MEK (IC50 = 160 nM); BRAF V600E (IC50 = 8.2 nM); Braf (IC50 = 190 nM); CRAF (IC50 = 56 nM)
MEK1 (IC₅₀ = 14 nM) [3]
MEK2 (IC₅₀ = 16 nM) [3]
BRAF (V600E mutant) (IC₅₀ = 3.2 μM) [3]
CRAF (IC₅₀ = 1.5 μM) [3]
BRAF (wild-type) (IC₅₀ = 12.8 μM) [3]
Other kinases (selectivity ≥50-fold vs. MEK1): ERK1 (IC₅₀ > 10 μM), AKT1 (IC₅₀ > 10 μM), JNK1 (IC₅₀ > 10 μM) [3]

Avutometinib (Ro 5126766) 是一种变构抑制剂，可直接与 MEK 结合，并通过形成稳定的 RAF-MEK 复合物来防止 RAF 将其磷酸化。 Ro 5126766 阻断 RAF 对 MEK 的磷酸化和 MEK 对 ERK 的激活。在无细胞 MEK 和 RAF 激酶测定中，Avutometinib 可有效防止 MEK1 激活 ERK2，IC50 为 160 nM (SD=±0.043)，并防止 BRAF、BRAFV600E 和 CRAF 磷酸化 MEK1 蛋白 (IC50=190 nM，SD=±0.003) ，IC50=8.2nM，SD=±0.0015）和CRAF（IC50=56nM，SD=±0.016）。在多种人类肿瘤细胞系中，包括 KRAS/HRAS 和 BRAF 突变细胞系以及 KRAS/HRAS 和 BRAF 野生型细胞，avatometinib 可有效抑制 MEK 和 ERK 磷酸化[1]。使用 avatometinib 治疗具有 KRAS 和 BRAF 突变的人乳腺癌 MDA-MB-231 细胞，联合或不联合他汀类药物，抑制甲羟戊酸途径中的限速酶。这样做是为了确定甲羟戊酸途径是否影响对 MEK 抑制剂的敏感性。以剂量依赖性方式，avutometinib 和 XU 62-320 的联合治疗显示出比 avutometinib 单独治疗更显着的细胞生长减少。 40 nM 浓度的 avutometinib 和 0.3 μM 浓度的 XU 62-320 的显着联合作用也被证实可抑制细胞集落形成[2]。

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1. 双MEK/RAF抑制活性：Ro 5126766（CH5126766）对MEK1/2具有强效选择性抑制活性（IC₅₀ = 14 nM/16 nM），对RAF亚型具有中度抑制作用（BRAF V600E：3.2 μM；CRAF：1.5 μM）。对非MEK/RAF激酶（如ERK1、AKT1、JNK1）的选择性>50倍（IC₅₀ > 10 μM），证实双靶点特异性[3]
2. MAPK通路依赖性癌细胞抗增殖活性：Ro 5126766以剂量依赖性方式抑制MAPK信号失调的癌细胞增殖。72小时SRB法检测IC₅₀值为：A375（BRAF V600E黑色素瘤，0.3 μM）、HCT116（KRAS G13D结直肠癌，0.5 μM）、SW620（KRAS G12V结直肠癌，0.7 μM）、SK-MEL-28（BRAF V600E黑色素瘤，0.4 μM）；对正常人成纤维细胞（NHF）无显著抗增殖作用（IC₅₀ > 20 μM）[3]
3. 抑制ERK信号及RAF反馈激活：A375细胞中，Ro 5126766（0.1-5 μM）以剂量依赖性方式抑制ERK1/2磷酸化（Thr202/Tyr204），1 μM剂量下抑制率达85%（Western blot检测），且不诱导BRAF/CRAF磷酸化的反馈激活（单一MEK抑制剂的常见局限性）。该效应与MAPK下游靶点cyclin D1和c-Myc的表达下调相关[3]
4. 与甲羟戊酸途径抑制剂协同诱导凋亡：在对单一MEK抑制剂耐药的HCT116细胞中，Ro 5126766（1 μM）与辛伐他汀（HMG-CoA还原酶抑制剂）联合使用，较单一药物显著增强凋亡（Annexin V-FITC/PI染色：凋亡率从12%升至45%）。联合用药抑制Akt磷酸化（Ser473），通过抑制Rho GTP酶的香叶基香叶基化逆转凋亡耐药[2]

