ALK (IC50 = 0.37 nM); ROS1 (IC50 = 1.9 nM); FLT3 (IC50 = 2.1 nM); IGF1R (IC50 = 24.9 nM); EGFR(C797S/del19) (IC50 = 39.9 nM)
Anaplastic Lymphoma Kinase (ALK): Wild-type ALK (IC50 = 1.6 nM), ALK L1196M (IC50 = 4.8 nM), ALK G1269A (IC50 = 2.7 nM), ALK C1156Y (IC50 = 6.4 nM), ALK T315I (IC50 = 16 nM); also inhibits EGFR (IC50 = 53 nM), ROS1 (IC50 = 19 nM) [2]
- ALK (focus on resistant mutants: ALK G1202R (IC50 = 12 nM), ALK F1174L (IC50 = 3.2 nM); no additional EGFR/ROS1 data) [1]
- ALK (neuroblastoma-derived ALK mutants: ALK F1174L (IC50 = 4.1 nM), ALK R1275Q (IC50 = 7.3 nM); no off-target kinase data) [3]

AP26113 对敏感和耐药 H3122 细胞均具有高度活性，以剂量依赖性方式减少细胞生长、抑制 ALK 磷酸化并诱导细胞凋亡。 AP26113 降低 H3122 和 H3122 CR 细胞中的 p-ALK，IC50 值分别为 7.4 和 16.8 nM。 AP26113 减少表达天然或突变 EML4-ALK 的 Ba/F3 细胞中的细胞数量，IC50 分别为 10 nM 和 24 nM。 AP26113 抑制 SU-DHL-1、H3122 和 Ba/F3-EML4-ALK v1 细胞系的细胞生长，GI50 分别为 9 nM、4 nM 和 13 nM。 AP26113 在 Karpas-299、SU-DHL-1 和 L-82 细胞系中抑制 ALK 磷酸化，IC50 分别为 3.2 nM、1.5 nM 和 2.1 nM。 AP26113 剂量依赖性地抑制 Karpas-299 和 H3122 细胞中 ALK 和 ERK 的磷酸化。 AP26113 抑制 Ba/F3 系（天然 EML4-ALK）和 Ba/F3 系（EML4-ALK G1269S 突变体）中的细胞生长，IC50 分别为 11 nM 和 16 nM。 AP26113 抑制 Ba/F3 系（天然 EML4-ALK）和 Ba/F3 系（EML4-ALK E1210K 突变体）中的 ALK 磷酸化，IC50 分别为 74 nM 和 335 nM。 AP26113 (10 mg/kg-75 mg/kg) 在 PF-02341066 耐药 EML4-ALK 突变小鼠异种移植模型中有效。 AP26113 分别以 25 mg/kg、50 mg/kg 和 50 mg/kg 剂量诱导表达天然 EML4-ALK 以及 G1269S 和 L1196M 突变体的肿瘤消退。在表达 EGFR-DEL 的 Ba/F3 细胞中，AP26113 抑制 EGFR 磷酸化和活力，IC50 分别为 75 nM 和 114 nM。在表达 EGFR-DEL/T790M 的 Ba/F3 细胞中，AP26113 抑制 EGFR 磷酸化和活力，IC50 分别为 15 和 281 nM。 AP26113 在表达 EGFR-DEL (HCC827) 的 NSCLC 系中抑制 EGFR 磷酸化，IC50 为 62 nM，抑制细胞生长，GI50 为 165 nM。 AP26113 在表达 EGFR-DEL/T790M 的 HCC827 细胞中抑制 EGFR 磷酸化，IC50 为 59 nM，抑制细胞生长，GI50 为 245 nM。 AP26113 在 HCC78 NSCLC 细胞中以剂量依赖性方式有效抑制 SLC34A2-ROS 驱动的信号传导和增殖。激酶测定：对 289 种激酶进行体外 HotSpotSM 激酶分析。该测定在 10 μM [33P]-ATP 存在下进行，使用 brigatinib 浓度范围为 0.05 nM 至 1 μM。细胞测定：使用 Cell Titer 96 Aqueous One Solution 细胞增殖测定或 CyQuant 细胞增殖测定评估细胞生长。铺板后 24 小时，用 AP26113 处理细胞并生长 72 小时。引起 50% 生长抑制 (GI50) 的浓度通过校正零时间（处理时间）的细胞计数并使用 XLfit 版本 4.2.2 for Microsoft Excel 将数据绘制为相对于媒介物 (DMSO) 处理的细胞的生长百分比来确定。使用 SU-DHL-1、H3122 和 Ba/F3-EML4-ALK v1 细胞系。

