Engineering an Effective Human SNAP-23 Cleaving Botulinum Neurotoxin A Variant.

Botulinum neurotoxin (BoNT) serotype A inhibits neurotransmitter release by cleaving SNAP-25 and represents an established pharmaceutical for treating medical conditions caused by hyperactivity of cholinergic nerves. Oversecretion from non-neuronal cells is often also the cause of diseases. Notably, excessive release of inflammatory messengers is thought to contribute to diseases such as chronic obstructive pulmonary disease, asthma, diabetes etc. The expansion of its application to these medical conditions is prevented because the major non-neuronal SNAP-25 isoform responsible for exocytosis, SNAP-23, is, in humans, virtually resistant to BoNT/A. Based on previous structural data and mutagenesis studies of SNAP-23 we optimized substrate binding pockets of the enzymatic domain for interaction with SNAP-23. Systematic mutagenesis and rational design yielded the mutations E148Y, K166F, S254A, and G305D, each of which individually increased the activity of LC/A against SNAP-23 between 3- to 23-fold. The assembled quadruple mutant showed approximately 2000-fold increased catalytic activity against human SNAP-23 in in vitro cleavage assays. A comparable increase in activity was recorded for the full-length BoNT/A quadruple mutant tested in cultivated primary neurons transduced with a fluorescently tagged-SNAP-23 encoding gene. Equipped with a suitable targeting domain this quadruple mutant promises to complete successfully tests in cells of the immune system.

Synthesis and structure-activity relationships of second-generation hydroxamate botulinum neurotoxin A protease inhibitors.

Botulinum neurotoxins are the most toxic proteins currently known. Based on a recently identified potent lead structure, 2,4-dichlorocinnamic acid hydroxamate, herein we report on the structure-activity relationship of a series of hydroxamate BoNT/A inhibitors. Among them, 2-bromo-4-chlorocinnamic acid hydroxamate, 2-methyl-4-chlorocinnamic acid hydroxamate, and 2-trifluoromethyl-4-chlorocinnamic acid hydroxamate displayed comparable inhibitory activity to that of the lead structure.

Antibodies and T cells against synthetic peptides of the C-terminal domain (Hc) of botulinum neurotoxin type A and their cross-reaction with Hc.

Seventeen peptides containing T cell and/or antibody (Ab) epitopes previously localized on Hc of botulinum neurotoxin type A were used in SJL and BALB/c mice as immunogens either individually or as an equimolar mixture of groups that contained epitopes of T cells, Abs or both, to determine their abilities to generate T cells and/or Abs that recognize intact Hc. In SJL, peptide 897-915 which included both T cell and Ab epitopes, elicited Abs that cross-reacted very strongly with Hc. In BALB/c, peptides 869-887, 883-901, 981-999 and 1275-1296 which contained Ab epitopes generated Abs that cross-reacted strongly with Hc. A mixture of peptides that contained T cell and Ab epitopes was effective in both strains in eliciting T cells and Abs that cross-reacted with Hc. This mixture form gave a quicker rise (after two injections) in cross-reactive (with Hc) Ab titer as compared to other peptide mixtures or the individual peptides, and sustained in BALB/c a high Ab titer upon further booster injections. Some of the regions that elicited crossreactive immunity to Hc have sequence similarity to other clostridial toxins, suggesting that one or more of these synthetic peptides might provide cross-protection against those toxins.

A randomized, double-masked, crossover comparison of the efficacy and safety of botulinum toxin type A produced from the original bulk toxin source and current bulk toxin source for the treatment of cervical dystonia.

In 1997, the US FDA approved a new bulk toxin source (now referred to as current) for the manufacture of botulinum toxin type A (BTX-A). The current BTX-A preparation has a lower neurotoxin complex protein load than the original BTX-A preparation, which may reduce antigenic potential. The present double-masked, multicenter study compared the efficacy and safety of BTX-A (BOTOX) produced from both original and current bulk toxin sources for the treatment of cervical dystonia. Patients (N = 133) were injected with BTX-A produced from original and current bulk toxin sources using a crossover design. Adverse events were assessed at each visit. Efficacy was assessed at 2 and 6 weeks post-injection using the severity and pain-disability subscales of the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS). Mean BTX-A doses were comparable (original: 155 U, current: 156 U). Both BTX-A preparations produced similar, statistically significant reductions in TWSTRS severity and pain-disability scores at weeks 2 and 6 post-injection. The original and current BTX-A preparations showed no significant differences in adverse events, including both treatment-related (34%, 31%) and treatment-unrelated (27%, 32%), respectively. BTX-A produced from the original and current bulk toxin sources showed comparable efficacy and safety in the treatment of cervical dystonia; both significantly reduced dystonia severity and pain.

Identifying and modulating disulfide formation in the biopharmaceutical production of a recombinant protein vaccine candidate.

Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.

Efficacy and safety of a new Botulinum Toxin Type A free of complexing proteins in the treatment of blepharospasm.

NT 201 is a new development of Botulinum Toxin Type A free of complexing proteins. In this double-blind Phase III trial, we compared the efficacy and safety of NT 201 and BOTOX in patients suffering from blepharospasm. Of 304 enrolled patients, 300 patients received study medication (intent-to-treat population), and 256 patients completed the study as planned (per-protocol population). At baseline, patients received a single injection of NT 201 or BOTOX (