキシＣ_１－６アルキル（Ｃ_１－６アルキル）アミノ、Ｃ_２－７アシルアミノ、Ｃ_１－６アルコキシカルボニルアミノ、Ｃ_１－６アルコキシカルボニル（Ｃ_１－６アルキル）アミノ、モノ（ジ）Ｃ_１－６アルキルカルバモイル、モノ（ジ）Ｃ_１－６アルコキシＣ_１－６アルキルカルバモイル、Ｃ_１－６アルコキシＣ_１－６アルキル（Ｃ_１－６アルキル）カルバモイル、モノ（ジ）Ｃ_１－６アルキルアミノカルボニルアミノ、Ｃ_１－６アルキルスルホニルアミノ又はＣ_１－６アルキルチオ；
   　（１０）Ｃ_３－８シクロアルキル、３～８員環ヘテロシクロアルキル、Ｃ_５－８シクロアルケニル又は５～８員環ヘテロシクロアルケニル；
   　（１１）Ｃ_６アリール、Ｃ_６アリールオキシ、Ｃ_６アリールカルボニル、５又は６員環ヘテロアリール、５又は６員環ヘテロアリールオキシ、５又は６員環ヘテロアリールカルボニル、Ｃ_６アリールアミノ、Ｃ_６アリール（Ｃ_１－６アルキル）アミノ、５又は６員環ヘテロアリールアミノ又は５又は６員環ヘテロアリール（Ｃ_１－６アルキル）アミノ；の何れかであり、
   （ｉｉ）　環Ｑの窒素原子に結合している場合は、（１２）～（１５）：
   　（１２）それぞれ独立して、置換基群αから選択される任意の基を有していてもよいＣ_１－６アルキル又はＣ_２－６アルケニル；
   　（１３）Ｃ_２－６アルキニル、Ｃ_１－６アルキルスルホニル、モノ（ジ）Ｃ_１－６アルキルスルファモイル、Ｃ_２－７アシル、Ｃ_１－６アルコキシカルボニル又はモノ（ジ）Ｃ_１－６アルキルカルバモイル；
   　（１４）Ｃ_３－８シクロアルキル又は３～８員環ヘテロシクロアルキル；
   　（１５）Ｃ_６アリール、５又は６員環ヘテロアリール、Ｃ_６アリールカルボニル又は５又は６員環ヘテロアリールカルボニル；の何れかであり；
   （ｎが２又は３であるとき、Ｒ^２はそれぞれ異なっていてもよく、更に、２つのＲ^２が、環Ｑ内の隣り合った原子に結合して存在し、それぞれＣ_１－６アルコキシを有していてもよいＣ_１－６アルキルである場合は、結合する環Ｑ内の原子と共に、５～８員環を形成してもよい。）；
   　　　置換基群αは、フッ素原子、水酸基、アミノ、カルボキシ、Ｃ_１－６アルコキシ、モノ（ジ）Ｃ_１－６アルキルアミノ、モノ（ジ）Ｃ_１－６アルコキシＣ_１－６アルキルアミノ、Ｃ_１－６アルコキシＣ_１－６アルキル（Ｃ_１－６アルキル）アミノ、Ｃ_１－６アルコキシカルボニルアミノ、Ｃ_２－７アシル、Ｃ_１－６アルコキシカルボニル、モノ（ジ）Ｃ_１－６アルキルカルバモイル、モノ（ジ）Ｃ_１－６アルコキシＣ_１－６アルキルカルバモイル、Ｃ_１－６アルコキシＣ_１－６アルキル（Ｃ_１－６アルキル）カルバモイル、Ｃ_１－６アルキルスルホニルアミノ、Ｃ_２－７アシルアミノ、Ｃ_１－６アルコキシカルボニルアミノ、Ｃ_３－８シクロアルキル、３～８員環ヘテロシクロアルキル、Ｃ_６アリール及び５又は６員環ヘテロアリールである〕で表される縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
4. Ｔがシアノである請求項１記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
5. Ｘ^１がＣＨである請求項１又は２記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
6. Ｘ^２がＣＨである請求項１～３の何れかに記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
7. 環Ｑがピリジン、ピリミジン、ピラジン、チアゾール、イミダゾール、ピラゾール、オキサゾール、イソチアゾール、イソオキサゾール、チオフェン、フラン又はピロール環である請求項１～４の何れかに記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
8. 環Ｑがピリジン、チオフェン又はピロール環である請求項５記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。
9. 環Ｕがベンゼン、ピリジン、チアゾール、ピラゾール又はチオフェン環である請求項１～６の何れかに記載の縮合複素環誘導体もしくはそのプロドラッグ又はその薬理学的に許容される塩。

http://www.google.co.jp/patents/WO2010044404A1?cl=ja


 