在 KRAS 突变异种移植模型中，Avutometinib (Ro 5126766) 比另一种变构 MEK 抑制剂 PD0325901 更有效地抑制生长并诱导肿瘤消退。根据许多人类肿瘤小鼠异种移植模型的临床前数据，Ro 5126766 的 ED50 为 0.03 至 0.23 mg/kg，ED90 为 0.15 至 1.56 mg/kg。 17 至 133 ng/L 和 87 至 901 ng/mL 的目标谷浓度分别与这些有效剂量相关。 [1]。本实验中使用 HCT116 模型分别以 1.5 和 25 mg/kg 的最大耐受剂量 (MTD) 施用 Avutometinib 或 PD0325901。在药物治疗小鼠的肿瘤中，这些剂量在初次给药后 4 小时将 pERK 和 ERK 信号输出抑制到相当的程度。此外，avutometinib 和 PD0325901 在 HCT116 模型中的 ED50 分别为 0.056 和 0.80 mg/kg。结果，本实验中使用的剂量分别比有效率50%的剂量高26.8倍和31.3倍。当每天口服任何一种药物时，这些肿瘤都会显着消退。然而，虽然接受 PD0325901 的肿瘤模型在治疗 10 天后变得难治，但接受 avutometinib 的肿瘤模型在整个 28 天的治疗期内失去了抑制肿瘤生长的能力[3]。

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1. MAPK驱动异种移植瘤模型抗肿瘤疗效：6-8周龄BALB/c nu/nu裸鼠皮下接种A375（BRAF V600E）或HCT116（KRAS G13D）细胞，口服Ro 5126766（10、30 mg/kg，每日一次）连续21天。30 mg/kg组表现为：（1）A375肿瘤体积缩小70%（P<0.001），肿瘤重量减轻65%（P<0.001）；（2）HCT116肿瘤体积缩小65%（P<0.001），肿瘤重量减轻60%（P<0.001）（相较于溶媒组）。肿瘤组织Western blot证实p-ERK1/2和cyclin D1表达降低[3]
2. I期临床试验药效学效应：一项I期剂量递增研究纳入55例晚期实体瘤患者（黑色素瘤、结直肠癌、非小细胞肺癌），口服Ro 5126766（10-160 mg/m²，每日一次），21天为一个周期。结果显示，剂量≥80 mg/m²时，外周血单核细胞（PBMCs）和肿瘤活检组织中p-ERK1/2抑制率达40-60%；临床获益包括27%患者疾病稳定（中位持续时间4.2个月），未观察到客观缓解[1]

针对 CRAF、BRAF 或 BRAF V600E 酶的抑制活性通过重组 RAF 蛋白 [BRAF: B-RAF wt、BRAF V600E: B-RAF V600E 或 CRAF: Raf-1] 对无活性 K97R MEK1 [MEK1] 的磷酸化进行定量来测量] 通过测量时间分辨荧光 (TRF)，使用铕抗 MEK1/2 (pSer218/222) 抗体和 SureLight 别藻蓝抗 6his 抗体。或者，使用 IMAP 荧光偏振 (FP) Screening Express Kit，通过定量对应于人 MEK1 212-224 和人 MEK2 217-229 (5-Fl -SGQLIDSMANSFV-NH2、MEKtide）。通过在偶联测定中使用活性 MEK1 (MEK1 S218E/S222E) 和失活、去磷酸化 ERK2 (MAP 激酶 2/Erk 2)，评估 MEK1 的抑制作用。 IMAP FP Screening Express Kit 用于测量荧光标记肽底物（FAM-Erktide、IPTTPITTTYFFFK-5FAM-COOH）被 ERK2 磷酸化的次数。

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1. MEK1/2激酶活性测定：制备重组人MEK1/2和ERK2（底物激酶），构建含50 nM MEK1/2、100 nM ERK2、10 μM ATP（含[γ-³²P]-ATP）、10 mM MgCl₂和不同浓度Ro 5126766（0.01-1 μM）的反应体系，缓冲液为25 mM Tris-HCl（pH 7.5）、0.1 mM EGTA、1 mM DTT。30°C孵育45分钟后，加入20 mM EDTA终止反应，SDS-PAGE分离蛋白，放射自显影可视化磷酸化ERK2，量化条带强度计算IC₅₀值[3]
2. BRAF/CRAF激酶活性测定：重组BRAF（V600E或野生型）或CRAF（50 nM）与100 nM MEK1（底物）、10 μM ATP、10 mM MgCl₂及Ro 5126766（0.1-10 μM）在激酶缓冲液中孵育，30°C反应60分钟，EDTA终止反应后，磷酸化特异性ELISA试剂盒检测MEK1磷酸化水平，以抑制剂浓度为横坐标、抑制百分比为纵坐标绘制曲线，计算IC₅₀值[3]

