Page 111 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Stabilized Sarabesin-3 for prostate imaging
Stability studies
To determine the stability of [111In]SB3 -/+ PA in vivo, healthy mice (n = 3 per group) were intravenously injected with 200 pmol/25 MBq or 2000 pmol/25 MBq [111In]SB3 (volume: 100 μL), co-injected with either 300 μg/100 μL PA (Peptides International Inc) in 0.5% ethanol or 100 μL saline solution. Five minutes after injection of [111In]SB3 -/+PA, blood (0.5 – 1 mL) was collected by cardiac puncture, while animals were under isoflurane/O2 anesthesia, and transferred to blood-collection tubes containing 90 USP units of lithium heparin (spray coated), HPLC grade ethanol was added in a 1:1 (v/v) ratio, and the tubes were placed on ice. Blood samples were centrifuged 5 minutes at 1250 G, and supernatant was collected. Next the collected supernatant was centrifuged again for 5 minutes at 12500 G. The secondary supernatant was collected and diluted with MilliQ down to <25% ethanol before injection on HPLC. The samples were analyzed by HPLC (Alliance, Waters) including a Symmetry C18 column (5 μm × 4.6 mm × 250 mm). Elution was performed in a 1 mL/min flow, completed in 30 min, with the gradient: 0–5 min 100% A, 5–5.01min 40% A 60% B, 5.01–20 min 20% A 80% B, 20–20.01min 100% B, 20.01–25min 100% B, 25.01–30 min 100% A, where A was 0.1% TFA in H2O and B was methanol. In one animal the sample volume was too low for further analysis, this animal was therefore excluded.
Biodistribution studies
Tumor-bearing mice received an intravenous injection of 200 pmol/2.5 MBq [111In]SB3, co-injected with either 300 μg/100 μL PA in 0.5% ethanol or 100 μL saline solution (n = 4 animals per group for each time point). In previous in vivo studies 200 pmol appeared to be the optimal peptide amount (regarding uptake in tumor and pancreas, data not shown). To determine specificity of tumor and organ uptake blocking experiments were performed; an additional group of animals (n = 3) was pre-injected with an excess (20 nmol) of unlabeled SB3 followed by injection of the radiotracer -/+ PA. At 3 different time points p.i. (1 h, 4 h and 24 h) animals were euthanized and blood, organs and tumors were collected, weighed and measured in a γ-counter (1480 WIZARD automatic γ-counter, Perkin Elmer). For γ-counter measurements an isotope specific energy window, a counting time of 60 sec and a counting error ≤ 5% was used. Results were expressed as the percentage of the injected dose per gram tissue (%ID/g tissue). Animals that had ≥ 7% ID in the tail were excluded.
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