Cilostazol As a Reagent involved in Rho Study 2013

IL-23 production was elevated by lipoteichoic acid (LTA), 
which increased the activation of RhoA in association with increased the nuclear translocation of NF-kB
and its DNA-binding activity.

Pretreatment of RA macrophages with the pharmacological inhibitors exoenzyme C3 (RhoA),
Y27632 (Rho-kinase) or BAY11-7082 (NF-kB)
inhibited IL-23 production by LTA.

Inhibition of the RhoA/Rho-kinase pathway by these drugs attenuated NF-kB activation.


Cilostazol suppressed the TLR2-mediated activation of RhoA,
decreased NF-kB activity with down-regulated IL-23 production, and these effects were reversed by RpcAMPS,
as an inhibitor of cAMP-dependent protein kinase.

 Abbreviations

• CIA, collagen induced arthritis;
• IL-23, interleukin-23;
• LTA, lipoteichoic acid;
• RA, rheumatoid arthritis;
• TLR2, toll like receptor 2

 Cilostazol suppressed the TLR2-mediated activation of RhoA, decreased NF-κB activity with down-regulated IL-23 production,

and these effects were reversed by Rp-cAMPS, as an inhibitor of cAMP-dependent protein kinase. The expression of IL-23, which colocalized with CD68(+) cells in knee joint of CIA mice, was significantly attenuated by cilostazol along with the decreased severity of arthritis.

Graphical abstract

Figure options

• Download full-size image
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Abbreviations

• CIA, collagen induced arthritis;
• IL-23, interleukin-23;
• LTA, lipoteichoic acid;
• RA, rheumatoid arthritis;
• TLR2, toll like receptor 2

 

 

Taken together, the RhoA/Rho-kinase pathway signals TLR2-stimulated IL-23 production in synovial fluid macrophages via activation of NF-κB.

Thus it is summarized that cilostazol suppresses TLR2-mediated IL-23 production by suppressing RhoA pathway via cAMP-dependent protein kinase activation.

http://www.sciencedirect.com.scopeesprx.elsevier.com/science/article/pii/S0167527314001703

2.1. Reagents and antibodies

Lipoteichoic acid (LTA),
BAY11-7082 and
Y27632, were obtained from Sigma (St. Louis, MO).


Rp-cAMPS was purchased
from Alexis (San Diego, CA).
Clostridium botulinum exoenzyme C3 transferase (exoenzyme C3) was from Upstate Biotechnology (Lake Placid, NY. Anti-TLR2 antibody was from Abcam (Cambridge, MA).


NF-kB p65, IkBa, Histone H1and
RhoA antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).


TLR2 neutralizing antibody was from eBioscience (San Diego, CA). IgG isotype control antibody (R&D systems, Minneapolis, MN).


Cilostazol (OPC-13013),
[6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2-(1H)-quinolinone, >98.5% purity by HPLC,
mean particle size, 14–28 (mean, 20) mm] was donated by Otsuka
Pharmaceutical Co. Ltd. (Tokushima, Japan),

and was dissolved in dimethyl sulfoxide to produce
a 10 mM stock solution.


https://www.blogger.com/blogger.g?blogID=4733967785175105509#editor/target=post;postID=2313110350264441265



Furthermore,

Cilostazol is a commercially available drug that has antiplatelet and vasodilatory activity and is indicated for peripheral artery disease (PAD)-related intermittent claudication [3].

 

 

We and other investigators have found that cilostazol may have beneficial effects on EPCs in vitro [3] and [4] and can provide vasculo-angiogenic effects in murine hindlimb ischemia [3] and [5].

