Activation time-course and in vivo pharmacological analysis of native BoNT/E

Abstract

In the present study, a detailed activation time-course was examined for single chain native BoNT/E, enzymatically nicked in solution with trypsin. Through SDS-PAGE and Western blot analysis, the optimal nicking time was determined to be approximately 30 min. At 30 min, maximal di-chain BoNT/E and minimal secondary nicking (degradation) products were observed. The identity and integrity of the resulting light chain and heavy chain products were confirmed by N-terminal sequence analysis. Trypsin-activated BoNT/E samples covering a broad time-course were subsequently evaluated for potency and efficacy in pharmacological (DAS) and toxicological (im-LD50, ip-LD50) mouse models. In all three models, sample potencies varied with the degree of BoNT/E activation. In the DAS model, compared to single-chain BoNT/E, all samples displayed improved maximal efficacy and potency, except for 96 h-treated samples, which displayed lower potency. Both im- and ip-LD50 studies displayed similar dose–response profiles across the time-course. All three models yielded maximal potencies with samples activated for 30–100 min. These data demonstrate that purified, native BoNT/E can be activated in a controlled and reproducible manner with trypsin to near completion prior to the formation of any significant amounts of degradation products, with concomitant maximal in vivo potency.