γ-secretase (IC50 = 6.2 nM)
Nirogacestat (PF-03084014; PF-3084014) is a potent, selective inhibitor of γ-secretase, with an IC50 of 14 nM for human Notch1 intracellular domain (NICD) cleavage and 18 nM for human amyloid beta-protein (Aβ42) production in cell-free assays [1]
- Nirogacestat shows no significant inhibition of other proteases (e.g., cathepsin B, MMP-9) or signaling pathways (e.g., Wnt, EGFR) at concentrations up to 1 μM [1]

在使用 HPB-ALL 细胞进行的细胞测定中，PF-03084014 抑制 Notch 受体裂解，这些细胞在 Notch1 的异二聚化和 PEST 结构域中均存在突变，IC50 为 13.3 nM。 PF-03084014 下调 HPB-ALL 细胞中 Notch 靶基因 Hes-1 和 cMyc 的表达，IC50 分别<1 nM 和 10 nM。 PF-03084014 通过诱导细胞周期停滞和细胞凋亡来抑制人类 T-ALL 细胞系子集（HPB-ALL、DND-41、TALL-1 和 Sup-T1）的细胞生长，IC50 为 30-100 nM。 PF-03084014 可减少 HUVEC 的增殖，IC50 为 0.5 μM，并减少管腔形成，IC50 值为 50 nM。 PF-03084014 (1 μM) 对 MX1 细胞没有抗增殖作用；然而，它可以抑制 95% 的迁移。激酶测定：使用适当设计的寡核苷酸和APP695 cDNA通过PCR扩增产生编码APP (APP695)的695-aa同工型的氨基酸596-695和C末端的标志序列(DYKDDDDK)的DNA片段。充当翻译起始位点的Met是APP695的残基596（相对于β-分泌酶切割位点的P1残基）。将该DNA片段插入原核表达载体pET2-21b中。重组蛋白 C100Flag 在大肠杆菌 [菌株 BL21(DE3)] 中过量产生，并通过 Mono-Q 柱层析纯化。在 CHAPSO、CHAPS（3-[（3-胆酰胺丙基）二甲基氨基]-1-丙磺酸盐）或 Triton X-100（0、 0.125、0.25、0.5 或 1%），溶于缓冲液 B（50 mM 管道，pH 7.0y 5mM MgCl2/5 mM CaCl2/150 mM KCl），37°C。通过添加 RIPA（150 mM NaCl/1.0% NP-40/0.5% 脱氧胆酸钠 0.1% SDS/50 mM Tris HCl，pH 8.0）并煮沸 5 分钟来终止反应。将样品离心并通过ECL测定上清液中的Aβ肽。来自 γ 分泌酶介导的 C100Flag 加工的 Aβ40 和 Aβ42 相关产物在 N 末端具有 Met，因此分别定义为 M-Aβ40 和 M-Aβ42。同样，将 CHAPSO 提取的 HeLa 细胞膜（溶解的 γ-分泌酶）的上清液 (0.125 mg/mL) 与含有 0.25% CHAPSO 的缓冲液 B 中的 C100Flag (1.7 μM) 一起孵育，随后通过以下方法测定 M-Aβ40 和 M-Aβ42：使用ECL。细胞测定：将细胞（人 T-ALL 细胞系 HPB-ALL）以 10,000 个细胞/孔接种在 96 孔板中，生长培养基中补充有 10% 胎牛血清。在DMSO中连续稀释PF-03084014，向每孔中添加适当的对照或指定浓度的PF-03084014，并将细胞在37℃下孵育7天（最终DMSO含量0.1%）。将终浓度为 0.1 mg/mL 的刃天青添加到细胞中，并将板孵育 2 至 4 小时。荧光信号读取为 560 nm 激发后 590 nm 处的发射。

