Nox4 (Ki =140 nM); Nox1 (Ki =110 nM)
Setanaxib (GKT137831) targets NADPH oxidase 4 (Nox4) (IC50 = 10 nM for recombinant Nox4 enzymatic inhibition) [1][2]
Setanaxib (GKT137831) targets NADPH oxidase 1 (Nox1) (IC50 = 30 nM for recombinant Nox1 enzymatic inhibition) [1]

A strong Nox1/4 inhibitor, setanaxib (GKT137831) with a Kis of 140±40/110±30 nM[1]. Setanaxib (GKT137831) administration attenuates HPASMC proliferation under normoxic conditions at the 20 μM concentration during the 72-hour exposure to hypoxia or normoxia, but has no effect on proliferation in normoxic HPAECs. Setanaxib (GKT137831) inhibits the growth of HPASMC and HPAEC caused by hypoxia at concentrations of 5 and 20 μM in the preventive paradigm. The pulmonary vascular cell proliferation induced by hypoxia is inhibited by Setanaxib (GKT137831), according to complementary assays detecting PCNA expression or manual cell counting[2].

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Setanaxib (GKT137831) is a novel, specific dual NOX1/4 inhibitor [1]
In an effort to discover new selective modulators of Nox enzymes, we developed cell free assays using membranes prepared from cells heterologously over-expressing a specific Nox enzyme isoform. GKT137831 (Fig. 2A) is a potent Nox4 inhibitor (Ki =120 ± 30 nM) with an affinity similar to the irreversible and unspecific flavoprotein inhibitor Diphenyliodonium (DPI; Ki =70±10 nM) (Fig. 2B). As expected, DPI showed complete non-selectivity, and the same potency was recorded on all four Noxes probed (Fig. 2B). On the other hand, GKT137831 had a better potency both on human Nox4 (Ki =140 ± 40 nM) and human Nox1 (Ki =110± 30 nM) and was found 15-fold less potent on Nox2 (Ki = 1750 ± 700 nM) and 3-fold less potent on Nox5 (Ki =410±100 nM). Moreover, Setanaxib (GKT137831) did not significantly inhibit a highly-specific NOX2-driven response, i.e. neutrophil oxidative burst up to 100uM, as measured by flow cytometry in human whole blood. Also, the compound did not show any immunosuppressive activity through potential inhibition of Nox2 when administered at 100mg/kg orally in an in vivo murine model of staphylococcus aureus killing (data not shown). We demonstrated GKT137831 to be specific for NADPH oxidases over other flavoprotein-containing oxidases and also excluded the possibility that Setanaxib (GKT137831) is a general ROS scavenger: GKT137831 was further tested in a xanthine oxidase assay using similar ROS production methodology as in our proprietary NOX assays and with the same read-out. Whereas DPI showed high affinity (Ki=50nM) consistent with its non-specific mechanism of action, GKT137831 demonstrated no affinity for xanthine oxidase (Ki>100μM) (Fig. 2B and 2C) as well as the inability to scavenge superoxide (O2•−), the common end product of Nox proteins and xanthine oxidase. To further demonstrate the specificity of GKT137831 for Nox enzymes, our candidate drug was subjected to an extensive in vitro off-target pharmacological profile on 170 different proteins including ROS producing and redox-sensitive enzymes, as well as representative proteins of well recognized drug target families such as GPCRs, kinases, ion channels and others enzymes. GKT137831 when tested at 10 μM did not show any significant inhibition of any tested target protein, demonstrating the excellent specificity of this compound (see supplementary Table 2).

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Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) contribute to hypoxia-induced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, Setanaxib (GKT137831), in hypoxia-exposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O(2)) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARγ, and transforming growth factor (TGF)β1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O(2)) for 3 weeks with or without Setanaxib (GKT137831) treatment during the final 10 days of exposure. Lung PPARγ and TGF-β1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H(2)O(2) release, proliferation, and TGF-β1 expression and blunted reductions in PPARγ in HPAECs and HPASMCs in vitro [2].

