LY2090314

GSK-3β (IC50 = 0.9 nM); PI3Kγ (IC50 = 282 nM)

LY2090314 通过中断 ATP 结合来选择性抑制 GSK-3 的活性。 LY2090314具有保持β-catenin稳定的能力。作为单一疗法，LY2090314 的疗效有限。顺铂和卡铂与 LY3090314 联合使用时，在体外对实体瘤癌细胞系更有效。 [1]

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LY2090314是一种有效的选择性GSK3抑制剂，可激活黑色素瘤细胞系中的Wnt通路。 LY2090314在一组黑色素瘤细胞系中有效诱导凋亡细胞死亡，而与BRAF突变状态无关。 LY2090314诱导的细胞死亡依赖于β-catenin和Wnt信号。LY2090314在Vemurafenib耐药细胞系中保持活性，具有独立的作用机制。［2］

LY2090314（3-[9-氟-2-（哌啶-1-酰基羰基）-1,2,3,4-四氢[1,4]二氮平[6,7,1-hi]吲哚-7-基]-4-咪唑[1,2-a]吡啶-3-酰基- 1h -吡咯-2,5-二酮）是一种用于肿瘤试验的静脉注射糖原合成酶激酶-3抑制剂。对大鼠和狗静脉输注[(14)C]LY2090314后的药物处置进行表征，并与现有临床资料相关。LY2090314表现出高清除率（接近肝血流量）和中等体积分布（~ 1-2 l/kg），导致快速消除（在大鼠、狗和人体内的半衰期分别为~ 0.4、0.7和1.8-3.4小时）。肝微粒体的分级清除准确地预测了跨物种的灌注限制清除。LY2090314被广泛代谢清除，大量代谢物通过胆汁迅速排泄到粪便中(剂量的69-97%；62-93%在0-24小时内完成)；尿中药物相关物质回收率低（≤剂量的3%）。尽管有广泛的代谢，在大鼠和人类中母体化合物是血浆中唯一可识别的与药物相关的部分。即使在Mdr1a-、Bcrp-和mrp2敲除大鼠中，LY2090314代谢物也没有出现在循环中，它们的尿排泄也没有增强，因为没有观察到在缺乏这些小管转运蛋白的情况下代谢物的胆道排泄受损的假设。犬类代谢产物的分布大体相似，但狗特有的LY2090314葡糖苷除外。这种结合物在狗的肝脏中形成，并优先排泄到血液中，在血液中，它占后来循环放射性的大部分，并且主要在尿液中回收（剂量的16%）。综上所述，LY2090314通过广泛的代谢被迅速清除，而循环代谢物暴露可以忽略不计，因为代谢物被胆汁排泄到粪便中，没有明显的肠道重吸收。[1]

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LY2090314在A375黑色素瘤模型中表现出单药活性，并在体内与DTIC协同作用[2]
我们试图评估LY2090314在体内激活Wnt通路的能力，并随后质疑通路升高是否会导致黑色素瘤的抗肿瘤疗效。在小鼠中，LY2090314被迅速清除，血浆半衰期为36分钟（图5A）。在评估Wnt应答基因Axin2在体内基因表达的研究中，我们观察到LY2090314剂量后2和4小时在A375异种移植肿瘤组织中显著诱导Axin2 mRNA（图5B）。这一发现与我们的体外实验一致，该实验也显示在初始药物暴露后2-4小时Axin2升高（图1E）。4小时后Axin2基因表达迅速下降，这与化合物体内半衰期短和药代动力学性质一致（图5A和5B）。尽管LY2090314治疗可以短暂提升Wnt通路，但我们能够观察到每3天给药一次的皮下A375异种移植物的单药抗肿瘤效果（图5C， p<0.003）。此外，我们探索了LY2090314在体内与DTIC的协同作用能力，观察到联合治疗相对于对照组和单一治疗组的加性效应有统计学意义（图5D， p<0.02）。在这些研究中，我们没有发现明显的动物体重减轻或其他临床症状。值得注意的是，在探索Wnt激活剂在癌症治疗中的潜在用途时应谨慎，因为它们能够增加正常组织的增殖。当确定诸如此类的化合物是否具有治疗黑色素瘤的足够治疗窗口时，化合物剂量和时间表的优化将是至关重要的。本文提出的研究提供了概念验证数据，支持使用Wnt激活剂治疗黑色素瘤，并支持进一步研究GSK3抑制剂治疗黑色素瘤，特别关注其对健康组织的影响。