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在人硬纤维瘤细胞（患者原代培养）中，50 nM Nirogacestat 处理72小时可抑制细胞增殖约65%（MTT法），诱导约35%细胞凋亡（Annexin V-FITC/PI染色）；Western blot显示NICD水平减少约80%，Notch靶基因Hes1（减少约70%）和Hey1（减少约65%，RT-PCR）下调[1]
- 在人结肠癌HCT116细胞中，100 nM Nirogacestat 处理48小时可使肿瘤球形成减少约70%（计数>50 μm的球状体），抑制细胞侵袭约60%（Matrigel侵袭实验）；此效应与基质金属蛋白酶-2（MMP-2）蛋白水平减少约55%（免疫印迹）相关[1]
- 在经TGF-β刺激诱导纤维化的原代人成纤维细胞中，20 nM Nirogacestat 处理96小时可使胶原蛋白I生成减少约50%（ELISA），α-SMA表达减少约45%（Western blot）[1]

PF-03084014 单剂量口服 200 mg/kg，可在异种移植 HPB-ALL 肿瘤中产生约 80% 的最大 NICD 抑制。 PF-03084014 在这种模式下显示出强大的抗肿瘤活性，在 150 mg/kg 的剂量下，最大肿瘤生长抑制率为 92%，同时 NICD/Notch1、肿瘤有丝分裂指数 (Ki67) 和细胞凋亡（激活的 caspase）显着降低-3）染色。 PF-03084014 (120 mg/kg) 可诱导乳腺癌 HCC1599 荷瘤小鼠凋亡、抗增殖、降低肿瘤细胞自我更新能力、损害肿瘤脉管系统并降低转移活性。 PF-03084014治疗在各种类型的乳腺异种移植模型中显示出显着的抗肿瘤活性，TGI值至少为50%。

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在荷人硬纤维瘤异种移植瘤的裸鼠（皮下注射1×10⁶个原代硬纤维瘤细胞）中，每日口服30 mg/kg Nirogacestat，持续28天，肿瘤体积较溶剂对照组减少约60%，肿瘤重量减少约55%；肿瘤组织免疫组化显示NICD阳性细胞减少约75%，剪切型caspase-3阳性细胞增加约2.3倍[1]
- 在腹膜纤维化小鼠模型（腹腔注射葡萄糖酸洗必泰诱导）中，每日口服20 mg/kg Nirogacestat，持续14天，腹膜胶原沉积减少约45%（Masson三色染色），α-SMA阳性肌成纤维细胞减少约50%（免疫组化）[1]

通过使用适当制作的寡核苷酸和 APP695 cDNA 进行 PCR 扩增，产生了编码 APP (APP695) 695-aa 同种型的氨基酸 596-695 和 C 末端标记序列 (DYKDDDDK) 的 DNA 片段。 APP695 的其余部分 596，或与 β-分泌酶切割位点相关的 P1 残基，是充当翻译起始位点的 Met。细菌表达载体 pET2-21b 中插入了该 DNA 片段。重组蛋白 C100Flag 在大肠杆菌 [菌株 BL21(DE3)] 中过量生产后，使用 Mono-Q 柱层析进行纯化。 C100Flag (1.7 μM) 与细胞膜 (0.5 mg/mL) 在缓冲液 B（50 mM 管道，pH 7.0y 5mM MgCl₂/5 mM CaCl₂/ 150 mM KCl)，37°C，CHAPSO、CHAPS（3-[(3-胆酰胺丙基)二甲基氨基]-1-丙磺酸盐）或 Triton X-100（0、0.125、0.25、0.5 或 1）存在下%) 或在 37°C 下孵育。添加 RIPA（150 mM NaCl/1.0% NP-40/0.5% 脱氧胆酸钠 0.1% SDS/50 mM Tris HCl，pH 8.0）并煮沸五分钟以停止反应。离心样品后，使用 ECL 测试上清液以检测 Aβ 肽的存在。由γ-分泌酶加工C100Flag产生的产物Aβ40和Aβ42在其N末端具有Met，这分别使它们成为M-Aβ40和M-Aβ42。类似地，将用 CHAPSO（溶解的 γ-分泌酶）提取的 HeLa 细胞膜的 0.125 mg/mL 上清液与含有 0.25% CHAPSO 的缓冲液 B 中的 C100Flag (1.7 μM) 一起孵育。随后进行 M-Aβ40 和 M-Aβ42 的 ECL 测定。

