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The safety of botulinum neurotoxin type A's intraarticular application in experimental animals.

ABSTRACT: In vivo studies of botulinum neurotoxin type A (BoNT-A) enabled characterization of its activity in the nociceptive sensory system separate from its preferred action in motor and autonomic nerve terminals. However, in the recent rodent studies of arthritic pain which employed high intra-articular (i.a.) doses (expressed as a total number of units (U) per animal or U/kg), possible systemic effects have not been conclusively excluded. Herein we assessed the effect of two pharmaceutical preparations, abobotulinumtoxinA (aboBoNT-A, 10, 20, and 40 U/kg corresponding to 0.05, 0.11, and 0.22 ng/kg neurotoxin) and onabotulinumtoxinA (onaBoNT-A, 10 and 20 U/kg corresponding to 0.09 and 0.18 ng/kg, respectively) injected into the rat knee, on safety-relevant readouts: digit abduction, motor performance and weight gain during 14 days post-treatment. The i. a. toxin produced dose-dependent impairment of the toe spreading reflex and rotarod performance, which was moderate and transient after 10 U/kg onaBoNT-A and ≤20 U/kg aboBoNT-A doses, and severe and long-lasting (examined up to 14 days) after ≥20 U/kg of onaBoNT-A and 40 U/kg aboBoNT-A. In addition, lower toxin doses prevented the normal weight gain compared to controls, while higher doses induced marked weight loss (≥20 U/kg of onaBoNT-A and 40 U/kg aboBoNT-A). Commonly employed BoNT-A formulations, depending on the doses, cause local relaxation of the surrounding muscles and systemic adverse effects in rats. Thus, to evade possible toxin unwanted local or systemic spread, careful dosing and motor testing should be mandatory in preclinical behavioral studies, irrespective of the sites and doses of toxin application.

SUBMITTER: Okrosa AD

PROVIDER: S-EPMC10121478 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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The safety of botulinum neurotoxin type A's intraarticular application in experimental animals.

Okroša Ana Dugonjić AD Muñoz-Lora Victor Ricardo Manuel VRM Matak Ivica I Bach-Rojecky Lidija L Kalinichev Mikhail M Lacković Zdravko Z

Toxicon: X 20230331

In vivo studies of botulinum neurotoxin type A (BoNT-A) enabled characterization of its activity in the nociceptive sensory system separate from its preferred action in motor and autonomic nerve terminals. However, in the recent rodent studies of arthritic pain which employed high intra-articular (i.a.) doses (expressed as a total number of units (U) per animal or U/kg), possible systemic effects have not been conclusively excluded. Herein we assessed the effect of two pharmaceutical preparation ...[more]

PMID: 37096009

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Project description:Experimental studies have demonstrated that botulinum neurotoxin serotype A (BoNT/A) causes flaccid paralysis by a multi-step mechanism. Following its binding to specific receptors at peripheral cholinergic nerve endings, BoNT/A is internalized by receptor-mediated endocytosis. Subsequently its zinc-dependent catalytic domain translocates into the neuroplasm where it cleaves a vesicle-docking protein, SNAP-25, to block neurally evoked cholinergic neurotransmission. We tested the hypothesis that mathematical models having a minimal number of reactions and reactants can simulate published data concerning the onset of paralysis of skeletal muscles induced by BoNT/A at the isolated rat neuromuscular junction (NMJ) and in other systems. Experimental data from several laboratories were simulated with two different models that were represented by sets of coupled, first-order differential equations. In this study, the 3-step sequential model developed by Simpson (J Pharmacol Exp Ther 212:16-21,1980) was used to estimate upper limits of the times during which anti-toxins and other impermeable inhibitors of BoNT/A can exert an effect. The experimentally determined binding reaction rate was verified to be consistent with published estimates for the rate constants for BoNT/A binding to and dissociating from its receptors. Because this 3-step model was not designed to reproduce temporal changes in paralysis with different toxin concentrations, a new BoNT/A species and rate (k(S)) were added at the beginning of the reaction sequence to create a 4-step scheme. This unbound initial species is transformed at a rate determined by k(S) to a free species that is capable of binding. By systematically adjusting the values of k(S), the 4-step model simulated the rapid decline in NMJ function (k(S) >or= 0.01), the less rapid onset of paralysis in mice following i.m. injections (k (S) = 0.001), and the slow onset of the therapeutic effects of BoNT/A (k(S) < 0.001) in man. This minimal modeling approach was not only verified by simulating experimental results, it helped to quantitatively define the time available for an inhibitor to have some effect (t(inhib)) and the relation between this time and the rate of paralysis onset. The 4-step model predicted that as the rate of paralysis becomes slower, the estimated upper limits of (t(inhib)) for impermeable inhibitors become longer. More generally, this modeling approach may be useful in studying the kinetics of other toxins or viruses that invade host cells by similar mechanisms, e.g., receptor-mediated endocytosis.

Project description:BackgroundClostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex.ResultsAn in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs--nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison.ConclusionsThe in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MS(E) data analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.

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The safety of botulinum neurotoxin type A's intraarticular application in experimental animals.

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The safety of botulinum neurotoxin type A's intraarticular application in experimental animals.

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