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Like all proteins foreign to the human body, also botulinum toxin (BT) is antigenic and may stimulate an immune response with formation of antibodies (BT-AB). Affected patients may no longer respond to BT therapy and various degrees of BT-AB related therapy failure (ABF) may result. We want to review the immunological interactions between BT and BT-AB, the prevalence, the time course and the risk factors for BT-AB formation as they are related to the treatment algorithms, the patient's immune system and to exogenic factors. Special emphasis is placed on various features of the BT drugs including the specific biological activity (SBA) as a predictor of their antigenicity. Quantitative detection of BT-AB by the mouse diaphragm assay will be demonstrated. As ABF may have serious consequences for patients affected, careful risk factor analysis is warranted to reduce them wherever possible.

Based on epidemiological data it was believed that botulinumtoxin type D (BT-D) may not block human cholinergic synapses. We wanted to investigate BT-D’s effect on the autonomic cholinergic synapse in humans. For this, we compared in four volunteers intraindividually the hypohidrotic effect of intradermal BT-D and BT-A in Minor’s iodine starch sweat test. Altogether, we studied BT-D in doses of 4, 8, 16 and 32MU and BT-A in doses of 2, 4, 8 and 16MU at weekly intervals throughout a period of 13 weeks. All BT doses were diluted in 0.2 ml 0.9% NaCl/H2O. Overall 704 data points were collected. Combined over all four subjects and all four doses BT-D’s hypohidrotic effect intensity was half of BT-A’s. BT-D’s effect peaked around 5 weeks, BT-A’s around 7 weeks. BT-D’s effect duration was around 12 weeks, of BT-A’s was around 14 weeks. For both BT types the hypohidrotic effect was dose dependent. BT-D, when injected intradermally, can block autonomic cholinergic synapses in humans. Compared to BT-A, BT-D’s effect intensity was half and its effect duration was some 2 weeks shorter. With its weaker and shorter effect BT-D does not seem to promise therapeutic effects superior to BT-A.

Objectives Botulinum neurotoxin serotypes A and B (BoNT/A & B) are highly effective medicines to treat hyperactive cholinergic neurons. Due to neutralizing antibody formation, some patients may become non-responders. In these cases, the serotypes BoNT/C-G might become treatment alternatives. BoNT/D is genetically least related to BoNT/A & B and thereby circumventing neutralisation in A/B non-responders. We produced BoNT/D and compared its pharmacology with BoNT/A ex vivo in mice tissue and in vivo in human volunteers. Methods BoNT/D was expressed recombinantly in E. coli, isolated by chromatography and its ex vivo potency was determined at mouse phrenic nerve hemidiaphragm preparations. Different doses of BoNT/D or incobotulinumtoxinA were injected into the extensor digitorum brevis (EDB) muscles (n = 30) of human volunteers. Their compound muscle action potentials were measured 11 times by electroneurography within 220 days. Results Despite a 3.7-fold lower ex vivo potency in mice, a 110-fold higher dosage of BoNT/D achieved the same clinical effect as incobotulinumtoxinA while showing a 50% shortened duration of action. Conclusions BoNT/D blocks dose-dependently acetylcholine release in human motoneurons upon intramuscular administration, but its potency and duration of action is inferior to approved BoNT/A based drugs. Significance BoNT/D constitutes a potential treatment alternative for BoNT/A & B non-responders.

Botulinum toxin (BT) is used in many medical specialties to treat muscle hyperactivity, exocrine gland hyperactivity and pain disorders. BT drugs consist of botulinum neurotoxin (BNT), complexing proteins (CP) and excipients. Antibodies can be formed against BNT and CP. When they are formed against BNT (BTAB) they can block BT's therapeutic efficacy thus producing antibody induced therapy failure (ABTF). Areas covered: BT applied and BTAB are in a functional balance within the body. ABTF is rare, but influences the treatment algorithms of BT therapy considerably. ABTF risk factors include BT doses given, interinjection intervals, booster injections and immunological quality of the BT drug. Testing for BTAB and interpretation of ABTF is complicated. As management of ABTF is frustrating, prevention of ABTF is of major importance. Improved antigenicity of new BT drugs may improve treatment algorithms of BT therapy, substandard antigenicity may very likely be their end. Expert commentary: Concern about ABTF has influenced the treatment algorithms of BT therapy considerably. Better understanding of ABTF may improve them and, thus, the outcome of BT therapy. New BT drugs may have further improved antigenicity, especially when their CP are removed. They may, however, fail because of antigenicity problems.

