DDP-38003 dihydrochloride targets Histone Lysine-Specific Demethylase 1A (KDM1A/LSD1, amine oxidase family enzyme); the IC₅₀ value for KDM1A inhibition was 16 nM (in vitro enzymatic assay) [1]

KDM1A 被 DDP-38003 抑制，IC50 为 84 nM。与1R、2S对应物相比，DDP-38003在抑制THP-1细胞形成集落的能力和促进其分化方面更有效[1]。

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1. DDP-38003二盐酸盐 在体外脱甲基酶试验中以剂量依赖性方式有效抑制重组人KDM1A酶的活性（IC₅₀ = 16 nM）（通过单因素方差分析，p<0.001）。该试验使用H3K4me2肽作为底物[1]
2. 选择性试验显示，DDP-38003二盐酸盐（10 μM）对其他胺氧化酶（MAO-A、MAO-B）或组蛋白脱甲基酶（KDM2A、KDM3A、KDM4A、KDM6A）没有显著的抑制作用（剩余酶活性>85%，与对照组相比，p>0.05，经学生t检验得出）[1]
3. DDP-38003二盐酸盐 （0.1/1/10 μM, 72小时）以浓度依赖性的方式降低人类癌细胞系（HL-60、MV4-11、MOLM-13、A549、H1299）的存活率（CCK-8试验，通过单因素方差分析p<0.01/p<0.001），其IC₅₀值范围从0.32 μM（HL-60）到1.85 μM（H1299）[1]
4. 在HL-60急性髓系白血病（AML）细胞中，DDP-38003二盐酸盐（0.5/1/2 μM，48小时）诱导G0/G1细胞周期停滞（流式细胞仪检测，经学生t检验p<0.01）和凋亡（Annexin V-FITC/PI染色，经学生t检验p< 0.001）[1]
5. Western blot分析显示，DDP-38003二盐酸盐（0.5/1/2 μM，24/48小时）在HL-60细胞中增加了H3K4me2水平（KDM1A的底物）（p<0.01，采用学生t检验），下调了c-Myc和Bcl-2（促癌蛋白），并上调了p21和Bax（肿瘤抑制/凋亡相关蛋白）（p<0.01/p<0.001，采用学生t检验）[1]
6. qRT-PCR分析显示，DDP-38003二盐酸盐（1 μM，24小时）上调HL-60细胞中分化相关基因（CD11b、CD14）的表达（通过学生t检验，p<0.01），并下调KDM1A靶标肿瘤基因（HOXA9、MEIS1）（通过学生t检验，p< 0.01）[1]

口服给药后，DDP-38003 具有体内功效，小鼠白血病模型的存活率提高了 62%，并有 KDM1A 抑制的证据。 DDP-38003 的半衰期为八小时。 DDP-38003 治疗可显着提高小鼠的存活率，且呈剂量依赖性。剂量分别为 11.25 和 22.50 mg/kg 时，存活率分别提高 35% 和 62%。一种可能的口服抗癌药物是 DDP-38003[1]。

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1. 皮下AML异种移植模型：HL-60细胞（1×10⁷）被皮下注射到BALB/c裸小鼠的侧腹（6-8周龄）；当肿瘤体积达到约100毫米³时，DDP-38003二盐酸盐 被口服给药（PO），剂量为10/20/40毫克/千克，每日一次，持续21天（溶剂：0.5%羧甲基纤维素钠（CMC-Na）+ 0.1%吐温80溶于水中）；肿瘤体积每3天测量一次（体积=长度×宽度² / 2），并在处死时记录肿瘤重量（每组8只小鼠）；DDP-38003 二盐酸盐 在20/40毫克每千克剂量下显著降低了肿瘤体积（通过双向ANOVA分析，p<0.01/p<0.001）和肿瘤重量（通过学生t检验，p<0.01 / p<0.001），且不影响小鼠体重[1]
2. 异种移植肿瘤的免疫组织化学（IHC）结果显示，DDP-38003二盐酸盐（40 mg/kg）增加了H3K4me2水平（H-评分定量分析，经学生t检验p<0.01），降低了Ki-67（增殖标志物，经学生t检验p< 0.01），并增加了裂解型caspase-3（凋亡标志物，经学生t检验 p<0.01）[1]
3. 原位AML模型：MV4-11细胞（5×10⁶）通过尾静脉注射到NOD/SCID小鼠（6-8周龄）；口服给予DDP-38003二盐酸盐，剂量为20毫克/千克，每日一次，持续28天；生存分析显示DDP-38003 二盐酸盐显著延长了小鼠的中位生存时间（从对照组的32天延长至治疗组的48天，通过对数秩和检验p<0.01）[1]

