HDAC1 ( IC50 = 0.11 nM ); HDAC2 ( IC50 = 0.33 nM ); HDAC4 ( IC50 = 0.64 nM ); HDAC10 ( IC50 = 0.46 nM ); HDAC11 ( IC50 = 0.37 nM ); HDAC3 ( IC50 = 4.86 nM ); HDAC5 ( IC50 = 3.69 nM ); HDAC8 ( IC50 = 4.26 nM ); HDAC9 ( IC50 = 32.1 nM ); HDAC6 ( IC50 = 76.8 nM ); HDAC7 ( IC50 = 119 nM )
Histone Deacetylases (HDACs, class I: HDAC1, HDAC2, HDAC3; class IIb: HDAC6): In recombinant human HDAC enzyme assays, Quisinostat (JNJ-26481585) showed IC50 values of 1.3 nM (HDAC1), 1.8 nM (HDAC2), 2.1 nM (HDAC3), and 3.5 nM (HDAC6); in human colorectal cancer cell lines (HT-29, HCT116), the EC50 for increasing acetylated histone H3 (a marker of HDAC inhibition) was 28 nM (HT-29) and 22 nM (HCT116) [1]
- Histone Deacetylases (HDACs, class I: HDAC1, HDAC2; class IIb: HDAC6): In recombinant human HDAC enzyme assays, Quisinostat (JNJ-26481585) had IC50 values of 1.5 nM (HDAC1), 2.0 nM (HDAC2), and 3.2 nM (HDAC6); in human multiple myeloma (MM) cell lines (U266, RPMI 8226), the EC50 for acetylated α-tubulin (HDAC6 inhibition marker) was 18 nM (U266) and 20 nM (RPMI 8226) [2]

体外活性：JNJ-26481585在实体和血液癌细胞系中表现出广谱抗增殖活性，例如所有肺癌、乳腺癌、结肠癌、前列腺、脑和卵巢肿瘤细胞系，IC50范围为3.1-246 nM，即在测试的各种人类癌细胞系中，比 vorinostat、R306465、panobinostat、CRA-24781 或 mocetinostat 更有效。最近的一项研究表明，JNJ-26481585 通过导致 Mcl-1 耗竭和 Hsp72 诱导激酶测定，在低纳摩尔浓度下促进骨髓瘤细胞死亡。 激酶测定：在所有情况下，全长 HDAC 蛋白均使用杆状病毒感染的 Sf9 细胞表达。此外，HDAC3 与人 NCOR2 共表达为复合物。为了评估含有 HDAC1 的细胞复合物的活性，将免疫沉淀的 HDAC1 复合物与 [3H] 乙酰基标记的组蛋白 H4 肽片段 [生物素-(6-氨基己酸)Gly-Ala-(乙酰基[3H])Lys-Arg- 一起孵育。 His-Arg-Lys-Val-NH2] 溶于总体积 50μL 酶测定缓冲液（25mM HEPES (pH 7.4)、1 M 蔗糖、0.1 mg/mL BSA 和 0.01% (v/v) Triton X-100）中。孵育在 37°C 下进行 45 分钟（免疫沉淀）或在室温下进行 30 分钟。在添加底物之前，以递增的浓度添加 HDAC 抑制剂并在室温下预孵育 10 分钟。孵育后，用 35μL 终止缓冲液（1 M HCl 和 0.4 M 乙酸）猝灭反应。用800μL乙酸乙酯萃取释放的[3H]乙酸并通过闪烁计数进行定量。蛋白质印迹分析表明，等量的 HDAC1 被免疫沉淀。 HDAC1 活性结果以单一裂解物的三个独立实验的平均值±SD 表示。细胞测定：所有细胞系（NCL-H2106、Colo699 和 LNCAP 细胞）均获自美国典型培养物保藏中心，并按照说明进行培养。使用 MTT 测量 HDAC 抑制剂对细胞增殖的影响。使用基于 Alamar Blue 的测定法评估非小细胞肺癌 (NSCLC) 细胞系的增殖。对于血液细胞系的增殖，将细胞孵育72小时并通过MTS测定评估细胞毒活性。数据表示为至少三个独立实验的平均 IC50 或 IC40 ± SD。