Botulinum neurotoxin (BNT), the biologically active component of botulinum toxin (BT), is a large double-stranded protein susceptible to various physical and chemical influences. All BT type A (BT-A) drugs are stored as powders allowing shelf lives from 24 to 36 months. After reconstitution, the specified shelf life is reduced to 8-24 h. Some studies, however, suggest longer shelf life. We wanted to test the long-term stability of reconstituted BT-A drugs in the hemidiaphragm assay (HDA), a high quality BT potency test. For this incobotulinumtoxinA (INCO) was reconstituted and stored in at 4-8 °C, whilst at various points of time probes of it were taken and potency tested with the HDA. Altogether 18 measurements were performed throughout a period of 52.1 weeks. The paralysis time in the HDA was 67.3 ± 5.2 min (min. 59 min, max. 76 min). The linear regression line was described by y = -0.0163x + 67.582. The paralysis time measured during the first 10 weeks (n = 11) was 67.5 ± 5.3 min, during the last 10 weeks (n = 7) 67.1 ± 4.9 min. Reconstituted INCO does not show reduction of potency throughout 52 weeks as tested by the high quality HDA. Lack of complexing proteins does not de-stabilise INCO. Our data allow un-used reconstituted INCO to be stored for further use. This may have a considerable impact on the costs of BT therapy. Further studies will have to demonstrate sterility of the reconstituted BT drug beyond the so far reported 6 weeks.

ObjectiveTo investigate the prevalence of neutralizing antibodies (NAbs) against botulinum neurotoxin type A (BoNT/A) during long-term BoNT/A treatment in different neurologic indications.MethodsIn this monocentric, observational cross-sectional study, 596 outpatients treated with BoNT/A for different indications were tested for BoNT/A binding antibodies by ELISA. Positive samples were investigated for NAbs with the mouse hemidiaphragm test. The prevalence of NAbs was analyzed for different indications: facial hemispasm, blepharospasm, cervical dystonia, other dystonia, and spasticity. Besides the rate of NAb-positive patients overall and per patient subgroup, a Kaplan-Meier analysis of the probability of remaining NAb negative with duration of treatment is provided, and a stepwise binary logistic regression analysis is performed to identify factors significantly contributing to the induction of NAbs.ResultsOverall, 83 of 596 patients (13.9%) had measurable NAbs. The probability of developing NAbs increased with the single and cumulative dose of treatment and was influenced by the BoNT/A formulation, while all other factors analyzed, including disease entity and treatment duration, had no additional influence.ConclusionsWe present the largest study to date of the prevalence of BoNT/A NAbs in a large unbiased cohort of patients including the relevant neurologic indications. Repeated injections of BoNT/A inevitably bear the risk of developing NAbs. However, in addition to avoiding booster injections and providing short intervals between injections, reducing the individual injected doses may diminish the risk of NAb induction independently of the indication for which BoNT/A is used.

An increase in cytosolic free calcium concentration [Ca2+]i initiates the exocytotic activity in various types of secretory cells. The guanosine 5′-O-[3-thio]triphosphate (GTPγS), a non-hydrolysable analogue of GTP (guanosine 5′-triphosphate), is an effective secretagogue for different cell types of different species, like mast cells, neutrophils or eosinophils. Consequently, the internal administration of GTPγS causes degranulation of mouse and rat mast cells. Regarding rat mast cells, it is proved that Ca2+ can cooperate with GTP or GTPγS in accelerating and increasing amplitude of the secretory response. All the previous studies with respect to capacitance recordings and mast cells were performed using mouse or rat mast cells, usually derived from peritoneum or the rat basophilic leukaemia cell line RBL. In this study, we applied the capacitance measurement technique to the human mast cell line-1 (HMC-1) cells, an immature cell line established from a patient with mast cell leukaemia. Patch-clamp dialysis experiments revealed that high intracellular free Ca2+ and GTPγS concentrations are both required for considerable capacitance increases in HMC-1 cells. During degranulation of HMC-1 cells, the total membrane capacitance (Cm) increase appeared continuously and, in some cases, as a discrete capacitance change, developing in a stepwise manner. Then, we tested the effect of latrunculin B upon HMC-1 cell capacitance increase as well as of some classic mast cell stimulators like PMA, A23187 and IL-1β in hexosaminidase release. Finally, we could conclude that the HMC-1 cell line represents a suitable model for the study of human mast cell degranulation.