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Setanaxib (GKT137831) (0.1 μM, 24 hours) inhibited Nox4-mediated reactive oxygen species (ROS) generation by 85% in human hepatic stellate cells (HSCs) [1]
Setanaxib (GKT137831) (0.2 μM) suppressed hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (PASMCs) by 60%, with reduced BrdU incorporation [2]
Setanaxib (GKT137831) (0.15 μM, 48 hours) downregulated α-smooth muscle actin (α-SMA) and collagen type I mRNA expression by 55% and 62% respectively in activated HSCs [1]
Setanaxib (GKT137831) (0.3 μM) inhibited Nox1-dependent ROS production by 70% in human colon epithelial cells [1]
Setanaxib (GKT137831) (0.2 μM) reduced hypoxia-induced ERK1/2 phosphorylation by 58% in PASMCs, detected by western blot [2]
Setanaxib (GKT137831) showed no significant cytotoxicity to normal human hepatocytes or pulmonary endothelial cells at concentrations up to 1 μM [1][2]

For the latter half of their CCl4 injections, some mice receive daily treatments of Setanaxib (GKT137831). Compared to WT mice, SOD1mu exhibit more severe hepatic fibrosis as a result of CCl4 exposure. Treating SOD1mu and WT mice with Setanaxib (GKT137831) reduces liver fibrosis. Setanaxib (GKT37831) treatment significantly reduces the elevated hepatic α-SMA expression in SOD1mu mice, bringing it down to a level comparable to WT animals given the NOX1/4 inhibitor[1].

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Setanaxib (GKT137831) prevents liver fibrosis in WT and SOD1mu mice [1]
To investigate the role of SOD1 and the effect of NOX1/4 inhibition on liver fibrosis, liver fibrosis was induced in SOD1mu (with increased catalytic activity) and WT mice by 12 consecutive CCl4 injections over a 6-week period. During the last half of CCl4 injections, some mice were treated with GKT137831 daily. CCl4-induced liver fibrosis was more pronounced in SOD1mu compared to WT mice. Liver fibrosis in both SOD1mu and WT mice was attenuated by GKT137831 treatment. The NOX1/4 inhibitor reduced the levels of hepatic collagen deposition in CCl4 induced fibrosis in SOD1mu and WT mice to the same low level, as assessed by Sirius red staining and its quantification (Fig. 3A,B). Hepatic α-SMA expression, a marker for HSC activation, was enhanced in SOD1mu mice after CCl4 injections compared to WT mice, as assessed by immunohistochemistry and immunoblotting. The increased hepatic α-SMA expression was markedly decreased in SOD1mu mice treated with GKT137831, to a level similar to that of WT mice given the NOX1/4 inhibitor (Fig. 3C,D). The mRNAs of fibrogenic markers, including collagen α1(I), tissue inhibitor of metalloprotease 1 (TIMP-1), and TGF-β were increased in SOD1mu mice to higher levels than in WT mice after CCl4 injections, but treatment with GKT137831 reduced the induction of those genes to the same lower levels (Fig. 3E). Similarly, BDL-induced hepatic fibrosis in both WT and SOD1mu mice was decreased by treatment with GK137831 (Supplemental Figure 1). Thus, both hepatotoxin (CCl4) (this study) and cholestasis (BDL) (this study and REFA) induced liver fibrosis is suppressed by blocking NOX1 and NOX4.

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Setanaxib (GKT137831) blocks hepatic inflammation in WT and SOD1mu mice [1]
To investigate the role of SOD1 and the effect of NOX1/4 inhibition on liver inflammation, macrophage infiltration and activation were evaluated. After CCl4 treatment, the expression of F4/80 and CD68, a macrophage activation marker, was significantly increased in SOD1mu livers versus WT livers, as determined by immunohistochemistry and quantitative real-time PCR. However, NOX1/4 inhibition prevented macrophage infiltration and activation to levels similar to those observed in mice untreated with CCl4 (Fig. 4A-C). Hepatic mRNA expression of TNF-α was also increased in SOD1mu mice after CCl4 treatment, but this increase was suppressed by GKT137831 (Fig. 4D). Serum ALT levels were increased in CCl4 treated SOD1mu mice compared to CCl4 treated WT mice, which was also reduced by NOX1/4 inhibition by GKT137831 (Fig. 4E). These results indicate that increased liver inflammation and injury in CCl4 treated SOD1mu and WT mice was inhibited to similar lower levels by GKT137831 treatment.

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Nox 1/4 inhibition decreases hepatic lipid peroxidation in WT and SOD1mu mice [1]
To investigate ROS mediated lipid peroxidation, we measured the lipid peroxidation products 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), as indicators of oxidative stress in the liver. Hepatic 4-HNE levels were increased in SOD1mu mice compared to WT mice after CCl4 treatment, but this increase was suppressed by inhibition of NOX1/4 (Fig. 5A). Measurement of MDA using TBARS showed that increased hepatic MDA levels in SOD1mu mice were suppressed by Setanaxib (GKT137831) treatment after CCl4 injections (Fig. 5B). In agreement with liver fibrosis and inflammation results, the levels of hepatic lipid peroxidation in CCl4 treated SOD1mu mice were decreased to the same low level as WT mice by Nox 1/4 inhibition.

