An in vitro cell-based potency assay for pharmaceutical type A botulinum antitoxins.

Botulism therapy relies on passive immunization with antitoxin. The mouse neutralization test is the only pharmacopeia assay to measure the potency of antitoxin preparations. Herein, we present an in vitro cell-based assay for the measurement of pharmaceutical type A antitoxin potency. Accuracy, reproducibility and compatibility with the mouse bioassay were demonstrated using different batches of standard antitoxin and toxin preparations. The established assay may substantially reduce the use of laboratory animals in the process of pharmaceutical antitoxin production.

Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins.

Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV < 13%. Most importantly, the antitoxin potency measured by the in vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p < 0.0001). Thus, this new in vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval.

The thioredoxin reductase--Thioredoxin redox system cleaves the interchain disulphide bond of botulinum neurotoxins on the cytosolic surface of synaptic vesicles.

Botulinum neurotoxins (BoNTs) are Janus toxins, as they are at the same time the most deadly substances known and one of the safest drugs used in human therapy. They specifically block neurotransmission at peripheral nerves through the proteolysis of SNARE proteins, i.e. the essential proteins which are the core of the neuroexocytosis machinery. Even if BoNTs are traditionally known as seven main serotypes, their actual number is much higher as each serotype exists in many different subtypes, with individual biological properties and little antigenic relations. Since BoNTs can be used as biological weapons, and the only currently available therapy is based on immunological approaches, the existence of so many different subtypes is a major safety problem. Nevertheless, all BoNT isoforms are structurally similar and intoxicate peripheral nerve endings via a conserved mechanism. They consist of two chains linked by a unique disulphide bond which must be reduced to enable their toxicity. We found that thioredoxin 1 and its reductase compose the cell redox system responsible for this reduction, and its inhibition via specific chemicals significantly reduces BoNTs activity, in vitro as well as in vivo. Such molecules can be considered as lead compounds for the development of pan-inhibitors.

Recent developments with metalloprotease inhibitor class of drug candidates for botulinum neurotoxins.

Botulinum Neurotoxins are the most poisonous of all toxins with lethal dose in nanogram quantities. They are potential biological warfare and bioterrorism agents due to their high toxicity and ease of preparation. On the other hand BoNTs are also being increasingly used for therapeutic and cosmetic purposes, and with that the chances of accidental overdose are increasing. And despite the potential damage they could cause to human health, there are no post-intoxication drugs available so far. But progress is being made in this direction. The crystal structures in native form and bound with substrate peptides have been determined, and these are enabling structure-based drug discovery possible. High throughput assays have also been designed to speed up the screening progress. Substrate-based and small molecule inhibitors have been identified. But turning high affinity inhibitors into clinically viable drug candidates has remained a challenge. We discuss here the latest developments and the future challenges in drug discovery for Botulinum neurotoxins.

Comparación de resultados obtenidos en el tratamiento del dolor miofascial de la cintura pélvica con toxina botulínica sola y asociada con lidocaína / Comparison of results obtained in the treatment of the myofascial pain of the pelvic girdle with botulinum toxin alone and associated with lidocaine

Objetivos: entre el 70 y el 85 % de la población adulta sufre de dolor de espalda alguna vez en su vida. El síndrome de dolor miofascial (SDM) ha sido descrito recientemente definiéndose como dolor musculoesquelético no inflamatorio, localizado, desarrollado sin causa aparente, refractario a tratamientos farmacológicos y físicos, y se acompaña de la presencia de puntos gatillos y de bandas tensas palpables en el músculo. Su prevalencia se estima que varía entre un 30 y un 85 %. Los músculos psoas, cuadrado lumbar y piramidal son los más frecuentemente implicados en el SDM de cintura pélvica. Una de las principales alternativas para tratar el SDM es la toxina botulínica tipo A (TB), que actúa en la membrana sináptica en la placa neuromuscular, inhibiendo la liberación de acetilcolina, produciendo relajación muscular y alivio del dolor, aunque, en muchas ocasiones, su efecto no se hace evidente hasta transcurridos varios días. La lidocaína es un anestésico local (AL) tipo amida con duración de acción intermedia que actúa impidiendo la propagación del impulso nervioso disminuyendo la permeabilidad de los canales de sodio. El objetivo de este estudio era comprobar si al añadir AL a ladosis de TB, conseguíamos un acortamiento en el tiempo dela reducción de la EVA y mejoría de la calidad de vida. Material y métodos: el diseño del estudio fue prospectivo, controlado, longitudinal y aleatorizado en el que se ha valorado la evolución de 20 pacientes divididos en dos grupos. Al primer grupo se les administró TB tipo A (grupo T). Al segundo grupo se les trató con TB tipo A y dosis adicional de lidocaína al 2% (grupo TL). Previamente, ambos grupos, habían respondido de forma positiva a un test con infiltración del músculo afecto con lidocaína al 2 %. El seguimiento de los pacientes se hizo secuencialmente a los 3, 7, 15 y 90 días de iniciado el tratamiento. Para el análisis estadístico se utilizó un análisis de la varianza, ANOVA, complementada por la prueba de Mauchly para comprobación de la esfericidad y la prueba de Greenhouse-Geisser, con un intervalo de confianza del 95 %, considerando una p<0,05 para establecer diferencias estadísticas. Resultados: hubo diferencia estadísticamente significativa entre la EVA del grupo TL Y TB en la valoración a los tres días, del mismo modo en la evaluación del índice de Lattinen. No hubo diferencias significativas en el resto de valoraciones. En ambos grupos hubo diferencia significativa en la reducción del EVA y mejoría del índice de Lattinen, al principio y final del estudio. Conclusiones: la TXB-A presenta una alternativa al tratamiento de este cuadro cuando la terapia conservadora ha fracasado. Los anestésicos locales producen una relajación previsible, breve y reversible de la musculatura provocada por el bloqueo de la conducción nerviosa en las terminaciones nerviosas, mientras que la TXB actúa en las terminaciones neuronales de la placa motora, impidiendo la liberación de la acetil colina. Su acción la ejercen en lugares distintos y con características diferentes. La acción de los anestésicos locales es casi instantánea y breve, la de la TXB es diferida y duradera en el tiempo, por lo que pueden ser complementarias y agonistas en su efecto final

