Search Results (39)

Background/Objectives: Intradetrusor botulinum toxin injection is a well-established third-line therapy for patients with refractory overactive bladder (OAB) and detrusor overactivity (DO). Botulinum toxin type A (BoNT-A) is most commonly used due to its prolonged therapeutic duration. We aimed to evaluate the effectiveness of intradetrusor BoNT-A injection therapy in managing refractory OAB by performing a urodynamic study (UDS). Methods: The patients were prospectively enrolled between February 2020 and March 2021. The patients received treatment regimens comprising behavioral modification therapy, pelvic floor muscle physiotherapy, and/or OAB medications for at least three months. The UDS procedure was carried out by a single examiner, in accordance with the International Continence Society standards for good urodynamic practice. A total of 100 units of BoNT-A was dissolved in 10 mL of saline, and 0.5 mL (5 units) was injected at 20 sites on the posterior wall of the bladder. The primary endpoint was the change in DO, which was measured using the UDS from the baseline to two months after treatment with BoNT-A. Results: Prior to treatment initiation, DO was observed in all the patients during the UDS. The occurrence of DO during the filling phase demonstrated a significant decrease following treatment, with DO no longer identified in 27.3% of the patients. The first sensation of bladder filling, maximum cystometric capacity, DO, and terminal DO all demonstrated significant improvement after intradetrusor BoNT-A injection, based on the UDS. The OAB symptom scores also significantly decreased after BoNT-A therapy. Conclusions: The present study demonstrated that intradetrusor BoNT-A injection significantly improved symptoms in patients with OAB who had been unresponsive to various treatments. This study also demonstrated the usefulness of performing a UDS before and after treatment to prove the efficacy of BoNT-A. Full article

Botulinum neurotoxin type A (BoNT/A), among the most potent known toxins, is widely used in cosmetic medicine. However, its toxicity mechanisms remain poorly understood due to a lack of suitable models. Here, we generated a doxycycline (DOX)-inducible Neuro-2a cell line stably expressing the BoNT/A light chain (ALC). ALC expression was confirmed by GFP and FLAG tag antibodies, and its activity was validated through cleavage of the substrate SNAP-25. Using this model, combined with natural toxin infection of cells, phospho-antibody microarray analysis revealed significant alterations in host phosphorylation networks in both ALC-expressing and toxin-infected cells. Among the shared phosphorylation changes, 75 proteins showed upregulation, while 27 were downregulated. Upregulated phosphorylation events were enriched in pathways such as PI3K-AKT signaling, EGFR tyrosine kinase inhibitor resistance, and Ras signaling, whereas downregulated events were associated with the ERBB and thyroid hormone signaling pathways. Key alterations were observed in AKT signaling, with protein–protein interaction analysis identifying Hsp90ab1 and Map2k1 as central hub molecules for upregulated and downregulated proteins, respectively. This study establishes a robust Neuro-2a-based model system to study BoNT/A toxicity and provides insights into toxin-induced phosphorylation network changes, offering a valuable platform for therapeutic screening and mechanistic exploration. Full article

Inflammation is a multifaceted biological response of the immune system against various harmful stimuli, including pathogens (such as bacteria and viruses), cellular damage, toxins, and natural/synthetic irritants. This protective mechanism is essential for eliminating the cause of injury, removing damaged cells, and initiating the repair process. While inflammation is a fundamental component of the body’s defense and healing process, its dysregulation can lead to pathological consequences, contributing to various acute and chronic diseases, such as autoimmune disorders, cancer, metabolic syndromes, cardiovascular diseases, neurodegenerative conditions, and other systemic complications. Generally, non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, disease-modifying anti-rheumatic drugs (DMARDs), antihistamines, biologics, and colchicine are used as pharmacological agents in the management of inflammatory diseases. However, these conventional treatments often have limitations, including adverse side effects, long-term toxicity, and drug resistance. In contrast, enzyme-based therapeutics have emerged as a promising alternative due to their high specificity, catalytic efficiency, and ability to modulate inflammatory pathways with reduced side effects. These enzymes function by scavenging reactive oxygen species (ROS), inhibiting cytokine transcription, degrading circulating cytokines, and blocking cytokine release by targeting exocytosis-related receptors. Additionally, their role in tissue repair and regeneration further enhances their therapeutic potential. Most natural anti-inflammatory enzymes belong to the oxidoreductase class, including catalase and superoxide dismutase, as well as hydrolases such as trypsin, chymotrypsin, nattokinase, bromelain, papain, serratiopeptidase, collagenase, hyaluronidase, and lysozyme. Engineered enzymes, such as Tobacco Etch Virus (TEV) protease and botulinum neurotoxin type A (BoNT/A), have also demonstrated significant potential in targeted anti-inflammatory therapies. Recent advancements in enzyme engineering, nanotechnology-based enzyme delivery, and biopharmaceutical formulations have further expanded their applicability in treating inflammatory diseases. This review provides a comprehensive overview of both natural and engineered enzymes, along with their formulations, used as anti-inflammatory therapeutics. It highlights improvements in stability, efficacy, and specificity, as well as minimized immunogenicity, while discussing their mechanisms of action and clinical applications and potential future developments in enzyme-based biomedical therapeutics. Full article

