Anacetrapib (MK-0859)   中文名称: 安塞曲匹

Cholesteryl ester transfer protein (CETP); recombinant human (rh) CETP (IC50 = 7.9 nM)[1]; CETPC13S (IC50 = 11.8 nM)[2]
Selective inhibitor of cholesteryl ester transfer protein (CETP) with the following inhibitory parameters:
- IC50 = 11 nM (recombinant human CETP), IC50 = 14 nM (mouse plasma CETP) [1]
- Ki = 8.5 nM (recombinant human CETP), showing high affinity and no significant binding to other lipid-related proteins (e.g., lipoprotein lipase, lecithin-cholesterol acyltransferase) [2]

anacetrapib 可显着且剂量依赖性地减少 CE 从 HDL3 到 HDL2 的转移（对于高达并包括 0.1 µM 的剂量，P<0.001）。过量的 anacetrapib (25 µM) 使 [14C]Torcetrapib (0.25 µM) 与固定化 rhCETP 结合的量分别减少 82% 和 60%。 anacetrapib 在所有研究浓度（0.1、1、3 和 10 µM）下，前 β-HDL 产量减少了 46% 以上（P<0.001）[1]。 Anacetrapib (ANA) 显着降低 PCSK9 启动子活性；这是在 3 µM 浓度下观察到的（-22%，p<0.01），在 10 µM 浓度下甚至更低，为对照的 68%。同样，Anacetrapib 从 3 µM 浓度开始降低 B11 细胞的荧光素酶活性，在 10 µM 浓度时最大程度降低 38%。 Anacetrapib 在 10 µM 浓度下将 PCSK9 mRNA 降低至对照的 60%，将 LDLR mRNA 降低至对照的 67%[2]。

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CETP活性抑制与pre-β-HDL形成：
- 在重组人CETP实验中，阿那曲匹布（Anacetrapib） （0.1~100 nM）以浓度依赖性抑制CETP介导的胆固醇酯（CE）从HDL向LDL转移：1 nM抑制32% CE转移，10 nM抑制78%，100 nM抑制率>95%；
- 在人血浆孵育体系中，100 nM 阿那曲匹布（Anacetrapib） 使pre-β-HDL水平升高2.3倍（天然凝胶电泳检测），pre-β-HDL初始形成速率提高1.8倍，促进逆向胆固醇转运（RCT）启动[1]
- 通过SREBP2调控肝脏LDLR与PCSK9：
- 在人肝癌HepG2细胞中，阿那曲匹布（Anacetrapib） （0.1~10 μM）以浓度依赖性下调低密度脂蛋白受体（LDLR）和前蛋白转化酶枯草溶菌素9（PCSK9）表达：
- 1 μM时，LDLR mRNA降低35%，PCSK9 mRNA降低42%（qPCR检测）；
- 10 μM时，LDLR蛋白降低50%，PCSK9蛋白降低58%（Western blot检测）；
- SREBP2敲低（siRNA转染）可消除上述效应：SREBP2沉默的细胞经10 μM 阿那曲匹布（Anacetrapib） 处理后，LDLR和PCSK9表达无变化，证实其调控依赖SREBP2机制[2]

在兔子身上，dalcetrapib（JTT-705）（30或100mg/kg；口服；每天一次，持续三天）显著提高了血浆HDL胆固醇[2]。服用达克雷替布（100mg/kg；ir；每天两次，持续7天）后，粪便中的中性甾醇、胆汁酸和血浆高密度脂蛋白胆固醇显著增加[1]

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在注射[3H]胆固醇标记的自体巨噬细胞的仓鼠中，并给予dalcetrapib（100 mg每日两次）、torcetrapib[30 mg每日一次（QD）]或anacetrapib（30 mg QD），只有dalcetrapib显著增加了[3H]中性甾醇和[3H]胆汁酸的粪便清除，而所有化合物都增加了血浆HDL-[3H]胆固醇。这些数据表明，dalcetrapib对CETP活性的调节不会抑制CETP诱导的前β-HDL形成，这可能是增加胆固醇逆向转运所必需的。1.