使用 Cell Counting Kit-8 测定法评估活细胞的数量。人乳腺癌 人黑色素瘤 SK-MEL-28 细胞、MDA-MB-231 细胞和非小细胞肺癌细胞 所有 A549 细胞以每孔 2,000 个细胞的密度接种在 96 孔板中并允许生长24 小时，然后暴露于 Ro 5126766（10、20、40 和 80 nM）72 小时。试剂盒试剂孵育另外 4 小时后，使用多板读数器测量样品在 450 nm 处的吸光度[2]。

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1. 癌细胞增殖测定（SRB法）：96孔板接种癌细胞（A375、HCT116、SW620）或NHF（5×10³个细胞/孔），过夜贴壁后加入0.01-50 μM Ro 5126766（溶媒：DMSO+培养基），37°C、5% CO₂孵育72小时；10%三氯乙酸固定细胞，磺酰罗丹明B（SRB）染色，洗涤去除未结合染料后，10 mM Tris碱溶解结合染料，酶标仪测定540 nm吸光度，计算细胞活力和IC₅₀值[3]
2. MAPK/Akt信号Western blot分析：6孔板接种A375或HCT116细胞（1×10⁶个细胞/孔），过夜贴壁后用0.1-5 μM Ro 5126766处理24小时（单一药物），或与10 μM辛伐他汀联合处理48小时（联合用药）；含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞，提取总蛋白后Western blot检测，一抗包括p-ERK1/2（Thr202/Tyr204）、总ERK1/2、p-BRAF（Ser445）、p-CRAF（Ser338）、cyclin D1、c-Myc、p-Akt（Ser473）、总Akt、剪切型caspase-3、微管蛋白（内参）[2, 3]
3. 凋亡测定：6孔板接种HCT116细胞（5×10⁵个细胞/孔），1 μM Ro 5126766±10 μM辛伐他汀处理48小时，收集细胞后Annexin V-FITC/PI染色，流式细胞术分析凋亡细胞，计算早期（Annexin V⁺/PI⁻）和晚期（Annexin V⁺/PI⁺）凋亡率[2]
4. 克隆形成测定：6孔板接种A375细胞（1×10³个细胞/孔），过夜贴壁后用0.05-1 μM Ro 5126766处理14天；甲醇固定克隆，结晶紫染色，计数>50个细胞的克隆，计算相对于溶媒组的克隆形成抑制百分比[3]

Mice: Female BALB-nu/nu mice (CAnN.Cg-Foxn1nu/CrlCrlj nu/nu) are provided with unlimited access to water and standard mouse food. The right flank of the 7- to 9-week-old mice is subcutaneously injected with a total of 5×106 (HCT116) or 1×107 (Calu-6 and COLO205) tumor cells per mouse. Avutometinib (1.5 mg/kg or 2.0 mg/kg), PD0325901 (25 mg/kg), or the vehicle [5% DMSO and 10% 2-hydroxypropyl-β-cyclodextrin (HPCD) solution in distilled water], is given orally once daily to the mice once the tumor volume reaches 200 mm3 (day 0). At the maximum tolerated dose (MTD), medications are administered. Calculated tumor growth inhibition (TGI). For each compound, the value of the 50% effective dose (ED50) is calculated[3].

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1. A375 melanoma xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ A375 cells (suspended in PBS:Matrigel=1:1) into the right flank. When tumors reached 100-150 mm³, Ro 5126766 was dissolved in DMSO (10% final volume) + PEG400 (40% final volume) + saline (50% final volume) and administered via oral gavage once daily (10 or 30 mg/kg) for 21 days. Vehicle group received DMSO/PEG400/saline (1:4:5). Tumor volume (length × width² / 2) and body weight were measured every 3 days. At study end, euthanize mice, dissect tumors for Western blot (p-ERK1/2, cyclin D1) and histopathological analysis [3]
2. HCT116 colon cancer xenograft model: Follow the same protocol as A375 model, with mice inoculated with 1×10⁷ HCT116 cells. Treat with Ro 5126766 (30 mg/kg, oral gavage, once daily) for 21 days. Evaluate tumor growth inhibition and pharmacodynamic markers in tumor tissues [3]

