Results of a BoNT/A antibody study in children and adolescents after onabotulinumtoxin A (Botox®) detrusor injection.

BACKGROUND: Onabotulinumtoxin A (OnaBoNT/A, Botox®) is effective in the treatment of neurogenic detrusor overactivity, however this therapy can fail. In a prospective study, we analyzed patient serum for BoNT/A antibodies (BoNT/A-AB) as a possible cause of therapy failure. METHODS: 17 patients (average age 14.5 years) who had neurogenic detrusor overactivity were admitted for repeated OnaBoNT/A injection into the detrusor muscle. We analyzed their serum for BoNT/A-AB. The clinical findings were correlated with the incidence of BoNT/A-AB. RESULTS: Positive BoNT/A-AB were clearly or marginally determined in 6 patients. Therapy had failed in all 6. In 4 of the 6, therapy might have failed because of a low-compliance bladder (3 patients) or tethered-cord syndrome (1), but BoNT/A-AB were found as the only possible cause in 2 patients. Thus, the incidence of BoNT/A-AB in the 17 patients was 35%, and the antibodies were clinically significant in 12%. All patients with BoNT/A-AB had a history of recurrent urinary tract infections. CONCLUSIONS: Patients who show a failure of therapy after OnaBoNT/A injections for which no other causes can be determined should have their serum checked for BoNT/A-AB. Recurrent urinary tract infection might be a predisposing factor for BoNT/A-AB.

A long-term follow-up of botulinum toxin A in cervical dystonia.

OBJECTIVE: Cervical dystonia (CD) is the most common form of adult-onset focal dystonia, and botulinum toxin A (BoNT-A) has become the first-line treatment for this condition. METHODS: In this work, we present data of 207 CD patients treated with BoNT-A for 6.7 +/- 3.5 years. One hundred and sixty-three patients were treated with Dysport (mean dose, 389 +/- 144 U) and 44 with Botox (mean dose, 145 +/- 44 U). RESULTS: The mean clinical benefit, based on a 0-3 scale (0=no effect, 1=slight, 2=moderate and 3=marked improvement) was similar for Dysport (2.5 +/- 0.3) and Botox (2.2 +/- 0.4). Adverse events were mild and similar for both products. Fewer than 2% of the patients developed neutralizing antibodies. DISCUSSION: These data confirm the efficacy and safety of BoNT-A treatment in CD over an extended period of up to 14 years.

Treatment of habitual snoring with botulinum toxin: a pilot study.

The objective of this study was to investigate whether injections of botulinum toxin into the soft palate reduce snoring in a subgroup of patients that present an active process causing habitual snoring. The study was conducted in eight patients with habitual snoring but without evidence of obstructive sleep apnea. Polysomnography was performed for diagnostic purposes and to monitor sleep quality before and after treatment. The patients and their partners completed a questionnaire before and after treatment. Recordings of snoring noise before and after treatment were evaluated on a visual analog scale by a blinded assessor. Doses of 20 U of botulinum toxin type A (Dysport) were injected unilaterally into the muscles of the soft palate. Snoring was reduced in eight cases. The patients reported no major adverse effects. These results justify further studies of botulinum toxin therapy in patients with habitual snoring. The scheme presented for injections of botulinum toxin into the levator veli palatini muscle provides a rational basis for the design of such studies. Therapy with botulinum toxin for habitual snoring is safe, non-invasive, easy to perform, fully reversible, and thus warrants investigation under placebo-controlled, double-blind conditions. This treatment is appropriate for a disorder that is of paramount social importance but does not pose a medical threat to the individuals affected.

Botulinum neurotoxin - from laboratory to bedside.

Botulinum neurotoxin (BoNT) has been used clinically since 1980, with an ever-increasing range of clinical applications. This has coincided with a period of massively expanded interest in the underlying biology of the neurotoxin. Tremendous advances have taken place in the scientific understanding of neurotoxin structure and function since the description of their endopeptidase activity in 1992. These developments have led to an increased understanding of the mechanisms underpinning the clinical use of the neurotoxins and also in the technologies available to support their clinical use. The expanding range of clinical applications, and use in increasing doses, has also generated challenges for the clinicians and manufacturers of BoNT preparations to ensure continuing efficacy and safety margins for these new clinical settings. To date the increased clinical use of BoNTs has occurred largely empirically, and not by application of the recent insights into neurotoxin structure and function. With the increased knowledge regarding the biology of the neurotoxins, however, there is the opportunity to select preferred forms of the toxin for particular clinical applications and even to consider engineering the neurotoxins to produce modified products more suited to specific clinical applications. These developments and opportunities that have arisen, particularly over the last decade, emphasise the increasing need to maintain an active two way dialogue between clinicians and basic scientists to ensure that the advances in the laboratory are translated into clinical benefit and that the clinical developments in use of neurotoxin are supported by the scientific research activity. This article is based upon presentations given in a workshop at the 5th International Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Toxin in Denver in June, 2005 seeking to address issues relating to the laboratory/clinic interface.