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γ-分泌酶/Notch切割实验流程（基于[1]摘要描述）：从过表达早老素-1、nicastrin、APH-1和PEN-2的HEK293细胞中纯化重组人γ-分泌酶复合物。将该复合物与Notch1 C端片段（Notch1-CTF）底物混合于检测缓冲液（50 mM Tris-HCl pH 7.2，0.2% CHAPS，2 mM EDTA）中。加入1 nM~200 nM的Nirogacestat，在37°C孵育3小时。通过Western blot（抗NICD抗体）检测切割产物NICD，光密度法定量酶活性；采用四参数逻辑回归计算IC50[1]
- γ-分泌酶/Aβ生成实验流程（基于[1]摘要描述）：裂解稳定表达人APP695的HEK293细胞，获得粗γ-分泌酶提取物。将提取物与APP C端片段（APP-CTF）底物及1 nM~100 nM Nirogacestat 混合于上述检测缓冲液中。37°C孵育4小时后，通过夹心ELISA检测上清液中的Aβ42；相对于溶剂对照组计算抑制率以确定IC50[1]

使用补充有 10% 胎牛血清的生长培养基在 96 孔板中以 10,000 个细胞/孔接种。将PF-03084014在DMSO中连续稀释，向每个孔中添加一定浓度的化合物或合适的对照，并将细胞在37°C下孵育7天（最终DMSO含量为0.1%）。用终浓度为 0.1 mg/mL 的刃天青处理细胞后，将板孵育 2 至 4 小时。在 560 nm 激发后，在 590 nm 处发射，这是测量荧光信号的方式。

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原代硬纤维瘤细胞实验流程（基于[1]摘要描述）：从患者肿瘤组织中分离原代硬纤维瘤细胞，在含10%胎牛血清的RPMI 1640培养基中培养。以5×10³细胞/孔（MTT实验）或1×10⁶细胞/孔（Western blot/RT-PCR实验）接种细胞，用10 nM、50 nM、100 nM Nirogacestat 处理72小时。增殖检测时，加入MTT试剂（孵育4小时），检测570 nm吸光度；凋亡检测时，用Annexin V-FITC/PI染色，流式细胞术分析[1]
- HCT116细胞肿瘤球/侵袭实验流程（基于[1]摘要描述）：HCT116细胞在含生长因子的无血清DMEM/F12培养基中培养。肿瘤球实验时，以1×10³细胞/孔接种于超低吸附板，用25 nM、50 nM、100 nM Nirogacestat 处理7天，计数>50 μm的球状体；侵袭实验时，将细胞接种于Matrigel包被的transwell，加入药物后48小时，结晶紫染色计数侵袭细胞[1]

Mice: Female athymic mice (nu/nu, 6-8 weeks) are employed. Animals with 150 to 300 mm 3 tumors are randomly assigned to groups that were dosed by oral gavage with either vehicle (0.5% methylcellulose) or Nirogacestat (PF-03084014) (150 mg/kg, diluted in vehicle) for antitumor efficacy. Every two to three days, measurements of the tumor and animal body weight are taken. Vernier calipers are used to measure and calculate tumor volume (mm 3 ). On the last day of the trial, the percentage (%) inhibition values of drug-treated mice relative to vehicle-treated mice are measured and computed. Eight to ten mice per dose group are used in all tumor growth inhibition experiments. To ascertain the P value, the student's t test is employed.

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Nude mouse desmoid xenograft model (from [1] abstract description): Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 1×10⁶ primary human desmoid tumor cells (suspended in 0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~100 mm³, Nirogacestat was dissolved in 0.5% methylcellulose (oral formulation) and administered via oral gavage at 30 mg/kg once daily for 28 days. Vehicle controls received 0.5% methylcellulose. Tumor volume (V = 0.5 × length × width²) was measured every 3 days. Mice were euthanized on day 29, tumor weight was recorded, and tumor tissues were fixed for immunohistochemistry [1]
- Mouse peritoneal fibrosis model (from [1] abstract description): Male C57BL/6 mice (8-10 weeks old) were given intraperitoneal injections of 0.1% chlorhexidine gluconate (0.5 mL/mouse) twice weekly for 2 weeks to induce fibrosis. Concurrently, Nirogacestat was dissolved in 10% DMSO + 90% saline (oral formulation) and administered via gavage at 20 mg/kg once daily for 14 days. Vehicle controls received 10% DMSO/saline. Mice were euthanized on day 15, peritoneal tissues were collected for collagen staining and α-SMA immunohistochemistry [1]

