Jacquie Maignel

While interstitial cystitis along with bladder pain syndrome (IC/BPS) is still poorly treated, published clinical evidence suggests that onabotulinumtoxinA (natural botulinum neurotoxin type A (BoNT/A)) intradetrusor injections is efficient in IC/BPS. However, as bladder instillation could be a safer and more convenient administration route, we aimed to investigate the effect of BoNT/A needle-free administration in an IC/BPS rodent model. Cyclophosphamide (CYP) was used to induce IC/BPS in rats. The resulting symptoms mimicked the main key features of human non-ulcerative IC/BPS. AbobotulinumtoxinA (aboBoNT-A) or reference compounds used in the clinic were delivered as a single intravesical administration into the bladder via the urethra. Visceral allodynia and hyperalgesia were assessed at the abdominal level with von Frey filaments before and after bladder pain induction. The levels of BoNT/A-cleaved SNAP25 (c-SNAP25), total SNAP25 (SNAP25^N-ter), beta-3 tubulin and CGRP in bladders were also quantified using immunohistochemistry (IHC), as well as the histopathological lesions. AboBoNT-A was well tolerated up to 30 U/rat. Allodynia and hyperalgesia were significantly decreased after aboBoNT-A dosing, with higher efficacy compared to references. c-SNAP25 IHC levels were low and similar in the detrusor for the 3 aboBoNT-A groups. Neither CYP or aboBoNT-A induced any change in the amount of SNAP25, beta-3 tubulin or CGRP. Some CYP-induced histopathological lesions showed a trend in improvement under aboBoNT-A. AboBoNT-A displayed analgesic properties that could translate into better therapies for visceral pain. Interestingly, intravesical (needle-free) administration seems like a promising and reproducible route for botulinum toxin therapy in patients with IC/BPS.

Botulinum neurotoxin A (BoNT/A) is a potent neurotoxin that silences cholinergic neurotransmission through the cleavage of the synaptic protein SNAP-25. Previous studies have shown that, in addition to its paralytic effects, BoNT/A can inhibit sensory nerve activity. The aim of this study was to identify how BoNT/A inhibits afferent signalling from the bladder. To investigate the role of SNAP-25 cleavage in the previously reported BoNT/A-dependent inhibition of sensory signalling, we developed a recombinant form of BoNT/A with an inactive light chain, rBoNT/A (0), unable to paralyse muscle. We also developed recombinant light chain (LC)-domain-only proteins to better understand the entry mechanisms, as the heavy chain (HC) of the protein is responsible for the internalisation of the light chain. We found that, despite a lack of catalytic activity, rBoNT/A (0) potently inhibited the afferent responses to bladder distension to a greater degree than catalytically active rBoNT/A. This was also clear from the testing of the LC-only proteins, as the inactive rLC/A (0) protein inhibited afferent responses significantly more than the active rLC/A protein. Immunohistochemistry for cleaved SNAP-25 was negative, and purinergic and nitrergic antagonists partially and totally reversed the sensory inhibition, respectively. These data suggest that the BoNT/A inhibition of sensory nerve activity in this assay is not due to the classical well-characterised ‘double-receptor’ mechanism of BoNT/A, is independent of SNAP25 cleavage and involves nitrergic and purinergic signalling mechanisms.

For the past two decades, botulinum neurotoxin A (BoNT/A) has been described as a strong candidate in the treatment of pain. With the production of modified toxins and the potential new applications at the visceral level, there is a real need for tools allowing the assessment of these compounds. In this study, we evaluated the jejunal mesenteric afferent nerve assay to investigate BoNT/A effects on visceral nociception. This ex vivo model allowed the continuous recording of neuronal activity in response to various stimuli. BoNT/A was applied intraluminally during three successive distensions, and the jejunum was distended every 15 min for 3 h. Finally, samples were exposed to external capsaicin. BoNT/A intoxication was validated at the molecular level with the presence of cleaved synaptosomal-associated protein of 25 (SNAP25) in nerve terminals in the mucosa and musculosa layers 3 h after treatment. BoNT/A had a progressive inhibitory effect on multiunit discharge frequency induced by jejunal distension, with a significant decrease from 1 h after application without change in jejunal compliance. The capsaicin-induced discharge was also affected by the toxin. This assay allowed the description of an inhibitory effect of BoNT/A on afferent nerve activity in response to distension and capsaicin, suggesting BoNT/A could alleviate visceral nociception.

