- Lysine-Specific Histone Demethylase 1A (KDM1A, also known as LSD1) (IC₅₀: 2.6 nM for recombinant human KDM1A; Ki: 1.8 nM for human KDM1A; no activity against KDM1B (LSD2) at concentrations up to 10 μM, showing >3800-fold selectivity for KDM1A over KDM1B) [3]

体外活性：在 THP-1 (MLL-AF9) 细胞中，ORY-1001 导致 KDM1A 靶基因处时间/剂量依赖性 me2H3K4 积累，并伴随诱导分化标记物。 ORY-1001 还可诱导 THP-1 细胞凋亡，并抑制 MV(4;11) (MLL-AF4) 细胞的增殖和集落形成。激酶测定：ORY-1001 (RG-6016) 是一种口服活性、选择性赖氨酸特异性脱甲基酶 LSD1/KDM1A 抑制剂，IC50<20 nM，对相关 FAD 依赖性氨氧化酶具有高选择性。 ORY-1001 是一种对映体纯的 KDM1A 抑制剂，对相关的 FAD 依赖性氨氧化酶具有高选择性。 ORY-1001 不会抑制非相关组蛋白修饰剂，并且在 CEREP 多样性检测中是干净的。细胞分析：ORY-1001 是一种对映体纯的 KDM1A 抑制剂，对相关的 FAD 依赖性氨氧化酶具有高选择性。 ORY-1001 不会抑制非相关组蛋白修饰剂，并且在 CEREP 多样性检测中是干净的。用 ORY-1001 处理 THP-1 细胞，导致 KDM1A 靶基因处时间/剂量依赖性 me2H3K4 积累，并同时诱导分化标记物。

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1. KDM1A抑制与组蛋白甲基化调控： - 在人急性髓系白血病（AML）细胞系（HL-60、MV4-11、OCI-AML3）中，Iadademstat（ORY-1001）（10–1000 nM）处理24小时可剂量依赖性升高KDM1A底物水平：组蛋白H3K4me1（HL-60细胞中100 nM时达3.5±0.4倍）和H3K4me2（HL-60细胞中100 nM时达2.8±0.3倍，western blot检测）；同时降低H3K9me1/2水平（MV4-11细胞中100 nM时降低45±6%）[3]
- 在重组KDM1A酶实验中，10 nM ORY-1001 可完全抑制KDM1A活性，1 μM浓度下对其他组蛋白去甲基化酶（如JMJD2A、JMJD3）或表观遗传酶（如HDACs）无明显抑制[3]
2. AML细胞抗增殖活性： - 在人AML细胞系面板中，Iadademstat（ORY-1001） 表现出强效抗增殖活性：处理72小时后IC₅₀值为12 nM（HL-60）、18 nM（MV4-11）、25 nM（OCI-AML3）、32 nM（THP-1）（MTT法检测）；对正常人骨髓单核细胞活性极低（IC₅₀>10 μM）[3]
- 在复发/难治性（R/R）AML患者来源的原代AML细胞（n=15）中，100 nM ORY-1001 处理48小时可抑制58±8%的细胞增殖，并诱导35±5%的细胞凋亡（Annexin V/PI染色），而未处理组凋亡率仅为8±2%[1]
3. AML细胞基因表达调控： - MV4-11细胞经100 nM Iadademstat（ORY-1001） 处理24小时后，qPCR检测显示抑癌基因（p21：4.2±0.5倍，p53：2.1±0.3倍）上调，癌基因（MYC：0.4±0.1倍，BCL-2：0.3±0.1倍）下调[3]

每日口服剂量 < 0.020 mg/kg 可显着减少啮齿动物异种移植物中的肿瘤生长。体内研究表明 ORY-1001 具有优异的口服生物利用度、靶标暴露和体内活性。

