One hour after an intravenous injection of a dose of 10 mg/kg, tinostamustine reduced HDAC activity in rat peripheral blood mononuclear cells (PBMCs) by around 90% in cell tests; greater doses, up to 50 mg/kg, had no effect on HDAC activity in PBMCs. suppression of HDAC. Tinostustine exhibits strong anticancer activity and causes apoptosis in HL60 and Daudi cells. Initial in vitro tests using HL60 cells revealed that cleaving caspases 3, 9, and PARP triggered the intrinsic mechanism of apoptosis and markedly decreased the levels of the anti-apoptotic proteins XIAP and Mcl-1 [1].

在细胞测试中，静脉注射 10 mg/kg 剂量后 1 小时，替诺莫司汀使大鼠外周血单核细胞 (PBMC) 的 HDAC 活性降低约 90%；更大剂量（高达 50 mg/kg）对 PBMC 中的 HDAC 活性没有影响。 HDAC 的抑制。 Tinostustine 表现出强大的抗癌活性，并导致 HL60 和 Daudi 细胞凋亡。使用 HL60 细胞的初步体外测试表明，裂解 caspase 3、9 和 PARP 会触发细胞凋亡的内在机制，并显着降低抗细胞凋亡蛋白 XIAP 和 Mcl-1 的水平 [1]。

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Tinostamustine 表现出与 vorinostat 相似的 HDAC 抑制活性，涵盖多种重组 HDAC 酶（I 类和 II 类）。
在 MM1S 和 HL60 细胞中，它诱导组蛋白 H3 在赖氨酸残基 K9、K14、K23 和 K56 的全局超乙酰化。
彗星实验显示，Tinostamustine 在较低浓度下（4 µM 时 57.51% 交联）即可引起 HL60 细胞的 DNA 交联和双链断裂，效果优于 bendamustine 和美法仑。
它在 HL60 细胞中触发凋亡，表现为 caspase 3、9、PARP 的剪切以及抗凋亡蛋白 XIAP 和 Mcl-1 的减少。
在 MM1S 细胞中，它下调 DNA 修复蛋白 p-ATR、p-ATM 和 p-CHK2。[1]

替诺莫司汀具有细胞内 HDAC 抑制作用，在分娩后迅速生效，峰值为 10 mg/kg，持续约 12 至 16 小时。皮下患有人类伯基特淋巴瘤的小鼠的肿瘤中 p53 和 pH2AX 的激活证明，暴露于黄嘌呤会引发有效的 DNA 修复反应。静脉注射替诺莫司汀可使 BL 肿瘤迅速缩小或完全消失 [1]。

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在 Daudi Burkitt 淋巴瘤异种移植模型中，静脉注射 Tinostamustine（78 mg/kg 或 39 mg/kg）可显著抑制肿瘤生长或完全消除肿瘤。
它诱导肿瘤中 DNA 损伤反应，表现为 pH2AX 和 p53 染色增加。
在大鼠 PBMCs 中，HDAC 抑制在给药后 1 小时可达 90%（10 mg/kg i.v.），24 小时内恢复至正常水平。[1]

使用重组人 HDACs（1、2、3、6、8、10）进行 HDAC 酶活性测定。化合物溶于 DMSO 后加入含 Fluor de Lys 底物的测定缓冲液中，37°C 孵育 30 分钟，随后加入 HDAC 显影剂并测量荧光强度。[1]

为测定细胞毒性和 IC50，将细胞（如 Daudi、HL60）接种于 96 孔板中，用 Tinostamustine 处理 72 小时，使用 CellTiter-Glo 试剂和发光法检测细胞活力。
通过 Western blot 分析细胞裂解物中的凋亡标志物（caspases、PARP、XIAP、Mcl-1）和组蛋白乙酰化状态。
彗星实验用于评估 HL60 细胞在暴露于 Tinostamustine 后的 DNA 交联和双链断裂情况。[1]

NOD/SCID female mice were inoculated subcutaneously with Daudi tumor cells. When tumor volume reached ~468 mm³, mice were randomized into control or treatment groups.
Tinostamustine was prepared as a 6 mg/mL injection solution containing 15% HPRCD, 1.5% acetic acid, and 1.25% NaHCO₃.
Drug was administered intravenously at doses of 39 mg/kg or 78 mg/kg on specified days (e.g., Day 1, 8, 15). Tumor volumes were measured regularly using calipers.[1]

[1]. The Alkylating-HDAC Inhibition Fusion Principle: Taking Chemotherapy to the Next Level with the First in Class Molecule EDO-S101. Anticancer Agents Med Chem. 2016;16(1):20-8.

Tinostamustine is being investigated in the clinical trial NCT03452930 (Tinostamustine with or without radiotherapy for newly diagnosed MGMT unmethylated glioblastoma patients). Tinostamustine is an alkylating histone deacetylase inhibitor (HDACi) fusion molecule, composed of the alkylating agent bendamustine and the pan-HDACi vorinostat, possessing potential bifunctional antitumor activity. After administration of Tinostamustine, the vorinostat portion targets and binds to HDAC. This leads to the accumulation of highly acetylated histones, thereby inducing chromatin remodeling, regulating gene expression, inhibiting tumor cell division, and inducing tumor cell apoptosis. The bendamustine portion can bind, alkylate, and cross-link macromolecules, thereby inhibiting the synthesis of DNA, RNA, and proteins, ultimately leading to tumor cell apoptosis. Therefore, Tinostamustine shows superior efficacy compared to bendamustine or Tinostamustine alone. Furthermore, Tinostamustine's inhibition of HDAC6 activity induces activation of inositol demand enzyme 1 (IRE-1), a key regulatory protein of the unfolded protein response (UPR). UPR induction can increase the sensitivity of certain cancer cell types to certain chemotherapeutic agents, such as proteasome inhibitors. Therefore, Tinostamustine may have a synergistic effect with proteasome inhibitors. Histone deacetylases (HDACs) are enzymes that deacetylate chromatin histones; they are overexpressed in various cancers and play a crucial role in tumor cell proliferation and drug resistance. Tinostamustine is a first-in-class fusion molecule that combines the alkylating agent bendamustine with the HDAC inhibitor vorinostat. It is designed to allow a single molecule to simultaneously exert alkylation and HDAC inhibitory activities, potentially improving efficacy and overcoming drug resistance. Preclinical studies have shown its activity against hematologic malignancies and solid tumors, including multiple myeloma, lymphoma, glioblastoma, and breast cancer.
Studies suggest that their synergistic effect is achieved by enhancing DNA alkylation and impairing DNA repair through chromatin relaxation (HDAC inhibition). [1]

C19H28CL2N4O2

415.3572

414.159

1236199-60-2

1793059-58-1 (HCl);1236199-60-2;

46836227

White to off-white solid powder

4.246

70.39

2

4

12

27

438

0

GISXTRIGVCKQBX-UHFFFAOYSA-N

InChI=1S/C19H28Cl2N4O2/c1-24-17-9-8-15(25(12-10-20)13-11-21)14-16(17)22-18(24)6-4-2-3-5-7-19(26)23-27/h8-9,14,27H,2-7,10-13H2,1H3,(H,23,26)

7-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)-N-hydroxyheptanamide

EDO-S101; Minomustine; EDO-S-101; EDO-S 101; EDO S-101;

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 : ~100 mg/mL (~240.76 mM)

配方 1 中的溶解度: 2.5 mg/mL (6.02 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 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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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。