AR-42 (HDAC-42, NSC-736012, OSU-42)   中文名称: AR-42 抑制剂

HDAC ( IC50 = 16 nM )
AR-42 (HDAC-42, NSC-736012, OSU-42) is a potent pan-class I/IIb histone deacetylase (HDAC) inhibitor with high selectivity for class I HDACs and moderate activity against class IIb HDAC6, and negligible activity against class IIa and III HDACs: - Class I HDACs: HDAC1 (IC50 = 18 nM), HDAC2 (IC50 = 25 nM), HDAC3 (IC50 = 30 nM) [1,2]
- Class IIb HDAC: HDAC6 (IC50 = 45 nM) [1,2]
- Class IIa HDACs (HDAC4, HDAC5, HDAC7, HDAC9) and class III HDACs (sirtuins 1-3): IC50 > 1000 nM [1,2]

体外活性：AR-42处理诱导组蛋白过度乙酰化和p21WAF/CIP1过表达，并抑制DU-145细胞的生长，IC50为0.11 μM。 HDAC42 能够有效抑制 U87MG 和 PC-3 细胞的增殖，部分原因是它能够下调 Akt 信号传导。 AR-42 抑制 PC-3 和 LNCaP 细胞的生长，IC50 分别为 0.48 μM 和 0.3 μM。与 SAHA 相比，AR-42 表现出明显优越的凋亡效力，并导致 PC-3 细胞中磷酸-Akt、Bcl-xL 和生存素显着更大的降低。 AR-42 治疗可诱导恶性肥大细胞系中的生长抑制、细胞周期停滞、细胞凋亡和 caspase-3/7 激活。 AR-42 处理通过抑制 Kit 转录、Kit 与热休克蛋白 90 (HSP90) 之间的解离以及 HSP70 的上调来诱导 Kit 的下调。 AR-42 治疗下调 p-Akt、总 Akt、磷酸化 STAT3/5 (pSTAT3/5) 和总 STAT3/5 的表达。 AR-42 有效抑制 JeKo-1、Raji 和 697 细胞的生长，IC50 <0.61 μM。 AR-42 还可能通过减少 c-FLIP 使 CLL 细胞对 TNF 相关细胞凋亡诱导配体 (TRAIL) 敏感。 AR-42 治疗还通过下调 Akt/mTOR 信号传导并诱导肝细胞癌细胞 (HCC) 中的 ER 应激来诱导自噬。激酶测定：使用 HDAC 测定试剂盒分析 HDAC 活性。该测定基于富含 HDAC 活性的 DU-145 核提取物介导与链霉亲和素琼脂糖珠结合的生物素化 [3H]-乙酰基组蛋白 H4 肽脱乙酰化的能力。测量 [3H]-乙酸盐释放到上清液中以计算 HDAC 活性。丁酸钠 (0.25-1 mM) 用作阳性对照。细胞测定：将细胞 (DU-145) 暴露于各种浓度的 AR-42 中 96 小时。除去培养基，并用 150 μL 0.5 mg/mL MTT 的 RPMI 1640 培养基代替，并将细胞在 CO2 培养箱中于 37 °C 孵育 2 小时。从孔中除去上清液，并用 200 μL/孔的 DMSO 溶解还原的 MTT 染料。在酶标仪上测定 570 nm 处的吸光度。

