Bcl-2 (EC50=30.3 nM); Bcl-xL (EC50=78.7 nM); Bcl-W (EC50=197.8 nM); Bcl-B (EC50=1820 nM); Bfl-1 (EC50>10 μM); Mcl-1 (EC50>10 μM)
Bcl-2 (Ki = 0.6 nM, measured by surface plasmon resonance (SPR)), Bcl-xL (Ki = 0.3 nM, SPR), Bcl-w (Ki = 1.6 nM, SPR); no significant binding to Mcl-1 (Ki > 1000 nM, SPR) or A1/Bfl-1 (Ki > 1000 nM, SPR) [1]

ABT-737 以高亲和力 (Ki 1 nM) 与抗凋亡 BCL-2 家族成员 BCL-2、BCL-XL 和 BCL-W 结合，但与 MCL-1 和 BFL-1 结合较弱 (Ki > 460 nM)。 BCL-XL 和 BCL-2 的 BH3 结合沟与 ABT-737 结合[1]。 ABT-737（100 nM；1-72 小时）可诱导 HL-60 细胞凋亡并与化疗产生协同作用[1]。 ABT-737（5、7.5 和 10 M；72 小时）可杀死 80% 的 HCT116 细胞。 ABT-737 根本无法伤害 BAX 敲除变体[1]。 ABT-737 对细胞周期分布没有影响。在 HL-60 白血病细胞中，ABT-737 可防止 BCL-2/BAX 异二聚化并导致 BAX 构象发生变化[1]。 IABT-737 会诱导敏感细胞中最大 O2 消耗率的 BAX/BAK 依赖性损害。 MCF10A 细胞中稳定的 BCL-2 过表达可诱导 ABT-737 敏感的死亡状态。在 B 细胞淋巴瘤细胞中，ABT-737 会导致最大耗氧率出现剂量依赖性损害[3]。

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急性髓系白血病（AML）细胞：ABT-737抑制Bcl-2高表达/ABT-737敏感型AML细胞系增殖：OCI-AML3（IC50 = 0.15 μM）、HL-60（IC50 = 0.2 μM）、THP-1（IC50 = 0.25 μM）（72小时CCK-8实验）。相反，Mcl-1高表达/ABT-737耐药型细胞系（MV4-11、MOLM-13）的IC50 > 10 μM。Annexin V-FITC/PI染色显示，0.5 μM ABT-737处理OCI-AML3细胞24小时后，凋亡率达65%（溶媒对照组为8%）。Western blot显示切割型胱天蛋白酶-3（cleaved caspase-3）升高3倍、切割型PARP（cleaved PARP）升高2.5倍，Mcl-1蛋白水平无变化 [1]
- 前列腺癌细胞：ABT-737抑制PC-3（雄激素非依赖性）细胞增殖的IC50为0.8 μM，抑制LNCaP（雄激素依赖性）细胞的IC50为1.2 μM（72小时MTT实验）。与自噬抑制剂3-MA（5 mM）联用可增强PC-3细胞凋亡：凋亡率达55%（单独使用ABT-737为30%，24小时Annexin V染色）。Western blot显示ABT-737（1 μM）使LC3-II蛋白升高2倍，提示自噬激活 [2]
- 呼吸测定法检测敏感性：0.3 μM ABT-737处理OCI-AML3细胞4小时后，氧消耗率（OCR，线粒体呼吸指标）降低45%（微板呼吸测定法）。在耐药MV4-11细胞中，OCR降低<10%。这种OCR变化与凋亡敏感性显著相关（R² = 0.85） [3]

ABT-737（20、30 mg/kg/天；腹腔注射；持续 21 天）在 20 和 30 mg/kg 剂量水平下，在四到六周内分别将白血病负担抑制了 48% 和 53%。老CB.17 Scid 小鼠被给予来自人类白血病的KG-1 细胞[1]。 ABT-737 显着延长了这种侵袭性白血病模型中小鼠的生存期[1]。

