DNA Alkylator/Crosslinker
Bendamustine HCl (SDX-105) targets tumor cell DNA (induces DNA alkylation and cross-linking)[1]
Bendamustine HCl (SDX-105) inhibits DNA repair-related enzymes (e.g., poly(ADP-ribose) polymerase, PARP) [5]

Bendamustine causes more extensive and much more durable single- and double-strand breaks in DNA than cyclophosphamide, cisplatinum, or carmustine. Bendamustine specifically modulates the transcription and posttranslation of genes related to mitotic checkpoints, DNA repair, and apoptosis. When compared to other alkylators, bendamustine regulates DNA repair pathways in non-Hodgkin'slymphomacells in a unique way. Bendamustine causes a mitotic catastrophe by blocking mitotic checkpoints. In SU-DHL-9 cells, bendamustine treatment causes a 60%–80% down-regulation of the mRNA expression of all three of these genes: cyclin B1, Aurora Kinase A, and polo-like kinase 1 (PLK–1). In contrast to the 6% of DMSO control cells, 26% of the MCF-7/ADR cells treated with bendamustine exhibited micronucleation.[1] After 48 hours, there was a dose- and time-dependent increase in cytotoxicity from 30.4% to 94.8% when bendamustine concentrations ranged from 1 μg/mL to 50 μg/mL. For both pretreated and untreated CLL cells, the LD50 is 4.4 μg/mL and 7.3 μg/mL, respectively. [2] Save for HL-60 cells, which show intermediate sensitivity, all myeloid and breast cancer cell lines are resistant to bendamustine. The clastogenic effect of bendamustine is found to be significantly lower than that of equimolar doses of lomustine.[3]

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Antiproliferative activity against multiple tumor cell lines: IC50 values were 15 μM for human CLL cell line MEC-1, 12 μM for HG-3, 8 μM for human NHL cell line SU-DHL-4, 10 μM for Raji, and 22 μM for human breast cancer cell line MCF-7[2]
- Induces tumor cell apoptosis: Treatment of MEC-1 cells with 10 μM Bendamustine HCl for 48 hours resulted in 45% apoptotic cells, accompanied by activation of caspase-3 and caspase-9, and increased PARP cleavage products[2]
- Alkylates DNA and inhibits DNA synthesis: Forms cross-link products with tumor cell DNA, leading to DNA strand breaks and G2/M cell cycle arrest; treatment of human ovarian cancer cell line A2780 with 15 μM for 24 hours increased the proportion of G2/M phase cells from 12% to 38%[5]
- Effective against drug-resistant tumor cells: IC50 value was 25 μM for cisplatin-resistant A2780/cis cell line and 18 μM for fludarabine-resistant CLL cell line, with no obvious cross-resistance[3]
- Synergistic effect with combination therapy: Combined treatment of Raji cells with Bendamustine HCl and rituximab reduced IC50 from 10 μM to 4 μM; combined treatment of MEC-1 cells with Bendamustine HCl and fludarabine increased apoptosis rate from 45% to 68% (each drug concentration was 5 μM)[1]
- Inhibits DNA repair: Treatment of human colon cancer cell line HCT116 with 20 μM Bendamustine HCl reduced PARP enzyme activity by 60%, decreased DNA damage repair, and enhanced cytotoxicity[5]

Bendamustine, at a dose of 25 mg/kg, shows considerable activity in DoHH-2, Granta 519, and RAMOS tumor lines after just one dose. With a tumor growth inhibition of 69% and an ORR of 30%, DoHH-2 is the most sensitive. Bendamustine also inhibits the growth of Granta 519 and RAMOS (%TGI of 74% and 81%, respectively), and the effect lasts longer in Granta 519 (%TGD of 124%) than it does in DoHH-2 or RAMOS (69% and 43%, respectively). [4]

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Human CLL cell xenograft model (NOD/SCID mice): Intraperitoneal injection of Bendamustine HCl at 25 mg/kg once weekly for 3 consecutive weeks reduced tumor volume by 72% compared with the control group, and prolonged the median survival time of mice from 35 days to 62 days[2]
- Human NHL cell xenograft model (BALB/c nude mice): Intravenous injection of Bendamustine HCl at 30 mg/kg once every two weeks for 2 consecutive times achieved a tumor inhibition rate of 68% without obvious weight loss[1]
- Rat Walker 256 sarcoma model: Intraperitoneal injection of Bendamustine HCl at 15 mg/kg once daily for 5 consecutive days reduced tumor weight from 1.8 g to 0.6 g, and decreased serum tumor marker CA125 level by 55%[3]
- Synergistic effect of combination therapy in vivo: Combined treatment of Raji cell-transplanted mice with Bendamustine HCl (20 mg/kg, intraperitoneal injection, once weekly) and rituximab (10 mg/kg, intravenous injection, once weekly) increased tumor inhibition rate from 68% to 85% and prolonged median survival time to 78 days[1]

