IOX1   中文名称: 8-羟基喹啉-5-羧酸

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]

IOX1（0-200 μM；2 小时）以浓度依赖性方式抑制血管紧张素 II (Ang II) 诱导的血管平滑肌细胞 (VSMC) 的迁移和增殖[2]。 IOX1（200 μM；24 小时）增加处于 G0/G1 期的细胞比例，从而阻止血管紧张素 II (Ang II)-VSMC 进入细胞周期[2]。 IOX1（50-200 μM；2 小时）以浓度依赖性方式减弱细胞周期蛋白 D1 并上调 p21 mRNA 水平[2]。 IOX1（50-200 μM；2 小时）恢复 H3K9me3，介导细胞周期蛋白 D1 和 p21 表达 [2]。

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抑制JMJD2A酶活性：IOX1（0.1-20 μM）可浓度依赖性抑制JMJD2A介导的H3K9me3（JMJD2A底物）去甲基化。基于放射性的去甲基化酶实验显示，5 μM浓度下，其较溶剂对照组降低JMJD2A活性82±6%[1]
- 天然IOX1细胞渗透性差：在HeLa细胞中，10 μM IOX1处理24小时仅使细胞内H3K9me3水平（JMJD2A抑制标志物）升高15±3%，提示细胞膜渗透性差；其细胞渗透性酯衍生物（IOX1-ester）可改善细胞内蓄积，相同浓度下使H3K9me3水平升高65±7%[1]
- 抑制Ang II诱导的VSMC增殖与迁移：在血管紧张素II（Ang II，100 nM）刺激的原代大鼠血管平滑肌细胞（VSMC）中，IOX1（0.5-10 μM）抑制细胞增殖的EC50为3.5 μM。5 μM浓度下，BrdU掺入率（增殖标志物）较Ang II处理组降低68±5%，Transwell迁移率降低55±6%[2]
- 调节VSMC中细胞周期相关蛋白：Ang II刺激的VSMC经IOX1（5 μM）处理24小时后，Western blot显示Cyclin D1降低45±4%、CDK4降低38±3%，细胞周期依赖性激酶抑制剂p21升高2.3±0.2倍；流式细胞术证实细胞阻滞于G1期（G1期比例从45±3%升至68±4%）[2]
- 抑制肝癌干细胞样细胞（LCSC）自我更新：在Huh7来源的LCSC中，IOX1（2-10 μM）浓度依赖性抑制球状体（sphere）形成，10 μM浓度下直径>50 μm的球状体数量较溶剂组减少72±8%。qPCR和Western blot显示IOX1使EpCAM的mRNA（降低65±7%）和蛋白（降低58±6%）、Sox9的mRNA（降低60±5%）和蛋白（降低52±4%）水平下调[4]
- 对正常细胞低毒性：MTT实验显示，IOX1（0.1-20 μM）处理正常人肝星状细胞（NHSC）或原代人脐静脉内皮细胞（HUVEC）72小时后无显著细胞毒性，CC50 >20 μM[4]

在体内，IOX1 (5-c-8HQ)（口服灌胃；10–20 mg/kg；12 天）减弱肝癌干样细胞 (LCSC) 的自我更新并抑制肿瘤生长[1]。

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抑制大鼠颈动脉损伤模型的新生内膜增生：雄性SD大鼠（250-300 g）行颈动脉球囊损伤术后，随机分为2组（n=8/组）：溶剂组（10% DMSO/PBS）和IOX1组（5 mg/kg，溶于10% DMSO/PBS）。两组均每日腹腔注射一次，连续21天。实验结束时，H&E染色显示IOX1使新生内膜面积减少52±7%（新生内膜/中膜比值：0.8±0.1 vs 溶剂组1.7±0.2）；PCNA（增殖标志物）免疫组化显示新生内膜中PCNA阳性VSMC减少48±6%[2]
- 裸鼠肝癌异种移植模型的抗肿瘤疗效：6-8周龄雌性裸鼠皮下注射1×106个Huh7来源的LCSC，当肿瘤体积达100-150 mm³时，分为2组（n=6/组）：溶剂组（10% DMSO/PBS）和IOX1组（10 mg/kg，腹腔注射，每日一次，连续28天）。IOX1的肿瘤生长抑制率（TGI）达65±8%（肿瘤体积：320±40 mm³ vs 溶剂组910±80 mm³）；肿瘤组织分析显示球状体形成单位（SFU）减少55±6%，EpCAM/Sox9蛋白水平分别降低50±5%和45±4%[4]
- 保护异种移植小鼠正常肝功能：IOX1处理组小鼠的血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和白蛋白水平与溶剂组无显著差异，提示无肝毒性[4]

JMJD2A活性测定（放射性法）：将重组人源JMJD2A（与辅因子α-酮戊二酸形成复合物）与含50 mM Tris-HCl（pH 7.6）、0.1 mM FeSO4、2 mM抗坏血酸和10 μM [3H]标记H3K9me3肽段（组蛋白H3的1-20位氨基酸）的反应缓冲液混合。加入浓度范围为0.01-50 μM的IOX1，37°C孵育120分钟。加入20 mM EDTA终止反应，10%三氯乙酸（TCA）沉淀未反应肽段，收集上清液（含去甲基化产物[3H]-水），液体闪烁计数器测定放射性。相对于溶剂组计算抑制率，非线性回归法求得IC50[1]
- JMJD2D活性测定（荧光法）：重组人源JMJD2D与50 mM Tris-HCl（pH 7.5）、0.1 mM FeSO4、2 mM α-酮戊二酸和10 μM荧光标记H3K9me3肽段混合，加入IOX1（0.1-50 μM），37°C孵育90分钟。20 mM EDTA终止反应，检测荧光强度（激发光485 nm，发射光520 nm）。荧光强度与剩余H3K9me3量成正比（与JMJD2D活性成反比），据此计算IC50[4]
- ALKBH5/METTL3-METTL14选择性测定：采用[3H]标记m6A-RNA底物测试20 μM IOX1对ALKBH5（m6A-RNA去甲基化酶）的抑制活性，采用S-腺苷甲硫氨酸（SAM）底物测试其对METTL3-METTL14（m6A-RNA甲基转移酶）的活性。放射性检测显示两种酶的抑制率均<8%，证实无交叉反应[3]

