Engineered botulinum neurotoxin B with improved efficacy for targeting human receptors.

Botulinum neurotoxin B is a Food and Drug Administration-approved therapeutic toxin. However, it has lower binding affinity toward the human version of its major receptor, synaptotagmin II (h-Syt II), compared to mouse Syt II, because of a residue difference. Increasing the binding affinity to h-Syt II may improve botulinum neurotoxin B's therapeutic efficacy and reduce adverse effects. Here we utilized the bacterial adenylate cyclase two-hybrid method and carried out a saturation mutagenesis screen in the Syt II-binding pocket of botulinum neurotoxin B. The screen identifies E1191 as a key residue: replacing it with M/C/V/Q enhances botulinum neurotoxin B binding to human synaptotagmin II. Adding S1199Y/W or W1178Q as a secondary mutation further increases binding affinity. Mutant botulinum neurotoxin B containing E1191M/S1199Y exhibits ~11-fold higher efficacy in blocking neurotransmission than wild-type botulinum neurotoxin B in neurons expressing human synaptotagmin II, demonstrating that enhancing receptor binding increases the overall efficacy at functional levels. The engineered botulinum neurotoxin B provides a platform to develop therapeutic toxins with improved efficacy.Humans are less sensitive to the therapeutic effects of botulinum neurotoxin B (BoNT/B) than the animal models it is tested on due to differences between the human and the mouse receptors. Here, the authors engineer BoNT/B to improve its affinity to human receptors and enhance its therapeutic efficacy.

High-throughput 3D whole-brain quantitative histopathology in rodents.

Histology is the gold standard to unveil microscopic brain structures and pathological alterations in humans and animal models of disease. However, due to tedious manual interventions, quantification of histopathological markers is classically performed on a few tissue sections, thus restricting measurements to limited portions of the brain. Recently developed 3D microscopic imaging techniques have allowed in-depth study of neuroanatomy. However, quantitative methods are still lacking for whole-brain analysis of cellular and pathological markers. Here, we propose a ready-to-use, automated, and scalable method to thoroughly quantify histopathological markers in 3D in rodent whole brains. It relies on block-face photography, serial histology and 3D-HAPi (Three Dimensional Histology Analysis Pipeline), an open source image analysis software. We illustrate our method in studies involving mouse models of Alzheimer's disease and show that it can be broadly applied to characterize animal models of brain diseases, to evaluate therapeutic interventions, to anatomically correlate cellular and pathological markers throughout the entire brain and to validate in vivo imaging techniques.

Does Reduction of Number of Intradetrusor Injection Sites of aboBoNTA (Dysport®) Impact Efficacy and Safety in a Rat Model of Neurogenic Detrusor Overactivity?

Intradetrusor injections of Botulinum toxin A-currently onabotulinumtoxinA-is registered as a second-line treatment to treat neurogenic detrusor overactivity (NDO). The common clinical practice is 30 × 1 mL injections in the detrusor; however, protocols remain variable and standardization is warranted. The effect of reducing the number of injection sites of Dysport(®) abobotulinumtoxinA (aboBoNTA) was assessed in the spinal cord-injured rat (SCI). Nineteen days post-spinalization, female rats received intradetrusor injections of saline or aboBoNTA 22.5 U distributed among four or eight sites. Two days after injection, continuous cystometry was performed in conscious rats. Efficacy of aboBoNTA 22.5 U was assessed versus aggregated saline groups on clinically-relevant parameters: maximal pressure, bladder capacity, compliance, voiding efficiency, as well as amplitude, frequency, and volume threshold for nonvoiding contractions (NVC). AboBoNTA 22.5 U significantly decreased maximal pressure, without affecting voiding efficiency. Injected in four sites, aboBoNTA significantly increased bladder capacity and compliance while only the latter when in eight sites. AboBoNTA significantly reduced NVC frequency and amplitude. This preclinical investigation showed similar inhibiting effects of aboBoNTA despite the number of sites reduction. Further studies are warranted to optimize dosing schemes to improve the risk-benefit ratio of BoNTA-based treatment modalities for NDO and further idiopathic overactive bladder.

IRC-082451, a novel multitargeting molecule, reduces L-DOPA-induced dyskinesias in MPTP Parkinsonian primates.

