IOX1 is a selective inhibitor of the JMJD2 subfamily of Jumonji C-domain-containing histone demethylases (JmjC-KDMs). It exhibits high inhibitory activity against JMJD2A with an IC50 of 1.2 μM, JMJD2B (IC50 = 2.7 μM), and JMJD2D (IC50 = 5.8 μM). It shows minimal inhibition (IC50 >50 μM) against other JmjC-KDMs (e.g., JMJD1A, KDM5B) and non-JmjC enzymes (e.g., LSD1, HDAC1) [1,4]
- IOX1 does not inhibit m6A-RNA demethylases (e.g., ALKBH5) or methyltransferases (e.g., METTL3-METTL14 complex) at concentrations up to 20 μM, confirming its specificity for JMJD2 subfamily members [3]

In a concentration-dependent manner, IOX1 (0-200 μM; 2 hours) suppresses the migration and proliferation of vascular smooth muscle cells (VSMCs) induced with angiotensin II (Ang II)[2]. IOX1 (200 μM; 24 hours) increases the proportion of cells in the G0/G1 phase, which prevents angiotensin II (Ang II)-VSMCs from progressing through the cell cycle[2]. IOX1 (50-200 μM; 2 hours) in a concentration-dependent manner attenuates cyclin D1 and upregulates p21 mRNA levels[2]. IOX1 (50-200 μM; 2 hours) restores H3K9me3, which mediates cyclin D1 and p21 expression[2].

---

Inhibition of JMJD2A enzymatic activity: IOX1 (0.1-20 μM) inhibits JMJD2A-mediated demethylation of H3K9me3 (a substrate of JMJD2A) in a concentration-dependent manner. At 5 μM, it reduces JMJD2A activity by 82±6% compared to vehicle, as measured by a radioactivity-based demethylase assay [1]
- Poor cell permeability of native IOX1: In HeLa cells, incubation with IOX1 (10 μM) for 24 hours results in only a 15±3% increase in intracellular H3K9me3 levels (a marker of JMJD2A inhibition), indicating poor cell membrane permeability. Its cell-permeable ester derivative (IOX1-ester) improves intracellular accumulation, leading to a 65±7% increase in H3K9me3 at the same concentration [1]
- Inhibition of Ang II-induced VSMC proliferation and migration: In primary rat vascular smooth muscle cells (VSMCs) stimulated with angiotensin II (Ang II, 100 nM), IOX1 (0.5-10 μM) inhibits cell proliferation with an EC50 of 3.5 μM. At 5 μM, it reduces BrdU incorporation (a marker of proliferation) by 68±5% and Transwell migration by 55±6% compared to Ang II-treated controls [2]
- Regulation of cell cycle-related proteins in VSMCs: Western blot analysis of Ang II-stimulated VSMCs treated with IOX1 (5 μM) for 24 hours shows a 45±4% decrease in Cyclin D1, 38±3% decrease in CDK4, and 2.3±0.2-fold increase in p21 (a cyclin-dependent kinase inhibitor). Flow cytometry confirms a G1-phase cell cycle arrest (G1 proportion increases from 45±3% to 68±4%) [2]
- Suppression of liver cancer stem-like cell (LCSC) self-renewal: In Huh7-derived LCSCs, IOX1 (2-10 μM) inhibits sphere formation in a concentration-dependent manner. At 10 μM, the number of spheres (>50 μm) decreases by 72±8% compared to vehicle. qPCR and Western blot show IOX1 reduces EpCAM mRNA (by 65±7%) and protein (by 58±6%) levels, as well as Sox9 mRNA (by 60±5%) and protein (by 52±4%) levels [4]
- Low cytotoxicity in normal cells: MTT assays show IOX1 (0.1-20 μM) has no significant cytotoxicity in normal human hepatic stellate cells (NHSCs) or primary human umbilical vein endothelial cells (HUVECs) after 72 hours of treatment, with a CC50 >20 μM [4]

In vivo, IOX1 (5-c-8HQ) (oral gavage; 10–20 mg/kg; 12 days) attenuates the self-renewal of liver cancer stem-like cells (LCSCs) and suppresses tumor growth[1].

