Topoisomerase; Camptothecins

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]

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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。孵育过夜后，加入每种ADC的连续稀释溶液。5天后，根据制造商的说明，使用Promega的CellTiter̴Glo发光细胞存活率测定法评估细胞存活率。在共培养研究中，将KPL-4和MDA-MB-468细胞分别以1×105个细胞和3×105个电池接种在2 mL/孔培养基中的6孔板中。孵育过夜后，从培养板中取出上清液，以6mL/孔的速度加入每种ADC稀释剂（10nM）。培养5天后，从培养板上分离活细胞，并使用细胞计数器测定每个孔中的细胞数量。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个染色细胞的荧光信号。根据每个处理孔中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]

Pharmacokinetics in cynomolgus monkeys [3] 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).

Safety profile of DS-8201a [3] 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]. 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.

[3]. 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.

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

1034.099

1033.39816

2270986-87-1

Exatecan mesylate dihydrate;197720-53-9

169450545

Brown to dark brown solid powder

-0.4

301 Ų

7

15

22

75

2360

3

CC[C@@]1(C2=C(COC1=O)C(=O)N3CC4=C5[C@@H](CCC6=C5C(=CC(=C6C)F)N=C4C3=C2)NC(=O)COCNC(=O)CNC(=O)[C@H](CC7=CC=CC=C7)NC(=O)CNC(=O)CNC(=O)CCCCCN8C(=O)C=CC8=O)O

WXNSCLIZKHLNSG-WAJAXPBZSA-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+/m1/s1

6-(2,5-dioxopyrrol-1-yl)-N-[2-[[2-[[(2S)-1-[[2-[[2-[[(10S,23R)-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

(1R)-Deruxtecan; SCHEMBL26807942;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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。