Objectives: between 70 and 85 % of the adult population suffers from back pain sometime in their life. Myofascial pain syndrome (MPS) has been described recently and defined as a localized non-inflammatory musculoskeletal pain, developed without apparent cause, being refractory to pharmacological and physical treatments, and is accompanied by the presence of trigger points and palpable taut bands in the muscle. Its prevalence is estimated to vary between 30 and 85 %. The psoas, quadratus lumborum and pyramidal muscles are the most frequently involved in the pelvic girdle MPS. One of the main alternatives to treat MPS is botulinum toxin type A (BT), which acts in the synaptic membrane at the neuromuscular junction, inhibiting the release of acetylcholine, producing muscle relaxation and pain relief, although in many cases its effect is not evident until several days have passed. Lidocaine is an amide type local anesthetic with an intermediate duration of action, which act by preventing the propagation of nerve impulses by decreasing the permeability of sodium channels. The objective of this study was to test whether adding LA to the BT dose, we got a shortening in the time of the reduction of EVA and improvement in quality of life. Material and methods: the study design was prospective, controlled, longitudinal and randomized in which we have evaluated the evolution of 20 patients randomly divided into two groups. The first group were given BT A type (group T). The second group was treated with BT A type and an additional dose of 2 % lidocaine (group TL). Previously, both groups had responded positively to a test with lidocaine 2 % infiltration of the affected muscle. Monitoring patients was sequentially to 3, 7, 15 and 90 days of treatment performed. For statistical analysis we used an analysis of variance, ANOVA, complemented by Mauchly test for sphericity check and by Greenhouse-Geisser test, with a confidence interval of 95 %, considering p < 0.05 to establish statistical differences. Results: there was statistically significant difference between group EVA TL and TB in the assessment on the third day, just as in the evaluation of Lattinen Index. No significant differences in the other reviews. In both groups there was significant difference in EVA reduction and Lattinen Index improvement at the beginning and end of the study. Conclusions: BT-A presents an alternative to the management of this condition when conservative therapy has failed. Local anesthetics cause a predictable, short and reversible muscle relaxation caused by blocking nerve conduction in nerve endings, while BT acts on the neuronal endings of the motor plate, preventing the release of acetylcholine. Its action is exercised in different places and with different characteristics. The action of local anesthetics is almost instantaneous and short, the TXB action is delayed and long lasting, so both can be complementary and agonists in their final effect

Prolonged prophylactic protection from botulism with a single adenovirus treatment promoting serum expression of a VHH-based antitoxin protein.

Current therapies for most acute toxin exposures are limited to administration of polyclonal antitoxin serum. We have shown that VHH-based neutralizing agents (VNAs) consisting of two or more linked, toxin-neutralizing heavy-chain-only VH domains (VHHs), each binding distinct epitopes, can potently protect animals from lethality in several intoxication models including Botulinum neurotoxin serotype A1 (BoNT/A1). Appending a 14 amino acid albumin binding peptide (ABP) to an anti-BoNT/A1 heterodimeric VNA (H7/B5) substantially improved serum stability and resulted in an effective VNA serum half-life of 1 to 2 days. A recombinant, replication-incompetent, adenoviral vector (Ad/VNA-BoNTA) was engineered that induces secretion of biologically active VNA, H7/B5/ABP (VNA-BoNTA), from transduced cells. Mice administered a single dose of Ad/VNA-BoNTA, or a different Ad/VNA, via different administration routes led to a wide range of VNA serum levels measured four days later; generally intravenous > intraperitoneal > intramuscular > subcutaneous. Ad/VNA-BoNTA treated mice were 100% protected from 10 LD50 of BoNT/A1 for more than six weeks and protection positively correlated with serum levels of VNA-BoNTA exceeding about 5 ng/ml. Some mice developed antibodies that inhibited VNA binding to target but these mice displayed no evidence of kidney damage due to deposition of immune complexes. Mice were also successfully protected from 10 LD50 BoNT/A1 when Ad/VNA-BoNTA was administered up to 1.5 hours post-intoxication, demonstrating rapid appearance of the protective VNA in serum following treatment. Genetic delivery of VNAs promises to be an effective method of providing prophylactic protection and/or acute treatments for many toxin-mediated diseases.