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抑制ALK阳性NSCLC耐药细胞系增殖：H3122-L1196M（克唑替尼耐药，IC50 = 7.2 nM）、H3122-G1202R（艾乐替尼耐药，IC50 = 18 nM）；在H3122-G1202R细胞中，100 nM处理2小时可降低p-ALK（Tyr1604）水平90%[1]
- 抑制ALK阳性细胞活力：NSCLC H2228（IC50 = 3.5 nM）、神经母细胞瘤SH-SY5Y（IC50 = 5.8 nM）、NSCLC H1975-ALK（IC50 = 6.1 nM）；抑制H1975细胞中EGFR驱动的增殖（IC50 = 62 nM）[2]
- 诱导神经母细胞瘤IMR-32细胞凋亡（ALK F1174L，IC50 = 8.3 nM）：200 nM Brigatinib（AP26113）处理48小时，caspase-3/7活性升高3.2倍；0.5 μM处理14天，集落形成减少75%[3]
- 穿透体外血脑屏障（BBB）模型：体外BBB渗透系数（Papp）= 22×10⁻⁶ cm/s（与艾乐替尼相当，后者Papp = 18×10⁻⁶ cm/s）[1]

AP26113 (< 50 mg/kg) 剂量依赖性地抑制 Karpas-299 异种移植小鼠模型肿瘤中的 p-ALK。 AP26113 (< 50 mg/kg) 剂量依赖性地抑制 Karpas-299 异种移植小鼠模型和 H3122 异种移植小鼠模型中的肿瘤生长。 AP26113 表现出良好的特性，包括中等的体外血浆蛋白结合（在人、大鼠、小鼠中为 47%、70% 和 76%），对主要 CYP 亚型的抑制可忽略不计。 AP26113 (10 mg/kg) 在大鼠中具有良好的耐受性，Cmax 为 2587 ng/mL，AUC 为 41120 hr.ng/mL。 AP26113 (25 mg/kg) 导致 HCC827(EGFR-DEL) 或 HCC827(EGFR-DEL/T790M) 异种移植小鼠模型中的肿瘤消退。

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携带H3122-G1202R异种移植瘤的裸鼠：口服Brigatinib（100 mg/kg/天），持续28天，肿瘤生长抑制率（TGI）达89%；克唑替尼/艾乐替尼TGI均<30%[1]
- 携带H2228脑转移瘤（颅内异种移植）的小鼠：口服Brigatinib（75 mg/kg/天），持续21天，脑肿瘤体积减少82%；中位存活期从溶剂组25天延长至56天[1]
- 携带SH-SY5Y神经母细胞瘤异种移植瘤的裸鼠：口服Brigatinib（50 mg/kg/天），持续21天，TGI达76%；通过免疫组化检测，肿瘤中p-ALK水平降低70%[3]
- 携带H460-ALK异种移植瘤的大鼠：口服Brigatinib（30 mg/kg/天），持续14天，TGI达68%；未观察到明显肿瘤消退[2]

在体外对 289 种激酶进行 HotSpot SM 激酶谱分析。该实验在 10 μM [ 33 P]-ATP 存在的情况下，使用浓度范围为 0.05 nM 至 1 μM 的布加替尼进行。

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ALK激酶活性实验（野生型/突变体）：重组人ALK激酶结构域（50 ng/孔）与10 μM ATP、荧光肽底物在反应缓冲液（25 mM HEPES pH 7.5，10 mM MgCl2，1 mM DTT）中于30°C孵育60分钟。加入ATP前15分钟，加入Brigatinib（0.01-100 nM）。通过HTRF（激发光340 nm，发射光665 nm）检测激酶活性；采用非线性回归计算IC50值[2]
- EGFR激酶活性实验：重组人EGFR激酶结构域（40 ng/孔）采用相同缓冲液体系，ATP浓度调整为20 μM。检测方法及IC50计算方式与ALK实验一致[2]

将指定的抑制剂连续稀释并添加到含有 15,000 个细胞的每个孔中。刃天青在 72 小时后测量细胞的活力。通过将数据拟合到对数（抑制剂浓度）与标准化响应（可变斜率）的方程中，使用 GraphPad Prism 6.0 确定 IC50 值。每个实验均进行两份，至少进行三次。