Pharmacology of botulinum toxin: differences between type A preparations.

Different types of botulinum neurotoxin (BoNT) block different proteins of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein complex within cholinergic nerve terminals, producing blockade of cholinergic neuromuscular and autonomic synapses. Animal studies indicate the longest duration of action for BoNT type A (BoNTA) followed by types B, F, and E. Diffusion to adjacent and remote muscles may be related to protein composition, dilutions, volume, target muscle selection, and injection technique. A review of head-to-head, randomized, controlled trials of BoNTA preparations (Botox and Dysport) suggests that Dysport tends to have higher efficacy, longer duration, and higher frequency of adverse effects. Conversion factors between the preparations varied, however, and remain controversial. In clinical settings, a Botox:Dysport conversion ratio of 1:3 may be appropriate. Animal studies suggest a conversion ratio of 1:2.5-3. When therapeutic effects between these preparations are attempting to be equalized, Dysport seems to produce more adverse effects. In mice, Botox appears to have a better safety margin than Dysport and BoNTB. In rats, diffusion margins are similar for Botox and Dysport. Jitter derived from stimulation single-fiber EMG of injected and remote muscles show no differences between Botox and Dysport. Atrophy of extrafusal muscle fibers of injected and remote muscles do not differ between the BoNTA preparations.

Botulism type B presenting as pure autonomic dysfunction.

Botulism nowadays is a rare mostly food-borne disease caused by the toxin of Clostridium botulinum. On the other hand, botulinum toxin blocking cholinergic transmission has become a most powerful treatment option for many focal movement disorders and is increasingly used to treat autonomic disorders [2, 7, 8]. Although muscle weakness is the hallmark of botulism, autonomic dysfunction may be the leading clinical symptom in rare cases and should be included in the differential diagnosis of pure dysautonomia. Here, we describe a patient with autonomic dysfunction as the leading symptom of botulism type B.

Botulinum A toxin: Dysport improvement of biological availability.

We investigated the efficacy and potency of Dysport, a botulinum neurotoxin type A complex approved for therapy, under various conditions. Conditions for maximal expression of biological activity were explored in vitro in the phrenic nerve-hemidiaphragm preparation, while conditions for optimal distribution of the toxin were tested in vivo in a double blind trial involving volunteers, using the foot Muscles extensor digitorum brevis. In contrast to the recommendations of the manufacturer, the biological availability of Dysport could be enhanced by (1) lowering its concentration, (2) supplementing with albumin, and (3) increasing the injection volume. On the basis of these experimental findings Dysport was diluted to a final concentration of 50 U/ml for therapeutic purposes. In a blind, single crossover study patients suffering from various forms of dystonia were treated with Dysport, first diluted and dosed as suggested by the manufacturer and then with doses cut by approximately 70% in accordance with the experimental findings. The low-dose treatment was as effective as the treatment with the recommended higher doses, but side effects were considerably less apparent. The benefits to be derived from these adjustments include a low risk of antibody formation, which could preclude continued or future treatment and substantial cost savings.

[Pharmacology of botulinum neurotoxin type A]. / Pharmakologie von Botulinum Neurotoxin Typ A.

The efficacies and potencies of Dysport and Botox, botulinum neurotoxin type A complexes approved for therapy, were investigated under various conditions. Conditions for maximal expression of biological activities of both commercial products were explored in vitro in the phrenic nerve-hemidiaphragm preparation whilst conditions for optimal distribution of Dysport were tested in vivo in a double blind trial involving volunteers, using the foot Muscles extensor digitorum brevis. Whilst Dysport and Botox expressed the same potency in vitro when albumin was added, their potencies markedly departed from each other when the products were reconstituted as recommended by the manufacturer. The biological availability of Dysport could be enhanced in vivo by (1) lowering its concentration, (2) supplementing with albumin and (3) increasing the injection volume. Thus, Dysport can be recovered more efficiently by deviating from the manufacturer's recommendations allowing a more economical use of Dysport.