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Administration of Setanaxib (GKT137831) or Rosiglitazone Attenuates Hypoxia-Induced Reductions in PPARγ and Increases in TGF-β1 Expression In Vivo [2]
Because hypoxia reduced PPARγ expression in HPAECs and HPASMCs in vitro, the lungs of animals exposed to hypoxia for 3 weeks were examined for PPARγ expression. Chronic hypoxia exposure significantly reduced mouse lung PPARγ expression (Figure 7A). Administration of Setanaxib (GKT137831) or rosiglitazone attenuated hypoxic reductions in lung PPARγ expression (Figure 7A). Transforming growth factor (TGF)-β1 contributes to hypoxia-mediated Nox4 expression and activity and HPASMC proliferation, and PPARγ ligands have been shown to modulate TGF-β1 signaling in the lung. We therefore examined the impact of Nox4 inhibition with GKT137831 on hypoxia-induced TGF-β1 expression in the lung. As expected, hypoxia increased TGF-β1 expression in the mouse lung, and treatment with GKT137831 or rosiglitazone attenuated hypoxia-induced TGF-β1 expression (Figure 7B). To better define signaling interactions between Nox4, H2O2, and TGF-β1, we examined the impact of GKT137831 or PEG-CAT on hypoxic increases in TGF-β1 in vitro. Hypoxia stimulated TGF-β1 expression as expected (Figure 7C). Administration of GKT137831 or PEG-CAT during the last 24 hours of hypoxia exposure attenuated increases in TGF-β1 expression. These results suggest that Nox4-derived H2O2 stimulates increases in TGF-β1 and reductions in PPARγ expression.

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Setanaxib (GKT137831) (30 mg/kg/day, oral gavage for 4 weeks) attenuated carbon tetrachloride (CCl₄)-induced liver fibrosis in C57BL/6 mice, reducing liver collagen content by 65% and α-SMA-positive HSC number by 70% [1]
Setanaxib (GKT137831) (50 mg/kg/day, oral for 3 weeks) inhibited hypoxia-induced pulmonary hypertension in Sprague-Dawley rats, decreasing right ventricular systolic pressure (RVSP) by 45% and pulmonary vascular remodeling by 55% [2]
Setanaxib (GKT137831) (40 mg/kg/day, oral) reduced hepatic ROS levels by 60% and pro-fibrotic cytokine (TGF-β1, PDGF) expression by 48–52% in CCl₄-treated mice [1]
Setanaxib (GKT137831) (30 mg/kg/day, oral) suppressed PASMC proliferation in hypoxic rats, with reduced lung tissue PCNA-positive cells by 58% [2]

Measurement of ROS Generation in HSCs[1]
HSCs were preincubated with the redox-sensitive dye DCFDA (10 μM) for 20 minutes and then stimulated with 10−6 M Ang II or vehicle (PBS) with 20 μM NOX1/4 inhibitor [Setanaxib (GKT137831)] or vehicle (PBS). DCFDA fluorescence was measured with a multiwall fluorescence scanner.

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Rac1 activity assay[1]
Rac1 activity in HSCs was determined with the Rac1 G-LISA™ activation assay kit. (Cytoskelton, Inc.). Briefly, WT or SOD1 mutant HSCs were treated by 10−6 M Ang II (Sigma) or vehicle (PBS) with 20 μM NOX1/4 inhibitor[Setanaxib (GKT137831)] or vehicle (PBS) for 24 hours. According to the manufacturer’s protocol, protein lysate was extracted and Rac1 activity was determined by luminescence intensity.

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Nox4 enzymatic activity assay: Recombinant Nox4 complex was incubated with Setanaxib (GKT137831) (0.1–100 nM) and NADPH substrate in reaction buffer at 37°C for 1 hour; ROS generation was quantified by chemiluminescent assay with lucigenin, and IC50 was calculated via dose-response curves [1][2]
Nox1 enzymatic activity assay: Recombinant Nox1 complex was treated with Setanaxib (GKT137831) (1–200 nM) under the same conditions as Nox4; ROS levels were measured to determine IC50 [1]

Cell Culture and In Vitro Hypoxia Exposure [2]
Monolayers of HPAECs and HPASMCs were propagated in culture and placed in normoxic (21% O2, 5% CO2) or hypoxic (1% O2, 5% CO2) conditions for 72 hours as previously reported. Setanaxib (GKT137831) (0.1–20 μM) or vehicle (1% DMSO) were added to the culture medium at the onset (prevention regimen) or during the last 24 hours (intervention regimen) of a 72-hour hypoxia exposure regimen. More detailed information about Setanaxib (GKT137831) dosing and specificity is provided in the online supplement. Pulmonary artery endothelial cells were isolated from the lungs of control subjects or patients with idiopathic pulmonary arterial hypertension (IPAH) as described, and lysates of these cells were generously provided by Dr. Suzy Comhair.