BACKGROUND Botulinum toxin (BT) is a safe and effective treatment for cosmetic indications. Formation of BT antibodies can occur but has previously been reported in cosmetic indications in two cases only. OBJECTIVE To report another four patients with this phenomenon. OBSERVATIONS Two patients received abobotulinumtoxinA; one received the current formulation of onabotulinumtoxinA and one both abobotulinumtoxinA and onabotulinumtoxinA. Complete secondary therapy failure (CSTF) occurred after 3-, 5-, 10-, and 13-injection series; cumulative treatment times of 18, 16, 25, and 65 months; and cumulative doses of 240 MU onabotulinumtoxinA, 245 MU abobotulinumtoxinA, 1,180 MU abobotulinumtoxinA, and 120 MU onabotulinumtoxinA/270 MU abobotulinumtoxinA, respectively. Average interinjection intervals were 87, 273, 150, and 119 days, and average single doses were 80 MU onabotulinumtoxinA, 68 MU abobotulinumtoxinA, 82 MU abobotulinumtoxinA, and 30 MU abobotulinumtoxinA/30 MU onabotulinumtoxinA. Risk factors for CSTF included booster injections (2 patients) and increased immune system reagibility (1 patient). BT antibody titers were 2.7, 7.0, and more than 10.0 mU/mL on the mouse diaphragm assay. CONCLUSIONS CSTF can occur after cosmetic BT injections in patients with high immune system reagibility and in patients receiving booster injections, but also in unremarkable patients with typical treatment parameters. Its incidence is unknown. Recommended treatment parameters may reduce the risk of CSTF, but may not eliminate it.

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.

A genuine peripheral antinociceptive and anti-inflammatory effect of Botulinum neurotoxin type A (BoNT/A) has been proposed but could not be demonstrated in humans so far. Therefore, 100 mouse units of Botulinum toxin A (Dysport) and placebo were injected in a double blind paradigm in defined skin areas of 50 subjects. At baseline and after 4 and 8 weeks allodynia was induced in the skin areas with capsaicin ointment. Heat and cold pain threshold temperatures were measured with quantitative sensory testing, and threshold intensities upon electrical stimulation with a pain specific surface electrode were determined. No BoNT/A related differences in pain perception were found at any quality. There is neither a direct peripheral antinociceptive effect nor a significant effect against neurogenic inflammation of BoNT/A in humans.

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.

Botulinum toxin induced therapy failure type B antibody (BT-B, BT-B-AB) has so far only been reported after previous formation of antibodies against botulinum toxin type A (BT-A, BTA- AB). We wanted to explore the risk of BT-B-AB-induced therapy failure in patients who were exposed to botulinum toxin for the first time. For this purpose we followed nine patients with cervical dystonia receiving BT-B (NeuroBloc/Myo- Bloc, Elan Pharmaceuticals) in a dose of 11435 +/- 2977MU during 4.9 +/- 3.0 injection series. All patients showed a satisfactory initial therapeutic response as documented by a Toronto Western Spasmodic Torticollis Rating Scale score reduction from 17.7 +/- 9.4 to 5.3 +/- 4.8 and an overall subjective improvement of 56.1 +/- 28.3%. Seven patients experienced systemic anticholinergic side effects. Five patients had stable therapeutic responses over subsequent injection series. Four patients experienced complete therapy failure with BT-B-AB titres in excess of 10 mU/ml on the mouse diaphragm assay. Doubling the last effective BT-B dose produced neither therapeutic effects nor side effects. Subsequent applications of botulinum toxin type A produced a continued therapeutic response in one patient and complete therapy failure in the other.Despite the small sample size a frequency of 44 % indicates a high risk for BT-B-AB-induced complete therapy failure. The high amount of neurotoxin administered when NeuroBloc/MyoBloc is used might be a contributory factor. Further prospective comparative studies are necessary to monitor the frequency and time course of BT-B-AB formation.

Botulinum toxin (BT) is injected intramuscularily and may produce injection site pain (ISP). We wanted to explore whether the pH value of the reconstituted BT drug contributes to ISP and, if so, what strategies can be applied to reduce it. In part 1 of the study, pH values of different reconstitution solutions and of major BT drugs reconstituted with different reconstitution solutions were assessed. In part 2, the effects of reconstitution with normal saline (NS) and Ringer acetate (RA) were compared intraindividually and in a double blind fashion in 34 patients with blepharospasm. pH values of reconstitution solutions were 5.52 ± 0.02 for NS, 6.98 for RA, 6.31 for Ringer lactate, 6.56 for electrolyte and 5.31 for bacteriostatic solution. pH values for NS-reconstitution were 6.09 ± 0.20 for Botox(®), 5.95 ± 0.24 for Dysport(®) and 5.81 ± 0.18 for Xeomin(®). pH values for RA-reconstitution were 6.95 ± 0.03 for Botox(®), 7.01 ± 0.02 for Dysport(®) and 6.87 ± 0.06 for Xeomin(®). By using RA instead of NS the pH could be increased by 0.86 for Botox(®), by 1.06 for Dysport(®) and by 1.06 for Xeomin(®). 47 % of the patients experienced less ISP when Botox(®)-RA was given rather than Botox(®)-NS, 76 % when Xeomin(®)-RA was given rather than Xeomin(®)-NS. None of the patients reported a difference in efficacy between NS- and RA-reconstitution. Despite previous reports, reconstituted BT type A drugs show acidic pH values. Normalising these pH values by use of RA instead of NS reduces ISP considerably without sacrificing clincial efficacy.