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细胞增殖实验（NSCLC/神经母细胞瘤）：将细胞接种于96孔板（5×10³个细胞/孔），用Brigatinib（0.01-100 nM）处理72小时。采用四唑盐类比色法检测细胞活力，记录570 nm处吸光度，通过四参数逻辑拟合计算IC50值[1][2][3]
- Western blot实验（p-ALK/p-ERK/p-AKT）：H3122-G1202R细胞（1×10⁶个细胞/孔）用Brigatinib（10-200 nM）处理2小时后，用RIPA缓冲液（含蛋白酶/磷酸酶抑制剂）裂解。裂解物（30 μg蛋白）经8% SDS-PAGE分离，转移至PVDF膜，用抗p-ALK（Tyr1604）、总ALK、p-ERK（Thr202/Tyr204）、总ERK、p-AKT（Ser473）、总AKT及GAPDH抗体孵育，通过化学发光检测信号[1]
- 凋亡实验（IMR-32）：细胞用Brigatinib（50-200 nM）处理48小时，用Annexin V-FITC/碘化丙啶染色，通过流式细胞术定量凋亡细胞（Annexin V阳性）[3]

Mice: (1) Female SCID/beige mice, aged eight to ten weeks, receive intravenous injections of 5x10 6 H3122 cells each. After the tumor size reaches approximately 300 mm 3 on day zero, the mice are randomized into ten treatment groups. Treatments are taken orally at a dose volume of 10 mL/kg for a maximum of 21 days in a row. Tumors under the skin are measured twice or three times a week. The formula (L×W 2 )/2 is used to calculate the tumor volume (in mm 3 ). The animal is put to sleep by CO₂ asphyxiation when a tumor weighs 10% of its body weight. (2) Female SCID/beige mice, aged eight to ten weeks, receive subcutaneous injections of 2.5 ×10 6 Karpas-299 cells per mouse. After the tumors reach approximately 180 mm 3 on day zero, the mice are randomly assigned to one of ten treatment groups. Oral treatment is given for 14 days in a row at a dose volume of 10 mL/kg. The measurement and computation of tumor volume follow the guidelines for the H3122 model.

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H3122-G1202R xenograft model (nude mice): 6-week-old female nude mice were subcutaneously injected with 5×10⁶ H3122-G1202R cells. When tumors reached 100-120 mm³, mice received Brigatinib (100 mg/kg/day, oral gavage) for 28 days. Drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80; tumor volume (length × width² / 2) was measured every 3 days [1]
- Intracranial xenograft model (nude mice): 1×10⁵ H2228 cells were injected into the right striatum. Seven days later, mice received Brigatinib (75 mg/kg/day, oral gavage) for 21 days. Brain tumor volume was assessed via MRI [1]
- SH-SY5Y neuroblastoma model (nude mice): 7-week-old male nude mice were subcutaneously injected with 1×10⁷ SH-SY5Y cells. When tumors reached 150 mm³, mice received Brigatinib (50 mg/kg/day, oral gavage) for 21 days. Drug was dissolved in 30% PEG400 + 5% ethanol + 65% water [3]
- H460-ALK xenograft model (rats): 8-week-old male Sprague-Dawley rats were implanted with 2×10⁷ H460-ALK cells subcutaneously. When tumors reached 200 mm³, rats received Brigatinib (30 mg/kg/day, oral gavage) for 14 days [2]

Absorption, Distribution and Excretion
Following a 90 mg dose of brigatinib, the peak plasma concentration (Cmax) was 552 ng/ml, and the area under the curve (AUC) was 8165 ng·h/ml. After a 180 mg dose, Cmax was 1452 ng/ml, and AUC was 20276 ng·h/ml. Exposure was dose-dependent, with a cumulative ratio of 1.9 to 2.4. The time to peak concentration (Tmax) after oral administration of brigatinib ranged from 1 to 4 hours. A high-fat meal decreased Cmax by 13% compared to an overnight fasting diet, but did not affect AUC. Brigatinib is excreted via feces (65%) and urine (25%). During elimination in both compartments, unchanged brigatinib accounted for 41% of total fecal output and 86% of total urinary output, respectively. The steady-state apparent volume of distribution was 153 L.
After oral administration of 180 mg brigatinib, the steady-state apparent oral clearance was 12.7 L/h.

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Metabolism/Metabolites
Brigatinib is metabolized in human liver microsomes and hepatocytes via CYP2C8 (72.4%) and CYP3A4 (27.6%). The two major metabolites produced are the N-demethylated form and the cysteine-bound form. Oral administration of radiolabeled brigatinib showed that 91.5% of the drug remained in vivo in its original form, with 3.5% as the major metabolite AP26123. The AUC of AP26123 is less than 10% of the AUC of brigatinib, and its inhibitory activity is 3-fold lower.

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Biological Half-Life
The steady-state half-life of brigatinib is 25 hours.

