Botulinum toxin therapy for pain and inflammatory disorders: mechanisms and therapeutic effects.

Botulinum toxin (BTX) injections are a well-recognised therapeutic modality for the treatment of regional involuntary muscle disorders and recently BTX has been used for treatment of pain and inflammatory disorders. The primary purpose of this review is to discuss the mechanism of action of therapeutic BTX in light of both the traditional understanding of BTX pharmacological effects as well as new observations. The review will deal with clinical observations and relevant animal experimentation. The data and hypotheses presented are not only relevant to botulinum toxin technology but will certainly prove important in the basic mechanisms of some of the diseases where botulinum toxin has been successfully applied. BTX used clinically comprises botulinum neurotoxin (BoNT) complexed with non-toxic proteins. The non-toxic components of the BTX complexes stabilise the labile BoNT during purification and formulation as a therapeutic. The complex proteins may also have unrecognised clinical significance such as slowing diffusion in tissues or imparting stability. The mechanisms of BTX formulations acting on SNARE proteins are briefly reviewed providing a basis for BTX clinical applications. The potential for design of improved botulinum toxins and formulations is addressed.

Giant cell angiofibroma of the orbit and eyelid.

PURPOSE: To report the clinicopathologic features of a newly recognized tumor, giant cell angiofibroma. DESIGN: Observational case series. MAIN OUTCOME MEASURES: Clinical and histopathologic features of giant cell angiofibroma. METHODS: Light and electron microscopy and immunohistochemistry of five cases of giant cell angiofibroma. RESULTS: A total of five patients (4 women and 1 man) are described: two presented with a painless mass in the eyelid, two with a mass in the orbit, and one presented with a conjunctival lesion. All lesions were well demarcated with no capsule and were composed of blood vessels, a patternless spindle-shaped cell proliferation with a solid and pseudovascular appearance, and multinucleated giant cells. Both spindle-shaped and giant tumor cells were intensely positive for CD34 and vimentin. CONCLUSION: Giant cell angiofibroma resembles solitary fibrous tumor and giant cell fibroblastoma and should be considered in the differential diagnosis of spindle-cell tumors in the eyelid, orbit, and conjunctiva.

Botulinum toxin therapy, immunologic resistance, and problems with available materials.

Botulinum toxin is a valuable technology for the treatment of regional movement disease. High-dose applications ( > 100 LD50 units per injection cycle) have been associated with sensitization that renders further therapeutic injections ineffective. The true incidence of sensitization is probably underestimated by the mouse bioassay. Other immunotypes of botulinum toxin have been effective in producing some therapeutic benefit; however, duration of action (botulinum toxin type F) and lower potencies may make these less attractive alternatives than botulinum type A. Increased specific activity botulinum toxin may be a method to reduce antigen exposure and mitigate against immunoresistance associated with dystonia therapy. Limiting the dose to < or = 100 LD50 units per injection cycle may limit this complication in the interim.

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.

Histologic assessment of dose-related diffusion and muscle fiber response after therapeutic botulinum A toxin injections.

Fiber diameter variability, acetylcholinesterase staining properties, and average fiber diameter were determined 5 weeks after varying doses of botulinum A toxin were administered into albino rabbit longissimus dorsi muscle. The average fiber diameter within the muscle appeared to be a function of the dose of botulinum toxin injected. Fiber diameter variability correlated with the dose of botulinum toxin administered. Both fiber diameter variability and acetylcholinesterase spread characteristics showed a distinct diffusion gradient over a defined field within a muscle. At lower doses (1 IU), collapse of the diffusion gradient occurred over a 15-30-mm segment of muscle. At higher doses (5-10 IU), diffusion of botulinum A toxin effect occurred throughout the entire muscle with no apparent end point. This study demonstrated that botulinum A toxin produces a gradient of denervation in a given muscle and that both the magnitude of denervation and the extent of the gradient are dose dependent. Furthermore, both muscle fiber diameter variability and acetylcholinesterase staining were useful as measures of chemodenervation.

Botulinum A toxin for treatment of aberrant facial nerve regeneration.

Twelve patients with involuntary synkinetic eyelid closure were given 40 injections of botulinum A toxin. Temporary improvement in involuntary eyelid closure was observed in all 12 patients. Eleven of the 12 patients desired repeated injections. Dose requirements for this indication were compared with doses used in 697 injections in 112 patients with essential blepharospasm and Meige syndrome. Additionally, dose comparisons were made with 269 injections in 71 patients with hemifacial spasm. Dose requirements needed to treat aberrant regeneration of the facial nerve were substantially less than needed to treat blepharospasm and Meige syndrome. The dose requirement was similar to that in hemifacial spasm treatment. The reason for the differences probably relates to existing muscular denervation associated with hemifacial spasm and aberrant facial nerve regeneration.

Botulinum B toxin as an alternative to botulinum A toxin: a histologic study.

Histochemical effects of botulinum B toxin were studied on fibers from longissimus dorsi muscle in Albino rabbits and compared to effects produced by botulinum A toxin. Acetylcholinesterase staining, muscle fiber size analysis, and ATPase staining indicated botulinum B toxin produced a denervation gradient and field similar to that produced by botulinum A toxin. At 5 weeks postinjection with botulinum B toxin, analysis showed muscle fiber size variability, and diffuse acetylcholinesterase fiber staining comparable to botulinum A toxin at the injection site. Muscle sections taken at 4.0 cm for analysis showed statistically significant decreased fiber size variability and contraction of acetylcholinesterase staining pattern for both immunotypes. In addition, the denervation reflected by histochemical staining and fiber size analysis appeared transient and lasted for approximately 3 months for both immunotypes. These findings suggest botulinum B toxin produces pharmacologic effects on innervation of striated muscle similar to botulinum A toxin. Because immunologic tolerance has been demonstrated after therapeutic botulinum A toxin injections, further clinical studies need to be conducted with other immunotypes of toxin with no cross-reactivity to type A.