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].

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].

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Tinostamustine exhibits HDAC inhibitory activity similar to vorinostat across multiple recombinant HDAC enzymes (Class I and II).
It induces global hyperacetylation of histone H3 at lysine residues K9, K14, K23, and K56 in MM1S and HL60 cells.
Tinostamustine causes DNA cross-linking and double-strand breaks in HL60 cells at lower concentrations than bendamustine and melphalan, as shown by comet assay (57.51% cross-linking at 4 µM).
It triggers apoptosis in HL60 cells, evidenced by cleavage of caspases 3, 9, PARP, and reduction of anti-apoptotic proteins XIAP and Mcl-1.
It downregulates DNA repair proteins p-ATR, p-ATM, and p-CHK2 in MM1S cells.[1]

Tenomustine has an intracellular HDAC inhibitory action that takes effect quickly after delivery, peaks at 10 mg/kg, and lasts for around 12 to 16 hours. Flavustine exposure triggers a potent DNA repair response, as evidenced by the activation of p53 and pH2AX in tumors from mice that had human Burkitt's lymphoma subcutaneously. Tinostamustine injections intravenously cause BL tumors to either rapidly shrink or disappear entirely [1].

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In Daudi Burkitt’s lymphoma xenograft models, Tinostamustine administered intravenously at 78 mg/kg or 39 mg/kg showed significant tumor regression or complete eradication.
It induced DNA damage response in tumors, indicated by increased pH2AX and p53 staining.
HDAC inhibition in rat PBMCs was observed up to 90% at 1 hour post-dosing (10 mg/kg i.v.), with recovery to normal levels within 24 hours.[1]

HDAC enzymatic assay was performed using recombinant human HDACs (1, 2, 3, 6, 8, 10). Compounds were dissolved in DMSO and added to assay buffer containing Fluor de Lys substrate. Reactions were incubated at 37°C for 30 minutes, followed by addition of HDAC developer and fluorescence measurement.[1]

For cytotoxicity and IC50 determination, cells (e.g., Daudi, HL60) were seeded in 96-well plates and treated with Tinostamustine for 72 hours. Viability was assessed using CellTiter-Glo reagent and luminescence measurement.
Western blotting was performed on cell lysates to analyze apoptosis markers (caspases, PARP, XIAP, Mcl-1) and histone acetylation.
Comet assay was used to evaluate DNA cross-linking and double-strand breaks in HL60 cells after exposure to Tinostamustine.[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

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)

Solubility in Formulation 1: 2.5 mg/mL (6.02 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)