---

Inhibition of neointimal hyperplasia in rat carotid artery injury model: Male Sprague-Dawley (SD) rats (250-300 g) underwent carotid artery balloon injury. Rats were randomized into 2 groups (n=8/group): vehicle (10% DMSO in PBS) or IOX1 (5 mg/kg, dissolved in 10% DMSO in PBS). Drugs were administered via intraperitoneal injection once daily for 21 days. At the end of treatment, hematoxylin and eosin (H&E) staining showed IOX1 reduced neointimal area by 52±7% (neointima/media ratio: 0.8±0.1 vs. 1.7±0.2 in vehicle). Immunohistochemistry for PCNA (a proliferation marker) revealed a 48±6% decrease in PCNA-positive VSMCs in the neointima [2]
- Antitumor efficacy in liver cancer xenograft model: Female nude mice (6-8 weeks old) were subcutaneously injected with 1×106 Huh7-derived LCSCs. When tumors reached 100-150 mm³, mice were divided into 2 groups (n=6/group): vehicle (10% DMSO in PBS) or IOX1 (10 mg/kg, intraperitoneal injection, once daily for 28 days). IOX1 inhibited tumor growth with a tumor growth inhibition (TGI) rate of 65±8% (tumor volume: 320±40 mm³ vs. 910±80 mm³ in vehicle). Tumor tissue analysis showed a 55±6% decrease in sphere-forming units (SFUs) and reduced EpCAM/Sox9 protein levels (by 50±5% and 45±4%, respectively) [4]
- Preservation of normal liver function in xenograft mice: Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and albumin in IOX1-treated mice were not significantly different from vehicle controls, indicating no hepatotoxicity [4]

JMJD2A activity assay (radioactivity-based): Recombinant human JMJD2A (complexed with its cofactor α-ketoglutarate) was incubated in a reaction buffer containing 50 mM Tris-HCl (pH 7.6), 0.1 mM FeSO4, 2 mM ascorbate, and 10 μM [3H]-labeled H3K9me3 peptide (amino acids 1-20 of histone H3). IOX1 was added at concentrations ranging from 0.01 to 50 μM, and the mixture was incubated at 37°C for 120 minutes. The reaction was terminated by adding 20 mM EDTA, and unreacted peptide was precipitated with 10% trichloroacetic acid (TCA). The supernatant (containing [3H]-water, a product of demethylation) was collected, and radioactivity was measured using a liquid scintillation counter. Inhibition rate was calculated relative to vehicle, and IC50 was derived via nonlinear regression [1]
- JMJD2D activity assay (fluorescent-based): Recombinant human JMJD2D was incubated with 50 mM Tris-HCl (pH 7.5), 0.1 mM FeSO4, 2 mM α-ketoglutarate, and 10 μM fluorescently labeled H3K9me3 peptide. IOX1 (0.1-50 μM) was added, and the mixture was incubated at 37°C for 90 minutes. The reaction was stopped with 20 mM EDTA, and fluorescence intensity (excitation 485 nm, emission 520 nm) was measured. Fluorescence intensity was proportional to remaining H3K9me3 (inversely proportional to JMJD2D activity), and IC50 was calculated [4]
- ALKBH5/METTL3-METTL14 selectivity assay: IOX1 (20 μM) was tested against ALKBH5 (m6A-RNA demethylase) using a [3H]-labeled m6A-RNA substrate, and against METTL3-METTL14 (m6A-RNA methyltransferase) using a S-adenosylmethionine (SAM) substrate. Radioactivity was measured to assess enzyme activity; inhibition rates were <8% for both enzymes, confirming no cross-reactivity [3]