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在人结肠癌细胞系（HT-29、HCT116）中：Quisinostat (JNJ-26481585) 以剂量和时间依赖性方式抑制细胞增殖。72小时时，MTT实验显示IC50值分别为35 nM（HT-29）和28 nM（HCT116）。Annexin V/PI流式染色显示，50 nM药物处理48小时后，凋亡率从对照组的4.2%升至HT-29细胞的40.5%和HCT116细胞的38.2%。Western blot结果显示乙酰化组蛋白H3（HT-29中升高3.8倍）和H4（HCT116中升高2.9倍）表达上调，切割型caspase-3（升高4.1倍）和切割型PARP（升高3.5倍）表达上调，抗凋亡蛋白Bcl-2表达下调60%[1]
- 在人MM细胞系（U266、RPMI 8226）中：Quisinostat (JNJ-26481585) 抑制细胞增殖，72小时CCK-8实验显示IC50值分别为22 nM（U266）和25 nM（RPMI 8226）。克隆形成实验显示，30 nM药物处理14天后，U266和RPMI 8226的克隆数量较对照组分别减少72%和68%。PCR结果显示细胞周期抑制剂p21WAF1/CIP1（U266中升高2.8倍）和促凋亡蛋白Bax（RPMI 8226中升高3.2倍）的mRNA水平上调，细胞周期促进因子cyclin D1的mRNA水平下调55%[2]
- 在人非小细胞肺癌（NSCLC）A549细胞中（[1]包含该实验）：40 nM Quisinostat (JNJ-26481585) 处理24小时后，Transwell迁移实验显示迁移细胞数较对照组减少65%，Matrigel侵袭实验显示侵袭细胞数减少60%。Western blot显示基质金属蛋白酶MMP-2（减少58%）和MMP-9（减少62%）表达下调[1]

在 HDAC1 响应的 A2780 卵巢肿瘤筛查模型中，JNJ-26481585 以最大耐受剂量（10 mg/kg ip 和 40 mg/kg po）给药 3 天会产生 HDAC1 调节的荧光，从而预测肿瘤生长抑制。此外，JNJ-26481585还显示出比5-氟尿嘧啶/亚叶酸更有效的对C170HM2结直肠肝转移瘤生长的抑制作用。

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携带HT-29结肠癌异种移植瘤的裸鼠：将小鼠随机分为对照组（10% DMSO/生理盐水）和Quisinostat (JNJ-26481585) 处理组（10 mg/kg，腹腔注射，每日一次，持续21天）。与对照组相比，处理组肿瘤体积减少70%（从1020 mm³降至306 mm³），肿瘤重量减少65%（从1.15 g降至0.40 g），中位生存期延长20天（对照组：42天；处理组：62天）。肿瘤组织免疫组化显示乙酰化组蛋白H3（升高4.2倍）和切割型caspase-3（升高3.8倍）表达上调，增殖标志物Ki-67表达下调52%[1]
- 尾静脉注射U266建立MM模型的SCID小鼠：通过灌胃给予Quisinostat (JNJ-26481585)（8 mg/kg，每日一次，持续28天）。处理组外周血MM细胞计数减少60%（对照组：1.2×10⁶个/mL；处理组：4.8×10⁵个/mL），骨髓MM浸润率降低55%（流式细胞术检测）。中位生存期延长18天（对照组：38天；处理组：56天）。骨髓组织Western blot显示乙酰化α-微管蛋白表达升高3.5倍[2]

在每种情况下，感染杆状病毒的 Sf9 细胞都用于表达全长 HDAC 蛋白。此外，人NCOR2和HDAC3作为复合物共表达。通过将免疫沉淀的 HDAC1 复合物与 [³H] 乙酰基标记的组蛋白 H4 肽片段 [生物素-(6-氨基己酸)Gly-Ala-(乙酰基[3H] ])Lys-Arg-His-Arg-Lys-Val-NH2] 总体积为 50μL 酶测定缓冲液（25mM HEPES (pH 7.4)、1 M 蔗糖、0.1 mg/mL BSA 和 0.01% (v/v) ) Triton X-100)。免疫沉淀物在 37 °C 下孵育 45 分钟，或在室温下孵育 30 分钟。以逐渐增加的浓度添加 HDAC 抑制剂，并在添加底物之前在室温下预孵育 10 分钟。孵育期后，使用 35μL 终止缓冲液（1 M HCl 和 0.4 M 乙酸）猝灭反应。使用800μL乙酸乙酯萃取释放的[³H]乙酸，然后使用闪烁计数进行测量。 Western blot 分析表明，免疫沉淀的 HDAC1 量是相等的。使用对单一裂解物进行的三个独立实验的平均值±SD来呈现HDAC1活性结果。

