Metformin exhibits radiation countermeasures efficacy when used alone or in combination with sulfhydryl containing drugs

Abstract

Metformin, a biguanide drug used in the treatment of type II diabetes, was evaluated alone and in combination with amifostine, captopril, MESNA or N-acetyl-cysteine (NAC) for its ability to protect when administered 24 h after irradiation. Mouse embryo fibroblasts (MEF), human microvascular endothelial cells (HMEC) and SA-NH mouse sarcoma cells were exposed to 4 Gy in vitro. C3H mice were exposed to 7 Gy and evaluated utilizing an endogenous spleen colony assay system. Amifostine and WR1065, administered 30 min prior to irradiation, were used as positive controls. Treatment of MEF, HMEC and SA-NH cells with metformin elevated survival levels by 1.4-, 1.5- and 1.3-fold compared to 1.9-, 1.8- and 1.6-fold for these same cells treated with WR1065, respectively. Metformin (250 mg/kg) was effective in protecting splenic cells from a 7 Gy dose in vivo (protection factor = 1.8). Amifostine (400 mg/kg), administered 30 min prior to irradiation resulted in a 2.6-fold survival elevation, while metformin administered 24 h after irradiation in combination with NAC (400 mg/kg), MESNA (300 mg/kg) or captopril (200 mg/kg) enhanced survival by 2.6-, 2.8- and 2.4-fold, respectively. Each of these agents has been approved by the FDA for human use and each has a well characterized human safety profile. Metformin alone or in combination with selected sulfhydryl agents possesses radioprotective properties when administered 24 h after radiation exposure comparable to that observed for amifostine administered 30 min prior to irradiation making it a potentially useful agent for radiation countermeasures use.

FIG. 1

The toxicity profile for metformin (Met) in SA-NH murine sarcoma cells treated with concentrations ranging of 0.5 to 20 mM after a 1 h exposure. Each experiment was performed three times and error bars represent the standard error of the mean (SEM).

FIG. 2

A time course of the radioprotective effects of metformin (Met) in SA-NH murine sarcoma cells at a concentration of 5 mM and an exposure time of 1 h administered from 1 h to 48 h after irradiation with 4 Gy. Amifostine's active free thiol form WR1065 (WR) was administered at a concentration of 4 mM 30 min prior to irradiation to serve as a positive control for radioprotective comparison. Each experiment was repeated three times and error bars represent the SEM. P values comparing cell survival at 4 Gy only to those following treatment with Met or WR are presented for comparison.

FIG. 3

A time course of the radioprotective effects of metformin (Met) in human microvascular endothelial cells (HMEC) at a concentration of 5 mM and an exposure time of 1 h administered from 1 h to 48 h after irradiation with 4 Gy. WR1065 (WR) was administered at a concentration of 4 mM 30 min prior to irradiation to serve as a positive control for radioprotective comparison. Each experiment was repeated three times and error bars represent the SEM. P values comparing cell survival at 4 Gy only to those following treatment with Met or WR are presented for comparison.

FIG. 4

A time course of the radioprotective effects of metformin (Met) in mouse embryo fibroblasts (MEF) at a concentration of 5 mM and an exposure time of 1 h administered from 1 h to 48 h after irradiation with 4 Gy. WR1065 (WR) was administered at a concentration of 4 mM either 30 min before irradiation as a positive control for radioprotective comparison or in combination with Met 1 h or 24 h after irradiation. Each experiment was repeated three times and error bars represent the SEM. P values comparing cell survival levels exposed to 4 Gy only with those exposed to Met and WR alone or in combination are presented for comparison.

FIG. 5

A comparison of the radioprotective effects of metformin (Met) at a dose of 250 mg/kg alone or in combination with amifostine (Ami) 400 mg/kg, captopril (Cap) 200 mg/kg, MESNA 300 mg/kg and N-acetyl-cysteine (NAC) 400 mg/kg administered 24 h after a 7 Gy dose of whole body ionizing radiation using a C3H mouse model and the endogenous spleen colony assay are presented. Ami alone (400 mg/kg), administered 30 min prior to or 24 h after 7 Gy irradiation are included as positive controls. The number of colonies growing on the surface of spleens from mice in each experimental group 13 days after radiation exposure were counted and serve as a measure of the relative radio-protectiveness of the various experimental treatments. Error bars represent the SEM. P values comparing the number of spleen colonies following the various treatment groups with that after 7 Gy only are presented for comparison.