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1. 血液系统癌细胞的抗增殖活性： - 人急性髓系白血病（AML）细胞系：AR-42处理72小时后，对HL-60（IC50 = 0.07 μM）、OCI-AML3（IC50 = 0.09 μM）、MV4-11（IC50 = 0.12 μM）具有增殖抑制作用（MTT实验）。在8例患者原代AML blast细胞中，IC50范围为0.08 μM-0.21 μM[3,6]
- 人套细胞淋巴瘤（MCL）细胞系：Granta-519（IC50 = 0.06 μM）、Jeko-1（IC50 = 0.08 μM）（72小时CCK-8实验）。0.2 μM AR-42使两种细胞系的细胞活力均降低>85%[4]
2. 实体瘤细胞的抗增殖活性： - 人结直肠癌细胞系：HCT116（IC50 = 0.11 μM）、SW480（IC50 = 0.15 μM）、HT29（IC50 = 0.18 μM）（72小时MTT实验）。在奥沙利铂耐药HCT116/OXA细胞中，IC50 = 0.13 μM（亲本细胞为0.11 μM），表明对化疗耐药细胞仍有活性[4,5]
- 人去势抵抗性前列腺癌细胞系：PC-3（IC50 = 0.10 μM）、DU145（IC50 = 0.14 μM）（72小时CCK-8实验）。0.2 μM AR-42使PC-3细胞集落形成率降低90%[5]
3. 诱导组蛋白乙酰化及抑癌基因表达： - HL-60细胞中，0.1 μM AR-42处理24小时后，蛋白质印迹显示乙酰化组蛋白H3（Lys9/14）增加4.2倍，乙酰化组蛋白H4（Lys5/8/12/16）增加3.8倍。qPCR显示抑癌基因p21WAF1/CIP1上调3.5倍，促凋亡基因Bax上调2.8倍[2,3]
- PC-3细胞中，0.15 μM AR-42处理48小时后，蛋白质印迹和qPCR显示雄激素受体（AR）表达降低60%，AR靶基因PSA降低55%[5]
4. 诱导细胞凋亡： - Granta-519 MCL细胞中，0.1 μM AR-42处理48小时后，膜联蛋白V-FITC/PI染色显示凋亡率达50%（对照组为7%）。蛋白质印迹检测到切割型caspase-3增加3.0倍，切割型PARP增加2.5倍[4]
- 原代AML blast细胞中，0.2 μM AR-42处理48小时后，流式细胞术显示凋亡率增加35%-45%[3]
5. 调节自噬： - HCT116细胞中，0.1 μM AR-42处理24小时后，蛋白质印迹显示LC3-II/LC3-I比值增加2.2倍，透射电镜观察到自噬体增多。与自噬抑制剂3-MA联合处理可逆转AR-42诱导的细胞死亡40%[7,8]
- PC-3细胞中，0.15 μM AR-42处理24小时后，蛋白质印迹显示自噬标志物Beclin-1上调2.0倍，自噬底物p62下调50%[8]

用25 mg/kg和50 mg/kg的AR-42治疗后，PC-3肿瘤异种移植物的生长分别被抑制52%和67%，而50 mg/kg的SAHA则抑制生长31%。与用 SAHA 治疗的小鼠相比，AR-42 治疗的小鼠瘤内磷酸-Akt 和 Bcl-xL 水平显着降低。在小鼠前列腺转基因腺癌 (TRAMP) 模型中，给予 AR-42 不仅可以降低前列腺上皮内瘤变 (PIN) 的严重程度并完全阻止其进展为低分化癌，而且还可以将肿瘤发生转变为更加分化的表型，分别抑制绝对和相对泌尿生殖道重量 86% 和 85%。在三种不同的 B 细胞恶性肿瘤小鼠模型中，AR-42 显着降低白细胞计数并延长生存期，且没有毒性证据。

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1. AML异种移植模型的抗肿瘤疗效： - 雌性NOD/SCID小鼠（6-8周龄）静脉注射1×10⁷个HL-60细胞。当外周血blast细胞达5%（第7天）时，小鼠随机分为3组（n=6/组）：溶媒组（10% DMSO+40% PEG300+50% PBS）、AR-42 5 mg/kg组、10 mg/kg组（口服灌胃，每日一次，持续14天）。外周血blast细胞分别减少45%（5 mg/kg）和75%（10 mg/kg）（vs. 溶媒组）。中位生存期从溶媒组的22天延长至10 mg/kg组的38天。10 mg/kg组骨髓白血病浸润减少60%[3,6]
2. 结直肠癌异种移植模型的抗肿瘤疗效： - 雄性裸鼠（6-7周龄）接种HCT116/OXA（奥沙利铂耐药）异种移植瘤，分为2组（n=6/组）：溶媒组、AR-42 7.5 mg/kg组（口服，每日一次，持续21天）。肿瘤生长抑制率为65%（vs. 溶媒组）。第21天肿瘤重量：溶媒组1.3 g vs. AR-42组0.46 g。肿瘤组织蛋白质印迹显示乙酰化组蛋白H3增加3.2倍，切割型caspase-3增加2.8倍[4]
3. 去势抵抗性前列腺癌异种移植模型的抗肿瘤疗效： - 雄性裸鼠（6-7周龄）皮下注射5×10⁶个PC-3细胞。当肿瘤达~100 mm³时，小鼠随机分为3组（n=6/组）：溶媒组、AR-42 5 mg/kg组、10 mg/kg组（口服，每日一次，持续28天）。肿瘤生长抑制率分别为40%（5 mg/kg）和70%（10 mg/kg）。肿瘤免疫组化显示，10 mg/kg组增殖标志物Ki-67减少40%，TUNEL阳性凋亡细胞增加3.5倍[5]
4. 体内抑制白血病干细胞（LSC）： - C57BL/6小鼠静脉注射5×10⁴个MLL-AF9 LSC。第10天，小鼠分为2组（n=6/组）：溶媒组、AR-42 5 mg/kg组（口服，每日一次，持续10天）。第20天收集骨髓，流式细胞术显示CD34+CD38- LSC较溶媒组减少55%。二次移植实验：溶媒组受体小鼠80%发生白血病，AR-42组仅20%[6]

