IAG933   中文名称: YAP-TEAD-抑制剂-3

YAP-TEAD (IC50 = 9 nM)[1]

IAG933及其类似物是YAP-TEAD蛋白-蛋白相互作用的强效一流和选择性干扰物，具有进入临床试验的合适特性。药理学上废除与所有四种TEAD副同源物的相互作用导致YAP从染色质中排出，并减少Hippo介导的转录和细胞死亡的诱导。[2]
IAG933是一种直接针对界面3的YAP-TEAD蛋白质相互作用干扰物（PPID），已于2021年进入临床试验[3]。

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IAG933通过诱导细胞凋亡改善对JDQ443的反应[2]
尽管选择性KRASG12C抑制剂对突变型癌症有影响，但它们的临床疗效通常不如RTK抑制剂明显。克服对KRASG12C抑制剂的耐药性仍然是一个挑战，促使正在进行的临床试验研究联合疗法。与变构TEAD抑制剂的研究结果一致，IAG933和诺华KRASG12C抑制剂JDQ443在KRASG12B突变的NSCLC和CRC细胞系中显示出很强的联合益处（图6a）。IAG933与其他JDQ443候选伴侣（如SHP2、MEK、ERK或PIKα抑制剂）相比具有优势，因为它导致细胞系间最大生长抑制的显著变化（扩展数据图9a）。在长期增殖试验中，我们观察到，当联合亚等效浓度的JDQ443和IAG933时，细胞生长受到强烈和持续的抑制，这适度延迟了细胞增殖（扩展数据图9b）。一致地，在体内，预先添加IAG933加深了NCI-H2122 NSCLC异种移植物对JDQ443的反应（图6b），这种组合优于JDQ443加SHP2抑制剂TNO155（扩展数据图9c）。在非小细胞肺癌的PDX模型中也观察到了这种抗肿瘤联合作用，治疗结束后30天内没有观察到肿瘤再生（图6c6c）。

IAG933和YTP-75具有剂量依赖性抗肿瘤疗效[2]
在小鼠MSTO-211H细胞衍生异种移植物（CDX）模型中，通过口服灌胃给药，单次剂量为每公斤体重30至240毫克（mg kg−1），对IAG933进行了评估。在最大浓度（Tmax）下观察到剂量相关的血液暴露时间约为1~2小时，与给药后约2小时开始的剂量/暴露依赖性TEAD靶基因抑制相关（图3a，b）。靶基因抑制的体内血液IC50为64 nM，略高于MSTO-211H细胞的体外IC50 11-26 nM（图1c）。在原位胸膜MSTO-211H肿瘤中，使用TEAD反应元件下的萤光素酶表达进行的体内报告分析显示，单剂量IAG933后，生物发光迅速而严重地丧失（图（图3c），3c），随后由于IAG933在小鼠体内的半衰期相对较短，在几个小时内反弹到基线。对IAG933类似物YTP-75观察到类似的PK/PD结果（扩展数据图5a，b），表明这两种化合物在体内具有深度和快速的TEAD转录抑制作用。

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IAG933在耐受剂量下消除大鼠模型中的肿瘤[2]
将我们的体内研究扩展到小鼠以外，我们还在MSTO-211H大鼠皮下异种移植物模型中评估了IAG933。单剂量IAG933后的靶基因抑制动力学与小鼠模型中观察到的动力学相似（扩展数据图7a，b），而20 nM的平均CCN2/ANKRD1/CCN1 IC50约低三倍，与体外IC50值相似。在每日给药2周后，在10 mg kg−1的剂量下观察到肿瘤停滞，在30 mg/kg的剂量下，五只动物中有四只出现完全消退（图（图3g）.3g）。暴露量与剂量成正比，在每天治疗的12天内没有检测到化合物积聚（扩展数据图7c）。没有观察到体重减轻，治疗耐受性良好。比较大鼠和小鼠模型反应曲线，确定大鼠每天一次30mg kg−1和小鼠每天一次240mg kg−2的剂量等效性（扩展数据图7d7d）。

