Gi-Hyeok Yang

Organization and nucleotide sequence of genes for hemagglutinin components of Clostridium botulinum type B progenitor toxin

The genes for hemagglutinin components (33 kD, 17 kD, and 21.5 kD) of Clostridium botulinum type B progenitor toxin were cloned and sequenced. Analysis of the nucleotide sequence showed that the 33 kD, 17 kD, and 21.5 kD hemagglutinin genes were organized into an operon in the 5′upstream region of the toxin gene and their ORF orientation were opposite to that of the toxin gene. A comparison of amino acid sequences between the hemagglutinin components in type B and type C progenitor toxin showed significant homology. Northern blot analysis also revealed that all of the genes for the hemagglutinin components were transcribed as a polycistronic RNA.

CLONING AND CHARACTERIZATION OF THE UPSTREAM REGION OF CLOSTRIDIUM BOTULINUM TYPE B NEUROTOXIN GENE

The upstream region of the gene coding for Clostridium botulinum type B (BoNT/B) neurotoxin was cloned and sequenced. There were two open reading frames, which were identified as a nontoxic‐nonhemagglutinin component (ntnh/B) and a 22 kDa adjacent open reading frame (orf22/B). Deduced primary structure of ntnh/B showed that it was composed of 1,197 amino acid residues. Pairwise comparisons of the ntnh/B component with other botulinum toxin types showed high degree of homology to ntnh/A (82% identity). Northern blot analysis revealed that toxin gene could be transcribed alone or co‐transcribed with the ntnh gene. The orf22/B gene encoding for 178 amino acids (M.W. 21.6 kDa) was located between the 33 kDa hemagglutinin gene and the ntnh gene. Orf22/B also showed high degree of homology to orf22/A (98.9% identity). These results suggested that the upstream region of the BoNT/B gene (containing the ntnh/B and orf22/B genes) might be evolutionarily closely related to the counterparts of the BoNT/A.

A new botulinum toxin potentially bioequivalent to onabotulinumtoxinA: are there any differences at all?

Botulinum toxins refer to a group of proteins classified into seven distinct serotypes (A–G) produced by different strains of Clostridium botulinum, which is a naturally occurring endosporeforming strict anaerobic eubacteria. There are more than six commercially available botulinum toxin products: onabotulinumtoxinA (Botox, Allergan Inc., Irvine, CA), BTXA (Lanzhou Biological Products Institute, China), abobotulinumtoxinA (Dysport, Ipsen Ltd., Wrexham, UK), rimabotulinumtoxinB (Neurobloc, Solstice Neurosciences, Louisville, KY), Neuronox (neu-BoNT/A, Medytox Inc., Cheonwon-gun, Korea), and incobotulinumtoxinA (Xeomin, Merz Pharmaceuticals, Frankfurt am Main, Germany).

A Pharmacodynamic Comparison Study of Different Botulinum Toxin Type A Preparations

BACKGROUND Because more botulinum toxin (BoNT) preparations have become available worldwide, there is a clinical need to compare the pharmacologic profiles of these products. OBJECTIVE We compared three different preparations: onabotulinumtoxinA (ona‐BoNT/A), abobotulinumtoxinA (abo‐BoNT/A), and Neuronox (neu‐BoNT/A), in a mouse model using a digit abduction scoring (DAS) assay. METHODS The efficacy, duration of effect, and safety margin of each preparation was determined after delivering a single injection to the right gastrocnemius (0–240 U/kg body weight of neu‐BoNT/A or ona‐BoNT/A; 0–600 Speywood Units/kg body weight of abo‐BoNT/A). RESULTS Neu‐BoNT/A (intramuscular (IM) median effective dose (ED50) 11.2 ± 2.7 U/kg) and ona‐BoNT/A (IM ED50 11.9 ± 2.4 U/kg) had similar effects in terms of muscle weakness at significantly lower doses than abo‐BoNT/A (IM ED50 41.2 ± 2.4 U/kg; p < .001). The safety margin (ratio between IM ED50 and IM median lethal dose (LD50)) of neu‐BoNT/A (10.7 ± 2.6 U/kg) was also similar to that of ona‐BoNT/A (10.3 ± 1.3 U/kg) but significantly higher than that of abo‐BoNT/A (5.9 ± 0.4 U/kg; p < .02). Neu‐BoNT/A and ona‐BoNT/A also produced comparable patterns of DAS response and body weight recovery by day 29. CONCLUSION Neu‐BoNT/A and ona‐BoNT/A may be interchangeable based on a simple dose ratio.

Mouse compound muscle action potential assay: An alternative method to conduct the LD50 botulinum toxin type A potency test

PURPOSE We performed a prevalidation of the compound muscle action potential (CMAP) assay to determine the potency of botulinum neurotoxin type A (BoNT/A) with the aim of substituting for the mouse lethality test (LD₅₀), which is used for quality control. METHODS Prevalidation experiments were performed to demonstrate the specificity, linearity, accuracy, precision, range, limit of quantitation (LOQ), and robustness of the assay. For specificity, toxin detection ability was determined in the presence of neutralizing antibodies (0.8 and 8 IU/mL). Linearity of this assay was determined by measuring CMAP amplitude using nine concentrations (n = 3) in the range of 1-100 U/mL (n = 3). Accuracy was assessed using five concentrations (n = 3) in the range of 4-40 U/mL. Intermediate precision was confirmed by analyzing individually prepared reagents on multiple days by one operator (n = 3). Different body weights (23-25 and 25-27 g) and measurement times (3-5 and 5-7 min) after anesthetic induction were tested to assess robustness. RESULTS This assay might have BoNT/A specificity, based on the CMAP amplitude recovery using a concentration of neutralizing antibodies. The calibration curves were linear over the range of 2-40 U/mL (R² = 0.982). The accuracy of 14 determinations was within the range of 89.8-118.6% compared to the theoretical values among 15 determinations, except one (131.3%). Assay variability was acceptable with coefficients of variation of 4.3-14.4%. The range of quantification and the LOQ were 4-40 U/mL and 4 U/mL, respectively. Different body weights and measurement times after inducing anesthesia had no effect on CMAP amplitude. CONCLUSIONS These results suggest that the mouse CMAP assay is an alternative method to the standard LD₅₀ potency test and meets the requirement of the three Rs (particularly refinement and reduction).