Implantable polymers for tirapazamine treatments of experimental intracranial malignant glioma.

Abstract

Malignant gliomas remain refractory to intensive radiotherapy and cellular hypoxia enhances clinical radioresistance. Under hypoxic conditions, the benzotriazine di-N-oxide (3-amino-1,2,4-benzotriazine 1,4-dioxide) (tirapazamine) is reduced to yield a free-radical intermediate that results in DNA damage and cellular death. For extracranial xenografts, tirapazamine treatments have shown promise. We therefore incorporated tirapazamine into the synthetic, biodegradable polymer, measured the release, and tested the efficacy both alone and in combination with external beam radiotherapy in the treatment of experimental intracranial human malignant glioma xenografts. The [(poly(bis(p-carboxyphenoxy)-propane) (PCPP):sebacic acid (SA) (PCPP:SA ratio 20:80)] polymer was synthesized. The PCPP:SA polymer and solid tirapazamine were combined to yield proportions of 20% or 30% (wt/wt). Polymer discs (3 x 2 mm) (10 mg) were incubated (PBS, 37 degrees C), and the proportion of the drug released vs. time was recorded. Male nu/nu nude mice were anesthetized and received intracranial injections of 2 x 10(5) U251 human malignant glioma cells. For single intraperitoneal (i.p.) drug and/or external radiation treatments, groups of mice had i.p. 0.3 mmol/kg tirapazamine, 5 Gy cranial irradiation, or combined treatments on day 8 after inoculation. For fractionated drug and radiation treatments, mice had i.p. 0.15 mmol/kg tirapazamine, 5 Gy radiation, or combined treatments on days 8 and 9 after inoculation. For intracranial (i.c.) polymer treatments, mice had craniectomies and intracranial placement of polymer discs at the site of cellular inoculation. The maximally tolerated percentage loading of tirapazamine in the polymer.disc was determined. On day 7 after inoculation, groups of mice had i.c. empty or 3% tirapazamine alone or combined with radiation (5 Gy x 2 doses) or combined with i.p. drug (0.15 mmol/kg x 2 doses on days 8 and 9). Survival was recorded. Polymers showed controlled, protracted in vitro release for over 100 days. The 5 Gy x 1 treatment resulted in improved survival; 28.5 +/- 3.7 days (P = 0.01 vs. controls), while the single i.p. 0.3 mmol/kg tirapazamine treatment, 17.5 +/- 1.9 days (P = NS) and combined treatments; 21.5 +/- 5.0 days (P = NS) were not different. The fractionated treatments: 5 Gy x 2, i.p. 0.15 mmol/kg tirapazamine x 2 and the combined treatments resulted in improved survival: 44.5 +/- 3.9 (P < 0.001), 24.5 +/- 2.3 (P = 0.05) and 50.0 +/- 6.0 (P < 0.001), respectively. Survival after intracranial empty polymer was 16.5 +/- 3.0 days and increased to 31.0 +/- 3.0 (P = 0.003) days when combined with the 5 Gy x 2 treatment. The survival after the polymer bearing 3% tirapazamine alone vs. combined with radiation was not different. The combined 3% tirapazamine polymer, i.p. tirapazamine, and radiation treatments resulted in both early deaths and the highest long-term survivorship. The basis for potential toxicity is discussed. We conclude that implantable biodegradable polymers provide controlled intracranial release for treatment of experimental glioma. For treatment of malignant gliomas, the combination of continuous polymer-mediated delivery and fractionated systemic delivery of tirapazamine with external beam radiotherapy warrants further exploration.