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间皮瘤和Hippo改变的异种移植物中的IAG933活性[2]
间皮瘤的发病机制通常涉及Hippo信号级联的肿瘤抑制基因的遗传改变，包括NF2和LATS1/LATS2，估计有32-50%的病例9,37-39。我们在每天用YTP-75治疗的九个人源性异种移植物（PDX）小鼠模型中探索了YTP在不同间皮瘤遗传背景下的抗肿瘤功效。在9个模型中，有7个观察到明显的肿瘤反应，3个NF2改变的模型出现深度肿瘤消退，另外4个模型出现持久的肿瘤停滞，但没有报告Hippo改变（图4a4a和扩展数据图7e）。7e）。有趣的是，两种没有反应的肿瘤模型显示TEAD靶基因的基础表达最低（图4a）。在其他实体瘤中也检测到NF2突变的低患病率（~1-2%）9,38,40。为了探索此类病例中的YTP活性，我们评估了三阴性乳腺癌症的NF2-替代PDX模型（5938-HX）中的IAG933和NF2-替代肺癌的CDX模型（NCI-H292）中的YTP-75。这两种模型都显示出对治疗的抗肿瘤反应，但5938-HX经历了肿瘤消退（图4b），而NCI-H292模型对肿瘤生长的抑制作用较小（图4c）。

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IAG933联合治疗提高RTK抑制剂疗效[2]
先前已证明，奥西咪替尼和VT104分别联合抑制EGFR和TEAD可增强非小细胞肺癌模型中的奥西咪替尼肿瘤反应43。HER2阳性癌症和复发性癌症的YAP活性升高3,8，YAP-TEAD激活与曲妥珠单抗耐药性有关8,44。此外，最近的数据表明，在RTK抑制剂治疗下，TEAD激活可以保持最小的残留疾病6,43。因此，共同抑制TEAD对于根除RTK介导的癌症和实现肿瘤消除至关重要。
与这一概念一致，IAG933联合奥西咪替尼显示出增强的抗肿瘤益处，导致EGFR突变的NCI-H1975 CDX NSCLC模型快速消退（图5a）。此外，IAG933加上MET抑制剂capmatinib在癌症的EBC-1 MET-放大CDX模型中诱导了严重的肿瘤收缩，而单独的IAG933没有活性（图（图5b）.5b）。尽管IAG933在来自各种癌症适应症的七个HER2扩增的细胞系中具有适度的单剂作用，但观察到IAG933与HER2抑制剂拉帕替尼的剂量依赖性组合活性（图（图5c），5c）；在更长的体外研究中观察到治疗结束后延长的组合活性（图图5d）.5d）。在体内，HER2扩增的NCI-N87胃癌异种移植物模型在YTP-75和曲妥珠单抗的组合下进行了完全的肿瘤消退（图（图5e）.5e）。因此，在由不同RTK驱动的癌症模型中观察到YTP的组合益处，这表明了共同的潜在机制，并提供了组合这些治疗剂的机会。

