Apabetalone (RVX-08, RVX-000222)   中文名称: 阿帕他隆

BD2 (IC50 = 510±41 nM), BD1 (IC50 = 87±10 μM)

Apabetalone (RVX-208) 与乙酰化组蛋白肽与四种 BET 蛋白的串联 BD1 BD2 蛋白结构的结合竞争，表现出 0.5 至 1.8 µM 之间的 IC50。 Apabetalone 在体外促进肝细胞中 ApoA-I 的形成，从而导致高密度脂蛋白胆固醇 (HDL-C) 增加。 Apabetalone 主要与 BET（溴结构域和额外末端）家族的溴结构域结合，竞争与内源配体乙酰化赖氨酸结合的位置，这解释了其药理作用。通过表观遗传机制增加载脂蛋白 AI (ApoA-I) 的合成，表明 BET 抑制可能是治疗动脉粥样硬化的一种有前途的新方法。 Apabetalone 促进肝细胞中 ApoA-I 的表达[2]。

在动脉粥样硬化预防性治疗的研究设计中，小鼠接受西方饮食以及 150 毫克/公斤/剂量 bid 的药物治疗，持续 12 周。治疗一年后，处死小鼠。媒介物处理组和Apabetalone (RVX-208)处理组均显示出体重逐渐增加。采用西方饮食 12 周后，Apabetalone 治疗组的体重增加了 4 克（从 24 克增至 28 克），而载体治疗组则增加了 9 克（从 25 克增至 34 克）。在用 Apabetalone 治疗的小鼠中观察到的体重增加显着减少并不是由于采食量减少，这表明该化合物具有有利的特性。在用媒介物或apibetalone治疗六周和十二周后，进行血浆脂质测量。治疗六周后，与载体对照动物相比，用 apibetalone 治疗的小鼠的 HDL-C 水平显着升高（约 200%）。这种增加一直持续到为期 12 周的研究得出结论[3]。

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尽管他汀类药物有助于降低心血管风险，但仍有相当一部分患者面临风险。由于HDL通过一个涉及前β颗粒形成的过程降低了CVD风险，该过程有助于从动脉中充满脂质的巨噬细胞中去除胆固醇，诱导前β颗粒可能会降低CVD的风险。据报道，一种新型BET溴结构域拮抗剂Apabetalone (RVX-208)可提高非人灵长类动物和人类的载脂蛋白A-I并增加前β-HDL颗粒。在本研究中，我们研究了Apabetalone (RVX-208)对高脂血症apoE（-/-）小鼠主动脉病变形成的影响。用150mg/kg b.i.d Apabetalone (RVX-208)口服apoE（-/-）小鼠12周，显著减少了主动脉病变的形成，同时循环HDL-C水平增加了2倍，LDL-C降低了约50%，尽管没有观察到血浆apoA-i的显著变化。循环粘附分子和细胞因子也显著减少。触珠蛋白是一种促炎蛋白，已知与HDL/apoA-I结合，在Apabetalone (RVX-208)治疗组中降低了2.5倍以上。采用治疗性给药方案，其中小鼠喂食西方饮食10周以形成病变，然后改用低脂饮食并同时用Apabetalone (RVX-208)治疗14周，RVX-208同样将整个主动脉的病变形成减少了39%，而血浆脂质参数没有显著变化。RVX-208显著降低了促炎细胞因子IP-10、MIP1（®）和MDC。这些结果表明，BET抑制剂RVX-208的抗动脉粥样硬化活性是通过脂质变化和抗炎活性的结合来实现的。

蛋白质稳定性变化分析（Tm分析）。[1]
如前所述，使用Mx3005p实时PCR机进行热熔实验。温度变化（保存图片、插图等的外部文件。 表S1总结了每种蛋白质/化合物三次独立测量的对象名称（pnas.1310658110i2.jpg）。[1]

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竞争性组蛋白置换试验（AlphaScreen试验）。[1]
实验在PHERAstar FS读板器上使用AlphaScreen 680激发/570发射滤光片组进行。在Prism 5中计算IC50值，并与相应的DMSO对照进行归一化。如前所述进行了检测，对制造商的方案进行了小幅修改。[1]