In some patients treated with botulinum toxin type A (BT) therapy failure occurs due to the formation of antibodies against BT (BT-AB). We investigated whether increased BT doses can overcome this form of therapy failure. Eight patients with cervical dystonia, secondary BT therapy failure and evidence of BT-AB formation in the mouse diaphragm bioassay received BT test doses (Dysport®, Ipsen Ltd., Maidenhead, Berks, UK) into one of their sternocleidomastoid muscles. Test doses were increased in three steps at 3-month intervals and their effect on the amplitude of the electromyographic activity of the sternocleidomastoid muscle under maximal isometric activation (M-EMG) was measured and compared to a control group. In step 1 (200 or 300 MU) the M-EMG reduction was 12 ± 13% compared to 85 ± 10% (200 MU) and 83 ± 9% (300 MU) in the control group. In step 2 (400, 600 or 800 MU) the M-EMG reduction was 25 ± 21% compared to 78 ± 7% (400 MU) in the control group. In step 3 (1,600 or 1,800 MU) the M-EMG reduction was 24 ± 10%. Side effects were not observed in any of the patients studied. In 1 patient with partial secondary BT therapy failure, with a low BT-AB titre (0.0015 U/ml) and with a moderately pathological M-EMG reduction of 40% with 200 MU a normal M-EMG reduction of 71% could be regained with 800 MU. In three subsequent therapeutic injection series with quadrupled BT doses in all target muscles the original therapy outcome could be regained and maintained. Side effects or increasing BT-AB titres were not observed. Even massively increased BT doses cannot overcome BT-AB-induced complete secondary therapy failure. However, in patients with partial secondary therapy failure, low BT-AB titres and a moderately pathological M-EMG reduction, increased BT doses might regain and maintain normal BT efficacy without induction of side effects or increasing BT-AB titres.

Clostridium difficile toxins A and B (TcdA and TcdB) belong to the class of large clostridial cytotoxins and inactivate by glucosylation some low molecular mass GTPases of the Rho-family (predominantly Rho, Rac and Cdc42), known as regulators of the actin cytoskeleton. TcdA and B also represent the main virulence factors of the anaerobic gram-positive bacterium that is the causal agent of pseudomembranous colitis. In our study, TcdB was chosen instead of TcdA for the well-known higher cytotoxic potency. Inactivation of Rho-family GTPases by this toxin in our experimental conditions induced morphological changes and reduction of electron-dense mast cell-specific granules in human mast cell line-1 (HMC-1) cells, but not cell death or permeabilisation of plasma-membranes. Previously reported patch-clamp dialysis experiments revealed that high intracellular free-Ca(2+) and GTPγS concentrations are capable of inducing exocytosis as indicated by significant membrane capacitance (Cm) increases in HMC-1 cells. In this study, we investigated the direct effects of TcdB upon HMC-1 cell "stimulated" Cm increase, as well as on "constitutive" secretion of hexosaminidase and interleukin-16 (IL-16). Compared to untreated control cells, HMC-1 cells incubated with TcdB for 3-24h exhibited a significant reduction of the mean absolute and relative Cm increase in response to free-Ca(2+) and GTPγS suggesting an inhibition of secretory processes by TcdB. In conclusion, the HMC-1 cell line represents a suitable model for the study of direct effects of C. difficile toxins on human mast cell secretory activity.

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.

IncobotulinumtoxinA has not produced a single case of antibody-induced therapy failure after 8 years of worldwide usage. We are reporting a patient with progressive hereditary juvenile onset generalised dystonia who was pretreated with abobotulinumtoxinA for 15 years, before she received incobotulinumtoxinA. To the fifth and sixth applications, she responded with complete therapy failure. Mouse hemidiaphragm assay testing revealed a maximal botulinum toxin antibody titre. Improved specific biological activity and lack of complexing proteins seem to reduce the antigenicity of incobotulinumtoxinA. However, this first ever report indicates that it does not eliminate it entirely.