Neutralizing botulinum toxin type a antibodies: clinical observations in patients with cervical dystonia.

Neutralization of antibodies poses a problem for a substantial number of cervical dystonia (CD) patients treated with botulinum toxin type A (BoNT/A). Presence of these antibodies may lead to a secondary nonresponse to BoNT/A treatment. In this study, we compared 6 antibody-positive (Ab+) with 12 antibody- negative (Ab-) CD patients treated with BoNT/A (Dysport) and matched for du- ration of treatment, number of BoNT/A injections, and severity of clinical symptoms. The two groups differed in cumulative BoNT/A dose (Ab+, 5984 mouse units [MU ], SD = 3151 MU; Ab-, 3143 MU, SD =1294 MU; P <.05), in addition, ab+ patients were significantly younger (ab+ mean age = 41.3 y, sd =5.9 y; ab - mean age = 56.8 y, sd = 15.3 y; p <.05), in or- der to avoid formation of neutralizing antibodies, doses of bont/a should be kept as low as possible, the risk of antibody formation seems to be higher in younger patients.

The sternocleidomastoid test: an in vivo assay to investigate botulinum toxin antibody formation in humans.

In a small number of patients treated with botulinum toxin (BT) antibody (Ab) formation occurs. BT Ab can be detected by the mouse protection assay (MPA) or by the mouse diaphragm assay (MDA). Both methods, however, have major drawbacks. We tested a method for detecting BT Ab which measures the BT-induced reduction in the electromyographic amplitude of the mean maximal voluntary activation (M-EMG) of the sternocleidomastoid muscle. The M-EMG reduction was compared in 17 patients with cervical dystonia and secondary BT therapy failure to the M-EMG reduction previously measured in controls. Values more than 2 SD below the mean of controls were considered abnormal. Six patients showed BT Ab on the MPA and MDA; all of these had abnormal M-EMG reductions. Eleven patients showed no BT Ab on MPA and MDA testing; in ten of these the M-EMG reduction was normal, and in one it was pathological, but MDA testing later changed to positive under continued BT therapy. The sternocleidomastoid test is easy to perform and produces quantitative results. Since its sensitivity and specificity are at least as good as those of the MDA and the MPA, it can replace them.

Botulinum toxin antibody testing: comparison between the mouse protection assay and the mouse lethality assay.

Conventionally, the standard test for detection of antibodies against botulinum toxin (BT-A) has been the mouse lethality assay (MLA). Because this test has a number of disadvantages, a novel mouse protection assay (MPA) was recently introduced. We sought to compare the results of both tests. Forty-three samples from 38 patients with cervical dystonia and complete or partial subjective BT-A therapy failure underwent simultaneous MPA and MLA testing. Twenty-seven samples showed concordant results in both tests. Eleven of them were MPA- and MLA-positive and 16 MPA- and MLA-negative, resulting in a significant association of the dichotomous test results (Fisher exact test, p <0.01). Sixteen samples showed discordant results. All of those were MPA-positive and MLA-negative. This excess of MPA-positive results was also significant (Wilcoxon signed-rank test, p <0.001). Of the patients with MPA-positive samples, 62% had complete and 38% had partial therapy failure. Of the patients with MLA-positive samples, 90% had complete and 10% had partial therapy failure. MPA and MLA results show significant association. Statistical analysis and predominance of partial therapy failure in MPA-positive patients demonstrate higher sensitivity of MPA. With its methodologic advantages, its test parameter being more relevant to BT-A therapy, and its higher sensitivity, the MPA appears to be superior to the MLA.

Acid secretion of parietal cells is paralleled by a redistribution of NSF and alpha, beta-SNAPs and inhibited by tetanus toxin.

Stimulation of parietal cells causes fusion of intracellular tubulovesicles with the canalicular plasma membrane thereby increasing the apical membrane area up to tenfold. The presence of the SNARE proteins synaptobrevin, syntaxin1, and SNAP25 in parietal cells and their intracellular redistribution after stimulation suggest a SNARE-mediated mechanism. Here we show that NSF and alpha, beta-SNAPs which are involved in the dissociation of the SNARE complex in neurons also occur in parietal cells exhibiting subcellular distributions similar to the ones obtained for SNARE proteins and for the H+, K(+)-ATPase. More importantly proteolytic cleavage of synaptobrevin by tetanus neurotoxin completely inhibits the cAMP-dependent increase of acid secretion further supporting the crucial role SNARE proteins play in parietal cells.

