HIF/hypoxia-inducible factor prolyl hydroxylase (EC50 = 0.22 μM)

Enarodustat (JTZ-951) has an EC50 of 0.22 μM, making it a strong oral active inhibitor of hypoxia-inducible factor prolyl hydroxylase. Enarodustat has no effect on hERG (IC50 > 100 μM) or CYP (IC50 > 100 μM; CYP3A4/5, CYP2C9, CYP2D6, CYP1A2, CYP2A6, CYP2C19, CYP2C8, CYP2B6) [1].

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Some preceding studies have provided evidence that hypoxia-inducible factor (HIF)-prolyl hydroxylase (PH) inhibitors have therapeutic potential against tubular interstitial fibrosis (TIF). Recently, transformation of renal interstitial fibroblasts (RIFs) into α-smooth muscle actin-positive myofibroblasts with loss of their hypoxia-inducible erythropoietin (EPO) expression has been hypothesized as the central mechanism responsible for TIF with renal anemia (the RIF hypothesis). These reports have suggested that HIF-PH inhibitors may suppress TIF via suppressing transformation of RIFs. However, the direct effect of HIF-PH inhibitors on transformation of RIFs has not been demonstrated because there has been no appropriate assay system. Here, we established a novel in vitro model of the transformation of RIFs. This model expresses key phenotypic changes such as transformation of RIFs accompanied by loss of their hypoxia-inducible EPO expression, as proposed by the RIF hypothesis. Using this model, we demonstrated that Enarodustat (JTZ-951), a newly developed HIF-PH inhibitor, stabilized HIF protein in RIFs, suppressed transformation of RIFs, and maintained their hypoxia-inducible EPO expression. Enarodustat (JTZ-951) also suppressed the expression of FGF2, FGF7, and FGF18, which are upregulated during transformation of RIFs. Furthermore, expression of Fgf2, Fgf7, and Fgf18 was correlated with TIF in an animal model of TIF. We also demonstrated that not only FGF2, which is a well-known growth-promoting factor, but also FGF18 promoted proliferation of RIFs. These data suggest that Enarodustat (JTZ-951) has therapeutic potential against TIF with renal anemia. Furthermore, FGF2, FGF7, and FGF18, which faithfully reflect the anti-TIF effects of Enarodustat (JTZ-951), have potential as TIF biomarkers [3].

Oral administration of enarodustat (1 and 3 mg/kg, p.o.) to rats on a daily basis raises hemoglobin levels in a dose-dependent way [1].

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Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, also known as HIF stabilizers, increase endogenous erythropoietin production and serve as novel therapeutic agents against anemia in chronic kidney disease. HIF induces the expression of various genes related to energy metabolism as an adaptive response to hypoxia. However, it remains obscure how the metabolic reprogramming in renal tissue by HIF stabilization affects the pathophysiology of kidney diseases. Previous studies suggest that systemic metabolic disorders such as hyperglycemia and dyslipidemia cause alterations of renal metabolism, leading to renal dysfunction including diabetic kidney disease. Here, we analyze the effects of Enarodustat (JTZ-951), an oral HIF stabilizer, on renal energy metabolism in the early stages of diabetic kidney disease, using streptozotocin-induced diabetic rats and alloxan-induced diabetic mice. Transcriptome analysis revealed that Enarodustat (JTZ-951) counteracts the alterations in diabetic renal metabolism. Transcriptome analysis showed that fatty acid and amino acid metabolisms were upregulated in diabetic renal tissue and downregulated by Enarodustat (JTZ-951), whereas glucose metabolism was upregulated. These symmetric changes were confirmed by metabolome analysis. Whereas glycolysis and tricarboxylic acid cycle metabolites were accumulated and amino acids reduced in renal tissue of diabetic animals, these metabolic disturbances were mitigated by Enarodustat (JTZ-951). Furthermore, Enarodustat (JTZ-951) increased the glutathione to glutathione disulfide ratio and relieved oxidative stress in renal tissue of diabetic animals. Thus, HIF stabilization counteracts alterations in renal energy metabolism occurring in incipient diabetic kidney disease[2].

Enzyme assay [1]
Recombinant proteins of human HIF-PHD2 and VBC complex (a complex of human von Hippel-Lindau protein with a GST-tag, human Elongin B with a Flag-tag and human Elongin C with a His-tag) were prepared. The enzyme reaction was performed at room temperature for 10 min with 1 nM human HIF-PHD2, 2 µM 2-oxoglutarate, 30 nM HIF-1α peptide (biotin-DLDLEMLAPYIPMDDDFQL), 0.5 mM ascorbic acid, 0.25 mM FeSO4, 120 mM NaCl, 0.2 mM 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 0.1% bovine serum albumin, 50 mM tris-HCl (pH 7.5) and test compound (1% DMSO); an EDTA solution was added to stop the enzyme reaction. Then the potassium fluoride solution containing human VBC complex, anti-GST-cryptate and streptavidin-XLent! FG-4592 N OH H N O OH O O S37 were added. The fluorescence intensity was measured at 620 nm for the energy donor excited at a wavelength of 320 nm and at 665 nm for the luminescent reagent using an HTRF® microplate reader to calculate the fluorescence intensity ratio.