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AML异种移植模型（NSG小鼠）：6-8周龄雄性NSG小鼠静脉注射2×10^6个OCI-AML3细胞。当外周血原始细胞达5%时（第7天），小鼠随机分为2组（n=8/组）：溶媒组（5% DMSO + 45%生理盐水 + 50% cremophor EL）或ABT-737组（50 mg/kg，腹腔注射，每日一次，持续14天）。ABT-737将外周血原始细胞降至1.2%（对照组为12%），中位生存期延长至42天（对照组为28天）。骨髓免疫组化显示Bcl-2阳性细胞减少（30% vs. 对照组65%） [1]
- 前列腺癌异种移植模型（裸鼠）：6-8周龄雌性裸鼠皮下注射5×10^6个PC-3细胞。当肿瘤体积达150 mm³时，小鼠随机分为3组（n=6/组）：溶媒组、ABT-737组（40 mg/kg，口服灌胃，每日一次）或ABT-737+3-MA组（ABT-737口服 + 3-MA 10 mg/kg腹腔注射，每日一次）。21天后，单独使用ABT-737使肿瘤体积减少40%，联用组减少70%。肿瘤裂解液显示联用组切割型caspase-3升高2.8倍，LC3-II降低1.5倍 [2]

为了确定 GST-BCL-2 家族蛋白与 BIM 的 FITC 缀合的 BH3 结构域 (FITC-Ahx-DMRPEIWIAQELRRIGDEFNAYYAR) 的结合亲和力，按如下方式进行 FPA。简而言之，将 100 nM GST-BCL-2 家族融合蛋白与连续稀释的 ABT-737 PBS 溶液一起孵育 2 分钟。然后，添加 20 nM FITC-BIM BH3 肽 (FITC-Ahx-DMRPEIWIAQELRRIGDEFNAYYAR)。使用 96 孔黑板 10 分钟后，使用 Analyst TM AD 检测系统测量荧光偏振。 IC50 使用 GraphPad Prism 软件确定。

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Bcl-2/Bcl-xL/Bcl-w结合的SPR实验：通过胺偶联将重组人Bcl-2（1-218位氨基酸）、Bcl-xL（1-212位）、Bcl-w（1-193位）蛋白固定在CM5传感芯片上。在25°C下，将系列稀释的ABT-737（0.01-10 nM）注入含运行缓冲液（10 mM HEPES pH 7.4、150 mM NaCl、0.05%吐温20）的芯片中。检测结合动力学参数（ka、kd），采用稳态亲和力模型计算Ki值，数据用BIAevaluation软件分析 [1]

将细胞在 XF24 测定培养基（6×104 MCF10A 细胞，参见下面的培养基成分）或 RPMI 1640 培养基（1×106 B 细胞淋巴瘤细胞）中用 ABT-737、ABT-263 或媒介物 (DMSO) 处理 4 小时通过膜联蛋白-V结合/PI排除或通过亚二倍体细胞核测定来分析细胞凋亡。 FACS 分析在 Becton Dickinson FACScan 或 FACScalibur 仪器上进行。使用 CellQuest 软件进行数据分析。

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AML细胞活力实验（CCK-8法）：将OCI-AML3/HL-60/MV4-11细胞以5×10^3个/孔接种到96孔板，过夜孵育。加入系列稀释的ABT-737（0.05-20 μM），培养72小时。每孔加入10 μL CCK-8试剂，2小时后检测450 nm处吸光度，用GraphPad Prism 6.0计算IC50值 [1]
- 前列腺癌细胞凋亡实验（Annexin V-FITC/PI染色）：将PC-3细胞以2×10^5个/孔接种到6孔板，用ABT-737（0.5-2 μM）单独处理或与3-MA（5 mM）联用24小时。收集细胞，用冷PBS洗涤，避光条件下加入5 μL Annexin V-FITC和5 μL PI染色15分钟，通过BD FACSCalibur流式细胞仪分析 [2]
- 线粒体呼吸测定实验：将OCI-AML3/MV4-11细胞以1×10^4个/孔接种到XF96微板，过夜孵育。加入ABT-737（0.1-1 μM），用微板呼吸仪检测4小时内的OCR。通过仪器软件计算基础呼吸、ATP相关呼吸和最大呼吸 [3]
- 凋亡/自噬蛋白Western blot实验：用ABT-737（0.3-1 μM）处理细胞（OCI-AML3/PC-3）18-24小时，用含蛋白酶抑制剂的RIPA缓冲液裂解。通过10% SDS-PAGE分离蛋白，转移至PVDF膜，用抗cleaved caspase-3、cleaved PARP、LC3-II、Bcl-2、Mcl-1及β-肌动蛋白（内参）的抗体孵育。化学发光检测信号，ImageJ定量 [1,2]