The development of dyskinesias following chronic L-DOPA replacement therapy remains a major problem in the long-term treatment of Parkinson's disease. This study aimed at evaluating the effect of IRC-082451 (base of BN82451), a novel multitargeting hybrid molecule, on L-DOPA-induced dyskinesias (LIDs) and hypolocomotor activity in a non-human primate model of PD. IRC-082451 displays multiple properties: it inhibits neuronal excitotoxicity (sodium channel blocker), oxidative stress (antioxidant) and neuroinflammation (cyclooxygenase inhibitor) and is endowed with mitochondrial protective properties. Animals received daily MPTP injections until stably parkinsonian. A daily treatment with increasing doses of L-DOPA was administered to parkinsonian primates until the appearance of dyskinesias. Then, different treatment regimens and doses of IRC-082451 were tested and compared to the benchmark molecule amantadine. Primates were regularly filmed and videos were analyzed with specialized software. A novel approach combining the analysis of dyskinesias and locomotor activity was used to determine efficacy. This analysis yielded the quantification of the total distance travelled and the incidence of dyskinesias in 7 different body parts. A dose-dependent efficacy of IRC-082451 against dyskinesias was observed. The 5 mg/kg dose was best at attenuating the severity of fully established LIDs. Its effect was significantly different from that of amantadine since it increased spontaneous locomotor activity while reducing LIDs. This dose was effective both acutely and in a 5-day sub-chronic treatment. Moreover, positron emission tomography scans using radiolabelled dopamine demonstrated that there was no direct interference between treatment with IRC-082451 and dopamine metabolism in the brain. Finally, post-mortem analysis indicated that this reduction in dyskinesias was associated with changes in cFOS, FosB and ARC mRNA expression levels in the putamen. The data demonstrates the antidyskinetic efficacy of IRC-082451 in a primate model of PD with motor complications and opens the way to the clinical application of this treatment for the management of LIDs.

Minimal effective dose of dysport and botox in a rat model of neurogenic detrusor overactivity.

BACKGROUND: Two botulinum toxins A have been evaluated for the treatment of refractory neurogenic detrusor overactivity (NDO) in humans: Dysport (abobotulinumtoxinA) and Botox (onabotulinumtoxinA). However, these two distinct commercialized products have different potency units and are not interchangeable. OBJECTIVE: Assessment of the dose response and determination of minimal effective dose (MED) for Dysport and Botox in spinal cord-injured (SCI) rats with NDO. DESIGN, SETTING, AND PARTICIPANTS: Female, adult, Sprague-Dawley rats (n=98) underwent T8-T9 spinal cord transection. Nineteen days after spinal cord injury, rats received intradetrusor injections (25µl injected, eight sites) of vehicle (V); Dysport 2, 5, 7.5, 10, and 12.5 U; and Botox 0.8, 2, 5, 7.5, and 10 U. Two days after injection, continuous cystometry was performed in conscious rats. MEASUREMENTS: Voiding contractions (VC) were assessed by duration of VC, intercontraction interval, voided volume, maximal pressure, pressure threshold change, and intravesical baseline pressure (BP), while nonvoiding contractions (NVC) were evaluated by amplitude, frequency, and volume threshold to elicit NVC. MEDs for Dysport and Botox were determined by analysis of variance step-down trend test. RESULTS AND LIMITATIONS: MEDs for Dysport and Botox were 10 U and 7.5 U, respectively. Regarding VC, only BP significantly decreased after 10 U Dysport and 7.5 U Botox compared to V (from 3.7±0.6 to 1.5±0.1 and 1.4±0.3mm Hg, respectively; p<0.01 and p<0.001, respectively). Dysport (10 U) and Botox (7.5 U) significantly inhibited NVC by decreasing their amplitude (from 7.4±1.1 to 5.8±0.5 and 5.4±0.6mm Hg, respectively; p<0.05); frequency (from 2.2±0.4 to 1.5±0.2 and 1.3±0.3 NVC per minute, respectively; p<0.01); and increasing volume threshold to elicit NVC (from 29.8±3.7 to 47.6±6.9 and 47.7±6.3%, respectively; p<0.05 and p<0.001, respectively). CONCLUSIONS: This is the first preclinical dose-ranging study with Dysport and Botox under standardized conditions showing similar inhibiting effects on NDO, albeit at different MEDs. It highlights the importance of distinguishing each preparation for predicted outcomes and doses to be used. Further studies in patients with NDO are warranted to confirm these experimental results.

