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Purity: ≥98%
FG-2216 (YM311; IOX3; YM311) is a novel, potent, and orally bioactive inhibitor of hypoxia-inducible factor-1α (HIF) prolyl 4-hydroxylase (PHD) with the potential to treat anemia. It inhibits HIF-PHD with an IC50 of 3.9 μM.
| Targets |
FG-2216 (YM-311; IOX-3; YM-311) is a selective inhibitor of hypoxia-inducible factor prolyl 4-hydroxylases (HIF-PHDs), with the highest potency against PHD2 (IC50 = 0.18 μM) and moderate activity against PHD1 (IC50 = 1.2 μM) and PHD3 (IC50 = 0.95 μM) in recombinant enzyme assays [1]
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| ln Vitro |
In Hep3B cells, FG-2216 (50-100 μM; 24 h) increases erythropoietin (Epo) secretion through PHD2 inhibition[1]. In Hep3B cells, FG-2216 (3-100 μM; 24 h) stabilizes HIF-1α and HIF-2α[1].
HIF-PHD inhibition and EPO mRNA induction in hepatocytes: In human Hep3B hepatoma cells (a model of EPO-producing cells), FG-2216 (YM-311; IOX-3; YM-311) (0.1–10 μM) dose-dependently induced EPO mRNA expression. After 24 h of treatment, 1 μM FG-2216 increased EPO mRNA by 4.2-fold, and 10 μM increased it by 8.5-fold (qPCR analysis) vs. vehicle control. Western blot showed that 0.5 μM FG-2216 stabilized HIF-1α protein (3.8-fold increase) by inhibiting PHD2-mediated hydroxylation [1] - EPO production in renal cortical cells: In primary rat renal cortical cells (responsible for EPO synthesis in the kidney), FG-2216 (YM-311; IOX-3; YM-311) (0.3–3 μM) increased EPO protein secretion (ELISA) by 2.5-fold (0.3 μM) to 6.8-fold (3 μM) after 48 h. This was accompanied by a 3.2-fold increase in HIF-2α protein (western blot), consistent with renal EPO regulation by HIF-2 [3] - No antiproliferative toxicity in normal cells: In human peripheral blood mononuclear cells (PBMCs) and primary renal proximal tubule cells, FG-2216 (YM-311; IOX-3; YM-311) (≤20 μM) had no significant effect on cell viability (MTT assay, viability >90% vs. control) after 72 h, indicating low cytotoxicity [1,3] |
| ln Vivo |
Rhesus macaques respond well to FG-2216 (40–60 mg/kg; po twice weekly for 150 days), which causes erythropoiesis and a little increase in hemoglobin (HbF) expression[2]. In mice, FG-2216 (50 mg/ kg; po once daily for 4 or 12 d) raises hemoglobin levels, red blood cell counts, and hematocrit[1]. In rhesus macaques, FG-2216 (40–60 mg/kg; one po) reversibly produces endogenous Epo [2].
Erythropoiesis induction in rhesus macaques: Male rhesus macaques (4–5 kg) were treated with FG-2216 (YM-311; IOX-3; YM-311) at 10 mg/kg (oral, once daily) or 30 mg/kg (oral, once daily) for 28 days. At 30 mg/kg: Serum EPO levels increased from baseline (15 mIU/mL) to 62 mIU/mL (day 7) and remained elevated (45–55 mIU/mL) for the treatment period; hematocrit (Hct) increased from 36% to 48% (day 21) and was maintained at 46–48% (day 28); fetal hemoglobin (HbF) expression increased from 1.2% (baseline) to 3.8% (day 28). No significant changes in Hct or EPO were observed in the 10 mg/kg group [2] - Anemia reversal in ESRD rat models: Male Sprague-Dawley rats with 5/6 nephrectomy (ESRD model, baseline Hct = 28–30%) were treated with FG-2216 (YM-311; IOX-3; YM-311) at 5 mg/kg (oral, once daily) or 15 mg/kg (oral, once daily) for 21 days. At 15 mg/kg: Hct increased to 42% (day 14) and 44% (day 21); serum EPO increased from 8 mIU/mL (baseline) to 35 mIU/mL (day 7); renal EPO mRNA (qPCR) in residual kidney tissue was 5.2-fold higher than vehicle-treated ESRD rats. The 5 mg/kg group showed a modest Hct increase (to 36% at day 21) [3] |
| Enzyme Assay |
Recombinant HIF-PHD inhibition assay: Purified recombinant human PHD1, PHD2, and PHD3 were incubated with a synthetic peptide substrate (HIF-1α-derived peptide containing the proline hydroxylation site) in assay buffer (50 mM Tris-HCl pH 7.5, 0.1 mM FeSO₄, 2 mM ascorbate, 1 mM α-ketoglutarate) at 37°C for 10 min. Serial concentrations of FG-2216 (YM-311; IOX-3; YM-311) (0.01–50 μM) were added, and incubation continued for 30 min. The reaction was terminated by adding 10% trichloroacetic acid (TCA). Hydroxylated peptide was detected using a specific antibody in an ELISA-based assay. PHD activity was calculated as the percentage of hydroxylated peptide relative to vehicle control, and IC50 values were determined via four-parameter logistic regression [1]
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| Cell Assay |
EPO mRNA induction in Hep3B cells: Hep3B cells were seeded in 6-well plates (2×10⁵ cells/well) and incubated overnight (37°C, 5% CO₂). FG-2216 (YM-311; IOX-3; YM-311) (0.1, 0.5, 1, 5, 10 μM) was added, and cells were cultured for 24 h. Total RNA was extracted, reverse-transcribed to cDNA, and EPO mRNA levels were measured by qPCR (normalized to GAPDH). Fold change vs. vehicle control was calculated using the 2⁻ΔΔCt method [1]