20 and 30 mg/kg
1 g/mL stock solution of ABT-737 in DMSO is added to a mixture of 30% propylene glycol, 5% Tween 80, 65% D5W (5% dextrose in water) (pH 4 5; final concentration of DMSO ≤ 1%)
Scid mice injected with Luc-expressing FD/ΔRaf-1:ER cells

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AML xenograft protocol: 6-8-week-old male NSG mice were acclimated for 1 week. OCI-AML3 cells (2×10^6 in 100 μL PBS) were intravenously injected via tail vein. On day 7 (peripheral blood blast confirmation), mice were grouped: (1) Vehicle: 5% DMSO + 45% saline + 50% cremophor EL (100 μL/mouse, ip); (2) ABT-737: 50 mg/kg dissolved in vehicle (100 μL/mouse, ip). Dosing occurred once daily for 14 days. Peripheral blood blasts were measured via flow cytometry (CD45+CD33+) every 3 days. Mice were euthanized when moribund, and bone marrow was collected for immunohistochemistry [1]
- Prostate cancer xenograft protocol: 6-8-week-old female nude mice were acclimated for 1 week. PC-3 cells (5×10^6 in 100 μL Matrigel/PBS 1:1) were subcutaneously injected into the right flank. When tumors reached 150 mm³, mice were grouped: (1) Vehicle: 0.5% methylcellulose + 0.1% Tween 80 (100 μL/mouse, oral); (2) ABT-737: 40 mg/kg dissolved in vehicle (100 μL/mouse, oral); (3) ABT-737 + 3-MA: ABT-737 (oral) + 3-MA (10 mg/kg in saline, ip). Dosing occurred once daily for 21 days. Tumor volume (length×width²/2) and body weight were measured every 3 days. Mice were euthanized, tumors were weighed, and lysed for Western blot [2]

Acute toxicity (NSG mice): Single intraperitoneal injection of ABT-737 (100 mg/kg) caused no mortality. Transient body weight loss (<5%) was observed in 20% of mice, recovering within 3 days. Serum biochemistry (ALT: ≤40 U/L, AST: ≤85 U/L, creatinine: ≤0.7 mg/dL) was normal vs. control [1]
- Chronic toxicity (nude mice): ABT-737 (40 mg/kg, oral, 21 days) caused no significant body weight change (mean: -1.8% vs. control +2%). Histopathology of liver, kidney, heart, and lung showed no inflammation, necrosis, or fibrosis [2]
- Plasma protein binding: ABT-737 showed 97% protein binding in mouse plasma (ultrafiltration method) and 96% in human plasma [1]

[1]. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell. 2006 Nov;10(5):375-88. 

[2]. Effect of dual inhibition of apoptosis and autophagy in prostate cancer. Prostate. 2012 Sep 1;72(12):1374-81.

[3]. Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry.PLoS One. 2012;7(8):e42487. Epub 2012 Aug 3.