Long-term treatment with standardized Ginkgo biloba extract (EGb 761) attenuates cognitive deficits and hippocampal neuron loss in a gerbil model of vascular dementia.

The standardized extract of Ginkgo biloba EGb 761 has been used to reduce cognitive dysfunction. The present study was designed to evaluate the effect of postischemic oral treatment with EGb 761 in a model of vascular dementia in gerbils. Daily oral posttreatment with EGb 761 led to a significant recovery of spatial memory assessed by the object location test, inhibited the decrease in plasma SOD activity and protected the hippocampal CA1 neurons, even when administered after the insult. These data provide further evidence for the therapeutic potential of EGb 761 in the treatment of vascular dementia.

BN82451 attenuates L-dopa-induced dyskinesia in 6-OHDA-lesioned rat model of Parkinson's disease.

The development of L-dopa-induced dyskinesia (LID) remains a major problem in the long-term treatment of Parkinson's disease (PD). This study aimed to assess the effect of the multitargeting molecule BN82451 on LID and to measure striatal mRNA expression of several genes in a rat model of PD. Rats were administered two unilateral injections of 6-OHDA in the striatum. After four weeks, the animals started a chronic daily treatment with increasing doses of L-dopa over a further four-week period. Over the course of L-dopa treatment, the rats developed abnormal involuntary movements (AIMs) classified as locomotive, axial, orolingual and forelimb dyskinesia. In animals rendered dyskinetic by L-dopa, administration of BN82451 at doses ranging from 1 to 10 mg/kg p.o. attenuated the severity of fully-established AIMs in a dose-related manner. This anti-dyskinetic effect could be achieved with lower doses of BN82451 administered sub chronically vs. acute single treatment. The improvement of AIMs is not due to a reduction in the general motor activity of dyskinetic rats. BN82451 treatment significantly reversed the overexpression of c-Fos, FosB and Arc mRNA associated with the dyskinesiogenic action of L-dopa. A significant correlation between the degree of overexpression of c-Fos, FosB and Arc mRNA and the dyskinesiogenic action of L-dopa was observed. The data demonstrate that BN82451 effectively attenuates LID and the associated molecular alterations in an animal model of PD and may represent a treatment option for managing dyskinesia.

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.

Different antinociceptive effects of botulinum toxin type A in inflammatory and peripheral polyneuropathic rat models.

In addition to inhibition of acetylcholine release in the neuromuscular junction botulinum toxin type A (BoNT-A) also inhibits the release of mediators involved in pain perception. We have investigated the effect of two types of BoNT-A on mechanical hyperalgesia in the rat models of carrageenan-induced hyperalgesia and of paclitaxel-induced peripheral neuropathy. A subplantar (s.p.) injection of BoNT-A in the ipsilateral hindpaw 3 days before carrageenan administration reduced hypersensitivity. Dysport and Botox elicited comparable antihyperalgesic effects. Dysport up to 30 U/kg and Botox up to 20 U/kg did not impair the rat withdrawal nociceptive reflex or the locomotor performance as assessed by the rotarod test. Intraperitoneal administration of the skeletal muscle relaxant dantrolene produced, in contrast to BoNT-A, more motor impairment than analgesia. Paclitaxel treatment resulted in a peripheral neuropathy that affected the two hindpaws. Injection of 20 U/kg (s.p.) Dysport produced a significant antihyperalgesic effect in the injected paw of neuropathic animals 3 days after administration. Unexpectedly, a similar analgesic effect was observed in the contralateral paw. The same results were also observed when Botox was used instead of Dysport. In contrast, a contralateral administration of Dysport in the carrageenan test was ineffective. We conclude that BoNT-A elicits antinociceptive effects independent of the effects on muscular relaxation. Our results suggest that different mechanisms of action are responsible for the effect of BoNT-A in inflammatory and peripheral polyneuropathic rat models.

The novel inhibitor of the heterotrimeric G-protein complex, BIM-46187, elicits anti-hyperalgesic properties and synergizes with morphine.