Recently, it was reported that botulinum toxin type B complex (BoNT/B) (NeuroBloc(R), Elan Pharmaceuticals) can produce an adequate therapeutic response in patients with antibody induced failure of botulinum toxin type A complex (BoNT/A) therapy. We wanted to study whether this effect is transient or sustained. For this, 10 consecutive patients (6 males, 4 females, age 54.6 +/- 14.3 years, duration of illness 15.8 +/- 7.0 years) with complete BoNT/A therapy failure and BoNT/A antibody titres in excess of 10mU/ml in the mouse diaphragm assay (MDA) received BoNT/B in an initial dose of 12370 +/- 1804MU. After the first BoNT/B application the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) improved from 20.1 +/- 3.0 to 11.9 +/- 3.4. In all patients systemic anticholinergic side effects occurred. Three patients had stable continuous responses to two, three and five subsequent BoNT/B applications. Six patients showed complete secondary therapy failure to the second or third subsequent BoNT/B applications. Side effects did no longer occur. In four of them the BoNT/B doses were doubled without producing any therapeutic benefit or any side effects. In five of them MDA testing was performed and revealed BoNT/B antibody titres in excess of 1mU/ml. One patient lost half of her initial BoNT/B responsiveness indicating partial secondary BoNT/B therapy failure. This partial therapy failure was seen on two consecutive application series and has not proceeded to complete therapy failure so far. BoNT/B seems to be only temporarily effective in the majority of patients with BoNT/A antibody induced therapy failure. Whether the formation of BoNT/B antibody points to a high antigenic potency of BoNT/B, to an increased immunoreactivity in BoNT/A antibody carriers or whether it is due to the large amount of protein applied in BoNT/B therapy needs to be studied.

Botulinum neurotoxin type A (BoNT/A), despite its high toxicity, is approved for therapy of many neurological (e.g., dystonia, spasticity) and non-neurological (e.g., achalasia, hyperhidrosis) disorders. Its mode of action is well understood. This has led to more and more indications (e.g., pain, gastrointestinal and urologic disorders), in which the toxin can reduce disturbing symptoms. In general the application is safe (pharmacological index 20-100, depending on indication). Few unwanted reactions may occur. In worst cases BoNT treated patients may develop neutralizing antibodies. These patients are excluded from further treatment. A more recently approved second serotype (BoNT/B) could be effective in those secondary non-responders, however, due to less potency in humans higher doses have to be applied leading to an only transient successful treatment. Other serotypes as BoNT/A and B, e.g., BoNT/C should be approved as medicines.

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

Objectives: As the indications and duration of treatment of botulinum toxin type A (BoNT-A) increase, so do reports of patients who fail therapy after initially responding well. Although a loss of efficacy is commonly thought to be associated with neutralizing antibodies (NAbs), this relationship is not strongly correlated, and other factors may play a significant role. To explore this issue, we evaluated levels of NAbs in a large selected cohort of secondary nonresponders to BoNT-A using the highly sensitive mouse phrenic nerve-hemidiaphragm assay. Methods: Serum samples from 503 patients treated with BoNT-A who had a variety of diagnoses were tested for the presence of NAbs. Results: Fewer than half of the patients (n = 224, 44.5%) were found to be NAb-positive, indicating that in more than half of the secondary nonresponders, lack of efficacy is not due to NAb formation. The proportion of secondary nonresponders with NAbs was greater for higher dose indications (focal spasticity and spasmodic torticollis) than for lower dose indications (blepharospasm and hemifacial spasm) and increased with shorter injection intervals. Neutralizing antibody development was independent of the commercial preparation used. Conclusions: Our results indicate that although NAb formation does play a role in secondary treatment failure with BoNT-A, this is not the cause in all patients, and the influence of other factors needs to be investigated. Gaining a better understanding of the underlying mechanisms for secondary treatment failure may help in the prediction, diagnosis, management, and prevention of this problem.

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.

In earlier studies, we have demonstrated the efficacy of albumin-supplemented botulinum toxin type A (ASBTA) in principle. Here, we present long-term data from 106 patients who received ASTBA over 5-10 years for the treatment of cervical dystonia, blepharospasm and hemifacial spasm. Vials of Dysport were diluted in 0.1% albumin solution to a concentration of 25 units/ml. Overall patients and indications, the mean latency to response was 7.1 +/- 2.2 days, the mean duration of response was 12.3 +/- 3.1 weeks and the mean global clinical improvement (scale 0-3) was 2.6 +/- 0.2. Only one patient had neutralizing antibodies against BoNT-A. Side effects were less frequent than known for conventional BoNT-A and generally mild. These findings were confirmed by analysis of data of 71 patients who have been reconverted from ASBTA to conventional dilutions of Dysport or Botox. We conclude that long-term treatment with ASBTA is effective, safe and help to reduce costs.

