Camptothecins; TOP I; topoisomerase I

抗体-药物偶联物可选择性、高效地将抗癌药物递送至肿瘤组织，具有显着的抗肿瘤功效和广泛的治疗窗[2]。使用具有不同生物学特性的五种 CRC 细胞系研究了 [fam-] trastuzumab deruxtecan 对 CRC 的抗肿瘤活性。首先检查了这些不同细胞系中 HER2 在 mRNA 和蛋白质水平上的表达。免疫印迹分析以及 RT 和实时聚合酶链反应 (PCR) 分析显示，所有 CRC 细胞系中 HER2 蛋白和 HER2 mRNA 的量均远小于 NCI-N87 细胞。 [fam-] trastuzumab deruxtecan 减弱了 NCI-N87 细胞的活力，与之前的结果一致，而所有五种 CRC 细胞系均表现出对该药物的耐药性。这些发现表明 HER2 蛋白的表达水平可能决定对 [fam-] 曲妥珠单抗 deruxtecan 的敏感性

测试了 [fam-] 曲妥珠单抗 deruxtecan 在表达 HER2 的异种移植肿瘤模型中的疗效。首次通过免疫组化（IHC）证实HCT116-Mock、HCT116-H2L或HCT116-H2H细胞裸鼠皮下肿瘤中HER2蛋白表达水平。以3.0 mg/kg剂量施用[fam-]曲妥珠单抗deruxtecan显着抑制由HCT116-H2L或HCT116-H2H细胞形成的肿瘤的生长，但不抑制由HCT116-Mock细胞形成的肿瘤的生长。第 24 天时，与 PBS 载体相比，[fam-] trastuzumab deruxtecan 对 HCT116-H2L 和 HCT116-H2H 细胞的抑制程度分别为 60% 和 93%。[fam-] trastuzumab deruxtecan 治疗对身体没有影响三组小鼠中任意一组的体重。因此，这些发现表明，异种移植模型中肿瘤对[fam-]曲妥珠单抗deruxtecan的敏感性取决于HER2表达水平，并且这种治疗与明显的毒性无关。

使用Freedom EVO200系统进行平行人工膜渗透性测定（PAMPA）。受体板的滤膜涂有GIT‐0脂质溶液。将DMSO（10mM）中的每种化合物溶液加入Prisma HT缓冲液中，获得5μM供体溶液（含有0.05%DMSO，pH 5.0和pH 7.4），然后放置在供体板上。受体板填充有受体沉缓冲液。将供体板堆叠在受体板上，并在25°C下孵育4小时。培养后，通过LC-MS/MS系统（API 4000）测量两个平板中的化合物浓度。使用PAMPA Evolution DP软件计算渗透系数（Peff；10−6 cm/s）。[1]

将细胞接种在96孔板中，KPL‐4为1000个细胞/孔，MDA‐MB‐468为2000个细胞/孔。培养过夜后，加入每个ADC的连续稀释溶液。5天后，根据制造商的说明，使用来自Promega的CellTiter‐Glo发光细胞活力测定法评估细胞活力。在共培养研究中，将KPL‐4和MDA‐MB‐468细胞分别以1×105细胞和3×105细胞接种在6孔板中的2 mL/孔培养基中。孵育过夜后，从板中取出上清液，并以6mL/孔的速度加入每种ADC稀释剂（10nM）。培养5天后，从平板上分离活细胞，并使用细胞计数器测定每个孔中的细胞数。为了确定KPL‐4和MDA‐MD‐468细胞在总活细胞中的比例，用抗HER2/nue-FITC对细胞进行染色，并在冰上孵育20分钟。洗涤后，使用流式细胞仪测量2×104染色细胞的荧光信号。根据每个处理井中HER2阳性和HER2阴性细胞的数量和比例，计算KPL‐4或MDA‐MB‐468细胞的数量。[1]

In vivo xenograft studies
All in vivo studies were carried out in accordance with the local guidelines of the Institutional Animal Care and Use Committee. Specific pathogen‐free female CAnN.Cg‐Foxn1nu/CrlCrlj mice (BALB/c nude mice) aged 5 weeks were used. All models were established by s.c. inoculation in the flanks of the mice. NCI‐N87 and MDA‐MB‐468‐Luc models were established by injecting 5 × 106 and 1 × 107 cells suspended in a Matrigel matrix, respectively. After 6 days for NCI‐N87, and 9 days for MDA‐MB‐468‐Luc models, the tumor‐bearing mice were randomized into treatment and control groups based on the tumor volume, and dosing initiated (day 0). Each ADC was given i.v. to the mice at a dose of 3 or 10 mg/kg, and a volume of 10 mL/kg. As a vehicle, ABS buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5) was given at the same volume as the ADCs. The tumor volume was defined as 1/2 × length × width2.[1]

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In vivo luciferase imaging
Seven days after inoculating the mixture of 5 × 10~6 NCI‐N87 cells and 1 × 107 MDA‐MB‐468‐Luc cells suspended in the Matrigel matrix into the right flank of the mice at a total volume of 100 μL, the tumor‐bearing mice were randomized into treatment and control groups based on the tumor volume, and dosing initiated (day 0). Each ADC or the vehicle was given i.v. to the mice. Luciferase activity of each mouse was measured using an in vivo imaging system twice a week in parallel with the measurement of tumor length 10 min after administering 150 mg/kg luciferin i.v. The amount of luminescence was analyzed using analysis software as average radiance (p/s/cm2/sr). For another study, MDA‐MB‐468‐Luc cells at a density of 1.5 × 107 cells were inoculated into the left flank of the mice in addition to the inoculation of the mixture into the right flank. Seven days after inoculation, dosing and evaluation were undertaken in a similar manner as described above.[1]

[1]. Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity. Cancer Sci. 2016 Jul;107(7):1039-46.