Low-dose treatment of cervical dystonia, blepharospasm and facial hemispasm with albumin-diluted botulinum toxin type A under EMG guidance. An open label study.

Several studies support the hypothesis that low-dose botulinum toxin treatment may be as beneficial as high-dose regimen. Therefore, we studied 115 patients (aged 27-84; mean 58.0, SD = 12.9 years; 68% females, 32% males) suffering from cervical dystonia (n = 66), blepharospasm (n = 28), and facial hemispasm (n = 21) over a period of 2 years in an open label, non-controlled pilot study. Patients received low-dose treatment with botulinum toxin type A (Dysport((R))). The toxin was diluted in 20 ml of 0.1% albumin solution to arrive at a concentration of 25 MU/ml and injected under EMG control. Patients responded to the treatment about 1 week after injection (mean 7.3 days, SD = 4.6). The mean duration of beneficial effects was 11.7 weeks (SD = 5.6). Patients evaluated the clinical global improvement on a scale ranging from 0 to 4. For the whole population, the mean was 2.7 points (SD = 1.1). In none of the subjects could antibodies to botulinum toxin type A be detected, and only a few side effects were observed. In conclusion, low-dose therapy with botulinum toxin A merits further controlled studies.

SNARE proteins and rab3A contribute to canalicular formation in parietal cells.

SNARE proteins - rab3A - parietal cells - H+/K+-ATPase When stimulated by histamine, acetylcholine, or gastrin the luminal compartments of oxyntic parietal cells display conspicuous morphological changes. The luminal plasma membrane surface becomes greatly expanded, while the cytoplasmic tubulovesicles are decreased in parallel. Due to these membrane rearrangements the H+/K(+)-ATPase obtains access to the luminal surface, where proton secretion occurs. The stimulation-induced translocation of H+/K(+)-ATPase involves a fusion process. Exocytotic membrane fusion in neurons is achieved by the highly regulated interaction of mainly three proteins, the vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 (synaptosomal-associated protein of 25 kDa), also referred to as SNARE proteins. Using immunofluorescence microscopy we analysed the subcellular distribution of neuronal synaptic proteins and rab3A in resting and stimulated parietal cells from pig and rat. In resting cells all synaptic proteins colocalized with the H+/ K(+)-ATPase trapped in the tubulovesicular compartment. After stimulation, translocated H+/K(+)-ATPase showed a typical canalicular distribution. Syntaxin, synaptobrevin, SNAP25 and rab3A underwent a similar redistribution in stimulated cells and consequently localized to the canalicular compartment. Using immunoprecipitation we found that the SNARE complex consisting of synaptobrevin, syntaxin and SNAP25, which is a prerequisite for membrane fusion in neurons, is also assembled in parietal cells. In addition the parietal cell-derived synaptobrevin could be proteolytically cleaved by tetanus toxin light chain. These data may provide evidence that SNARE proteins and rab3A are functionally involved in the stimulation-induced translocation of the H+/K(+)-ATPase.

Depletion of neutralising antibodies resensitises a secondary non-responder to botulinum A neurotoxin.

The objective was to evaluate whether removal of neutralising antibodies potentially resensitises a secondary non-responder to botulinum neurotoxin A (BoNT/A). Neutralising antibodies directed against BoNT/A are produced during long term treatment with BoNT/A-hemagglutinin complex in up to 10% of patients with cervical dystonia. These patients become secondary non-responders. Other serotypes of BoNT are not yet generally available and may also bear the risk of inducing antibody formation. Plasma exchange (PE) (one treatment cycle) and immunoadsorption on a protein A column (IA-PA; three treatment cycles) was employed over 15 months to remove neutralising antibodies from a severely disabled secondary non-responder with cervical dystonia. After plasma exchange or IA-PA, BoNT/A was reinjected. Antibodies were measured with a sensitive functional toxin neutralising test. Repeated use of plasma exchange and IA-PA depleted neutralising antibodies to below the detection limit and subsequently allowed successful BoNT/A injection into dystonic muscles. No serious side effects were found related to the depletion of IgG. In conclusion PE or IA-PA performed before BoNT/A readministration may provide an alternative strategy in treating selected secondary non-responders who are severely disabled.

