GSK360A

HIF-PHD

GSK360A是一种强效（纳摩尔）HIF PHDs抑制剂（PHD1>PHD2=PHD3），能够在包括新生大鼠心室肌细胞和H9C2细胞在内的多种细胞类型中激活HIF-1α通路。[1]
GSK360A增加了红细胞生成素（EPO）、血红素加氧酶-1（HO1）和葡萄糖转运蛋白1（Glut1）转录物，所有HIF1α靶基因，并促进了OGD后神经元和少突胶质细胞的存活。[2]

在完善的心室衰竭模型中，GSK360A（30 mg/kg ig）可改善死亡率、血管分布和长期心室功能[1]。

白细胞计数与血浆EPO测定[2]
从大鼠身上采集血液，并将其转移到含有EDTA的埃彭多夫管中。使用Hemavet 1500血液分析仪测量新鲜血液中的白细胞计数。对于血浆制备，将血液以1000 g离心15分钟，并将血浆上清液储存在−80°C的冰箱中以备进一步使用。如前所述，通过基于Luminex珠的多重ELISA测量血浆EPO，并通过Bio-Plex Manager软件定量。

氧-葡萄糖剥夺（OGD）模型和细胞活力测定[2]
通过细胞特异性神经元（MAP2）或少突胶质细胞（Rip）标记物（Sun等人，2010），证实了第17天胚胎大鼠大脑的原代培养皮层神经元和出生后第0-2天新生大鼠大脑中的少突胶质母细胞（OLs）的细胞纯度（>85%）。在无葡萄糖和脱氧的细胞培养基中，用GSK360A（3和30μM）预处理体外10天（DIV）的神经元和5-DIV的OLs 30分钟，并将其转移到厌氧室中，在37°C下通过连续流量的气体混合物（5%CO2和95%N2）预平衡1%的氧浓度2小时。用常规细胞培养基代替培养基，并将培养基放回5%CO2培养箱中。神经元和OLs的存活率通过细胞计数试剂盒-8在三个以上的独立实验中测量。

Animal/Disease Models: Male Lewis rat ventricular dysfunction model [1]
Doses: 30 mg/kg
Route of Administration: po (oral gavage)
Experimental Results: Circulating levels of erythropoietin and hemoglobin and blood oxygenation in the heart and skeletal muscle of male rats Enzyme-1 expression is increased.

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GSK360A was delivered by intracerebroventricular (ICV, 12μg /5μg/ 10 g body-weight), intranasal (IN, 30 or 60μg/ 10 g) or intraperitoneal (IP, 100~500μg/ 10 g) as previously described (Bao et al., 2010; Yang et al., 2013a; 2013b; 2013c; Zhou et al., 2017). GSK360A powder were dissolved in 30% hydroxypropyl-β-cyclodextrin (HBC) solution to a final concentration of 2.5μg/ μg. ICV or IP administration of GSK360A was performed at 30 min after HI, and IN-GSK360A delivery was given twice at 30 and 60 min after HI. The ICV injection of 5μg saline or GSK360A with the speed 1μg/min was performed with a Hamilton syringe at right hemisphere as previously described (Yang et al., 2009). The stereotaxic coordinates were 2.0 mm rostral and 1.5 mm lateral to the right from the lambda point, and at a depth of 2.0 mm from the surface of brain. The Rice-Vannucci model of neonatal HI was performed in 7-day-old (P7) Wistar rats, as described (Yang et al., 2009). Briefly, pups of both genders were anesthetized by 2% isoflurane mixed with compressed air when the right common carotid artery was ligated. After a 1 h recovery period, pups were exposed to 10% O2 balanced by 90% nitrogen for 90 min in glass chambers submerged in a 37°C water bath. The pups were returned to dams after hypoxia induction for recovery. The C57BL/6 mice were obtained from Charles River Laboratories and applied in the experiments of HIF1α immunoblots and tissue staining (Fig. 1, Fig. 2C-​-E,E, Fig. 5A). The ODD-luciferase (HIF1α-Luc) mice were obtained from the Jackson Laboratories (Stock# 006206) and the brains were harvested for luciferase activity assay using the Dual-Luciferase® reporter assay system. Whole blood from P7 rats were collected for complete blood counts measurement by a Hemavet 1500 blood analyzer (1500 R series). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) and conducted according to the National Institutes of Health Guide for Care and Use of Laboratory Animals. Experiments are performed and reported in accordance with the ARRIVE.[2]

[1]. Chronic inhibition of hypoxia-inducible factor prolyl 4-hydroxylase improves ventricular performance, remodeling, and vascularity after myocardial infarction in the rat. J Cardiovasc Pharmacol. 2010 Aug;56(2):147-55.

