Evaluation of Recombinant Botulinum Neurotoxin Type A1 Efficacy in Peripheral Inflammatory Pain in Mice.

Well-established efficacy of botulinum neurotoxin type A (BoNT/A) in aesthetic dermatology and neuromuscular hyperactivity disorders relies on canonical interruption of acetylcholine neurotransmission at the neuromuscular junction at the site of the injection. The mechanisms and the site of activity of BoNT/A in pain, on the other hand, remain elusive. Here, we explored analgesic activity of recombinant BoNT/A1 (rBoNT/A1; IPN10260) in a mouse model of inflammatory pain to investigate the potential role of peripheral sensory afferents in this activity. After confirming analgesic efficacy of rBoNT/A1 on CFA-induced mechanical hypersensitivity in C57Bl6J mice, we used GCaMP6s to perform in vivo calcium imaging in the ipsilateral dorsal root ganglion (DRG) neurons in rBoNT/A1 vs. vehicle-treated mice at baseline and following administration of a range of mechanical and thermal stimuli. Additionally, immunohisochemical studies were performed to detect cleaved SNAP25 in the skin, DRGs and the spinal cord. Injection of CFA resulted in reduced mechanical sensitivity threshold and increased calcium fluctuations in the DRG neurons. While rBoNT/A1 reduced mechanical hypersensitivity, calcium fluctuations in the DRG of rBoNT/A1- and vehicle-treated animals were similar. Cleaved SNAP25 was largely absent in the skin and the DRG but present in the lumbar spinal cord of rBoNT/A1-treated animals. Taken together, rBoNT/A1 ameliorates mechanical hypersensitivity related to inflammation, while the signal transmission from the peripheral sensory afferents to the DRG remained unchanged. This strengthens the possibility that spinal, rather than peripheral, mechanisms play a role in the mediation of analgesic efficacy of BoNT/A in inflammatory pain.

New Modified Recombinant Botulinum Neurotoxin Type F with Enhanced Potency.

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.

Recombinant botulinum neurotoxin serotype A1 in vivo characterization.

Clinically used botulinum neurotoxins (BoNTs) are natural products of Clostridium botulinum. A novel, recombinant BoNT type A1 (rBoNT/A1; IPN10260) has been synthesized using the native amino acid sequence expressed in Escherichia coli and has previously been characterized in vitro and ex vivo. Here, we aimed to characterize rBoNT/A1 in vivo and evaluate its effects on skeletal muscle. The properties of rBoNT/A1 following single, intramuscular administration were evaluated in the mouse and rat digit abduction score (DAS) assays and compared with those of natural BoNT/A1 (nBoNT/A1). rBoNT/A1-injected tibialis anterior was assessed in the in situ muscle force test in rats. rBoNT/A1-injected gastrocnemius lateralis (GL) muscle was assessed in the compound muscle action potential (CMAP) test in rats. The rBoNT/A1-injected GL muscle was evaluated for muscle weight, volume, myofiber composition and immunohistochemical detection of cleaved SNAP25 (c-SNAP25). Results showed that rBoNT/A1 and nBoNT/A1 were equipotent and had similar onset and duration of action in both mouse and rat DAS assays. rBoNT/A1 caused a dose-dependent inhibition of muscle force and a rapid long-lasting reduction in CMAP amplitude that lasted for at least 30 days. Dose-dependent reductions in GL weight and volume and increases in myofiber atrophy were accompanied by immunohistochemical detection of c-SNAP25. Overall, rBoNT/A1 and nBoNT/A1 exhibited similar properties following intramuscular administration. rBoNT/A1 inhibited motoneurons neurotransmitter release, which was robust, long-lasting, and accompanied by cleavage of SNAP25. rBoNT/A1 is a useful tool molecule for comparison with current natural and future modified recombinant neurotoxins products.

Optimization of the rat digit abduction score (DAS) assay: Evaluation of botulinum neurotoxin activity in the gastrocnemius lateralis, peronei, and extensor digitorum longus.