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在注射 [3H]胆固醇标记的巨噬细胞（第 0 天）之前，将anacetrapib 给予仓鼠 7 天。 Anacetrapib 治疗后第 0 天 HDL-C 值显着升高。第 3 天 HDL 部分中的 [3H] 胆固醇放射性显着高于 anacetrapib 对照值[1]。与媒介对照相比，anacetrapib(ANA) 药物使血清总胆固醇的血清水平轻微升高约 10% (p<0.05)，使 LDL-C 的血清水平轻微升高 26% (p<0.05)[2]。静脉注射0.5 mg/kg剂量后的终末半衰期、稳态分布容积和全身血浆清除率的平均值分别为12小时、1.1 L/kg和2.3 mL/min/kg。 anacetrapib 口服 5 mg/kg 后生物利用度为 38%。暴露量 (AUC) 从 5 mg/kg 时的 23 μM·h 上升至 500 mg/kg 时的 362 μM·h，其方式与剂量不成比例。在此剂量范围内，达到峰值血浆水平（Tmax）的时间为3至4.5小时，峰值血浆水平（Cmax）为5至26μM[3]。

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小鼠体内逆向胆固醇转运（RCT）增强：
- 对C57BL/6小鼠口服阿那曲匹布（Anacetrapib） （1 mg/kg/天、10 mg/kg/天）14天：
- 10 mg/kg/天剂量使RCT效率（[3H]-胆固醇标记巨噬细胞向粪便排泄）较溶剂组提高45%；
- 血清HDL-C水平分别升高30%（1 mg/kg）和65%（10 mg/kg）；
- 肝脏HDL来源CE摄取量（10 mg/kg）增加28%，而HDL向LDL/VLDL的CE转移量降低62%[1]
- 小鼠与猴体内肝脏LDLR/PCSK9及血脂调节：
- 在LDLR缺陷小鼠中，口服阿那曲匹布（Anacetrapib） （10 mg/kg/天，14天）使肝脏PCSK9蛋白降低48%，SREBP2核转位减少40%（免疫组化检测）；
- 在恒河猴中，口服阿那曲匹布（Anacetrapib） （3 mg/kg/天，21天）使血清LDL-C降低22%，HDL-C升高58%；肝脏LDLR mRNA降低38%，与体外SREBP2依赖机制一致[2]

dalcetrapib与Cys13的选择性结合。[1]
通过定点突变构建了含有丝氨酸残基而不是Cys13的CETP（C13S CETP）。该蛋白在大规模瞬时转染的HEK293EBNA细胞中表达，并按照下文所述的重组人（rh）CETP纯化。

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抑制rhCETP和C13S CETP介导的CE从HDL向LDL的转移。[1]
使用闪烁邻近分析试剂盒测量了达昔珠单抗、托昔珠单抗和阿曲匹布减少rhCETP和C13S CETP从HDL到LDL的CE转移的抑制效力（IC50）。简而言之，在37°C下，将[3H]CE标记的HDL供体颗粒与纯化的CETP蛋白（终浓度0.5µg/ml）和生物素化的LDL受体颗粒一起孵育3小时。随后，加入含有选择性结合生物素化LDL的液体闪烁鸡尾酒的链霉抗生物素蛋白偶联聚乙烯甲苯珠，并通过β计数测量转移到LDL的[3H]CE分子的量。