BIM-46187 (7-[2-amino-1-oxo-3-thio-propyl]-8-cyclohexylmethyl-2-phenyl-5,6,7,8-tetrahydro-imidazo-[1,2a]-pyrazine dimer, hydrochloride) is an inhibitor of the heterotrimeric G-protein complex signalling. Since many mediators of pain act through G-protein coupled receptors, the anti-hyperalgesic effects of BIM-46187 were assessed on experimental models of pain. In addition since opioids are widely used in pain management and act through specific G-protein-coupled receptors, the effects of BIM-46187 on the analgesic properties of morphine have also been investigated. BIM-46187 elicited a dose dependent analgesic effect in the models of carrageenan-induced hyperalgesia (0.1-1 mg/kg; i.v.) and chronic constriction injury (0.3-3 mg/kg; i.v.) in rats. BIM-46187, however, up to 10 mg/kg did not modify the paw oedema induced by carrageenan excluding an anti-inflammatory effect. In addition, at these doses, the compound was not sedative as shown by the lack of effect on the motor performance in the rotarod test. The combination of BIM-46187 and morphine (ratio 1/1) resulted in an unexpected synergistic effect in the model of carrageenan-induced hyperalgesia and in the chronic constriction injury model in rats when evaluated by isobolographic analysis. This synergy allowed a reduction of at least 20 fold in the dose of each compound. Conversely, the drug combination did not increase the side effects of morphine as assessed in the rotarod test. In conclusion, BIM-46187 elicits a potent anti-hyperalgesic effect and strongly synergizes with morphine. This work highlights the role of heterotrimeric G-protein complexes in pain and supports further investigations of the use of BIM-46187 alone, or in combination with low doses of morphine, in the management of pain.

Pharmacological properties of BN82451: a novel multitargeting neuroprotective agent.

BN82451 belongs to a new family of small molecules designated as multitargeting or hybrid molecules. BN82451 is orally active, has good central nervous system penetration, and elicits potent neuronal protection and antiinflammatory properties. Neuronal protection is due to Na+ channel blockade, antioxidant properties, and mitochondria-protecting activity, whereas inhibition of cyclooxygenases is mostly responsible for its antiinflammatory activity. BN82451 has been shown to exert a potent neuroprotective effect in various in vitro and in vivo animal models. BN82451 was found to exert a significant protection in experimental animal models mimicking aspects of cerebral ischemia, Parkinson disease, Huntington disease, and more particularly amyotrophic lateral sclerosis. Collectively, its pharmacological properties designate BN82451 as a promising neuroprotective agent.

A novel dual inhibitor of calpains and lipid peroxidation (BN82270) rescues the cochlea from sound trauma.

Free radical and calcium buffering mechanisms are implicated in cochlear cell damage that has been induced by sound trauma. Thus in this study we evaluated the therapeutic effect of a novel dual inhibitor of calpains and of lipid peroxidation (BN 82270) on the permanent hearing and hair cell loss induced by sound trauma. Perfusion of BN 82270 into the scala tympani of the guinea pig cochlea prevented the formation of calpain-cleaved fodrin, translocation of cytochrome c, DNA fragmentation and hair cell degeneration caused by sound trauma. This was confirmed by functional tests in vivo, showing a clear dose-dependent reduction of permanent hearing loss (ED50 = 4.07 microM) with almost complete protection at 100 microM. Furthermore, BN82270 still remained effective even when applied onto the round window membrane after sound trauma had occurred, within a therapeutic window of 24 h. This indicates that BN 82270 may be of potential therapeutic value in treating the cochlea after sound trauma.

Butyl 2-(4-[1.1'-biphenyl]-4-yl-1H-imidazol-2-yl)ethylcarbamate, a potent sodium channel blocker for the treatment of neuropathic pain.

A series of 4-arylimidazole carbamates was synthesized and their binding affinities to the site-2 sodium (Na+) channel were determined. SAR studies led to the identification of compound 10, a potent Na+ channel blocker which was efficacious in pain models in vivo.

Calpain inhibitors and antioxidants act synergistically to prevent cell necrosis: effects of the novel dual inhibitors (cysteine protease inhibitor and antioxidant) BN 82204 and its pro-drug BN 82270.

Cell death is a common feature observed in neurodegenerative disorders, and is often associated with calpain activation and overproduction of reactive oxygen species (ROS). This study investigated the use of calpain inhibitors and antioxidants in combination to protect cells against necrosis. Maitotoxin (MTX), which induces a massive influx of calcium, was used to provoke neuronal cell death. This toxin increased, in a concentration-dependent manner, both calpain activity and ROS formation. Calpain inhibitors or antioxidants inhibited MTX-induced necrosis only marginally (below 20%), whereas their association protected against cell death by 40-66% in a synergistic manner. BN 82204, which possesses both calpain-cathepsin L inhibitory and antioxidant properties, and its acetylated pro-drug BN 82270, totally protected cells at 100 microm. The pro-drug BN 82270, which had better cell penetration, was twice as effective as the active principle BN 82204 in protecting glioma C6 or neuroblastoma SHSY5Y cells against death. These results suggest the potential therapeutic relevance of using a single molecule with multiple activities (cysteine protease inhibitor/antioxidant), and warrant further in vivo investigations in models of neuronal disorders.