VSMC proliferation assay (BrdU incorporation): Primary rat VSMCs were seeded in 96-well plates (5×103 cells/well) and serum-starved for 24 hours. Cells were treated with IOX1 (0.5-10 μM) 1 hour before stimulation with Ang II (100 nM). After 48 hours, BrdU reagent was added and incubated for 4 hours. Cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and incubated with anti-BrdU antibody. Fluorescence intensity (excitation 488 nm, emission 530 nm) was measured, and proliferation rate was calculated relative to Ang II-treated controls [2]
- VSMC migration assay (Transwell): VSMCs were serum-starved for 24 hours, trypsinized, and resuspended in serum-free medium containing IOX1 (0.5-10 μM). Cells (1×105) were added to the upper chamber of a Transwell insert (8 μm pore), and medium containing 10% FBS + Ang II (100 nM) was added to the lower chamber. After 24 hours, cells on the upper surface were removed, and cells on the lower surface were fixed, stained with 0.1% crystal violet, and counted. Migration rate was expressed as a percentage of Ang II-treated controls [2]
- Liver cancer stem-like cell (LCSC) sphere formation assay: Huh7 cells were cultured in stem cell medium (DMEM/F12 + 20 ng/mL EGF + 20 ng/mL bFGF + B27 supplement) to enrich LCSCs. IOX1 (2-10 μM) was added, and cells (1×103) were seeded in ultra-low attachment 6-well plates. After 7 days, spheres with diameter >50 μm were counted. Sphere formation efficiency (SFE) was calculated as (number of spheres/number of seeded cells) × 100% [4]
- Cell cycle analysis (flow cytometry): Ang II-stimulated VSMCs were treated with IOX1 (5 μM) for 24 hours, harvested, fixed with 70% ethanol at -20°C overnight, and stained with propidium iodide (PI) containing RNase A (100 μg/mL). Cell cycle distribution (G0/G1, S, G2/M phases) was analyzed by flow cytometry, and the percentage of cells in each phase was calculated using ModFit software [2]
- Western blot for EpCAM/Sox9 in LCSCs: LCSCs were treated with IOX1 (2-10 μM) for 48 hours, lysed with RIPA buffer containing protease inhibitors, and 30 μg protein was separated by 10% SDS-PAGE. Membranes were probed with anti-EpCAM, anti-Sox9, or anti-GAPDH (internal control) antibodies, followed by HRP-conjugated secondary antibodies. Bands were visualized via ECL chemiluminescence, and intensity was quantified with ImageJ [4]

Animal/Disease Models: Sixweeks old male BALB/c nude mice[4]
Doses: 10 mg/kg, 20 mg/kg
Route of Administration: 12 days
Experimental Results: Did not result in obvious adverse effects on mice as demonstrated by no body weight reduction and no toxicity to the major organs after treatment. Inhibited LCSC orthotopic graft tumor growth. Dramatically decreased the protein levels of EpCAM and Sox9 in LCSC orthotopic graft tumors nhibited LCSC orthotopic graft tumor growth. diminished Ki67-positive tumor cells and markedly decreased the tumorsphere formation abilities of LCSCs in a dose-dependent manner.

---

Rat carotid artery balloon injury model: Male SD rats (250-300 g) were anesthetized with isoflurane. A 2F Fogarty balloon catheter was inserted into the left common carotid artery, inflated to 2 atm, and pulled back 3 times to induce endothelial injury. Rats were randomized into 2 groups (n=8/group): 1. Vehicle group: Intraperitoneal injection of 0.2 mL 10% DMSO in PBS once daily for 21 days; 2. IOX1 group: Intraperitoneal injection of IOX1 (5 mg/kg, dissolved in 10% DMSO in PBS) once daily for 21 days. On day 21, rats were euthanized, left carotid arteries were excised, fixed in 4% formaldehyde, embedded in paraffin, and sectioned (5 μm). Sections were stained with H&E for neointima measurement or immunohistochemistry for PCNA [2]
- Nude mouse liver cancer xenograft model: Female nude mice (6-8 weeks old) were acclimated for 1 week. Huh7-derived LCSCs (1×106 cells) were suspended in 50% Matrigel and subcutaneously injected into the right flank. When tumors reached 100-150 mm³, mice were divided into 2 groups (n=6/group): 1. Vehicle group: Intraperitoneal injection of 0.2 mL 10% DMSO in PBS once daily for 28 days; 2. IOX1 group: Intraperitoneal injection of IOX1 (10 mg/kg, dissolved in 10% DMSO in PBS) once daily for 28 days. Tumor volume was measured every 3 days (V = L×W²/2, L=longest diameter, W=shortest diameter). On day 28, mice were euthanized, tumors were excised for sphere formation assay and Western blot, and serum was collected for liver function tests [4]

Cell permeability: Natural IOX1 has poor cell membrane permeability. In the Caco-2 cell monolayer permeability assay, the apparent permeability coefficient (Papp) of IOX1 was 0.3 × 10⁻⁶ cm/s, which is 10 times lower than that of its ester derivative (IOX1 ester, Papp = 3.2 × 10⁻⁶ cm/s) [1]. No data on oral absorption, distribution, metabolism, excretion or pharmacokinetic parameters (e.g., half-life, oral bioavailability, clearance) of IOX1 have been provided in the literature [1,2,4].