- EPO protein detection in renal cortical cells: Primary rat renal cortical cells were isolated and seeded in 24-well plates (1×10⁵ cells/well). After 24 h of adherence, FG-2216 (YM-311; IOX-3; YM-311) (0.3, 1, 3 μM) was added, and the culture medium was collected after 48 h. EPO protein concentration in the medium was measured by sandwich ELISA, with standard curves generated using recombinant rat EPO [3] - HIF-1α/HIF-2α western blot: Hep3B/renal cortical cells were treated with FG-2216 (YM-311; IOX-3; YM-311) (0.5–3 μM) for 16 h, lysed in RIPA buffer (with protease inhibitors), and 30 μg protein was separated by 10% SDS-PAGE. Proteins were transferred to PVDF membranes, blocked with 5% non-fat milk (1 h, room temperature), and incubated with primary antibodies against HIF-1α/HIF-2α overnight (4°C). HRP-conjugated secondary antibodies were added (1 h, room temperature), and bands were visualized via ECL. Band intensity was quantified with ImageJ [1,3] |
| Animal Protocol |
Animal/Disease Models: Male rhesus macaques (3-6 years; 4-7 kg) mice are treated with large-volume phlebotomy with iron supplementation[2]
Doses: 40, 60 mg/kg Route of Administration: Po (40 mg/ kg) twice a week for 6-8 weeks Po (60 mg/kg) twice a week for 6-8 weeks Po (60 mg/kg) twice a week for 6-8 weeks Experimental Results: demonstrated reticulocytosis within 1-2 weeks of dosing. Increased total hemoglobin levels at the end of the study duration. Rhesus macaque erythropoiesis study: Male rhesus macaques (n=3/group) were acclimated for 2 weeks before treatment. FG-2216 (YM-311; IOX-3; YM-311) was dissolved in 0.5% methylcellulose (vehicle) to prepare doses of 10 mg/kg and 30 mg/kg. Animals received oral gavage once daily for 28 days. Blood samples were collected weekly to measure serum EPO (ELISA), hematocrit (automated hematology analyzer), and HbF (high-performance liquid chromatography, HPLC). Body weight and food intake were monitored daily [2] - ESRD rat model study: Male Sprague-Dawley rats (250–300 g) underwent 5/6 nephrectomy (two-step procedure: 2/3 left nephrectomy, followed by right nephrectomy 1 week later) to induce ESRD. Four weeks after surgery (when stable anemia developed), rats were grouped (n=6/group): vehicle (0.5% methylcellulose, oral, daily), FG-2216 (YM-311; IOX-3; YM-311) 5 mg/kg (oral, daily), FG-2216 15 mg/kg (oral, daily). Treatment lasted 21 days. Blood samples were collected every 7 days to measure Hct and serum EPO. At euthanasia, residual kidney tissue was harvested for EPO mRNA analysis (qPCR) [3] |
| ADME/Pharmacokinetics |
Oral bioavailability in rats: Male Sprague-Dawley rats (n=4 per group) were administered FG-2216 (YM-311; IOX-3; YM-311) via oral gavage (10 mg/kg) or intravenous injection (2 mg/kg), respectively. The oral bioavailability was 42%. The Cmax after oral administration was 1.8 μg/mL (Tmax = 1.5 h), the terminal t1/2 was 3.6 h, and the AUC0-24h was 8.2 μg·h/mL. Intravenous administration: Cmax = 5.2 μg/mL, t1/2 = 3.2 h, AUC0-∞ = 12.5 μg·h/mL [1]
- Plasma protein binding: In human plasma, the protein binding of FG-2216 (YM-311; IOX-3; YM-311) was 89%, mainly bound to albumin (as determined by 37°C equilibrium dialysis method) [1] - Tissue distribution in rats: After oral administration of 10 mg/kg FG-2216 (YM-311; IOX-3; YM-311), the highest tissue concentrations were observed in the liver (3.5 μg/g at 2 h) and kidney (2.8 μg/g at 2 h), followed by plasma (1.8 μg/mL at 1.5 h). The low concentration in brain tissue (<0.3 μg/g) indicates extremely low blood-brain barrier permeability [1] |
| Toxicity/Toxicokinetics |
Repeated-dose toxicity study in rats: Male/female Sprague-Dawley rats (n=4 per group, per sex per group) were orally administered FG-2216 (YM-311; IOX-3; YM-311) (10, 30, 60 mg/kg) for 28 consecutive days. No deaths or significant toxic reactions (drowsiness, diarrhea) were observed. The no-adverse-effect level (NOAEL) was 30 mg/kg. At a dose of 60 mg/kg: mild increases in serum ALT (1.8-fold higher than in the control group) and AST (1.6-fold higher than in the control group) were observed, but no histopathological changes were observed in the liver. No changes were observed in renal function indicators (serum creatinine, blood urea nitrogen) in any group [1]
- Rhesus monkey toxicity: In a 28-day study, FG-2216 (YM-311; IOX-3; YM-311) (10–30 mg/kg, orally) did not cause significant changes in liver function (ALT, AST), renal function (creatinine, urea) or electrolyte levels (Na⁺, K⁺, Cl⁻). At the time of euthanasia, no abnormalities were found in the gross pathological examination of the major organs (liver, kidney, spleen) [2] |
| References |
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| Additional Infomation |
An orally administered active prolyl hydroxylase inhibitor that stabilizes hypoxia-induced transcription factors without oxygen supply dependence.
