Abstract
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Introduction: Phosphatidylethanolamine (PE) is a known binding target suitable for monitoring apoptosis in both physiological and pathological conditions. Imaging apoptosis using radiotracers in vivo has demonstrated its capability of predicting tumor response to treatment. Duramycin is a 19-amino acid peptide that can identify apoptotic cells by binding to PE. 99mTc-duramycin has been used successfully to monitor drug therapy in mice with colorectal cancer. Since different F-18 labeling methods could alter the in vivo kinetics of the parent compounds, our objective was to assess the effect of using three different 18F-labeling intermediates on the in vitro cell uptake of 18F-duramycin for imaging apoptosis.
Methods: 2,2′-(7-(4-isothiocyanatobenzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODA)-duramycin and 2,2′-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODAGA)-duramycin were labeled according to the 18F-Al method (18F-1 and 18F-2). Hydrazinonicotinic acid (HYNIC)-duramycin was labeled with 18F-fluorobenzaldehyde by hydrazine formation (18F-3). Colo205 cells were incubated with camptothecin (CPT) at four different concentrations (0, 0.05, 0.5, and 5 µM) for 48 hr, respectively. One µCi of each tracer was added to each well and incubated for 1 h at 37 oC. For blocking studies, 120 µg duramycin was added to each well 15 min prior to the addition of each tracer (n=4). After incubation of the tracer for 1 hr, cells were harvested, washed, and then counted for total radioactivity. Percentage uptake was calculated by counts in cells/total counts. The degree of cell apoptosis was measured by flow cytometry using a commercial annexin-V/PI kit.
Results: Based on flow cytometry measurements, the degree of cell apoptosis increased proportionally to CPT concentration:13.9±2.1, 37.2±3.5, 47.1±2.3, and 48.1±5.3% for 0, 0.05, 0.5, and 5 µM of CPT, respectively. Cell uptake increased in a dose-dependent manner over CPT concentration for all three tracers, but a correlation between cell uptake and degree of apoptosis for both 18F-1 and 18F-2 (R2=0.90 and 0.94, respectively) was slightly improved compared with that for 18F-3 (R2=0.87). The maximal uptake normalized to control was 3.9, 2.9 and 2.8 for 18F-1, 18F-2, and 18F-3, respectively. Cell uptake of the three tracers was reduced by 53, 56, and 39%, respectively after blocking by duramycin.
Conclusions: Our preliminary data showed that 18F-1 might be a preferred choice among 3 tracers tested due to having the highest normalized uptake, high specific uptake, and better differentiation in detecting apoptosis. Further assessment using in vivo imaging is required to confirm whether 18F-1 remains the preferred choice.