Additionally, methylpropylamine shields DNA double-strand breaks from ionizing radiation [1]. Bystander cells are shielded from radiation-induced DNA damage by methylpropylamine [2]. Radioprotective properties of mmethylproamine are concentration-dependent[3].

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1. In human A549 lung carcinoma cells, pretreatment with Methylproamine (0.1–10 μM) 15 min prior to γ-irradiation (2–8 Gy) dose-dependently enhanced cell survival, with the maximum protective effect observed at 1 μM (survival rate increased by ~30% after 4 Gy irradiation); at concentrations ≥10 μM, the compound showed mild cytotoxicity and reduced radioprotective efficacy; colony formation assays also demonstrated that Methylproamine (1 μM) increased the surviving fraction of A549 cells after 2–6 Gy irradiation, with the dose reduction factor (DRF) calculated to be 1.3 [6]
2. In Chinese hamster ovary (CHO) cells, pretreatment with Methylproamine (1 μM) 15 min before γ-irradiation (4 Gy) significantly reduced the number of DNA double-strand breaks (DSBs), as evidenced by decreased γ-H2AX foci formation (γ-H2AX foci count per cell decreased from ~12 to ~4 at 30 min post-irradiation); the compound also inhibited the formation of ionizing radiation-induced Rad51 foci (a marker of homologous recombination repair), indicating that its radioprotective effect is associated with preventing DSB generation rather than promoting DSB repair [1]
3. In normal human keratinocytes (NHK), pretreatment with Methylproamine (0.1–5 μM) 15 min before γ-irradiation (2–6 Gy) improved cell viability in a dose-dependent manner (cell viability increased by 25–40% after 4 Gy irradiation at 1 μM); the compound also reduced the level of radiation-induced DNA damage, as shown by decreased comet assay tail moment (tail moment decreased from ~15 to ~6 at 4 Gy) and reduced γ-H2AX expression (protein level downregulated by ~50% at 1 μM and 4 Gy) [3]
4. In targeted (directly irradiated) and bystander (co-cultured with irradiated cells) human keratinocyte HaCaT cells, pretreatment with Methylproamine (1 μM) 15 min before 2 Gy α-particle irradiation of targeted cells protected both targeted and bystander cells from radiation-induced cytotoxicity (viability of targeted cells increased from ~55% to ~80%, and bystander cells from ~60% to ~78%); the compound also reduced γ-H2AX foci formation in both cell populations (foci count in targeted cells decreased from ~10 to ~3, and bystander cells from ~6 to ~2) and inhibited the release of reactive oxygen species (ROS) from irradiated cells, which is the key mediator of bystander effect [2]

Cytotoxicity assay[3]
Cell Types: Keratinocytes
Tested Concentrations: 10, 20 μM
Incubation Duration: 60 min
Experimental Results: Did not show any detectable cytotoxicity at 10 μM, Significant cytotoxicity at 20 μM.

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1. For A549 cell survival and colony formation assays: Cells were seeded in 96-well plates (for viability) or 6-well plates (for colony formation) and incubated until 70–80% confluent; Methylproamine at concentrations of 0.1, 1, 5, 10 μM was added to the culture medium 15 min prior to γ-irradiation (doses of 2, 4, 6, 8 Gy); cell viability was measured by MTT assay 72 h post-irradiation, and colony formation was evaluated by fixing and staining colonies with crystal violet after 10–14 days of incubation, with colonies containing ≥50 cells counted as viable clones [6]
2. For CHO cell DSB detection assay: CHO cells were plated on coverslips and treated with 1 μM Methylproamine 15 min before 4 Gy γ-irradiation; at 30 min, 2 h, 6 h post-irradiation, cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and incubated with anti-γ-H2AX or anti-Rad51 primary antibodies followed by fluorescent secondary antibodies; foci were visualized under a fluorescence microscope, and the number of foci per cell was counted for at least 100 cells per group [1]
3. For NHK cell DNA damage and viability assays: NHK cells were cultured in keratinocyte growth medium until logarithmic growth phase; Methylproamine (0.1–5 μM) was added 15 min before γ-irradiation (2–6 Gy); cell viability was detected by trypan blue exclusion assay 48 h post-irradiation; DNA damage was assessed by comet assay (cells were embedded in agarose, lysed, subjected to electrophoresis, stained with DNA dye, and tail moment was analyzed by image software) and western blot (cells were lysed, protein samples were separated by SDS-PAGE, transferred to membranes, and probed with anti-γ-H2AX and anti-GAPDH antibodies, with protein expression quantified by densitometry) [3]
4. For HaCaT cell targeted-bystander effect assay: HaCaT cells were divided into targeted and bystander groups, with targeted cells seeded in inserts with porous membranes; Methylproamine (1 μM) was added to both groups 15 min before targeted cells were exposed to 2 Gy α-particle irradiation; after 24 h of co-culture, cell viability was measured by CCK-8 assay; γ-H2AX foci were detected by immunofluorescence as described above; ROS levels were measured by adding fluorescent ROS probe to the culture medium and detecting fluorescence intensity with a microplate reader [2]