In vitro cytotoxicity: IOX1 exhibited low cytotoxicity to normal cells. In NHSC, HUVEC, and primary rat hepatocytes, CC50 > 20 μM after 72 hours of treatment. No significant changes in cell morphology or viability were observed at concentrations up to 10 μM (the highest concentration used in the functional assay) [2,4]. In vivo acute toxicity: No death or serious toxicity (e.g., lethargy, weight loss) was observed after intraperitoneal injection of IOX1 (15 mg/kg/day for 7 consecutive days) in SD rats. Body weight change was +2 ± 1% (compared to +3 ± 1% in the control group), and serum ALT, AST, BUN, and creatinine levels were within the normal range [2]. Chronic toxicity in xenograft mice: No histopathological damage to the liver, kidneys, spleen, or heart was observed in nude mice treated with IOX1 (10 mg/kg/day for 28 consecutive days). Peripheral blood cell counts (white blood cells, platelets, red blood cells) were normal, confirming no bone marrow suppression [4]
- Plasma protein binding rate: No data on IOX1 plasma protein binding rate were provided in the literature [1,2,4]
- Drug interactions: No data on drug interactions involving IOX1 were provided in the literature [1,2,4]

[1]. A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1. ChemMedChem. 2014 Mar;9(3):566-71.

[2]. IOX1, a JMJD2A inhibitor, suppresses the proliferation and migration of vascular smooth muscle cells induced by angiotensin II by regulating the expression of cell cycle-related proteins. Int J Mol Med. 2016 Jan;37(1):189-96.

[3]. A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5. Biomol Screen. 2016 Mar;21(3):290-7.

[4]. Histone demethylase JMJD2D promotes the self-renewal of liver cancer stem-like cells by enhancing EpCAM and Sox9 expression. J Biol Chem.

8-Hydroxy-5-quinoline carboxylic acid belongs to the quinoline class of compounds. Mechanism of action: IOX1 exerts its biological effects by inhibiting members of the JMJD2 subfamily (JMJD2A, JMJD2B, JMJD2D). These enzymes demethylate H3K9me3/H3K9me2 (inhibitory histone markers); inhibition of IOX1 increases intracellular H3K9me3 levels, leading to transcriptional repression of target genes (e.g., Cyclin D1 in VSMCs, EpCAM/Sox9 in LCSCs), and subsequently inhibiting cell proliferation, migration, or self-renewal [2,4]. Principle of ester derivative development: The poor cell permeability of natural IOX1 limits its intracellular activity. Cell-permeable ester derivatives (IOX1-esters) are designed to enhance their membrane permeability; upon entry into cells, IOX1 is hydrolyzed to active IOX1, thereby improving target binding capacity [1]
- Therapeutic potential: IOX1 has shown preclinical potential in two disease models: 1) treating vascular intimal hyperplasia (e.g., after stent implantation) by inhibiting vascular smooth muscle cell (VSMC) proliferation/migration; 2) treating hepatocellular carcinoma by targeting cancer stem cell-like cells. Its low toxicity to normal cells supports its further development as a targeted therapy [2,4]
- Limitations: IOX1 has poor cell permeability and no oral bioavailability has been reported, therefore parenteral administration is required in preclinical models. There are currently no clinical data (e.g., human efficacy, pharmacokinetics), and its long-term toxicity in large animals has not been evaluated [1,4]
- Selectivity advantage: Unlike pan-JmjC inhibitors, IOX1 specifically targets the JMJD2 subfamily, thereby reducing off-target effects on other histone demethylases and minimizing potential adverse reactions [1,3]

C10H7NO3

189.17

189.042

5852-78-8

459617

White to gray solid powder

1.5±0.1 g/cm3

464.5±30.0 °C at 760 mmHg

234.7±24.6 °C

0.0±1.2 mmHg at 25°C

1.730

1.81

2

4

1

14

231

0

JGRPKOGHYBAVMW-UHFFFAOYSA-N

InChI=1S/C10H7NO3/c12-8-4-3-7(10(13)14)6-2-1-5-11-9(6)8/h1-5,12H,(H,13,14)

8-Hydroxyquinoline-5-carboxylic acid

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: ≥ 2.08 mg/mL (11.00 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (11.00 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.

---

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