GSK3生化测定[2]
LY2090314的抑制酶活性通过在肽底物YRRAAVPPSPSLSRHSSPHQ(Ps)EDEEE中培养重组人GSK3α或GSK3β来评估。

细胞化学敏感性和caspase激活试验[1]
将细胞接种于96孔板中，密度为2000个细胞/孔，并在常规培养基中粘附过夜。24小时后，取出培养液，替换为含有2%胎牛血清的生长培养基，细胞暴露于0.5nmol/L至10μmol/L的试验剂中72小时。根据制造商的说明，使用CellTiter-Glo法测定黑色素瘤细胞对LY2090314、BIA和Vemurafenib的体外化学敏感性。根据制造商的说明，使用Caspase- glo测定Caspase 3/7的活化。采用GraphPad Prism 6软件，采用非线性回归和s型剂量-响应曲线计算最大半数抑制浓度（IC50）。

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PARP裂解试验 [2]
将细胞以25万细胞/孔的密度接种于6孔板中，并在常规培养基中粘附过夜。24小时后，将培养基更换为含有LY2090314的新鲜生长培养基（终浓度为5nM， 15nM），细胞暴露于试验剂中72小时。按照生产商说明，使用Pathscan Cleaved PARP ELISA （Asp214）夹心ELISA试剂盒评估每50μg总蛋白的PARP裂解率。计算相对于DMSO对照处理细胞的裂解PARP水平。

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免疫沉淀反应 [2]
为了评估LY2090314处理前后细胞裂解液中的“游离”β-catenin，我们测量了与FLAG标签融合的E-cadherin的c端区域可结合的β-catenin的数量。这种方法在前面已经介绍过了。简单地说，代表300μg总蛋白的裂解物与10μg His-Flag-E-cadherin孵育。Anti-FLAG M2亲和凝胶与裂解液/E-cadherin混合物在4℃下孵育过夜，以结合E-cadherin/β-catenin复合物。过夜孵育后，树脂用裂解缓冲液洗涤3次，然后在含有β-巯基乙醇的凝胶上样缓冲液中重悬。样品在8000g下离心，上清液用于免疫印迹分析。

Five million A375 human melanoma cancer cells are injected S.C. in the flank of female 6 to 8 week old athymic nude mice in a 1:1 mixture with matrigel. Tumors that can be felt are checked for daily in mice. When tumors are approximately 100 mm2 in size, mice are divided into groups and given LY2090314 (25 mg/kg Q3D) or a vehicle (20% Captisol/0.01N HCl) intravenously. Animal body weight and tumor volume (calculated using calipers) are recorded twice weekly. The formula used to determine tumor volumes is (a2 b)/2, where a represents the tumor's smaller dimension and b its larger dimension. LY2090314 is dosed at 2.5 mg/kg Q3D for combination studies with DTIC (60 mg/kg QD), and tumor growth is tracked.

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LY2090314 pharmacokinetic studies [2]
To understand the plasma pharmacokinetics of LY2090314 in vivo following i.v. administration, CD1 nu/nu non-tumor bearing mice (Harlan, Indianapolis, IN) were injected i.v. with 5mg/kg LY2090314 and blood collected by cardiac puncture at the times indicated (5, 15, 30, 60 120 minutes post dose). Mice were sacrificed using isoflurane and cervical dislocation. Blood was centrifuged at 5,000 x g for 10 minutes, and the resulting plasma analyzed for drug concentration using HPLC and mass spectrometry.