 

 

Therefore, we hypothesized that cilostazol can enhance mobilization and proliferation of EPCs and collateral formation by modifying vasculo-angiogenic biomarkers in PAD.

 

 

 

This prospective, double-blind, randomized placebo-controlled trial consecutively enrolled 44 patients with mild-to-moderate PAD who had ankle-brachial index less than 0.9 in one or both legs without obvious intermittent claudication. Exclusion criteria were listed in the registered study protocol (Clinicaltrials.org registration number NCT01952756).

 

 

All study participants provided signed informed consent, and this study followed the regulation of the ethics committee of the National Cheng Kung University Hospital, where all data were collected.

Eligible subjects were randomly assigned to cilostazol 200 mg (n = 24) or dummy placebo (n = 20) daily for 12 weeks using unrestricted randomization and sealed envelopes for allocation concealment. Serum concentrations of biomarkers were measured by enzyme-linked immunosorbent assay [6].

 

 

The isolation of human early EPCs was performed according to standard protocols [3].

 

 

The quantification of colony formation by EPCs was performed and the chemotactic motility, proliferation/viability (XTT) and apoptosis of EPCs were measured as previously described [3].


http://www.sciencedirect.com.scopeesprx.elsevier.com/science/article/pii/S0167527314001703

シロスタゾール（２） ２０１３

Probucol and cilostazol exert a combinatorial anti-atherogenic effect in cholesterol-fed rabbits

 

 

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Abstract

Introduction

Probucol (PB) and cilostazol (CZ) both exhibit anti-atherogenic effects. However, their combinatorial effects are unclear. This study was designed to investigate their combinatorial anti-atherogenic effect in cholesterol-fed rabbits.

Materials and Methods

Rabbits were fed a cholesterol diet with PB or CZ alone or both PB and CZ for 16 weeks. The plasma levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, C-reactive protein, superoxide dismutase, malondialdehyde, and nitric oxide (NO) were measured. The aortic atherosclerotic lesions were grossly and microscopically evaluated. Additionally, in vitro experiments were conducted using human umbilical vein endothelial cells.

Results and Conclusion

We found that the PB group and the PB + CZ group exhibited a reduction in the lesion areas (70% in the PB + CZ group, 56% in the PB group) compared with the vehicle group. However, although PB alone and PB + CZ led to a reduction in the lesion size, the histological analysis revealed that only PB + CZ significantly decreased the macrophage accumulation and smooth muscle cell proliferation in the lesions compared with the vehicle group. The plasma levels of total cholesterol in the PB + CZ group were decreased compared with the vehicle group, Moreover, PB + CZ exerted obvious anti-oxidant and anti-inflammatory effects. Interestingly, the PB + CZ treatment led to a marked increase in the levels of plasma NO. The in vitro experiments showed that the combinatorial treatment up-regulated the levels of NO and protein S-nitrosylation in endothelial cells treated with oxidized LDL. In summary, these results suggest that PB and CZ exert combinatorial anti-atherogenic effects.

Abbreviations

• CRP, C-reactive protein;
• CZ, cilostazol;
• eNOS, endothelial nitric oxide synthase;
• HCD, high cholesterol diet;
• HDL, high-density lipoproteins;
• HDL-C, high-density lipoprotein cholesterol;
• HUVECs, human umbilical vein endothelial cells;
• LDL-C, low-density lipoprotein cholesterol;
• MDA, malondialdehyde;
• MФ, macrophages;
• NO, nitric oxide;
• PB, probucol;
• ROS, reactive oxygen species;
• SMC, smooth muscle cell;
• SOD, superoxide dismutase;
• TC, total cholesterol

Keywords

• Probucol;
• Cilostazol;
• Atherosclerosis;
• S-nitrosylation;
• Rabbits

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Introduction

Atherosclerosis is a major cause of mortality in both developed and developing countries [1], and hypercholesterolemia is an important risk factor for the development of atherosclerosis. The extensive use of lipid-lowering agents, such as statins, has led to a marked reduction in the number of cardiovascular events in recent years. Despite this achievement, many patients with cardiovascular disease cannot be effectively treated with the use of statins alone [2]. Therefore, it is necessary to develop new therapeutics for these patients.