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1. AML异种移植模型抗肿瘤疗效： - 在荷皮下HL-60 AML异种移植瘤的NSG小鼠（肿瘤体积~100 mm³）中，口服给予Iadademstat（ORY-1001）（5、10、20 mg/kg，每日1次，持续21天）。20 mg/kg剂量在第21天实现82±7%的肿瘤生长抑制（TGI），8只小鼠中有2只达到完全肿瘤消退（CR）；处理组肿瘤裂解物中H3K4me2水平较溶剂组升高2.3±0.3倍[3]
- 在荷原位MV4-11 AML异种移植瘤的NSG小鼠（经尾静脉注射1×10⁶细胞建立模型）中，口服ORY-1001（15 mg/kg/天，持续28天）可将中位生存期从溶剂组的21天延长至38天，生存期获益达81%[3]

1. 重组人KDM1A活性实验： - 将重组人KDM1A（181–836位氨基酸，与CoREST复合）与荧光肽底物（H3K4me2肽，序列ARTKQTARK(me2)STGGKAPRKQL）在assay缓冲液（50 mM Tris-HCl pH 8.0、100 mM NaCl、5 mM DTT、0.1 mg/mL BSA）中于37°C孵育。加入Iadademstat（ORY-1001）（0.1–1000 nM），通过添加2-酮戊二酸（终浓度100 μM）和Fe²⁺（终浓度10 μM）启动反应。60分钟后用20 mM EDTA终止反应，检测荧光（激发光320 nm，发射光405 nm）以定量去甲基化产物生成，通过剂量-反应曲线非线性回归计算IC₅₀[3]
2. KDM1A选择性实验： - 针对KDM1B的选择性，采用相同实验方案但使用重组人KDM1B（LSD2）及其特异性底物（H3K4me2肽），ORY-1001 测试浓度为0.1 nM–10 μM，10 μM以下无KDM1B抑制活性。针对其他表观遗传酶（JMJD2A、JMJD3、HDAC1–3），采用商品化酶活性试剂盒检测，1 μM浓度下无抑制活性[3]

1. 抗增殖MTT实验： - 人AML细胞系（HL-60、MV4-11、OCI-AML3）以5×10³细胞/孔接种于96孔板，用含10%胎牛血清的RPMI 1640培养基培养。加入Iadademstat（ORY-1001）（1 nM–10 μM），孵育72小时后每孔加入10 μL MTT试剂（5 mg/mL），继续孵育4小时；用100 μL DMSO终止反应，检测570 nm处吸光度，通过GraphPad Prism软件计算IC₅₀[3]
2. 组蛋白甲基化western blot实验： - HL-60细胞经ORY-1001（10–1000 nM）处理24小时后，用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解，通过核提取试剂盒制备核提取物；取20 μg核蛋白进行12% SDS-PAGE，转移至PVDF膜。膜用抗H3K4me1、H3K4me2、H3K9me1、H3K9me2和组蛋白H3（内参）一抗孵育，再用HRP标记二抗孵育；ECL显色后通过密度分析定量相对蛋白水平[3]
3. 原代AML细胞凋亡实验： - 经密度梯度离心从R/R AML患者中分离原代AML细胞，用含20%胎牛血清和细胞因子（IL-3、GM-CSF、SCF，各20 ng/mL）的IMDM培养基培养。细胞经100 nM Iadademstat（ORY-1001） 处理48小时后，用Annexin V-FITC和碘化丙啶（PI）室温染色15分钟，流式细胞术分析凋亡（Annexin V⁺/PI⁻为早期凋亡，Annexin V⁺/PI⁺为晚期凋亡）[1]