DNA cross-linking activity assay: Purified calf thymus DNA was incubated with serial concentrations of Bendamustine HCl at 37°C for 2 hours. After adding DNA helicase, the degree of DNA strand unwinding was detected by agarose gel electrophoresis. Results showed that the DNA cross-linking rate reached 58% at 20 μM, with a concentration-dependent increase[5]
- PARP enzyme activity inhibition assay: Recombinant PARP enzyme was incubated with different concentrations of the drug for 30 minutes, and fluorescence-labeled ADP-ribose substrate was added to detect changes in fluorescence intensity to reflect enzyme activity. Results showed that the PARP enzyme activity inhibition rate was 60% at a drug concentration of 20 μM[5]
- DNA polymerase activity assay: Bendamustine HCl was added to the reaction system containing DNA template, primers, and dNTP substrates. After incubation at 37°C for 1 hour, the amount of DNA synthesis products was detected by autoradiography. DNA polymerase activity decreased by 45% at 15 μM[4]

Bendamustine and melphalan both exhibit cytotoxicity on multiple myeloma (MM) cells, which is quantified as an inhibition of cell viability based on the MTS assay's percentage of cell survival. In summary, 96-well plates are seeded with 1 × 10 4 cells per well, and the drug is added in increasing concentrations. The cells are then incubated for 24, 48, 72, and 96 hours before analysis. In order to achieve this, 1 μg/mL of MTS solution is added to each well. The dark blue formazan crystals are then dissolved by isopropanol 1 N and HCl (24:1, vol/vol) after 1 hour at 37 °C. Ultimately, a 96-well plate reader is used to measure the absorbance at 490 nm. For each test, triplicates are used, and the percentage of untreated control absorbance is used to estimate cell survival. Bendamustine and melphalan equitoxic concentrations are used for parallel testing. The inhibitory concentrations 50 (IC50) and 25 (IC25) of each drug are determined, which represent the amounts able to reduce cell growth to 50% and 25%, respectively, of that of untreated control cells. By dividing the IC50 of 8226-LR5 by the IC50 of RPMI-8226 cells, one can calculate the relative resistance index (RRI).

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Cell proliferation inhibition assay (MTT method): Different tumor cell lines (MEC-1, Raji, MCF-7, etc.) were seeded in 96-well plates at 1×10⁴ cells per well. After incubation for 24 hours, serial concentrations of Bendamustine HCl (0.1~50 μM) were added, and incubation continued for 72 hours. MTT reagent was added for incubation for 4 hours, and the absorbance at 570 nm was measured to calculate IC50 values[2]
- Apoptosis detection assay (Annexin V-FITC/PI double staining method): MEC-1 cells were seeded in 6-well plates, treated with 10 μM Bendamustine HCl for 48 hours, collected, stained with Annexin V-FITC and PI, and the proportion of apoptotic cells was detected by flow cytometry[2]
- Cell cycle analysis assay: Raji cells were treated with 5 μM Bendamustine HCl for 24 hours, fixed with ethanol, stained with PI, and the proportion of cells in each cycle was detected by flow cytometry to analyze cell cycle arrest[1]
- Western blot assay: A2780 cells were treated with 15 μM Bendamustine HCl for 24 hours, total cellular protein was extracted and subjected to SDS-PAGE electrophoresis. After membrane transfer, incubation with caspase-3 and PARP primary antibodies was performed, followed by secondary antibody incubation and development to detect the expression and cleavage of target proteins[5]
- Colony formation assay: HCT116 cells were seeded in 6-well plates at 500 cells per well, treated with 5~20 μM Bendamustine HCl, stained with crystal violet after 14 days of culture, and the number of colonies was counted to calculate the colony formation inhibition rate[4]

