HIF-PHD

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. [1]
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. [2]

In well-established models of ventricular failure, GSK360A (30 mg/kg ig) improves mortality, vascularity, and long-term ventricular function [1].

Leukocyte count and measurement of plasma EPO[2]
Blood was collected from rat and transffer into an eppendorf tube containing EDTA. The leukocyte counts in fresh blood were measured using a Hemavet 1500 blood analyzer. For plasma preparation, blood was centrifuged at 1000 g for 15 min, and the supernatants of plasma were stored at −80 °C freezer for the further use. Plasma EPO was measured by the Luminex bead-based multiplex ELISA and quantified by the Bio-Plex Manager Software as described previously.[2]

Oxygen-glucose deprivation (OGD) model and cell viability assay[2]
The primary cultured cortical neurons from embryonic rat brains at day 17 and oligodendrocytes (OLs) from postnatal day 0–2 neonatal rat brains were confirmed the cell purity (>85%) by cell-specific-neuron (MAP2) or OL (Rip) marker (Sun et al., 2010). The neurons at 10 day-in-vitro (DIV) and OLs at 5-DIV were pretreated with GSK360A (3 and 30μM) for 30 min in glucose-free and deoxygenized cell medium, and transferred to an anaerobic chamber with pre-equilibration the 1% oxygen concentration by a continuous flux of gas mixture (5% CO2 and 95% N2) at 37°C for 2 h. The culture medium was replaced with regular cell culture medium and placed back to 5% CO2 incubator. Survival rate of neurons and OLs was measured by a cell counting kit-8 in more than three independent experiments for each.

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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)