HIF-PHI/hypoxia-inducible factor-prolyl-hydroxylase

In PC12 cells, roxadustat (5-50 μM; 6 hours) dramatically reduces TBHP-induced apoptosis[2]. In PC12 cells, roxadustat (50 μM; 6 hours) stabilizes HIF-1α protein expression[2].

Improved recovery from spinal cord injury and protection of motor neuron survival are two benefits of roxadustat (50 mg/kg; ip; daily for 7 days)[2].

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FG-4592/roxadustat administration also improved recovery and increased the survival of neurons in spinal cord lesions in the mice model. Combination therapy including the specific HIF-1α blocker YC-1 down-regulated the HIF-1α expression and partially abolished the protective effect of FG-4592. Taken together, our results revealed that the role of FG-4592 in SCI recovery is related to the stabilization of HIF-1α and inhibition of apoptosis. Overall, our study suggests that PHDIs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans[2].

Apoptosis Analysis[2]
Cell Types: PC12 cells
Tested Concentrations: 5, 20, 50 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: Dramatically inhibited TBHP-induced apoptosis.

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Western Blot Analysis[2]
Cell Types: PC12 cells
Tested Concentrations: 50 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: stabilized HIF-1α protein expression.

Animal/Disease Models: 12-week female C57BL/6 mice[2]
Doses: 50 mg/ kg
Route of Administration: intraperitoneal (ip)injection; daily for 7 days
Experimental Results: Protected the survival of motor neurons and improved recovery from spinal cord injury.

Absorption, Distribution and Excretion
Roxadustat plasma exposure (AUC and Cmax) increases dose-proportionally within the recommended therapeutic dose range. In a three times per week dosing regimen, steady-state roxadustat plasma concentrations are achieved within one week (three doses) with minimal accumulation. Maximum plasma concentrations (Cmax) are usually achieved at two hours post dose in the fasted state. Administration of roxadustat with food decreased Cmax by 25% but did not alter AUC as compared with the fasted state.
Following oral administration of radiolabelled roxadustat in healthy subjects, the mean recovery of radioactivity was 96% (50% in feces, 46% in urine). In feces, 28% of the dose was excreted as unchanged roxadustat. Less than 2% of the dose was recovered in urine as unchanged roxadustat.
The blood-to-plasma ratio of roxadustat is 0.6. The apparent volume of distribution at steady-state is 24 L.
The apparent total body clearance (CL/F) of roxadustat is 1.1 L/h in patients with CKD not on dialysis and 1.4 L/h in patients with CKD on dialysis.

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Metabolism / Metabolites
_In vitro_, roxadustat is a substrate for CYP2C8 and UGT1A9 enzymes. Roxadustat is primarily metabolized to hydroxy-roxadustat and roxadustat O-glucuronide. Unchanged roxadustat was the major circulating component in human plasma and detectable metabolites in human plasma constituted less than 10% of total drug-related material exposure. No human-specific metabolites were observed but roxadustat O-glucuronide was detected in human urine sample.

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Biological Half-Life
The mean effective half-life of roxadustat is approximately 15 hours in patients with CKD.

Protein Binding
Roxadustat is highly bound to human plasma proteins (approximately 99%), mainly to albumin.

[1]. Roxadustat (FG-4592) Versus Epoetin Alfa for Anemia in Patients Receiving MaintenanceHemodialysis: A Phase 2, Randomized, 6- to 19-Week, Open-Label, Active-Comparator, Dose-Ranging, Safety and Exploratory Efficacy Study. Am J Kidney Dis. 2016 Jun;67(6):912-24.

[2]. Stabilization of HIF-1α by FG-4592 promotes functional recovery and neural protection in experimental spinal cord injury. Brain Res. 2016 Feb 1;1632:19-26.