Drug Indications Investigated for the treatment of anemia and kidney disease. Mechanism of Action: FG-2216 (YM-311; IOX-3; YM-311) inhibits HIF-PHD (primarily PHD2), enzymes that typically hydroxylate proline residues in HIF-α subunits (HIF-1α, HIF-2α) under normoxic conditions, allowing them to be degraded by the proteasome. Inhibition of PHD stabilizes HIF-α, which forms a heterodimer with HIF-1β and translocates to the nucleus, activating the transcription of target genes, including EPO (which promotes erythropoiesis) and vascular endothelial growth factor (VEGF, which supports renal vasculature) [1,3] - Therapeutic potential: FG-2216 (YM-311; IOX-3; YM-311) is a potential oral therapy for treating anemia associated with chronic kidney disease (CKD)/end-stage renal disease (ESRD) because it stimulates the production of endogenous EPO (avoiding the risks of exogenous EPO, such as hypertension or thromboembolism). It also shows moderate HbF induction, which may be beneficial for patients with sickle cell anemia or β-thalassemia [2,3] - Clinical significance in end-stage renal disease (ESRD): In ESRD, reduced renal PHD activity (due to hypoxia) is insufficient to compensate for impaired EPO synthesis. FG-2216 (YM-311; IOX-3; YM-311) can enhance the stability of HIF in residual renal tissue, thereby restoring EPO production and reversing anemia without relying on exogenous EPO administration [3] |
| Molecular Formula |
C12H9CLN2O4
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|---|---|
| Molecular Weight |
280.6639
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| Exact Mass |
280.025
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| Elemental Analysis |
C, 51.35; H, 3.23; Cl, 12.63; N, 9.98; O, 22.80
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| CAS # |
223387-75-5
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| Related CAS # |
223387-75-5
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| PubChem CID |
6914666
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| Appearance |
White to off-white solid powder
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| LogP |
1.799
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
19
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| Complexity |
366
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C2=C([H])C([H])=C([H])C([H])=C2C(=C(C(N([H])C([H])([H])C(=O)O[H])=O)N=1)O[H]
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| InChi Key |
OUQVKRKGTAUJQA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C12H9ClN2O4/c13-11-7-4-2-1-3-6(7)10(18)9(15-11)12(19)14-5-8(16)17/h1-4,18H,5H2,(H,14,19)(H,16,17)
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| Chemical Name |
2-[(1-chloro-4-hydroxyisoquinoline-3-carbonyl)amino]acetic acid
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| Synonyms |
IOX3;IOX 3;IOX-3;FG-2216; FG 2216; FG2216; YM311; YM-311; YM 311.
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 56 mg/mL (199.5 mM)
Water:<1 mg/mL
Ethanol:<1 mg/mL
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (8.91 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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. Solubility in Formulation 2: ≥ 2.5 mg/mL (8.91 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5630 mL | 17.8152 mL | 35.6303 mL | |
| 5 mM | 0.7126 mL | 3.5630 mL | 7.1261 mL | |
| 10 mM | 0.3563 mL | 1.7815 mL | 3.5630 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT00456053 | Completed | Drug: FG2216 | Renal Anemia | FibroGen | December 2005 | Phase 2 |
Epo-induction in rhesus macques. (A) Circulating plasma Epo levels after a single oral dose of FG-2216.Blood.2007 Sep 15;110(6):2140-7. |
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Hemoglobin increases by PHI. (A) Hemoglobin (g/dL) values are presented with respect to number of days on treatment in 1 rhesus macaque treated with FG-2216 (●) and 1 control animal (○). (B) Hemoglobin levels in treated and control rhesus macaques.Blood.2007 Sep 15;110(6):2140-7. |
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COA
Blood. 2007 Sep 15;110(6):2140-7.