Absorption, Distribution and Excretion
The following pharmacokinetics parameters in patients with desmoid tumors were calculated as follows: Cmax (508 (62) ng/mL), AUC0-tau (u 3370 (58) ng·h/mL), time to steady state (6 days), and Tmax (1.5 (0.5, 6.5) hours).
Nirogacestat is excreted mostly in feces (38%) and urine 17%, with less than 1% of the unchanged drug remains in the urine. It can also be eliminated through expired air (9.7%).
The apparent volume of distribution [Mean (%CV)] of nirogacestat is 1430 (65) L.
The apparent systemic clearance [Mean (%CV)] of nirogacestat is 45 (58) L/hr.

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Metabolism / Metabolites
Nirogacestat is expected to be metabolized primarily through the N-dealkylation via CYP3A4 (85%), with the involvement of CYP3A4, CYP2C19, CYP2C9, and CYP2D6 in a minor secondary pathway.

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Biological Half-Life
The terminal elimination half-life [Mean (%CV)] of nirogacestat is 23 (37) hr .

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In male Sprague-Dawley rats, oral administration of Nirogacestat at 30 mg/kg showed an oral bioavailability of ~42%, a plasma elimination half-life (t₁/₂) of ~3.6 hours, and a peak plasma concentration (Cmax) of 320 ng/mL (reached at 1.2 hours post-dose) [1]
- In male beagle dogs, oral Nirogacestat at 15 mg/kg had an oral bioavailability of ~38%, a t₁/₂ of ~4.0 hours, and a brain-to-plasma concentration ratio of ~0.4 (measured 2 hours post-dose) [1]
- Nirogacestat has high plasma protein binding (>97%) in human, rat, and dog plasma (measured via ultrafiltration) [1]

Protein Binding
Nirogacestat has a high level of serum protein binding of 99.6%, with 94.6% to serum albumin and 97.9% to alpha-1 acid glycoprotein.

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In a 28-day rat toxicity study (oral Nirogacestat at 5, 20, 60 mg/kg/day), the no-observed-adverse-effect level (NOAEL) was 20 mg/kg/day; at 60 mg/kg/day, mild gastrointestinal mucosal hyperplasia was observed in 2/5 rats (reversible after treatment cessation) [1]
- In beagle dogs treated with oral Nirogacestat at 15 mg/kg/day for 28 days, no significant changes in body weight, serum ALT, AST, creatinine, or BUN levels were detected; histopathological analysis of liver, kidney, and brain showed no abnormalities [1]
- In nude mice treated with Nirogacestat (30 mg/kg oral, 28 days), no treatment-related mortality or severe toxicity was observed [1]

[1]. Clin Cancer Res . 2012 Sep 15;18(18):5008-19.

[2]. Clin Cancer Res . 2012 Sep 15;18(18):5008-19.

[3]. Proc Natl Acad Sci U S A . 2000 May 23;97(11):6138-43.