Mice: Inoculation of 1 × 10 6 (DoHH-2, RAMOS), 3 × 10 6 (SuDHL-4) or 5 × 10 6 (Granta 519) cells s.c. in the right flank is administered to C.B.-17 scid-bg mice (SuDHL-4, RAMOS) or C.B.-17 scid-bg mice (DoHH-2, Granta 519). The inoculation volume for flank xenografts is 0.2 mL, and it consists of a 50:50 blend of cells in growth medium and Matrigel. Two to three weekly measurements of the tumor's length and width using electronic calipers are used to estimate the tumor's volume, which is then calculated using the formula V=L×W 2 /2. When the tumors reach about 250 mm 3 , mice are matched for size on day 0 and placed into treatment and control groups. In the case of systemic Granta 519 tumor models, treatment commences on day 14 after 2 × 10 6 cells are injected via the tail vein in 0.1 mL of cell medium on day 0. Every animal in the experiment has an ear tag, and they are all kept under close observation. Once daily, Navitoclax is given by oral gavage in a Phosal 50PG: PEG400: ethanol mixture. On day 1, rituximab (10 mg/kg) and bendamustine (25 mg/kg) are given intravenously. About two hours prior to the administration of bendamustine and rituximab, navigateoclax is given. Ten mice per group are used in each trial. When tumors grow to a size of more than 2000 mm 3 or when any signs of distress are observed, mice are humanely killed. Breathing difficulties, loss of mobility, or weight loss greater than 20% of the average body weight per cage are indicators of distress.

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Human CLL cell xenograft model (NOD/SCID mice): 1×10⁷ MEC-1 cells were inoculated via tail vein injection. Drug administration started on day 7 after inoculation. Bendamustine HCl was dissolved in normal saline to prepare a 5 mg/mL solution, administered by intraperitoneal injection at 25 mg/kg once weekly for 3 consecutive weeks. Mouse body weight and tumor volume were measured weekly, and survival time was recorded[2]
- Human NHL cell xenograft model (BALB/c nude mice): 2×10⁶ Raji cells were subcutaneously inoculated into the right back of mice. Drug administration started when the tumor volume reached 100 mm³. The drug was dissolved in 5% glucose solution, administered by intravenous injection at 30 mg/kg once every two weeks for 2 consecutive times. Tumor volume was measured every 3 days after administration, and tumors were excised and weighed at the end of the experiment[1]
- Rat Walker 256 sarcoma model: 5×10⁶ Walker 256 cells were subcutaneously inoculated into the axilla of rats. Drug administration started on day 5 after inoculation. The drug was dissolved in normal saline, administered by intraperitoneal injection at 15 mg/kg once daily for 5 consecutive days. Rat body weight was monitored during the experiment, and serum CA125 level was detected and tumors were excised and weighed at the end of the experiment[3]
- Combination therapy model (BALB/c nude mice): After subcutaneous inoculation of Raji cells, when the tumor volume reached 100 mm³, Bendamustine HCl (20 mg/kg, intraperitoneal injection, once weekly) was combined with rituximab (10 mg/kg, intravenous injection, once weekly) for 3 consecutive weeks. Tumor volume was measured every 3 days, and mouse survival time was recorded[1]

Absorption: The oral bioavailability in rats is 35%~40%; the peak plasma concentration (Cmax) after intravenous injection of 20 mg/kg is 12 μg/mL[4]
- Distribution: After intravenous injection of 20 mg/kg, the drug concentration in tumor tissue is high, reaching 2.8 times the concentration in mouse plasma; it is mainly distributed in the liver, spleen, kidney and tumor tissue, and the concentration in brain tissue is low[4]
- Metabolism: It is mainly metabolized in the liver by the cytochrome P450 enzyme system, and the main metabolite is γ-hydroxybenzamidostin, which has no cytotoxicity[4]
- Excretion: Within 72 hours after administration to rats, 45% of the administered dose is excreted in urine, 30% in feces, and the remainder is excreted in bile[4]
- Half-life: The elimination half-life (t1/2β) after intravenous injection in rats is 4.2 hours; the elimination half-life after oral administration is 5.8 hours[4]
- Plasma protein binding rate: In vitro experiments showed that the drug had a plasma protein binding rate of 94%~96% in human plasma, mainly binding to albumin [5]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information regarding the use of bendamustine during lactation. Most sources consider breastfeeding contraindicated during maternal treatment with anti-tumor drugs, especially alkylating agents such as bendamustine. Depending on the half-life of the drug and its metabolites, the drug should be eliminated from breast milk within 24 to 48 hours after the last dose. The manufacturer recommends discontinuing breastfeeding during bendamustine treatment and for at least one week after the last dose.
◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found.
◉ Effects on Lactation and Breast Milk
Some evidence suggests that carmustine, a drug closely associated with bendamustine, can increase serum prolactin levels.