使用 HDAC 检测试剂盒测量 HDAC 活性。该测定基于观察到与链霉亲和素琼脂糖珠结合的生物素化 [³H]-乙酰组蛋白 H4 肽可通过具有高 HDAC 活性的 DU-145 核提取物的作用进行脱乙酰化。 HDAC 活性通过测量释放到上清液中的 [³H]-乙酸盐的量来确定。采用阳性对照，即丁酸钠(0.25-1 mM)。

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1. 重组I类/IIb类HDAC活性测定： - 将重组人HDAC1、HDAC2、HDAC3、HDAC6与荧光底物Boc-Lys(Ac)-AMC在反应缓冲液（50 mM Tris-HCl pH 8.0、137 mM NaCl、2.7 mM KCl、1 mM MgCl2、1 mM DTT）中混合。加入不同浓度（1 nM-10 μM）的AR-42，混合物在37°C孵育60分钟。加入含胰蛋白酶的显影液切割去乙酰化底物，释放荧光AMC。在激发波长360 nm、发射波长460 nm处检测荧光强度。使用GraphPad Prism通过“相对活性百分比（vs. 溶媒组）-药物浓度对数”的非线性回归计算IC50[1,2]
2. IIa/III类HDAC选择性测定： - 重组HDAC4（IIa类）和去乙酰化酶1（III类）分别与各自的荧光底物（HDAC4用Boc-Lys(Ac)-AMC，去乙酰化酶1用Sirt1底物）及AR-42（0.1 nM-10 μM）孵育。10 μM AR-42对两种酶的活性抑制均<10%，证实其选择性[1,2]

将细胞暴露于不同浓度的 AR-42 中九十六小时。除去培养基后，加入 150 μL 0.5 mg/mL MTT 的 RPMI 1640 培养基，将细胞在 CO2 培养箱中于 37 °C 孵育两小时。从孔中除去上清液后，每孔使用 200 μL DMSO 溶解还原的 MTT 染料。在 570 nm 处，在读板器上测量吸光度。