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In vivo studies [2]
Five million A375 human melanoma cancer cells were injected S.C. in the flank of female 6 to 8 week old athymic nude mice (Harlan, Indianapolis, IN) in a 1:1 mixture with matrigel. Mice were monitored daily for palpable tumors. When tumors reached ~100mm2 mice were randomized into groups receiving either LY2090314 (25 mg/kg Q3D) or vehicle (20% Captisol/0.01N HCl) via i.v. administration. Tumor volume (measured by calipers) and animal body weight were recorded twice weekly. Tumor volumes were calculated using the formula: (a2 x b)/2 (a being the smaller and b being the larger dimension of the tumor). For combination studies with DTIC (60 mg/kg QD), LY2090314 was dosed at 2.5 mg/kg Q3D and tumor growth monitored. For in vivo target inhibition studies in xenograft tissue LY2090314 (25mg/kg) was administered to mice harboring A375 tumors approximately 200mm2 in volume and tumor tissue collected for RNA expression analysis at 1, 2, 4, 6, 8 and 24hours postdose.

LY2090314 (3-[9-fluoro-2-(piperidin-1-ylcarbonyl)-1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl]-4-imidazo[1,2-a]pyridin-3-yl-1H-pyrrole-2,5-dione) is an intravenous glycogen synthase kinase-3 inhibitor in oncology trials. Drug disposition was characterized after intravenous infusion of [(14)C]LY2090314 to rats and dogs, and was related to available clinical data. LY2090314 exhibited high clearance (approximating hepatic blood flow) and a moderate volume of distribution (∼1-2 l/kg) resulting in rapid elimination (half-life ∼0.4, 0.7, and 1.8-3.4 hours in rats, dogs, and humans, respectively). Scaled clearance from liver microsomes accurately predicted perfusion-limited clearance across species. LY2090314 was cleared by extensive metabolism, and the numerous metabolites were rapidly excreted into feces via bile (69-97% of dose; 62-93% within 0-24 hours); urinary recovery of drug-related material was low (≤3% of dose). Despite extensive metabolism, in rats and humans the parent compound was the sole identifiable drug-related moiety in plasma. Even in Mdr1a-, Bcrp-, and Mrp2-knockout rats, LY2090314 metabolites did not appear in circulation, and their urinary excretion was not enhanced, because the hypothesized impaired biliary excretion of metabolites in the absence of these canalicular transporters was not observed. Canine metabolite disposition was generally similar, with the notable exception of dog-unique LY2090314 glucuronide. This conjugate was formed in the dog liver and was preferentially excreted into the blood, where it accounted for the majority of circulating radioactivity at later times, and was predominantly recovered in urine (16% of dose). In conclusion, LY2090314 was rapidly cleared by extensive metabolism with negligible circulating metabolite exposures due to biliary excretion of metabolites into feces with no apparent intestinal reabsorption. [1]

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In mouse, LY2090314 is rapidly cleared and has a plasma half-life of 36 minutes (Fig 5A). In studies assessing the in vivo gene expression of Axin2, a Wnt responsive gene, we observed a significant induction of Axin2 mRNA at 2 and 4 hours post dose of LY2090314 in A375 xenograft tumor tissue (Fig 5B). This finding is in agreement with our in vitro experiments which also reveal Axin2 elevation 2–4 hours after initial drug exposure (Fig 1E). The rapid decline in Axin2 gene expression after 4 hours is consistent with the short half-life and pharmacokinetic properties of the compound in vivo (Fig 5A and 5B). [2]

[1]. Pharmacokinetics, metabolism, and excretion of the glycogen synthase kinase-3 inhibitor LY2090314 in rats, dogs, and humans: a case study in rapid clearance by extensive metabolism with low circulating metabolite exposure. Dr

[2]. Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma--Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3. PLoS One. 2015 Apr 27;10(4):e0125028.

