Increased tumor oxygenation and drug uptake during anti-angiogenic weekly low dose cyclophosphamide enhances the anti-tumor effect of weekly tirapazamine

Metronomic cyclophosphamide treatment is associated with anti-angiogenic activity and is anticipated to generate exploitable hypoxia using hypoxia-activated prodrugs. Weekly administration of tirapazamine (TPZ; 5 mg/kg body weight i.p.) failed to inhibit the growth of 9L gliosarcoma tumors grown s.c. in scid mice. However, the anti-tumor effect of weekly cyclophosphamide (CPA) treatment (140 mg/kg BW i.p.) was substantially enhanced by weekly TPZ administration. An extended tumor free period and increased frequency of tumor eradication without overt toxicity were observed when TPZ was given 3, 4 or 5 days after each weekly CPA treatment. Following the 2^nd CPA injection, Electron Paramagnetic Resonance (EPR) Oximetry indicated significant increases in tumor pO₂, starting at 48 hr, which further increased after the 3^rd CPA injection. pO₂ levels were, however, stable in growing untreated tumors. A strong negative correlation (-0.81) between tumor pO₂ and tumor volume during 21 days of weekly CPA chemotherapy was observed, indicating increasing tumor pO₂ with decreasing tumor volume. Furthermore, CPA treatment resulted in increased tumor uptake of activated CPA. CPA induced increases in VEGF RNA, which reached a maximum on day 1, and in PLGF RNA which was sustained throughout the treatment, while anti-angiogenic host thrombospondin-1 increased dramatically through day 7 post-CPA treatment. Weekly cyclophosphamide treatment was anticipated to generate exploitable hypoxia. However, our findings suggest that weekly CPA treatment induces a functional improvement of tumor vasculature, which is characterized by increased tumor oxygenation and drug uptake in tumors, thus counter-intuitively, benefiting intratumoral activation of TPZ and perhaps other bioreductive drugs.