Hsia Chien-Chung, Huang Fu-Lei, Lin Cheng-Hsien, Shen Lie-Hang, Wang Hsin-Ell
Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taiwan.
Appl Radiat Isot. 2010 Sep;68(9):1610-5. doi: 10.1016/j.apradiso.2010.03.008. Epub 2010 Mar 15.
(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.
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.
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.
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.
（99m）Tc-HL91（Prognox，通用电气医疗集团）是首个用于评估肿瘤、中风和心肌梗死区域缺氧分数的非硝基芳基放射性示踪剂。然而，（99m）Tc-HL91的高亲水性可能会阻碍其进入细胞。在本研究中，我们制备了一种新的配体4,4,11,11-四甲基-5,10-二氮杂十四烷-3,12-二酮二肟（HL91-ET），其亲脂性更高，但结构与HL91相似。使用中风小鼠模型对（99m）Tc-HL91-ET作为缺氧闪烁显像探针进行了化学和生物学特性研究。
合成HL91-ET，并与氯化亚锡和缓冲液配制成试剂盒。与高锝酸盐（99m）Tc混合后，可高产率、高放射化学纯度（均>96%）制备（99m）Tc-HL91-ET。在正辛醇/磷酸盐缓冲液（PBS）体系中测定（99m）Tc-HL91-ET的分配系数。在氧气控制的二氧化碳培养箱中进行常氧和缺氧条件下的细胞摄取试验。通过电灼大脑中动脉诱导小鼠模型发生脑中风。将（99m）Tc-HL91-ET静脉注射到患有脑中风的Balb/c小鼠体内后，在指定时间点采集小动物单光子发射计算机断层扫描（SPECT）图像，并对脑切片进行放射自显影。在相同条件下对（99m）Tc-HL91进行平行研究以作比较。
（99m）Tc-HL91-ET的分配系数较高（0.294±0.007），表明其亲脂性高于（99m）Tc-HL91（0.089±0.005）。（99m）Tc-HL91-ET制剂在室温下24小时稳定。细胞摄取试验表明，（99m）Tc-HL91-ET在缺氧细胞中的选择性保留低于（99m）Tc-HL91。注射（99m）Tc-HL91-ET和（99m）Tc-HL91后2小时，中风小鼠脑放射自显影片得出的靶脑与正常脑比值分别为1.31±0.02和17.47±0.10（n = 3）。
本研究表明，（99m）Tc-HL91-ET虽然亲脂性高于（99m）Tc-HL91，但在缺氧细胞或组织中的特异性积聚并不比（99m）Tc-HL91更好。（99m）Tc-HL91的摄取机制至少不完全是被动扩散。在设计用于缺氧成像的HL91衍生物时，亲脂性不应是主要考虑因素。
