Development and evaluation of a novel 99mTc-labeled annexin A5 for early detection of response to chemotherapy

金沢大学疾患モデル総合研究センター99mTc-HYNIC-annexin A5 can be considered as a benchmark in the field of apoptosis imaging. However, 99mTc-HYNIC-annexin A5 has characteristics of high uptake and long retention in non-target tissues such as kidney and liver. To minimize this problem, we developed a novel 99mTc-labeled annexin A5 using a bis(hydroxamamide) derivative [C3(BHam)2] as a bifunctional chelating agent, and evaluated its usefulness as an imaging agent for detecting apoptosis. The amino group of C3(BHam)2 was converted to a maleimide group, and was coupled to thiol groups of annexin A5 pretreated with 2-iminothiolane. 99mTc labeling was performed by a ligand exchange reaction with 99mTc-glucoheptonate. Biodistribution experiments for both 99mTc-C3(BHam)2-annexin A5 and 99mTc-HYNIC-annexin A5 were performed in normal mice. In addition, in tumor-bearing mice, the relationship between the therapeutic effects of chemotherapy (5-FU) and the tumor accumulation of 99mTc-C 3(BHam)2-annexin A5 just after the first treatment of 5-FU was evaluated. 99mTc-C3(BHam)2-annexin A5 was prepared with a radiochemical purity of over 95%. In biodistribution experiments, 99mTc-C3(BHam)2-annexin A5 had a much lower kidney accumulation of radioactivity than 99mTc-HYNIC- annexin A5. In the organs for metabolism, such as liver and kidney, radioactivity after the injection of 99mTc-HYNIC-annexin A5 was residual for a long time. On the other hand, radioactivity after the injection of 99mTc-C3(BHam)2-annexin A5 gradually decreased. In therapeutic experiments, tumor growth in the mice treated with 5-FU was significantly inhibited. Accumulation of 99mTc-C 3(BHam)2-annexin A5 in tumors significantly increased after 5-FU treatment. The accumulation of radioactivity in tumor correlated positively with the counts of TUNEL-positive cells. These findings suggest that 99mTc-C3(BHam)2-annexin A5 may contribute to the efficient detection of apoptotic tumor response after chemotherapy. © 2013 Ogawa et al.CC-BY 4.

Development and Evaluation of a Novel 99mTc-Labeled Annexin A5 for Early Detection of Response to Chemotherapy

99mTc-HYNIC-annexin A5 can be considered as a benchmark in the field of apoptosis imaging. However, 99mTc-HYNIC-annexin A5 has characteristics of high uptake and long retention in non-target tissues such as kidney and liver. To minimize this problem, we developed a novel 99mTc-labeled annexin A5 using a bis(hydroxamamide) derivative [C3(BHam)2] as a bifunctional chelating agent, and evaluated its usefulness as an imaging agent for detecting apoptosis. The amino group of C3(BHam)2 was converted to a maleimide group, and was coupled to thiol groups of annexin A5 pretreated with 2-iminothiolane. 99mTc labeling was performed by a ligand exchange reaction with 99mTc-glucoheptonate. Biodistribution experiments for both 99mTc-C3(BHam)2-annexin A5 and 99mTc-HYNIC-annexin A5 were performed in normal mice. In addition, in tumor-bearing mice, the relationship between the therapeutic effects of chemotherapy (5-FU) and the tumor accumulation of 99mTc-C 3(BHam)2-annexin A5 just after the first treatment of 5-FU was evaluated. 99mTc-C3(BHam)2-annexin A5 was prepared with a radiochemical purity of over 95%. In biodistribution experiments, 99mTc-C3(BHam)2-annexin A5 had a much lower kidney accumulation of radioactivity than 99mTc-HYNIC- annexin A5. In the organs for metabolism, such as liver and kidney, radioactivity after the injection of 99mTc-HYNIC-annexin A5 was residual for a long time. On the other hand, radioactivity after the injection of 99mTc-C3(BHam)2-annexin A5 gradually decreased. In therapeutic experiments, tumor growth in the mice treated with 5-FU was significantly inhibited. Accumulation of 99mTc-C 3(BHam)2-annexin A5 in tumors significantly increased after 5-FU treatment. The accumulation of radioactivity in tumor correlated positively with the counts of TUNEL-positive cells. These findings suggest that 99mTc-C3(BHam)2-annexin A5 may contribute to the efficient detection of apoptotic tumor response after chemotherapy

