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]. It was found that the proliferation of L929 cells was inhibited and the production of collagen I, collagen III, prolyl hydroxylase domain protein 2 (PHD2), HIF-1α, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and p-Smad3 were reduced relative to that in the CoCl2 or BLM group after roxadustat treatment. [3]

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Effects of roxadustat on L929 cell proliferation and protein expression in vitro [3]
L929 cells were stimulated with CoCl2 (50 nM) so as to mimic the pro-fibrotic environment under hypoxia (Pardo et al., 2005). Cells proliferation was analyzed by using the EdU cell proliferation kit with TMB after 72 h of CoCl2 induction of L929 cells. The CoCl2-stimulated group showed significantly higher proliferation rates than the control group. The group of L929 cells treated with varying concentrations of roxadustat (0.3, 1,3, 10 μM) showed significantly inhibited cell proliferation rates than the CoCl2-stimulated group (Fig. 5A). For proteins analysis, the L929 cells were treated as that in the proliferation assay. Briefly, the proteins were extracted and examined, as shown in Fig. 5. As compared to the control cells, the protein expression of collagen I, collagen III, and α-SMA were significantly increased in cells-stimulated with CoCl2 (50 nM). However, the expression of collagen I, collagen III and α-SMA were significantly inhibited with roxadustat treatment than with CoCl2 stimulation (Fig. 5B, E). Relative to that in the control group, the protein expression of TGF-β1, CTGF, and p-Smad3, significantly increased after CoCl2-stimulated pro-fibrosis, but decreased after roxadustat treatment in the CoCl2-stimulated group (Fig. 5D, G). Particularly, CoCl2 or roxadustat treatment showed no effect on the Smad3 expression (Fig. 5D and G). [3]
Under normoxia, increased HIF activity increases the production of endogenous erythropoietin for anemia treatment. Roxadustat prevents HIF breakdown and promotes the HIF activity in normoxia (Malyszko, 2016). The effect of roxadustat on HIF-1α is different under normoxia and hypoxia; therefore, the effect of roxadustat on the expression of HIF-1α and PHD2 in L929 cells during normoxia and hypoxia was investigated. Our results showed that roxadustat increased the HIF-1α activity and reduced PHD2 under normoxia, whereas it decreased the HIF-1α activity under hypoxia in CoCl2-stimulated L929 cells (Fig. 5C, F). [3]
To determine the mechanism underlying TGF-β1 activation, SB525334 was used to inhibit TGF-β1 activation. L929 cell proliferation was analyzed, and the protein expression levels of TGF-β1, CTGF, Smad3, p-Smad3, HIF-1α, PHD2, α-SMA, collagen I, and collagen III were examined after the incubation of the cells with roxadustat (3 μM) without or with 1 μM SB525334 for 72 h (Fig. 6B-G). Our results showed that the Roxadustat-treated group showed decreased expression levels for all proteins, except for Smad3 (Fig. 6D and G), as well as reduced L929 cell proliferation without or with SB525334 than the CoCl2-stimulated group (Fig. 6A). In addition, the SB525334 group showed reduced expression of proteins and cell proliferation; the SB525334+roxadustat group did not show reduction in the protein expression levels or cell proliferation rates any further than the SB525334 group (Fig. 6; P > 0.05). These findings indicate that roxadustat attenuates experimental lung fibrosis by inhibiting TGF-β1 activation. [3]
To determine the mechanism underlying Smad3 activation, SIS3 was used to inhibit Smad3 activation. L929 cell proliferation was analyzed, and the protein expression levels of TGF-β1, CTGF, Smad3, p-Smad3, HIF-1α, PHD2, α-SMA, collagen I, and collagen III were examined after incubating the cells for 72 h with roxadustat (3 μM) without or with 0.5 μM SIS3 (Fig. 7B-7I). These results suggest that the roxadustat-treated group showed reduced expression levels for all proteins, except for TGF-β1 and Smad3 (Fig. 7E and I), as well as reduced L929 cell proliferation without or with SIS3 than the CoCl2–stimulated group (Fig. 7A). Treatment with SIS3 alone reduced the expression of proteins and the extent of cell proliferation. However, treatment with SIS3+Roxadustat did not reduce the protein expression levels or cell proliferation rates any further when compared to that by SIS3 alone (P > 0.05; Fig. 7). These findings indicate that roxadustat attenuates experimental lung fibrosis by inhibiting the p-Smad3 expression [3].

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].