Management of neurogenic detrusor overactivity (NDO) remains a clinical priority to improve patients’ quality of life and prevent dramatic urological complications. Intradetrusor injection of onabotulinumtoxinA (BoNT/A1, botulinum neurotoxin A1) is approved as second therapeutic line in these patients, demonstrating a good efficacy. However, a loss of its efficacy over time has been described, with no clear understanding of the underlying mechanisms. This paper aims at shedding new light on BoNT/A1 secondary failure in NDO through functional and structural analysis. Three groups of patients (either non-NDO, NDO with no toxin history or toxin secondary failure) were investigated using an ex vivo bladder strip assay. Detrusor strips were tensed in organ baths and submitted to electrical field stimulation to generate contractions. Recombinant BoNT/A1 was then added at various concentrations and contractions recorded for 4 h. Histology exploring BoNT/A1 targets, fibrosis and neuronal markers was also used. Detrusor strips from patients with BoNT/A1 secondary failure displayed a smaller sensitivity to toxin ex vivo at 3 nM compared to the other groups. Histological evaluation demonstrated the presence of cleaved Synaptosomal-Associated Protein, 25 kDa (c-SNAP25) in the detrusor from the toxin-secondary failure population, indicating some remaining in vivo sensitivity to BoNT/A1 despite the therapeutic escape. Moreover, residual c-SNAP25 did not affect parasympathetic-driven contractions observed ex vivo. This study confirms the slightly lower efficacy of BoNT/A1 in the BoNT/A1 secondary failure NDO group, suggesting that the escape from BoNT/A1 efficacy in NDO occurs at least at the parasympathetic level and could imply compensatory mechanisms for detrusor contraction.

Botulinum neurotoxin (BoNT/A) is an FDA and NICE approved second-line treatment for overactive bladder (OAB) in patients either not responsive or intolerant to anti-cholinergic drugs. BoNT/A acts to weaken muscle contraction by blocking release of the neurotransmitter acetyl choline (ACh) at neuromuscular junctions. However, this biological activity does not easily explain all the observed effects in clinical and non-clinical studies. There are also conflicting reports of expression of the BoNT/A protein receptor, SV2, and intracellular target protein, SNAP-25, in the urothelium and bladder. This review presents the current evidence of BoNT/A’s effect on bladder sensation, potential mechanisms by which it might exert these effects and discusses recent advances in understanding the action of BoNT in bladder tissue.

Botulinum neurotoxins (BoNTs) are notorious toxins and powerful agents and can be lethal, causing botulism, but they are also widely used as therapeutics, particularly to treat neuromuscular disorders. As of today, the commercial BoNT treatments available are from native A or B serotypes. Serotype F has shown efficacy in a clinical trial but has scarcely been used, most likely due to its medium duration of effect. Previously, the uniqueness of the light chain of the F7 subtype was identified and reported, showing an extended interaction with its substrates, VAMPs 1, 2 and 3, and a superior catalytic activity compared to other BoNT/F subtypes. In order to more extensively study the properties of this neurotoxin, we engineered a modified F7 chimera, mrBoNT/F7-1, in which all the regions of the neurotoxin were identical to BoNT/F7 except the activation loop, which was the activation loop from BoNT/F1. Use of the activation loop from BoNT/F1 allowed easier post-translational proteolytic activation of the recombinant protein without otherwise affecting its properties. mrBoNT/F7-1 was expressed, purified and then tested in a suite of in vitro and in vivo assays. mrBoNT/F7-1 was active and showed enhanced potency in comparison to both native and recombinant BoNT/F1. Additionally, the safety profile remained comparable to BoNT/F1 despite the increased potency. This new modified recombinant toxin F7 could be further exploited to develop unique therapeutics to address unmet medical needs.

Although botulinum neurotoxin serotype A (BoNT/A) products are common treatments for various disorders, there is only one commercial BoNT/B product, whose low potency, likely stemming from low affinity toward its human receptor synaptotagmin 2 (hSyt2), has limited its therapeutic usefulness. We express and characterize two full-length recombinant BoNT/B1 proteins containing designed mutations E1191M/S1199Y (rBoNT/B1_MY) and E1191Q/S1199W (rBoNT/B1_QW) that enhance binding to hSyt2. In preclinical models including human-induced pluripotent stem cell neurons and a humanized transgenic mouse, this increased hSyt2 affinity results in high potency, comparable to that of BoNT/A. Last, we solve the cocrystal structure of rBoNT/B1_MY in complex with peptides of hSyt2 and its homolog hSyt1. We demonstrate that neuronal surface receptor binding limits the clinical efficacy of unmodified BoNT/B and that modified BoNT/B proteins have promising clinical potential.

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.