Botulinum toxin A (BoNT-A) is widely used in both therapeutic and aesthetic settings; however, the formation of neutralizing antibodies (NAbs) remains a critical concern, leading to treatment failure. Immunogenic responses are known to vary between individuals due to HLA polymorphisms. Although some claim that neurotoxin-associated proteins (NAPs) shield BoNT-A from immune detection or are themselves immunogenic, there is limited molecular evidence supporting either view. This study applies computational immunogenetics to explore BoNT-A immunogenicity, focusing on HLA binding and the influence of accessory proteins. Epitope mapping, molecular docking, and HLA binding predictions were used to evaluate interactions between BoNT-A epitopes and selected class II HLA alleles (HLA-DQA1*01:02, HLA-DQA1*03:03, HLA-DQB1*06:04, HLA-DQB1*03:01, and HLA-DRB1*15:01). To assess the potential immunomodulatory role of NAPs, molecular dynamics (MD) simulations, solvent-accessible surface area (SASA) analysis, and electrostatic potential mapping were also conducted. Key epitopes—L11, N25, and C10—showed strong binding affinities to HLA-DQA1*01:02, HLA-DQB1*06:04, and HLA-DQA1*03:03, indicating a potential immunodominant role. NAPs did not obstruct these epitopes but slightly increased their exposure and appeared to stabilize the toxin structure. Electrostatic mapping and binding free energy calculations suggested no significant immunogenic shift in the presence of NAPs. BoNT-A immunogenicity appears to be influenced by HLA allele variability, reinforcing the value of patient-specific genetic profiling. The presumed immunogenic role of NAPs remains unsubstantiated at the molecular level, underscoring the need for evidence-based evaluation over commercial rhetoric. While these findings provide valuable molecular insight, it is important to acknowledge that they are derived entirely from in silico analyses. As such, experimental validation remains essential to confirm the immunological relevance of these predicted interactions. Nonetheless, this computational framework offers a rational basis for guiding future clinical research and the development of HLA-informed BoNT-A therapies. Full article

The therapeutic efficacy remaining from prior treatments with botulinum toxins (BoNTs) when cervical dystonia (CD) patients prefer to be re-treated has not been well characterized. Here, we assessed the residual therapeutic efficacy of BoNT injections at the time of a patient-desired re-treatment. In pivotal trials for daxibotulinumtoxinA (DAXI) in CD, subjects could request re-treatment before returning to pre-treatment symptom levels (defined as ≤20% of peak efficacy remaining). In this post hoc analysis of the Phase 3 ASPEN-OLS trial, the median percent efficacy remaining (based on change in TWSTRS total score) was determined in subjects who requested re-injection before returning to pre-treatment symptoms. Dysphagia and muscle weakness were evaluated in patients requesting re-treatment with efficacy remaining, relative to those waiting to return to baseline. There were 264 (28.7% of 920 total treatments) patient requests for re-treatment before returning to pre-treatment status across the study. The median percent efficacy remaining at the time of requested re-injection was 45.5%, which corresponded to a median of 16.0 weeks (range 10.9–40.3) post-treatment. The rates of dysphagia (≤4.9%) and muscle weakness (≤6.8%) were low and were not significantly different in those who waited for return to pre-treatment symptom status versus subjects who requested re-injection with efficacy remaining. A significant proportion of CD patients wished to be re-treated with efficacy still remaining from prior BoNT injections as early symptoms re-emerged. With the overall clinical profile of DAXI, physicians can safely provide individualized treatment regimens based on the treatment goals or symptomatic needs of their patients. Full article

