HIF-PHD
HIF prolyl hydroxylase (PHD) enzymes (PHD1, PHD2, PHD3). GSK360A is an orally active HIF-PHD inhibitor.

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]

---

GSK360A inhibits all three HIF-PHD isoforms (PHD1, PHD2, and PHD3) with IC50 values of 10 nM, 100 nM, and 126 nM, respectively, in a 2-OG-competitive manner. GSK360A inhibits PHD2 with an IC50 of 100 nM, whereas roxadustat (FG-4592) demonstrates a significantly higher IC50 of 591.4 nM for the same enzyme. It promotes cardiomyocyte mitochondrial aerobic glycolysis under normoxic conditions by stabilizing cellular HIF-1 and upregulating HIF-1α target genes transcription. GSK360A activates the HIF-1 alpha pathway.

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

---

In vivo, GSK360A (30 mg/kg i.g.) improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. It protects the failing heart after myocardial infarction (MI). GSK360A provided rapid exposure in plasma (7734 ng/ml), kidney (45-52% of plasma level), and brain (1-4% of plasma level), and increased kidney EPO mRNA (80-fold) and brain VEGF mRNA (2-fold). The compound is orally active. Pharmacokinetic and pharmacodynamic studies were conducted including plasma, kidney, and brain GSK360A concentration measurements at 5 and 24 hours after oral administration of 30 mg/kg GSK360A. GSK360A decreases post-stroke brain injury and sensory, motor, and cognitive behavioral deficits.

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]

---

HIF-PHD inhibition is assessed using in vitro enzyme assays with recombinant PHD isoforms (PHD1, PHD2, PHD3) and a substrate (e.g., HIF-1α peptide). 2-oxoglutarate consumption is measured by a coupled enzyme assay or by detecting the release of succinate or CO2. HIF-α hydroxylation is measured by detecting hydroxylated HIF-1α using mass spectrometry or antibody-based methods. IC50 values are calculated from dose-response curves using nonlinear regression analysis. Selectivity is evaluated by testing against related 2-OG-dependent dioxygenases.

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.

---

Cellular activity is evaluated in cardiomyocytes, endothelial cells, or other HIF-responsive cells. Cells are treated with GSK360A at various concentrations (typically 0.001-100 μM) for appropriate time periods (4-24 hours for HIF stabilization, 24-72 hours for gene expression). HIF-1α protein levels are measured by Western blot (with normalization to loading controls). HIF target gene expression (VEGF, GLUT1, EPO, PDK1, BNIP3) is measured by qRT-PCR. Mitochondrial aerobic glycolysis is assessed by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using Seahorse analyzers. Metabolic flux analysis using [U-13C]-glucose or [U-13C]-glutamine may be performed.

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.

---

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]

---

In vivo efficacy is studied in animal models of myocardial infarction (MI), heart failure, and stroke. GSK360A is administered orally at doses of 30 mg/kg. Ventricular function is assessed by echocardiography (ejection fraction, fractional shortening). Cardiac remodeling is assessed by measuring infarct size, ventricular wall thickness, and fibrosis (Masson's trichrome staining). Vascularity is assessed by CD31 immunohistochemistry. Pharmacodynamic markers including HIF-1α stabilization, EPO mRNA (kidney), VEGF mRNA (brain), and other HIF target genes are measured in tissue samples by qRT-PCR or Western blot. Pharmacokinetics and organ distribution are determined by measuring compound concentrations in plasma, kidney, and brain by LC-MS/MS. Brain injury and behavioral deficits are assessed in stroke models.

GSK360A has a molecular formula of C20H17F3N4O3 and a molecular weight of 418.37 g/mol. CAS Number: 931399-19-8. It is an orally active HIF-PHD inhibitor. Purity: typically ≥98% by HPLC. The compound is soluble in DMSO and should be stored at -20°C for long-term stability, protected from light and moisture. Detailed pharmacokinetic parameters including half-life, oral bioavailability, volume of distribution, and plasma protein binding are available from the primary literature. GSK360A provided rapid exposure in plasma (7734 ng/ml).

Safety data for GSK360A indicate it is a research compound for laboratory use only. As a HIF-PHD inhibitor, it may have effects on erythropoiesis, angiogenesis, and metabolism. Specific toxicological profiles including acute toxicity, organ toxicity, genotoxicity, and reproductive toxicity are not extensively published. Standard laboratory safety practices should be followed during handling, including the use of personal protective equipment and adequate ventilation. The compound is for research use only and not for human therapeutic applications without appropriate regulatory approval. Consult the material safety data sheet (MSDS) for detailed safety information.