A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model.

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V(H) (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.

Binding activity and immunogenic characterization of recombinant C-terminal quarter and half of the heavy chain of botulinum neurotoxin serotype A.

In the present study, we explored and compared the binding activity and immunogenic characterization of the most effective part corresponding to C-terminal quarter of heavy chain of botulinum neurotoxin serotype A (AHc-C) with C-terminal half of heavy chain of botulinum neurotoxin serotype A (AHc). Firstly, the fully soluble AHc-C protein successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx) was shown to bind with ganglioside as the AHc, indicating that the recombinant AHc-C protein retains a functionally active conformation. Furthermore, a solid-phase assay showed that the anti-AHc-C sera effectively inhibited the binding of AHc or AHc-C to the ganglioside GT1b, the first step in BoNT/A intoxication of neurons, as good as the anti-AHc sera. Finally, although the recombinant AHc-C protein still induced a high serum antibody titers and afforded protection level as the mice challenged with active botulinum neurotoxin serotype A, the immunization with AHc protein induced stronger protective potency than the AHc-C protein. The data presented in the report shows that there are the same ganglioside binding activity and different immunogenic characterization between the C-terminal quarter and half of heavy chain of botulinum neurotoxin serotype A. Therefore, the recombinant AHc-C protein can not only be developed into a minimal subunit candidate vaccine for prophylaxis against botulinum neurotoxin serotype A but also be used as a promising tool in the search for binding inhibitors and chimeric vaccines.

Molecular bases of protective immune responses against botulinum neurotoxin A--how antitoxin antibodies block its action.

In studies from this laboratory, we localized the regions on the H chain of botulinum neurotoxin A (BoNT/A) that are recognized by anti-BoNT/A antibodies (Abs) and block the activity of the toxin in vivo. These Abs were obtained from cervical dystonia patients who had been treated with BoNT/A and had become unresponsive to the treatment, as well as blocking Abs raised in mouse, horse, and chicken. We also localized the regions involved in BoNT/A binding to mouse brain synaptosomes (snp). Comparison of spatial proximities in the three-dimensional structure of the Ab-binding regions and the snp binding showed that except for one, the Ab-binding regions either coincide or overlap with the snp regions. It should be folly expected that protective Abs when bound to the toxin at sites that coincide or overlap with snp binding would prevent the toxin from binding to nerve synapse and therefore block toxin entry into the neuron. Thus, analysis of the locations of the Ab-binding and the snp-binding regions provides a molecular rationale for the ability of protecting Abs to block BoNT/A action in vivo.

Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin.

Lectins from Anguilla anguilla, Artocarpus integrifolia, Canavalia ensiformis, Datora stramonium, Glycine max, Limax flavus, Ricinus communis and Triticum vulgaris were tested for their abilities to antagonize the binding of botulinum neurotoxin and tetanus toxin to rat brain membranes and to antagonize the ability of these toxins to block neuromuscular transmission in mouse phrenic nerve-hemidiaphragm preparations. Lectins from Limax flavus and Triticum vulgaris, both of which have affinity for sialic acid, were antagonists of the various serotypes of botulinum neurotoxin and tetanus toxin. When tested against the high affinity binding site for botulinum neurotoxin type B, the lectin from Limax flavus had a Ki of 3.1 x 10(-7) M and the lectin from Triticum vulgaris had a Ki of 3.75 x 10(-7) M. When tested against the high affinity binding site for tetanus toxin, the lectins from Limax flavus and Triticum vulgaris had Ki values of 1.5 x 10(-7) and 1 x 10(-6) M, respectively. In all cases the lectins behaved as competitive antagonists. In reverse experiments, neither botulinum toxin nor tetanus toxin was a very effective antagonist of lectin binding to brain membranes. Studies on isolated neuromuscular preparations showed that the lectin from Triticum vulgaris did not affect transmission at concentrations of 10(-6) to 10(-3) M, but at a concentration of 3 x 10(-5) M the lectin produced highly statistically significant antagonism of the neuromuscular blocking properties of botulinum neurotoxin types A, B, C, D, E and F as well as tetanus toxin. The lectin did not antagonize beta-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)