| Objecctives: To evaluate the feasibility of 99Tcm direct labelling of Annexin V recombinant with ten consecutive histidines(His10-Annexin V) and observe its biodistribution and pharmacokinetics in normal mice. To evaluate the feasibility of 99Tcm-His10-Annexin V for single photon emission computed tomography(SPECT) detection of apoptosis in non-small cell lung cancer(NSCLC) after paclitaxel inducement and determinate the ideal apoptosis imaging time. To establish a non-invasion in vivo apoptosis imaging for real-time monitoring the apoptosis level of tumor cell after chemotherapy. It is very helpful that to predict and estimate the effect of tumor therapy early and individualize the treatment plan.Methods: 99Tcm-Annexin V derivate(99Tcm-His10-Annexin V) was prepared with direct-labelling method and the radiochemical purity(RCP) and colloid content were measured with paper chromatography(PC) method. The biodistribution in normal mice was studied and pharmacokinetics parameters were determined with DAS2.0 soft ware. Nude mice bearing H460 non-small cell lung cancer were established and assigned to 4 groups of 5 mice each, one group was no treatment as control, others were groups which received chemotherapy including 24h, 48h, 72h after treatment with a dose of 100mg equivalent paclitaxel/kg were injected through tail veins. 18.5MBq 99mTc-Annexin V were injected via tail vein of nude mice and planar static images at 2h, 4h, 6h after injection were acquired using dual head SPECT equipped with pinhole collimator. Region of interest(ROI) were drawn in tumor area and contralateral normal tissue at the 6h image and the ratio of T/NT(activities of tumor to non- tumor) were calculated. Imaing of 99Tcm-IgG was used as control to determine the non-specific accumulation in tumor. At the end of imaging session, the mice were killed and tumors were removed for measuring radioactivity, the tumor uptake of 99Tcm-His10-Annexin V were calculated, histopathologic analysis and TUNEL assay were performed for apoptotic index and flow cytometry analysis were also performed for analyzing activated caspase-3. The software of SPSS 12.0 were utilized for data analysis. Statistical comparisons between untreated control group and induced groups were calculated using one-way Anova. The Pearson correlation coefficient was used to evaluate correlation between their T/NT, tumor uptake of 99mTc-His10-Annexin V, activated caspase-3 and apoptotie index respectively.Results: The radiochemical purity(RCP) of the labeling product was (98.01±1.67)％, the colloid content was (3.31±1.37)％, and the radiochemicai purity of (95.45±1.34)％was remained at 3h after preparation. The biodistribution showed high uptake of the tracer in kidneys but low uptake in liver and spleen. The pharmacokinetics indicates the feature of two-component model with the distribution phase half-life (T1/2a) of (21.18±5.95)min and the elimination phase half-life (T1/2β) of (69.32±0.10)min. Focal uptakes of 99Tcm-His10-Annexin V were visualized in tumor position at 2h after injection. It was notable that the uptake of the radiotracer in the tumor region gradually increased with the time after injection. The values of T/NT at 6h in chemotherapy groups were 2.63±0.76, 3.41±0.90 and 3.85±0.62, respectively, much higher than that of control group(1.42±0.19). The difference between two groups was significant(P＜0.01). Tumor uptake in chemotherapy groups at 24h, 48h and 72h were, (2.55±0.73)％ID/g, (3.60±1.09)％ID/g and (3.73±0.97)％ID/g, respectively, control group was (1.09±0.18)％ID/g, paclitaxel induced groups were significant higher than untreated group(P＜0.01). Mice imaging of 99Tcm-IgG showed non-specific distribution, tumor uptake were relatively low in both induced group at 48h after chemotherapy[(1.19±0.12)％ID/g] and control group[(1.13±0.14)％ID/g], no significant difference between them(P＞0.05), and T/NT ratio in two groups were 1.53±0.26 and 1.31±0.12(P=0.132＞0.05). Quantitative analysis of activated caspase-3 revealed that the activity increased significantly after chemotherapy. The percentage of activated caspase-3 of control group was (3.70±0.74)％, and that were (23.46％2.23)％, (62.85±6.13)％and (70.44±6.09)％in induced groups at 24h, 48h, 72h after chemotherapy. The differences between two groups were significant(P＜0.01). More importantly, there were significant positive correlation betweem their T/NT(r=0.847, P＜0.01) and tumor uptake of 99mTc-His10-Annexin V(r=0.774, P＜0.01). Histological analysis of tumor with HE stained and TUNEL assay revealed that paclitaxel treatment significantly increased the percentage of apoptotic cells. The appearance of apoptotic bodies were seldom in untreated group and the apoptotic index was(3.31±0.61)%, and was elevated to (32.90±6.64)%,(70.42±7.54)%, (83.23±9.71)% at 24h, 48h, and 72h after paclitaxel inducement, respectively. The differences between two groups were significant(P＜0.01). Significant correlation were found between apoptotic index with T/NT(r=0.833, P＜0.01) and tumor uptake of 99mTc-His10-Annexin V (r=0.850, P＜0.01).Conclusions: His10-Annexin V is easy to be labeled, and has good stability in vivo and in vitro. The biodistribution showed its high uptake in kidney but lower uptake in liver and spleen. 99Tcm-His10-Annexin V may be a promising agent for apoptosis imaging. The ideal apoptosis imaging time of NSCLC is 48-72h after paclitaxel inducement, and focal uptakes of 99Tcm-His10-Annexin V were visualized in tumor position at 2h after injection. In vivo detection of apoptosis in NSCLC after paclitaxel inducement was feasible, T/NT and tumor uptake of 99mTc-His10-Annexin V were correlated with TUNEL positive cells and activated caspase-3. The His10-Annexin V holds promise as a molecular probe to early predict cancer treatment efficacy.
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