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IAG933联合治疗BRAFV600E改变的肿瘤显示出益处[2]
由于激活BRAF中的突变也会驱动对MAPK通路的致癌依赖，我们通过将IAG933与BRAF抑制剂dabrafenib、MEK1/MEK2抑制剂曲美替尼和/或ERK1/ERK2抑制剂LTT462联合使用，探索了YTPs在BRAFV600E突变疾病中的联合潜力。dabrafenib+IAG933、dabrafenib+LTT462+IAG933和dabrafenibi+曲美替尼+IAG93的组合在短期细胞存活率测定中显示出益处（图7a）。与TEAD活性对MAPK通路抑制的适应性作用相一致，在没有YTP的情况下，dabrafenib+曲美替尼治疗后，TEAD反应基因的表达增加，这可以通过IAG933类似物YTP-10同时抑制TEAD来预防（图7b）。在BRAFV600E突变的CRC CDX模型HT-29中，与单药治疗相比，dabrafenib+LTT462+IAG933的三重组合具有更强的抗肿瘤反应（图7c）。同样，在BRAFV600E突变的CRC异种移植物模型5238-HX中，dabrafenib+trametinib+YTP-75的三重组合显示出比dabrafenib+trametnib或dabrafenik+trametiib+西妥昔单抗更强的抗肿瘤活性，导致在21天的研究期间肿瘤持续消退（图（图7d7d）。 TEAD和RAF/MAPK阻断对非KRASG12C PDAC有益 除了临床上可靶向的G12C变体外，KRAS驱动的肿瘤发生的治疗抑制仍然具有挑战性52。为了解决非KRASG12C-突变肿瘤，有效抑制下游RAF、MEK和/或ERK效应器可能提供潜在的治疗选择。在此背景下，考虑到突变体特异性抑制剂所取得的令人鼓舞的组合结果，IAG933可能代表着一个有前景的组合机会（图10）（图66和77以及扩展数据图10）。我们在携带各种KRAS等位基因的PDAC细胞中研究了这一假设。在23个PDAC细胞系中，将YTP-75添加到曲美替尼和RAF抑制剂萘帕非尼中显著增强了生长抑制作用（图8a），这与小鼠临床试验53的结果一致，其中包括12个具有不同KRAS突变的PDAC PDX（7 G12D、2 G12V、2 Q61H和1 G12R），其中8个模型（66%）显示三重组合的肿瘤消退或接近停滞（图8b、c）。在SUIT-2 PDAC细胞中，基于萤光素酶的报告系统中观察到并通过YTP-13共处理，观察到曲美替尼+萘普生对TEAD转录活性的强烈诱导（图8d），并且这种三重组合被证明可以抑制三个PDAC系中DUSP6和TEAD反应性ANKRD1基因的表达（图8e8e）。

表面等离子体共振分析[2]
如前所述，使用人类TEAD1209-426、TEAD2221-447、TEAD3218-435和TEAD4217-434进行表面等离子体共振测定。用AviTag标记四种N-生物素化的TEAD蛋白并将其固定在传感器芯片上，在298 K下测量不同浓度YTP-3和YTP-32的结合。使用Biacore T200评估软件用1:1相互作用模型对数据进行全局拟合，以确定平衡时测得的解离常数（Kd）。

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TR-FRET检测[2]
如前所述，在TR-FRET测定中测试了不同的化合物24。靶向肉豆蔻酸/棕榈酰口袋的脂质粘合剂化合物（K-975和VT104）在TR-FRET中没有活性，因为该测定中使用的TEAD4蛋白完全酰化。

SF-268细胞克隆集落形成试验的选择性评估[2]
对SF-268细胞系进行工程改造，并按如下方式建立了TEAD1双突变克隆（V406A/E408A）。将TEAD1的靶向序列（gtgcattcgctgtttcaaat）克隆到pNGx_006载体中（pUC/ori，tracrRNA/嵌合体的U6启动子，SPyCas9和嘌呤霉素选择的CMV启动子）。使用具有以下参数的Neon转染系统，用1.5μg pNGx_006_sgTEAD1和0.5μg TEAD1V406A和TEAD1E408的单链寡核苷酸（ttaacaggtgtggtaaacaaacaggagaagaaactctctgcatggcctggatcgtgtgtgtgttcagaccacatcatatttacaggtgtaaaggactg）对SF-268细胞（2×105）进行电穿孔：电压1300 V，脉冲20 ms和脉冲数2。选择嘌呤霉素后接种单个克隆，并通过Sanger测序进行鉴定。对于集落形成试验，在处理前24小时以低密度（六孔板中每孔1000个细胞）接种SF-268克隆18和23。将试验化合物（IAG933）以最高浓度10μM的五点三倍连续稀释分配到测定板中。DMSO用作对照，DMSO含量标准化为所有化合物处理孔中的最高体积。含有化合物的培养基每周更新两次。在常规细胞培养条件下（37°C，5%CO2）孵育11天后，用3.7%甲醛固定细胞10分钟，并用结晶紫对菌落进行染色。