[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 key regulator of cellular adaptation to hypoxia and oxidative stress. Recent advancements in prolyl-4-hydroxylase (P4H) inhibitors have provided powerful clinical tools for stabilizing HIF1α. However, previous studies have not clearly defined whether HIF1α induces or resists neonatal hypoxic-ischemic (HI) brain injury. We hypothesized that systemic and brain-targeted HIF1α stabilization might produce different effects. To test this hypothesis, we compared the effects of the potent P4H inhibitor GSK360A in an in vitro oxygen-glucose deprivation (OGD) model and an in vivo neonatal HI model, administered via intraventricular (ICV), intraperitoneal (IP), and intranasal (IN) routes of administration. We found that GSK360A increases the transcriptional levels of erythropoietin (EPO), heme oxygenase-1 (HO1), and glucose transporter 1 (Glut1), all of which are target genes of HIF1α and promote the survival of neurons and oligodendrocytes after hypoxia-glucose-deficiency (OGD). Neonatal hypoxic-ischemic (HI) injury can stabilize HIF1α in the ipsilateral hemisphere for up to 24 hours. Intraventricular (ICV) or intranasal (IN) administration of GSK360A after HI can increase the transcriptional levels of HIF1α target genes and reduce brain injury. Conversely, intraperitoneal injection of GSK360A failed to reduce HI brain injury, but at high doses it increased the risk of death, which may be related to elevated EPO levels in the kidneys and plasma, leukocytosis, and high expression of vascular endothelial growth factor (VEGF) mRNA in the brain. These results suggest that brain-targeted HIF1α stabilization may be a potential approach to treating hypoxic-ischemic brain injury in newborns, while systemic P4H inhibition may cause adverse effects. [2]

---

Background: Hypoxia-inducible factors (HIFs) are a class of transcription factors whose activity is regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play important roles in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that the novel orally administered active HIF-PHD inhibitor GSK360A could promote local and systemic HIF-1α signaling and protect the failing heart after myocardial infarction (MI). [1]
Methods and Results: GSK360A is a potent (nanomolar) HIF-PHD (PHD1>PHD2=PHD3) inhibitor that activates the HIF-1α pathway in multiple cell types, including neonatal rat ventricular myocytes and H9C2 cells. After oral administration of GSK360A (30 mg/kg/d) to male rats, the levels of circulating erythropoietin and hemoglobin were continuously elevated, and the expression of heme oxygenase-1 in the heart and skeletal muscle was also increased. In a rat model of systolic dysfunction heart failure induced by ligation of the left anterior descending coronary artery, continuous 28-day treatment with GSK360A prevented progressive decline in ejection fraction, ventricular dilation, and increased lung weight for up to 3 months, symptoms observed in animals receiving the carrier therapy. In addition, the microvessel density in the periinfarct area of the GSK360A-treated group was increased (>2-fold). The treatment was well tolerated (survival rate was 89% in the GSK360A group and 82% in the placebo group). [1] Conclusion: In established models of ventricular dysfunction, long-term treatment after myocardial infarction with the selective HIF PHD inhibitor (GSK360A) can exert systemic and local effects by stabilizing the HIF-1α signaling pathway and improve long-term ventricular function, remodeling, and angiogenesis. These results suggest that HIF-PHD inhibitors may be suitable for treating post-myocardial infarction remodeling and heart failure. [1]

---

GSK360A is a research tool compound for studying HIF-PHD biology and cardioprotection. It is not approved for clinical use. The compound has been investigated for its potential to protect the failing heart after myocardial infarction by activating the HIF-1 alpha pathway. GSK360A also decreases post-stroke brain injury and improves sensory, motor, and cognitive behavioral deficits. Reference: GSK360A (GSK1120360A) is an orally active HIF-prolyl 4-hydroxylase (HIF-PHD) inhibitor that targets all three HIF-PHD isoforms with high potency (IC50 from 10-120 nM) in a 2-oxoglutarate-competitive manner. The compound's ability to stabilize HIF-1α and upregulate HIF target genes makes it a valuable tool for studying the role of HIF signaling in ischemia, cardioprotection, and neuroprotection.

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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