The ultimate radiochemical nightmare: upon radio-iodination of Botulinum neurotoxin A, the introduced iodine atom itself seems to be fatal for the bioactivity of this macromolecule

Botulinum neurotoxin A (BoNT-A) is a highly neurotoxic drug and frequently used in patients. Knowledge on the optimal way of administration of BoNT-A and its subsequent distribution is still rather limited. An accurate method for monitoring these processes might be the use of radiolabelled BoNT-A. In this paper, we report our feasibility study on labelling BoNT-A with high-dose iodine-125 (¹²⁵I) via IODOGEN-coated BoNT-A method.

Using cetuximab as model substrate for BoNT-A, a miniaturization of the IODOGEN-coated mAb method was developed with special attention to the minimum required amount of the oxidant IODOGEN, while the amount of substrate, reaction volume and reaction time were downsized. Labelling efficiency and radiochemical purity were determined by TLC, integrity by SDS-PAGE and HPLC and immunoreactivity by cell-binding assay. BoNT-A (50 μg) was labelled with ¹²⁵I by coating with 2.5 μg IODOGEN, in a total reaction volume of 250 μL and a reaction time of 90 s. ¹²⁵I-BoNT-A was purified by size exclusion chromatography (PD10 column) using ascorbic acid solution (5 mg/ml, pH = 5) as eluent. Quality analysis of ¹²⁵I-BoNT-A was performed by an in vitro bladder strip model, an electrochemiluminescence assay and an Endopep assay.

Cetuximab (50 μg) labelling with ¹²⁵I (15 to 150 MBq) resulted in a labelling efficiency of 70% to 80%, a radiochemical purity of >99%, an immunoreactivity of >95% and a retained integrity on SDS; HPLC analysis revealed partly affected integrity when 110 to 150 MBq ¹²⁵I was used, i.e. when the averaged I/mAb molar ratio exceeded 3. Addition of HEPES (20 mM) and lactose (1.25%) (lyophilized BoNT-A contains HEPES and lactose) decreased the labelling efficiency to 44% to 54%. BoNT-A (50 μg) labelling with ¹²⁵I (97.2 to 98.3 MBq) resulted in labelling efficiency of 51% to 52% with a radiochemical purity >98.5%, a specific activity of 150.5 to 152.9 MBq/nmol and an I/BoNT-A molar ratio of 1.86 to 1.90. The in vitro bladder strip model showed no bioactivity of ¹²⁵I-BoNT-A when compared to unlabelled BoNT-A. The electrochemiluminescence and Endopep assay demonstrated around 10% and 15% bioactivity of ¹²⁵I-BoNT-A compared to unlabelled BoNT-A, respectively. The remaining bioactivity correlates within the Poisson distribution with the amount of BoNT-A molecules that does not bear an iodine atom.