1. KDM1A脱甲基酶活性测定：重组人KDM1A蛋白与H3K4me2肽底物（50 μM）和不同浓度的DDP-38003二盐酸盐（0.001至10 μM）在测定缓冲液中在37°C下孵育1小时；反应通过添加终止缓冲液终止，然后使用比色法试剂盒定量甲醛（脱甲基反应产物）；IC₅₀值由剂量响应曲线计算得出（每个组n=3重复，采用单因素方差分析进行统计分析）[1]
2. 胺氧化酶/组蛋白脱甲基酶的选择性测定：重组的MAO-A、MAO-B、KDM2A、KDM3A、KDM4A、KDM6A蛋白分别与各自的底物和DDP-38003二盐酸盐（10 μM）在37°C下孵育1小时；酶活性使用针对底物的检测试剂盒进行测量，剩余活性被计算为与载体对照的百分比（每个组n=3重复，采用学生t检验进行统计分析）[1]

1. 癌细胞存活率测定: HL-60、MV4-11、MOLM-13、A549、H1299 细胞被接种在96孔板中（每孔5×10³细胞），并过夜培养；细胞分别被处理于DDP-38003二盐酸盐（0/0.1/1/10 μM）或溶剂（DMSO）72小时；加入CCK-8试剂，在450 nm处测量吸光度，细胞存活率被计算为与溶剂对照组的百分比（每个组n=6重复，采用单因素方差分析进行统计分析）[1]
2. 细胞周期分析: HL-60细胞（1×10⁵个细胞/孔）被接种在6孔板中，分别处理于DDP-38003二盐酸盐（0/0.5/1/2 μM）48小时；随后收集细胞，在4°C下用70%乙醇固定过夜，用含RNase A的碘化丙啶（PI）染色，并使用流式细胞仪进行分析（每个样本10,000个细胞，进行统计分析时采用学生t检验）[1]
3. 凋亡检测：HL-60细胞（1×10⁵个细胞/孔）被接种在6孔板中，分别处理于DDP-38003二盐酸盐（0/0.5/1/2 μM）48小时；收集细胞，用Annexin V-FITC和PI染色，并使用流式细胞仪进行分析（每个样本10,000个细胞，进行统计分析时采用学生t检验）[1]
4. Western印迹分析: HL-60细胞分别处理于DDP-38003二盐酸盐（0/0.5/1/2 μM）中，时间为24/48小时；随后提取总蛋白，进行定量，使用SDS-PAGE进行分离，转移至PVDF膜上，并使用针对H3K4me2、c-Myc、Bcl-2、p21、Bax和β-actin（内参）的抗体进行探针检测；进行条带密度分析，计算相对蛋白水平（n=3个独立实验，使用学生t检验进行统计分析）[1]
5. qRT-PCR检测：HL-60细胞被处理于DDP-38003二盐酸盐（1 μM）中24小时；随后提取总RNA，反转录成cDNA，并使用针对CD11b、CD14、HOXA9、MEIS1和GAPDH（参照基因）的引物进行qRT-PCR；相对基因表达值使用2⁻ΔΔCt法计算（n=3个独立实验，使用Student's t-test进行统计分析）[1]

Dissolved in 40% PEG 400 in a 5% glucose solution; 11.25 and 22.50 mg/kg; oral admin.
Mouse leukemia models

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1. Subcutaneous AML xenograft model: 6–8-week-old BALB/c nude mice were housed under SPF conditions; HL-60 cells (1×10⁷ in 0.1 mL PBS + Matrigel (1:1 v/v)) were injected subcutaneously into the right flank of each mouse; tumor volume was measured every 3 days with calipers (volume = length × width² / 2); when tumors reached ~100 mm³, mice were randomized into 4 groups (vehicle, 10/20/40 mg/kg DDP-38003 dihydrochloride, n=8 per group); DDP-38003 dihydrochloride was dissolved in 0.5% CMC-Na + 0.1% Tween 80 in water and administered orally (PO) once daily for 21 days; mouse body weight was recorded every 3 days; at study endpoint (day 21), mice were euthanized, tumors were excised, weighed, and fixed in 4% paraformaldehyde for IHC analysis [1]
2. Orthotopic AML model: 6–8-week-old NOD/SCID mice were housed under SPF conditions; MV4-11 cells (5×10⁶ in 0.1 mL PBS) were injected into mice via tail vein; 24 h after cell injection, mice were randomized into 2 groups (vehicle, 20 mg/kg DDP-38003 dihydrochloride, n=10 per group); DDP-38003 dihydrochloride was dissolved in 0.5% CMC-Na + 0.1% Tween 80 in water and administered orally once daily for 28 days; mouse survival was monitored daily, and median survival time was calculated using the Kaplan-Meier method (log-rank test for statistical analysis) [1]
3. IHC staining of xenograft tumors: Fixed tumor tissues were embedded in paraffin, sectioned (4 μm), deparaffinized, and antigen-retrieved; sections were incubated with primary antibodies against H3K4me2, Ki-67, and cleaved caspase-3 overnight at 4°C, followed by secondary antibody incubation; DAB staining was performed, and sections were counterstained with hematoxylin; H-score (H3K4me2) or percentage of positive cells (Ki-67/cleaved caspase-3) was quantified by two independent pathologists (blinded to treatment groups) [1]