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Effects of roxadustat on lung coefficients and histopathological changes in lung tissues [3]
Histopathological changes were assessed by HE staining using the semi-quantitative method. Intact and clear alveoli, normal interstitium, and a few inflammatory cells were noted in the sham mice (Fig. 1A1). Inflammatory and fibrotic changes such as the destruction of lung alveoli and inflammatory cell infiltration were detected in the lung tissues of the BLM-induced mice (Fig. 1A2). However, as compared with the BLM-induced mice, the roxadustat-treated mice showed great improvements in inflammatory cell infiltration and thickening of the lung interstitium as well as a significant decrease in the pathology score (Fig. 1A3). The lung coefficient is the ratio of lung weight to the body weight, and it reflects the degree of pulmonary fibrosis. During the development of pulmonary fibrosis, the increase in lung mass at an early stage can be attributed to factors such as cell swelling and capillary congestion, while, at the later stage, it is mostly caused by collagen fiber formation. In the BLM-induced mice, the body weight increased gradually at the early stage due to the state of the disease, although some mice showed continual decline in the body weight, which directly contributed to increase in lung coefficient. The weight of the mice was recorded before their sacrificed, and the weight of the lung tissues was recorded and the lung coefficient was calculated. As compared to that in BLM-induced mice, the lung coefficient decreased in roxadustat-treated mice.

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Effects of roxadustat on the collagen levels in lung tissues [3]
Masson’s trichrome staining and western blotting were employed to examine the amount of collagen deposition. Collagen I and III were quantified by western blotting (Fig. 2A, B), while the histochemical quantification of collagen was performed with Masson’ s trichrome staining (Fig. 2C1–C3; D). A large amount of collagen deposition was observed in the pulmonary interstitium by Masson’s trichrome staining in BLM-induced mice as compared to that in sham-operated mice. However, the collagen content was significantly reduced after 21 consecutive days of roxadustat administration (blue collagen deposition in Fig. 2C1–C3). HYP–a characteristic amino acid that accounts for approximately 13 % of the total amino acids in collagen is an essential marker indicating collagen accumulation. HYP content was significantly decreased in roxadustat-treated mice than in BLM-induced mice (Fig. 2E). The expression of collagen I and III were higher in the BLM group than in the sham group (p < 0.01). Nevertheless, the expression of collagen I and III was lower in the roxadustat-treated group than in the BLM -induced group.

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Effects of roxadustat on the in vivo protein expression [3]
The protein expression of HIF-1α, PHD2, α-SMA, TGF-β1, p-Smad3, Smad3, and CTGF in the lung tissues was measured by western blotting. Compared to that in the sham mice, the expression of HIF-1α, PHD2, and α-SMA were higher in the BLM-induced mice, but lower in the roxadustat-treated mice (Fig. 3A, C; p < 0.01). As compared to that in the sham mice, the expression of TGF-β1, p-Smad3 and CTGF were higher in the BLM-induced mice, but lower in the roxadustat-treated mice (Fig. 3B, D; p < 0.01). Notably, the expression of Smad3 remained unchanged in the BLM-induced and roxadustat-treated groups.

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.

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Analysis of L929 proliferation [3]
To assess the cell proliferation of L929 cells, the cells were initially seeded into 96-well plates at a density of 2 × 103 cells/well and then incubated at 37 °Covernight. The medium was removed, followed by the addition of either the medium alone (control) or the medium withvarying concentrations of roxadustat (0.3, 1, 3, or 10 μM) without or with CoCl2 (50 nM) and incubated for 72 h. To further verify the mechanism of cell proliferation, the cells were treated with CoCl2 for 72 h without or with 1 μM SB525334 (a TGF-β1 inhibitor) or 0.5 μM SIS3 (a Smad inhibitor). Cell proliferation was assessed using the BeyoClick™ 5-ethynyl-2′-deoxyuridine (EdU) Cell Proliferation Kit with TMB, which is based on EdU as a novel alternative for 5-bromo-2’-deoxyuridine (BrdU) assay to directly measure active DNA synthesis or S-phase synthesis of a cell cycle via reaction with fluorescent azides in a Cu(I)-catalyzed [3 + 2] cycloaddition. The absorbance was measured at 630 nm and calculated as a ratio against untreated cells.

