Pharmacologic characterization of botulinum toxin for basic science and medicine.

The use of Botulinum neurotoxin (BoNT) is increasing in both clinical and basic science. Clinically, intramuscular injection of nanogram quantities of BoNT is fast becoming the treatment of choice for a spectrum of disorders including movement disorders such as torticollis, blepharospasm, Meige Disease, and hemifacial spasm (Borodic et al., 1991, 1994a; Jankovic and Brin, 1991; Clarke, 1992). Neuroscientists are using BoNTs as tools to develop a better understanding of the mechanisms underlying the neurotransmitter release process. Consequently, our ability to accurately and reliably quantify the biologic activity of botulinum toxin has become more important than ever. The accurate measurement of the pharmacologic activity of BoNTs has become somewhat problematic with the most significant problems occurring with the clinical use of the toxins. The biologic activity of BoNTs has been measured using a variety of techniques including assessment of whole animal responses to in vitro effects on neurotransmitter release. The purpose of this review is to examine the approaches employed to characterize, quantify and investigate the actions of the BoNTs and to provide a guide to aid investigators in determining which of these methods is most appropriate for their particular application or use.

The median paralysis unit: a more pharmacologically relevant unit of biologic activity for botulinum toxin.

Although the LD50 has been used to quantify the biologically active toxin in clinical preparations of botulinum A toxin (Botox and Dysport), a discrepancy exists between the clinical potency of equivalent international units of different formulations of botulinum A toxin for multiple clinical indications. Our laboratory previously reported that a regional chemodenervation assay in the mouse could be utilized to detect the difference in the potencies of the clinical preparations of toxin [Pearce et al. (1994) Toxic. appl. Pharmac. 128, 69-77]. The purpose of this study was to quantify the regional paralysis produced by botulinum toxin and define a new pharmacologic/biologic unit of activity that more accurately reflects the mechanism of action of botulinum toxin in the clinical setting. Quantal analysis of regional paralysis revealed that the ED50, defined as the median paralysis unit (MPU) for Botox and Dysport, was 0.41 +/- 0.01 and 1.00 +/- 0.02 LD50 units, respectively. Differences in the potencies found in retrospective clinical studies comparing Botox and Dysport were accurately reflected, for the first time, by the dose of toxin expressed in terms of the MPU (median paralysis unit). The data suggested that the MPU may be a more appropriate measure of the biologic activity in therapeutic formulations of botulinum toxin.

Measurement of botulinum toxin activity: evaluation of the lethality assay.

The use of the mouse lethality assay for the estimation of the biologic activity of botulinum toxin was evaluated. The relationship between the number of animals, number of doses, and duration of the assay used to estimate the LD50 and the precision of the assay was investigated. The results of these studies demonstrated that the LD50 for botulinum toxin can be estimated with a high degree of precision (+/- 5%). The precision of the assay is not increased by using more than a 5-dose 50-animal assay or extending the duration of the assay beyond 72 hr. Estimates of the LD50 obtained at 48 hr were only slightly less precise but underestimated the LD50 by 15%. Analysis of the commercially available preparations of botulinum toxin with the mouse LD50 assay revealed significant discrepancies between the units of toxin in these preparations. In addition, a 2.67-fold difference in the relative potency of the two preparations of botulinum A toxin was observed using a regional chemodenervation assay that measures paralysis. The mouse LD50 assay could not detect this large difference in the potency of the two approved clinical preparations of botulinum toxin. The results of these studies demonstrate that although the mouse LD50 assay can be used to estimate the number of units of botulinum toxin with a high degree of precision this assay alone is not an adequate method for assessing the preclinical biological potency of botulinum toxin.