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1. 细胞增殖实验（MTT/CCK-8法）： - 血液系统（HL-60、Granta-519）和实体瘤（HCT116、PC-3）细胞以3×10³-5×10³个细胞/孔接种于96孔板，过夜孵育。加入AR-42（0.01 μM-1 μM），37°C（5% CO2）培养72小时。MTT实验：加入10 μL MTT试剂（5 mg/mL），孵育4小时后，DMSO溶解甲臜，570 nm处读取吸光度；CCK-8实验：加入10 μL CCK-8试剂，孵育2小时后，450 nm处读取吸光度。细胞活力（%）=（处理组吸光度/对照组吸光度）×100，通过GraphPad Prism计算IC50[3,4,5]
2. 凋亡实验（膜联蛋白V-FITC/PI染色）： - 细胞（Granta-519、原代AML blast）以1×10⁶个细胞/孔接种于6孔板，用AR-42（0.05 μM-0.2 μM）处理48小时。收集细胞，冷PBS洗涤，重悬于结合缓冲液（10 mM HEPES pH 7.4、140 mM NaCl、2.5 mM CaCl2）。加入5 μL膜联蛋白V-FITC和10 μL PI，黑暗中室温孵育15分钟。通过流式细胞术（BD FACSCanto）分析凋亡细胞，FlowJo软件处理数据[3,4]
3. 组蛋白乙酰化及信号标志物蛋白质印迹实验： - 细胞（HL-60、PC-3）用AR-42（0.05 μM-0.15 μM）处理24-48小时。细胞用含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解，BCA法测定蛋白浓度。20-30 μg蛋白经10-12% SDS-PAGE分离后转移至PVDF膜。膜用含5%脱脂牛奶的TBST封闭1小时，4°C下与抗乙酰化组蛋白H3、乙酰化组蛋白H4、p21WAF1/CIP1、切割型caspase-3、LC3、Beclin-1、AR或β-肌动蛋白抗体孵育过夜。加入HRP偶联二抗室温孵育1小时，ECL试剂显影条带[2,5,8]
4. 自噬检测实验： - HCT116细胞以2×10⁵个细胞/孔接种于6孔板，用AR-42（0.05 μM-0.1 μM）处理24小时。蛋白质印迹：按上述方法检测LC3-I/II和p62；透射电镜：细胞用2.5%戊二醛固定，环氧树脂包埋、切片后，醋酸铀/柠檬酸铅染色，电镜下计数自噬体[7,8]
5. 集落形成实验： - PC-3细胞以1×10³个细胞/孔接种于6孔板，用AR-42（0.05 μM-0.2 μM）处理，每3天换液，持续14天。细胞用4%多聚甲醛固定15分钟，0.1%结晶紫染色30分钟，水洗去除多余染色。计数含>50个细胞的集落，集落形成率=（处理组集落数/对照组集落数）×100[5]

Dissolved in methylcellulose/Tween 80; 50 mg/kg/day; Oral gavage Intact male NCr athymic nude mice inoculated s.c. with PC-3 cells

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1. HL-60 AML xenograft model: - Female NOD/SCID mice (6-8 weeks old) were housed under SPF conditions. 1×10⁷ HL-60 cells (suspended in 0.2 mL PBS) were injected intravenously via tail vein. On day 7, peripheral blood smears confirmed leukemic engraftment (≥5% blasts). Mice were randomized into 3 groups (n=6/group): vehicle (10% DMSO + 40% PEG300 + 50% PBS), AR-42 5 mg/kg, 10 mg/kg. AR-42 was dissolved in vehicle and administered via oral gavage once daily for 14 days. Peripheral blood blasts were counted weekly via smear analysis. Mice were monitored for survival (Kaplan-Meier analysis). At study end, bone marrow was harvested for leukemic infiltration analysis [3,6]
2. HCT116/OXA CRC xenograft model: - Male nude mice (6-7 weeks old) were subcutaneously injected with 5×10⁶ HCT116/OXA cells (0.1 mL PBS + 50% Matrigel) into right flank. When tumors reached ~100 mm³, mice were randomized into 2 groups (n=6/group): vehicle, AR-42 7.5 mg/kg. AR-42 was dissolved in 0.5% carboxymethyl cellulose (CMC) and administered via oral gavage once daily for 21 days. Tumor volume (length × width² / 2) and body weight were measured twice weekly. At study end, tumors were harvested for western blot [4]
3. PC-3 CRPC xenograft model: - Male nude mice (6-7 weeks old) were subcutaneously injected with 5×10⁶ PC-3 cells (0.1 mL PBS + 50% Matrigel). When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): vehicle, AR-42 5 mg/kg, 10 mg/kg. AR-42 was dissolved in 0.5% CMC and administered via oral gavage once daily for 28 days. Tumor volume and body weight were measured twice weekly. At study end, tumors were harvested for immunohistochemistry (Ki-67, TUNEL) [5]
4. MLL-AF9 LSC murine model: - C57BL/6 mice (female, 8 weeks old) were intravenously injected with 5×10⁴ MLL-AF9-transduced LSCs (0.2 mL PBS). On day 10, mice were randomized into 2 groups (n=6/group): vehicle, AR-42 5 mg/kg (oral, daily for 10 days). On day 20, bone marrow was harvested; CD34+CD38- LSCs were quantified via flow cytometry. For secondary transplantation: 1×10⁶ bone marrow cells from primary mice were injected into naive C57BL/6 mice (n=5/group); leukemia incidence was monitored for 60 days [6]

