Do complexing proteins provide mechanical protection for botulinum neurotoxins?

Botulinum toxin (BT) consists of botulinum neurotoxin and complexing proteins (CPs). CPs might provide mechanical protection for botulinum neurotoxin. As incobotulinumtoxinA (INCO, Xeomin®) does not contain CPs, we wanted to compare its mechanical stability to that of onabotulinumtoxinA (ONA, Botox®) containing CPs. For this, ONA and INCO were reconstituted without mechanical stress (NS) and with mechanical stress (WS) generated by a recently introduced stress test. Potencies were then measured by the paralysis times (PTs) in the mouse diaphragm assay. ONA-PT was 75.8 ± 10.3 min (n = 6) under NS and 116.7 ± 29.8 min (n = 6) under WS (two-tailed t test, p = 0.002). Mechanical stress increased the ONA-PT by 35.0% on the Growth Percentage Index. INCO-PT was 66.0 ± 7.0 min for NS and 76.0 ± 1.0 min for WS (t test, p = 0.129). Mechanical stress increased the INCO-PT by 13.2% on the Growth Percentage Index. Our data show that mechanical stress inactivates a CP-containing BT drug, but not a CP-free BT drug. We conclude that CPs do not provide protection against mechanical stress, supporting the view that CPs are not necessary for therapeutic purposes.

Comparing lanbotulinumtoxinA (Hengli®) with onabotulinumtoxinA (Botox®) and incobotulinumtoxinA (Xeomin®) in the mouse hemidiaphragm assay.

LanbotulinumtoxinA (LAN) is manufactured and registered in China since 1994. Despite its widespread use in China and its increasing use in other Asian countries and in South America, it is not yet well known elsewhere. We wanted to compare its potency labelling using the mouse diaphragm assay (MDA), an isolated muscle model for botulinum toxin (BT) potency measurements, which is superior to clinical tests and which was recently refined as an alternative batch release assay for BT manufacturing. We also wanted to estimate LAN manufacturing quality by testing its inter-batch potency consistency. Potencies of 20, 60 and 100 MU of LAN, onabotulinumtoxinA (ONA) and incobotulinumtoxinA (INCO) were measured by the inversely related paresis time (PT) in the MDA. The PT (M ± SD) of all doses of LAN, ONA and INCO was 90.4 ± 27.0 min, 114.9 ± 46.5 min and 94.3 ± 29.9 min, respectively. Statistical analysis demonstrated indistinguishable potency labelling of LAN and INCO, but revealed a slightly lower potency of ONA compared to LAN and INCO. PT of LAN batch 1 and LAN batch 2 was 86.9 ± 21.2 min and 94.0 ± 32.8 min, respectively (no statistically significant difference), suggesting an adequate LAN manufacturing consistency. The MDA is an appropriate instrument for potency testing of BT drugs, including new ones currently under development. Our results allow comparing therapeutic effects, adverse effects and economics of LAN, ONA and INCO. They also suggest adequate manufacturing consistency of LAN.

Botulinum toxin type D blocks autonomic cholinergic synapses in humans: discussion of a potential therapeutic use.

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.

Comparing incobotulinumtoxinA (Xeomin®) and onabotulinumtoxinA (Botox®): identical potency labelling in the hemidiaphragm assay.

Botulinum toxin (BT) is provided by several manufacturers producing a number of different drugs. Their potency is given in internationally standardised mouse units (MU). Clinical practise, however, reveals that the potency labelling of different BT drugs may not be identical. We wanted to use the mouse diaphragm assay (MDA) to compare the two BT drugs onabotulinumtoxinA (ONA) and incobotulinumtoxinA (INCO). For this, we measured the paresis time (PT) of different ONA or INCO doses. All BT came from several different and unexpired drug batches. PT for 20MU were 169.7 ± 28.9 min (ONA) and 132.3 ± 1.5 min (INCO) (p = 0.089), for 60MU 105.3 ± 10.1 min and 84.7 ± 4.2 min (p = 0.031), for 100MU 69.7 ± 1.5 min and 66.0 ± 7.0 min (p = 0.462) and for 140MU 74.7 ± 0.6 min and 62.3 ± 2.1 min (p = 0.100), respectively. The overall PT were 104.8 ± 12.5 and 86.3 ± 8.5 min (p = 0.178). Results presented here do not reveal differences in potency labelling between ONA and INCO, even when the full range of therapeutic doses are examined, although there was a trend towards stronger INCO effects. Data confirm previous reports on identical potency labelling of ONA and INCO. The MDA seems to be an appropriate instrument to test the potency labelling of other BT drugs as well, including new BT drugs currently under development.

