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      Characterization of SNARE Cleavage Products Generated by Formulated Botulinum Neurotoxin Type-A Drug Products

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          Abstract

          The study evaluated substrate cleavage product(s) generated by three botulinum neurotoxin serotype A (BoNT/A) medicinal drug products utilizing a novel and highly specific, light-chain activity, high-performance liquid chromatography (LCA-HPLC) method. Samples were reacted with a commercially available BoNT/A fluorescent substrate derived from the SNAP-25 sequence. Reaction products were separated by reversed-phase HPLC. The method detected an atypical cleavage pattern by one of the formulated drug products. IncobotulinumtoxinA produced two cleavage fragments rather than the single fragment typically generated by BoNT/A. Identification confirmed the secondary cleavage at a position corresponding to SNAP-25 Arg198–Ala199 (normal BoNT/A cleavage is Gln197–Arg198). Arg198–Ala199 is also the cleavage site for trypsin and serotype C toxin. Normal cleavage was observed for all other BoNT/A drug product samples, as well as 900-kD and 150-kD bulk toxin BoNT/A. The reason for this unexpected secondary cleavage pattern by one formulated BoNT/A drug product is unknown. Possible explanations include a contaminating protease and/or damage to the 150-kD type-A toxin causing nonspecific substrate recognition and subsequent cleavage uncharacteristic of type-A toxin. The BoNT/A drug products were also analyzed via the LCA-HPLC assay using a commercial BoNT/C fluorescent substrate derived from the syntaxin sequence. Cleavage of the serotype C substrate by incobotulinumtoxinA was also confirmed whilst neither of the other drug products cleaved the syntaxin substrate.

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          How botulinum and tetanus neurotoxins block neurotransmitter release.

          B Poulain, F Doussau, Y Humeau … (2000)
          Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M(r) approximately 50) disulfide linked to a heavy chain (M(r) approximately 100). By inhibiting neurotransmitter release at distinct synapses, these toxins cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin), syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins.
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            Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage.

            V Vaidyanathan, M Jahnz, S Bade … (1998)
            Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg198 and Ala199, depends on the presence of regions Asn93 to Glu145 and Ile156 to Met202, and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex (KD = 1.9 x 10(-7) M) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met146-Gln197, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile156-Asp186. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys185Asp or Pro182Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.
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              Potency evaluation of a formulated drug product containing 150-kd botulinum neurotoxin type A.

              Terrence Hunt, Kenneth Clarke (2008)
              To assess the potency of a formulated drug product containing 150-kd botulinum toxin type A (BoNT/A) as the active pharmaceutical ingredient. Potencies of 3 unexpired lots of a commercially available BoNT/A drug product, reportedly devoid of complexing proteins (Xeomin), were determined using an approved in-house potency bioassay by injecting mice intraperitoneally and recording percent-mortality across dilutions. For each test session, duplicate sets of dilutions were performed for each lot alongside a 900-kd BoNT/A (BOTOX) potency reference standard. A relative potency for each 150-kd BoNT/A preparation was determined using this potency reference standard. A standard normalized potency estimate for each lot of 150-kd BoNT/A was calculated by multiplying the relative potency by the nominal value for the reference standard. The average potency for each 150-kd BoNT/A lot was calculated using a weighted combination of assay results and compared against the labeled potency of 100 U per vial. Similar follow-on testing was performed 1 year later to assess stability. The average potencies for the 3 lots of 150-kd BoNT/A product were 69 (95% confidence interval [CI], 65-73), 75 (95% CI, 70-80), and 78 (95% CI, 70-87) U per vial. Follow-on testing produced even lower potency results for all 3 lots. The potency of the drug product containing 150-kd BoNT/A (Xeomin) measured substantially lower than the labeled 100 U per vial when tested in a potency assay approved for release testing of an established drug product containing 900-kd BoNT/A (BOTOX).
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                Author and article information

                Journal
                Journal ID (nlm-ta): Toxins (Basel)
                Journal ID (publisher-id): toxins
                Title: Toxins
                Publisher: MDPI
                ISSN (Electronic): 2072-6651
                Publication date (Electronic): 19 August 2010
                Publication date Collection: August 2010
                Volume: 2
                Issue: 8
                Pages: 2198-2212
                Affiliations
                Author notes
                [* ] Author to whom correspondence should be addressed; Email: hunt_terrence@ 123456allergan.com ; Tel.: +1-714-246-6268; Fax: +1-714-246-5883.
                Article
                Publisher ID: toxins-02-02198
                DOI: 10.3390/toxins2082198
                PMC ID: 3153282
                PubMed ID: 22069680
                SO-VID: f9240461-cd2d-4e5b-86d4-e00ed6d44d26
                Copyright © © 2010 by the authors; licensee MDPI, Basel, Switzerland
                License:

                This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                Date received : 09 July 2010
                Date revision received : 10 July 2010
                Date accepted : 18 August 2010
                Categories
                Subject: Article

                ScienceOpen disciplines: Molecular medicine
                Keywords: abotulinumtoxina,botulinum toxin,botox®,dysport®,incobotulinumtoxina,onabotulinumtoxin a,snap-25,snare,syntaxin,xeomin®
                Data availability:
                ScienceOpen disciplines: Molecular medicine
                Keywords: abotulinumtoxina, botulinum toxin, botox®, dysport®, incobotulinumtoxina, onabotulinumtoxin a, snap-25, snare, syntaxin, xeomin®

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