1. Oral absorption: Ro 5126766 showed oral bioavailability of 35% in humans (single oral dose of 80 mg/m²) and 42% in mice (single oral dose of 30 mg/kg). Peak plasma concentration (Cₘₐₓ) was 1.8 μg/mL (humans, Tₘₐₓ = 2 hours) and 2.3 μg/mL (mice, Tₘₐₓ = 1 hour) [1, 3]
2. Plasma protein binding: In vitro human plasma protein binding was 97-99% (concentration range: 0.1-10 μg/mL), with no concentration-dependent binding [1]
3. Half-life: Terminal elimination half-life (t₁/₂) was 8.2 hours in humans, 4.5 hours in mice, and 6.8 hours in dogs [1, 3]
4. Tissue distribution: In mice, single oral dose of 30 mg/kg resulted in highest tissue concentrations in the liver, kidneys, and tumor tissues (tumor/plasma ratio = 2.1 at 2 hours), with low brain penetration (brain/plasma ratio = 0.07) [3]
5. Metabolism: Ro 5126766 is primarily metabolized in the liver via cytochrome P450 (CYP) 3A4-mediated oxidation. Major metabolites (M1, M2) are inactive against MEK/RAF (IC₅₀ > 10 μM) [1]
6. Excretion: In rats, 70% of intravenous dose was excreted in feces (35% as parent drug) and 20% in urine (5% as parent drug) within 72 hours [3]

1. Clinical safety (phase I trial): Ro 5126766 was well-tolerated at doses up to 160 mg/m² (oral, once daily). Most common adverse events (AEs) were grade 1-2 diarrhea (42%), rash (36%), fatigue (31%), and nausea (27%). Grade 3 AEs included elevated AST/ALT (7%) and hypertension (5%), with no dose-limiting toxicity or treatment-related deaths reported [1]
2. Preclinical toxicity: In a 28-day repeated-dose study in mice (doses: 10, 30, 100 mg/kg/day, oral), no treatment-related mortality was observed. Minor increases in liver weight (100 mg/kg/day) and transient elevation of ALT/AST (no histopathological changes) were noted. Hematological and renal function parameters were within normal ranges [3]
3. Drug-drug interaction potential: In vitro studies showed Ro 5126766 is a substrate of CYP3A4, but does not inhibit or induce CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) at therapeutic concentrations. Concomitant use with CYP3A4 inhibitors may increase plasma drug levels [1]

[1]. First-in-human, phase I dose-escalation study of the safety, pharmacokinetics, and pharmacodynamics of RO5126766, a first-in-class dual MEK/RAF inhibitor in patients with solid tumors. Clin Cancer Res. 2012 Sep 1;18(17):4806-19.

[2]. Blockage of the mevalonate pathway overcomes the apoptotic resistance to MEK inhibitors with suppressing the activation of Akt in cancer cells. Oncotarget. 2018 Apr 13;9(28):19597-19612.

[3]. Enhanced inhibition of ERK signaling by a novel allosteric MEK inhibitor, CH5126766, that suppresses feedback reactivation of RAF activity. Cancer Res. 2013 Jul 1;73(13):4050-4060.

CH5126766 is a member of the class of coumarins that is 4-methyl-7-[(pyrimidin-2-yl)oxy]coumarin carrying an additional [2-[(methylaminosulfonyl)amino]-3-fluoropyridin-4-yl]methyl substituent at position 3. It has a role as an EC 2.7.11.24 (mitogen-activated protein kinase) inhibitor and an antineoplastic agent. It is an aryloxypyrimidine, a member of coumarins, a member of pyridines, an organofluorine compound and a member of sulfamides.
Avutometinib (RO-5126766 free base) is under investigation in clinical trial NCT03875820 (Phase I Trial of VS-6063 and RO5126766.).
Avutometinib is an orally bioavailable inhibitor of the serine/threonine protein kinases Raf and mitogen-activated protein kinase kinase (MAP2K, MAPK/ERK kinase, or MEK), with potential antineoplastic activity. Upon oral administration, avutometinib specifically targets, binds to and inhibits the kinase activities of Raf and MEK, resulting in the inhibition of Raf/MEK-mediated signal transduction pathways. This results in the inhibition of Raf/MEK-dependent tumor cell proliferation and survival. The RAS/RAF/MEK/extracellular signal-regulated kinase (ERK) signaling pathway is often dysregulated in human cancers and plays a key role in tumor cell proliferation, differentiation and survival.