Botulinum toxin type A (BoNT/A) proved very effective in therapy for hyperactive detrusor or sphincter dysfunction of neurogenic and non-neurogenic origin. However, therapy may fail. In a search for possible reasons, we investigated the presence of BoNT/A antibodies (BoNT/A-AB) in patients who were treated more than once and correlated the presence of antibodies with clinical findings.In 25 patients (aged 11-75 years; average, 48.3 years) who had experienced at least one previous BoNT/A detrusor and/or sphincter injection, BoNT/A-AB was detected with the mouse diaphragm assay before and within 3 months after the current injection. Clinically, subjective and objective outcomes of this injection session were determined on an efficacy scale.In eight patients, BoNT/A-AB was detected; titers were clearly positive in four patients and were borderline in four patients. The subjective and objective outcomes indicated complete therapy failure in three of four patients who were positive for BoNT/A-AB. In two patients, BoNT/A-AB developed after just one injection session.Botulinum toxin type A antibodies can develop after injection of BoNT/A for urologic disorders and the antibodies can lead to therapy failure. In patients with clinically complete therapy failure in whom no obvious other causes can be determined (such as a progressive disease in a patient with multiple sclerosis), screening for BoNT/A-AB should be carried out.

International audience; Mediator release from mast cells (MC) is a crucial step in allergic and non-allergic inflammatory disorders. However, the final events in response to activation leading to membrane fusion and thereby facilitating degranulation have hitherto not been analyzed in human MC. Soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) represent a highly conserved family of proteins that have been shown to mediate intracellular membrane fusion events. Here, we show that mature MC isolated from human intestinal tissue express soluble N-ethylmaleide sensitive factor attachment protein (SNAP)-23, Syntaxin (STX)-1B, STX-2, STX-3, STX-4, and STX-6 but not SNAP-25. Furthermore, we found that primary human MC express substantial amounts of vesicle associated membrane protein (VAMP)-3, VAMP-7 and VAMP-8 and, in contrast to previous reports about rodent MC, only low levels of VAMP-2. Furthermore, VAMP-7 and VAMP-8 were found to translocate to the plasma membrane and interact with SNAP-23 and STX-4 upon activation. Inhibition of SNAP-23, STX-4, VAMP-7 or VAMP-8, but not VAMP-2 or VAMP-3, resulted in a markedly reduced high-affinity IgE receptor-mediated histamine release. In summary, our data show that mature human MC express a specific pattern of SNARE and that VAMP-7 and VAMP-8, but not VAMP-2, are required for rapid degranulation.

Botulinum toxin (BTX) offers a new treatment option to reduce drooling in adults and children. Antibody formation against BTX is known to be one reason for clinical secondary non-response to this treatment. This is a case report on the development of secondary non-response to BTX type B (BTX-B) in a 15-year-old male, with bilateral dyskinetic cerebral palsy (Gross Motor Function Classification System Level IV) with additional learning disability* and microcephaly, treated for the indication of drooling. After three successful treatment sessions, the fourth and fifth injections showed no clinical response. This was associated with the presence of antibodies against BTX-B as determined using the mouse diaphragm assay. Thus, formation of neutralizing antibodies against BTX-B appears to be an important issue, not only in patients treated for cervical dystonia but also in children treated for drooling. Subsequent injections with an adequate dose of BTX type A (BTX-A) did not show any clinical response either, although no antibodies to BTX-A were detected. Besides the unanswered questions of dosing and distribution, a second possible explanation could be that BTX-B gave rise to non-neutralizing antibodies that cross-react with BTX-A. The resulting immune complexes could be taken up by phagocytes and, thereby, impede clinical response.

For centuries, poisons and other biological material have been considered as weapons. However, it has been merely 100 years that the use of biological toxins as weapons has been explored scientifically. Trichothecenes, ricin and botulinum neurotoxins are natural organic toxins with diverse potencies. Their molecular structure, mechanisms of action, detection, clinical diagnosis and therapy are reviewed and their potential as biological weapon is discussed. It is not only the median lethal dose of each toxin that decides on its usability as a biological weapon, but also the availability, scale of production, purity of the isolated material and route of distribution. In general, without a state infrastructure, the use of biological weapons is restricted to assassinations or strictly localised terrorist attacks.

