Metformin (1,1-Dimethylbiguanide hydrochloride from Sigma-Aldrich, St. Louis, MO) was dissolved in PBS and sterilized using a 0.22 μm syringe filter. Concentrations of metformin for studies with cells in culture ranged from 1–20 mM. No cytotoxicity was observed at any of these concentrations. A 5 mM concentration of metformin was chosen for routine use in all subsequent in vitro studies. WR1065 (Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD) was used either alone prior to irradiation as a positive control at its maximum cytoprotective concentration of 4 mM (18 (link)) or in combination with metformin at select times after irradiation.
For in vivo studies, mice were injected i.p. with a final volume of 0.2 ml for all single and drug combinations used. Metformin was administered at a final concentration of 250 mg/kg body weight. Captopril was injected at a concentration of 200 mg/kg body weight, MESNA was injected at a concentration of 300 mg/kg body weight and NAC was injected at a concentration of 400 mg/kg body weight. These concentrations were chosen because they were nontoxic. Amifostine (Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute) was administered i.p. 30 min prior to irradiation at a concentration of 400 mg/kg body weight, a dose known to afford maximum radioprotection in the C3H mouse model (4 ).
For in vivo studies, mice were injected i.p. with a final volume of 0.2 ml for all single and drug combinations used. Metformin was administered at a final concentration of 250 mg/kg body weight. Captopril was injected at a concentration of 200 mg/kg body weight, MESNA was injected at a concentration of 300 mg/kg body weight and NAC was injected at a concentration of 400 mg/kg body weight. These concentrations were chosen because they were nontoxic. Amifostine (Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute) was administered i.p. 30 min prior to irradiation at a concentration of 400 mg/kg body weight, a dose known to afford maximum radioprotection in the C3H mouse model (4 ).