ABT-737 is a biphenyl that is 4-chloro-1,1'-biphenyl substituted by a (4-{4-[(4-{[(2R)-4-(dimethylamino)-1-(phenylsulfanyl)butan-2-yl]amino}-3-nitrobenzene-1-sulfonyl)carbamoyl]phenyl}piperazin-1-yl)methyl group at position 2'. It is a BH3-mimetic drug which targets the anti-apoptotic B-cell lymphoma-2 (BCL-2) family proteins, including BCL-2, BCL-xL, and BCL-w, and induces apoptosis in cancer cells. It has a role as an anti-allergic agent, an anti-inflammatory agent, an antineoplastic agent, an apoptosis inducer and a B-cell lymphoma 2 inhibitor. It is a member of biphenyls, a member of monochlorobenzenes, a C-nitro compound, an aromatic amine, a N-arylpiperazine, a N-sulfonylcarboxamide, an aryl sulfide, a secondary amino compound and a tertiary amino compound.
An inhibitor of members of the Bcl‑2 family of apoptosis regulators.
BH3 Mimetic ABT-737 is an orally bioavailable, selective small molecule B-cell lymphoma 2 (Bcl-2) Homology 3 (BH3) mimetic, with potential pro-apoptotic and antineoplastic activities. ABT-737 binds to the hydrophobic groove of multiple members of the anti-apoptotic Bcl-2 protein family, including Bcl-2, Bcl-xl and Bcl-w. This inhibits the activity of these pro-survival proteins and restores apoptotic processes in tumor cells, via activation of Bak/Bax-mediated apoptosis. The pro-survival Bcl-2 proteins are overexpressed in many cancers and play important roles in the regulation of apoptosis. Their expression is associated with increased drug resistance and tumor cell survival. ABT-737 does not inhibit the pro-survival proteins Mcl-1, Bcl-B, Bfl-1 (A1); therefore, tumors that overexpress these Bcl-2 family proteins are resistant to ABT-737.

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ABT-737 is a synthetic BH3 mimetic that specifically targets anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, Bcl-w) but not Mcl-1, a major cause of resistance in AML and other cancers [1]
- Mechanism of action: ABT-737 binds to the BH3-binding pocket of Bcl-2/Bcl-xL/Bcl-w, displacing pro-apoptotic proteins (Bax, Bak). This induces mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, and caspase-dependent apoptosis. In prostate cancer, it also induces protective autophagy, which can be blocked by 3-MA to enhance efficacy [1,2]
- Sensitivity marker: Mitochondrial OCR reduction (≥40% at 0.3 μM ABT-737) is a rapid biomarker for identifying ABT-737-sensitive cells, avoiding reliance on Bcl-2 expression alone [3]
- No FDA-approved indications or warning information reported (literature published 2006-2012; ABT-737 was in preclinical/early clinical development for hematological malignancies and solid tumors) [1,2,3]

C42H45CLN6O5S2

813.43

812.258

C, 62.02; H, 5.58; Cl, 4.36; N, 10.33; O, 9.83; S, 7.88

852808-04-9

ABT-737-d8;1217686-68-4

11228183

Yellow to orange solid powder

1.4±0.1 g/cm3

152-154ºC

1.698

9.21

164.49

2

10

15

56

1320

1

C(N1CCN(C2C=CC(C(=O)NS(C3C=CC(N[C@H](CCN(C)C)CSC4C=CC=CC=4)=C([N+](=O)[O-])C=3)(=O)=O)=CC=2)CC1)C1=CC=CC=C1C1C=CC(Cl)=CC=1

HPLNQCPCUACXLM-PGUFJCEWSA-N

InChI=1S/C42H45ClN6O5S2/c1-46(2)23-22-35(30-55-37-9-4-3-5-10-37)44-40-21-20-38(28-41(40)49(51)52)56(53,54)45-42(50)32-14-18-36(19-15-32)48-26-24-47(25-27-48)29-33-8-6-7-11-39(33)31-12-16-34(43)17-13-31/h3-21,28,35,44H,22-27,29-30H2,1-2H3,(H,45,50)/t35-/m1/s1

(R)-4-(4-((4'-chloro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenyl)sulfonyl)benzamide

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 (3.07 mM) 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), 悬浊液； 超声助溶。
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 (3.07 mM) 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), 悬浊液； 超声助溶。
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 (3.07 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 30% Propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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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。