A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice
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.
References (25)
The structure of botulinum neurotoxin
- et al.
Ocular involvement in benign botulism B
Am. J. Ophthalmol.
(1975) - et al.
Therapeutic botulinum type A toxin: factors affecting potency
Toxicon
(1996) - et al.
The median paralysis unit: a more pharmacologically relevant unit of biologic activity for botulinum toxin
Toxicon
(1995) - et al.
Dose standardisation of botulinum toxin
Lancet
(1989) - et al.
Double-blind study of botulinum toxin in spasmodic torticollis
Lancet
(1986) Preclinical update on BOTOX® (botulinum toxin type A)-purified neurotoxin complex relative to other botulinum neurotoxin preparations
Eur. J. Neurol.
(1999)- et al.
Interaction of 125-I labelled botulinum neurotoxins with nerve terminals— I. Ultrastructural autoradiographic localization and quantitation of distinct membrance acceptors for types A and B on motor nerves
J. Cell Biol.
(1986) Botulinum toxin: chemistry, pharmacology, toxicity, and immunology
Muscle & Nerve
(1997)- et al.
Safety and efficacy of NeuroBloc (botulinum toxin type B) in type A-resistant cervical dystonia
Neurology
(1999)