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等温滴定量热法。[1]
在MicroCal的ITC200微量热计上，在15°C、50 mM Hepes、pH 7.5（25°C）、150 mM NaCl的条件下，通过将蛋白质滴定到配体溶液中（反向滴定）进行了实验，并使用MicroCal Origin软件对数据进行了稀释和去卷积的蛋白质热校正，如前所述。表S2和S3列出了离解常数和热力学参数。[1]

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光漂白后的荧光恢复。[1]
使用与倒置蔡司Axio Observer连接的蔡司LSM 710扫描头，在转染了编码BRD3 GFP嵌合体的哺乳动物过表达构建体的U2OS细胞中进行了光漂白后荧光恢复（FRAP）研究。Z1显微镜配备了一个高数值孔径（N.a.1.3）的40倍油浸物镜，其加热室设置为37°C，使用了之前研究中修改的协议。[1]

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蛋白质热变性试验[2]
在快速96孔光学板中，在100µM化合物或DMSO（0.2%）的存在下，将5µM纯化的溴结构域蛋白与最终浓度为20 mM HEPES pH 7.4的5X SYPRO®Orange、100 mM NaCl一起孵育。样品在室温下孵育30分钟，并在ViiA7实时PCR机中从25°C升至95°C。使用Protein Thermal Shift™软件v1.0分析所得荧光数据并计算熔融温度。[2]

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时间分辨荧光共振能量转移（TR-FRET）分析[2]
200 nM N-末端His标记的溴结构域或BRD4（BD1BD2）和25-50 nM生物素化的四乙酰化组蛋白H4肽在白色96孔微量滴定板中，在铕Cryptate标记的链霉抗生物素蛋白和XL665标记的单克隆抗His抗体的存在下孵育。对于抑制试验，将连续稀释的化合物以0.2%终浓度的DMSO加入到这些反应中。最终缓冲液浓度为30 mM HEPES pH 7.4、30 mM NaCl、0.3 mM CHAPS、20 mM PO4 pH 7.0、320 mM KF、0.08%BSA）。在室温下孵育2小时后，通过SynergyH4平板读数器在665和620nm处测量FRET的荧光。IC50值由剂量反应曲线确定。[2]

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等温量热法[2]
将50 mM HEPES pH 7.5、150 mM氯化钠和0.05%DMSO中的150µM BRD2[BD1]、BRD2[BD 2]、BRD4[BD1]或BRD4[BD 2]注入25°C下同一缓冲液中含有10µM RVX-208的溶液中，并在Microcal Auto ITC仪器中测量相关的热量变化。

细胞培养[2]
Huh7细胞以23000/孔的速度在DMEM+10%FBS的96孔板中铺板，然后生长过夜。细胞在含有或不含有5µM放线菌素D的0.1%DMSO中用化合物处理48小时。U937细胞在60 ng/mL PMA中分化3天，96孔格式为32000个细胞/孔。然后用RPMI培养基+10%FBS中0.1%DMSO的化合物处理细胞，1小时后，以1µg/mL的浓度向细胞中加入脂多糖（LPS）3小时。[2]

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RT-PCR[2]
通过mRNA Catcher PLUS试剂盒收获细胞，然后使用RNA UltraSense一步法qRT-PCR系统进行实时PCR。在同一样本中相对于内源性对照丝氨酸蛋白酶A1或亲环素测量ApoA-I、IL-6和TNFαmRNA水平。使用7500实时PCR系统获取数据。[2]

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细胞培养和RNA提取。处理HepG2细胞，使DMSO的终浓度达到0.1%。使用标准方案收集、洗涤和原位裂解细胞。使用RNeasy柱提取和制备总RNA，使用Nanodrop分光光度计对RNA进行定量和质量控制。[1]

The test article Apabetalone (RVX-208), 2-(4-(2-Hydroxymethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one, was prepared in formulation EA006, and stored in refrigerator at 4 °C for up to 1 week. Formulation EA006 includes an in situ HCl salt formation of Apabetalone (RVX-208) followed by suspension in a Polyethylene glycol-300/polysorbate-80 based vehicle after pH adjustment to 2.5–3.0. Each preparation of dosing solution was subjected to verification of the test compound concentration by collecting 0.2 mL of aliquot in duplicate.[3]