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Evaluation of protein expression in CoCl2-stimulated L929 cells[3]
The mouse lung fibroblasts L929 cells were cultured in MEM supplemented with 10 % (v/v) fetal bovine serum (FBS) under a 5% CO2 and 95 % N2 humidified atmosphere at 37 °C. The cells were grown to approximately 60 % confluency and treated with 3 μM roxadustat without or with CoCl2 (50 nM) for 72 h. Then, the protein expression levels of TGF-β1, CTGF, Smad3, p-Smad3, HIF-1α, PHD2, α-SMA, collagen I, and collagen III were assessed by western blotting. To investigate the possible mechanism of lung fibrosis, the cells were treated with CoCl2 (50 nM) for 72 h without or with 1 μM SB525334 or 0.5 μM SIS3, and the expression of TGF-β1, CTGF, Smad3, p-Smad3, HIF-1α, PHD2, α-SMA and collagen I/III expression were evaluated by western blotting.

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.

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Bleomycin (BLM)-induced pulmonary fibrosis model in mice [3]
A total of 40 adult male C57BL/6 mice were housed in a standard animal laboratory at consistent temperature (22 °C ± 2 °C) and humidity (60 ± 10 %) condition, with free access to chow and water. After 7 days of adaptation, an animal model was established. Ten mice were randomly selected to form the control group. Another 30 mice were intraperitioneally injected with BLM (Invitrogen, Carlsbad, CA, USA) in 0.2 mL saline (50 mg/kg) at days 1, 4, 8, 11, 15, 18, 22 and 25 of the experiment. The control mice were intraperitoneally injected with an equal volume of saline, without BLM. The mice were maintained under good conditions for 2 weeks after BLM exposure, and the body weights of the mice were recorded every week. At day 39, 20 mice with better exposure to BLM were selected and randomly grouped in the BLM and the BLM + roxadustat groups. The BLM + group waroxadustats intragastrically with 20 mg/kg/day roxadustat (dose selection based on the daily dosage in anemia and based on the data from a pre-experiment on the anti-BLM-induced fibrosis in mice) (Zhang et al., 2019). The control and BLM mice were intragastrically administered with an equal amount of saline. At day 60, the lung tissues were collected, and the lung coefficients were determined using the following formula: the lung coefficient = Wet lung weight/body weight × 100 %. Next, the tissues were categorized into two portions: the left lungs tissues were fixed in 4% paraformaldehyde for histological examination and the right lungs tissue were stored in liquid nitrogen for western blotting. Before starting the main experiment, an exploratory preliminary expeiment was conducted on 5 groups (sham, BLM, BLM + Roxadustat 10 mg/kg/day, 20 mg/kg/day or 40 mg/kg/day) of mice, with 10 mice in each group. The method and duration of administration were the same as those in the main experiment. Indexes such as lung weight, lung coefficient, and hydroxyproline (HYP) levels of the mice were monitored. As roxadustat at the dosage of 20 mg/kg/day was found to reduce the lung coefficients and HYP levels, this dosage was used in the main experiment.

Absorption, Distribution and Excretion
Within the recommended therapeutic dose range, roxadustat plasma exposure (AUC and Cmax) increases proportionally with the dose. With a three-week dosing regimen, steady-state plasma concentrations of roxadustat are reached within one week (three doses), with minimal accumulation. In the fasting state, maximum plasma concentrations (Cmax) are typically reached two hours after administration. Taking roxadustat after food reduces Cmax by 25% compared to the fasting state, but does not alter AUC. In healthy subjects, the mean recovery rate of radiolabeled roxadustat after oral administration is 96% (50% in feces, 46% in urine). In feces, 28% of the dose is excreted unchanged as roxadustat. Less than 2% of the dose is recovered unchanged as roxadustat in urine. The plasma-to-plasma concentration ratio of roxadustat is 0.6. The steady-state apparent volume of distribution is 24 L.
The apparent systemic clearance (CL/F) of roxadustat in patients with chronic kidney disease not receiving dialysis was 1.1 L/h, while that in patients with chronic kidney disease receiving dialysis was 1.4 L/h.
Metabolism/Metabolites
In vitro studies have shown that roxadustat is a substrate of CYP2C8 and UGT1A9 enzymes. Roxadustat is primarily metabolized to hydroxyroxadustat and roxadustat O-glucuronide. The unchanged roxadustat is the main circulating component in human plasma, and detectable metabolites in human plasma account for less than 10% of total drug-related substance exposure. No human-specific metabolites were observed, but roxadustat O-glucuronide was detected in human urine samples.
Biological Half-Life
The mean effective half-life of roxadustat in patients with chronic kidney disease is approximately 15 hours.

Protein Binding
Roxadustat has a high binding rate to human plasma proteins (approximately 99%), primarily binding 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.

[3]. Roxadustat attenuates experimental pulmonary fibrosis in vitro and in vivo. Toxicol Lett. 2020 Oct 1;331:112-121.