Long-term stability of reconstituted incobotulinumtoxinA: how can we reduce costs of botulinum toxin therapy?

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.

Reconstituting botulinum toxin drugs: shaking, stirring or what?

Most botulinum toxin (BT) drugs are stored as powders which need to be reconstituted with normal saline before clinical use. As botulinum neurotoxin (BNT), the therapeutically active ingredient, is a large double-stranded protein the process of reconstitution should be performed with special attention to mechanical stress applied. We wanted to test the mechanical stability of BNT during the reconstitution process. For this, 100 MU onabotulinumtoxinA (Botox(®), Irvine, CA, USA) was reconstituted with 2.0 ml of NaCl/H2O. Gentle reconstitution (GR) was performed with a 5 ml syringe, a 0.90 × 70 mm injection needle, one cycle of injection-aspiration-injection and two gentle shakes of the vial. Aggressive reconstitution (AR) was performed with a 5 ml syringe, a 0.40 × 40 mm injection needle, ten injection-aspiration-injection cycles and 30 s of continuous shaking of the vial. AR increased the time to paralysis in the mouse hemidiaphragm assay (HDA) from 72.0 ± 4.6 to 106.0 ± 16.0 min (*p = 0.002, two-tailed t test after Kolmogorov-Smirnova test with Lilliefors correction for normal distribution). Construction of a calibration curve revealed that the increase in the time to paralysis was correlated with a loss of potency of from 100 to 58 MU (-42 %). BT users should use large diameter injection needles for reconstitution, apply two or three injection-aspiration-injection cycles and, maybe, shake the vials a few times to rinse the entire glass wall. Aggressive reconstitution with small diameter needles, prolonged injection-aspiration-injection and violent shaking should be avoided.

Botulinum toxin therapy: reduction of injection site pain by pH normalisation.

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.

Botulinum toxin: application, safety, and limitations.

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.

An enzyme-linked immunosorbent assay for detection of botulinum toxin-antibodies.

INTRODUCTION: Antibodies against botulinum neurotoxin (BNT-AB) can be detected by the mouse protection assay (MPA), the hemidiaphragm assay (HDA), and by enzyme-linked immunosorbent assays (ELISA). Both MPA and HDA require sacrifice of experimental animals, and they are technically delicate and labor intensive. We introduce a specially developed ELISA for detection of BNT-A-AB and evaluate it against the HDA. METHODS: Thirty serum samples were tested by HDA and by the new ELISA. Results were compared, and receiver operating characteristic analyses were used to optimize ELISA parameter constellation to obtain either maximal overall accuracy, maximal test sensitivity, or maximal test specificity. When the ELISA is optimized for sensitivity, a sensitivity of 100% and a specificity of 55% can be reached. When it is optimized for specificity, a specificity of 100% and a sensitivity of 90% can be obtained. RESULTS: We present an ELISA for BNT-AB detection that can be-for the first time-customized for special purposes. Adjusted for optimal sensitivity, it reaches the best sensitivity of all BNT-AB tests available. CONCLUSIONS: Using the new ELISA together with the HDA as a confirmation test allows testing for BNT-AB in large numbers of patients receiving BT drugs in an economical, fast, and more animal-friendly way.

IncobotulinumtoxinA (Xeomin(®)) can produce antibody-induced therapy failure in a patient pretreated with abobotulinumtoxinA (Dysport(®)).

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.

SNARE tagging allows stepwise assembly of a multimodular medicinal toxin.

