Purpose: Efficient extravascular penetration is essential for the optimal activity of most anticancer drugs and is particularly relevant to bioreductive cytotoxins which target hypoxic cells that can be located distal to functional blood vessels within tumours. Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide; Triazone; SR 259075; formerly SR 4233) is a lead bioreductive cytotoxin currently undergoing clinical evaluation. It exhibits preferential cytotoxicity towards cells at reduced oxygen tension, and could complement existing anticancer therapies where hypoxic cells are believed to constitute a refractory population. We assessed the ability of tirapazamine to penetrate tumour tissue using an in vitro multilayered cell culture (MCC) model.
Methods: Diffusion of tirapazamine through oxic and hypoxic multilayered cell cultures composed of SiHa. human cervical carcinoma cells, was measured using a dual reservoir diffusion apparatus from which samples were quantified via HPLC. Drug concentration kinetics from both reservoirs were analysed using a mathematical model for diffusion and metabolism within the MCC. Results were then applied to a second mathematical model which described extravascular drug penetration within a tumour cord, the sheath of cells surrounding a blood vessel.
Results: The diffusion coefficient of tirapazamine within SiHa MCCs was determined as 7.0+/-0.5 x 10(-7) cm2/s and the maximal metabolic rate for hypoxic MCCs, Vmax, as 1.5+/-0.4 microM/s. The thickness of individual tissue cultures was determined by diffusion of tritiated water (HTO). A linear relationship was shown to exist between tissue thickness and the inverse of permeability to HTO. Experimental results were used to simulate drug distribution within a tumour cord. These simulations indicate that, when tirapazamine is administered via intravenous infusion, a stable tirapazamine distribution throughout the cord occurs within 15 min with cells most peripheral to the blood vessel exposed to only 10% of the blood drug concentration. Under these conditions, the simulations predict cell kill to be limited to the first 75 microm of tissue surrounding a blood vessel.
Conclusion: This study indicates that extravascular penetration of tirapazamine to peripheral cells existing at low oxygen tension may be limited by the metabolism of tirapazamine by more proximal cells existing at moderate oxygen tension. Simulations found that tirapazamine reached only 10% of the blood concentration at cells most peripheral to blood vessels. These results indicate that tirapazamine would be significantly cytotoxic only to cells located within approximately 75 microm of blood vessels. Further MCC-based modelling of extravascular drug penetration would serve as a means of identifying new antitumour agents with location-specific activity.