Topoisomerase I ( IC50 = 0.31 μM ); Camptothecins

Dxd（ADC 的 Exatecan 衍生物）是一种有效的 DNA 拓扑异构酶 I 抑制剂，用作与 HER2 靶向 ADC (DS-8201a) 的缀合药物，IC50 为 0.31 μM。 Dxd 的 IC50 范围为 1.43 nM 至 4.07 nM，对 KPL-4、NCI-N87、SK-BR-3 和 MDA-MB-468 人类癌细胞系具有细胞毒性；然而，对照 IgG-ADC（其中 Dxd 为有效负载）对四种细胞系（表达 HER2）没有表现出抑制作用。 DS-8201a（有效负载为 Dxd）的 IC50 值分别为 26.8、25.4 和 6.7 ng/mL，对 HER2 阳性 KPL-4、NCI-N87 和 SK-BR-3 细胞系表现出显着抑制作用，但 MDA-MB-468 没有观察到这种抑制作用（IC50，>10,000 ng/mL）[1]。

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DS-8201a的结构如图1A所示。DS-8201a是一种靶向HER2的ADC，由抗HER2抗体和拓扑异构酶I抑制剂DX-8951的衍生物（DXd）组成，它们通过马来酰亚胺-甘油基-苯丙氨酸-甘油基（GGFG）肽接头结合在一起。在用还原剂三（2-羧乙基）膦盐酸盐（TCEP HCl）还原链间二硫键后，接头有效载荷通过半胱氨酸残基与抗体偶联。由于四肽被溶酶体酶（如组织蛋白酶B和L）分解，这些酶在肿瘤细胞中高度表达（30-34），因此推测DS-8201a被溶酶体酶切割并释放出DXd，在与HER2受体结合并内化于肿瘤细胞后，它会特异性地攻击肿瘤细胞中的靶分子。通过使用RPC，DS-8201a的DAR被确定为约8，这是传统链间半胱氨酸偶联的理论最大载药量。因此，在HIC图中观察到均匀的药物分布（图1B）。我们证实，通过拓扑异构酶I介导的DNA松弛试验（图1C）测量，DXd在拓扑异酶I的抑制活性方面比SN-38和DX-8951f更有效。

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DS-8201a对癌症细胞生长的抑制作用[1]
将DS-8201a对癌症细胞生长的抑制活性与体外结合DXd的抗HER2 Ab和对照IgG–ADC对各种人类癌症细胞系的抑制活性进行比较。首先通过流式细胞术分析评估细胞系KPL-4、NCI-N87、SK-BR-3和MDA-MB-468细胞表面HER2的表达（图2A）。KPL-4、NCI-N87和SK-BR-3的相对MFI分别为95.7、101.6和56.2，表明HER2在细胞表面上明确表达，而MDA-MB-468的相对MFI为1.0，表明MDA-MB-465中没有表达。DS-8201a对HER2阳性KPL-4、NCI-N87和SK-BR-3具有显著的细胞生长抑制活性，IC50值分别为26.8、25.4和6.7 ng/mL，而对MDA-MB-468没有这种抑制作用，IC50值>10000 ng/mL（图2B）。尽管抗HER2抗体对NCI-N87和SK-BR-3显示出细胞生长抑制活性，但这些活性比DS-8201a弱得多；NCI-N87和SK-BR-3的IC50值分别为204.2和65.9 ng/mL（图2B）。此外，对照IgG ADC在四种细胞系中的任何一种都没有显示出细胞生长抑制活性（图2B），尽管所有四种细胞株都对有效载荷敏感，DXd（IC50:1.43 nmol/L-4.07 nmol/L）。这些结果表明，药物与抗HER2抗体结合显著增强了DS-8201a的细胞生长抑制活性，并且DS-8201a对HER2阳性细胞系表现出靶向特异性生长抑制作用。

DS-8201a（Dxd 为有效负载，10 mg/kg，静脉注射）在具有 HER2 IHC 1+/FISH 阴性表达的 HER2 低表达 ST565 和 ST313 模型以及具有 KPL4 的 HER2 阳性模型中表现出有效的抗肿瘤活性、JIMT-1 和 Capan-1[1]。