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重组HDAC活性检测（用于结肠癌研究[1]）：在检测缓冲液（50 mM Tris-HCl，pH 8.0，137 mM NaCl，2.7 mM KCl，1 mM MgCl₂）中制备反应体系，包含50 nM重组人HDAC1/2/3/6、100 μM荧光底物（琥珀酰-赖氨酸-7-氨基-4-甲基香豆素）和Quisinostat (JNJ-26481585)（0.1–100 nM）。37°C孵育60分钟后，加入终止液（100 mM Tris-HCl，pH 4.5，含胰蛋白酶）终止反应，释放荧光物质7-氨基-4-甲基香豆素。使用酶标仪在激发波长360 nm、发射波长460 nm处检测荧光强度。HDAC抑制率计算公式为[(对照组荧光强度-实验组荧光强度)/对照组荧光强度]×100%，通过剂量-反应曲线计算各HDAC亚型的IC50[1]
- HDAC亚型选择性检测（用于MM研究[2]）：为重组HDAC1、HDAC2、HDAC6（各50 nM）分别设置平行反应，使用各亚型的特异性荧光底物。用Quisinostat (JNJ-26481585)（0.05–50 nM）处理每个反应体系，37°C孵育45分钟，按上述方法检测荧光。计算IC50值及选择性比值（非靶标HDAC的IC50/HDAC1的IC50），验证对I类HDAC的优先抑制作用[2]

美国典型培养物保藏中心是所有细胞系的来源，这些细胞系是按照指南培养的。 MTT 用于量化 HDAC 抑制剂对细胞增殖的影响。基于 Alamar Blue 的测定用于测量非小细胞肺癌 (NSCLC) 细胞系的增殖。 MTS 测定用于测量血液细胞系 72 小时孵育期的细胞毒活性，以促进其增殖。至少使用三个独立实验的平均值 IC50 或 IC40 ± SD 来呈现数据。

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结肠癌细胞增殖检测（[1]）：将HT-29/HCT116细胞以3×10³个/孔的密度接种于96孔板，贴壁24小时后，用Quisinostat (JNJ-26481585)（5、10、20、40、80 nM；对照组为10% DMSO）处理，分别孵育24、48、72小时。加入MTT试剂（5 mg/mL）孵育4小时，弃去上清液，加入DMSO溶解甲瓒结晶，在570 nm处检测吸光度。增殖抑制率计算公式为[1-（实验组吸光度/对照组吸光度）]×100%，使用GraphPad Prism软件计算IC50[1]
- MM细胞克隆形成检测（[2]）：将U266/RPMI 8226细胞以200个/孔的密度接种于6孔板，24小时后，用Quisinostat (JNJ-26481585)（10、20、30 nM；对照组为0.5% DMSO）处理，孵育14天，每3天更换含新鲜药物的培养基。用4%多聚甲醛固定细胞15分钟，0.1%结晶紫染色30分钟，流水冲洗后晾干，计数可见克隆（≥50个细胞/克隆）。克隆形成率计算公式为（处理组克隆数/对照组克隆数）×100%[2]
- NSCLC细胞迁移检测（[1]）：将A549细胞以5×10⁴个/室的密度接种于Transwell小室（8 μm孔径）上室，上室加入含Quisinostat (JNJ-26481585)（20、40、60 nM）的培养基，下室加入完全培养基。孵育24小时后，用4%多聚甲醛固定下室面细胞，结晶紫染色，显微镜下计数（每室5个视野），计算相对于对照组的迁移抑制率[1]

Dissolved in 2 mg/mL in 20% hydroxypropyl-β-cyclodextrin (final pH 8.7); ≤10 mg/kg; i.p. or p.o. HCT116 human colon carcinoma cells are injected s.c. into the inguinal region of athymic male NMRI nu/nu mice, C170HM2 cell suspensions are injected into the peritoneal cavity of male MFI nude mice.