1. In A549 cells, methylpropylamine at concentrations ≤1 μM did not show significant cytotoxicity (cell survival >95% after 72 hours without irradiation); at concentrations ≥5 μM, mild cytotoxicity was observed (cell survival decreased to ~85% at 5 μM and ~70% at 10 μM after 72 hours) [6]
2. In NHK cells, methylpropylamine at concentrations up to 5 μM did not show significant cytotoxicity (cell survival >90% after 48 hours without irradiation); no apoptotic morphological changes or increased caspase-3 activity were detected in cells treated with this compound alone [3]

[1]. Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks.

[2]. Radioprotection of targeted and bystander cells by methylproamine. Strahlenther Onkol. 2015 Mar;191(3):248-55.

[3]. Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage. Int J Radiat Biol. 2011 Mar;87(3):274-83.

[4]. Lobachevsky PN, Vasireddy RS, Broadhurst S, Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage. Int J Radiat Biol. 2011 Mar;87(3):274-83.

[5]. Sprung CN, Vasireddy RS, Karagiannis TC, Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks. Mutat Res. 2010 Oct 13;692(1-2):49-52.

[6]. Martin RF, Broadhurst S, Reum ME, In vitro studies with methylproamine: a potent new radioprotector. Cancer Res. 2004 Feb 1;64(3):1067-70.

1. Methamphetamine is a novel synthetic radioprotective agent with a unique mechanism of action: it does not promote the repair of existing DNA double-strand breaks (DSBs), but rather prevents the formation of radiation-induced DSBs, which distinguishes it from traditional radioprotective agents (such as amifostine) that mainly function by scavenging free radicals or enhancing DNA repair [1]. 2. Methamphetamine's radioprotective effect on bystander cells is achieved by inhibiting the release of reactive oxygen species (ROS) from directly irradiated cells, thereby blocking the intercellular transmission of radiation-induced damage signals [2]. 3. In human keratinocytes, the radioprotective efficacy of methacin is positively correlated with its ability to reduce DNA damage, and at low concentrations, it has a better protective effect on normal cells (NHK) than on some cancer cell lines, indicating that it has the potential to protect normal tissues during radiotherapy [3]. 4. Methamphetamine has a rapid onset of action, and the best radioprotective effect is achieved when administered 15 minutes before irradiation; extending the interval between administration and irradiation to 60 minutes reduces its protective efficacy, indicating that its biological half-life in cells is short [6].

C28H31N7

465.593

465.264

188247-01-0

448201

Light yellow to pink solid powder

4.902

2

5

4

35

709

0

ADKLMOJIJGHCCD-UHFFFAOYSA-N

InChI=1S/C28H31N7/c1-18-15-20(33(2)3)6-8-22(18)28-30-23-9-5-19(16-25(23)32-28)27-29-24-10-7-21(17-26(24)31-27)35-13-11-34(4)12-14-35/h5-10,15-17H,11-14H2,1-4H3,(H,29,31)(H,30,32)

N,N,3-trimethyl-4-[6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazol-2-yl]aniline

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 : ≥ 41 mg/mL (~88.06 mM)

Solubility in Formulation 1: ≥ 0.62 mg/mL (1.33 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 6.2 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: ≥ 0.62 mg/mL (1.33 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 6.2 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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 (Please use freshly prepared in vivo formulations for optimal results.)