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体内抗肿瘤活性[1]
在HER2阳性NCI-N87异种移植物模型中评估DS-8201a的体内抗肿瘤活性。DS-8201a以剂量依赖的方式诱导肿瘤生长抑制，单次剂量超过1mg/kg时肿瘤消退，而不会诱导小鼠的一般状况或体重变化出现任何异常（图2C）。在同一模型中，4 mg/kg的抗HER2抗体给药部分抑制了肿瘤生长，表明在第21天与对照组相比，肿瘤生长抑制率（TGI）为31%（图2D）。另一方面，DS-8201a明显显示出更有效的抗肿瘤疗效，表明在相同的4 mg/kg剂量下，TGI为99%，因此在体内和体外模型中都观察到药物偶联的疗效增强（图2D）。此外，DS-8201a的体内疗效取决于其HER2结合，因为对照IgG-ADC没有抑制肿瘤生长（图2D）。

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DS-8201a在低HER2表达肿瘤中的抗肿瘤活性[1]
T-DM1已被批准用于HER2阳性转移性癌症患者，根据当前指南（45），其定义为HER2 IHC 3+或IHC 2+/FISH阳性，并且在FISH阴性、HER2 1+和2+人群中，HER2靶向治疗的临床需求仍有待满足。因此，在具有不同HER2表达水平的各种小鼠异种移植物模型中评估了DS-8201a的抗肿瘤活性；KPL-4（强阳性）、JIMT-1（中度阳性）、Capan-1（弱阳性）和GCIY（阴性）（图4A和B）。还评估了具有与DS-8201a相同的药物接头和约一半DAR（DAR 3.4）的抗-HER2 ADC，以研究DAR对抗肿瘤活性的影响。虽然T-DM1仅对KPL4模型有效，但DS-8201a对所有具有KPL4、JIMT-1和Capan-1的HER2阳性模型有效。两种ADC在GCIY模型中均无效。抗HER2 ADC（DAR 3.4）抑制了所有HER2阳性模型的肿瘤生长，其疗效是HER2表达依赖性的。在HER2弱阳性Capan-1模型中，DS-8201a的疗效明显强于抗HER2 ADC（DAR 3.4）。这些结果表明，高DAR ADC DS-8201a能够将足够的有效载荷量递送到癌症细胞中，表明即使在低HER2水平下也具有细胞毒性。在HER2强阳性模型的情况下，即使是低DAR ADC也能够为细胞死亡提供足够的有效载荷。DS-8201a对HER2水平较高的肿瘤有效，因为其DAR约为8。为了在HER2低表达模型中确认DS-8201a的HER2特异性，在HER2高CFPAC-1模型中进行了竞争性抑制研究（图4C）。DS-8201a的疗效被抗HER2抗体的先前治疗所抵消，对照IgG ADC在比DS-8201a高3倍的剂量下没有抑制肿瘤生长。从这些结果中，证实了DS-8201a在HER2低表达模型中的HER2特异性。

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与PDX模型中的T-DM1的比较[1]
除了基于细胞系的异种移植物模型外，还进行了几次PDX评估，以更精确地评估临床效益。在癌症PDX模型中，NIBIO G016，DS-8201a表现出强大的抗肿瘤活性，并伴有肿瘤消退，但T-DM1没有（图5A）。由于该模型中的HER2状态为IHC 3+/FISH+，因此假设DS-8201a和T-DM1之间抗肿瘤疗效的差异是基于有效载荷的不同敏感性，这是由于每种有效载荷的作用机制不同。在癌症PDX模型中，尽管DS-8201a和T-DM1在HER2 IHC 2+/FISH阳性ST225模型中均有效，但在第21天，用DS-8201a治疗的5只小鼠中有3只观察到肿瘤完全消退，而不是T-DM1（图5B）。此外，DS-8201a在HER2 IHC 1+/FISH阴性表达的HER2低表达ST565和ST313模型中显示出抗肿瘤活性（图5C和D），但T-DM1没有。这一结果表明了与基于细胞系的异种移植物模型（如Capan-1和CFPAC-1）类似的趋势（图4B和C）。因此，在具有几种HER2表达水平的所有4种模型中，DS-8201a显示出比T-DM1更有效的抗肿瘤活性。这些结果表明，DS-8201a与T-DM1具有可区分的潜力，T-DM1在T-DM1不敏感和HER2低表达的肿瘤中显示出有效性，这是由于结合药物的不同作用机制和DS-8201a的高DAR造成的。