Roxadustat is an N-acylglycine compound formed by the condensation of the amino group of glycine with the carboxyl group of 4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylic acid. It is an inhibitor of hypoxia-inducible factor prolyl hydroxylase (HIF-PH). It is also an inhibitor of EC 1.14.11.2 (procollagen-proline dioxygenase) and EC 1.14.11.29 (hypoxia-inducible factor-proline dioxygenase). It belongs to the isoquinoline, aromatic ether, and N-acylglycine classes. Roxadustat is a first-in-class hypoxia-inducible factor prolyl hydroxylase inhibitor used to treat anemia associated with chronic kidney disease. Roxadustat works by reducing the breakdown of hypoxia-inducible factor (HIF). HIF is a transcription factor that stimulates erythropoiesis under hypoxic conditions. 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. After administration, roxadustat binds to HIF-PHI and inhibits its activity. HIF-PHI is an enzyme responsible for degrading HIF family transcription factors under normoxic conditions. This prevents HIF degradation and promotes HIF activity. Enhanced HIF activity leads to increased production of endogenous erythropoietin, thereby enhancing erythropoiesis. It can also reduce the expression of the peptide hormone hepcidin, improve iron utilization, and increase hemoglobin (Hb) levels. HIF regulates gene expression in response to hypoxia, including genes required for erythropoiesis and iron metabolism.

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Drug Indications
Roxadustat is indicated for the treatment of adult patients with symptomatic anemia associated with chronic kidney disease (CKD).
Evrenzo is indicated for the treatment of adult patients with symptomatic anemia associated with chronic kidney disease (CKD).
Treatment of Anemia Caused by Chronic Diseases

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Mechanism of Action
Anemia is a common complication of chronic kidney disease. Its causes may include decreased renal erythropoietin (EPO) production, functional iron deficiency due to elevated hepcidin levels, blood loss, shortened erythrocyte survival time, and inflammation. Hypoxia-inducible factor (HIF) is a transcription factor that induces the expression of various oxygen-sensitive genes in response to low oxygen levels (i.e., hypoxia) in the cellular environment. Target genes are involved in erythropoiesis, such as erythropoietin (EPO), EPO receptors, and proteins that promote iron uptake, iron transport, and heme synthesis. HIF pathway activation is an important adaptive response of cells to hypoxia, increasing erythropoiesis. HIF is a heterodimer containing an oxygen-regulated α subunit. This α subunit contains an oxygen-dependent degradation (ODD) domain, which is regulated and hydroxylated by HIF-prolyl hydroxylase (HIF-PHD) under normoxic cellular conditions. HIF-PHD enzymes play a crucial role in maintaining the balance between oxygen supply and HIF activity. Roxadustat is a reversible and potent HIF-PHD enzyme inhibitor: inhibition of HIF-PHD leads to the accumulation of functional HIF, increases plasma endogenous EPO production, enhances erythropoiesis, and indirectly inhibits hepcidin (an iron-regulating protein whose expression is elevated during inflammation in chronic kidney disease). Roxadustat also modulates iron transporters and regulates iron metabolism by increasing serum transferrin, intestinal iron absorption, and the release of stored iron, thereby improving the condition of patients with anemia associated with dialysis-dependent or non-dialysis-dependent chronic kidney disease (CKD). Overall, roxadustat improves iron bioavailability, increases hemoglobin production, and increases red blood cell count.

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Pharmacodynamics
Roxadustat dose-dependently improves iron bioavailability, increases hemoglobin production, and increases red blood cell count in patients with anemia. In patients with anemia in non-dialysis-dependent CKD, roxadustat can maintain hemoglobin levels for up to 2 years. Its efficacy in raising hemoglobin levels is comparable to that of erythropoietin. Roxadustat lowers cholesterol levels regardless of the use of statins or other lipid-lowering drugs. Roxadustat is the first oral small-molecule hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor to have submitted a marketing application to the U.S. Food and Drug Administration (FDA) for the treatment of secondary anemia in chronic kidney disease. Its use is also considered to be related to pulmonary fibrosis; however, its corresponding therapeutic effects remain to be studied. This study investigated the in vitro effects of roxadustat on pulmonary fibrosis in L929 mouse fibroblasts stimulated by cobalt chloride (CoCl2), and its effects on an in vivo pulmonary fibrosis model induced by bleomycin (BLM; intraperitoneal injection, 50 mg/kg, twice weekly for 4 weeks). The study found that, compared with the CoCl2 group or the bleomycin group, roxadustat treatment inhibited L929 cell proliferation and reduced the expression of collagen I, collagen III, prolyl hydroxylase domain protein 2 (PHD2), HIF-1α, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1), and phosphorylated Smad3 (p-Smad3). Roxadustat improved pulmonary fibrosis by reducing pathological scores and collagen deposition, as well as reducing the expression of collagen I, collagen III, PHD2, HIF-1α, α-SMA, CTGF, TGF-β1, and p-Smad3/Smad3. Our cumulative results indicate that roxadustat can alleviate experimental pulmonary fibrosis by inhibiting TGF-β1/Smad activation. [3]