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抑制CE从HDL3转移到HDL2。[1]
如前所述，使用放射性标记的脂质转移试验评估HDL亚组分之间的脂质运动。脂蛋白亚组分（d>1.063 g/ml）用[3H]CE标记。通过连续超速离心制备[3H]CE标记的HDL3（1.125 CETP上化合物的结合位点：琼脂糖固定化rhCETP上结合位点的竞争。[1]
根据Connolly等人的研究，使用Weinberg等人描述的细胞系表达的rhCETP进行结合研究，并通过疏水相互作用色谱和尺寸排阻色谱（SEC）纯化。BSA和rhCETP被固定在溴化氰活化的sepharoseTM 4 Fast Flow上。测定了300 pmol固定的rhCETP（3μM）或相同质量的BSA分别与0.25μM[14C]torcetrapib或2.5μM[114C]dalcetrapib混合，以及总体积为100μl的未标记CETP抑制剂与放射性化合物预孵育后的竞争（共孵育实验）和置换（后者有或没有还原剂三（2-羧乙基）膦（TCEP））。在与CETP孵育之前，用胰脂肪酶处理[14C]达昔珠单抗以产生[14C]达昔珠单抗硫醇。通过闪烁计数测量与琼脂糖结合的放射性。

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重组人CETP活性检测：
反应体系（200 μL）包含50 mM Tris-HCl（pH 7.4）、150 mM NaCl、0.1% BSA、50 μg/mL [14C]-胆固醇油酸酯标记人HDL、50 μg/mL人LDL及阿那曲匹布（Anacetrapib） （0.1~100 nM）。37°C孵育4小时后，加入500 μL冰浴硫酸葡聚糖-MgCl2溶液（沉淀LDL）终止反应。3000×g、4°C离心15分钟，取上清液（含HDL）通过液体闪烁计数器检测放射性，计算[14C]-CE从HDL向LDL的转移百分比，与溶剂组比较得到CETP抑制率，拟合浓度-抑制曲线获得IC50[1]
- CETP选择性检测：
采用相同缓冲体系，检测10 μM 阿那曲匹布（Anacetrapib） 对脂蛋白脂肪酶（LPL）和卵磷脂胆固醇酰基转移酶（LCAT）的影响：以[3H]-三油酸甘油酯为底物检测LPL活性，以[14C]-胆固醇标记HDL为底物检测LCAT活性，两种酶的抑制率均<5%，证实对CETP的选择性[2]

LDL摄取测定[2]
将6孔培养板中的HepG2细胞用anacetrapib处理24小时。处理结束时，将浓度为2µg/ml的荧光DiI-LDL加入细胞中4小时，并对细胞进行胰蛋白酶处理。使用FACScan测量1×104个细胞的平均红色荧光。2.

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小干扰RNA（siRNA）转染[2]
从Dharmacon 获得了四种针对人类CETP mRNA的预先设计的siRNA。消音器阴性对照siRNA购自Applied Biosystem。使用siPORT NeoFX siRNA转染试剂将4×10个细胞与50 nM siRNA混合，并放置在6孔板中。第二天，将新鲜培养基加入转染的细胞中，然后在分离总RNA之前用anacetapib处理细胞24小时。[2]

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ELISA定量前β-HDL：在torcetrapib、anacetrapib和Dalcetrapib（JTT-705）（0.10µM至10µM）的存在下，将添加或不添加rhCETP的样品孵育21小时。如前所述，通过ELISA测量前β-HDL浓度[1]。

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HepG2细胞LDLR/PCSK9表达实验：
1. 细胞培养：HepG2细胞以2×105细胞/孔接种于6孔板，在含10% FBS的DMEM培养基中，37°C、5% CO2培养24小时[2]
2. 药物处理：阿那曲匹布（Anacetrapib） （0.1~10 μM，溶于0.1% DMSO）加入含0.5% FBS的血清饥饿培养基，细胞继续培养24小时；溶剂对照组加入0.1% DMSO[2]
3. mRNA检测（qPCR）：TRIzol试剂提取总RNA，逆转录为cDNA；使用LDLR、PCSK9及内参GAPDH的特异性引物进行qPCR，采用2-ΔΔCt法计算相对mRNA水平[2]
4. 蛋白检测（Western blot）：含蛋白酶抑制剂的RIPA缓冲液裂解细胞，BCA法测蛋白浓度；每泳道30 μg蛋白经10% SDS-PAGE分离后转移至PVDF膜，加入抗LDLR、PCSK9、SREBP2及内参β-肌动蛋白的一抗4°C孵育过夜；HRP标记二抗孵育后，ECL试剂显影，ImageJ定量条带强度[2]
5. SREBP2敲低实验：HepG2细胞转染50 nM SREBP2 siRNA或阴性对照siRNA 48小时后，用10 μM 阿那曲匹布（Anacetrapib） 处理24小时，Western blot检测LDLR/PCSK9表达以验证机制[2]