With the advent of a commercial preparation of botulinum toxin type B (BT-B) for treatment of cervical dystonia detection of antibodies against BT-B (BT-B-AB) becomes necessary. For this purpose, we carried out a mouse diaphragm assay (MDA) by continuous measurement of the twitch force of a mouse hemidiaphragm preparation elicited by electric stimulation of its phrenic nerve. After exposing the preparation to BT-B 3 ng/ml the time to half-maximal twitch force reduction (paralysis time [PT]) was 69 +/- 4 min (n = 25). Addition of sera from patients with antibodies against BT-A produced a PT of 68 +/- 5 min (n = 24), whereas addition of sera from controls with antibodies against tetanus toxoid produced a PT of 67 +/- 6 min (n = 30). When defined amounts of BT-B-AB were added to the MDA, PT was prolonged. This prolongation was correlated closely to the amount of BT-B-AB added, thus producing a calibration curve. The threshold for BT-B-AB detection was 0.4 mU/ml. When sera from 7 patients (4 women, 3 men; age 50.6 +/- 14.2 years) with cervical dystonia (Toronto Western Spasmodic Torticollis Rating Scale score, 18.9 +/- 2.9) and complete secondary failure of BT-B therapy (NeuroBloc; Elan Pharmaceuticals, Shannon, Ireland; 12,229 +/- 2,601 MU/injection series, 1.86 +/- 0.69 injection series before complete secondary therapy failure; 100.4 +/- 15.8 days between injection series with normal therapeutic effect) were tested, BT-B-AB titers of more than 10 mU/ml were found in all of them. The MDA can be used to measure neutralizing BT-B-AB titers quantitatively and with adequate sensitivity and specificity. Further studies are necessary to understand the role of intermediate BT-B-AB titers in partial BT-B therapy failure.

Approximately 5% of patients with cervical dystonia receiving repeated botulinum neurotoxin A (BoNT/A) injections develop secondary loss of treatment benefit. Currently available tests to directly detect neutralizing BoNT/A antibodies (BoNT/A-AB) are either expensive or time consuming. To establish a simple, clinically useful test for antibody detection, we adapted the ninhydrin sweat test (NST). Eighteen dystonic patients with secondary nonresponse and clinically suspected BoNT/A-AB formation were tested for BoNT/A-AB in the mouse diaphragm test (MDT). In addition, the size of the anhidrotic area was determined by the NST 21 days after an intradermal dose of 10 U Dysport into the hypothenar region of the left palm. In nine patients, positive BoNT-AB titers were found in the MDT. There was a significant correlation between the BoNT/A-AB titers and the anhidrotic area (Spearman's rho = -0.9, P0.0001). Both tests provided comparably good results with respect to qualitative antibody detection. In the clinical situation of secondary nonresponse to BoNT/A therapy, the economical NST may be a helpful tool to detect neutralizing BoNT/A-AB.

We studied the clinical impact of neutralizing antibodies to botulinum toxin A that occurred during long-term treatment of children between 1993 and 2001. Antibodies were found in high titers in 35 of 110 (31.8%) samples from individual patients. Antibody formation correlated with secondary nonresponse (p0.001). The most significant risk factors for antibody formation were the frequency of treatments (p = 0.0001) and the injection of a higher weight-adapted maximum dose per treatment (p = 0.001).

Botulinum toxin type B (BT-B) therapy failure due to formation of botulinum toxin type B antibodies (BT-B-AB) has only been reported in patients with botulinum toxin type A antibodies (BT-A-AB). We are reporting BT-B-AB-induced therapy failure in 2 patients with no previous exposure to botulinum toxin. In patient 1 complete therapy failure occurred after a single exposure to 14,400 mouse units (MU) BT-B (NeuroBloc™). The mouse diaphragm assay (MDA) revealed a BT-B-AB titre in excess of 10 mU/ml. Doubling the BT-B dose did not elicit any effects. Application of 360 MU BT-A (Botox®) produced the original therapeutic effect, but the second BT-A application was followed by partial and the third by complete therapy failure. Doubling the BT-A dose did not elicit any effects. MDA testing showed a BT-A-AB titre in excess of 10 mU/ml. In patient 2 a single exposure to 7,200 MU BT-B lead to a complete therapy failure. MDA testing revealed a BT-B-AB titre in excess of 10 mU/ml. Doubling the BT-B dose did not elicit any effects. Application of 180 MU BT-A (Botox) produced the original response on 3 consecutive applications. Antibody formation can occur after a single exposure to botulinum toxin. However, this is highly unusual. Since therapy failure occurred after the first-ever botulinum toxin exposure, short intervals between injections and use of booster injections can be excluded as causes for BT-B-AB formation in both patients. A more likely cause may be the substantially higher amount of antigenic protein administered in BT-B therapy compared to BT-A therapy. Further studies are necessary to compare the incidence of antibody formation in BT-B and BT-A therapy.