Animal treatments[2]
Most compounds were administered at the indicated doses by oral gavage with the following formulations. IAG933 was formulated in 0.5% methylcellulose and 0.1% Tween-80 in 100 mM phosphate buffer (pH adjusted to 8). VT104 and K-975 were formulated in 100% Maisine CC. YTP-75 was formulated in 30% PEG300 and 50 mM acetate buffer (pH adjusted to 5.5). YTP-13 was formulated in 5% PEG300 and 50 mM acetate buffer (pH adjusted to 4.8). LTT462, dabrafenib and trametinib were formulated in 20% MEPC4 in water. JDQ443, TNO155, osimertinib and capmatinib were formulated in 0.5% methylcellulose and 0.1% Tween-80 in water. Other compounds were administered by intraperitoneal injection. Antibodies to trastuzumab and cetuximab and MRTX1133 compound were formulated in Dexolve.

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PD in vivo studies[2]
Animals were assigned into groups of n = 3–5 per time point and treatment. Blood, plasma and tumor samples for PK and PD analyses were collected. Blood samples were collected on ice and stored at –20 °C until further processing. Plasma and tumor samples were snap-frozen on dry ice and stored frozen at –80 °C until further processing. The in vivo TEAD reporter assay was performed with the MSTO-211H STB-Luc orthotopic pleural mesothelioma tumor model. For each measurement, mice were injected intraperitoneally with luciferin (150 mg kg−1). Exactly 20 min later, the mice were imaged with an IVIS Spectrum while conscious and restrained for less than 1 min.

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In vivo efficacy studies[2]
Treatment was initiated when the tumors engrafted in the flank were at least 100 mm3, and random enrollment was applied. Efficacy studies, tumor response and relapse were reported with the measures of tumor volumes at the start of treatment. For efficacy studies on ectopic models, animals were randomized into treatment groups based on tumor volume. Tumor size was measured using a caliper and calculated using the formula length × width2 × π/6. As a measure of efficacy, the percent T/C value was sometimes calculated at the end of the experiment or at best response using the formula (Δtumor volume treated/Δtumor volume control) × 100. In the case of tumor regression, the tumor response was quantified using the formula –(Δtumor volume treated/tumor volume treated at start) × 100. Statistical analyses were performed using GraphPad Prism. For efficacy studies on pleural orthotopic models, viable tumor burden was assessed by measurements of GLuc from 20 μl of blood collected in microvette EDTA-coated tubes, and samples were stored at –20 C. Coelentrazine (Nanolight) substrate solution was added (100 μl of a 100 mM solution) to each well of 96-well white plates, and 5 μl of blood was added in triplicate. Bioluminescence was measured with a CentroXS LB960 Luminometer for 2 s.

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Bioanalytical method for detection of compounds in blood, plasma and tumors[2]
Concentrations of IAG933 and YTP-75 in total blood, plasma and tissues were determined by a ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) assay. Frozen tissue samples were pulverized to powder using CryoPrep according to manufacturer’s instructions or homogenized in an equal volume of HPLC water using the Fast Prep-24 system. Samples (about 25 mg, exact weight collected) of blood, plasma or tissue (in the form of powder or homogenate) were mixed with 25 µl of internal standard (1 µg ml–1) and extracted by the addition of 200 µl of acetonitrile to precipitate proteins. After sonication for 5 min, samples were centrifuged, and supernatants (70 µl) were mixed with 60 µl of HPLC water before the analysis of 5-µl aliquots by UPLC–MS/MS. Samples were injected onto a reverse-phase column using formic acid in water and formic acid in acetonitrile as mobile phases. The column eluent was directly introduced into the ion source of the triple quadrupole mass spectrometer. Electrospray positive ionization multiple reaction monitoring was used for MS/MS detection of the analyte. PK parameters were calculated from the mean values with the linear trapezoidal rule by using a noncompartmental model for extravascular dosing.

