Tirapazamine (SR-4233) combined with radiations and cis-platinum: cytotoxicity in vivo and in vitro on 2 human cell lines

Purpose/objective: Solid human tumors contain areas with low oxygen tension (pO2) and such hypoxia is a limiting factor for the biological efficacy of ionizing radiations. However the relative decrease in tumor oxygenation compared to normal tissues could be used as a target for the activation of cytotoxic molecules. Bioreductive drugs, such as tirapazamine (SR-4233), are metabolized at low oxygen pressures and for drug concentration which are clinically relevant. For one murine cell line, published data have shown a marked increase in cell kill when tirapazamine and radiations were combined to cisplatinum (c-DDP). This study was performed to assess the cytotoxicity obtained in vivo and in vitro on two human cell lines with ionizing radiations, tirapazamine and cis-platinum given alone or combined. Material and Methods: - In vitro (cell survival curves): Two human cell lines were used: a rectocolic adenocarcinoma (HRT 18) and a melanoma (Na11+). The percentage of radio biologically hypoxic cells was around 15% for HRT 18 and 60% for NA11+. The pO2 dependence of cell survival was studied at four different oxygen concentrations: air (20.9 % O2), 10, 2 and 0.2 % O2 (hypoxia). Tumor cells were irradiated after one hour of incubation at the defined pO2, in medium, tirapazamine (50 μM) and c-DDP (2 μg/ml) alone or combined. After 2 weeks of incubation (37 deg. C, 5% carbon dioxide humidified atmosphere), the colonies were fixed, stained with a solution of crystal violet and counted. - In vivo (excision assay): Tumors were xenografted into the flank of nude mice (3x106 cells in 0.5 ml of medium). Tirapazamine (36 mg kg-1 for 4 days, LD10) was injected I.P. 3 hours before or at the same time than c-DDP (I.P., 8 mg kg-1 for 4 days). The total volume injected daily was below 0.6 ml. The mice were killed 24 h after the last drug injection and the tumors were excised, minced and dissociated with an enzyme cocktail. The cell suspension was filtered and the cells plated for clonogenic assay. After 2 weeks of incubation (37 deg. C, 5% carbon dioxide humidified atmosphere), the colonies were fixed, stained with a solution of crystal violet and counted. Results: - In vitro: For the 2 cell lines, tirapazamine alone was not cytotoxic in air or at 10%, but there was a marked decrease in cell survival at 2 and 0.2 % O2 (p < 0.001). For HRT 18, no cell killing was found with c-DDP alone at 20.9 and 10% oxygen tension, but at low pO2 (2 and 0.2%), a significant increase (p=0.05) in cell survival was found compared to control. At the 5 pO2, cell survival was always superior (non significant) with the combination of tirapazamine and c-DDP than with tirapazamine alone. For NA 11+ and at the 5 pO2, cell survival was identical when tirapazamine and c-DDP where combined, compared to tirapazamine alone. - In vivo: For NA 11+, there was a decrease in plating efficiency (P.E.) when tirapazamine and c-DDP were given at the same time (P.E.: 0.01, p=0.04), compared to c-DDP (P.E.: 0.05) or tirapazamine (P.E.: 0.10) alone. This effect was not significant with a 3 h interval between the injections (P.E.: 0.03). For HRT 18, the effect (P.E.) of the combination of tirapazamine and c-DDP was comparable to the P.E. obtained with tirapazamine alone. However, a significant increase in cell yield was observed in that cell line with c-DDP alone compared to controls. Conclusion: The activation of tirapazamine at a low concentration and at pO2, found mainly in tumors (below 2% oxygen), could yield a very beneficial therapeutic ratio when ionizing radiations and tirapazamine are combined. The combination to c-DDP did not give a significant benefit in cell killing in vitro, due to the absence of sensitivity of the cell lines used to this molecule. However, a decrease in plating efficiency was observed in vivo only in the cell line with a significant hypoxic fraction (NA11+). Furthermore, the timing between the injections of tirapazamine and c-DDP was not found to be an important issue in vivo for these cell lines