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Atherosclerosis prophylactic study design[3]
Eight week old apoE−/− mice were first acclimated for a week on rodent chow diet and following a 4 h fast blood was drawn under ether anesthesia and lipid measurements were done. Based on the body weight and lipid values, mice were divided into 2 groups (n = 12): group 1, vehicle; and group 2, test agent, Apabetalone (RVX-208). Mice were then switched to Western diet (0.15% cholesterol and 42% calories from fat) and concurrently treated orally by gavage with either vehicle or the test agent, Apabetalone (RVX-208) (150 mg/kg/dose b.i.d) for 12 weeks. After 6 week of treatment, an interim blood draw was done to monitor serum lipid levels. After 12 weeks of treatment mice were sacrificed to measure blood lipid parameters, aortic lesion, and liver and aortic RNA. Eight mice were used for enface (aortic plaque) analysis, 4 mice for tissue collection for mRNA and all 12 mice used for aortic sinus lesion area measurement.

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Atherosclerosis therapeutic study design[3]
Eight week old apoE−/− mice were first acclimated for a week on rodent chow diet. Mice were then switched to Western diet (0.15% cholesterol and 42% calories from fat) ad libitum for 10 weeks in order to allow the development of lesion formation. Baseline lipid measurements after feeding Western diet for 10 weeks were carried out following 4 h fast, and all mice were switched to rodent chow diet and concurrently treated with either vehicle or Apabetalone (RVX-208) (150 mg/kg/dose, b.i.d) by oral gavage for 14 weeks. After 6 week of treatment, an interim blood draw was done to monitor serum lipid levels. After 14 weeks of treatment mice were sacrificed to measure blood lipid parameters, aortic plaque and sinus lesion, liver and aortic mRNA as described above in section.

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Dissolved in 1N HCl and carboxymethyl cellulose; 60mg/kg; i.v. injection or p.o.

Na ve adult male AGMs

[1]. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain. Proc Natl Acad Sci U S A. 2013 Dec 3;110(49):19754-9.

[2]. RVX-208, an inducer of ApoA-I in humans, is a BET bromodomain antagonist. PLoS One. 2013 Dec 31;8(12):e83190.

[3]. A novel BET bromodomain inhibitor, RVX-208, shows reduction of atherosclerosis in hyperlipidemic ApoE deficient mice. Atherosclerosis. 2014 Sep;236(1):91-100.

Apabetalone has been investigated for the treatment of Diabetes, Atherosclerosis, and Coronary Artery Disease.

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Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.[1]

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Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.[2]

C20H22N2O5

370.4

370.152

C, 64.85; H, 5.99; N, 7.56; O, 21.60

1044870-39-4

135564749

Typically exists as White to yellow solids at room temperature

1.3±0.1 g/cm3

1.596

2.04

93.67

2

6

6

27

543

0

O(C([H])([H])C([H])([H])O[H])C1C(C([H])([H])[H])=C([H])C(=C([H])C=1C([H])([H])[H])C1=NC2C([H])=C(C([H])=C(C=2C(N1[H])=O)OC([H])([H])[H])OC([H])([H])[H]

NETXMUIMUZJUTB-UHFFFAOYSA-N

InChI=1S/C20H22N2O5/c1-11-7-13(8-12(2)18(11)27-6-5-23)19-21-15-9-14(25-3)10-16(26-4)17(15)20(24)22-19/h7-10,23H,5-6H2,1-4H3,(H,21,22,24)

2-(4-(2-hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one

RVX-000222; RVX208; RVX 000222; RVX 208; RVX000222; RVX-208; 2-(4-(2-Hydroxyethoxy)-3,5-dimethylphenyl)-5,7-dimethoxyquinazolin-4(3H)-one; RVX-000222; RVX 208; Apabetalone [INN]; RVX000222; Apabetalone.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.75 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.75 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.75 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (6.75 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: ≥ 2.5 mg/mL (6.75 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 6 中的溶解度: ≥ 0.5 mg/mL (1.35 mM) (饱和度未知) in 1% DMSO 99% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 7 中的溶解度: 0.5% CMC Na (1N HCl, PH 2.5-3.0):8 mg/mL

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配方 8 中的溶解度: 15.15 mg/mL (40.90 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。