 

 

Probucol (PB) not only is a lipid-lowering agent but also possesses strong antioxidant properties against low-density lipoproteins (LDL). To date, many studies have shown that PB exhibits anti-atherogenic effects [3]. It has been shown that PB exerts its anti-atherogenic effects through diverse molecular mechanisms, including anti-inflammatory effects, the inhibition of smooth muscle cell (SMC) proliferation, the enhancement of the expression of scavenger receptor class B type I, and the improvement of the functions of high-density lipoproteins (HDL) to enhance reverse cholesterol transport [4], [5], [6] and [7].

 

 

Cilostazol (CZ) is a specific phosphodiesterase type III inhibitor that is currently used for the treatment of thrombotic vascular disease due to its anti-platelet aggregation functions [8]. As an anti-thrombotic drug, CZ is a preferred alternative to aspirin because CZ has fewer side effects, such as increased bleeding time [9]. Previous studies have shown that CZ also exhibits anti-atherogenic effects [10]. In vitro studies have revealed that CZ suppresses the production of intracellular reactive oxygen species (ROS) [11], and increases the production of nitric oxide (NO) [12]. Furthermore, CZ promotes reverse cholesterol transport and inhibits the inflammation and proliferation of SMCs [13], [14], [15] and [16].

 

 

Because both PB and CZ have anti-atherogenic effects, we hypothesized that the combination of minimal doses of PB and CZ may exert additional beneficial effects and may result in a greater athero-protective response. If so, this combinatorial therapy may provide a novel strategy for the treatment and prevention of atherosclerosis for those patients who cannot be effectively treated by statins.

 

 

In the current study, we compared the anti-atherogenic effects of the combination of PB and CZ in cholesterol-fed rabbits with those obtained with either PB or CZ alone. Our results showed that the combination of PB and CZ clearly reduced the aortic atherosclerotic lesion area, and markedly inhibited macrophage (MФ) accumulation and SMC proliferation in the lesions compared with the vehicle group.

 

 

The anti-atherogenic effects of PB and CZ may be collectively mediated by multiple mechanisms, including anti-oxidant effects, anti-inflammatory effects, a decrease in the plasma lipid levels, an increase in the levels of NO, and the maintenance of the endothelial protein S-nitrosylation.

 

 

シロスタゾール & ROCK （１）

RhoA/ROCK-dependent pathway is required for TLR2-mediated IL-23 production in human synovial macrophages: Suppression by cilostazol

• So Youn Park^a,
• Sung Won Lee^c,
• Won Suk Lee^{a, b},
• Byung Yong Rhim^b,
• Seung Jin Lee^a,
• Sang Mo Kwon^d,
• Ki Whan Hong^a,
• Chi Dae Kim^{a, b, ,}

• ^a Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Gyeongnam, South Korea
• ^b Department of Pharmacology, School of Medicine, Pusan National University, Gyeongnam, South Korea
• ^c Department of Internal Medicine, College of Medicine, Dong-A University, Busan, South Korea
• ^d Department of Physiology, School of Medicine, Pusan National University, Gyeongnam, South Korea

Received 20 June 2013, Accepted 13 August 2013, Available online 22 August 2013

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DOI: 10.1016/j.bcp.2013.08.017

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  Open Access

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Abstract

IL-23 is produced by antigen presenting cells and plays critical roles in immune response in rheumatoid arthritis (LA).

 

 

In this study, we investigated whether the RhoA/Rho-kinase pathway is required to elevate TLR2-mediated IL-23 production in synovial macrophages from patients with rheumatoid arthritis (RA),

and then examined the suppressive effect of cilostazol on these pathways.

 

 

 

IL-23 production was elevated by lipoteichoic acid (LTA), a TLR2 ligand, and this elevation was more prominent in RA macrophages than in those from peripheral blood of normal control.

 

 

LTA increased the activation of RhoA in association with increased the nuclear translocation of NF-κB and its DNA-binding activity.

 

 

Pretreatment of RA macrophages with the pharmacological inhibitors exoenzyme C3 (RhoA), Y27632 (Rho-kinase) or BAY11-7082 (NF-κB) inhibited IL-23 production by LTA.

 

 

Inhibition of the RhoA/Rho-kinase pathway by these drugs attenuated NF-κB activation.

