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.

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.

Distribution of the molecular forms of acetylcholinesterase in human brain: alterations in dementia of the Alzheimer type.

Acetylcholinesterase (AChE), the enzyme that degrades acetylcholine, is a heterogeneous enzyme that can be separated into multiple molecular forms. A tetrameric membrane-bound form (G4) and a monomeric soluble form (G1) are the two predominant enzyme species in mammalian brain. The distribution of AChE molecular forms was defined by sucrose density gradients of 11 anatomical regions of postmortem brains from 10 patients with dementia of the Alzheimer type (DAT) and 14 nondemented controls of similar ages. The results demonstrate an overall loss of protein and enzyme activity in all areas of the DAT brains studied and a selective loss of the G4 form of AChE in Brodmann areas 9, 10, 11, 21, 22, and 40, and the amygdala. There was no change in the G4/G1 ratio in areas 17 and 20, in the hippocampus, or in the cerebellum. There was a high regional correlation of the G4/G1 ratios with published values for choline acetyltransferase activity but lower correlation with total AChE activity. We propose that there is a predominant loss of the G4 form of AChE in DAT and that this loss is correlated with the degeneration of presynaptic elements.