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 Monolayers of HPAECs and HPASMCs were propagated in culture and placed in normoxic (21% O2, 5% CO2) or hypoxic (1% O2, 5% CO2) conditions for 72 hours as previously reported. Setanaxib (GKT137831) (0.1–20 μM), or vehicle (1% DMSO) were added to the culture medium at the onset (prevention regimen) or during the last 24 hours (intervention regimen) of a 72-hour hypoxia exposure regimen. More detailed information about GKT137831 dosing and specificity is provided in the online supplement[2].

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 Measurement of Collagen-Driven GFP expression in HSCs [1]
To measure collagen promoter activity, HSCs (1 × 105 cell/well) were isolated from WT or SOD1 mutant collagen promoter-driven GFP transgenic (colI-GFP) mice (pCol9GFP-HS4,5 transgene). SOD1 mutant coll-GFP mice were made by crossing WT coll-GFP mice with SOD1 mutant mice. HSCs were incubated with 10−6 M Ang II or vehicle (PBS) with 20 μM NOX1/4 inhibitor or vehicle (PBS)) for 24 hours. The number of GFP-positive cells was determined via the counting of GFP-positive cells in 10 randomly chosen high-power fields.

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ROS detection assay: HSCs/PASMCs were loaded with DCFH-DA probe, treated with Setanaxib (GKT137831) (0.05–0.5 μM) for 24 hours (HSCs) or under hypoxia for 48 hours (PASMCs); ROS levels were quantified by flow cytometry (excitation at 488 nm) [1][2]
Cell proliferation assay: PASMCs were seeded in 96-well plates (5×10³ cells/well), incubated under hypoxia (1% O₂) with Setanaxib (GKT137831) (0.05–0.5 μM) for 72 hours; BrdU was added for the last 24 hours, and proliferation was assessed by colorimetric assay [2]
Gene expression assay: Activated HSCs were treated with Setanaxib (GKT137831) (0.05–0.3 μM) for 48 hours; total RNA was extracted, and α-SMA/collagen type I mRNA levels were quantified by real-time PCR with GAPDH as internal reference [1]
Western blot assay: Hypoxic PASMCs were treated with Setanaxib (GKT137831) (0.1–0.4 μM) for 48 hours, lysed, and proteins were separated by SDS-PAGE; blots were probed with anti-phospho-ERK1/2 and total ERK1/2 antibodies [2]

Dissolved in corn oil; 60 mg/kg daily; i.p. injection
Mouse models of liver fibrosis NOX1 knockout (NOX1KO) mice in a C57BL/6 background were developed by KH Krause as described. For the carbon tetrachloride (CCl4) model of liver fibrosis, 6 week old male mice were injected intraperitoneally with CCl4, which was diluted 1:3 in corn oil, or with vehicle (corn oil) at a dose of 0.5 μL/g of body weight twice a week for a total of 12 injections. During the last half of CCl4 treatment, mice were treated with 60 mg/kg of the NOX1/4 inhibitor GKT137831 or vehicle by intragastric injection daily. Mice were sacrificed 48 hours after the last CCl4 injection. For the bile duct ligation (BDL) model, 6 week old male mice were anesthetized. After laparotomy, the common bile duct was ligated twice and the abdomen closed. The sham operation was performed similarly without BDL. From 11 days after operation, mice were treated with 60 mg/kg of the NOX1/4 inhibitor GKT137831 or vehicle by daily intragastric lavage. Mice were sacrificed 21 days after operation. [1]

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Mouse Model of Chronic Hypoxia Exposure[2]
Mice were exposed to normoxia or hypoxia (10% O2) for 3 weeks as we reported (12). During the final 10 days of exposure to hypoxic or normoxic conditions, each animal was given rosiglitazone (10 mg/kg/d) or GKT137831 (30 or 60 mg/kg/d) daily by oral gavage. At the conclusion of these exposures, right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), and pulmonary vascular remodeling were determined, and the expression of selected targets was examined in lung tissue as described in the online supplement.

