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

Abstract

Background 213Bismuth (213Bi, T1/2 = 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.ResultsAt 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.Conclusion 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s41181-016-0014-4) contains supplementary material, which is available to authorized users.

Improved safety and efficacy of 213Bi-DOTATATE-targeted alpha therapy of somatostatin receptor-expressing neuroendocrine tumors in mice pre-treated with l-lysine

“…The resultant elution containing 213 Bi (630–740 MBq) was used for labeling with 10 μg DOTATATE (BioSynthema) in a reaction vial including 0.15 M TRIS buffer and 2.6 mM ascorbic acid at pH 8.4. The reaction was incubated for 5 min at 95 °C and cooled to ambient temperature for 2 min before adding 50 mM DTPA [17]. Instant thin-layer chromatography (ITLC-SG, Varian) was performed using 0.9% NaCl as mobile phase to determine the radionuclide-peptide incorporation yield.…”

From octreotide to shorter analogues: Synthesis, radiolabeling, stability

“…19 The absence of same tendency for Bi 3+ might be a consequence of the higher stability constant of the Bi-DOTA complex in comparison with Sc-DOTA (logK Bi-DOTA = 30, logK Sc-DOTA = 27). 24,25 Furthermore, we observed the formation of intermediate species M-H x DOTA-P, which was demonstrated by autoradiograms of Bi-DOTA-P1 ( Figure 6) and Eu-DOTA-P1 ( Figure S4). The Bi-H x DOTA-P species immediately forms in the first minutes of the reaction (R f = 0.2-0.4) ( Figure 6).…”

“…It is shown that at pH 2.3 to 2.6, the labeling yield of DOTA‐P1 is at most 17% for Bi 3+ and 11% for Eu 3+ (Figure 5B,C). For Bi 3+ and Eu 3+ , an increase in pH raises the maximum yield of labeling; at higher pH, faster binding of the cation occurs (Figure 5B,C) …”

“…From this point of view, Sc 3+ is intermediate between Bi 3+ (highly hydrolysable cation, logβ BiOH = −1.1) and Eu 3+ (much less hydrolysable cation among studied) . The absence of same tendency for Bi 3+ might be a consequence of the higher stability constant of the Bi‐DOTA complex in comparison with Sc‐DOTA (logK Bi‐DOTA = 30, logK Sc‐DOTA = 27) …”

“…For Bi 3+ and Eu 3+ , an increase in pH raises the Figure 5B,C). 24 Interestingly, for scandium, the highest reaction yield was obtained at pH 4.0 to 5.0 ( Figure 5A). An increase in pH up to six causes a fast growth of the labeling yield at the beginning of the reaction due to binding by partly deprotonated DOTA, and then the fraction of bound cation decreases.…”