Botulinum neurotoxin (BoNT) is a major therapeutic agent that is licensed in neurological indications, such as dystonia and spasticity. The BoNT family, which is produced in nature by clostridial bacteria, comprises several pharmacologically distinct proteins with distinct properties. In this review, we present an overview of the current therapeutic landscape and explore the diversity of BoNT proteins as future therapeutics. In recent years, novel indications have emerged in the fields of pain, migraine, overactive bladder, osteoarthritis, and wound healing. The study of biological effects distal to the injection site could provide future opportunities for disease-tailored BoNT therapies. However, there are some challenges in the pharmaceutical development of BoNTs, such as liquid and slow-release BoNT formulations; and, transdermal, transurothelial, and transepithelial delivery. Innovative approaches in the areas of formulation and delivery, together with highly sensitive analytical tools, will be key for the success of next generation BoNT clinical products.

Botulinum neurotoxins (BoNTs) are highly successful protein therapeutics. Over 40 naturally occurring BoNTs have been described thus far and, of those, only 2 are commercially available for clinical use. Different members of the BoNT family present different biological properties but share a similar multi-domain structure at the molecular level. In nature, BoNTs are encoded by DNA in producing clostridial bacteria and, as such, are amenable to recombinant production through insertion of the coding DNA into other bacterial species. This, in turn, creates possibilities for protein engineering. Here, we review the production of BoNTs by the natural host and also recombinant production approaches utilised in the field. Applications of recombinant BoNT-production include the generation of BoNT-derived domain fragments, the creation of novel BoNTs with improved performance and enhanced therapeutic potential, as well as the advancement of BoNT vaccines. In this article, we discuss site directed mutagenesis, used to affect the biological properties of BoNTs, including approaches to alter their binding to neurons and to alter the specificity and kinetics of substrate cleavage. We also discuss the target secretion inhibitor (TSI) platform, in which the neuronal binding domain of BoNTs is substituted with an alternative cellular ligand to re-target the toxins to non-neuronal systems. Understanding and harnessing the potential of the biological diversity of natural BoNTs, together with the ability to engineer novel mutations and further changes to the protein structure, will provide the basis for increasing the scope of future BoNT-based therapeutics.

Botulinum neurotoxin (Bo NT ) is a major therapeutic agent. Of seven native Bo NT serotypes (A to G), only A and B are currently used in the clinic. Here we compared the potency of commercially available purified native serotypes A1 to F1 across in vitro, ex vivo, and in vivo assays. Bo NT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. Bo NT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm ( PNHD ) assay, measuring muscle contractility. In vivo , Bo NT ‐induced muscle relaxation in mice and rats was assessed in the Digit Abduction Score ( DAS ) test, while effects on body weight (BW) gain were used to assess tolerability. In all assays, all Bo NT serotypes were potent toxins, except serotype D1 in vivo which failed to produce significant muscle flaccidity in mice and rats. In rats, all serotypes were well‐tolerated, whereas in mice, reductions in BW were detected at high doses. Serotype A1 was the most potent serotype across in vitro, ex vivo, and in vivo assays. The rank order of potency of the serotypes revealed differences among assays. For example, species‐specificity was seen for serotype B1, and to a lesser extent for serotype C1. Serotypes F1 and C1, not currently in the clinic, showed preference for sensory over motor models and therefore could be considered for development in conditions involving the somatosensory system.

Botulinum neurotoxins (BoNTs) are important therapeutic agents. The in vivo median lethal dose (LD₅₀) assay has been commonly used to measure the potency of BoNT commercial preparations. As an alternative, we developed cell-based assays for abobotulinumtoxinA in both powder (Dysport^®, Azzalure^®) and liquid (Alluzience^®) formulations using the in vitro BoCell^® system. The assays demonstrated linearity over 50–130% of the expected relative potency, with a correlation coefficient of 0.98. Mean recoveries of 90–108% of the stated potency were observed over this range. The coefficients of variation for powder and liquid formulations, respectively, were 3.6% and 4.0% for repeatability and 8.3% and 5.0% for intermediate precision. A statistically powered comparability assessment of the BoCell^® and LD₅₀ assays was performed. Equivalence was demonstrated between the assays for the liquid formulation at release and end of shelf life using a paired equivalence test with predefined equivalence margins. For the powder formulation, the assays were also shown to be equivalent for release samples and when determining loss of potency following thermal degradation. The BoCell^® assay was approved for establishing the potency of abobotulinumtoxinA for both powder and liquid formulations in Europe and for the powder formulation only in the USA.

Botulinum neurotoxins (BoNTs) are highly potent multi-domain proteins, responsible for botulism in animals and humans. The modular structural organization of BoNTs has led to the development of novel engineered bio-therapeutic proteins called targeted secretion inhibitors (TSIs). We report here that botulinum neurotoxin A (BoNT/A) and a TSI/A in which the neuronal binding domain of BoNT/A has been substituted by an epidermal growth factor (EGF) ligand, named EGFR-targeted TSI/A, exploit different routes to gain entry in the same in vitro neuroblastoma cell system, SiMa cells. We found that the EGF ligand conferred the affinity to the EGFR-targeted TSI/A at the EGF receptor when compared to an untargeted TSI/A and also the ability to internalize into the cells and cleave its cytosolic target protein SNAP-25. Using high content analysis we found that both BoNT/A and the EGFR-targeted TSI/A enter the cell in a concentration-dependent manner and in compartments which are able to translocate the proteins into the cytosol within 4 h. The EGFR-targeted TSI/A internalized into a compartment which gave a punctate staining pattern by immunofluorescence and partially overlapped with structures positive for the early endosomal marker EAA1; whereas BoNT/A did not internalize into a punctate compartment but did so in an acidifying compartment consistent with local synaptic vesicle recycling. These findings show that the BoNT/A translocation domain, common to both BoNT/A and the EGFR-targeted TSI/A, is a versatile tool for cytosolic delivery from distinct intracellular vesicular compartments.

Botulinum neurotoxin (BoNT) is a major therapeutic agent licensed in neurological indications such as dystonia and spasticity. In recent years, its use has steadily increased in other neurological areas and new therapeutic areas and also in the aesthetic setting. Paradoxically, BoNT is also the causative agent of the disease botulism and a potential bioterrorism toxin. The BoNT family of toxins comprised more than 40 individual members, classified into 7 serotypes and are produced by Gram-positive obligate anaerobic bacteria. BoNTs are enzymatic multi-modular proteins with a complex multistep mechanism of action. Their target site is at peripheral neurons, particularly the neuromuscular junction, at which they inhibit acetylcholine neurotransmission. Despite intense activity in the BoNT field, today there are still gaps in knowledge both in clinical practice and in basic research. The discovery of the structure-function of BoNT and its domains has allowed rational design of new features using molecular engineering. The diversity of BoNT molecules, both natural and engineered, is an invaluable pool from which to design future new therapeutics with unique pharmacological properties for current and novel indications.