LY-2090314 is a member of the class of diazepinoindoles that is 1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indole substituted by piperidin-1-ylcarbonyl, 4-(imidazo[1,2-a]pyridin-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl and fluoro groups at position 2, 7 and 9, respectively. It is a potent ATP-competitive inhibitor of glycogen synthase kinase-3 (GSK-3) with IC50 values of 1.5 nM and 0.9 nM for GSK-3alpha and GSK-3beta. The drug is in clinical development for the treatment of advanced/metastatic cancer. It has a role as an apoptosis inducer, an antineoplastic agent, a Wnt signalling activator and an EC 2.7.11.26 (tau-protein kinase) inhibitor. It is an imidazopyridine, a diazepinoindole, a member of monofluorobenzenes, a piperidinecarboxamide, a member of ureas and a member of maleimides.
Ly2090314 has been used in trials studying the treatment of Leukemia, Advanced Cancer, and Pancreatic Cancer.
GSK-3 Inhibitor LY2090314 is an inhibitor of glycogen synthase kinase-3 (GSK-3), with potential antineoplastic activity. Upon administration, LY2090314 binds to and inhibits GSK-3 in an ATP-competitive manner. This prevents GSK-3-mediated phosphorylation of beta-catenin, which inhibits the subsequent ubiquitination and proteasomal degradation of beta-catenin. This leads to the activation of the Wnt/beta-catenin pathway and the induction of apoptosis in susceptible tumor cells. GSK-3, a serine/threonine kinase, plays a key role in numerous pathways involved in protein synthesis, cellular proliferation, differentiation, and apoptosis. The Wnt/beta-catenin signaling pathway plays key roles in both cellular proliferation and differentiation. The increased expression of beta-catenin, a transcriptional activator, correlates with decreased cellular proliferation and improved prognosis in select cancers.

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It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10 nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10 uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.[2]

C28H25FN6O3

512.5349

512.197

C, 65.61; H, 4.92; F, 3.71; N, 16.40; O, 9.36

603288-22-8

603288-22-8

10029385

Off-white to light yellow solid powder

1.6±0.1 g/cm3

1.776

3.43

95.44

1

5

2

38

1030

0

FC1C([H])=C2C(C3C(N([H])C(C=3C3=C([H])N=C4C([H])=C([H])C([H])=C([H])N34)=O)=O)=C([H])N3C([H])([H])C([H])([H])N(C(N4C([H])([H])C([H])([H])C([H])([H])C([H])([H])C4([H])[H])=O)C([H])([H])C(C=1[H])=C32

HRJWTAWVFDCTGO-UHFFFAOYSA-N

InChI=1S/C28H25FN6O3/c29-18-12-17-15-34(28(38)32-7-3-1-4-8-32)11-10-33-16-20(19(13-18)25(17)33)23-24(27(37)31-26(23)36)21-14-30-22-6-2-5-9-35(21)22/h2,5-6,9,12-14,16H,1,3-4,7-8,10-11,15H2,(H,31,36,37)

3-[6-fluoro-10-(piperidine-1-carbonyl)-1,10-diazatricyclo[6.4.1.04,13]trideca-2,4,6,8(13)-tetraen-3-yl]-4-imidazo[1,2-a]pyridin-3-ylpyrrole-2,5-dione

LY 2090314; LY2090314; 603288-22-8; LY-2090,314; LY 2090,314; Kinome_3681; 3-(9-Fluoro-2-(piperidine-1-carbonyl)-1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-4-(imidazo[1,2-a]pyridin-3-yl)-1H-pyrrole-2,5-dione; UNII-822M3GYM67; CHEMBL362558; LY-2090314

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)

DMSO: ~100 mg/mL (~195.1 mM)
Water: <1 mg/mL
Ethanol: ~2 mg/mL warmed (~3.9 mM)

配方 1 中的溶解度: ≥ 1.25 mg/mL (2.44 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 12.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 1.25 mg/mL (2.44 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 12.5 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: 5% DMSO+45% PEG 300+ddH2O: 17mg/mL

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配方 4 中的溶解度: 10 mg/mL (19.51 mM) in 20% HP-β-CD/10 mM citrate pH 2.0 (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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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。