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

Abstract

Background: 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.

Results: 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.

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.

Keywords: 213Bismuth; Absorbed dose; Ascorbic acid; Radiochemical purity; Somatostatin; Targeted alpha therapy.

Fig. 1

MCNP geometry input for 1 mL reaction vial with 0.8 mL radioactive fluid (in blue), water with density 1 g/mL. The vial wall (in grey) had a thickness of 0.65 mm and was polyethylene with density 0.9 g/mL

Fig. 2

The incorporation (%) of ²¹³Bi-DOTATATE 71 ± 28 MBq determined by ITLC analysis as function of peptide amount in a reaction volume of 800 μL. An S-shaped cureve has been fitted through the data with an incorporation of 50 % at 2.4 ± 0.09 nmol and a slope of 0.25 ± 0.03 nmol peptide, n ≥ 3

Fig. 3

HPLC chromatogram of ²¹³Bi-DOTATATE in presence a and absence b of ascorbic acid during labelling (n ≥ 3). Peak A; RT = 13.0 min and peak B; RT = 16.3 min. The y-axis represents the detected radioactive signals (mV) and x-axis represents the RT in minutes

Fig. 4

Stability as function of time of ²¹³Bi-DOTATATE with different concentration ascorbic acid added during labelling, y-axis is RCP expressed in mean percentage ± SD and x-axis time in minutes after labelling (n ≥ 3)

Fig. 5

Time-integrated activity of ²¹³Bi, ²¹³Po, ²⁰⁹Tl and ²⁰⁹Pb determined by Bateman equations a and the resultant total absorbed dose rate b as a function of time, see Table 2. The majority of the absorbed dose rate (96 % at 60 min) is caused by the α-particles from ²¹³Bi and ²¹³Po

Fig. 6

Survival (%) ± SD of CA20948 after exposure to the compounds used in standard labelling procedure, n = 3. Concentration in incubation medium was; [TRIS] = 16 mmol.L⁻¹, [AA] = ascorbic acid, 3.4 mmol.L⁻¹, [NaI] = 3.6 mmol.L⁻¹, [HCl] = 3.6 mmol.L⁻¹ and [DTPA] = 6.1 × 10⁻³ mmol.L⁻¹

Fig. 7

Survival (%) ± SD of CA20948 as function of concentration of ascorbic acid in DMEM incubation medium containing 30 mmol.L^-1 HEPES and 0.25 % BSA (total volume was 8.4 mL), n = 3