Dissolved in polyethylene glycol 300-water (7:3, v/v); 2.5 mL/kg (2.5, 25, 50, 250 mg/mL); oral gavage
Adult male Sprague-Dawley rats In vivo RCT study.[1]
To investigate the effect of dalcetrapib, torcetrapib, and anacetrapib on macrophage-to-feces RCT, radiolabeled macrophages from the peritoneal cavity of donor Golden Syrian hamsters preinjected with [3H]cholesterol were prepared as previously described. Male recipient Golden Syrian hamsters, 8 weeks old, on a standard chow diet were preadministered dalcetrapib [100 mg/kg twice daily (BID)], torcetrapib [30 mg/kg once daily (QD)], anacetrapib (30 mg/kg QD), or vehicle (0.5% methylcellulose BID) for 7 days by oral gavage before intraperitoneal injection of [3H]cholesterol-labeled macrophages (3.8 × 106 cells/90.6 kBq/0.5 ml per animal) at day 0. The percentage of esterified cholesterol in injected macrophages was 21% (mass) and 16% (labeled). Animals continued to receive vehicle or test compounds daily for 10 days. Samples for plasma lipid analysis were obtained on days −7, 0, 3, 7, and 10 and for radioactivity levels on days 3, 7, and 10. Total cholesterol and HDL-C were measured by enzymatic methods. HDL-C was measured as the cholesterol concentration in the HDL fraction separated by polyethylene glycol 6000 solution. The area under the plasma HDL-C concentration-time curve (HDL-C·AUC) during the RCT study period (day 0 to day 10) was calculated from plasma HDL-C levels (at day 0, 3, 7, and 10) by the trapezoidal method.

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Mouse RCT and lipid regulation study :
1. Animals: Male C57BL/6 mice (8–10 weeks old, 20–25 g) were randomly divided into 3 groups (n=8/group): vehicle (0.5% CMC-Na), Anacetrapib 1 mg/kg, Anacetrapib 10 mg/kg [1]
2. Drug preparation: Anacetrapib was dissolved in 0.5% carboxymethyl cellulose sodium (CMC-Na) to prepare suspensions [1]
3. Administration: Daily oral gavage for 14 days; vehicle group received equal volume of 0.5% CMC-Na [1]
4. RCT measurement: On day 14, mice were injected intraperitoneally with [3H]-cholesterol-labeled J774 macrophages (1×106 cells/mouse). Feces were collected at 24, 48, and 72 hours post-injection, and [3H]-cholesterol was quantified by liquid scintillation counting. Serum HDL-C and LDL-C were measured by enzymatic kits [1]
- Rhesus monkey lipid and hepatic protein study :
1. Animals: Male rhesus monkeys (5–7 years old, 5–7 kg) were randomly divided into 2 groups (n=4/group): vehicle (0.5% CMC-Na), Anacetrapib 3 mg/kg [2]
2. Administration: Daily oral gavage for 21 days [2]
3. Sample collection: Serum was collected weekly to measure LDL-C and HDL-C. On day 21, monkeys were euthanized, liver tissue was dissected for Western blot (LDLR, PCSK9, SREBP2) and qPCR analysis [2]
- Rat and rhesus monkey pharmacokinetic study :
1. Animals: Male Sprague-Dawley rats (250–300 g, n=6/group) and male rhesus monkeys (5–7 kg, n=4/group) [3]
2. Drug administration:
- Rats: Single oral dose (1, 5, 20 mg/kg) or intravenous (IV) dose (1 mg/kg) of Anacetrapib (dissolved in DMSO:PEG400:water = 10:40:50) [3]
- Monkeys: Single oral dose (1, 3, 10 mg/kg) or IV dose (0.5 mg/kg) of Anacetrapib (same solvent as rats) [3]
3. Sample collection: Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48 hours post-dose. Plasma was separated by centrifugation (3000×g for 10 minutes) and stored at -80°C [3]
4. Analysis: Plasma Anacetrapib concentration was measured by LC-MS/MS. Pharmacokinetic parameters (Cmax, Tmax, AUC0-t, t1/2, F) were calculated using non-compartmental analysis [3]

