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.

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.

Contralateral injections of botulinum A toxin for the treatment of hemifacial spasm to achieve increased facial symmetry.

Six patients noted facial asymmetry after botulinum toxin injection for hemifacial spasm. Each patient was injected on the side contralateral to the spasms with 10 to 15 IU over the zygomatic major and minor muscles. Each patient noted improvement in facial symmetry in the resting position and dynamic facial movements. Five of the six patients desired this approach with subsequent injections. This injection method variation proved helpful in the managing of hemifacial weakness created by botulinum A toxin for this condition.

Effects of repeated botulinum toxin injections on orbicularis oculi muscle.

Histologic evaluation was conducted on 12 orbicularis oculi specimens from 11 patients with essential blepharospasm and Meige's disease who had received an average of 11.3 injections of botulinum A toxin over 3.5 years. Denervation was demonstrated by the spread of acetylcholinesterase staining on muscle fibers when specimens were evaluated within 11 weeks of the last injection. When specimens were taken after 12 weeks, spread of acetylcholinesterase was confined to the neuromuscular junctions, with little fiber size variability resembling normal muscle. Fibrosis seen in three specimens could be correlated to prior surgery. Repeated injections of botulinum toxin into human muscle do not appear to cause irreversible muscle atrophy or other degenerative changes. Denervation changes (fiber size variability, acetylcholinesterase spread) appear to correlate to the time interval since the last injection.

Botulinum a toxin for spasmodic torticollis: multiple vs single injection points per muscle.

Eighty-six injections in 49 patients with adult onset spasmodic torticollis were evaluated for efficacy with respect to single point per muscle versus multiple point per muscle injection techniques. Parameters of the syndrome assessed were pain, posture deformity, range of cervical motion, disfigurement, cervical muscle hypertrophy, activity limitation, and degree of involuntary movement. The multiple point per muscle injection strategy appeared superior to the single injection per muscle technique with respect to pain (p less than 0.002, chi-square), posture deformity (p less than 0.001), range of motion (p less than 0.001), and improvement in activity endurance (p less than 0.001). No significant differences were noted with respect to cervical muscle hypertrophy or degree of involuntary movements, although the injections were considered beneficial in both groups to these syndrome components.

Botulinum A toxin for (expressionistic) ptosis overcorrection after frontalis sling.

Botulinum A toxin was injected into the frontalis muscle in two patients with complete third nerve palsies to limit intermittent upper lid retraction after a frontalis sling procedure. This form of lid retraction is noted during periods of active facial movement with occipitofrontalis muscle contraction. Although upper lid position may be symmetric when the facial muscles are adynamic, the upper lid may retract during periods of active facial expression. This type of lid retraction was corrected using Botulinum A toxin injections into the frontalis muscles, without affecting the lid position when the facial muscles are adynamic. Both improvement in appearance and intermittent exposure were noted in both cases. Additionally, a blunting of the transverse forehead creases occurred over a defined area after this injection, representing a clinical example of a denervation field produced by a point injection of botulinum toxin.

Botulinum A toxin for the treatment of adult-onset spasmodic torticollis.

Thirty-five patients with adult-onset idiopathic torticollis were treated by local injections of botulinum A toxin into dystonic cervical muscles. Substantial improvement with respect to reduction and elimination of pain was found in 81 percent, improvement in posture deformity and involuntary spasms in 70 percent, increased range of motion of the neck in 78 percent, reduction in visible sternocleidomastoid hypertrophy in 86 percent, and improvement in tremor in 65 percent. The syndrome was divided into four subtypes based on pattern of dystonic muscle groups involved in the dystonia, head and shoulder posture, and sternocleidomastoid muscle hypertrophy. Injection strategy based on this subdivision is described.

Innervation zone of orbicularis oculi muscle and implications for botulinum A toxin therapy.

Motor points (areas of maximal sensitivity to electrical stimulation) were found in constant locations over orbicularis oculi when measured in both eyes of six normal subjects. All subjects had a motor point at the lateral terminus of the upper lid crease and the medial extent of the lower lid crease. A study of the innervation zone [distribution of neuromuscular junctions (NMJ)] was conducted on strips of pretarsal and preseptal portions of the upper eyelid orbicularis that had been removed routinely during involutional ptosis surgery. There was no significant difference in NMJ concentration between the medial and lateral sections, as determined by cholinesterase staining. Therefore, we concluded that the innervation zone is diffuse for the orbicularis muscle within this portion of the upper eyelid. Single-point injections of botulinum toxin were then compared to the conventional multiple injection sites on separate eyes in 10 patients with benign essential blepharospasm. Eight of the 10 patients reported greater relief on the side given injections into multiple points; the other two patients experienced no difference between the two methods. Both histologic data and clinical observation of response to botulinum toxin injection suggest the innervation zone for the upper orbicularis is diffuse. Thus, we conclude that multiple injections are superior to the injection of a single motor point.

Botulinum A toxin for the treatment of spasmodic torticollis: dysphagia and regional toxin spread.

Chemodenervation of cervical muscles with botulinum A toxin, although useful in treating spasmodic torticollis, has been associated with dysphagia. Retrospective analysis of dose and injection site (sternomastoid vs. posterior cervical muscle groups) in 26 patients (49 injections) suggested that dysphagia was related to the quantity of toxin injected into the sternomastoid muscle: dysphagia, median 150 IU (7 injections); and no dysphagia, median 100 IU (42 injections; p = 0.026 Wilcoxon test). In a prospective study (31 injections to 24 patients), limiting the dose administered to the sternomastoid to 100 IU, substantially reduced the incidence of dysphagia (0 of 31, p = 0.27, Fisher's exact test). Denervation of human orbicularis muscle fibers, 5 weeks to 4 months after injection of botulinum A toxin for the treatment of blepharospasm, was successfully demonstrated by intense, diffuse acetylcholinesterase staining. A weight-adjusted dose similar to that given for torticollis was injected into longissimus dorsi muscle in 6 albino rabbits. Using the acetylcholinesterase stain as a marker, a diffusion gradient was noted over a distance of 30 to 45 mm from the point of injection and in contralateral muscle 15 to 25 mm from this point. Thus, denervation was demonstrated to occur within a definable area which crossed anatomic barriers, such as fascia and bone. These clinical and laboratory data suggest that dysphagia following botulinum toxin therapy results from toxin spread to pharyngeal musculature from the sternocleidomastoid injection site. Limiting of the injection dose to 100 IU or less to the sternomastoid substantially decreases the incidence of this complication.