 

 

Cilostazol suppressed the TLR2-mediated activation of RhoA, decreased NF-κB activity with down-regulated IL-23 production, and these effects were reversed by Rp-cAMPS, as an inhibitor of cAMP-dependent protein kinase.

 

 

The expression of IL-23, which colocalized with CD68(+) cells in knee joint of CIA mice, was significantly attenuated by cilostazol along with the decreased severity of arthritis.

 

 

Taken together, the RhoA/Rho-kinase pathway signals TLR2-stimulated IL-23 production in synovial fluid macrophages via activation of NF-κB. Thus it is summarized that cilostazol suppresses TLR2-mediated IL-23 production by suppressing RhoA pathway via cAMP-dependent protein kinase activation.

 

 

 

---

Graphical abstract

Figure options

• Download full-size image
• Download as PowerPoint slide

Abbreviations

• CIA, collagen induced arthritis;
• IL-23, interleukin-23;
• LTA, lipoteichoic acid;
• RA, rheumatoid arthritis;
• TLR2, toll like receptor 2

Keywords

• RA synovial macrophages;
• RhoA;
• TLR2;
• IL-23;
• Cilostazol

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1. Introduction

Rheumatoid arthritis (RA) is a common autoimmune and chronic inflammatory joint disease characterized by increased infiltration of macrophages, proliferation of synovial fibroblast with joint destruction [1]. When abundant monocytes/macrophages in the synovial fluid of RA patients are activated, they produce high levels of cytokines and chemokines, such as interleukin-1β (IL-β), tumor necrosis factor α (TNFα), IL-6, and MCP-1, which contribute to chronic inflammation and joint destruction [2] and [3].

Toll-like receptors (TLRs) are conserved receptors that recognize pathogen-associated molecular patterns, and play important roles in innate and adaptive immunity [4]. TLR2 are mainly expressed on cells, such as macrophages and dendritic cells; and act as primary sensors by recognizing diverse ranges of stimuli [5]. The lipoteichoic acid (LTA) and peptidoglycan are recognized mainly by TLR-2 [6]. It has been reported that TLR2 stimulation causes the preferential induction of IL-8 and IL-23 p19 [7]. IL-23 is involved in autoimmune diseases like RA and psoriasis, in which the cellular function of IL-23 is associated with the self-reactive productions of IL-17, IL-6, and TNF-α, and thus IL-23 plays a critical role in development of autoimmune inflammation [8]. Furthermore, they reported that mice deficient in IL-23 (p19^−/−) were relatively resistant to the development of joint and bone inflammation in a collagen-induced arthritis (CIA) model.

The Rho-GTPase family of monomeric RhoA, Rac1 and Cdc42 is known to cycle between the inactivated GDP-bound state and the activated GTP-bound state [9]. In the active state, Rho is implicated in various cellular processes, such as the cell cycle, cytoskeletal regulation, cellular growth and apoptosis [10] and [11]. Furthermore, it has been reported that RhoA is a key regulator of transcription factors, NF-κB [12], and that inhibition of Rho-kinase reduces the severity of synovial inflammation in rats with CIA [13].

Although it has been demonstrated that IL-23 production plays a crucial role in inflammatory reactions associated with rheumatoid arthritis [14], the signal pathway by which TLR2 induces IL-23 production in RA synovial macrophages has not been defined. On the other hand, cilostazol, a type-III phosphodiesterase inhibitor, has been reported to have anti-inflammatory effects due to the cAMP-dependent protein kinase activation-coupled suppression of NF-κB gene transcription [15].

Therefore, in the present study, we undertook to investigate the signal transduction pathways responsible for TLR2-mediated IL-23 production in synovial fluid macrophages from RA patients: in particular, the present study highlighted implication of the RhoA/ROCK signal pathway in the regulation of TLR2-mediated IL-23 production in RA macrophages. We found that increased IL-23 production by TLR2 involves the activation of NF-κB via a RhoA/ROCK pathway. Further, cilostazol was found to inhibit TLR2-mediated IL-23 production by suppressing RhoA activity via the activation of cAMP-dependent protein kinase, and to suppress the expression of IL-23 in the knee joints of CIA mice.