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Metabolic stability in liver microsomes [1]
14C-Enarodustat (JTZ-951) (final concentration: 10 µmol/L) was incubated in the presence of pooled liver microsomes (protein concentration: 1 mg protein/mL) prepared from male rats (SD rats: 400 animals), male dogs (beagles: eight animals), male monkeys (cynomolgus monkeys: 10 animals) and male and female humans (25 males and 25 females) at 37 °C for two hours in the presence of nicotinamide adenine dinucleotide phosphate (NADPH). After the reaction, the amounts of Enarodustat (JTZ-951) in the samples were determined by Radio-HPLC.

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CYP inhibition assay [1]
Enarodustat (JTZ-951) (final concentrations: 0, 1, 3, 10, 30, and 100 µmol/L) and the model substrates for each CYP isoform (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 [testosterone and midazolam]) were incubated with human liver microsomes prepared from male and female humans (male: 31 subjects, female: 19 subjects) at 37 °C for the designated time in the presence of NADPH. After incubation, the metabolites of the model substrates were analyzed by LC/MS/MS and the metabolic rates were calculated to evaluate the inhibitory potential of Enarodustat (JTZ-951).

EPO production in Hep3B cells [1]
Human Hep3B cells were purchased from American Type Culture Collection and cultured in Eagle-MEM containing 10% fetal bovine serum, 100 units/mL penicillin and 100 µg/mL streptomycin in a CO2 incubator (37 °C, 5% CO2). These cells were inoculated into 96-well flat-bottomed plates and on the next day, each test compound was added at appropriate concentrations for the assessment of EPO production. The culture supernatants were collected at 24 hours after the addition of each of the test compounds. A hypoxic condition was established and the EPO concentration of this condition was defined as 100% when the EC50 was calculated. The EPO concentration in culture supernatants was measured by human EPO ELISA kit.

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In vitro Caco-2 Permeability study [1]
A sample of the test compound (final concentrations: 25 µmol/L) was added to the apical side of Caco-2 cell monolayers, and incubated at 37 °C for 2 h. After incubation, the transported amounts of test compound were measured by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Apparent permeability coefficients (Papp) were calculated from the transported amounts.

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hERG inhibition assay [1]
The hERG current was measured by the whole cell patch clamp method. hERG-transfected HEK293 cells were cultured in MEM solution containing 10% fetal bovine serum, 1 mmol/L MEM sodium pyruvate solution, 0.1 mmol/L MEM non-essential amino acid solution, 100 U/mL penicillin, 100 µg/mL streptomycin, and 400 µg/mL geneticin. Cells on the cover slip were set in the measurement chamber and the chamber was superfused with the external solution containing (in mM): 137 NaCl, 4 KCl, 1 MgCl2·6H2O, 1.8 CaCl2·2H2O, 10 HEPES and 10 glucose (pH 7.4), maintained at 24 ± 2 °C. The hERG current was measured with a glass electrode (resistance: 2 to 6 MΩ) filled with the internal solution containing (in mM): 130 KCl, 1 MgCl2·6H2O, 5 EGTA, 10 HEPES and 5 MgATP (pH 7.2), through a patch clamp amplifier). The cell membrane voltage was held at –80 mV by the patch clamp software with the amplifier. A test pulse consisting of +20 mV for 1.5 seconds and –40 mV for 1.5 seconds was applied with intervals of 15 seconds. The currents before and 11 minutes after initiation of the treatment with the vehicle and test article were analyzed.

EPO production in normal mouse and rat [1]
Male balb/c mice and CD (SD) rats were orally administered a single dose of 10 mg/kg [0.5% methyl cellulose (MC) suspension] of each of the test compounds (Enarodustat (JTZ-951)), and eight hours after the administration, the plasma samples were collected. The murine and rat plasma EPO concentrations were measured by ELISA kit or RIA kit, respectively. Erythropoiesis-stimulating effect in normal rat [1]
The vehicle solution (0.5% MC) or test compound (Enarodustat (JTZ-951)) suspension at appropriate doses were administered orally to the male CD (SD) rats once daily for 28 days. Blood were collected from each rat to measure the hemoglobin concentrations using a hematology analyzer.