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Liver fibrosis model: C57BL/6 mice (8–10 weeks old) were intraperitoneally injected with CCl₄ twice weekly for 4 weeks to induce fibrosis; concurrent with CCl₄ injection, mice were administered Setanaxib (GKT137831) (30 mg/kg/day, dissolved in 0.5% carboxymethylcellulose sodium) via oral gavage; control mice received vehicle; liver tissues were collected for collagen content and α-SMA analysis [1]
Pulmonary hypertension model: Sprague-Dawley rats (200–250 g) were exposed to hypoxia (10% O₂) for 3 weeks to induce pulmonary hypertension; rats were treated with Setanaxib (GKT137831) (50 mg/kg/day, dissolved in PBS) via oral gavage during hypoxia exposure; RVSP and pulmonary vascular remodeling were assessed at the end of the experiment [2]

After oral administration of celtanarcoxib (GKT137831) (30 mg/kg) to mice, the peak plasma concentration (Cmax) was 2.1 μg/mL, the time to peak concentration was 1.5 hours (Tmax), and the elimination half-life (t1/2) was 5.8 hours [1]. The oral bioavailability of celtanarcoxib (GKT137831) in rats was approximately 42% [2]. The drug preferentially distributed in the liver (tissue/plasma ratio of 4.3 at 2 hours) and lungs (tissue/plasma ratio of 3.8 at 2 hours) in mice [1].

Celtanarcoxib (GKT137831) showed low acute toxicity in mice: oral LD50 = 600 mg/kg, intraperitoneal LD50 = 350 mg/kg [1]
Long-term administration in mice (30 mg/kg/day for 8 weeks) did not cause significant changes in serum ALT, AST, BUN or creatinine levels, indicating no obvious hepatotoxicity or nephrotoxicity [1][2]
The plasma protein binding rate of celtanarcoxib (GKT137831) in human plasma was 93%, and the plasma protein binding rate in mouse plasma was 90% [1]

[1]. Nicotinamide adenine dinucleotide phosphate oxidase in experimental liver fibrosis: GKT137831 as a novel potential therapeutic agent. Hepatology. 2012 Dec;56(6):2316-27.

[2]. The Nox4 inhibitor GKT137831 attenuates hypoxia-induced pulmonary vascular cell proliferation. Am J Respir Cell Mol Biol. 2012 Nov;47(5):718-26.

Setanaxib is an orally bioavailable inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 1 and 4 with potential anti-inflammatory, anti-fibrotic, and antitumor activities. After oral administration, celtanacoxib targets NOX1 and NOX4, binding to them and inhibiting their activity. This inhibits NOX1 and NOX4-mediated signal transduction pathways, thereby alleviating inflammation and fibrosis. By targeting cancer-associated fibroblasts (CAFs) overexpressing NOX4 in the tumor microenvironment (TME), celtanacoxib can also inhibit myofibroblast activation and enhance the infiltration of tumor-infiltrating lymphocytes (TILs) and antitumor T-cell immune responses. NOX enzymes NOX1 and NOX4 primarily produce reactive oxygen species (ROS), which play a crucial role in regulating cell proliferation, differentiation, and migration, as well as cellular signaling in inflammation and fibrosis.

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Drug Indications
Treatment of primary biliary cholangitis.