2-Alkyl-4-arylimidazoles: structurally novel sodium channel modulators.

A series of 2-alkyl-4-arylimidazoles were prepared and their binding affinities to the site-2 sodium (Na+) channel were determined. SAR studies led to highly potent Na+ channel blockers.

Increased survival and neuroprotective effects of BN82451 in a transgenic mouse model of Huntington's disease.

There is substantial evidence that excitotoxicity and oxidative damage may contribute to Huntington's disease (HD) pathogenesis. We examined whether the novel anti-oxidant compound BN82451 exerts neuroprotective effects in the R6/2 transgenic mouse model of HD. Oral administration of BN82451 significantly improved motor performance and improved survival by 15%. Oral administration of BN82451 significantly reduced gross brain atrophy, neuronal atrophy and the number of neuronal intranuclear inclusions at 90 days of age. These findings provide evidence that novel anti-oxidants such as BN82451 may be useful for treating HD.

Neuroprotective effects of (S)-N-[4-[4-[(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2-thiophenecarboximid-amide (BN 80933), an inhibitor of neuronal nitric-oxide synthase and an antioxidant, in model of transient focal cerebral ischemia in mice.

Nitric oxide (NO) and reactive oxygen species are both implicated in neuronal death due to cerebral ischemia. BN 80933, an original compound associating an inhibitor of neuronal NO synthase with an antioxidant, has been shown to reduce functional and histological damage in rat submitted to cerebral ischemia. The aim of the present study was to confirm these results in mice and to further examine the effects of BN 80933 on inflammatory response, including blood-brain barrier (BBB) disruption, brain edema, and neutrophil infiltration after transient middle cerebral artery occlusion (MCAO). Intravenous administration of BN 80933 at 3 and 10 mg/kg 3 h after MCAO significantly reduced by 26 to 36% the infarct volume evaluated 24 and 48 h after ischemia, and improved the neurological score. Furthermore, BN 80933 at both dosages decreased by 42 to 75% the extravasation of Evans blue in brain parenchyma observed 24 h after ischemia. This reduction in BBB disruption was associated with decreased brain edema as demonstrated by the 37% reduction in brain water content induced by BN 80933 at 3 mg/kg 24 h after MCAO. Neutrophil infiltration in brain parenchyma, evaluated by the myeloperoxidase activity, was also reduced by 45 to 56% in animals treated with BN 80933 at 3 and 10 mg/kg. Together, these results extend the protective capacity of BN 80933 against brain ischemic injury and confirm that BN 80933 represents a promising treatment for stroke.

Severe muscle dysfunction precedes collagen tissue proliferation in mdx mouse diaphragm.

After extensive necrosis, progressive diaphragm muscle weakness in the mdx mouse is thought to reflect progressive replacement of contractile tissue by fibrosis. However, little has been documented on diaphragm muscle performance at the stage at which necrosis and fibrosis are limited. Diaphragm morphometric characteristics, muscle performance, and cross-bridge (CB) properties were investigated in 6-wk-old control (C) and mdx mice. Compared with C, maximum tetanic tension and shortening velocity were 37 and 32% lower, respectively, in mdx mice (each P < 0.05). The total number of active CB per millimeter squared (13.0 +/- 1.2 vs. 18.4 +/- 1.7 x 10(9)/mm(2), P < 0.05) and the CB elementary force (8.0 +/- 0.2 vs. 9.0 +/- 0.1 pN, P < 0.01) were lower in mdx than in C. The time cycle duration was lower in mdx than in C (127 +/- 18 vs. 267 +/- 61 ms, P < 0.05). Percentages of fiber necrosis represented 2.8 +/- 0.6% of the total muscle fibers, and collagen surface area occupied 3.6 +/- 0.7% in mdx diaphragm. Our results pointed to severe muscular dysfunction in mdx mouse diaphragm, despite limited necrotic and fibrotic lesions.