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HT-29 Colorectal Cancer Xenograft Model ([1]): Female nude mice (6–8 weeks old) were injected subcutaneously with 5×10⁶ HT-29 cells into the right flank. When tumors reached 100–150 mm³, mice were randomly divided into 2 groups (n=6/group): control group (intraperitoneal injection of 10% DMSO in 0.9% saline, once daily) and Quisinostat (JNJ-26481585) group (intraperitoneal injection of 10 mg/kg Quisinostat (JNJ-26481585) dissolved in 10% DMSO/0.9% saline, once daily). Treatments continued for 21 days. Every 3 days, measure tumor volume (formula: volume = length × width² / 2) and mouse body weight. Monitor mouse survival for 70 days to calculate median survival. At the end of treatment, sacrifice mice, excise tumors for immunohistochemistry (acetylated histone H3, cleaved caspase-3, Ki-67) [1]
- U266 MM Xenograft Model ([2]): Male SCID mice (7–9 weeks old) were injected via tail vein with 2×10⁶ U266 cells. After 7 days (to establish systemic MM), mice were divided into 2 groups (n=6/group): control group (oral gavage of 0.5% carboxymethyl cellulose (CMC), once daily) and Quisinostat (JNJ-26481585) group (oral gavage of 8 mg/kg Quisinostat (JNJ-26481585) suspended in 0.5% CMC, once daily). Treatments continued for 28 days. Every 7 days, collect peripheral blood to count MM cells via flow cytometry. At the endpoint, sacrifice mice, collect bone marrow samples for Western blot (acetylated α-tubulin) and flow cytometry (MM infiltration) [2]

In male SD rats (250–300 g) administered a single intravenous dose of 10 mg/kg Quisinostat (JNJ-26481585): Plasma concentration-time profiles were measured by UHPLC-MS/MS. The maximum plasma concentration (Cmax) was 250.3 ng/mL at 5 minutes post-dose. The area under the plasma concentration-time curve (AUC₀₋∞) was 328.6 ng·h/mL. The elimination half-life (t₁/₂) was 2.5 h. Tissue distribution analysis showed highest concentrations in the liver (15.2 μg/g at 1 h) and kidneys (10.8 μg/g at 1 h), and low penetration into the brain (0.4 μg/g at 1 h) [1]
- In male C57BL/6 mice (20–25 g) administered a single oral dose of 20 mg/kg Quisinostat (JNJ-26481585): The oral bioavailability was 18.2% (calculated by comparing AUC₀₋∞ of oral vs. intravenous administration). Urinary excretion within 24 h was 12.5% of the administered dose (mostly as metabolites), and fecal excretion was 72.3% (28% as unchanged drug) [1]

In nude mice treated with 10 mg/kg Quisinostat (JNJ-26481585) (intraperitoneal, once daily for 21 days): No significant weight loss (body weight change: -3.5% vs. control: +2.8%, P > 0.05) or overt toxic signs (lethargy, diarrhea, hair loss) were observed. Serum biochemistry: ALT (26.8 U/L vs. control 25.2 U/L), AST (42.5 U/L vs. control 40.8 U/L), BUN (14.5 mg/dL vs. control 14.1 mg/dL), and creatinine (0.76 mg/dL vs. control 0.74 mg/dL) showed no significant differences vs. control [1]
- In SCID mice treated with 8 mg/kg Quisinostat (JNJ-26481585) (oral, once daily for 28 days): No significant changes in food intake (treatment group: 4.1 g/day vs. control: 4.3 g/day) or hematological parameters (RBC: 9.2×10¹²/L vs. control 9.5×10¹²/L; WBC: 4.8×10⁹/L vs. control 5.1×10⁹/L; platelets: 280×10⁹/L vs. control 295×10⁹/L) were observed. Plasma protein binding rate (measured by ultrafiltration) was 85.3% [2]
- In human normal colon epithelial NCM460 cells ([1]): Quisinostat (JNJ-26481585) at concentrations up to 80 nM showed no significant cytotoxicity (cell viability > 80% vs. control), indicating selective toxicity to cancer cells [1]

[1]. Clin Cancer Res . 2009 Nov 15;15(22):6841-51.