[2]. METHOD FOR SELECTIVELY MANUFACTURING ANTIBODY-DRUG CONJUGATE. WO2017002776A1.

Antibody-drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window. DS-8201a is a human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugate prepared using a novel linker-payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX-8951 derivative, DXd). It was effective against trastuzumab emtansine (T-DM1)-insensitive patient-derived xenograft models with both high and low HER2 expression. In this study, the bystander killing effect of DS-8201a was evaluated and compared with that of T-DM1. We confirmed that the payload of DS-8201a, DXd (1), was highly membrane-permeable whereas that of T-DM1, Lys-SMCC-DM1, had a low level of permeability. Under a coculture condition of HER2-positive KPL-4 cells and negative MDA-MB-468 cells in vitro, DS-8201a killed both cells, whereas T-DM1 and an antibody-drug conjugate with a low permeable payload, anti-HER2-DXd (2), did not. In vivo evaluation was carried out using mice inoculated with a mixture of HER2-positive NCI-N87 cells and HER2-negative MDA-MB-468-Luc cells by using an in vivo imaging system. In vivo, DS-8201a reduced the luciferase signal of the mice, indicating suppression of the MDA-MB-468-Luc population; however, T-DM1 and anti-HER2-DXd (2) did not. Furthermore, it was confirmed that DS-8201a was not effective against MDA-MB-468-Luc tumors inoculated at the opposite side of the NCI-N87 tumor, suggesting that the bystander killing effect of DS-8201a is observed only in cells neighboring HER2-positive cells, indicating low concern in terms of systemic toxicity. These results indicated that DS-8201a has a potent bystander effect due to a highly membrane-permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T-DM1.[1]

C52H56FN9O13

1034.05195617676

1,033.40

C, 60.40; H, 5.46; F, 1.84; N, 12.19; O, 20.11

1599440-13-7

Exatecan mesylate;169869-90-3;Deruxtecan-d6;2760715-89-5;Deruxtecan-d5;Exatecan mesylate dihydrate;197720-53-9

118305111

White to yellow solid powder

1.48±0.1 g/cm3

1491.1±65.0 °C

-0.4

301Ų

7

15

22

75

2360

3

FC1=CC2=C3C(=C1C)CC[C@@H](C3=C1C(C3=CC4[C@](C(=O)OCC=4C(N3C1)=O)(CC)O)=N2)NC(COCNC(CNC([C@H](CC1C=CC=CC=1)NC(CNC(CNC(CCCCCN1C(C=CC1=O)=O)=O)=O)=O)=O)=O)=O

WXNSCLIZKHLNSG-MCZRLCSDSA-N

InChI=1S/C52H56FN9O13/c1-3-52(73)33-19-38-48-31(24-62(38)50(71)32(33)25-75-51(52)72)47-35(14-13-30-28(2)34(53)20-36(60-48)46(30)47)58-43(67)26-74-27-57-41(65)22-56-49(70)37(18-29-10-6-4-7-11-29)59-42(66)23-55-40(64)21-54-39(63)12-8-5-9-17-61-44(68)15-16-45(61)69/h4,6-7,10-11,15-16,19-20,35,37,73H,3,5,8-9,12-14,17-18,21-27H2,1-2H3,(H,54,63)(H,55,64)(H,56,70)(H,57,65)(H,58,67)(H,59,66)/t35-,37-,52-/m0/s1

Glycinamide, N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]glycylglycyl-L-phenylalanyl-N-[[2-[[(1S,9S)-9-ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9-hydroxy-4-methyl-10,13-dioxo-1H,12Hbenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino]-2-oxoethoxy]methyl]-

Deruxtecan; DS-8201a; DS8201a; DX-8951 derivative; Trastuzumab deruxtecan; DS 8201a; exatecan derivative; DX 8951; DX8951; Deruxtecan; 1599440-13-7; Mc-ggfg-dxd(1); 5SEB972CO4; N-((S)-10-Benzyl-1-(((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)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide; 6-(2,5-dioxopyrrol-1-yl)-N-[2-[[2-[[(2S)-1-[[2-[[2-[[(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]amino]-2-oxoethoxy]methylamino]-2-oxoethyl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-2-oxoethyl]amino]-2-oxoethyl]hexanamide; Deruxtecan [USAN]; UNII-5SEB972CO4;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

注意： (1) 该产品在溶液状态不稳定，请现配现用。 (2) 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~35 mg/mL (~33.85 mM)
H2O : Insoluble (< 1 mg/mL)

配方 1 中的溶解度: 1.75 mg/mL (1.69 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；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 17.5 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: 1.75 mg/mL (1.69 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 17.5mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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: ≥ 1.75 mg/mL (1.69 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 17.5 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: 10% DMSO+ 40% PEG300+ 5% Tween-80+ 45% saline: 1.75 mg/mL (1.69 mM)

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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。