Roxadustat is an N-acylglycine resulting from the formal condensation of the amino group of glycine with the carboxy group of 4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylic acid. It is an inhibitor of hypoxia inducible factor prolyl hydroxylase (HIF-PH). It has a role as an EC 1.14.11.2 (procollagen-proline dioxygenase) inhibitor and an EC 1.14.11.29 (hypoxia-inducible factor-proline dioxygenase) inhibitor. It is a member of isoquinolines, an aromatic ether and a N-acylglycine.
Roxadustat is a first-in-class hypoxia-inducible factor prolyl hydroxylase inhibitor used to treat anemia associated with chronic kidney disease. It works by reducing the breakdown of the hypoxia-inducible factor (HIF), which is a transcription factor that stimulates red blood cell production in response to low oxygen levels. Roxadustat was first approved by the European Commission in August 2021.
Roxadustat is an orally bioavailable, hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), with potential anti-anemic activity. Upon administration, roxadustat binds to and inhibits HIF-PHI, an enzyme responsible for the degradation of transcription factors in the HIF family under normal oxygen conditions. This prevents HIF breakdown and promotes HIF activity. Increased HIF activity leads to an increase in endogenous erythropoietin production, thereby enhancing erythropoiesis. It also reduces the expression of the peptide hormone hepcidin, improves iron availability, and boosts hemoglobin (Hb) levels. HIF regulates the expression of genes in response to reduced oxygen levels, including genes required for erythropoiesis and iron metabolism.

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Drug Indication
Roxadustat is indicated for the treatment of adult patients with symptomatic anemia associated with chronic kidney disease (CKD).
Evrenzo is indicated for treatment of adult patients with symptomatic anaemia associated with chronic kidney disease (CKD).
Treatment of anaemia due to chronic disorders

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Mechanism of Action
Anemia is a common complication of chronic kidney disease that may be caused by reduced production of renal erythropoietin (EPO), functional iron deficiency due to increased levels of hepcidin, blood loss, reduced erythrocyte survival duration, and inflammation. Hypoxia-inducible factor (HIF) is a transcription factor that induces several target oxygen-sensitive genes in response to low oxygen levels in the cellular environment, or hypoxia. Target genes are involved in erythropoiesis, such as those for EPO, EPO receptor, proteins promoting iron absorption, iron transport, and haem synthesis. Activation of the HIF pathway is an important adaptive responsive to hypoxia to increase red blood cell production. HIF is heterodimeric and contains an oxygen-regulated α-subunit. The α-subunit houses an oxygen-dependent degradation (ODD) domain that is regulated and hydroxylated by HIF-prolyl hydroxylase (HIF-PHD) enzymes under normoxic cellular conditions. HIF-PHD enzymes play a crucial role in maintaining a balance between oxygen availability and HIF activity. Roxadustat is a reversible and potent inhibitor of HIF-PHD enzymes: inhibition of HIF-PHD leads to the accumulation of functional HIF, an increase in plasma endogenous EPO production, enhanced erythropoiesis, and indirect suppression of hepcidin, which is an iron regulator protein that is increased during inflammation in chronic kidney disease. Roxadustat can also regulate iron transporter proteins and regulates iron metabolism by increasing serum transferrin, intestinal iron absorption and the release of stored iron in patients with anemia associated with dialysis-dependent or dialysis-independent CKD. Overall, roxadustat improves iron bioavailability, increases Hb production, and increases red cell mass.

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Pharmacodynamics
Roxadustat dose-dependently improves iron bioavailability, increases hemoglobin production, and increases red blood cell mass in patients with anemia. In non-dialysis-dependent CKD patients with anemia, roxadustat maintained Hb for up to 2 years. It has a comparable efficacy to erythropoietin-stimulating agents in achieving Hb response. Roxadustat also reduces cholesterol levels from baseline, regardless of the use of statins or other lipid-lowering agents.

C19H16N2O5

352.34100

352.105

C, 64.77; H, 4.58; N, 7.95; O, 22.70

808118-40-3

Roxadustat-d5;2043026-13-5; 1537179-95-5 (potassium); 808118-40-3 (free); 1537180-01-0 (HCl); 1537179-94-4 (sodium); 1537180-03-2 (mesylate)

11256664

Light yellow to green yellow solid powder

1.4±0.1 g/cm3

684.3±55.0 °C at 760 mmHg

199-215°C

367.6±31.5 °C

0.0±2.2 mmHg at 25°C

1.674

3.9

3

6

5

26

508

0

YOZBGTLTNGAVFU-UHFFFAOYSA-N

InChI=1S/C19H16N2O5/c1-11-15-9-13(26-12-5-3-2-4-6-12)7-8-14(15)18(24)17(21-11)19(25)20-10-16(22)23/h2-9,24H,10H2,1H3,(H,20,25)(H,22,23)

(4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carbonyl)glycine

Roxadustat; ASP1517; ASP 1517; Roxadustat (FG-4592); N-[(4-Hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl)carbonyl]glycine; ASP-1517; FG-4592; FG4592; FG-4592;

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 (~283.82 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.10 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 4: 5 mg/mL (14.19 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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