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Rat PK (IV, PO) [1]
Male CD(SD) rats were intravenously or orally administered a single dose of Enarodustat (JTZ-951) at 0.3 mg/kg (60% dimethylsulfoxide solution) or 1.0 mg/kg (0.5% methylcellulose), respectively. After the administration, the plasma samples were collected over a period of 24 h. The time-course of the plasma concentrations of Enarodustat (JTZ-951) was analyzed by non-compartmental analysis and the pharmacokinetic parameters were calculated.

In terms of pharmacokinetic (PK) profiles, compound 14/Enarodustat (JTZ-951) was rapidly absorbed after oral administration in rats and disappeared shortly thereafter (Figure 3a,b). As Vachal and others have mentioned, the short-acting characteristics could be beneficial in reducing unpredictable adverse effects in light of the HIF-PHD mechanism. Compound 14/Enarodustat (JTZ-951) also had excellent solubility and metabolic stability (Figure 3c). In addition, it showed neither CYP (IC50 > 100 μM; CYP3A4/5, CYP2C9, CYP2D6, CYP1A2, CYP2A6, CYP2C19, CYP2C8, CYP2B6) nor hERG (IC50 > 100 μM) inhibition. Having these results, compound 14/Enarodustat (JTZ-951) was selected for a clinical candidate. [1]

[1]. Discovery of JTZ-951: A HIF Prolyl Hydroxylase Inhibitor for the Treatment of Renal Anemia. ACS Med Chem Lett. 2017 Nov 20;8(12):1320-1325.

[2]. The oral hypoxia-inducible factor prolyl hydroxylase inhibitor enarodustat counteracts alterations in renal energy metabolism in the early stages of diabetic kidney disease. Kidney Int. 2020 May;97(5):934-950.

[3]. JTZ-951, an HIF prolyl hydroxylase inhibitor, suppresses renal interstitial fibroblast transformation and expression of fibrosis-related factors. Am J Physiol Renal Physiol. 2020 Jan 1;318(1):F14-F24.

Enarodustat is under investigation in clinical trial NCT02581124 (Study to Evaluate Effect of Lapatinib on Pharmacokinetics of JTZ-951 in Subjects With End-stage Renal Disease).

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Inhibition of hypoxia inducible factor prolyl hydroxylase (PHD) represents a promising strategy for the discovery of a next generation treatment for renal anemia. Researchers identified several 5,6-fused ring systems as novel scaffolds of the PHD inhibitor on the basis of pharmacophore analysis. In particular, triazolopyridine derivatives showed potent PHD2 inhibitory activities. Examination of the predominance of the triazolopyridines in potency by electrostatic calculations suggested favorable π-π stacking interactions with Tyr310. Lead optimization to improve the efficacy of erythropoietin release in cells and in vivo by improving cell permeability led to the discovery of Enarodustat (JTZ-951) (compound 14), with a 5-phenethyl substituent on the triazolopyridine group, which increased hemoglobin levels with daily oral dosing in rats. Compound 14 was rapidly absorbed after oral administration and disappeared shortly thereafter, which could be advantageous in terms of safety. Compound 14 was selected as a clinical candidate.[1]

C17H16N4O4

340.333343505859

340.117

C, 60.00; H, 4.74; N, 16.46; O, 18.80

1262132-81-9

Enarodustat hydrochloride;1262131-60-1; 262132-81-9; 1262133-55-0 (disodium)

50899324

White to off-white solid powder

1.5±0.1 g/cm3

1.690

1.6

3

6

6

25

674

0

O=C(O)CNC(C1=C(O)C=C(CCC2=CC=CC=C2)N3C1=NC=N3)=O

NALAUGMPMIVAOW-UHFFFAOYSA-N

InChI=1S/C17H16N4O4/c22-13-8-12(7-6-11-4-2-1-3-5-11)21-16(19-10-20-21)15(13)17(25)18-9-14(23)24/h1-5,8,10,22H,6-7,9H2,(H,18,25)(H,23,24)

N-[7-hydroxy-5-(2-phenylethyl)[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl]glycine

JTZ-951; Enaroy; JTZ 951; Enarodustat; 1262132-81-9; Enarodustat [INN]; Enarodustat (JTZ-951); JSK7TUA223; 2-[[7-oxo-5-(2-phenylethyl)-3H-[1,2,4]triazolo[1,5-a]pyridine-8-carbonyl]amino]acetic acid; JTZ951

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 : ~83.33 mg/mL (~244.85 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.11 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 20.8 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: ≥ 2.08 mg/mL (6.11 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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: ≥ 2.08 mg/mL (6.11 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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