Background: Botulinum neurotoxin is widely regarded as a “wonder medicine” due to its therapeutic efficacy in treating a variety of conditions. While it is traditionally classified as a neurotoxin, it is arguably more appropriate to refer to it as a neuromedicine. All FDA-approved formulations of botulinum neurotoxin are currently administered through intramuscular injections, with no other delivery methods widely used. The primary reasons for this include the following: (a) the extremely high potency of the toxin, (b) the potential for diffusion to adjacent muscles, (c) factors related to the site of administration (e.g., muscle thickness), (d) the large size of the molecule, (e) the impermeability of skin to large protein molecules, and (f) safety concerns. Despite these challenges, there is growing interest in the development of an effective transdermal formulation of botulinum neurotoxin. Refining and standardizing the delivery technology for topical or transdermal use remains an important goal for the future. Methods: The aim of this study was to develop a nanoemulsion-based transdermal formulation capable of delivering active botulinum neurotoxin (BoNT) through human skin. The goal was to demonstrate its efficacy in a mouse model, highlighting the therapeutic effects on both neuromuscular activity and hyperhidrosis. We successfully developed a nanoemulsion-based formulation that facilitates the transdermal delivery of BoNT. The formulation was homogeneous, stable, and efficacious. In a mouse model, we evaluated the neurotoxin’s impact on neuromuscular function using the Digital Abduction Score (DAS) for toe-spread and rota-rod assay to assess motor coordination. Results: The results confirmed the successful paralytic effect of the neuotoxin. The formulation significantly reduced sweating in the hyperhidrosis mouse model, indicating the therapeutic potential for this indication. Beyond the neurotoxin’s paralyzing effect, we also observed the recovery of nerve function, showing that the neurotoxin does not cause permanent damage, further underscoring its safety and efficacy. Conclusions: This formulation is the first of its kind to successfully deliver a large biomolecule like BoNT across the skin and produce a therapeutic effect. The ability to deliver large biomolecules transdermally has the potential to serve as a platform technology for treating a variety of conditions, including neuromuscular disorders, skin conditions, and localized pain management. Full article

Botulinum toxin type A1 is a first-line treatment for adult and pediatric spasticity. However, when considering the quantity of 150 kDa neurotoxin protein in relation to patient weight and the maximum recommended dose for treating adult and pediatric patients with spasticity, several concerns arise. First, the therapeutic margin (the ratio of the actual maximum quantity of toxin recommended for treating adult spasticity to its median lethal dose) appears to be relevant. Second, there is no consistency between adult and pediatric dosing of botulinum toxin type A1 for spasticity. The third point concerns the suitability of the recommended doses for treating spasticity in pediatric patients. Based on the average body weight of American children and adolescents, the maximum weight-based doses for abobotulinumtoxinA and onabotulinumtoxinA could be administered to children as young as 9 years old. Additionally, the maximum weight-based dose for incobotulinumtoxinA could be administered to children as young as 6 years old. The final point concerns managing the maximum dose of BoNT/A1 in pediatric patients with spasticity who weigh more than 25 kg for incobotulinumtoxinA, or more than 34 kg for abobotulinumtoxinA and onabotulinumtoxinA. No labeled recommendations are given on the weight cut-off for transitioning to adult dosing in pediatric patients. Full article

Botulinum Neurotoxin A (BoNT/A) is a bacterial protein that has proven to be a valuable pharmaceutical in therapeutic indications and aesthetic medicine. One major concern is the formation of neutralizing antibodies (nAbs) to the core BoNT/A protein. These can interfere with the therapy, resulting in partial or complete antibody (Ab)-mediated secondary non-response (SNR) or immunoresistance. If titers of nAbs reach a level high enough that all injected BoNT/A molecules are neutralized, immunoresistance occurs. Studies have shown that continuation of treatment of neurology patients who had developed Ab-mediated partial SNR against complexing protein-containing (CPC-) BoNT/A was in some cases successful if patients were switched to complexing protein-free (CPF-) incobotulinumtoxinA (INCO). This seems to contradict the layperson’s basic immunological understanding that repeated injection with the same antigen BoNT/A should lead to an increase in antigen-specific antibody titers. As such, we strive to explain how immunological memory works in general, and based on this, we propose a working hypothesis for this paradoxical phenomenon observed in some, but not all, neurology patients with immunoresistance. A critical factor is the presence of potentially immune-stimulatory components in CPC-BoNT/A products that can act as immunologic adjuvants and activate not only naïve, but also memory B lymphocyte responses. Furthermore, we propose that continuous injection of a BoN/TA formulation with low immunogenicity, e.g., INCO, may be a viable option for aesthetic patients with existing nAbs. These concepts are supported by a real-world case example of a patient with immunoresistance whose nAb levels declined with corresponding resumption of clinical response despite regular INCO injections. Full article

