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

Aim: (99m)Tc-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 (99m)Tc-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 (99m)Tc-HL91-ET as a scintigraphic probe for hypoxia were performed with a stroke-bearing mouse model.

Materials And Methods: HL91-ET was synthesized and formulated with stannous chloride and buffer to afford kits. After mixing with (99m)Tc-pertechnetate, (99m)Tc-HL91-ET can be prepared in high yield and high radiochemical purity (both >96%). The partition coefficient of (99m)Tc-HL91-ET was determined in n-octanol/PBS system. Cellular uptake assays under normoxic and hypoxic conditions were performed in an oxygen-controlled CO(2) incubator. Brain stroke in the mouse model was induced by the electrocautery of the middle cerebral artery. After intravenous injection of (99m)Tc-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 (99m)Tc-HL91 were also conducted at the same conditions for comparison.

Results: The higher partition coefficient of (99m)Tc-HL91-ET (0.294+/-0.007) indicated higher lipophlicity compared with that of (99m)Tc-HL91 (0.089+/-0.005). The (99m)Tc-HL91-ET preparation was stable at ambient temperature for 24h. Cellular uptake assay showed that (99m)Tc-HL91-ET was less selectively retained in hypoxic cells than (99m)Tc-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 2h post injection of (99m)Tc-HL91-ET and (99m)Tc-HL91.

Conclusions: This study revealed that (99m)Tc-HL91-ET, though with higher lipophilicity than (99m)Tc-HL91, did not suggest better specific accumulation in hypoxic cells or tissues than (99m)Tc-HL91. The uptake mechanism of (99m)Tc-HL91 was at least not solely by passive diffusion. Lipophilicity should not be the major consideration in designing HL91-derivatives for hypoxia imaging.