The use of technetium tc 99m annexin V for in vivo imaging of apoptosis during cardiac allograft rejection

AbstractObjective: Apoptosis, or programmed cell death, has been suggested as a mechanism of immunologic injury during cardiac allograft rejection. We tested the hypothesis that technetium Tc 99m annexin V, a novel radiopharmaceutical used to detect apoptosis, can be used to detect cardiac allograft rejection by nuclear imaging. Methods: Untreated ACI rats served as recipients of allogeneic PVG rat (n = 66) or syngeneic ACI rat (n = 30) cardiac grafts. Untreated recipient animals underwent 99mTc-annexin V imaging daily for 7 days. Region of interest analysis was used to quantify the uptake of 99mTc-annexin V. Immediately after imaging grafts were procured for histopathologic analysis and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate–biotin nick-end labeling of apoptotic nuclei. One group was treated with 10 mg/kg/d cyclosporine (INN: ciclosporin) commencing on day 4 after transplantation (n = 6). Results: Untreated allografts showed histologic signs of rejection 4 days after transplantation. Apoptotic nuclei could be demonstrated in myocytes, endothelial cells, and graft-infiltrating cells of all rejecting allografts. Nuclear imaging revealed a significantly greater uptake of 99mTc-annexin V in rejecting allogeneic grafts than in syngeneic grafts on day 4 (P = .05), day 5 (P < .001), day 6 (P < .001), and day 7 (P = .013) after transplantation. A correlation between the histologic grade of acute rejection and uptake of 99mTc-annexin V was observed (r2 = 0.87). After treatment of rejection with cyclosporine, no apoptotic nuclei could be identified in allografts and uptake of 99mTc-annexin V decreased to baseline. Conclusions: Apoptosis occurs during acute cardiac allograft rejection and disappears after treatment of rejection. 99mTc-annexin V can be used to detect and monitor cardiac allograft rejection. (J Thorac Cardiovasc Surg 1998;116:844-53

Correlation between TUNEL-positive cells and radioacitvity in tumor section.

<p>Correlation between the number of TUNEL-positive cells in each grid (0.45 mm×0.55 mm) of a tumoral section and ^99mTc-C₃(BHam)₂-annexin A5 accumulation (%dose) determined by autoradiography in each corresponding grid of an adjacent section from mice treated with 5-FU (A) or non-treatment mice (B).</p

Tumor grouth curves.

<p>Curves depicting inhibition of the growth of colon-26 in treatment with 150 mg/kg of 5-FU (closed circles) or 100 mg/kg of 5-FU (closed diamonds) compared with untreated control group (open circles). Data are expressed as tumor volume relative to that on the day of treatment (mean ± SEM for 5 mice). Significance was determined using one-way ANOVA followed by Dunnett's post hoc test (**<i>p</i><0.01, *<i>p</i><0.05 vs. control group).</p

Autoradiography.

<p>Representative autoradiographic images (A, D) and TUNEL-staining images (B, C, E, F) for adjacent tumor sections from mice treated with 5-FU (A, B, C) or non-treatment mice (D, E, F). Scale bar  = 1 mm.</p

TUNEL-stained images.

<p>Representative TUNEL-stained images of tumor specimen in control mouse (A), 100 mg/kg of 5-FU-treated mouse (B), and 150 mg/kg of 5-FU-treated mouse (C). Scale bar  = 100 µm.</p

Tumore uptake.

<p>Comparison of tumor uptake of (A) ^99mTc-C₃(BHam)₂-annexin A5 (mean ± SD for 4-6 mice) and (B) ^99mTc-HYNIC-annexin A5 (mean ± SD for 6 mice) at 4 h after injection after 5-FU treatment or non-treatment. In the case of ^99mTc-C₃(BHam)₂-annexin A5, significance was determined using one-way ANOVA followed by Dunnett's post hoc test (**<i>p</i><0.01, *<i>p</i><0.05 vs. control group). In the case of ^99mTc-HYNIC-annexin A5, significance was determined using Students' t test (*<i>p</i><0.05 vs. control group).</p