Pre-Clinical Study of a Novel Recombinant Botulinum Neurotoxin Derivative Engineered for Improved Safety.

Edwin Vazquez-Cintron,Philip Band,Aurelia Syngkon,Christopher Angeles,Victoria Liublinska,Konstantin Ichtchenko,Luis Tenezaca

doi:10.1038/srep30429

Abstract

Cyto-012 is a recombinant derivative of Botulinum neurotoxin Type A (BoNT/A). It primarily differs from wild type (wt) BoNT/A1 in that it incorporates two amino acid substitutions in the catalytic domain of the light chain (LC) metalloprotease (E224 > A and Y366 > A), designed to provide a safer clinical profile. Cyto-012 is specifically internalized into rat cortical and hippocampal neurons, and cleaves Synaptosomal-Associated Protein 25 (SNAP-25), the substrate of wt BoNT/A, but exhibits slower cleavage kinetics and therefore requires a higher absolute dose to exhibit pharmacologic activity. The pharmacodynamics of Cyto-012 and wt BoNT/A have similar onset and duration of action using the Digital Abduction Assay (DAS). Intramuscular LD50 values for Cyto-012 and wt BoNT/A respectively, were 0.63 ug (95% CI = 0.61, 0.66) and 6.22 pg (95% CI = 5.42, 7.02). ED50 values for Cyto-012 and wt BoNT/A were respectively, 0.030 ug (95% CI = 0.026, 0.034) and 0.592 pg (95% CI = 0.488, 0.696). The safety margin (intramuscular LD50/ED50 ratio) for Cyto-012 was found to be improved 2-fold relative to wt BoNT/A (p < 0.001). The DAS response to Cyto-012 was diminished when a second injection was administered 32 days after the first. These data suggest that the safety margin of BoNT/A can be improved by modulating their activity towards SNAP-25.

Highlights

Botulinum neurotoxins (BoNT) are widely used as neuromodulators to treat spastic disorders[1]
The light chain (LC) of Cyto-012 has two amino acid substitutions (E224 >A and Y366 >A), which have been previously shown to reduce the toxicity of recombinant BoNT (rBoNT) derivatives by 100,000-fold compared to wt BoNT/A9
This is the first report of a recombinant Botulinum neurotoxin Type A (BoNT/A) derivative that retains intramuscular pharmacologic activity in vivo, despite amino acid substitutions that reduce its systemic toxicity by approximately 100,000-fold

Summary

Introduction

Botulinum neurotoxins (BoNT) are widely used as neuromodulators to treat spastic disorders[1]. We provide proof-of-principle studies demonstrating that the pharmacologic activity of recombinant derivatives of BoNT can be modulated by molecular design to achieve an improved safety margin. At higher doses of both Cyto-012 and wt BoNT/A, mice exhibited muscle weakening as early as 2 hours post im injection.

Results

Conclusion