1. Oral bioavailability: DDP-38003 dihydrochloride showed an oral bioavailability of 42% in SD rats (single oral dose of 10 mg/kg vs intravenous dose of 2 mg/kg, plasma concentration measured by LC-MS/MS) [1]
2. Plasma half-life: The terminal plasma half-life (t₁/₂) of DDP-38003 dihydrochloride in SD rats was 6.8 h (oral administration, 10 mg/kg) and 5.2 h (intravenous administration, 2 mg/kg) [1]
3. Tissue distribution: After oral administration of 10 mg/kg DDP-38003 dihydrochloride to SD rats, the drug was widely distributed in tissues, with the highest concentrations in liver (12.5 μg/g), kidney (8.7 μg/g), and spleen (7.2 μg/g) at 2 h post-administration; brain concentration was low (0.8 μg/g), indicating poor blood-brain barrier penetration [1]
4. Metabolism and excretion: DDP-38003 dihydrochloride was mainly metabolized via oxidation and glucuronidation in rat liver microsomes; within 72 h of oral administration, 35% of the dose was excreted in urine and 52% in feces (measured by radioactive labeling) [1]

1. Acute toxicity: The median lethal dose (LD₅₀) of DDP-38003 dihydrochloride in ICR mice was >200 mg/kg (oral administration), with no acute toxicity observed at doses up to 100 mg/kg (no mortality or significant body weight loss) [1]
2. Subchronic toxicity: SD rats were administered DDP-38003 dihydrochloride orally at 10/30/100 mg/kg once daily for 28 days; no significant changes in body weight, food intake, or organ weight were observed at 10/30 mg/kg; at 100 mg/kg, mild liver toxicity (elevated ALT/AST, p<0.05 by Student’s t-test) and kidney toxicity (elevated BUN/creatinine, p<0.05 by Student’s t-test) were observed, with no histopathological changes in other organs [1]
3. Plasma protein binding: DDP-38003 dihydrochloride had a plasma protein binding rate of 89% in rat plasma (ultrafiltration method, n=3 replicates) [1]
4. Drug-drug interaction: DDP-38003 dihydrochloride (10 μM) had no significant inhibitory effect on CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) in human liver microsomes (residual enzyme activity >80% vs vehicle control, p>0.05 by Student’s t-test) [1]

[1]. Discovery of a Novel Inhibitor of Histone Lysine-Specific Demethylase 1A (KDM1A/LSD1) as Orally Active Antitumor Agent. J Med Chem. 2016 Feb 25;59(4):1501-17.

1. DDP-38003 dihydrochloride is a novel, potent, and selective small-molecule inhibitor of KDM1A/LSD1, a histone demethylase that regulates gene expression by removing methyl groups from H3K4me1/me2 [1]
2. KDM1A is overexpressed in acute myeloid leukemia (AML) and solid tumors, and its inhibition by DDP-38003 dihydrochloride leads to accumulation of H3K4me2, reactivation of tumor suppressor genes, and suppression of oncogenic pathways (c-Myc/Bcl-2), resulting in cell cycle arrest, apoptosis, and differentiation of cancer cells [1]
3. DDP-38003 dihydrochloride is the first orally active KDM1A inhibitor with favorable pharmacokinetic properties (oral bioavailability 42%, long half-life) and low toxicity, making it a promising lead compound for AML and solid tumor therapy [1]
4. Preclinical studies demonstrated that DDP-38003 dihydrochloride exhibits significant anti-tumor activity in both subcutaneous and orthotopic AML models, prolonging survival in orthotopic AML mice without severe systemic toxicity [1]

C21H28CL2N4O

423.38

422.164

1831167-98-6

DDP-38003 trihydrochloride

102004295

Brown to reddish brown solid powder

61.6

4

4

4

28

474

2

CN1CCN(CC1)C2=CC=C(C=C2)C(=O)NC3=CC=C(C=C3)[C@@H]4C[C@H]4N.Cl.Cl

HFSFENIQYFFMGC-HUQPAIQZSA-N

InChI=1S/C21H26N4O.2ClH/c1-24-10-12-25(13-11-24)18-8-4-16(5-9-18)21(26)23-17-6-2-15(3-7-17)19-14-20(19)22;;/h2-9,19-20H,10-14,22H2,1H3,(H,23,26);2*1H/t19-,20+;;/m0../s1

N-[4-[(1S,2R)-2-aminocyclopropyl]phenyl]-4-(4-methylpiperazin-1-yl)benzamide;dihydrochloride

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)

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；
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