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In mice: Brigatinib oral bioavailability = 55% (10 mg/kg dose); plasma t1/2 = 6.5 h; Cmax 1.2 h after oral administration = 4.2 μM [2]
-In rats: oral bioavailability = 48% (5 mg/kg); t1/2 = 8.1 h; Vss = 1.2 L/kg [2]
-In dogs: oral bioavailability = 62% (2 mg/kg); t1/2 = 11.3 h; plasma protein binding = 97.6% (ultrafiltration) [2]
-Humans (preclinical prediction): predicted oral bioavailability = 50-60%; predicted half-life = 12-14 h [2]

Hepatotoxicity
In pre-registration trials of brigatinib, up to 40% of patients experienced elevated ALT levels, but only 1% to 3% had ALT values exceeding five times the upper limit of normal (ULN). Brigatinib treatment was also associated with frequent elevations in alkaline phosphatase (15% to 29%), but these elevations were usually mild to moderate, asymptomatic, and transient. No clinically significant liver injury with jaundice was reported in pre-marketing studies of brigatinib, and no such reports have been seen since its approval. Generally, ALK kinase inhibitors are associated with higher rates of serum enzyme elevation during treatment, but cases of specific, clinically significant liver injury caused by them are very rare. Most case reports are related to crizotinib (approved in 2011), which is also the most commonly used anti-ALK kinase inhibitor. Cases of liver injury with jaundice have been reported in clinical trials of alectinib (2015) and ceritinib (2014), but details were not provided. Therefore, brigatinib may cause acute liver injury with jaundice, but even if it occurs, it should be very rare. Probability score: E (Unproven, but suspected cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the clinical use of brigatinib during lactation. The manufacturer recommends discontinuing breastfeeding during brigatinib treatment and for one week after the last dose. ◉ Effects on Breastfed Infants No published information found as of the revision date. ◉ Effects on Lactation and Breast Milk No published information found as of the revision date.

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Protein Binding 66% of the brigatinib dose was bound to plasma proteins, with a blood concentration to plasma concentration ratio of 0.69.

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In a 28-day H3122-G1202R xenograft study (100 mg/kg/day, orally): no significant weight loss (>8%) was observed; serum ALT = 29 ± 5 U/L, AST = 51 ± 7 U/L (normal range: ALT 20-40 U/L, AST 40-60 U/L) [1]
- In a 14-day rat study (30 mg/kg/day, orally): 1 out of 6 rats experienced mild diarrhea (resolved on day 7); no histopathological changes were observed in the liver/kidneys [2]
- In a 21-day SH-SY5Y mouse model (50 mg/kg/day, orally): no treatment-related deaths were observed; 2 out of 8 mice experienced mild alopecia (recovered after treatment) [3]

[1]. The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models. Clin Cancer Res. 2016 Nov 15;22(22):5527-5538.

[2]. Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase. J Med Chem. 2016 May 26;59(10):4948-64.

[3]. Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice. Oncotarget. 2016 May 17;7(20):29011-22.

Brigatinib, formerly known as AP26113, is a reversible dual inhibitor of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR). It is selective for EGFR mutants compared to wild-type EGFR. Furthermore, it is selective for nine different crizotinib-resistant EML4-ALK fusion gene mutants, which play a crucial role in the transformation of susceptible lung parenchyma. Brigatinib was developed by Ariad Pharmaceuticals, a subsidiary of Takeda Pharmaceutical Company Limited, and was approved by the U.S. Food and Drug Administration (FDA) on April 28, 2017. Brigatinib is a kinase inhibitor. Its mechanism of action is as a tyrosine kinase inhibitor and a cytochrome P450 3A inducer. Brigatinib is a tyrosine kinase receptor inhibitor and anti-tumor drug used to treat certain types of advanced non-small cell lung cancer. A moderate, transient increase in serum transaminase levels may occur during brigatinib treatment, but it has not been found to be associated with clinically significant cases of acute liver injury. Brigatinib is an oral receptor tyrosine kinase inhibitor that inhibits anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR), possessing potential antitumor activity. Brigatinib binds to and inhibits the activity of ALK kinase and its fusion protein, as well as EGFR and its mutants. This leads to inhibition of ALK and EGFR kinase activity, thereby disrupting their signaling pathways and ultimately inhibiting the growth of susceptible tumor cells. Furthermore, AP26113 appears to overcome mutation-based resistance. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development; ALK dysregulation and gene rearrangements are associated with a range of tumors. EGFR is overexpressed in multiple cancer cell types.

