Elsevier

Toxicon

Volume 39, Issue 12, December 2001, Pages 1815-1820
Toxicon

A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice

https://doi.org/10.1016/S0041-0101(01)00101-5Get rights and content

Abstract

This study compared the respective intramuscular (IM) safety margins of two preparations of botulinum toxin (BTX) serotype A and one preparation each of BTX serotypes B and F in mice. Mice received an IM injection (0–200 U kg−1 body weight) of BTX-A (BOTOX® or DYSPORT®), an experimental preparation of BTX-B (WAKO Chemicals, Inc.), or an experimental preparation of BTX-F (WAKO). An observer who was masked to treatment scored muscle weakness using the Digit Abduction Scoring (DAS) assay. Peak DAS responses were plotted and IM ED50 values calculated. The safety margin for each BTX preparation was calculated as a ratio of the IM median lethal dose after hind limb injection to the median effective dose in the DAS assay (IM LD50/IM ED50). Experiments were repeated 4–6-times for each preparation (10 mice/dose). Mean safety margin values were highest for BTX-F (WAKO; 16.7±3.9) and one of the BTX-A preparations (BOTOX®; 13.9±1.7). Mean safety margins values for the other BTX-A preparation (DYSPORT®) and BTX-B (WAKO) were significantly lower (7.6±0.9 and 4.8±1.1, respectively). Thus, the BTX preparations exhibited different safety margins in mice. These results support the hypothesis that the preparations are unique therapeutics and are not interchangeable based on a simple dose ratio.

Introduction

Botulinum neurotoxins reduce muscular contractions by temporarily inhibiting acetylcholine release at the neuromuscular junction (Simpson, 1981). The muscle weakening properties of botulinum neurotoxin type A were first used for therapeutic purposes in the treatment of strabismus (Scott, 1980). Over the past several decades, worldwide use of botulinum neurotoxin type A (BTX-A) has expanded to dozens of indications, including the treatment of hemifacial spasm, cervical dystonia, and spasticity (Grazko et al., 1995, Jankovic and Orman, 1987, Jankovic et al., 1990).

Botulinum neurotoxin is synthesized in seven different serotypes (A, B, C1, D, E, F, G) by various strains of Clostridium botulinum. Although all serotypes inhibit acetylcholine release, they differ in neurotoxin complex size, activation level (‘nicking’), intracellular site of action, acceptor/receptor sites, muscle weakening efficacy, duration of action, and target affinity (Simpson, 1981, DasGupta, 1989, Black and Dolly, 1986, Hughes, 1991, Brin, 1997, Sloop et al., 1997). Indeed, many of these properties differ even between botulinum neurotoxin preparations containing the same serotype (McLellan et al., 1996).

For the past decade, the only commercially available botulinum neurotoxin preparations have been based on the A serotype. However, a preparation containing botulinum toxin type B has recently been approved for the treatment of cervical dystonia in the US and Europe. Several studies indicate that botulinum toxin type F may be useful for focal dystonias as well (Mezaki et al., 1995, Greene and Fahn, 1996).

It is likely that differences among botulinum neurotoxin preparations, both within and between serotypes, will lead to differences in clinical performance. In support of this, direct comparison of serotypes A and B in humans have shown that type A is more potent and has a longer duration than type B (Sloop et al., 1997). Studies have also shown that type A has a longer duration of action than type F (Mezaki et al., 1995). Even within the same serotype, the clinically effective unit doses differ between the two type A preparations (Quinn and Hallett, 1989).

It is possible that botulinum toxin preparations may also exhibit differences in safety, antigenicity, and specificity. However, the comparative properties of botulinum neurotoxin preparations have not been well studied. The present study was therefore designed to compare the safety margins of two preparations of botulinum toxin type A (BOTOX® and DYSPORT®), and one laboratory preparation each of types B and F, following intramuscular injections in mice. These serotypes were selected because they have been used clinically, and intramuscular injections were employed in order to mimic the clinical route of administration. Such preclinical studies may help identify factors that are important in the clinical setting and assist in the development of key research questions. Preliminary data from the DAS IM ED50 portion of this study have been previously reported (Aoki, 1999).

Section snippets

Experimental animals

Female, Swiss Webster mice (17–21 g) were used in all experiments. Mice were housed in groups of 10 and allowed ad libitum access to food and water. All experiments were conducted in accordance with the guidelines of the American Association for the Accreditation of Laboratory Animal Care (AAALAC).

Injection procedure

Each mouse received a single intramuscular injection of neurotoxin or vehicle into the head of the right gastrocnemius muscle. Injections were made in a fixed volume of 5 μl using a 30 gauge needle

DAS reliability

The correlation coefficient for the 944 observations made by the two raters was r=0.96. Of the 944 observations, 83% were given identical scores by the two raters, 17% were scored by a difference of 1, and 0.3% were scored by a difference of 2. The consistency and reproducibility of scoring is also supported by the low standard errors obtained in the comparative studies reported in this manuscript.

Muscle weakening (DAS)

All serotypes tested produced dose-related increases in the magnitude and duration of muscle

Discussion

The most important finding of the present study was that the botulinum toxin preparations tested showed significantly different safety margins in mice following intramuscular injection. Higher safety margins indicate greater separation between the IM effective and lethal doses. In the present study, BTX-F (WAKO) and one of the BTX-A preparations (BOTOX®) had the highest safety margins, which were approximately twice as high as the other BTX-A preparation (DYSPORT®) and 3-times as high as the

Acknowledgements

The author gratefully acknowledges the excellent technical assistance of Lan Do, Wei-Jen Lin, PhD; Garrett Merlino; Katie Peng; John Rubino; Alan Satorius and editorial assistance of Mary Ann Chapman, PhD.

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