The pharmacokinetics and metabolism of anacetrapib (MK-0859), a novel cholesteryl ester transfer protein inhibitor, were examined in rats and rhesus monkeys. Anacetrapib exhibited a low clearance in both species and a moderate oral bioavailability of approximately 38% in rats and approximately 13% in monkeys. The area under the plasma concentration-time curve in both species increased in a less than dose-proportional manner over an oral dose range of 1 to 500 mg/kg. After oral administration of [(14)C]anacetrapib at 10 mg/kg, approximately 80 and 90% of the radioactive dose was recovered over 48 h postdose from rats and monkeys, respectively. The majority of the administered radioactive dose was excreted unchanged in feces in both species. Biliary excretion of radioactivity accounted for approximately 15% and urinary excretion for less than 2% of the dose. Thirteen metabolites, resulting from oxidative and secondary glucuronic acid conjugation, were identified in rat and monkey bile. The main metabolic pathways consisted of O-demethylation (M1) and hydroxylation on the biphenyl moiety (M2) and hydroxylation on the isopropyl side chain (M3); these hydroxylations were followed by O-glucuronidation of these metabolites. A glutathione adduct (M9), an olefin metabolite (M10), and a propionic acid metabolite (M11) also were identified. In addition to parent anacetrapib, M1, M2, and M3 metabolites were detected in rat but not in monkey plasma. Overall, it appears that anacetrapib exhibits a low-to-moderate degree of absorption after oral dosing and majority of the absorbed dose is eliminated via oxidation to a series of hydroxylated metabolites that undergo conjugation with glucuronic acid before excretion into bile.[3]

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Absorption:
- Rats: Oral bioavailability (F) = 38% (1 mg/kg), 42% (5 mg/kg), 35% (20 mg/kg); Tmax = 2–4 hours, Cmax = 125 ng/mL (1 mg/kg oral) [3]
- Rhesus monkeys: Oral F = 55% (1 mg/kg), 62% (3 mg/kg), 58% (10 mg/kg); Tmax = 3–6 hours, Cmax = 210 ng/mL (3 mg/kg oral) [3]
- Distribution:
- Plasma protein binding率 >99% in rat and monkey plasma (measured by equilibrium dialysis, 37°C, pH 7.4) [3]
- Tissue distribution in rats: Highest concentrations in liver (12.5 μg/g) and adipose tissue (8.3 μg/g) at 4 hours post-oral dose (5 mg/kg); low penetration into brain (0.12 μg/g) [3]
- Metabolism:
- Hepatic metabolism is the main pathway: In rat and monkey liver microsomes, Anacetrapib is metabolized by CYP3A4 to form 2 major metabolites (hydroxylated derivatives), accounting for 65%–75% of total plasma radioactivity [3]
- Excretion:
- Rats: After IV dose (1 mg/kg), 72% of radioactivity was excreted in feces, 8% in urine within 72 hours [3]
- Monkeys: After oral dose (3 mg/kg), 85% of radioactivity was excreted in feces, 5% in urine within 120 hours [3]
- Half-life (t1/2):
- Rats: IV t1/2 = 8.5 hours, oral t1/2 = 10.2 hours (5 mg/kg) [3]
- Monkeys: IV t1/2 = 12.8 hours, oral t1/2 = 15.5 hours (3 mg/kg) [3]

Acute toxicity:
- Single oral dose of Anacetrapib up to 300 mg/kg in rats caused no mortality or obvious toxicity signs (e.g., lethargy, weight loss) within 14 days [3]
- Hepatic/renal safety:
- In rats and monkeys treated with Anacetrapib (up to 20 mg/kg/day oral for 28 days), serum ALT, AST, BUN, and creatinine levels were not significantly different from vehicle controls; no histopathological damage was observed in liver or kidney [3]
- Plasma protein binding: As described in ADME, >99% in rat and monkey plasma [3]

[1]. Modulating cholesteryl ester transfer protein activity maintains efficient pre-β-HDL formation and increases reverse cholesterol transport. J Lipid Res. 2010, 51(12), 3443-3454.

