The preparation and biological characterization of a new HL91-derivative for hypoxic imaging on stroke mice

Aim99mTc-HL91 (Prognox, GE-Healthcare) was the first nonnitro-aryl-based radiotracer for evaluating hypoxic fraction in neoplasm, stroke and myocardium infarction regions. However, the high hydrophilicity of 99mTc-HL91 might hamper its penetration into cells. In this study, we prepared a new ligand 4,4,11,11-tetramethyl- 5,10-diazatetradecane- 3,12-dionedioxime (HL91-ET) with higher lipophilicity but structurally similar compared with that of HL91. The chemical and biological characterizations of 99mTc-HL91-ET as a scintigraphic probe for hypoxia were performed with a stroke-bearing mouse model.Materials and MethodsHL91-ET was synthesized and formulated with stannous chloride and buffer to afford kits. After mixing with 99mTc-pertechnetate, 99mTc-HL91-ET can be prepared in high yield and high radiochemical purity (both >96%). The partition coefficient of 99mTc-HL91-ET was determined in n-octanol/PBS system. Cellular uptake assays under normoxic and hypoxic conditions were performed in an oxygen-controlled CO2 incubator. Brain stroke in the mouse model was induced by the electrocautery of the middle cerebral artery. After intravenous injection of 99mTc-HL91-ET into the Balb/c mouse suffering brain stroke, small-animal SPECT images were acquired at designated time points and autoradiography of the brain slides was conducted. Parallel studies of 99mTc-HL91 were also conducted at the same conditions for comparison.ResultsThe higher partition coefficient of 99mTc-HL91-ET (0.294±0.007) indicated higher lipophlicity compared with that of 99mTc-HL91 (0.089±0.005). The 99mTc-HL91-ET preparation was stable at ambient temperature for 24 h. Cellular uptake assay showed that 99mTc-HL91-ET was less selectively retained in hypoxic cells than 99mTc-HL91. The target-to-normal brain ratios derived from the autoradiograms of the brains of stroke mice were 1.31±0.02 and 17.47±0.10 (n=3), respectively, at 2 h post injection of 99mTc-HL91-ET and 99mTc-HL91.ConclusionsThis study revealed that 99mTc-HL91-ET, though with higher lipophilicity than 99mTc-HL91, did not suggest better specific accumulation in hypoxic cells or tissues than 99mTc-HL91. The uptake mechanism of 99mTc-HL91 was at least not solely by passive diffusion. Lipophilicity should not be the major consideration in designing HL91-derivatives for hypoxia imaging.