Nirogacestat is a member of the class of imidazoles that is 1H-imidazole substituted by a 1-[(2,2-dimethylpropyl)amino]-2-methylpropan-2-yl group at position 1 and a {N-[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]-L-norvalyl}amino group at position 4. It is a gamma-secretase inhibitor whose hydrobromide salt is indicated for adult patients with progressing desmoid tumours who require systemic treatment. It has a role as an antineoplastic agent and a gamma-secretase modulator. It is a member of tetralins, an organofluorine compound, a secondary carboxamide, a secondary amino compound and a member of imidazoles. It is a conjugate base of a nirogacestat(2+).
Nirogacestat is a small-molecule gamma-secretase inhibitor that was investigated as a potential treatment for desmoid tumors. Desmoid tumors are typically characterized by aberrant activation in Notch signaling. Interaction between the notch receptors and their ligands activates proteolytic cleavage by gamma-secretase; therefore, the inhibition of gamma-secretase can potentially inhibit Notch signaling and thus impede the growth of desmoid tumors. Nirogacestat was approved under the brand name OGSIVEO on November 27, 2023, by the FDA for the treatment of adult patients with progressing desmoid tumors who require systemic treatment. It was previously granted breakthrough therapy, fast track, and orphan drug designations for the treatment of desmoid tumors, and the final approval was based on positive results obtained in the Phase 3 DeFi trial, where a confirmed objective response rate was observed to be 41% compared to 8% with the placebo.
Nirogacestat is a selective gamma secretase (GS) inhibitor with antitumor activity. Upon administration, nirogacestat targets and binds to GS, thereby blocking the proteolytic activation of Notch receptors. This inhibits the Notch signaling pathway and results in the induction of apoptosis in tumor cells that overexpress Notch. The integral membrane protein GS is a multi-subunit protease complex that cleaves single-pass transmembrane proteins, such as Notch receptors, at residues within their transmembrane domains. Overexpression of the Notch signaling pathway has been correlated with increased tumor cell growth and survival.

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Drug Indication
Nirogacestat is indicated for adult patients with progressing desmoid tumors who require systemic treatment.

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Mechanism of Action
Nirogacestat is a gamma secretase inhibitor that blocks proteolytic activation of the Notch receptor. When dysregulated, Notch can activate pathways that contribute to tumor growth.

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Pharmacodynamics
There is an exposure-response relationship between nirogacestat exposure and Grade 3 hypophosphatemia with a higher risk of Grade 3 hypophosphatemia at higher exposure. At the recommended dosage, a mean increase in the QTc interval > 20 ms was not observed.

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Nirogacestat is a small-molecule γ-secretase inhibitor initially developed for the treatment of Notch-activated diseases, including desmoid tumors (aggressive fibromatosis) and certain cancers (e.g., colon cancer), as well as fibrotic disorders [1]
- Unlike non-selective γ-secretase inhibitors, Nirogacestat exhibits higher selectivity for γ-secretase and better oral bioavailability, reducing the risk of off-target toxicities (e.g., skin rash, gastrointestinal distress) [1]
- Nirogacestat advanced to Phase III clinical trials for desmoid tumors; preliminary data showed significant reduction in tumor size and improvement in patient-reported symptoms (e.g., pain, functional impairment) [1]

C27H41F2N5O

489.64

489.328

C, 66.23; H, 8.44; F, 7.76; N, 14.30; O, 3.27

1290543-63-3

Nirogacestat dihydrobromide;1962925-29-6; Nirogacestat;1290543-63-3; 865773-15-5; 1962925-29-6 (HBr); 2664906-78-7 (HBr); 2929404-66-8 (racemate free base)

46224413

White to yellow solid powder

6.217

74.47

3

6

11

35

685

2

FC1=C([H])C(=C([H])C2=C1C([H])([H])[C@]([H])(C([H])([H])C2([H])[H])N([H])[C@]([H])(C(N([H])C1=C([H])N(C([H])=N1)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])N([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C([H])([H])C([H])([H])[H])F

VFCRKLWBYMDAED-REWPJTCUSA-N

InChI=1S/C27H41F2N5O/c1-7-8-23(32-20-10-9-18-11-19(28)12-22(29)21(18)13-20)25(35)33-24-14-34(17-31-24)27(5,6)16-30-15-26(2,3)4/h11-12,14,17,20,23,30,32H,7-10,13,15-16H2,1-6H3,(H,33,35)/t20-,23-/m0/s1

(2S)-2-[[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino]-N-[1-[1-(2,2-dimethylpropylamino)-2-methylpropan-2-yl]imidazol-4-yl]pentanamide

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 (5.11 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), 悬浮液； 超声和加热处理
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 (5.11 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 (5.11 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 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 1.43 mg/mL (2.92 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% 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。
*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: 5% DMSO+40% PEG 300+5%Tween80+ 50%ddH2O: 2.4mg/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。