The biological characterization of 99mTc-BnAO-NI as a SPECT probe for imaging hypoxia in a sarcoma-bearing mouse model

Objectives: Tumor growth beyond the region where vascular oxygen can reach creates a hypoxic domain. In this study, BnAO, a ligand that had been labeled with 99mTc-pertechnetate for hypoxia imaging, was conjugated with 2-nitroimidazole to give 3,3,10,10-tetramethyl-1-(2-nitro-1H-imidazo-1-y1)-4,9-diazadodecane-2,11- dionedioxime (BnAO-NI) as a potential ligand for hypoxia detection. Pentoxifylline is a peripheral vasodilator and has been used as a radiosensitizer in tumor radiotherapy. 99mTc-BnAO-NI/SPECT was applied to noninvasively assess the pharmacological effect of pentoxifylline in reducing tumor hypoxia in vivo. Methods: BnAO-NI was synthesized and formulated with methylene diphosphonate (MDP), stannous chloride and carbonate buffer to afford kits. After mixing with 99mTc-pertechnetate, 99mTc-BnAO-NI injection can be readily prepared. The partition coefficient, radiochemical purity and in vitro stability were determined. Cellular uptake of radiotracers in KHT cells under hypoxia was conducted in a CO2 incubator at 37 oC under hypoxia or normoxia. A biodistribution study after intravenous injection of 99mTc-BnAO-NI in KHT sarcoma-implanted C3H mice was performed. The effect of pentoxifylline (100 mg/kg) on reducing tumor hypoxia was also studied. Results: The radiochemical purity (RCP) of the 99mTc-BnAO-NI preparation was greater than 96% and stable at ambient temperature for 24 h (RCP>90%). The accumulation of 99mTc-BnAO-NI and 99mTc-BnAO in KHT cells under hypoxia were 3.57 and 4.13-fold higher than those under normoxic environment, indicating unambiguous oxygen-dependent uptakes of these two probes. The distribution of 99mTc-BnAO-NI in KHT sarcoma-bearing mice revealed rapid clearance from the blood circulation. The tumor uptake peaked at 2 h post-injection (0.32±0.05%ID/g) with tumor-to-blood and tumor-to-muscle ratios of 10.32 and 3.96, respectively. The effect of pentoxifylline on the tumor blood perfusion was obvious. The tumor-to-muscle ratios at 2 h post-injection of 99mTc-BnAO-NI with and without pentoxifylline pretreatment were 1.67±0.38 and 2.59±0.25, respectively (p=0.025, n=3). Conclusion: This study demonstrates that 99mTc-BnAO-NI is a hypoxia-sensitive radio probe for monitoring hypoxic regions in a malignant neoplasm. However, 99mTc-BnAO-NI, though with higher lipophilicity than 99mTc-BnAO, did not achieve better specific accumulation in hypoxic tissues. 99mTc-BnAO-NI/SPECT could be applied in clinics to noninvasively evaluate the feasibility of using pentoxifylline as a radiosensitizer by reducing tumor hypoxia in vivo.