拓扑异构酶I抑制试验[1]
SN-38、DX-8951f和DX-8951衍生物（DXd）是在内部合成的。根据之前的报告，SN-38、DX-8951f和DXd对人拓扑异构酶I的抑制活性是通过拓扑异酶I介导的DNA松弛试验进行评估的。简而言之，重组人拓扑异构酶I与每种药物一起孵育5分钟。然后，加入超螺旋DNA pBR322，在25°C下孵育60分钟。在琼脂糖凝胶上电泳混合物后，用CCD成像仪测量超螺旋DNA的量。

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DS-8201a在血浆中的体外稳定性[1]
在小鼠、大鼠、猴子和人血浆中评估了DS-8201a在37°C下以10μg/mL的浓度在21天内释放DXd的速率。

96 孔板每孔接种 1,000 个细胞。孵育一晚后添加 Dxd。六天后，使用 CellTiter-Glo 发光细胞活力测定法评估细胞活力。为了鉴定每个细胞系中的 HER2 表达，将 FITC 小鼠 IgG1、κ 同型对照或抗 HER2/neu FITC 在冰上孵育 30 分钟。洗涤后，使用 FACSCalibur 分析标记的细胞。完成相对平均荧光强度（rMFI）的计算[1]。

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细胞毒性试验[1]
将细胞以每孔1000个细胞的速度接种到96孔板上。孵育过夜后，加入每种稀释物质。根据制造商的说明，在6天后使用CellTiter Glo发光细胞存活率测定法评估细胞存活率。为了检测每个细胞系中的HER2表达，将细胞与FITC小鼠IgG1、κ同种型对照或抗HER2/neu FITC在冰上孵育30分钟，用FACSCalibur分析标记的细胞。相对平均荧光强度（rMFI）通过以下方程式计算：

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ELISA[1]
对于结合试验，免疫板在包被缓冲液中用2.5μg/mL His标记的HER2-ECD蛋白包被，并在4°C下保持过夜。洗涤后，将板封闭，并将每种连续稀释的物质加入孔中。在37°C下孵育1.5小时后，清洗平板，并在37°C下用HRP偶联的抗人IgG二抗孵育1小时。洗涤后，加入TMB溶液，用微孔板读数器测量每个孔中的A450。为了检测磷酸化Akt（pAkt），将SK-BR-3细胞在96孔板中预孵育4天，然后与每种物质孵育24小时。孵育后，根据制造商的说明，裂解细胞，并使用PathScan Phospho-Akt1（Ser473）夹心ELISA试剂盒和PathScan total-Akt1夹心ELISA试剂袋检测细胞间pAkt和总Akt。通过将处理过的归一化pAkt值除以未处理的归一化pAk值来计算每个样品孔的相对pAkt。

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ADCC评估[1]
使用来源于供体的人外周血单核细胞（PBMC）作为效应细胞，SK-BR-3细胞作为靶细胞，评估抗体依赖性细胞介导的细胞毒性（ADCC）活性。效应细胞（2×105个细胞）和51Cr标记的靶细胞（1×104个细胞）与每种物质一起孵育，指示效应：靶（E:T）比为20:1。孵育4小时后，通过培养上清液中的放射性测量ADCC活性。

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免疫印迹[1]
用每种物质处理KPL-4细胞。24、48或72小时后，收获细胞并用含有Halt蛋白酶和磷酸酶抑制剂混合物的M-PER裂解缓冲液裂解。 通过SDS-PAGE对样品进行装载和分离，并将其印迹到聚偏二氟乙烯膜上。将膜封闭，并用抗磷酸化Chk1（Ser345；133D3）兔单克隆抗体、抗Chk1（2G1D5）小鼠单克隆抗体、反切割PARP（Asp214）抗体、抗β-肌动蛋白（8H10D10）小鼠mAb、抗磷酸化组蛋白H2A探测过夜。X（Ser139）抗体和抗组蛋白H2A。4°C下的X抗体。然后，用SNAP皮内洗涤膜并用荧光标记的二抗孵育10分钟。使用Odyssey成像系统检测荧光信号。

Mice: In brief, specific pathogen-free female nude mice are subcutaneously injected with each cell suspension or tumor fragment. Dosing begins on day 0 and the tumor-bearing mice are randomized into treatment and control groups based on the tumor volumes once the tumor has grown to an appropriate size. The mice receive intravenous injections of DS-8201a (1 or 10 mg/kg, i.v.; Dxd is the payload). One calculates tumor growth inhibition (TGI, %)[1].