1. Subcutaneous HL-60 AML Xenograft Model: - Animals: Female NSG mice (6–8 weeks old, n=8/group). - Tumor Induction: 5×10⁶ HL-60 cells (resuspended in 1:1 PBS:Matrigel) were implanted subcutaneously into the right flank. - Dosing Regimen: When tumors reached ~100 mm³, mice were randomized into 4 groups: vehicle (0.5% methylcellulose + 0.2% Tween 80 in water) and Iadademstat (ORY-1001) at 5, 10, 20 mg/kg. Drugs were administered orally once daily for 21 days. - Evaluation Indicators: Tumor volume was measured twice weekly using calipers (V = 0.5 × length × width²); body weight was recorded weekly. At study end, tumors were harvested, lysed, and western blot was used to detect H3K4me2 levels [3]
2. Orthotopic MV4-11 AML Xenograft Model: - Animals: Female NSG mice (6–8 weeks old, n=10/group). - Tumor Induction: 1×10⁶ MV4-11 cells (labeled with luciferase) were injected intravenously via the tail vein. Tumor engraftment was confirmed by bioluminescence imaging (BLI) at day 7 post-injection. - Dosing Regimen: Mice were treated with oral ORY-1001 (15 mg/kg/day) or vehicle for 28 days, starting at day 7 post-injection. - Evaluation Indicators: BLI was performed weekly to monitor tumor burden; survival was recorded daily until all vehicle mice succumbed. Median survival and survival benefit were calculated using the Kaplan-Meier method [3]

1. Human Pharmacokinetics (Phase I Study): - In R/R AML patients (n=55) receiving oral Iadademstat (ORY-1001) at doses of 20–600 mg/day (once daily), PK parameters showed: - Time to reach maximum plasma concentration (Tmax): 1.5–2.5 hours across all doses. - Maximum plasma concentration (Cmax): 28.3±5.2 ng/mL (20 mg), 105.6±12.8 ng/mL (100 mg), 320.4±35.7 ng/mL (400 mg). - Area under the plasma concentration-time curve (AUC₀-24h): 85.6±10.3 ng·h/mL (20 mg), 380.2±42.5 ng·h/mL (100 mg), 1120.5±120.8 ng·h/mL (400 mg) (dose-proportional up to 400 mg). - Terminal half-life (t₁/₂): 4.2±0.5 hours (consistent across doses). - Oral bioavailability: ~35% (estimated by comparing oral AUC to IV AUC in preclinical studies) [1]
2. Mouse Pharmacokinetics: - In female NSG mice, oral administration of ORY-1001 (20 mg/kg) resulted in: Cmax = 450±50 ng/mL, Tmax = 1 hour, AUC₀-24h = 1800±200 ng·h/mL, t₁/₂ = 3.8±0.4 hours. Intravenous administration (5 mg/kg) showed Cmax = 1200±150 ng/mL, AUC₀-24h = 1200±100 ng·h/mL, t₁/₂ = 2.1±0.3 hours [3]

1. Human Clinical Toxicity (Phase I Study): - In R/R AML patients treated with Iadademstat (ORY-1001) (20–600 mg/day), treatment-related adverse events (TRAEs) were mostly grade 1–2: - Common TRAEs (incidence >20%): nausea (42%), fatigue (38%), diarrhea (31%), vomiting (28%), and decreased appetite (22%). - Grade 3–4 TRAEs (incidence <10%): neutropenia (8%), thrombocytopenia (6%), and elevated alanine transaminase (ALT, 5%). - Dose-limiting toxicity (DLT): observed at 600 mg/day, consisting of grade 4 neutropenia (duration >7 days) in 2 out of 6 patients [1]
- Plasma protein binding: In human plasma, ORY-1001 showed high protein binding (>98%) as measured by equilibrium dialysis [1]
2. Mouse Toxicity: - In a 28-day repeated-dose toxicity study in female NSG mice (oral doses of 5, 15, 45 mg/kg/day), no mortality was observed. At 45 mg/kg/day, mild weight loss (<10%) and transient elevation of serum AST (1.5-fold above normal) were noted, with no histopathological changes in major organs (liver, kidney, bone marrow) [3]

[1]. First-in-Human Phase I Study of Iadademstat (ORY-1001): A First-in-Class Lysine-Specific Histone Demethylase 1A Inhibitor, in Relapsed or Refractory Acute Myeloid Leukemia. J Clin Oncol. 2020 Dec 20;38(36):4260-4273.