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Combination assays in matrix format[2]
The effect of compound combinations on cell proliferation was assessed by ATP quantification using CellTiter-Glo reagent. Cells were seeded at 300–700 cells per well in white-walled, clear-bottomed 384-well plates and incubated overnight at 37 °C before the addition of serial compound dilutions or vehicle control in a matrix format using an HP300 digital dispenser, and treatments were applied in triplicate. After incubation for 5–7 days in the presence of compounds, cell viability was monitored using CellTiter-Glo following the supplier’s instructions. Data were analyzed using the in-house program Combination Analysis Module. To enable differentiation of cytotoxic from cytostatic compound effects, the number of viable cells on the day of compound addition (day 0) was also assessed in a separate cell plate and used to calculate the extent of cell viability suppression. Depending on whether the CellTiter-Glo signal for a given point in the concentration matrix was above or below day 0, the latter suggesting cell death due to compound treatment, a ‘growth inhibition’ (GI) value was calculated as follows: T < D0: GI = 100 × {1 – [(S – D0)/D0]}; T ≥ D0: GI = 100 × [1 – (S – D0)/(V – D0)], where D0 is day 0, V is vehicle control, and S is signal. This formula leads to a scale where 0 corresponds to no compound effect compared to vehicle, 100 corresponds to growth arrest (that is, signal on endpoint equal to signal on day 0), and 200 corresponds to complete cell killing. In Fig. ​Fig.6a,6a, threefold dilutions were used for IAG933 starting from 5.595 µM for NSCLC and 3 µM for CRC cell lines and fourfold dilutions for JDQ443 starting with 1.6 µM as the highest compound concentrations.

[1]. Preparation of biaryl derivatives as YAP/TAZ-TEAD protein-protein interaction inhibitors. World Intellectual Property Organization, WO2021186324 A1. 2021-09-23.

The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway.[2]

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The Hippo signaling pathway is a highly conserved pathway that plays important roles in the regulation of cell proliferation and apoptosis. Transcription factors TEAD1-4 and transcriptional coregulators YAP/TAZ are the downstream effectors of the Hippo pathway and can modulate Hippo biology. Dysregulation of this pathway is implicated in tumorigenesis and acquired resistance to therapies. The emerging importance of YAP/TAZ-TEAD interaction in cancer development makes it a potential therapeutic target. In the past decade, disrupting YAP/TAZ-TEAD interaction as an effective approach for cancer treatment has achieved great progress. This approach followed a trajectory wherein peptidomimetic YAP-TEAD protein-protein interaction disruptors (PPIDs) were first designed, followed by the discovery of allosteric small molecule PPIDs, and currently, the development of direct small molecule PPIDs. YAP and TEAD form three interaction interfaces. Interfaces 2 and 3 are amenable for direct PPID design. One direct YAP-TEAD PPID (IAG933) that targets interface 3 has entered a clinical trial in 2021. However, in general, strategically designing effective small molecules PPIDs targeting TEAD interfaces 2 and 3 has been challenging compared with allosteric inhibitor development. This review focuses on the development of direct surface disruptors and discusses the challenges and opportunities for developing potent YAP/TAZ-TEAD inhibitors for the treatment of cancer.[3]

C27H26CLF2N3O4

529.962852954865

529.157

2714434-21-4

156855755

White to off-white solid powder

3.7

92.7Ų

3

8

7

37

793

2

CNC(=O)C1=CN=C(C(=C1C2=C3C[C@@](OC3=CC(=C2Cl)F)([C@@H]4CCCN4)C5=CC=CC=C5)F)OCCO

HUVOYQMXUNTUAI-DCFHFQCYSA-N

InChI=1S/C27H26ClF2N3O4/c1-31-25(35)17-14-33-26(36-11-10-34)24(30)22(17)21-16-13-27(20-8-5-9-32-20,15-6-3-2-4-7-15)37-19(16)12-18(29)23(21)28/h2-4,6-7,12,14,20,32,34H,5,8-11,13H2,1H3,(H,31,35)/t20-,27-/m0/s1

4-[(2S)-5-chloro-6-fluoro-2-phenyl-2-[(2S)-pyrrolidin-2-yl]-3H-1-benzofuran-4-yl]-5-fluoro-6-(2-hydroxyethoxy)-N-methylpyridine-3-carboxamide

YAP-TEAD-IN-3; IAG933; 2714434-21-4; IAG-933; NVP-IAG933; SCHEMBL23834952; GTPL13367; IAG933?;

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: 125 mg/mL (235.87 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。