Investigation of Tc-99m-labelling of recombinant human interleukin-2 via hydrazinonicotinamide

Introduction: Interleukin-2 (IL-2) when radiolabelled with Tc-99m has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of Tc-99m-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation.Methods: Various molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The Tc-99m-labelling was optimized using various amounts of HYNIC rhIL-2, Tc-99m, SnCl2, tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats.Results: Generally, the highest radiolabelling yields were achieved when the HYNIC rhIL-2 conjugates of ca. 2-4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of Tc-99m-HYNIC rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabel ling, the Tc-99m-HYNIC rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield lea. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 mu g HYNIC rhIL-2, coligands, buffer and antioxidant; the second vial contained tricine and SnCl2. The monomer of Tc-99m-HYNIC rhIL-2 was obtained by gel chromatography on a PD-ID column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed.Conclusions: Our study shows that rhIL-2 can be efficiently radiolabelled with Tc-99m via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use Tc-99m-HYNIC(tricine,NA)-rhIL-2 within 1 h. (C) 2010 Elsevier Inc. All rights reserved.