Botulinum neurotoxin serotypes A and C do not affect motor units survival in humans: an electrophysiological study by motor units counting Roberto Eleopra a, * , Valeria Tugnoli a , Rocco Quatrale a , Ernesto Gastaldo a , Ornella Rossetto b , Domenico De Grandis c , Cesare Montecucco b a Department of Clinical Neuroscience, Neurology Section, S.Anna University Hospital, corso Giovecca 203, 44100 Ferrara, Italy b Biomedical Science Department, University of Padua, Padua, Italy c Department of Clinical Neuroscience, Neurology Section, Hospital of Rovigo, Rovigo, Italy Accepted 21 March 2002 Abstract Objectives: Botulinum neurotoxin serotype A (BoNT/A) is a valid therapy for dystonia but repeated BoNT/A injections may induce a clinical immuno-resistance that could be overcome by using other BoNT serotypes. In vitro experiments and our preliminary investigations in vivo, indicate that botulinum neurotoxin serotype C (BoNT/C) could be an effective alternative to BoNT/A. Moreover, in cultured neurons ‘in vitro’ BoNT/C has been reported to be more toxic than BoNT/A. Methods: To verify this possibility, we compare the effect of BoNT/C and BoNT/A on the motor units count in humans by using the electrophysiological motor unit number estimation (MUNE) technique (‘multiple point nerve stimulation’). Preliminarily, BoNT/C and BoNT/A dosage was calibrated in a mouse hemidiaphragm neuromuscular junction preparation. Subsequently, 8 volunteers were treated with 3 IU of BoNT/C in the extensor digitorum brevis muscle of one foot and 3 IU of BoNT/A in the contralateral one. Other 4 subjects were similarly injected at higher doses (10 IU of BoNT/C or BoNT/A) to detect a possible dose-toxic effect. Results: In both groups, no statistically significant variations in MUNE counting or single motor unit potential size were detected after 4 months from injections, when it was evident a recovery from the BoNTs blockade. Conclusions: We conclude that BoNT/C, similarly to BoNT/A, is safe and effective in humans and it could be proposed for a clinical use. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Botulinum toxin C; Botulinum toxin A; Electrophysiology; Motor unit number estimation; Dystonia 1. Introduction Seven different serotypes of botulinum neurotoxins (BoNTs), denoted by letters from A to G, are the sole responsible of botulism (Humeau et al., 2000) following their persistent inhibition of cholinergic fibers of the periph- eral nervous system (Cherington, 1998; Schiavo et al., 2000). BoNTs act in the neuronal cytosol by preventing the fusion of synaptic vesicles with the presynaptic membrane and therefore the release of acetylcholine. In humans, the injections of BoNT/A in muscles of subjects affected by focal dystonia is now considered a valid and useful therapy (Blackie and Lees, 1990; Greene et al., 1990; Elston, 1990, 1992; Grandas et al., 1991; Lees et al., 1992; Poewe et al., 1992; Jankovic and Hallett, 1994). Selected neuromuscular junctions are partially inhibited for many weeks, and this is followed by a progressive recovery of the muscular functions. Repeated injections, particularly when high doses are required, may lead to immunization with production of protective antibodies (Borodic et al., 1996). Indeed, when a BoNT/A specific immune response is proven as the cause of resistance to BoNT/A (Zuber et al., 1993; Jankovic and Schwartz, 1995; Brin, 1997; Hanna and Jankovic, 1998; Sankhla et al., 1998), the injection of a different BoNT serotype could overcome the problem posed by patients not responding to BoNT/A. BoNT/F was tested in humans but, its beneficial effects are of rather short time duration with respect to BoNT/A (Mezaki et al., 1995; Chen et al., 1998), whereas BoNT/B appears to have a similar clinical action only if injected at very high doses (Lew et al., 1997; Sloop et al., 1997; Brashear et al., 1999; Brin et al., 1999). BoNT/C is unique among the various BoNTs because it Clinical Neurophysiology 113 (2002) 1258–1264 1388-2457/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S1388-2457(02)00103-7 www.elsevier.com/locate/clinph CLINPH 2001747 * Corresponding author. Tel.: 139-0532-236544; fax: 139-0532- 237102. E-mail address: releopra@global.it (R. Eleopra).