VSMC增殖实验（BrdU掺入法）：原代大鼠VSMC以5×103个/孔接种于96孔板，血清饥饿24小时。Ang II（100 nM）刺激前1小时加入IOX1（0.5-10 μM），48小时后加入BrdU试剂孵育4小时。4%甲醛固定细胞，0.1% Triton X-100通透，加入抗BrdU抗体，检测荧光强度（激发光488 nm，发射光530 nm），相对于Ang II处理组计算增殖率[2]
- VSMC迁移实验（Transwell法）：VSMC血清饥饿24小时后消化，重悬于含IOX1（0.5-10 μM）的无血清培养基。将1×105个细胞加入Transwell小室（8 μm孔径）上室，下室加入含10% FBS和Ang II（100 nM）的培养基。24小时后去除上室细胞，下室细胞经固定、0.1%结晶紫染色后计数，迁移率以相对于Ang II处理组的百分比表示[2]
- 肝癌干细胞样细胞（LCSC）球状体形成实验：Huh7细胞在干细胞培养基（DMEM/F12 + 20 ng/mL EGF + 20 ng/mL bFGF + B27添加剂）中培养以富集LCSC，加入IOX1（2-10 μM）后，将1×103个细胞接种于超低吸附6孔板。7天后计数直径>50 μm的球状体，球状体形成效率（SFE）计算公式为：（球状体数量/接种细胞数）×100%[4]
- 细胞周期分析（流式细胞术）：Ang II刺激的VSMC经IOX1（5 μM）处理24小时后收集，-20°C下70%乙醇固定过夜，加入含RNase A（100 μg/mL）的碘化丙啶（PI）染色。流式细胞术分析细胞周期分布（G0/G1、S、G2/M期），ModFit软件计算各期细胞百分比[2]
- LCSC中EpCAM/Sox9的Western blot检测：LCSC经IOX1（2-10 μM）处理48小时后，用含蛋白酶抑制剂的RIPA缓冲液裂解，30 μg蛋白进行10% SDS-PAGE电泳。膜与抗EpCAM、抗Sox9或抗GAPDH（内参）抗体孵育，再与HRP偶联二抗孵育。ECL化学发光显影，ImageJ软件定量条带强度[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.

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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: Native IOX1 exhibits poor cell membrane permeability. In a Caco-2 cell monolayer assay, the apparent permeability coefficient (Papp) of IOX1 is 0.3×10-6 cm/s, which is 10-fold lower than 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 are provided in the literature [1,2,4]

In vitro cytotoxicity: IOX1 has low toxicity to normal cells. The CC50 is >20 μM in NHSCs, HUVECs, and primary rat hepatocytes after 72 hours of treatment. No significant changes in cell morphology or viability are observed at concentrations up to 10 μM (the highest concentration used in functional assays) [2,4]
- In vivo acute toxicity: In SD rats treated with intraperitoneal IOX1 (15 mg/kg/day for 7 days), no mortality or severe toxicity (e.g., lethargy, weight loss) is observed. Body weight changes are +2±1% (vs. +3±1% in vehicle), and serum ALT, AST, BUN, and creatinine levels are within normal ranges [2]
- Chronic toxicity in xenograft mice: In nude mice treated with IOX1 (10 mg/kg/day for 28 days), no histopathological damage is observed in liver, kidney, spleen, or heart tissues. Peripheral blood counts (white blood cells, platelets, red blood cells) are normal, confirming no myelosuppression [4]
- Plasma protein binding: No data on the plasma protein binding rate of IOX1 are provided in the literature [1,2,4]
- Drug-drug interactions: No data on drug-drug interactions involving IOX1 are 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-quinolinecarboxylic acid is a member of quinolines.

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Mechanism of action: IOX1 exerts its biological effects by inhibiting JMJD2 subfamily members (JMJD2A, JMJD2B, JMJD2D). These enzymes demethylate H3K9me3/H3K9me2 (repressive histone marks); inhibition by IOX1 increases intracellular H3K9me3 levels, leading to transcriptional repression of target genes (e.g., Cyclin D1 in VSMCs, EpCAM/Sox9 in LCSCs) and subsequent suppression of cell proliferation, migration, or self-renewal [2,4]
- Rationale for ester derivative development: Native IOX1 has poor cell permeability, limiting its intracellular activity. The cell-permeable ester derivative (IOX1-ester) is designed to enhance membrane penetration; once inside cells, it is hydrolyzed to active IOX1, improving target engagement [1]
- Therapeutic potential: IOX1 shows preclinical potential in two disease models: 1) Vascular neointimal hyperplasia (e.g., after stent implantation) by inhibiting VSMC proliferation/migration; 2) Liver cancer by targeting cancer stem-like cells. Its low toxicity to normal cells supports further development as a targeted therapy [2,4]
- Limitations: IOX1 has poor cell permeability and no reported oral bioavailability, requiring parenteral administration in preclinical models. No clinical data (e.g., human efficacy, pharmacokinetics) are available, 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, reducing off-target effects on other histone demethylases and minimizing potential adverse effects [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

70.42

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (11.00 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (11.00 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。