Botulinum A toxin therapy: neutralizing and nonneutralizing antibodies--therapeutic consequences.

Although muscle-relaxant doses of botulinum A toxin (BoNT/A) are generally lower than doses stimulating the immune system, specific antibodies are raised in a substantial number of patients. As a rule, this necessitates the termination of treatment. Therefore, a reliable determination of specific anti-BoNT/A antibodies is helpful and we introduced, for this purpose, a novel in vitro toxin-neutralizing assay based on a nerve-muscle preparation. We measured the antibody titers in four groups of subjects: Group 1 comprised 75 randomly selected patients of a total of 295 who responded to treatment with Dysport in our local clinic. Five patients, in group 2, were nonresponders. Group 3 consisted of 32 untreated volunteers and group 4 of 8 subjects immunized with a toxoid more than 10 years ago. Two of the responders had marginal titers of neutralizing antibodies, while they were present in all nonresponders. The sera of all responders were also tested for nonneutralizing antibodies by ELISA. Their occurrence, however, was of no consequence to the therapeutic success. The blood samples of volunteers were free from specific antibodies, whereas antibodies persisted in the immunized subjects for longer than a decade. Patients from various clinics who had been treated unsuccessfully with the toxin-14 patients had received BOTOX, 7 had been treated with Dysport, and 7 with both products-all had neutralizing antibodies. Whether there was an antibody response depended on the amount of toxin administered. We believe, however, the effective toxin dose can be reduced by so much as to make antibody production highly improbable.

Botulinum A toxins: units versus units.

We investigated the efficacies and potencies of two commercial preparations of botulinum neurotoxin type A (BoNt/A) reputed to differ in potency. Tests were conducted in vitro using the mouse phrenic nerve-hemidiaphragm which is an approved tool for measuring clostridial toxicity. In addition, in a double-blind trial on volunteers, varying amounts of one product were injected into the Musculus extensor digitorum brevis of the left foot, while equal amounts, i.e. units, of the other preparation were injected into the same muscle of the right foot. Compound muscle action potentials (CMAPs) were recorded before and at various points in time after the injections. As opposed to wide-spread anecdotal reports, no difference in effectiveness was found. The dose-response curves obtained from the mouse organ preparation with both commercial products equalled one another in potency (number of units) and corresponded to previous toxicity tests in mice conducted elsewhere. Dose-response curves from volunteers were also identical for both commercial preparations. The time course of paralysis and recovery of muscle function did not differ either. At lower concentrations of toxin, however, restoration of muscle function was more rapid than at higher concentrations. Since the results obtained from man and the animal organ preparation are in excellent accord, we conclude that 1 unit of Botox corresponds to 1 unit of Dysport.

Molecular aspects of tetanus and botulinum neurotoxin poisoning.

Clostridial neurotoxins, tetanus and the botulinum toxins A-G, are high molecular weight proteins consisting of a heavy chain which is responsible for the internalisation and a light chain possessing a zinc-dependent proteolytic activity. They exclusively proteolyse either the vesicle membrane protein, synaptobrevin or two integral plasma membrane proteins, SNAP 25 and syntaxin. Together with cytosolic proteins these proteins form the SNARE complex involved in vesicle exocytosis, and their cleavage blocks the latter process. Clostridial neurotoxins have now become powerful tools to investigate the final events occurring during secretion in neuronal, endocrine, and non-neuronal cells. They are applied to dissect the specific interactions of the SNARE protein complex with cytosolic fusogens and other modulators of exocytosis. Whereas exocytosis is not essential for the survival of cells, the organism as a whole will fall victim to a few nanograms since interneuronal and neuromuscular transmission is vital to muscular control, especially in respiration. Although all clostridial neurotoxins by their light chains attack proteins of the SNARE complex, tetanus toxin and the various botulinum toxins differ dramatically in their clinical symptoms. The biological information for this difference resides on the respective heavy chains which select different transport routes carrying the light chain from the place of entrance to the final compartment of action. So far the different transport vesicles used either by the various botulinum neurotoxins or by tetanus toxin are not yet defined. Nevertheless at least one of the botulinum toxins serves as a beneficial drug in the treatment of severe neuromuscular spasms.