The mouse digit abduction score (DAS) assay is commonly used to measure muscle flaccidity-inducing effects of botulinum neurotoxin (BoNT) in vivo. Adapting the assay to rats has been challenging, as injection of onabotulinumtoxinA (onaBoNT-A) into the gastrocnemius muscle, as performed in mice, or into the tibialis anterior leads to sub-optimal sensitivity of the test (Broide et al., 2013). To optimize the experimental design of the rat DAS assay, we evaluated the effects of research-grade, purified, native BoNT serotype A1 (BoNT-A) in three muscles: the gastrocnemius lateralis, peronei, and extensor digitorum longus using female animals. Following injection, animals were tested daily for the digit abduction and body weight. BoNT-A caused dose-dependent inhibition of digit abduction when injected into the gastrocnemius lateralis or peronei. BoNT-A was six-fold more potent when injected into the peronei in comparison to the gastrocnemius lateralis. As injection of BoNT-A into the extensor digitorum longus muscle resulted in an all-or-none digit abduction response and therefore prevented calculation of the ED50, it was considered unsuitable for the rat DAS assay. At equipotent doses, peronei- and extensor digitorum longus-injected animals showed normal body weight gain, while those injected with BoNT-A into the gastrocnemius lateralis gained less weight in comparison to vehicle-treated controls. Thus, injecting the peronei muscles of female rats offers optimized conditions for evaluating the biological properties of BoNTs in the rat DAS assay; for assessing the potency, onset, and duration of action across natural and recombinant BoNT in a robust and reproducible manner.

WITHDRAWN: Optimization of the rat digit abduction score (DAS) assay: Evaluation of botulinum neurotoxin activity in the gastrocnemius lateralis, peronei, and extensor digitorum longus.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.toxcx.2020.100029. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

The use of the dynamic weight bearing test to assess the effects of acute, intramuscularly administered botulinum neurotoxin type A1 in rats.

Assessing the efficacy of botulinum neurotoxin (BoNT) in vivo is essential given the growing number of BoNT products used in the clinic. Here, we evaluated the dynamic weight bearing (DWB) test for sensitivity to paralytic effects of BoNT-A following intramuscular administration. The toxin was administered into the gastrocnemius lateralis as a single bolus or into the gastrocnemius lateralis and medialis as two boluses. The effects of BoNT-A in DWB were compared to those in the compound muscle action potential (CMAP) and the Digit Abduction Score (DAS) tests. Female Sprague-Dawley rats received an acute, intramuscular (i.m.) injection of BoNT-A1 (0.1, 1, 10 pg/rat) into the right gastrocnemius muscle, while the left received vehicle. The DWB and CMAP tests were performed one-two days after the injection in order to detect the onset of sub-maximal BoNT-A activity. Both tests were preceded by the DAS test. BoNT-A produced dose-related reductions in both the weight-bearing and surface-bearing outcomes of up to 60% while showing moderate activity in the DAS. BoNT-A effects in the DWB test were well-aligned with those in the CMAP test, which showed dose-dependent reductions in CMAP amplitude and the area under the curve (AUC; up to 100%) as well as increases in latency (up to 130%). The efficacy of BoNT-A in DWB and CMAP was more pronounced with two boluses. Thus, the DWB test can be used to assess the properties of BoNTs following i.m. administration. It can be used to assess the candidate therapies and is more ethical than the mouse lethality assay.

Engineered botulinum neurotoxin B with improved binding to human receptors has enhanced efficacy in preclinical models.

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/B1MY) and E1191Q/S1199W (rBoNT/B1QW) 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/B1MY 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.

A comparison of biological activity of commercially available purified native botulinum neurotoxin serotypes A1 to F1 in vitro, ex vivo, and in vivo.

Botulinum neurotoxin (BoNT) is a major therapeutic agent. Of seven native BoNT 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. BoNT potency in vitro was assessed in rat primary cells (target protein cleavage and neurotransmitter release assays) in supraspinal, spinal, and sensory systems. BoNT potency ex vivo was measured in the mouse phrenic nerve hemidiaphragm (PNHD) assay, measuring muscle contractility. In vivo, BoNT-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 BoNT 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.