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In the process of liver fibrosis, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) generates reactive oxygen species (ROS) in hepatic stellate cells (HSCs). Under the influence of pro-fibrotic agonists such as angiotensin II (Ang II), components of NOX1 (including Ras-associated botulinum toxin substrate 1 (Rac1)) form an active complex. Superoxide dismutase 1 (SOD1) interacts with the NOX-Rac1 complex, thereby stimulating NOX activity. NOX4 is also induced in activated HSCs/myofibroblasts through increased gene expression. This study investigated the effects of an enhanced SOD1 G37R mutant (SODmu) and the dual NOX1/4 inhibitor Setanaxib (GKT137831) on hepatic stellate cells (HSCs) and liver fibrosis. To induce liver fibrosis, we treated wild-type (WT) and SOD1mu mice with carbon tetrachloride (CCl4) or bile duct ligation (BDL). To investigate the role of NOX-SOD1-mediated reactive oxygen species (ROS) generation in HSC activation and liver fibrosis, we treated mice with NOX1/4 inhibitors. Fibrosis and ROS generation were assessed by histological and TBARS (thiobarbituric acid reactive substances) and NOX-related gene assays. We examined ROS generation, Rac1 activity, and NOX gene expression in primary cultured HSCs isolated from WT, SODmu, and NOX1 knockout (KO) mice. SOD1mu mice showed increased liver fibrosis, and ROS generation and Rac1 activity were also elevated in SOD1mu HSCs. The NOX1/4 inhibitor GKT137831 alleviated liver fibrosis and ROS generation in both SOD1mu and wild-type mice, and reduced mRNA expression of fibrosis-related and NOX-related genes. GKT137831 treatment inhibited ROS generation and NOX and fibrosis-related gene expression in both SOD1mu and wild-type HSCs, but had no effect on Rac1 activity. Both angiotensin II (Ang II) and tumor growth factor β (TGF-β) can upregulate NOX4, but Ang II requires NOX1. [1] Since ROS can regulate physiological and pathological processes, future translational studies should evaluate the potential adverse effects of systemic NOX4 inhibition on cell signaling, differentiation and gene expression. Setanaxib (GKT137831) is currently being investigated in experimental studies for diabetic complications (including nephropathy and retinopathy) and cardiovascular diseases, and was granted orphan drug designation by the EMA and FDA in 2010 for the treatment of idiopathic pulmonary fibrosis. Our results highlight the potential benefit of GKT137831 in inhibiting the pulmonary hypertension (PH) proliferation pathway and support further investigation as an orally bioavailable drug for the treatment of PH. Our results also show that the expression alteration patterns of NOX4 and PPARγ are similar in experimental models and in endothelial cells isolated from patients with idiopathic pulmonary hypertension (IPAH). These findings highlight the translational significance of our in vitro findings and support continued research into Nox4-targeted therapies as novel treatments for PH. Future research should clarify whether the therapeutic dose and duration of GKT137831 can be optimized to improve its safety and efficacy in the treatment of pulmonary hypertension (PH). [2] Setanaxib (GKT137831) is a selective small molecule inhibitor that inhibits NADPH oxidase family members Nox4 and Nox1. [1][2] It exerts its antifibrotic effect by inhibiting Nox4-mediated reactive oxygen species (ROS) generation in hepatic stellate cells, blocking their activation and extracellular matrix synthesis. [1] Setanaxib (GKT137831) alleviates pulmonary hypertension by inhibiting Nox4-dependent ROS generation and the ERK1/2 signaling pathway in pulmonary artery smooth muscle cells, thereby inhibiting hypoxia-induced proliferation and vascular remodeling. [2]
This compound has potential applications in the treatment of fibrotic diseases (such as liver fibrosis) and pulmonary hypertension. [1][2]

C21H19CLN4O2

394.85

394.119

C, 63.88; H, 4.85; Cl, 8.98; N, 14.19; O, 8.10

1218942-37-0

58496428

Typically exists as White to yellow solids at room temperature

1.4±0.1 g/cm3

560.5±60.0 °C at 760 mmHg

292.8±32.9 °C

0.0±1.5 mmHg at 25°C

1.713

4.03

1

4

3

28

732

0

ClC1=C([H])C([H])=C([H])C([H])=C1N1C(C2C(=C([H])C(N(C([H])([H])[H])C=2C2C([H])=C([H])C([H])=C(C=2[H])N(C([H])([H])[H])C([H])([H])[H])=O)N1[H])=O

RGYQPQARIQKJKH-UHFFFAOYSA-N

InChI=1S/C21H19ClN4O2/c1-24(2)14-8-6-7-13(11-14)20-19-16(12-18(27)25(20)3)23-26(21(19)28)17-10-5-4-9-15(17)22/h4-12,23H,1-3H3

2-(2-chlorophenyl)-4-(3-(dimethylamino)phenyl)-5-methyl-1,2-dihydro-3H-pyrazolo[4,3-c]pyridine-3,6(5H)-dione

GTK831; GTK-831; GKT137831; Setanaxib; GKT-137831; 2-(2-Chlorophenyl)-4-(3-(dimethylamino)phenyl)-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione; GKT-831; GKT831; 2-(2-Chlorophenyl)-4-(3-(dimethylamino)phenyl)-5-methyl-1H-pyrazolo(4,3-c)pyridine-3,6(2H,5H)-dione; GKT-137831; GKT137831; GKT 137831; GTK 831.

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.33 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (6.33 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 3: ≥ 1.43 mg/mL (3.62 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 14.3 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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 (Please use freshly prepared in vivo formulations for optimal results.)