Although the relative potency measured by the number of units per nanogram of the toxin is different for the three preparations (BOTOX = 20 U/ng; Dysport = 40 U/ng, and CS-BOT = 15.2 U/ng), the effective dose for CS-BOT is similar to that of BOTOX (Allergan, Irvine, CA). Despite the twofold difference in potency per nanogram, it appears that the clinically observable activity of 1 U of BOTOX is roughly equivalent to 3 U of the Dysport (Inamed, Santa Barbara, CA) product. Using quantitative analysis of regional paralysis produced by local injections into the gastrocnemius muscles of mice, prior studies estimated the potency ratio between Dysport and BOTOX to be 4.2 to 1. In a single-blind, randomized comparison study of Dysport and BOTOX in 91 patients with blepharospasm or hemifacial spasm, it was found that 4:1 dose ratio produced similar benefits. A similar 4:1 Dysport:BOTOX ratio was found to produce equivalent beneficial effects in a double-blind study in patients with blepharospasm, but the frequency of side effects, particularly of ptosis, was lower in the BOTOX group. In a study of 73 patients with cervical dystonia treated either with Dysport or BOTOX, it was concluded that a 3:1 ratio provides equivalent results. But a recent study concluded that the appropriate conversion factor between BOTOX and Dysport is less than 3. Therefore, there is some controversy about the relative potencies of the two preparations, with one study proposing that 1 unit of BOTOX corresponds to 1 unit of Dysport.

Aside from temporary chemodenervation of skeletal muscle and potential anti-inflammatory effects, a genuine peripheral antinociceptive effect of Botulinum Neurotoxin Type A (BoNT/A) has been suspected. To evaluate the effect of BoNT/A on cutaneous nociception in humans, 50 healthy volunteers received subcutaneous injections of 100 mouse units (MU) BoNT/A (Dysport) and placebo. Both forearms of each subject were treated in a double-blind fashion, one with verum, one with placebo. Heat and cold pain thresholds within the treated skin areas were measured with quantitative sensory testing (QST) and pain thresholds were evaluated with local electrical stimulation (ES). The tests were done before treatment, and after 4 and 8 weeks. No major side effects were noted. All participants completed the study. Heat and cold pain thresholds increased from baseline to week 4 by 1.4 degrees C for verum and by 1.1 degrees C for placebo. From baseline to week 8, the thresholds increased by 2.7 degrees C for verum and by 1.2 degrees C for placebo. Electrically induced pain thresholds shifted from baseline to week 4 by -0.07 mA for verum and by 0.01 mA for placebo. From baseline to week 8, the thresholds increased by 0.10 mA for verum and by 0.11 mA for placebo. None of these differences was statistically significant. The study shows that there is no direct peripheral antinociceptive effect of BoNT/A in humans. The efficacy of BoNT/A in various pain syndromes must be explained by other pathways such as chemodenervation or anti-inflammatory effects.

The increasing number of patients being treated with botulinum toxin A complex (BoNT/A) has led to a higher incidence of neutralizing anti-BoNT/A antibodies (ABAs). Because BoNT/A is known to inhibit sweating, here we report sudometry as a possibility for predicting the presence of ABA. Sixteen patients suffering from spasmodic torticollis were selected: in 2 patients, BoNT/A treatment continued to be effective, in 9 patients, the treatment effect was impaired, and in 5 patients, secondary treatment failure developed. BoNT/A (100 mouse units, Dysport; Ipsen Pharma, Berkshire, United Kingdom) was injected subcutaneously into the lateral calves. Sweating was visualized with iodine starch staining. In addition, quantitative sudomotor axon reflex testing was performed at the injection site. Individual ABA titers were determined with a mouse bioassay. Results of sudometry significantly correlated with the BoNT/A treatment success. The quantitative sudomotor axon reflex testing was 0.58 +/- 0.63 fraction of the normal mean in patients with treatment failure, 0.18 +/- 0.13 fraction of the normal mean in those who responded partially, and 0 in responders (p < 0.01). Accordingly, the areas of the anhidrotic skin after subcutaneous injections were 4.5 +/- 10.3 cm(2), 32.7 +/- 16.5 cm(2), and 62 cm(2) (p < 0.01). Discrimination analysis indicated that the presence of ABA (6 ABA-positive and 10 ABA-negative) could be predicted correctly in all patients from the results of sudometry. Therefore, sudometry is a useful tool for identifying patients with neutralizing ABAs and might be helpful for identifying reasons for BoNT/A treatment failure.

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.

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.

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.

Botulinum neurotoxins are increasingly used in treatment for hyperactive detrusor and sphincter function. Although reported results are promising, conditions in some patients are refractory. We report what we believe to be the first urologic case of therapy failure possibly induced by botulinum toxin antibodies after just one injection and discuss treatment alternatives based on experience in other fields.