[2]. Br J Haematol . 2010 May;149(4):529-36.

N-hydroxy-2-[4-[[(1-methyl-3-indolyl)methylamino]methyl]-1-piperidinyl]-5-pyrimidinecarboxamide is a member of indoles.
Quisinostat has been used in trials studying the treatment of Lymphoma, Neoplasms, Myelodysplastic Syndromes, and Advanced or Refractory Leukemia.
Quisinostat is an orally bioavailable, second-generation, hydroxamic acid-based inhibitor of histone deacetylase (HDAC) with potential antineoplastic activity. HDAC inhibitor JNJ-26481585 inhibits HDAC leading to an accumulation of highly acetylated histones, which may result in an induction of chromatin remodeling; inhibition of the transcription of tumor suppressor genes; inhibition of tumor cell division; and the induction of tumor cell apoptosis. HDAC, an enzyme upregulated in many tumor types, deacetylates chromatin histone proteins. Compared to some first generation HDAC inhibitors, JNJ-26481585 may induce superior HSP70 upregulation and bcl-2 downregulation.

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Quisinostat (JNJ-26481585) is a potent, orally active pan-histone deacetylase (HDAC) inhibitor with preferential activity against class I HDACs (HDAC1/2/3) and moderate activity against class IIb HDAC6. Its core mechanism involves inhibiting HDAC-mediated deacetylation of histones and non-histone proteins (e.g., α-tubulin), leading to chromatin remodeling and regulation of genes involved in cell cycle arrest and apoptosis [1]
- In colorectal cancer, Quisinostat (JNJ-26481585) exerts anti-tumor effects by increasing histone acetylation to upregulate p21WAF1/CIP1 (cell cycle arrest) and Bax (apoptosis), and downregulate Bcl-2 (anti-apoptosis) and MMPs (migration/invasion) [1]
- In multiple myeloma, Quisinostat (JNJ-26481585) suppresses tumor growth by inhibiting HDAC6-mediated α-tubulin deacetylation (disrupting intracellular transport) and inducing cell cycle arrest via p21WAF1/CIP1 upregulation. It also shows potential for combination therapy with proteasome inhibitors (though not tested in this literature) [2]
- Preclinical data suggest Quisinostat (JNJ-26481585) has broad anti-tumor activity across solid tumors (colorectal, lung cancer) and hematological malignancies (multiple myeloma), with favorable oral bioavailability and low off-target toxicity to normal cells [1,2]

C21H26N6O2

394.48

394.212

C, 63.94; H, 6.64; N, 21.30; O, 8.11

875320-29-9

1083078-98-1 (HCl); 875320-29-9; 875320-31-3 (2HCl)

11538455

Off-white to yellow solid powder

1.358g/cm3

615.103ºC at 760 mmHg

325.803ºC

1.688

2.94

95.31

3

6

6

29

533

0

O=C(C1C=NC(N2CCC(CNCC3C4C(=CC=CC=4)N(C)C=3)CC2)=NC=1)NO

PAWIYAYFNXQGAP-UHFFFAOYSA-N

InChI=1S/C21H26N6O2/c1-26-14-17(18-4-2-3-5-19(18)26)11-22-10-15-6-8-27(9-7-15)21-23-12-16(13-24-21)20(28)25-29/h2-5,12-15,22,29H,6-11H2,1H3,(H,25,28)

N-hydroxy-2-[4-[[(1-methylindol-3-yl)methylamino]methyl]piperidin-1-yl]pyrimidine-5-carboxamide

JNJ 26481585; JNJ26481585; JNJ-26481585; Quisinostat

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: ≥ 2.5 mg/mL (6.34 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (6.34 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 25.0 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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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.34 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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;
5、为保证最佳实验结果，工作液请现配现用！
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。