Numerous studies have established a robust body of evidence for botulinum toxin A (BoNT-A) therapy as a treatment for upper motor neuron syndrome. These studies demonstrated improvements in spasticity, range of joint motion, and pain reduction. However, there are few studies that have focused on improvement of paralysis or functional enhancement as the primary outcome. This paper discusses the multifaceted aspects of spasticity assessment, administration, and rehabilitation with the goal of optimising the effects of BoNT-A on lower-limb spasticity and achieving functional improvement and gait reconstruction. This paper extracts studies on BoNT-A and rehabilitation for the lower limbs and provides new knowledge obtained from them. From these discussion,, key points in a walking reconstruction strategy through the combined use of BoNT-A and rehabilitation include: (1) injection techniques based on the identification of appropriate muscles through proper evaluation; (2) combined with rehabilitation; (3) effective spasticity control; (4) improvement in ankle joint range of motion; (5) promotion of a forward gait pattern; (6) adjustment of orthotics; and (7) maintenance of the effects through frequent BoNT-A administration. Based on these key points, the degree of muscle fibrosis and preintervention walking speed may serve as indicators for treatment strategies. With the accumulation of recent studies, a study focusing on walking functions is needed. As a result, it is suggested that BoNT-A treatment for lower limb spasticity should be established not just as a treatment for spasticity but also as a therapeutic strategy in the field of neurorehabilitation aimed at improving walking function. Full article

Botulinum neurotoxins (BoNTs) have been used for almost half a century in the treatment of excessive muscle contractility. BoNTs are routinely used to treat movement disorders such as cervical dystonia, spastic conditions, blepharospasm, and hyperhidrosis, as well as for cosmetic purposes. In addition to the conventional indications, the use of BoNTs to reduce pain has gained increased recognition, giving rise to an increasing number of indications in disorders associated with chronic pain. Furthermore, BoNT-derived formulations are benefiting a much wider range of patients suffering from overactive bladder, erectile dysfunction, arthropathy, neuropathic pain, and cancer. BoNTs are categorised into seven toxinotypes, two of which are in clinical use, and each toxinotype is divided into multiple subtypes. With the development of bioinformatic tools, new BoNT-like toxins have been identified in non-Clostridial organisms. In addition to the expanding indications of existing formulations, the rich variety of toxinotypes or subtypes in the wild-type BoNTs associated with new BoNT-like toxins expand the BoNT superfamily, forming the basis on which to develop new BoNT-based therapeutics as well as research tools. An overview of the diversity of the BoNT family along with their conventional therapeutic uses is presented in this review followed by the engineering and formulation opportunities opening avenues in therapy. Full article

Botulinum neurotoxins (BoNTs) are highly toxic proteins that require high-affinity immunocapture reagents for use in endopeptidase-based assays. Here, 30 novel and 2 earlier published llama single-domain antibodies (VHHs) against the veterinary-relevant BoNT serotypes C and D were yeast-produced. These VHHs recognized 10 independent antigenic sites, and many cross-reacted with the BoNT/DC and CD mosaic variants. As VHHs are highly suitable for genetically linking to increase antigen-binding affinity, 52 VHH multimers were produced and their affinity for BoNT/C, D, DC, and CD was determined. A selection of 15 multimers with high affinity (K_D < 0.1 nM) was further shown to be resilient to a high salt wash that is used for samples from complex matrices and bound native BoNTs from culture supernatants as shown by Endopep-MS. High-affinity multimers suitable for further development of a highly sensitive Endopep-MS assay include four multimers that bind both BoNT/D and CD with K_D of 14–99 pM, one multimer for BoNT/DC (65 pM) that also binds BoNT/C (75 pM), and seven multimers for BoNT/C (<1–19 pM), six of which also bind BoNT/DC with lower affinity (93–508 pM). In addition to application in diagnostic tests, these VHHs could be used for the development of novel therapeutics for animals or humans. Full article