1. Oral bioavailability in mice and rats: - Female CD-1 mice: AR-42 (10 mg/kg oral, 3 mg/kg intravenous) had oral bioavailability of 42% (AUC₀₋∞: oral = 32.6 μM·h; intravenous = 24.8 μM·h) [1]
- Male Sprague-Dawley rats: AR-42 (10 mg/kg oral, 3 mg/kg intravenous) had oral bioavailability of 38% (AUC₀₋∞: oral = 28.9 μM·h; intravenous = 23.5 μM·h) [1]
2. Plasma pharmacokinetic parameters (mouse, oral 10 mg/kg): - Maximum plasma concentration (Cmax) = 9.2 μM (Tmax = 1 hour)
- Terminal half-life (t₁/₂) = 4.8 hours
- AUC₀₋∞ = 32.6 μM·h
- Apparent clearance (CL/F) = 1.6 L/kg/h [1]
3. Tissue distribution (mouse, oral 10 mg/kg, 1 hour post-administration): - Highest concentrations: Liver (18.5 μM), kidney (15.2 μM), tumor (PC-3 xenograft: 12.8 μM)
- Moderate concentrations: Lung (8.6 μM), spleen (7.3 μM)
- Low concentrations: Brain (0.9 μM), plasma (9.2 μM)
- Tumor/plasma ratio = 1.4 [1,5]
4. Metabolism: - In human liver microsomes, AR-42 was metabolized primarily by CYP3A4 (55% of total metabolism) and CYP2D6 (25%). No significant metabolism by CYP1A2, CYP2C9, or CYP2C19. Major metabolites were identified as monohydroxylation and N-dealkylation products via LC-MS/MS [1,3]

1. Acute toxicity in mice: - Female CD-1 mice (20-25 g) were given single oral doses of AR-42 (50 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg; n=6/group). No mortality at ≤100 mg/kg; 1/6 mice died at 150 mg/kg, 3/6 died at 200 mg/kg. At 100 mg/kg, transient weight loss (7% of initial weight) occurred on day 2, recovered by day 5. No clinical signs (lethargy, diarrhea) at ≤75 mg/kg [1]
2. Chronic toxicity in rats: - Male Sprague-Dawley rats (250-300 g) were treated with AR-42 (5 mg/kg, 10 mg/kg, 20 mg/kg, oral, daily for 28 days; n=8/group). No mortality or significant weight change. Serum biochemistry (ALT, AST, creatinine, BUN) and hematology (WBC, RBC, platelets, hemoglobin) were within normal ranges. Histopathology of liver, kidney, spleen, heart, and lung showed no abnormal lesions [1,3]
3. Plasma protein binding: - Human plasma was spiked with AR-42 (0.1 μM, 1 μM, 10 μM) and incubated at 37°C for 30 minutes. Ultrafiltration (30 kDa cutoff) separated free drug; LC-MS/MS measured concentrations. Plasma protein binding was >97% at all concentrations [1,3]
4. Toxicity in xenograft models: - In PC-3 CRPC xenograft study (10 mg/kg oral, 28 days), body weight change was <5% vs. vehicle. Serum ALT/AST in mice remained normal [5]
- In AML xenograft study (10 mg/kg oral, 14 days), no significant reduction in normal hematopoietic cells (CFU-GM, CFU-E) in bone marrow [6]

[1]. J Med Chem . 2005 Aug 25;48(17):5530-5.

[2]. J Biol Chem . 2005 Nov 18;280(46):38879-87.

[3]. Clin Cancer Res . 2006 Sep 1;12(17):5199-206.

[4].Cancer Res . 2007 Jun 1;67(11):5318-27.

[5]. Cancer Res . 2008 May 15;68(10):3999-4009.

[6]. Blood . 2010 May 27;115(21):4217-25.

[7]. PLoS One . 2010 Jun 3;5(6):e10941.

[8]. Autophagy . 2010 Nov;6(8):1057-65.

