Optimizing labelling conditions of ²¹³Bi-DOTATATE for preclinical applications of peptide receptor targeted alpha...

²¹³Bismuth (²¹³Bi, T_1/2 = 45.6 min) is one of the most frequently used α-emitters in cancer research. High specific activity radioligands are required for peptide receptor radionuclide therapy. The use of generators containing less than 222 MBq ²²⁵Ac (actinium), due to limited availability and the high cost to produce large-scale ²²⁵Ac/²¹³Bi generators, might complicate in vitro and in vivo applications though.Here we present optimized labelling conditions of a DOTA-peptide with an ²²⁵Ac/²¹³Bi generator (< 222 MBq) for preclinical applications using DOTA-Tyr³-octreotate (DOTATATE), a somatostatin analogue. The following labelling conditions of DOTATATE with ²¹³Bi were investigated; peptide mass was varied from 1.7 to 7.0 nmol, concentration of TRIS buffer from 0.15 mol.L^-1 to 0.34 mol.L^-1, and ascorbic acid from 0 to 71 mmol.L^-1 in 800 μL. All reactions were performed at 95 °C for 5 min. After incubation, DTPA (50 nmol) was added to stop the labelling reaction. Besides optimizing the labelling conditions, incorporation yield was determined by ITLC-SG and radiochemical purity (RCP) was monitored by RP-HPLC up to 120 min after labelling. Dosimetry studies in the reaction vial were performed using Monte Carlo and in vitro clonogenic assay was performed with a rat pancreatic tumour cell line, CA20948.

At least 3.5 nmol DOTATATE was required to obtain incorporation ≥ 99 % with 100 MBq ²¹³Bi (at optimized pH conditions, pH 8.3 with 0.15 mol.L^-1 TRIS) in a reaction volume of 800 μL. The cumulative absorbed dose in the reaction vial was 230 Gy/100 MBq in 30 min. A minimal final concentration of 0.9 mmol.L^-1 ascorbic acid was required for ~100 MBq (t = 0) to minimize radiation damage of DOTATATE. The osmolarity was decreased to 0.45 Osmol/L.Under optimized labelling conditions, ²¹³Bi-DOTATATE remained stable up to 2 h after labelling, RCP was ≥ 85 %. In vitro showed a negative correlation between ascorbic acid concentration and cell survival.

²¹³Bismuth-DOTA-peptide labelling conditions including peptide amount, quencher and pH were optimized to meet the requirements needed for preclinical applications in peptide receptor radionuclide therapy.