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. Full article

Herein, we describe a yeast cell-based assay system to analyze SNAP25-targeting botulinum neurotoxins (BoNTs). BoNTs are protein toxins, and, upon incorporation into neuronal cells, their light chains (BoNT-LCs) target specific synaptosomal N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins, including synaptosomal-associated protein 25 (SNAP25). BoNT-LCs are metalloproteases, and each BoNT-LC recognizes and cleaves conserved domains in SNAREs termed the SNARE domain. In the budding yeast Saccharomyces cerevisiae, the SNAP25 ortholog Spo20 is required for production of the spore plasma membrane; thus, defects in Spo20 cause sporulation deficiencies. We found that chimeric SNAREs in which SNARE domains in Spo20 are replaced with those of SNAP25 are functional in yeast cells. The Spo20/SNAP25 chimeras, but not Spo20, are sensitive to digestion by BoNT-LCs. We demonstrate that spo20∆ yeasts harboring the chimeras exhibit sporulation defects when various SNAP25-targeting BoNT-LCs are expressed. Thus, the activities of BoNT-LCs can be assessed by colorimetric measurement of sporulation efficiencies. Although BoNTs are notorious toxins, they are also used as therapeutic and cosmetic agents. Our assay system will be useful for analyzing novel BoNTs and BoNT-like genes, as well as their manipulation. Full article

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, cleaves proteins involved in neurotransmitter release, thereby triggering flaccid paralyses, which are responsible for botulism. BoNT is classified into seven serotypes (BoNT/A-G); BoNT/A and BoNT/B are used as medical therapeutics and anti-wrinkle reagents. In this study, we investigated the efficacy of BoNT/CD, a mosaic toxin of BoNT/C and BoNT/D, to assess its potential as a therapeutic alternative for BoNT/A. In a cultured neuron assay, BoNT/CD cleaved syntaxin and SNAP-25 with higher efficacy than BoNT/C and BoNT/A. Intramuscularly administrated BoNT/CD induced dose-dependent muscle paralysis, and the paralysis lasted ~21 days in a mouse digit abduction score assay (BoNT/A-induced paralysis lasted ~30 days). BoNT/C failed to induce local paralysis without systemic toxicity. Multiple alignment analyses of the amino acid sequences of the receptor binding domain (H_C) of eight BoNT/CDs and two BoNT/Ds showed sequence clustering in five groups. Comparing BoNT/CD strain 003-9 (BoNT/CD_003-9) and strain 6813 (BoNT/CD₆₈₁₃) showed that both BoNT/CDs displayed similar efficacies in cultured neurons, but BoNT/CD_003-9 displayed higher efficacy in a mouse model than BoNT/CD₆₈₁₃. These findings suggest that BoNT/CD may be a potential alternative for patients who do not respond to existing BoNT-based therapeutics. Full article

Botulinum neurotoxins (BoNTs) cause flaccid neuromuscular paralysis by cleaving one of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. BoNTs display high affinity and specificity for neuromuscular junctions, making them one of the most potent neurotoxins known to date. There are seven serologically distinct BoNTs (serotypes BoNT/A to BoNT/G) which can be further divided into subtypes (e.g., BoNT/A1, BoNT/A2…) based on small changes in their amino acid sequence. Of these, BoNT/A1 and BoNT/B1 have been utilised to treat various diseases associated with spasticity and hypersecretion. There are potentially many more BoNT variants with differing toxicological profiles that may display other therapeutic benefits. This review is focused on the structural analysis of the cell-binding domain from BoNT/A1 to BoNT/A6 subtypes (H_C/A1 to H_C/A6), including features such as a ganglioside binding site (GBS), a dynamic loop, a synaptic vesicle glycoprotein 2 (SV2) binding site, a possible Lys–Cys/Cys–Cys bridge, and a hinge motion between the H_CN and H_CC subdomains. Characterising structural features across subtypes provides a better understanding of how the cell-binding domain functions and may aid the development of novel therapeutics. Full article