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Cell line and patient-derived xenograft studies [1]
Detailed study procedures are written in the supplement. Briefly, each cell suspension or tumor fragment was inoculated subcutaneously into specific pathogen-free female nude mice. When the tumor had grown to an appropriate volume, the tumor-bearing mice were randomized into treatment and control groups based on the tumor volumes, and dosing was initiated on day 0. Each substance was administered intravenously to the mice. Tumor growth inhibition (TGI, %) was calculated according to the following equation:

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Pharmacokinetics of DS-8201a in cynomolgus monkeys [1]
Concentrations of DS-8201a and the total antibody in plasma were determined with a validated ligand-binding assay; the lower limit of quantitation was 0.100 μg/mL. Concentrations of DXd in plasma were determined with a validated liquid chromatography-tandem mass spectrometry (LC/MS-MS) method; the lower limit of quantitation was 0.100 ng/mL. DS-8201a was intravenously administered at 3.0 mg/kg to male cynomolgus monkeys. Plasma concentrations of DS-8201a, total antibody, and DXd were measured up to 672 hours postdose.

Pharmacokinetics in cynomolgus monkeys[1]
The plasma DS-8201a concentrations decreased exponentially after a single intravenous administration of DS-8201a. The volume of distribution at steady state (Vss) of DS-8201a and total antibody was close to the plasma volume (data not shown). No clear difference was observed in the pharmacokinetic profile between DS-8201a and the total antibody, indicating that the peptide-linker of DS-8201a is stable in plasma even at DAR 8 (Fig. 2E). A low level of DXd was detected only at the limited time points (Fig. 2E).

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In vitro stability in plasma[1]
The release rates of DXd from DS-8201a ranged from 1.2% to 3.9% on day 21 in mouse, rat, monkey, and human plasma (Fig. 2F), and these were comparable or rather lower than those of other ADCs, such as T-DM1, SGN-35 (Brentuximab vedotin), and inotuzumab ozogamicin (35–37). These results indicate that DS-8201a is stable in plasma.

Safety profile of DS-8201a [1]
A repeated intravenous dosing (every 3 weeks for 3 doses) study was conducted in cynomolgus monkeys, the cross-reactive species for DS-8201a, and in rats (antigen–non-binding species; Table 1). In the rat study, no deaths or life-threatening toxicities were found at dose levels up to 197 mg/kg, the maximum dose. Therefore, the severely toxic dose of 10% in animals (STD10) was considered to be >197 mg/kg. In the monkey study, one female at the highest dose of 78.8 mg/kg was euthanized due to moribundity on day 26. The cause of the moribundity appeared to be the deteriorated condition of the animal, which included decreased body weight and food consumption, as well as bone marrow toxicity and intestinal toxicity. Microscopic findings in the intestines, bone marrow and lungs in the surviving monkeys are shown in Supplementary Table S1. Gastrointestinal toxicity and bone marrow toxicity are typical dose-limiting factors in the clinical use of topoisomerase I inhibitors. The effects of DS-8201a on the intestines were very slight, and severe changes were not pronounced in any animal at up to 78.8 mg/kg. The bone marrow toxicity was produced only at 78.8 mg/kg, and was accompanied by decreases in reticulocyte ratios. No abnormalities in leukocyte and erythrocyte counts were observed in monkeys at 10 and 30 mg/kg. The repeated dose of DS-8201a caused moderate pulmonary toxicity in monkeys at 78.8 mg/kg, and findings graded as slight or very slight after the 6-week recovery period at ≥30 mg/kg. On the basis of the mortality and severity of the findings above, the highest non-severely toxic dose (HNSTD) for monkeys was considered to be 30 mg/kg. DS-8201a was well tolerated at the doses up to 197 mg/kg in rats and 30 mg/kg in monkeys following the repeated administration corresponding to the clinical regimen, and the nonclinical safety profile was acceptable for entry into human trials.

[1]. DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1. Clin Cancer Res. 2016 Oct 15;22(20):5097-5108.