Hypoxia-inducible factor-1α (HIF1α) is a major regulator of cellular adaptation to hypoxia and oxidative stress, and recent advances of prolyl-4-hydroxylase (P4H) inhibitors have produced powerful tools to stabilize HIF1α for clinical applications. However, whether HIF1α provokes or resists neonatal hypoxic-ischemic (HI) brain injury has not been established in previous studies. We hypothesize that systemic and brain-targeted HIF1α stabilization may have divergent effects. To test this notion, herein we compared the effects of GSK360A, a potent P4H inhibitor, in in-vitro oxygen-glucose deprivation (OGD) and in in-vivo neonatal HI via intracerebroventricular (ICV), intraperitoneal (IP), and intranasal (IN) drug-application routes. We found that GSK360A increased the erythropoietin (EPO), heme oxygenase-1 (HO1) and glucose transporter 1 (Glut1) transcripts, all HIF1α target-genes, and promoted the survival of neurons and oligodendrocytes after OGD. Neonatal HI insult stabilized HIF1α in the ipsilateral hemisphere for up to 24 h, and either ICV or IN delivery of GSK360A after HI increased the HIF1α target-gene transcripts and decreased brain damage. In contrast, IP-injection of GSK360A failed to reduce HI brain damage, but elevated the risk of mortality at high doses, which may relate to an increase of the kidney and plasma EPO, leukocytosis, and abundant vascular endothelial growth factor (VEGF) mRNAs in the brain. These results suggest that brain-targeted HIF1α-stabilization is a potential treatment of neonatal HI brain injury, while systemic P4H-inhibition may provoke unwanted adverse effects.[2]

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Background: Hypoxia inducible factors (HIFs) are transcription factors that are regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play an important role in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that GSK360A, a novel orally active HIF-PHD inhibitor, could facilitate local and systemic HIF-1 alpha signaling and protect the failing heart after myocardial infarction (MI).[1]
Methods and results: GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1>PHD2 = PHD3) capable of activating the HIF-1 alpha pathway in a variety of cell types including neonatal rat ventricular myocytes and H9C2 cells. Male rats treated orally with GSK360A (30 mg x kg x d) had a sustained elevation in circulating levels of erythropoietin and hemoglobin and increased hemoxygenase-1 expression in the heart and skeletal muscle. In a rat model of established heart failure with systolic dysfunction induced by ligation of left anterior descending coronary artery, chronic treatment with GSK360A for 28 days prevented the progressive reduction in ejection fraction, ventricular dilation, and increased lung weight, which were observed in the vehicle-treated animals, for up to 3 months. In addition, the microvascular density in the periinfarct region was increased (>2-fold) in GSK360A-treated animals. Treatment was well tolerated (survival was 89% in the GSK360A group vs. 82% in the placebo group).[1]
Conclusions: Chronic post-myocardial infarction treatment with a selective HIF PHD inhibitor (GSK360A) exerts systemic and local effects by stabilizing HIF-1 alpha signaling and improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. These results suggest that HIF-PHD inhibitors may be suitable for the treatment of post-MI remodeling and heart failure.[1]

C17H17FN2O5

348.3304

348.112

C, 58.62; H, 4.92; F, 5.45; N, 8.04; O, 22.97

931399-19-8

54685147

Typically exists as white to off-white solids at room temperature

2.035

112.12

3

6

6

25

616

0

C1CC1CCN2C3=C(C=C(C=C3)F)C(=C(C2=O)C(=O)NCC(=O)O)O

TYHRZQVUPPODPT-UHFFFAOYSA-N

InChI=1S/C17H17FN2O5/c18-10-3-4-12-11(7-10)15(23)14(16(24)19-8-13(21)22)17(25)20(12)6-5-9-1-2-9/h3-4,7,9,23H,1-2,5-6,8H2,(H,19,24)(H,21,22)

{[1-(2-Cyclopropyl-ethyl)-6-fluoro-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carbonyl]-amino}-acetic acid

GSK-360A; GSK 360A; GSK360A

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。