Bendamustine HCl (SDX-105)

Alias: SDX-105 (Cytostasane) HCl; EP-3101; SDX105; EP 3101; SDX 105; SDX-105; EP3101; DD6304600; Bendamustinum; Bendamustina; Ribomustin. Brand name: Treanda.
Cat No.:V1466 Purity: ≥98%
Bendamustine HCl (formerly also called SDX-105;EP-3101; Cytostasane; DD6304600; Treanda), the hydrochloride salt of bendamustine which is a bifunctional mechlorethamine derivative, is a potent DNA-alkylating/ cross-linking / damaging agent (IC50 = 50 μM in cell-free assay) approved for cancer treatment.
Bendamustine HCl (SDX-105) Chemical Structure CAS No.: 3543-75-7
Product category: DNA(RNA) Synthesis
This product is for research use only, not for human use. We do not sell to patients.
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Purity & Quality Control Documentation

Purity: ≥98%

Product Description

Bendamustine HCl (formerly also called SDX-105; EP-3101; Cytostasane; DD6304600; Treanda), the hydrochloride salt of bendamustine which is a bifunctional mechlorethamine derivative, is a potent DNA-alkylating/ cross-linking / damaging agent (IC50 = 50 μM in cell-free assay) approved for cancer treatment. It has been documented that apoptosis and DNA damage stress activate bendamustine. By suppressing a number of genes linked to mitosis, including Cyclin B1, Aurora kinase A, and Polo-like kinase 1, bendamustine prevents mitotic checkpoints and causes a mitotic catastrophe. Bendamustine caused G2 cell cycle arrest in myeloma cell lines by cleaving caspase 3, which triggered apoptosis.

Biological Activity I Assay Protocols (From Reference)
Targets
DNA Alkylator/Crosslinker
ln Vitro

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]

ln Vivo
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]
Cell Assay
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 × 104 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).
Animal Protocol
Mice: Inoculation of 1 × 106 (DoHH-2, RAMOS), 3 × 106 (SuDHL-4) or 5 × 106 (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×W2/2. When the tumors reach about 250 mm3, 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 × 106 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 mm3 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.
References

[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.

These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C16H21CL2N3O2.HCL
Molecular Weight
394.72
Exact Mass
393.08
Elemental Analysis
C, 48.68; H, 5.62; Cl, 26.95; N, 10.65; O, 8.11
CAS #
3543-75-7
Related CAS #
16506-27-7; 3543-75-7(HCl)
Appearance
Solid powder
SMILES
CN1C2=C(C=C(C=C2)N(CCCl)CCCl)N=C1CCCC(=O)O.Cl
InChi Key
ZHSKUOZOLHMKEA-UHFFFAOYSA-N
InChi Code
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
Chemical Name
4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid;hydrochloride
Synonyms
SDX-105 (Cytostasane) HCl; EP-3101; SDX105; EP 3101; SDX 105; SDX-105; EP3101; DD6304600; Bendamustinum; Bendamustina; Ribomustin. Brand name: Treanda.
HS Tariff Code
2934.99.9001
Storage

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.
Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO: 78~100 mg/mL (197.6~253.3 mM)
Water: ~2 mg/mL (~5.0 mM)
Ethanol: ~17 mg/mL (~43.1 mM)
Solubility (In Vivo)
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.

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.


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

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).

 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 2.5334 mL 12.6672 mL 25.3344 mL
5 mM 0.5067 mL 2.5334 mL 5.0669 mL
10 mM 0.2533 mL 1.2667 mL 2.5334 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

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What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:
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Note: Chemical formula is case sensitive: C12H18N3O4  c12h18n3o4
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In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
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Calculation results

Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
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Clinical Trial Information
NCT Number Recruitment interventions Conditions Sponsor/Collaborators Start Date Phases
NCT02996773 Active
Recruiting
Drug: Bendamustine
Drug: Cyclophosphamide
Lymphoma, Hodgkin
Lymphoma, Follicular
University of Arizona November 29, 2016 Phase 1
NCT03834688 Active
Recruiting
Drug: Bendamustinee
Drug: Venetoclax
Mantle Cell Lymphoma PrECOG, LLC. January 13, 2020 Phase 2
NCT04083898 Active
Recruiting
Drug: Bendamustine
Drug: Prednisone
Multiple Myeloma Washington University School
of Medicine
April 3, 2020 Phase 1
NCT03872180 Active
Recruiting
Drug: Bendamustine
Biological: Obinutuzumab
CCND1 Positive
Mantle Cell Lymphoma
Emory University April 11, 2019 Phase 2
NCT03311126 Active
Recruiting
Drug: Bendamustine
Drug: Obinutuzumab
Mantle Cell Lymphoma
Non-hodgkin Lymphoma
University of Wisconsin, Madison October 19, 2017 Phase 2
Biological Data
  • Xenograft sensitivity to treatment with navitoclax and bendamustine. Br J Pharmacol . 2012 Oct;167(4):881-91.
  • Molecular analysis of bendamustine in the absence and presence of navitoclax in Granta 519 flank tumours. Br J Pharmacol . 2012 Oct;167(4):881-91.
  • Structures of bendamustine, cyclophosphamide, chlorambucil, and melphalan. Clin Cancer Res . 2008 Jan 1;14(1):309-17.
  • Enhanced apoptosis signaling by bendamustine when compared with the cyclophosphamide metabolite, phosphoramide mustard, and chlorambucil. Clin Cancer Res . 2008 Jan 1;14(1):309-17.
  • Bendamustine differentially modifies genes involved in base excision repair. Clin Cancer Res . 2008 Jan 1;14(1):309-17.
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