Purpose: An anti-HER2 antibody-drug conjugate with a novel topoisomerase I inhibitor, DS-8201a, was generated as a new antitumor drug candidate, and its preclinical pharmacologic profile was assessed. [1]
Experimental design: In vitro and in vivo pharmacologic activities of DS-8201a were evaluated and compared with T-DM1 in several HER2-positive cell lines and patient-derived xenograft (PDX) models. The mechanism of action for the efficacy was also evaluated. Pharmacokinetics in cynomolgus monkeys and the safety profiles in rats and cynomolgus monkeys were assessed. [1]
Results: DS-8201a exhibited a HER2 expression-dependent cell growth-inhibitory activity and induced tumor regression with a single dosing at more than 1 mg/kg in a HER2-positive gastric cancer NCI-N87 model. Binding activity to HER2 and ADCC activity of DS-8201a were comparable with unconjugated anti-HER2 antibody. DS-8201a also showed an inhibitory activity to Akt phosphorylation. DS-8201a induced phosphorylation of Chk1 and Histone H2A.X, the markers of DNA damage. Pharmacokinetics and safety profiles of DS-8201a were favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys, supporting DS-8201a as being well tolerated in humans. DS-8201a was effective in a T-DM1-insensitive PDX model with high HER2 expression. DS-8201a, but not T-DM1, demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. [1]
Conclusions: DS-8201a exhibited a potent antitumor activity in a broad selection of HER2-positive models and favorable pharmacokinetics and safety profiles. The results demonstrate that DS-8201a will be a valuable therapy with a great potential to respond to T-DM1-insensitive HER2-positive cancers and low HER2-expressing cancers. Clin Cancer Res; 22(20); 5097-108. ©2016 AACR.

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Most of the ADCs currently in the market and in clinical development carry tubulin polymerization inhibitors such as T-DM1 and SGN-35 (Brentuximab vedotin; ref. 13). We synthesized a novel ADC with a topoisomerase I inhibitor, which has a different mechanism of action from tubulin polymerization inhibitors, and a novel self-immolative linker system using an aminomethylene (AM) moiety. Although other cleavable linker systems applied to SGN-35 (Brentuximab vedotin) and several ADCs release amino group–containing payloads, this AM self-immolative linker system is able to release DXd containing the hydroxyl group from DS-8201a. Moreover, this novel linker-payload system enables a reduction in the hydrophobicity of the ADC and helps increase its DAR. In the case of T-DM1, lysine conjugation and noncleavable systems are used, and it is quite a different system from DS-8201a. DS-8201a showed potent HER2-specific efficacy both in vitro and in vivo, and by drug conjugation maintained the functional effects of trastuzumab equal to those of T-DM1. Furthermore, the safety profiles of DS-8201a in rats and cynomolgus monkeys showed DS-8201a as being well tolerated.[1]

C26H24FN3O6

493.48367023468

493.16

C, 63.28; H, 4.90; F, 3.85; N, 8.52; O, 19.45

1599440-33-1

Exatecan;171335-80-1;Exatecan mesylate;169869-90-3;Dxd-d5;Exatecan mesylate dihydrate;197720-53-9

117888634

Off-white to light yellow solid powder

0

129

3

8

3

36

1080

2

CC[C@@]1(C2=C(COC1=O)C(=O)N3CC4=C5[C@H](CCC6=C5C(=CC(=C6C)F)N=C4C3=C2)NC(=O)CO)O

PLXLYXLUCNZSAA-QLXKLKPCSA-N

InChI=1S/C26H24FN3O6/c1-3-26(35)15-6-19-23-13(8-30(19)24(33)14(15)10-36-25(26)34)22-17(28-20(32)9-31)5-4-12-11(2)16(27)7-18(29-23)21(12)22/h6-7,17,31,35H,3-5,8-10H2,1-2H3,(H,28,32)/t17-,26-/m0/s1

N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]-2-hydroxyacetamide

DX-8951 derivative; DX-8951; DX8951; 1599440-33-1; Dxd; N-[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.02,14.04,13.06,11.020,24]tetracosa-1,6(11),12,14,16,18,20(24)-heptaen-23-yl]-2-hydroxyacetamide; OQM5SD32BQ; N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide; N-((1S,9S)-9-Ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9-hydroxy-4-methyl-10,13-dioxo-1H,12H-benzo(de)pyrano(3',4':6,7)indolizino(1,2-b)quinolin-1-yl)-2-hydroxyacetamide; Acetamide, N-((1S,9S)-9-ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9-hydroxy-4-methyl-10,13-dioxo-1H,12H-benzo(de)pyrano(3',4':6,7)indolizino(1,2-b)quinolin-1-yl)-2-hydroxy-; Acetamide, N-[(1S,9S)-9-ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9-hydroxy-4-methyl-10,13-dioxo-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxy-; Exatecan derivative; Trastuzumab Deruxtecan (DS-8201a).

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: 8~40 mg/mL (16.2~81.1 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。