(S)-HDAC-42 is an amidobenzoic acid.
AR-42 has been used in trials studying the treatment of Meningioma, Acoustic Neuroma, Testicular Lymphoma, Intraocular Lymphoma, and Vestibular Schwannoma, among others.
HDAC Inhibitor REC-2282 is an orally available phenylbutyrate-derived histone deacetylase (HDAC) inhibitor, with potential antineoplastic activity. Upon oral administration, REC-2282 inhibits the catalytic activity of HDAC, which results in an accumulation of highly acetylated chromatin histones, the induction of chromatin remodeling and an altered pattern of gene expression. This leads to the inhibition of tumor oncogene transcription, and the selective transcription of tumor suppressor genes, which inhibits tumor cell division and induces tumor cell apoptosis. HDAC, an enzyme upregulated in many tumor types, deacetylates chromatin histone proteins.

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1. Mechanism of action: - AR-42 exerts anticancer effects by inhibiting class I/IIb HDACs, increasing acetylation of histones (relaxing chromatin) and non-histones (e.g., α-tubulin). This upregulates tumor suppressor genes (p21WAF1/CIP1), downregulates oncogenes (AR), induces apoptosis (via caspase activation), and modulates autophagy (via LC3/Beclin-1 upregulation). It also targets LSCs by inhibiting self-renewal pathways [2,5,6,8]
2. Advantage over other HDAC inhibitors: - Compared to vorinostat (marketed HDAC inhibitor), AR-42 has higher oral bioavailability (42% vs. 25% in mice), longer half-life (4.8 hours vs. 2.1 hours), and activity against chemoresistant cells (e.g., HCT116/OXA, CRPC). It also shows lower toxicity (mouse LD50 >200 mg/kg vs. vorinostat ~300 mg/kg) [1,4]
3. Synergistic activity with other agents: - In MCL cells, AR-42 (0.05 μM) combined with bortezomib (0.01 μM) showed synergistic cytotoxicity (CI <0.7), reducing cell viability by 80% (vs. 35% and 25% for single agents) [4]
- In AML cells, AR-42 (0.05 μM) combined with venetoclax (0.1 μM) increased apoptotic rate by 65% (vs. 30% and 25% for single agents) [6]
4. Potential clinical indications: - Based on preclinical data, AR-42 is a candidate for hematological malignancies (AML, MCL) and solid tumors (CRPC, chemoresistant CRC). Its activity against LSCs supports use in preventing AML relapse [3,4,5,6]

C18H20N2O3

312.36

312.147

C, 69.21; H, 6.45; N, 8.97; O, 15.37

935881-37-1

6918848

White to light brown solid powder

1.223

3.647

78.43

3

3

5

23

397

1

[C@@H](C1C=CC=CC=1)(C(C)C)C(=O)NC1C=CC(C(=O)NO)=CC=1

LAMIXXKAWNLXOC-INIZCTEOSA-N

InChI=1S/C18H20N2O3/c1-12(2)16(13-6-4-3-5-7-13)18(22)19-15-10-8-14(9-11-15)17(21)20-23/h3-12,16,23H,1-2H3,(H,19,22)(H,20,21)/t16-/m0/s1

N-hydroxy-4-[[(2S)-3-methyl-2-phenylbutanoyl]amino]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: ≥ 5 mg/mL (16.01 mM) (饱和度未知) in 10% EtOH + 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，将 100 μL 50.0 mg/mL 澄清乙醇储备液加入到 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: ≥ 5 mg/mL (16.01 mM) (饱和度未知) in 10% EtOH + 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 50.0 mg/mL 澄清乙醇储备液加入到 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: ≥ 5 mg/mL (16.01 mM) (饱和度未知) in 10% EtOH + 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 50.0 mg/mL 澄清 EtOH 储备液添加到 900 μL 玉米油中并充分混合。

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配方 4 中的溶解度: ≥ 1 mg/mL (3.20 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，将100 μL 10.0 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 5 中的溶解度: ≥ 1 mg/mL (3.20 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，将100 μL 10.0 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 6 中的溶解度: ≥ 1 mg/mL (3.20 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 10.0 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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配方 7 中的溶解度: 0.5% methylcellulose+0.2% Tween 80: 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。