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Drug Indications
Anaplastic lymphoma kinase-positive metastatic non-small cell lung cancer (ALK+ NSCLC) accounts for only 3-5% of NSCLC cases, but ALK mutations, overexpression, and the presence of various oncogenic fusion proteins in solid tumors and hematologic malignancies highlight its importance and potential as a cancer therapeutic target. ALK-related NSCLC cases are associated with the presence of the fusion gene EML4-ALK, which fuses the ALK protein with echinoderm microtubule-associated protein-like protein 4 (EML4). The original function of EML4 is to promote the proper formation of microtubules. The presence of abnormal fusion proteins leads to aberrant signaling, thereby promoting cell growth, proliferation, and survival. Crizotinib is suitable for treating these cases, but the presence of mutations in the ALK kinase domain leads to resistance. Therefore, brigatinib is indicated for the treatment of ALK-positive non-small cell lung cancer (NSCLC) patients who are intolerant to crizotinib.
FDA Label
Alunbrig is indicated as monotherapy for the treatment of adult patients with anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC) who have not previously received ALK inhibitor therapy. Alunbrig is indicated as monotherapy for adult patients with ALK-positive advanced NSCLC who have previously received crizotinib therapy.
Treatment of anaplastic large cell lymphoma, treatment of inflammatory myofibroblastoma, treatment of non-small cell lung cancer.
Mechanism of Action
Brigitanib is a tyrosine kinase inhibitor active against multiple kinases, including ALK, ROS1, insulin-like growth factor 1 receptor, and EGFR deletions and point mutations. Its mechanism of action is through inhibition of ALK phosphorylation and activation of downstream signaling proteins.
Pharmacodynamics
Brigitanib inhibits the proliferation and in vitro viability of cells expressing the EML4-ALK fusion protein and 17 crizotinib-resistant ALK mutants. Its range of action extends to cells expressing EGFR deletion, ROS1-L2026M, FLT3-F691L, and FLT3-D835Y. In an EML4-ALK xenograft mouse model, Brigatinib dose-dependently inhibited tumor growth, reduced tumor burden, and prolonged survival. The time course and exposure-response studies of Brigatinib remain unclear. Brigatinib (AP26113) is an ATP-competitive dual ALK/EGFR inhibitor designed to overcome resistance to first-generation (crizotinib) and second-generation (alectinib) ALK inhibitors through high affinity for ALK-resistant mutants (e.g., G1202R) [1][2]. It has strong blood-brain barrier penetration (mouse cerebrospinal fluid/plasma concentration ratio of 0.72), which explains its efficacy in ALK-positive non-small cell lung cancer with brain metastases [1].
- In neuroblastoma models, Brigatinib showed synergistic effects with the MEK inhibitor trametinib (quantitative data were not available in this literature) [3].
- Preclinical data support Brigatinib as a candidate drug for the treatment of acquired resistant or brain metastatic ALK-positive cancers. [1][2][3]

C₂₉H₃₉CLN₇O₂P

584.09

583.259

C, 59.63; H, 6.73; Cl, 6.07; N, 16.79; O, 5.48; P, 5.30

1197953-54-0

Brigatinib-13C6

68165256

Light yellow solid powder

1.3±0.1 g/cm3

781.8±70.0 °C at 760 mmHg

426.6±35.7 °C

0.0±2.7 mmHg at 25°C

1.641

0.43

95.67

2

9

8

40

835

0

ClC1=C([H])N=C(N=C1N([H])C1=C([H])C([H])=C([H])C([H])=C1P(C([H])([H])[H])(C([H])([H])[H])=O)N([H])C1C([H])=C([H])C(=C([H])C=1OC([H])([H])[H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H]

AILRADAXUVEEIR-UHFFFAOYSA-N

InChI=1S/C29H39ClN7O2P/c1-35-15-17-37(18-16-35)21-11-13-36(14-12-21)22-9-10-24(26(19-22)39-2)33-29-31-20-23(30)28(34-29)32-25-7-5-6-8-27(25)40(3,4)38/h5-10,19-21H,11-18H2,1-4H3,(H2,31,32,33,34)

5-chloro-4-N-(2-dimethylphosphorylphenyl)-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]pyrimidine-2,4-diamine

AP-26113; AP 26113; Brigatinib-analog; AP26113; Brigatinib; Alunbrig.

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: ≥ 1 mg/mL (1.71 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 10.0 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: ≥ 1 mg/mL (1.71 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 10.0 mg/mL 澄清乙醇储备液加入 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: ≥ 1 mg/mL (1.71 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 10.0 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: ≥ 0.5 mg/mL (0.86 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 5.0 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: ≥ 0.5 mg/mL (0.86 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 5.0 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: ≥ 0.5 mg/mL (0.86 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 5.0 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: NMP+polyethylene glycol 300 (10+90, v+v): 1 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。