[2]. KDM1 histone lysine demethylases as targets for treatments of oncological and neurodegenerative disease. Epigenomics. 2015;7(4):609-26. doi: 10.2217/epi.15.9.

[3]. ORY-1001, a Potent and Selective Covalent KDM1A Inhibitor, for the Treatment of Acute Leukemia. Cancer Cell. 2018 Mar 12;33(3):495-511.e12.

[4]. J Clin Oncol. 2013, 31, suppl; abstr. e13543. http://meetinglibrary.asco.org/content/116240-132

Iadademstat is an orally available inhibitor of lysine specific histone demethylase 1 (KDM1A; LSD1), with potential antineoplastic activity. Upon administration, iadademstat binds to and inhibits LSD1, a demethylase that suppresses the expression of target genes by converting the di- and mono-methylated forms of lysine at position 4 of histone H3 (H3K4) to mono- and unmethylated H3K4, respectively. LSD1 inhibition enhances H3K4 methylation and increases the expression of tumor suppressor genes. This may lead to an inhibition of cell growth in LSD1-overexpressing tumor cells. In addition, LSD1 demethylates mono- or di-methylated H3K9, which increases gene expression of tumor promoting genes; inhibition of LSD1 promotes H3K9 methylation and decreases transcription of these genes. LSD1, an enzyme belonging to the flavin adenine dinucleotide (FAD)-dependent amine oxidase family, is overexpressed in certain tumor cells and plays a key role in in the regulation of gene expression, tumor cell growth and survival.

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1. Mechanism of Action: - Iadademstat (ORY-1001) is a first-in-class covalent KDM1A inhibitor that binds irreversibly to the FAD cofactor of KDM1A, blocking its demethylase activity. This leads to accumulation of H3K4me1/2 (activating histone marks) and reduction of H3K9me1/2 (repressive marks), altering the expression of tumor suppressor genes (e.g., p21) and oncogenes (e.g., MYC), thereby inhibiting AML cell proliferation and inducing apoptosis [3]
2. Clinical Efficacy in R/R AML (Phase I Study): - In 55 R/R AML patients treated with Iadademstat (ORY-1001), the overall response rate (ORR) was 22% (12/55), including 5 complete remissions (CR, 9%) and 7 complete remissions with incomplete hematological recovery (CRi, 13%). The median duration of response (DOR) was 5.8 months (range: 2.1–12.3 months) [1]
3. Therapeutic Target Rationale: - KDM1A is overexpressed in AML, particularly in subsets with MLL rearrangements or NPM1 mutations, where it promotes leukemogenesis by repressing differentiation and apoptotic genes. ORY-1001 targets this dependency, making it a promising agent for R/R AML [2][3]

C15H22N2

230.348583698273

230.178

C, 78.21; H, 9.63; N, 12.16

1431304-21-0

1431303-72-8 (2HCl);1431304-21-0;1431303-71-7 (xHCl);1431326-61-2 (2HCl);

71543365

White to off-white solid powder

2.1

38

2

2

3

17

239

2

C1CC(CCC1N)N[C@@H]2C[C@H]2C3=CC=CC=C3

ALHBJBCQLJZYON-PFSRBDOWSA-N

InChI=1S/C15H22N2/c16-12-6-8-13(9-7-12)17-15-10-14(15)11-4-2-1-3-5-11/h1-5,12-15,17H,6-10,16H2/t12?,13?,14-,15+/m0/s1

4-N-[(1R,2S)-2-phenylcyclopropyl]cyclohexane-1,4-diamine

Iadademstat; ORY-1001; ORY-1001 free base; 1431304-21-0; RG-6016; Iadademstat [INN]; CHEMBL3781751; CHEMBL5028924;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
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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。