Intraoperative abobotulinumtoxinA alleviates pain after surgery and improves general wellness in a translational animal model.

Pain after surgery remains a significant healthcare challenge. Here, abobotulinumtoxinA (aboBoNT-A, DYSPORT) was assessed in a post-surgical pain model in pigs. Full-skin-muscle incision and retraction surgery on the lower back was followed by intradermal injections of either aboBoNT-A (100, 200, or 400 U/pig), vehicle (saline), or wound infiltration of extended-release bupivacaine. We assessed mechanical sensitivity, distress behaviors, latency to approach the investigator, and wound inflammation/healing for 5-6 days post-surgery. We followed with immunohistochemical analyses of total and cleaved synaptosomal-associated protein 25 kD (SNAP25), glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor protein-1(Iba1), calcitonin gene-related peptide (CGRP) and substance P (SP) in the skin, dorsal root ganglia (DRG) and the spinal cord of 400 U aboBoNT-A- and saline-treated animals. At Day 1, partial reversal of mechanical allodynia in aboBoNT-A groups was followed by a full reversal from Day 3. Reduced distress and normalized approaching responses were observed with aboBoNT-A from 6 h post-surgery. Bupivacaine reversed mechanical allodynia for 24 h after surgery but did not affect distress or approaching responses. In aboBoNT-A-treated animals cleaved SNAP25 was absent in the skin and DRG, but present in the ipsilateral dorsal horn of the spinal cord. In aboBoNT-A- versus saline-treated animals there were significant reductions in GFAP and Iba1 in the spinal cord, but no changes in CGRP and SP. Analgesic efficacy of aboBoNT-A appears to be mediated by its activity on spinal neurons, microglia and astrocytes. Clinical investigation to support the use of aboBoNT-A as an analgesic drug for post-surgical pain, is warranted.

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.

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.

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.

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.

An improved model to investigate the efficacy of antidyskinetic agents in hemiparkinsonian rats.

A number of experimental models of L-DOPA-induced dyskinesia have been proposed, but these models result in a low to medium rate of dyskinetic animals with mild to severe symptoms. The objective of this study was to combine a model of 6-OHDA-induced parkinsonism and of L-DOPA-induced dyskinesia in rats to establish a reliable preclinical model. Two stereotaxic injections of 6-OHDA were administered in the left striatum. This model led to 90-100% of rats with a marked contralateral circling behaviour, significant limb use asymmetry (20%), a decrease in ipsilateral striatal dopamine content (70%) and degeneration of dopamine neurons in the substantia nigra (70%). Chronic treatment with L-DOPA was administered for 35 days and consisted of three phases with incremental daily doses. The third phase resulted in 83-90% of rats developing severe abnormal involuntary movements (AIMs) which included limb and locomotive dyskinesia, axial dystonia and orolingual dyskinesia. Reproducibility of the model, criteria of strict blinding, placebo-controlled design, randomization of study subjects and pretrial determination of sample size were used to measure efficacy of amantadine and istradefylline and to validate the protocol design. Acute or subchronic post-treatment with amantadine reduced the severity of dyskinesia while istradefylline punctually attenuated AIMs. Our experimental conditions using gradual development of dyskinesia induced by increasing doses of L-DOPA resulted in a reliable model of L-DOPA-induced dyskinesia with a high rate of dyskinetic rats.

Lack of evidence of direct mitochondrial involvement in the neuroprotective effect of minocycline.

Minocycline has been reported to exert neuroprotection through inhibition of inflammatory processes and of mitochondrial cell death pathway. To further characterize the neuroprotective effect of minocycline, we determined its efficacy in different neuronal damage paradigms involving inflammation or mitochondrial dysfunction. In transient global ischaemia in gerbils, minocycline reduced hippocampal neuronal damage measured by peripheral type benzodiazepine binding sites density, a marker of microglial activation. The antiinflammatory properties of minocycline were confirmed on the model of carrageenan-induced paw oedema in rats. The use of two experimental animal models involving administration of mitochondrial toxins inhibiting a different complex of the mitochondrial respiratory chain permitted the exploration of the mitochondrial impact of minocycline. Although minocycline exhibited a marked efficacy in 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP; complex I inhibitor)-induced neurotoxicity in mice, it was ineffective in malonate (complex II inhibitor)-induced striatal lesion in rats. In vitro investigations on energized mitochondria isolated from rat liver showed that minocycline (1 microM) did not inhibit the swelling induced by MPP+(1-methyl-4-phenylpyridinium). Moreover, higher concentrations of minocycline induced swelling. From these experiments, the neuroprotective activity of minocycline appears more related to its antiinflammatory activity than to a direct beneficial action on mitochondria.

Phenolic thiazoles as novel orally-active neuroprotective agents.

Novel phenolic thiazoles compounds were prepared which demonstrated potent antioxidant activity and potent in vivo neuroprotection in mitochondrial toxin models and also possess good oral bioavailability.