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Hematologic toxicity: After rats were intraperitoneally injected with 15 mg/kg bendamustine for 5 consecutive days, the white blood cell count decreased from 12×10⁹/L to 4.5×10⁹/L and the platelet count decreased from 350×10⁹/L to 120×10⁹/L. The counts returned to normal 2 weeks after the drug was discontinued [3]
- Effects on liver and kidney function: After mice were intravenously injected with 30 mg/kg bendamustine, the serum ALT and AST levels increased by 30%~40% compared with the control group, while the serum creatinine and blood urea nitrogen levels did not change significantly; no histopathological damage to liver and kidney tissues was observed after long-term administration (once a week for 4 consecutive weeks) [4]
- Gastrointestinal toxicity: After dogs were orally administered 25 mg/kg, mild vomiting and diarrhea occurred in about 20% of cases. No serious gastrointestinal bleeding or ulceration occurred [4]
- Median lethal dose (LD50): The LD50 for mice was 120 mg/kg after intravenous injection. The intraperitoneal LD50 was 150 mg/kg, and the oral LD50 was 280 mg/kg [3]
- Drug interactions: In vitro experiments showed that the drug had no significant inhibitory or inducing effects on enzymes such as CYP3A4 and CYP2D6, and no significant pharmacokinetic interactions were observed when it was used in combination with rituximab or fludarabine [5]

[1]. Clin Cancer Res . 2008 Jan 1;14(1):309-17.

[2]. Leukemia . 2002 Oct;16(10):2096-105.

[3]. J Cancer Res Clin Oncol . 2002 May;128(5):271-8.

[4]. Br J Pharmacol . 2012 Oct;167(4):881-91.

[5]. Biochem Pharmacol . 2003 Sep 1;66(5):711-24.

Bendamustine hydrochloride is the hydrochloride salt of bendamustine, a bifunctional nitrogen mustard derivative with alkylating and antimetabolizing activities. Bendamustine contains three active moieties: an alkylating group; a benzimidazole ring (potentially acting as a purine analog); and a butyrate side chain. Although its exact mechanism of action is not fully understood, the drug appears to act primarily as an alkylating agent. Bendamustine metabolites can alkylate and cross-link macromolecules, thereby inhibiting the synthesis of DNA, RNA, and proteins, ultimately leading to apoptosis. Bendamustine differs from other alkylating agents in that it may more effectively activate the p53-dependent stress pathway and induce apoptosis; it may induce mitotic catastrophe; and it may activate the base excision repair pathway rather than the alkyltransferase DNA repair mechanism. Therefore, compared to other alkylating agents, this drug may be more effective and less prone to developing resistance.
A nitrogen mustard compound, used as an alkylating antitumor drug for the treatment of chronic lymphocytic leukemia and non-Hodgkin's lymphoma. See also: Bendamustine (with active component).

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Mechanism of action: It has the dual effects of alkylating agent and antimetabolite. It forms cross-linked products by alkylating DNA, destroying the structure and function of DNA, while inhibiting the activity of DNA repair enzymes and inducing tumor cell apoptosis[5]
-Clinical related studies: For patients with relapsed/refractory chronic lymphocytic leukemia, the objective response rate (ORR) of bendamustine hydrochloride monotherapy is 60%~70%; the objective response rate (ORR) of rituximab combination therapy exceeds 80%[1]
-Drug resistance mechanism: Some tumor cells develop drug resistance by upregulating the expression of DNA repair enzymes (such as PARP, DNA polymerase), and the drug resistance can be reversed by combination with DNA repair inhibitors[5]
-Dosage related: In vitro experiments show that continuous low concentration administration (5 μM, for 72 hours) has stronger cytotoxicity than pulse administration (20 μM, for 24 hours), and the cell apoptosis rate is increased by 30%[2]

C16H21CL2N3O2.HCL

394.72

393.077

C, 48.68; H, 5.62; Cl, 26.95; N, 10.65; O, 8.11

3543-75-7

77082

Solid powder

149-151°C

4.066

2

4

9

24

380

0

ClC([H])([H])C([H])([H])N(C([H])([H])C([H])([H])Cl)C1C([H])=C([H])C2=C(C=1[H])N=C(C([H])([H])C([H])([H])C([H])([H])C(=O)O[H])N2C([H])([H])[H].Cl[H]

ZHSKUOZOLHMKEA-UHFFFAOYSA-N

InChI=1S/C16H21Cl2N3O2.ClH/c1-20-14-6-5-12(21(9-7-17)10-8-18)11-13(14)19-15(20)3-2-4-16(22)23;/h5-6,11H,2-4,7-10H2,1H3,(H,22,23);1H

4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid;hydrochloride

SDX-105 (Cytostasane) HCl; EP-3101; SDX105; EP 3101; SDX 105; SDX-105; EP3101; DD6304600; Bendamustinum; Bendamustina; Ribomustin. Brand name: Treanda.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.27 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% DMSO +30% polyethylene glycol+1% Tween 80 : 30 mg/mL

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Solubility in Formulation 5: 5.88 mg/mL (14.90 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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