TrenibotulinumtoxinE (trenibotE), a botulinum neurotoxin serotype E (BoNT/E), is being developed for clinical use, and can fill a unique treatment gap for patients who are seeking neurotoxin treatment with a rapid onset and short duration of effect. This preclinical study characterized the pharmacological activity of trenibotE using the mouse Digit Abduction Score (DAS) assay. A comparative analysis was also performed between trenibotE and an equi-efficacious dose of the botulinum neurotoxin serotype A (BoNT/A) onabotulinumtoxinA (onabotA). TrenibotE showed a dose-dependent increase in peak DAS and duration of effect. A comparison of onabotA and trenibotE in this assay at approximate equi-efficacious doses showed trenibotE to have a faster onset of effect (trenibotE yielded a significantly greater effect as early as 6 h post-injection), shorter time to peak effect (24–27 h vs. 2 days), and an overall shorter duration of response (3 days vs. 14 days). The unique temporal characteristics of trenibotE and pharmacological differentiation from onabotA observed in this preclinical assay support the clinical development of this molecule.

Objective: The goal of this study was to compare the unit-to-unit biological activity of the vacuum-dried formulation of prabotulinumtoxinA (prabotA) and onabotulinumtoxinA (onabotA) in preclinical assays. Methods: Reconstituted 100 U vials of prabotA and onabotA were tested in 3 distinct assays: plate-capture light chain activity (PC-LCA), measuringlight chain enzymatic activity after recovery of toxin from reconstituted product using a proprietary toxin capture step; cell-based potency assay (CBPA), measuring the intoxication steps of binding, translocation, and light chain activity (synaptosomal-associated protein 25 [SNAP25] cleavage); and mouse Digit Abduction Score (DAS), evaluating muscle paresis. Each assay tested 3 separate prabotA and onabotA lots on several independent test dates. Results: Multiple orthogonal assays established that when assessed on a unit-to-unit basis, the biological activity of prabotA is lower than that of onabotA. In the PC-LCA and CBPA assays, onabotA displayed 1.51 ± 0.14–fold higher (mean ± SD) and 1.33 ± 0.07–fold higher (mean of pooled lots ± SEM) activity than prabotA, respectively. Similarly, the mouse DAS data showed that onabotA had 1.4 ± 0.1–fold higher (mean ± SEM) potency than prabotA. Results of all 3 assays demonstrated differences in potency, efficacy, and duration of action between onabotA and prabotA on a unit-to-unit basis. Conclusion: Preclinical assays established differences in the biological activity of onabotA and prabotA, supporting that the units of biological activity are not interchangeable.

Aim: Cortical spreading depression (CSD), the neural correlate of migraine aura, has been shown to cause activation of dural nociceptive neurons as well as immune cells, among which macrophages (MPs) are the most abundant and reactive. OnabotulinumtoxinA (onbotA) is used to treat chronic migraine but the mechanism of action is not fully understood. Here we investigate the role of meningeal MPs in a model of migraine activation and evaluate whether onabotA has an effect on their response. Methods: We use our previously developed method to determine meningeal MP activation based on shape changes using time-lapse in vivo multiphoton microscopy. Results: We found that a small subset (∼10%) of MPs contracted their processes in response to CSD induction, but only in female mice. A similar subset of MPs contracted with lipopolysaccharide injection, suggesting that this is an M1-like response. Together this may provide insight into the phenotypic differences of migraine across males and females. We also found a small subset of MPs (∼10%) that expanded their processes in response to IL-10 (presumably an M2-like response), but were not affected by CSD. In female mice, pre-treatment with onabotA (i) reduces overall MP number in the dura, (ii) reduces pro-inflammatory M1 MP response and (iii) increases anti-inflammatory M2 response post-CSD compared to pretreatment with saline. Conclusion: This suggests that the mechanism of action of onabotA may not be simply due to its effects on nociceptors, but also due to an additional anti-inflammatory effect on the environment of the dura.

Objectives: There has been a long-standing debate over the presence or absence of receptors for botulinum toxin A (BoNT/A) in cephalic areas relevant to migraine pathophysiology and onabotulinumtoxinA (onabotA) sites of action in migraine prevention. To address this issue, we sought to investigate for the first time whether synaptic vesicle protein 2C (SV2C), one member of the SV2 receptor family, is present in axons innervating the dura and periosteum. Methods: Single- and double- labeling immunohistochemical techniques were used to map and characterize the distribution of axons containing SV2C, the third isoform of the SV2 glycoprotein, in the mouse dura and periosteum. Results: Dense networks of axons containing SV2C receptors were distributed throughout all regions of the dura and periosteum. In the dura, SV2C-LIR axons were found in 43% of all peripherin-LIR fibers, 49% of all CGRP-LIR fibers, and 75% of all NaV1.8-LIR fibers. In the periosteum, SV2C-LIR was found in 38% of all peripherin-LIR fibers, 53% of all CGRP-LIR fibers, and 68% of all NaV1.8-LIR fibers. Conclusions: We interpret these findings as suggesting that many of the labeled axons are peripheral nerve axons (peripherin-positive) of unmyelinated sensory and possibly parasympathetic origin (CGRP-positive), and that some of these sensory axons are nociceptors (NaV1.8-positive). Clinically, these findings demonstrate an abundance of axons containing onabotA receptors in the vicinity of scalp structures commonly injected with onabotA for the treatment of chronic migraine. Dense labeling in the periosteum provides another rationale for the possibility that onabotA injections in this layer of the scalp may be advantageous.