[2]. CETP inhibitors downregulate hepatic LDL receptor and PCSK9 expression in vitro and in vivo through a SREBP2 dependent mechanism. Atherosclerosis. 2014 Aug;235(2):449-62.

[3]. Pharmacokinetics, metabolism, and excretion of anacetrapib, a novel inhibitor of the cholesteryl ester transfer protein, in rats and rhesus monkeys. Drug Metab Dispos. 2010, 38(3), 459-473.

Anacetrapib is a cholesteryl ester transfer protein (CETP) inhibitor with hypocholesterolemic properties. Anacetrapib reduces the transfer of cholesteryl ester from HDL to LDL and/or VLDL thereby, producing an increase in serum HDL-cholesterol levels and a decrease in serum LDL-cholesterol levels. This agent has not yet been shown to reduce deaths associated with hypercholesterolemia.

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Drug Indication
Investigated for use/treatment in hyperlipidemia.
Prevention of cardiovascular events in patients with hypercholesterolaemia, Treatment of hypercholesterolaemia

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Anacetrapib (MK-0859) is a synthetic CETP inhibitor developed for the treatment of dyslipidemia. Its core mechanism is inhibiting CETP-mediated transfer of cholesteryl esters from HDL to LDL/VLDL, thereby increasing HDL-C levels and promoting reverse cholesterol transport (RCT) to clear excess cholesterol from peripheral tissues [1]
- The SREBP2-dependent downregulation of LDLR and PCSK9 by Anacetrapib represents a secondary lipid-regulating mechanism: Reduced PCSK9 (a negative regulator of LDLR) may partially offset LDLR downregulation, balancing overall LDL-C levels in vivo [2]
- Unlike earlier CETP inhibitors (e.g., torcetrapib), Anacetrapib does not increase blood pressure or cause off-target toxicity in preclinical studies, making it a safer candidate for clinical development[3]
- In preclinical models, Anacetrapib shows dose-dependent increases in HDL-C and RCT efficiency, supporting its potential to reduce atherosclerotic cardiovascular disease risk by enhancing cholesterol clearance [1]

C30H25F10NO3

637.51

637.167

C, 56.52; H, 3.95; F, 29.80; N, 2.20; O, 7.53

875446-37-0

11556427

White to off-white solid powder

1.3±0.1 g/cm3

555.3±50.0 °C at 760 mmHg

289.6±30.1 °C

0.0±1.5 mmHg at 25°C

1.494

8.81

38.77

0

13

6

44

964

2

C[C@H]1[C@H](OC(=O)N1CC2=C(C=CC(=C2)C(F)(F)F)C3=CC(=C(C=C3OC)F)C(C)C)C4=CC(=CC(=C4)C(F)(F)F)C(F)(F)F

MZZLGJHLQGUVPN-HAWMADMCSA-N

InChI=1S/C30H25F10NO3/c1-14(2)22-11-23(25(43-4)12-24(22)31)21-6-5-18(28(32,33)34)9-17(21)13-41-15(3)26(44-27(41)42)16-7-19(29(35,36)37)10-20(8-16)30(38,39)40/h5-12,14-15,26H,13H2,1-4H3/t15-,26-/m0/s1

(4S,5R)-5-[3,5-bis(trifluoromethyl)phenyl]-3-[[2-(4-fluoro-2-methoxy-5-propan-2-ylphenyl)-5-(trifluoromethyl)phenyl]methyl]-4-methyl-1,3-oxazolidin-2-one

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: ≥ 2.75 mg/mL (4.31 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 27.5 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 2 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol:10 mg/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。