Generation of supramolecular architectures through controlled linking of suitable building blocks can offer new perspectives to medicine and applied technologies. Current linking strategies often rely on chemical methods that have limitations and cannot take full advantage of the recombinant technologies. Here we used SNARE proteins, namely, syntaxin, SNAP25, and synaptobrevin, which form stable tetrahelical complexes that drive fusion of intracellular membranes, as versatile tags for irreversible linking of recombinant and synthetic functional units. We show that SNARE tagging allows stepwise production of a functional modular medicinal toxin, namely, botulinum neurotoxin type A, commonly known as BOTOX. This toxin consists of three structurally independent units: Receptor-binding domain (Rbd), Translocation domain (Td), and the Light chain (Lc), the last being a proteolytic enzyme. Fusing the receptor-binding domain with synaptobrevin SNARE motif allowed delivery of the active part of botulinum neurotoxin (Lc-Td), tagged with SNAP25, into neurons. Our data show that SNARE-tagged toxin was able to cleave its intraneuronal molecular target and to inhibit release of neurotransmitters. The reassembled toxin provides a safer alternative to existing botulinum neurotoxin and may offer wider use of this popular research and medical tool. Finally, SNARE tagging allowed the Rbd portion of the toxin to be used to deliver quantum dots and other fluorescent markers into neurons, showing versatility of this unique tagging and self-assembly technique. Together, these results demonstrate that the SNARE tetrahelical coiled-coil allows controlled linking of various building blocks into multifunctional assemblies.

Botulinum toxin antibody titres: measurement, interpretation, and practical recommendations.

Botulinum toxin (BT) therapy may be blocked by antibodies (BT-AB) resulting in BT-AB induced therapy failure (ABF). BT-AB may be detected by the mouse lethality assay (MLA), the mouse diaphragm assay (MDA) and the sternocleidomastoid test (SCMT). For the first time, we wanted to compare all three BT-AB tests and correlate them to subjective complaint of complete or partial secondary therapy failure in 37 patients with cervical dystonia (25 females, 12 males, age 51.2 ± 11.4 years, disease duration 12.4 ± 6.3 years). Complaint of therapy failure was not correlated with any of the BT-AB tests. MDA and MLA are closely correlated, indicating that the MDA might replace the MLA as the current gold standard for BT-AB measurement. The SCMT is closely correlated with MDA and MLA confirming that BT-AB titres and BT's paretic effect are in a functional balance: low BT-AB titres are reducing BT's paretic effect only marginally, whereas high BT-AB titres may completely block it. When therapy failure is classified as secondary and permanent, BT-AB evaluation is recommended and any BT-AB test may be applied. For MDA > 10 mU/ml, MLA > 3 and SCMT < 25%, ABF is highly likely. MDA < 0.6 mU/ml are therapeutically irrelevant. They are neither correlated with pathologic MLA nor with pathologic SCMT. They should not be the basis for treatment decisions, such as switching dystonia therapy to deep brain stimulation. All other results are intermediate results. Their interactions with therapy efficacy is unpredictable. In these cases, BT-AB tests should be repeated or one or two additional test methods should be applied.

Significantly lower antigenicity of incobotulinumtoxin than abo- or onabotulinumtoxin.

BACKGROUND: For many indications, BoNT/A is repetitively injected with the risk of developing neutralizing antibodies (NABs). Therefore, it is important to analyze whether there is a difference in antigenicity between the different licensed BoNT/A preparations. METHODS: In this cross-sectional study, the prevalence of NABs was tested by means of the sensitive mouse hemidiaphragm assay (MHDA) in 645 patients. Patients were split into those having exclusively been treated with the complex protein-free incoBoNT/A preparation (CF-MON group) and those having started BoNT/A therapy with a complex protein-containing BoNT/A preparation (CC-I group). This CC-I group was split into those patients who remained either on abo- or onaBoNT/A (CC-MON group) and those who had been treated with at least two BoNT/A preparations (CC-SWI group). To balance treatment duration, only CC-MON patients who did not start their BoNT/A therapy more than 10 years before recruitment (CC-MON-10 group) were further analyzed. The log-rank test was used to compare the prevalence of NABs in the CF-MON and CC-MON-10 group. RESULTS: In the CF-MON subgroup, no patient developed NABs. In the CC-I group, 84 patients were NAB-positive. NABs were found in 33.3% of those who switched preparations (CC-SWI) and in 5.9% of the CC-MON-10 group. Kaplan-Meier curves for remaining NAB-negative under continuous BoNT/A therapy were significantly different (p < 0.035) between the CF-MON and CC-MON-10 group. CONCLUSION: Frequent injections of a complex protein-containing BoNT/A preparation are associated with significantly higher risks of developing NABs than injections with the same frequency using the complex protein-free incoBoNT/A preparation.