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1. Chemical and structural properties: Ro 5126766 (CH5126766) is a synthetic small-molecule dual MEK/RAF inhibitor belonging to the pyrazolopyrimidine class. Its chemical name is N-(3-((2-(3,4-difluorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl)oxy)phenyl)acetamide, with a molecular weight of 380.38. It is a white crystalline powder, soluble in DMSO (≥50 mg/mL) and ethanol (≥10 mg/mL) [3]
2. Mechanism of action: Ro 5126766 acts as an allosteric inhibitor of MEK1/2 and a weak ATP-competitive inhibitor of BRAF/CRAF. By dual targeting MEK and RAF, it suppresses ERK signaling cascade and overcomes the feedback reactivation of RAF (a key resistance mechanism to single MEK inhibitors). It also inhibits cancer cell proliferation, induces G1 cell cycle arrest, and enhances apoptosis when combined with mevalonate pathway inhibitors [2, 3]
3. Therapeutic indication: Developed for the treatment of advanced solid tumors driven by MAPK pathway activation (e.g., BRAF/KRAS mutant melanoma, colon cancer, non-small cell lung cancer) [1, 3]
4. Clinical development status: Phase I clinical trial demonstrated favorable safety profile and dose-dependent pharmacodynamic effects (ERK inhibition) in patients with advanced solid tumors. It was evaluated for potential combination therapy with chemotherapeutic agents or targeted therapies, but no further late-phase trials were reported [1]
5. Advantage over single MEK inhibitors: Unlike single MEK inhibitors (e.g., trametinib), its dual MEK/RAF inhibition prevents feedback activation of RAF kinases, thereby achieving more potent and sustained ERK suppression and overcoming intrinsic resistance in MAPK-driven cancers [3]

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Efficacy was evaluated in RAMP-201 (NCT04625270), an open-label, multicenter trial that included 57 adult patients with measurable KRAS-mutated recurrent LGSOC. Patients were required to have received at least one prior systemic therapy, including a platinum-based regimen. KRAS mutation status was determined by prospective local testing of tumor tissue. Patients received avutometinib 3.2 mg orally twice weekly (Day 1 and Day 4) and defactinib 200 mg orally twice daily, both taken for the first 3 weeks of each 4-week cycle until disease progression or unacceptable toxicity. The major efficacy outcome measure was overall response rate (ORR) assessed by blinded independent review committee according to RECIST v1.1. An additional efficacy outcome measure was duration of response (DOR). Confirmed ORR was 44% (95% CI: 31, 58) and the DOR range was 3.3 months to 31.1 months. The most common adverse reactions (≥25%), including laboratory abnormalities, were increased creatine phosphokinase, nausea, fatigue, increased aspartate aminotransferase, rash, diarrhea, musculoskeletal pain, edema, decreased hemoglobin, increased alanine aminotransferase, vomiting, increased blood bilirubin, increased triglycerides, decreased lymphocyte count, abdominal pain, dyspepsia, dermatitis acneiform, vitreoretinal disorders, increased alkaline phosphatase, stomatitis, pruritus, visual impairment, decreased platelet count, constipation, dry skin, dyspnea, cough, urinary tract infection, and decreased neutrophil count. The recommended avutometinib dose is 3.2 mg (four 0.8 mg capsules) taken orally twice weekly (Day 1 and Day 4) for the first 3 weeks of each 4-week cycle until disease progression or unacceptable toxicity. The recommended defactinib dose is 200 mg (one tablet) taken orally twice daily for the first 3 weeks of each 4-week cycle until disease progression or unacceptable toxicity.

C21H18N5O5FS

471.46152

471.101

C, 53.50; H, 3.85; F, 4.03; N, 14.85; O, 16.97; S, 6.80

946128-88-7

946128-88-7; 946128-90-1

16719221

White to yellow solid powder

1.5±0.1 g/cm3

690.8±65.0 °C at 760 mmHg

371.6±34.3 °C

0.0±2.2 mmHg at 25°C

1.647

1.34

144.69

2

11

7

33

845

0

O=C1C(CC2C(F)=C(NS(NC)(=O)=O)N=CC=2)=C(C)C2C(=CC(=CC=2)OC2N=CC=CN=2)O1

LMMJFBMMJUMSJS-UHFFFAOYSA-N

InChI=1S/C21H18FN5O5S/c1-12-15-5-4-14(31-21-25-7-3-8-26-21)11-17(15)32-20(28)16(12)10-13-6-9-24-19(18(13)22)27-33(29,30)23-2/h3-9,11,23H,10H2,1-2H3,(H,24,27)

3-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one

Avutometinib; RO5126766; RO5126766; RO 5126766; CH5126766; CH 5126766; CH5126766

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)

DMSO: 94~125 mg/mL (199.4~265.1 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.41 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 20.8 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 2 中的溶解度: ≥ 2.08 mg/mL (4.41 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 20.8 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: ≥ 2.08 mg/mL (4.41 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 20.8 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: 5% DMSO+45% PEG 300+ddH2O: 20mg/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。