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Background: Roxadustat (FG-4592) is an oral hypoxia-inducible factor prolyl hydroxylase inhibitor that promotes erythropoiesis by increasing endogenous erythropoietin, improving iron regulation, and decreasing hepcidin. Study Design: Phase II, randomized (3:1), open-label, active-controlled, safety and efficacy study. Subjects and Participants: End-stage renal disease patients undergoing hemodialysis who had previously used erythropoietin to maintain stable hemoglobin (Hb) levels. Intervention: Part 1: A 6-week dose-range study in 54 subjects compared the efficacy of oral roxadustat three times weekly versus continued intravenous erythropoietin. Part 2: 90 individuals in 6 cohorts received 19 weeks of treatment with different starting doses and adjustment rules (1.0–2.0 mg/kg or weight-based), and these individuals responded differently to erythropoietin α. Intravenous iron was contraindicated. Results: The primary endpoint was hemoglobin level response, defined as a decrease in hemoglobin level (ΔHb) of 0.5 g/dL or more from baseline at the end of treatment (Part 1), and a mean hemoglobin level ≥11.0 g/dL over the last 4 weeks of treatment (Part 2). Measurements included hepcidin, iron parameters, cholesterol, and plasma erythropoietin (the latter was measured only in a subset of subjects). Results: In Part 1, subjects were randomized to the roxadustat group and the erythropoietin group at baseline erythropoietin α doses of 138.3 ± 51.3 (SD) and 136.3 ± 47.7 U/kg/week, respectively; in Part 2, the baseline erythropoietin α doses were 152.8 ± 80.6 and 173.4 ± 83.7 U/kg/week, respectively. In Part 1, the hemoglobin level response rate was 79% in the roxadustat 1.5–2.0 mg/kg group and 33% in the erythropoietin α control group (P = 0.03). The reduction in hepcidin levels was greater in the roxadustat 2.0 mg/kg group than in the erythropoietin α group (P<0.05). In Part II of the study, the mean dose of roxadustat required to maintain hemoglobin levels was approximately 1.7 mg/kg. The least-squares mean ΔHb in the roxadustat treatment group was comparable to that in the erythropoietin α treatment group (approximately -0.5 g/dL), with a least-squares mean difference of -0.03 g/dL between the two groups (95% CI, -0.39 to 0.33 g/dL) (mixed-effects model - repeated measures). Roxadustat significantly reduced mean total cholesterol levels, a phenomenon not observed with erythropoietin α. No safety issues were identified. Limitations: Short treatment duration and small sample size. Conclusion: In this phase II study of anemia treatment in end-stage renal disease patients undergoing maintenance hemodialysis, roxadustat was well-tolerated and effectively maintained hemoglobin levels. [1] Previous studies have shown that prolyl hydroxylase (PHD) inhibitors can stabilize the hypoxia-inducible factor 1α subunit (HIF-1α), improve the body's tolerance to hypoxia, and improve the prognosis of various diseases. However, the role of PHD inhibitors (PHDIs) in spinal cord injury recovery remains controversial. This study investigated the protective effect of a novel PHDI, FG-4592, both in vivo and in vitro. FG-4592 treatment stabilized HIF1α expression in PC12 cells and the spinal cord. FG-4592 treatment significantly inhibited tert-butyl hydroperoxide (TBHP)-induced apoptosis and improved the survival rate of PC-12 neurons. In a mouse spinal cord injury model, FG-4592 administration also improved post-injury recovery and increased neuronal survival. Combined use with the specific HIF-1α blocker YC-1 downregulated HIF-1α expression and partially eliminated the protective effect of FG-4592. In summary, our results indicate that the role of FG-4592 in spinal cord injury recovery is related to the stabilization of HIF-1α and the inhibition of apoptosis. Overall, our study suggests that PHDIs may be potential drug candidates for the treatment of human spinal cord injury and central nervous system diseases. [2]

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

O=C(O)CNC(C1=C(O)C2=C(C(C)=N1)C=C(OC3=CC=CC=C3)C=C2)=O

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.)