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites.

The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins.

The immunology of botulinum toxin therapy: A brief summary.

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.

Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner.

The extraordinarily high toxicity of botulinum neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven BoNT (botulinum neurotoxin) serotypes A-G inhibit acetylcholine release leading to flaccid paralysis. Uptake of BoNT/A, B, E, F and G requires a dual interaction with gangliosides and the synaptic vesicle proteins synaptotagmin or SV2 (synaptic vesicle glycoprotein 2), whereas little is known about the cell entry mechanisms of the serotypes C and D, which display the lowest amino acid sequence identity compared with the other five serotypes. In the present study we demonstrate that the neurotoxicity of BoNT/D depends on the presence of gangliosides by employing phrenic nerve hemidiaphragm preparations derived from mice expressing the gangliosides GM3, GM2, GM1 and GD1a, or only GM3 [a description of our use of ganglioside nomenclature is given in Svennerholm (1994) Prog. Brain Res. 101, XI-XIV]. High-resolution crystal structures of the 50 kDa cell-binding domain of BoNT/D alone and in complex with sialic acid, as well as biological analyses of single-site BoNT/D mutants identified two carbohydrate-binding sites. One site is located at a position previously identified in BoNT/A, B, E, F and G, but is lacking the conserved SXWY motif. The other site, co-ordinating one molecule of sialic acid, resembles the second ganglioside-binding pocket (the sialic-acid-binding site) of TeNT (tetanus neurotoxin).

Long-term adherence and response to botulinum toxin in different indications.

OBJECTIVE: The objective of the study was the analysis of adherence and self-perceived treatment response to long-term botulinum neurotoxin type A (BoNT-A) treatment in different neurological indications. METHODS: In this retrospective, monocentric, observational study, cross-sectional and longitudinal data of 1351 patients documenting 20705 injection appointments at the BoNT outpatient clinic of Heinrich Heine University Duesseldorf between 1989 and 2014 were retrospectively analyzed. Patients had been treated with BoNT for neurological conditions, including cervical dystonia (CD), blepharospasm (BSP), other dystonia (ODT), hemifacial spasm (HFS), and spasticity (SPAS). The parameters longitudinally analyzed for the entire cohort were therapy duration as well as the mean and cumulative BoNT-A dose. Cross-sectionally, for subgroups of at least 721, patients' global self-perceived quality of health and life, global self-perceived reduction of symptoms by BoNT-A treatment as well as the clinical global impression were evaluated. Furthermore, mouse hemidiaphragm assay antibodies (MHDA-ABs) were analyzed in a subgroup. RESULTS: The mean treatment duration was 4.58 years (95% CI 4.32-4.84), and 678 (50.2%) therapy dropouts of 1351 patients occurred within the first 8 years. Therapy adherence and self-perceived symptom reduction in long-term BoNT-A treatment over the years were significantly longer in BSP, HFS, and CD patients than in ODT and SPAS patients. INTERPRETATION: The treatment indication determines long-term adherence and self-perceived symptom reduction in BoNT-A therapy, which are better in BSP, HFS, and CD patients than in ODT and SPAS patients. MHDA-ABs had a significant impact on global self-perceived symptom reduction, but with only a limited degree.

Antibody-induced failure of botulinum toxin a therapy in cosmetic indications.

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.

Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor.

The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A-G (BoNT/A-G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.

Experience with long-term treatment with albumin-supplemented botulinum toxin type A.

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.